Research ArticleThe Correlation between Chemical Composition asDetermined by UPLC-TOF-MS and Acute Toxicity ofVeratrum nigrum L and Radix paeoniae alba

Xianxie Zhang Yuguang Wang Qiande Liang Zengchun Ma Chengrong XiaoHongling Tan and Yue Gao

Department of Pharmacology and Toxicology Beijing Institute of Radiation Medicine Taiping Road 27 Beijing 100850 China

Correspondence should be addressed to Yuguang Wang wangygbmiaccn and Yue Gao gaoyuebmiaccn

Received 6 March 2014 Revised 7 May 2014 Accepted 12 May 2014 Published 9 June 2014

Academic Editor Yuping Tang

Copyright copy 2014 Xianxie Zhang et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The eighteen incompatible medicaments is an important theory in traditional Chinese medicine The theory suggests that drugsin the eighteen incompatible medicaments can be toxic when used together Veratrum nigrum L and Radix paeoniae alba belongto the eighteen incompatible medicaments and have been prohibited for thousands of years This study offers preliminary insightinto the mechanism and chemical constituents responsible for the incompatibility and toxicity of these two agents Specifically weperformed toxicology studies to identify and quantify the constituent substances of the two agents Experiments revealed that acutetoxicity increases when the dose ofV nigrum L is higher than or equal to RPA UPLC-TOF-MS analysis showed that although thevolumes ofV nigrum L were the same the content of some veratrum alkaloids changed significantly and had a trend toward a highlypositive correlation (|119903| ge 08) with toxicity This suggests that the increased toxicity of the V nigrum L and RPA combination wasdue mainly to increased content of the special veratrum alkaloids The cytotoxicity of veratridine in SH-SY5Y cells was decreasedwith increasing paeoniflorin concentrations This study provides insight into the mechanism behind the incompatibility theory ofTCM

1 Introduction

Traditional Chinese medicine (TCM) is an important part ofChinese culture and makes significant contributions to theprosperity and health of the Chinese population TCM hasbecome more popular worldwide because of its efficacy andcurative effects However it is important to ensure that thesetreatments are safe Confirming the compatibility of TCMis currently the major method used to ensure its safety andefficacy [1] TCMhas formed a unique incompatibility theoryover thousands of years and the typical principles of prescrip-tions include the eighteen incompatible medicaments Thismeans that these specific agents can be toxic when used incombination [2] However some studies have used combi-nations of drugs in the eighteen incompatible medicamentsto treat incurable diseases [2] Therefore it is important todetermine whether these agents are incompatible when usedin combination and the reasons behind any incompatibility

In this study we used Veratrum nigrum L (V nigrumL) and Radix paeoniae alba (RPA) which are two agentsbelonging to the eighteen incompatible medicaments whoseconcurrent use has been prohibited for thousands of yearsAlthough there are no cases describing the compatibility ofV nigrum L and RPA in modern Chinese medicine fewerthan 20 prescriptions using a combination of V nigrum Land RPA have been described to treat incurable diseases suchas tumors hemorrhoids carbuncle and breast carbuncle [3]Therefore it is necessary to determine whether the use of Vnigrum L and RPA in combination should be prohibited aswell as the reasons for any incompatibility [4]

V nigrum L is the dried roots and rhizomes ofVeratrum nigrum L It has been used for medicinal purposesfor thousands of years in China and was used in Europeduring theMiddle Ages [5] despite its well-known poisonouscharacteristics [4] It is used to treat hypertension strokeexcessive phlegm and epilepsy However aqueous extracts

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2014 Article ID 892797 13 pageshttpdxdoiorg1011552014892797

2 Evidence-Based Complementary and Alternative Medicine

Table 1 Doses of V nigrum L and RPA in the 12 decoction groups

Group V nigrum L dose (g) RPA dose (g) Water (mL) Final volume (mL) Drug concentration ratio(V nigrum L RPA)

A 6415 06415 700 100 10 1B 6415 0802 700 100 8 1C 6415 1069 700 100 6 1D 6415 1604 700 100 4 1E 6415 3208 700 100 2 1F 6415 6415 700 100 1 1G 6415 1283 700 100 1 2H 6415 2566 700 100 1 4I 6415 3849 700 100 1 6J 6415 5132 700 100 1 8K 6415 6415 700 100 1 10L 6415 0 700 100 mdash

are toxic and irritate the digestive tract mucosa nucleus nervivagi and central nervous system [6 7]

RPA the dried root of Paeonia lactiflora Pall withoutbark has been used as a medicinal herb in traditionalChinese medicine for centuries and exerts a wide range ofpharmacological activities In ancient pharmacology RPAwas used to calm liver wind relieve pain nourish bloodregulate menstrual functions and suppress sweating [8] Inmodern pharmacology RPA decoctions could be used totreat rheumatoid arthritis systemic lupus erythematosushepatitis dysmenorrhea muscle cramping and spasms andlong-standing fever [4 9 10]

In this study UPLCTOF MS multivariate statisticalanalysis and typical metabolomics approach were used toidentify the key chemical markers that are responsible for theincreased toxicity and the combination of V nigrum L andRPA In addition cellular and mouse acute toxicity assayswere used to identify the toxic effects of this combinationThe compatibility of V nigrum L and RPA has not yet beenvalidated by modern science therefore it is important toprovide preliminary insight into the mechanism of theirreported incompatibility as well as to identify and quantifytheir toxic chemical constituents

2 Materials and Methods

21 Chemicals andMaterials RPA from Panan County (Zhe-jiang China lot 100714) andV nigrumL fromHuajia County(Changchun China lot 100701) were purchased from AnhuiBBCA Tongling Chinese herbal medicine company Formicacid (CNW Technologies GmbH) and acetonitrile (FisherScientific Fair LawnNJUSA)were both of chromatographicpurity Deionized water was prepared using aMillipore waterpurification system Veratridine (D00114760) was purchasedfrom Merck (Merck KGaA Germany) and paeoniflorin waspurchased from Sigma-Aldrich The MTS cell proliferationassay kit was purchased from Promega Roswell Park Memo-rial Institute 1640 (RPMI-1640) and fetal bovine serum (FBS)were purchased from Gibco BRL (Invitrogen USA)

22 Preparation of Decoctions Based on a previous studythe LD

50of aqueous extracts of V nigrum L and RPA after

intragastric administration were 2566 gkg and 160 gkgrespectively [11] In this study the dose of V nigrum L wasfixed at 2566 gkg and the RPA dose varied from 02566 to2566 gkg

The doses ofV nigrum L andRPA in each of 12 groups areshown in Table 1 Each group was extracted using deionizedwater (700mL) for 1 h during microboiling under refluxThe extracts were filtered through three layers of gauzeand the drug extraction was then repeated The filtrateswere combined and were then concentrated to 100mL at60minus70∘C under reduced pressure Samples were then shakencalibrated and stored at 4∘C

All decoctions were centrifuged at 13000 rpm for 10minusing a Heraeus Labofuge 400R refrigerated centrifuge(Thermo Scientific USA) The supernatants were then fil-tered through a 022120583M aqueous microporous membraneand were stored at 4∘C for UPLC-TOF-MS analysis Groupswere prepared in triplicate [12 13]

23 Animals and Experimental Design Seven-week-oldKun-ming (KM) mice (18minus22 g) were obtained from the Experi-mental Animal Center of the Academy of Military MedicalSciences with the certificate of conformity SCXK-(Army)2007-004 Animal subjects were housed at the SPF animalcenter of the Academy of Military Medical Sciences (BeijingChina) according to the regulations of the animal carecommittee Mice were housed at a constant temperature of25 plusmn 1

∘C with 50 plusmn 20 humidity with a 12 h lightdark cycleand 10ndash15 air changes per hour Mice were allowed free accessto water and food during the experimental period

Mice were acclimatized to the facilities and environmentfor 3 days before the experiments Two hundred and fortymice were randomized into 12 groups (AndashL) with 10 miceper group Food was removed from all animals 12 h beforethe experiments Mice received decoctions at 04mL10 g viagavage and were observed for 14 days

Evidence-Based Complementary and Alternative Medicine 3

Table 2 Mice mortality after the intragastric administration of different proportion decoctions of V nigrum L and RPA

Group V nigrum L dose (g) RPA dose (g) Crude drug concentration ratio Female mortality Male mortality Total mortalityA 6415 06415 10 1 100 90 95B 6415 0802 8 1 100 100 100C 6415 1069 6 1 100 60 80D 6415 1604 4 1 100 60 80E 6415 3208 2 1 90 70 80F 6415 6415 1 1 90 100 95G 6415 1283 1 2 50 70 60H 6415 2566 1 4 60 60 60I 6415 3849 1 6 0 20 10J 6415 5132 1 8 0 0 0K 6415 6415 1 10 0 0 0L 6415 0 0 70 70 70

24 UPLC-MS

241 Liquid Chromatography UPLC was performed on aWaters Acquity UPLC system (Waters Milford MA USA)equipped with a binary solvent delivery system an autosam-pler and a photodiode-array detection (PDA) system Chro-matography was performed using a Waters ACQUITYBEHC

18column (100mm times 21mm 17 120583m) [12] The mobile

phase consisted of (A) water containing 01 formic acidand (B) acetonitrile containing 01 formic acid The elutingconditions were as follows isocratic 2 B (0-1min) lineargradients of 2minus5 B (1-2min) 5ndash20 B (2ndash5min) 20ndash30B (5ndash7min) 30ndash33 B (7ndash10min) 33ndash36 B (10ndash13min)36ndash40 B (13ndash17min) 40ndash100 B (17-18min) 100ndash2 B (18-19min) and 2 B (19-20min)The flow rate was 05mLminThe sample chamber was maintained at 4∘C with the columnat 45∘CThe injection volume was 5 120583L [12 14 15]

242 Mass Spectrometry Mass spectrometry was performedusing aWaters SYNAPTmass spectrometer equippedwith anelectrospray ionization (ESI) source Samples were injectedtwice once in positive ESI mode and once in negative ESImode The data acquisition range was 100ndash1500Da The lockspray reference scan frequencywas 20 s with a reference conevoltage of 30V The MS source temperature was set at 100∘Cand the desolvation temperature was 450∘C with a gas flowof 900 LhThe lock mass compound was leucine enkephalin(200 pg120583L) with anmz of 5562771 in the positive ionmodeand 5542615 in negative ion mode The capillary voltageswere set to 29 kV for ESI+ and 3 kV for ESIminus The conevoltage was 40 kV and the collision energies were 6V (trap)and 4V (transfer) with 200mLmin trap gas flow [16]

25 SH-SY5Y Cell Cultures and Cell Proliferation Assay SH-SY5Y cells (ATCC Manassas USA) were cultured in RPMI-1640 supplemented with 10 FBS Cells were maintained at37∘C in an incubator with a saturated humidity atmosphereof 95 air and 5 CO

2 They were cultured in a 96-well plate

and treated with different drug combinations An MTS cellproliferation assay was performed and theOD at 490 nmwasread using a VICTOR X plate reader (Perkin Elmer USA)

26 Data Analysis The UPLC-TOF-MS data of all sampleswere analyzed using MassLynx41 software (Waters Manch-ester UK) and principal component analysis (PCA) wasused for data analysis Pearson correlation coefficients wereused to identify relationships between the study parame-ters and mortality The chemical markers in each groupwere identified using the V nigrum L and RPA chemicaldatabases [12] For data analyses we compared the correlationbetween chemical composition and the acute toxicity of Vnigrum L and RPA between the 12 groups Experimentalvalues are expressed as means plusmn standard deviations (SD)Statistical analyses were performed using two-tailed Studentrsquos119905-tests A value of 119875 lt 001 was considered statisticallysignificant

3 Results and Discussion

31 Mouse Acute Toxicity Mice in the 12 groups wereadministered the decoctions by intragastric administrationDeath occurred 5minus10min after administration Toxicity wasmanifested predominantly as trembling convulsions andspasms The major organs showed no obvious lesions atnecropsy by the naked eye [6 17 18] Mouse mortality isshown in Table 2

Group L (with aV nigrum L dose of the LD502566 gkg)

had a mortality of 70 Groups AminusF were treated with thesame dose of V nigrum L but the groups fed a lower ratioof RPA had a higher mortality than group L Groups GminusKhad a higher proportion of RPA and lower mortality thangroup L suggesting that toxicity was minimized Groups Band F were the most toxic with mortalities of 100 and95 respectively Overall analyses showed that mortalityincreasedwhen the dose ofV nigrum L was less than the doseof RPA Conversely toxicity decreased when the proportionof RPA increased The trends in mortality are shown inFigure 1

Female mouse mortality decreased with increasing pro-portions of RPA whereas males mortality exhibited twopeaks at groups B and F which both had 100 (Figure 2)

4 Evidence-Based Complementary and Alternative Medicine

0

20

40

60

80

100

120

Mor

talit

y ra

te (

)

A B C D E F G H I J K LGroup

Figure 1 Total mortality of mice after the intragastric administra-tion of decoctions containing different proportions of V nigrum Land RPA The groups represent the combinations shown in Table 1

FemaleMale

0

20

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60

80

100

120

Mor

talit

y ra

te (

)

A B C D E F G H I J K LGroup

Figure 2 Mortality of male and female mice after the intragastricadministration of decoctions containing different proportions of Vnigrum L and RPA The groups represent the combinations shownin Table 1

32 Identification and Quantification of the Chemical Com-ponents of V nigrum L and RPA To explain the cause ofacute toxicity of the combined use of V nigrum L andRPA we compared the chemical composition of the 12groups using a supervised orthogonal partial least squareddiscriminant analysis (OPLS-DA) After Pareto scaling withmean centering the data from both the positive and negativeion modes were displayed as scores plots (Figure 3) Thescores plots clearly revealed that the samples were clustered

minus40

minus20

0

20

40

minus100 0 100

A BC DE FG HI JK L

t[1]

t[2]

Scores comp[1] versus comp[2] colored by sample group

(a)

minus50

0

50

minus200 minus100 0 100 200

A BC DE FG HI JK L

Scores comp[1] versus comp[2] colored by sample group

t[1]

t[2]

(b)

Figure 3 BBThe OPLS-DA score plot of 36 samples obtained usingPareto scaling with mean centering (a) Positive ion mode (b)Negative ion mode The same score plots represent the same groupBoth the positive and negative ion modes showed that the sampleswere clearly clustered into 12 groups Replicates of the 12 groupscould be distinguished easily

into 12 groups replicates of same group were comparable butthe 12 groups could be distinguished easilyThis suggests thatthe changes in chemical compositions of the 12 groups wereconsistent and the experiment was reproducible

UPLC-TOF-MS is a rapid specific and sensitive methodused to identify and quantify individual components Repre-sentative BPI chromatograms of the 36 samples in the positiveand negative mode ESI are shown in Figure 4

The contents of some special alkaloids change signif-icantly in different proportion decoctions To determinewhich constituents contributed to the differences in toxicity

Evidence-Based Complementary and Alternative Medicine 5

F-P-2

95

()

minus5

2481128

4763010

4603062

44632764422959

5363228

4443088

4263004

4103042

5763557

6763667

7163999

108

9

7

3

2

1

465

200 400 600 800 1000 1200 1400 1600 1800

Time

BPI103e5

1 TOF MS ES+

(a)

43909591690150 2550515

5431194

5671754

5251649

4491475

2270695

6311725

6311760

6291927

3292323

10

8

97

3

2

1

4

65

200 400 600 800 1000 1200 1400 1600 1800

Time

BPI430e495

minus5

()

5-2 neg1 TOF MS ESminus

(b)

Figure 4 Representative BPI chromatograms (a) Positive ionmode (b) Negative ionmode Some of identified constituents (1ndash10) are labeledin the BPI chromatogram and the numbers correspond to Tables 3 and 4

observed with the 12 decoctions statistical analyses wereperformed using Pearsonrsquos correlation and the correlationcoefficient (119903) described the degree of linear correlationbetween two variables total mouse mortality and the chem-ical composition of the 12 decoctions The 119903 values werebetween minus1 and +1 119903 gt 0 indicates that two variables werepositively correlated whereas 119903 lt 0 indicates a negativecorrelation The larger the absolute value the higher thecorrelation therefore we defined 119903 = 090ndash100 as extremelyrelated and 119903 = 080ndash089 as highly related We extractedthe relevant chemical component data when 119903 ge 08 andidentified the individual components using the RPA and Vnigrum L chemical composition databases

According to Pearsonrsquos correlation coefficient 131 compo-sitions had an extremely positive relationship (119903 ge 09) withtoxicity However only 14 of these could be identified andall came from V nigrum L In addition 35 components werehighly positively correlated (119903 ge 08) to acute toxicity andthese constituents contributed most of the toxicity identifiedin the 12 decoctions Therefore we hypothesized that thesecomponents might play important roles in the acute toxicity

of this drug combination We identified that these com-poundswere all veratrum alkaloids that were detected in eachsample in various ionized forms The chemical componentswith 119903 ge 08 are shown in Table 3 [7 19ndash24]

3-Veratroylgermine jervine veratramine germanitrinegermidine and germerine were the main chemical compo-nents that were associated with acute toxicity with Pearsonrsquoscorrelation coefficients of 09779 09661 09589 0958109402 and 09393 respectively These compounds are allveratrum alkaloids and the changes in their content areshown in Figure 5The other 29 components that were highlypositively correlated (119903 ge 08) with acute toxicity are shownin Figure 6

Interestingly Pearsonrsquos correlation coefficient identified24 RPA components that were extremely negatively corre-lated (119903 le minus09) with toxicityThe chemical components with119903 le minus09 are shown in Table 4 [25ndash33]

The concentrations of these components increased withincreasing amounts of RPA Paeoniflorin sulfonate ben-zoyloxy paeoniflorin galloyl paeoniflorin paeonilacto-neBpaeonilacto-neC and tetragalloyglucose were the major RPA

6 Evidence-Based Complementary and Alternative Medicine

Table 3 The chemical components that were highly positively correlated with toxicity (119903 ge 08)

Number 119903 tR (min) Assigned identity Molecularformula

Mean measuredmass (Da)

Theoretical exactmass (Da)

Mass accuracy(ppm)

1 09779 1298 3-Veratroylgermine C36H53NO11 6763667 6758061 018052 09661 1239 Jervine C27H39NO3 4263004 4256035 236243 09589 1261 Veretramine C27H39NO2 4103042 4096041 297514 09581 1343 Germanitrine C39H59NO11 7184169 7178859 250155 09402 1220 Germidine C34H53NO10 6363412 6357853 057556 09393 1340 Germerine C37H59NO11 7163999 6938645 034087 09390 1291 3-Angeloylzygadenine C32H49NO8 5763557 5757334 127088 09311 1035 11-Deoxojervine C27H41NO2 4123189 4116199 64392

9 09240 157 11205733120572-Dihydroxy-5120573-jervanin-12-en-11-one C27H41NO4 4443088 4436187 24040

10 09237 696 Jervinone C27H37NO3 4242860 4235876 2014011 09182 1125 Neogermbudine C37H59NO12 7323971 7098639 4916912 09143 425 Germine C27H43NO8 5103059 5096322 15549

13 09131 418 NeogermineVeramanine C27H43NO5 4623213 4616340 14522

14 09034 1158 Veraline B C27H45NO3 4323465 4316511 2964615 08978 1116 Verdine C27H41NO5 4603056 4596181 1524916 08903 1163 Stenophylline A C36H51NO11 6743474 6737902 9865517 08870 1035 Germitrine C39H61NO12 7364278 7359011 0828818 08805 914 Polydatin C20H22O8 4290937 3903839 3484919 08738 1081 Cervdine C32H49NO9 5923498 5917328 20608

20 08675 1234 3-Veratrum acylprotoveratrine C36H51NO12 6903482 6897896 10609

21 08671 1289 Zygadenitic acid120575-lactone-16-angelate C32H47NO8 5743408 5737175 48884

22 08652 1232 Maackinine C39H59NO11 7184171 7178859 06797

23 08641 1238Rubijervine rubivirineetioline epirubijervine

IsorubijervineC27H43NO2 4143357 4136358 36827

24 08639 1049 Solanidine C27H43NO3 4303308 4296352 3049425 08492 1057 Pseudojervine C32H49NO8 5883525 5877441 1971026 08444 1181 Neojerminalanine C39H61NO13 7524244 7519005 3001427 08379 843 Zygacine C29H45NO8 5363223 5356695 0000028 08358 1320 Neoverataline B C27H42N2O9 5563260 5386304 407929 08201 1280 3-Veratroylzygadenine C36H51NO10 6963187 6577908 5346930 08176 583 Zygadenine C27H43NO7 4943110 4936328 1543431 08176 054 120574-Aminobutyric acid C4H9NO2 2460978 1031198 1612132 08160 1273 Veratrosine C33H49NO7 6163528 5717447 6832933 08128 1322 Stenophylline A C37H55NO10 6743901 6738333 05430

34 08119 13347-Acetyl-15-

methylbutyryl-3-veratroylgermine

C43H61NO13 8383791 7999433 13104

35 08017 1213 Vanilloylzygadenine C35H49NO10 6443444 6437643 15156

components that were identified with Pearsonrsquos correlationcoefficients of minus09811 minus09799 minus09793 minus09737 minus09736and minus09734 respectively The changes in content of the 24components that were highly negatively correlated (119903 le minus09)with acute toxicity are shown in Figure 7

33 Cell ProliferationAssay To further validate the toxicity ofthe combination ofV nigrumL andRPAonneurons we usedveratridine and paeoniflorin the major pharmacologicallyactive components of V nigrum L and peony for in vitroexperimentsThe effect of these two chemical components on

Evidence-Based Complementary and Alternative Medicine 7

Table 4 The chemical components that were highly negatively correlated with toxicity (119903 le minus09)

Number 119903 tR (min) Assigned identity Molecularformula

Mean measuredmass (Da)

Theoretical exactmass (Da)

Mass accuracy(ppm)

1 minus09811 495 Paeoniflorinsulfonate C23H28O13S 5431194 5445256 337142 minus09799 1220 Benzoyloxy paeoniflorin C30H32O13 5991785 6001843 030463 minus09793 977 Galloyl paeoniflorin C30H32O15 6311725 6321741 038564 minus09737 1479 Paeonilacto-ne B C17H18O6 3171750 3181103 392095 minus09736 056 Paeonilacto-ne C C10H12O4 1950496 1960736 531926 minus09734 809 Tetragalloyglucose C34H28O22 7871008 7885729 566077 minus09728 230 Vanillic acid C7H6O5 1690161 1701195 374718 minus09716 1391 Desbenzoyl paeoniflorin C16H24O10 3751868 3761369 445059 minus09708 1352 Benzoyl paeoniflorin C30H32O12 5831848 5845679 06258

10 minus09684 415 6-O-120573-D-Glucopyranosyllactinolide C16H26O9 3611537 3623722 93435

11 minus09670 1037 Paeoniflorin albiflorin R1albiflorin mudanpioside I C23H28O11 5191261 4804618 15284

12 minus09655 1167 Gallic acid C9H10O2 1510750 1501745 5959013 minus09640 493 Paeonol C9H10O3 1650545 1661739 4067614 minus09625 1167 Lactiflorin C23H26O10 4801872 4624465 0444915 minus09609 667 Isomaltopaeoniflorin C29H38O16 6432221 6426024 2656916 minus09589 852 Albiflorin C23H27O11 9573118 4794539 9372317 minus09582 679 11015840-O-Benzoylsucrose C19H26O12 4641757 4464025 2366818 minus09567 1057 Pentagalloylglucose C41H32O26 9391166 9406772 6629219 minus09562 802 Oxypaeoniflorin C23H28O12 5141934 4964612 1899220 minus09544 1158 Ethyl gallate C9H10O5 2210433 1981727 3244521 minus09478 1011 Eugeniin C41H30O26 9371010 9386613 6708622 minus09395 585 Paeonilactinone C9H14O2 1530904 1542063 7575023 minus09352 113 Lactinolide C10H16O4 2011111 2002316 7890724 minus09173 1373 Palbinone C22H30O4 3572047 3584712 52115

3-Veratroylgermine250

200

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50

0A B C D E F G H I J K L

Ion

inte

nsity

Sample group

Ion

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Sample group

0010203040506070809 Veratramine

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A B C D E F G H I J K LSample group

Jervine

A B C D E F G H I J K L

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Sample group

020406080100120140160180200

Grmidine

A B C D E F G H I J K L

Germanitrine

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A B C D E F G H I J K L

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06 Germerine

minus01

Figure 5 Ion intensity trend plots for 3-veratroylgermine jervine veratramine germanitrine germidine and germerine which were highlypositively correlated (119903 ge 09393) with toxicity

8 Evidence-Based Complementary and Alternative Medicine

0100200300400500600700800

A B C D E F G H I J K L A B C D E F G H I J K L

Ion

inte

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A B C D E F G H I J K L

A B C D E F G H I J K L

A B C D E F G H I J K LSample groupSample group

Sample group A B C D E F G H I J K LSample group

Sample groupA B C D E F G H I J K L

Sample group

3-Angeloylzygadenine

0

01

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07

Ion

inte

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Ion

inte

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11-Deoxojervine

0

50

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Sample group

Ion

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Sample group

A B C D E F G H I J K L0

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06 Jervinone

0

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15

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3 Neogermbudine

0020406081

121416182 Germine

0051

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35 Veraline B

0

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6 Verdine

Ion

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A B C D E F G H I J K LSample group

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n in

tens

ity

A B C D E F G H I J K LSample group

0

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Polydatin

minus05

minus05

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Ion

inte

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A B C D E F G H I J K LSample group

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Stenophylline A

minus05

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Neogermineveramanine

jervanin-12-en-11-one

0

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A B C D E F G H I J K L

Ion

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Cervdine

A B C D E F G H I J K L

Ion

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Sample group

0

02

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08

1

12 Maackinine

Ion

inte

nsity

A B C D E F G H I J K LSample group

0123456789 Rubijervine

A B C D E F G H I J K L

Ion

inte

nsity

Sample group

0020406081

12141618 3-Veratrum acyl protoveratrine

Ion

inte

nsity

A B C D E F G H I J K LSample group

0051

152

253

354

Solanidine

minus05

A B C D E F G H I JK L

Ion

inte

nsity

Sample group

0020406081

121416182

Zygadenitic acid 120575-Lactone-16-angelate

11205733a-Dihydroxy-5120573-

(a)

Figure 6 Continued

Evidence-Based Complementary and Alternative Medicine 9

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

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Ion

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A B C D E F G H I J K LSample group

0

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12

14 Neojerminalanine

Ion

inte

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A B C D E F G H I J K LSample group

0

02

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1

12 Zygacine

Ion

inte

nsity

A B C D E F G H I J K LSample group

012345678 Zygadenine

Ion

inte

nsity

A B C D E F G H I J K LSample group

000501015020250303504045 Neoverataline B

minus005

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

01

02

03

04

05

06 3-Veratroylzygadenine

minus01

0

05

1

15

2

25 Vanilloylzygadenine

A B C D E F G H I J K LSample group

Ion

inte

nsity

A B C D E F G H I J K LSample group

Ion

inte

nsity

0

2

4

6

8

10

12 Veratrosine

A B C D E F G H I J K LSample group

Ion

inte

nsity

0

2

4

6

8

10

12 Stenophylline A

A B C D E F G H I J K LSample group

Ion

inte

nsity

0020406081

1214 120574-Aminobutyric acid

minus02

A B C D E F G H I J K LSample group

Ion

inte

nsity

0

02

04

06

08

1

12 7-Acetyl-15-methylbutyryl-3-veratroylgermine

(b)

Figure 6 Ion intensity trend plots for the remaining 29 chemical components that were highly positively correlated (119903 ge 08) with toxicity

the viability of SH-SY5Y cells was tested using MTS assaysAs shown in Figures 8(a) and 8(b) veratridine had an LD

50

of 450 whereas paeoniflorin exerted no significant toxicityat doses of 0ndash2000120583M Therefore the dose of veratridinewas fixed at 400120583M and the RPA dose was varied from50 to 2000120583M SH-SY5Y cell cytotoxicity was increasedwhen the concentration of paeoniflorin was decreased inthe combination (Figure 8(c)) Therefore paeoniflorin couldoffset the cytotoxicity caused by veratridine in SH-SY5Y cells[25]

4 Discussion

The eighteen incompatible medicaments (Shi Ba Fan) is awell-known traditional Chinese medicine (TCM) theory It

basically listed eighteen pairs of TCM herbal medicine thatare not to be used in combination as they may cause fatalconsequences This principal has been used as guidancefor general TCM practices for hundreds of years with littlechemical comprehension In this work for the first time weused the UPLC-TOFMS coupled with multivariate statisticalanalysis in an effort to gain scientific understanding for thecauses of the incompatibility

We previously reported that the LD50of aqueous extracts

of V nigrum L and RPA were 2566 gkg and 160 gkgrespectively after intragastric administration [11] V nigrumL extract was severely toxic whereas RPA had minimaltoxicity Although the amount of V nigrum L in each groupwas unchanged the mortality changed significantly withaltered ratios of V nigrum L to RPA When the doses of

10 Evidence-Based Complementary and Alternative Medicine

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

1

2

3

4

5

6 Tetragalloyglucose

Ion

inte

nsity

A B C D E F G H I J K LSample group

0020406081

121416182 Vanillic acid

Ion

inte

nsity

A B C D E F G H I J K LSample group

minus2

0

2

4

6

8

10

12 Benzoylpaeoniflorin

000501015020250303504

A B C D E F G H I J K L

Ion

inte

nsity

Sample groupminus005

Paeoniflorin sulfonate

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

05

1

15

2 Benzoyloxypaeoniflorin

minus05

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

05

1

15

2 Gallic acid

minus05

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

05

1

15

2

253 Galloylpaeoniflorin

minus05

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

005

01

015

02

025

03

035 Paeonilacto-neC

Ion

inte

nsity

A B C D E F G H I J K LSample group

0123456789 Paeonilacto-neB

minus1

Ion

inte

nsity

A B C D E F G H I J K LSample group

Vanillic acid

0

1

2

3

4

5

6

7

minus1

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

01

02

03

04

05

06

07

minus01

6-O-120573-D-Glucopyranosyllactinolide

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

05

1

15

2

25

3

35

minus05

Paeoniflorinalbiflorin R1albiflorinmudanpioside I

A B C D E F G H I J K L

Ion

inte

nsity

Sample group

Paeonol

0

02

04

06

08

1

12

minus02

Ion

inte

nsity

A B C D E F G H I J K LSample group

01020304050607080 Pentagalloylglucose

minus10

Ion

inte

nsity

A B C D E F G H I J K LSample group

00102030405060708 Lactiflorin

minus01

Ion

inte

nsity

A BC D E F G H I J K LSample group

0020406081

121416 Isomaltopaeoniflorin

minus02

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

05

1

15

2

25

3

minus05

Albiflorin

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

05

1

15

2

25 1998400-O-Benzoylsucrose

(a)

Figure 7 Continued

Evidence-Based Complementary and Alternative Medicine 11

Ion

inte

nsity

Ion

inte

nsity

Ion

inte

nsity

A B C D E F G H I J K LSample group A B C D E F G H I J K L

Sample group

A B C D E F G H I J K LSample group

00102030405060708 Oxypaeoniflorin

001020304050607 Eugeniin

Ion

inte

nsity

A B C D E F G H I J K LSample group

0020406081

12141618 Paeonia lactinone

00102030405060708091 Ethyl gallate

minus01

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

01

02

03

04

05

Lactinolide06

minus01

0

5

10

15

20

25 Palbinone

Ion

inte

nsity

A B C D E F G H I J K LSample group

minus5

minus01

(b)

Figure 7 Ion intensity trend plots for the chemical components that were highly negatively correlated with toxicity (119903 ge 09)

OD

val

ue

25 50 100 200 400 800

lowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

mdashVeratridine (120583M)

10

12

08

06

04

02

0

(a)

OD

val

ue

02

04

06

08

12

Paeoniflorin (120583M) 100 500 1500 2000mdash 1000

10

00

(b)

OD

val

ue

Paeoniflorin (120583M)

mdashmdash

Veratridine (120583M) 400 400 400 400 400 400 400 400 400 400

50 100 250 500 750 1000 1250 1500 1750 2000

02

04

06

08

12

10

00

lowastlowastlowast

lowastlowast

lowastlowast lowastlowast

lowastlowast

lowast

(c)

Figure 8 SH-SY5Y cell viability SH-SY5Y cells were cultured with (a) veratridine (25ndash800120583M) (b) paeoniflorin (50minus2000120583M) and (c)400 120583M veratridine in combination with 50minus2000120583M paeoniflorin for 24 h Cell viability was then assessed using MTS assays Data arepresented as mean plusmn SD Means of three independent experiments Asterisks indicate a statistically significant difference compared withuntreated cells lowast119875 lt 005 lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001 versus controL

12 Evidence-Based Complementary and Alternative Medicine

V nigrum L were equal to or higher than RPA particularlyratios of 8 1 and 1 1 mouse mortality increased significantlyTherefore components of V nigrum L were the majorcausative factors of toxicity In addition RPA modulated thetoxicity ofV nigrum LTherefore clinical applications shouldbe monitored carefully

Mice died 5minus10min after drug administration Toxicitymanifested predominantly as trembling convulsions andspasms but the major organs showed no obvious lesionsat necropsy by the naked eye Based on the toxic reactionsand the time of death in the current study and accord-ing to the guidelines for Chinese natural medicine acutetoxicity testing we speculate that the toxicity was causedpredominantly by suppression of the central nervous systemand that V nigrum L was the major cause of death Vnigrum L water extracts exert toxic effects on the digestivetract mucosa nucleus nervi vagi and central nervous system[6 17 18] Pharmacological studies of veratrum alkaloidshave been performed previously and reported that thesealkaloids stimulate the central nervous system and inhibit thebrain This results in spasms convulsions coma drowsinessand other symptoms of consciousness in animals which isconsistent with the symptoms of themice in the current study[6 17]

Veratrum alkaloid is the toxic component of V nigrum LWe previously investigated the differently expressed genes inSH-SY5Y cells treated with veratridine using cDNAmicroar-rays [34] Data revealed that veratridine treatment alteredthe expression of MMP caused Ca2+ concentration over-load increased reactive oxygen species production enhancedLDH release damaged the cell membrane reduced SH-SY5Y cell viability and induced apoptosis Under oxidizingconditions the MAPK signaling pathway is activated whichincreases apoptosisThis study illustrates the neurotoxicity ofthe combination of V nigrum L and reveals the molecularmechanism behind the neurotoxic effects of V nigrum L(data not shown)

The amount of V nigrum L in each group was the samethe dose of RPA is in ascending order and the chemicalcomponents of RPA are also increased but the mortalityof each group changed significantly with the increase ofRPA so we conclude that higher amount of RPA can haveprotection effect which can offset the toxicity of combinationof V nigrum L and RPA And pharmacological studies havedemonstrated that RPA exerts a wide range of pharmacolog-ical activities including analgesic sedative anticonvulsantand antispasmodic effects on the central nervous systemRPA could also inhibit writhing reactions and antagonizepentylenetetrazol-induced convulsions The most importantactive ingredient in RPA is paeoniflorin which has significantneuroprotective effects [26ndash34]

In this study mouse acute toxicity experiments revealedthat the mortality of mice changed significantly when thedose of V nigrum L was higher than or equal to that ofRPA particularly at ratios of V nigrum L RPA of 8 1and 1 1 UPLC-Q-TOFMS with automated data analysisused to identify and quantify the specific chemical compo-nents According to Pearsonrsquos correlation coefficient most of

the chemical components that were positively correlated withtoxicity were from V nigrum L We conclude that the majorveratrum alkaloids of V nigrum L caused acute toxicity anddeath in mice When the doses of V nigrum L were higherthan or equal to RPA the main chemical components of Vnigrum L and the toxicity of the decoctions were increasedThis suggests that V nigrum L and RPA have opposingroles Additional experiments revealed that the cytotoxicityof veratridine in SH-SY5Y cells was decreasedwith increasingconcentrations of paeoniflorin Therefore the clinical use ofthese agents should be considered carefully This study alsoprovides information to help improve the incompatibilitytheory of TCM and introduces novel ideas for further studieson the development and application of TCM

When preparing V nigrum L and RPA decoctions alower amount of RPA contributes to the dissolution of vera-trum alkaloids Conversely higher amounts of RPA can exertneuroprotective effects which can offset the toxicity causedby the combination of V nigrum L and RPA Under theseconditions dissolution is the most important factor for Vnigrum L toxicity The results of cell-based experiments alsodemonstrated that paeoniflorin could offset the neurotoxiceffects of veratridine when treated in combination whichexcluded the factor of dissolution in preparation

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors are grateful for financial support from NationalNature Science Foundation of China (Grant no 81073149)National Basic Research Program of China (Grant nos2011CB505304 and 2012CB518402) and Natural ScienceFoundation of Beijing (Grant no 7112110)

References

[1] H Guo W Li X Y Wang and G W Fan ldquoAdvance of researchon toxic attenuation by compatibility of traditional Chinesemedicine prescriptionsrdquo Zhongguo Zhongyao Zazhi vol 37 no1 pp 120ndash123 2012

[2] Y Zhang H Hua X Fan C Wang and J Duan ldquoExplorationon eighteen incompatible medicaments of chest pain prescrip-tions based on association rules miningrdquo Zhongguo ZhongyaoZazhi vol 36 no 24 pp 3544ndash3547 2011

[3] C-H Xu P Wang Y Wang et al ldquoPharmacokinetic compar-isons of two different combinations of Shaoyao-Gancao Decoc-tion in rats competing mechanisms between paeoniflorin andglycyrrhetinic acidrdquo Journal of Ethnopharmacology vol 149 no2 pp 443ndash452 2013

[4] D H TianDictionary of Practical Traditional Chinese MedicineII Peoplersquos Medical Beijing China 2002

[5] Y Gaillard and G Pepin ldquoLC-EI-MS determination of ver-atridine and cevadine in two fatal cases of Veratrum albumpoisoningrdquo Journal of Analytical Toxicology vol 25 no 6 pp481ndash485 2001

Evidence-Based Complementary and Alternative Medicine 13

[6] H Li G-Y Gao S-Y Li W-J Zhao Y-T Guo and Z-J Liang ldquoEffects of Veratrum nigrum alkaloids on centralcatecholaminergic neurons of renal hypertensive ratsrdquo ActaPharmacologica Sinica vol 21 no 1 pp 23ndash28 2000

[7] L Wang W Li and Y Liu ldquoHypotensive effect and toxicologyof total alkaloids and veratramine from roots and rhizomesof Veratrum nigrum L in spontaneously hypertensive ratsrdquoPharmazie vol 63 no 8 pp 606ndash610 2008

[8] Y P Jiang Y G Liu and H C Chen ldquoEffect of aqueous extractof Radix Paeoniae Rubra against Carbon Tetrachloride inducedliver fibrosis in ratrdquo Herald of Medicine vol 23 pp 527ndash5292004

[9] D-Y He and S-M Dai ldquoAnti-inflammatory and immunomod-ulatory effects of Paeonia lactiflora Pall a traditional Chineseherbal medicinerdquo Frontiers in Pharmacology 2011

[10] C Feng M Liu X Shi et al ldquoPharmacokinetic properties ofpaeoniflorin albiflorin and oxypaeoniflorin after oral gavage ofextracts of Radix Paeoniae Rubra and Radix Paeoniae Alba inratsrdquo Journal of Ethnopharmacology vol 130 no 2 pp 407ndash4132010

[11] X X Zhang Y G Wang Z C Ma Y Gao and C R XiaoldquoUniform designed research on the compatibility toxicity ofVeratridine and peonyrdquo Zhong Hua Zhong Yi Yao Za Zhi vol28 no 10 pp 2901ndash2904 2013

[12] Q D Liang Y Gao J Ma et al ldquoChemical comparison of driedrehmannia root and prepared rehmannia root by UPLC-TOFMS andHPLC-ELSDwithmultivariate statistical analysisrdquoActaPharmaceutica Sinica B vol 3 no 1 pp 55ndash64 2013

[13] Y Wang S He X Cheng Y Lu Y Zou and Q Zhang ldquoUPLC-Q-TOF-MSMS fingerprinting of traditional Chinese formulaSiJunZiTangrdquo Journal of Pharmaceutical and Biomedical Analy-sis vol 80 pp 24ndash33 2013

[14] L-P Kang K Yu Y Zhao et al ldquoCharacterization of steroidalglycosides from the extract of Paris Polyphylla var Yunnanensisby UPLCQ-TOFMSErdquo Journal of Pharmaceutical and Biomed-ical Analysis vol 62 pp 235ndash249 2012

[15] A Kaufmann P Butcher K Maden and M Widmer ldquoUltra-performance liquid chromatography coupled to time of flightmass spectrometry (UPLC-TOF) a novel tool for multiresiduescreening of veterinary drugs in urinerdquoAnalytica Chimica Actavol 586 no 1-2 pp 13ndash21 2007

[16] S Zhou Z C Ma Q D Liang et al ldquoUPLCQ-TOF-MS basedchemical profiling approach to evaluate chemical compositionof augmentation toxicity in combination of Radix aconiti andPinellia praeparatardquoActa Chimica Sinica vol 70 no 3 pp 284ndash290 2012

[17] N Lin and X Gao ldquoExperimental observation on the absorp-tion of Veratrum nigrum L in the upper digestive tractrdquoZhongguo Zhong Yao Za Zhi vol 17 no 1 pp 43ndash45 1992

[18] Z-Z Wang W-J Zhao X-S Zhang et al ldquoProtection ofVeratrum nigrum L var ussuriense Nakai alkaloids againstischema-reperfusion injury of the rat liverrdquo World Journal ofGastroenterology vol 13 no 4 pp 564ndash571 2007

[19] V Christov B Mikhova A Ivanova et al ldquoSteroidal alka-loids of Veratrum lobelianum Bernh and Veratrum nigrum LrdquoZeitschrift fur Naturforschung C Journal of Biosciences vol 65no 3-4 pp 195ndash200 2010

[20] V Christov BMikhova andD Selenge ldquo(-)-Veranigrine a newsteroidal alkaloid fromVeratrum nigrum Lrdquo Fitoterapia vol 80no 1 pp 25ndash27 2009

[21] Y Cong J-H Wang R Wang Y-M Zeng C-D Liu and XLi ldquoA study on the chemical constituents of Veratrum nigrum

L processed by rice vinegarrdquo Journal of Asian Natural ProductsResearch vol 10 no 7-8 pp 619ndash624 2008

[22] H-L Li J Tang R-H Liu C Zhang and W-D Zhang ldquoTwonew flavanone glycosides from Veratrum nigrum Lrdquo NaturalProduct Research vol 23 no 2 pp 122ndash126 2009

[23] Y Cong Y-B Zhou J Chen Y-M Zeng and J-H WangldquoAlkaloid profiling of crude and processed Veratrum nigrum Lthrough simultaneous determination of ten steroidal alkaloidsby HPLC-ELSDrdquo Journal of Pharmaceutical and BiomedicalAnalysis vol 48 no 3 pp 573ndash578 2008

[24] L-S Wang D-Q Zhao H-M Tao and Y-H Liu ldquoDetermi-nation of vetatramine in Veratrum nigrum by HPLC-ELSDrdquoZhongguo Zhongyao Zazhi vol 33 no 7 pp 791ndash792 2008

[25] Q Wang H-Z Guo C-H Huo et al ldquoChemical constituentsin root of Paeonia lactiflorardquo Chinese Traditional and HerbalDrugs vol 38 no 7 pp 972ndash976 2007

[26] J-J TanQ-C Zhao L Yang Z-P Shang Z-QDu andMYanldquoChemical constituents in roots of Paeonia lactiflorardquo ChineseTraditional and Herbal Drugs vol 41 no 8 pp 1245ndash1248 2010

[27] Q Zhang Z Z Wang W Xiao et al ldquoUPLC characteristicchromatographic profile of Paeoniae Radix Albardquo ZhongguoZhongyao Zazhi vol 36 no 6 pp 712ndash714 2011

[28] X R Gao and Y Tian ldquoActive principles of Paeonia lactifloraPallrdquo Chinese Journal of New Drugs vol 15 pp 416ndash418 2006

[29] N-C Luo W Ding J Wu et al ldquoUPLC-Q-TOFMS coupledwith multivariate statistical analysis as a powerful techniquefor rapidly exploring potential chemicalmarkers to differentiatebetween radix paeoniae alba and radix paeoniae rubrardquoNaturalProduct Communications vol 8 no 4 pp 487ndash491 2013

[30] W K Zhao Y Guo T Yasuhiro and K Tohru ldquoIsolation andstructure determination of echinuline from Veratrum nigrumL var ussuriense Nakairdquo Zhongguo Zhong Yao Za Zhi vol 16no 7 pp 425ndash426 1991

[31] W Zhao Y Guo T Yasuhirb and T Kikuchi ldquoIsolation andstructure determination of aurantiamide acetate fromVeratrumnigrum Lvarussuriense Nakairdquo Zhongguo Zhongyao Zazhi vol23 no 1 p 41 1998

[32] L Yang X H Xia Q Zhu and X P Tan ldquoStudy on therule of influence of two purification methods on the chemicalcompositions in aqueous solution of paeoniae radix albardquoZhong Yao Cai vol 36 pp 118ndash121 2012

[33] X Y Zang and X Li ldquoDevelopment of the study on chemicalconstituentsof Paeonia lact if lora Pallrdquo Journal of ShenyangPharmaceutical University vol 19 pp 70ndash73 2002

[34] Y-L Wang Y-G Wang Q-D Liang et al ldquoUsing cDNAmicroarray to screen differently expressed genes in SH-SY5Ytreated by veratridinerdquo Chinese Pharmacological Bulletin vol29 no 5 pp 643ndash647 2013

Submit your manuscripts athttpwwwhindawicom

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

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Diabetes ResearchJournal of

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Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

2 Evidence-Based Complementary and Alternative Medicine

Table 1 Doses of V nigrum L and RPA in the 12 decoction groups

Group V nigrum L dose (g) RPA dose (g) Water (mL) Final volume (mL) Drug concentration ratio(V nigrum L RPA)

A 6415 06415 700 100 10 1B 6415 0802 700 100 8 1C 6415 1069 700 100 6 1D 6415 1604 700 100 4 1E 6415 3208 700 100 2 1F 6415 6415 700 100 1 1G 6415 1283 700 100 1 2H 6415 2566 700 100 1 4I 6415 3849 700 100 1 6J 6415 5132 700 100 1 8K 6415 6415 700 100 1 10L 6415 0 700 100 mdash

are toxic and irritate the digestive tract mucosa nucleus nervivagi and central nervous system [6 7]

RPA the dried root of Paeonia lactiflora Pall withoutbark has been used as a medicinal herb in traditionalChinese medicine for centuries and exerts a wide range ofpharmacological activities In ancient pharmacology RPAwas used to calm liver wind relieve pain nourish bloodregulate menstrual functions and suppress sweating [8] Inmodern pharmacology RPA decoctions could be used totreat rheumatoid arthritis systemic lupus erythematosushepatitis dysmenorrhea muscle cramping and spasms andlong-standing fever [4 9 10]

In this study UPLCTOF MS multivariate statisticalanalysis and typical metabolomics approach were used toidentify the key chemical markers that are responsible for theincreased toxicity and the combination of V nigrum L andRPA In addition cellular and mouse acute toxicity assayswere used to identify the toxic effects of this combinationThe compatibility of V nigrum L and RPA has not yet beenvalidated by modern science therefore it is important toprovide preliminary insight into the mechanism of theirreported incompatibility as well as to identify and quantifytheir toxic chemical constituents

2 Materials and Methods

21 Chemicals andMaterials RPA from Panan County (Zhe-jiang China lot 100714) andV nigrumL fromHuajia County(Changchun China lot 100701) were purchased from AnhuiBBCA Tongling Chinese herbal medicine company Formicacid (CNW Technologies GmbH) and acetonitrile (FisherScientific Fair LawnNJUSA)were both of chromatographicpurity Deionized water was prepared using aMillipore waterpurification system Veratridine (D00114760) was purchasedfrom Merck (Merck KGaA Germany) and paeoniflorin waspurchased from Sigma-Aldrich The MTS cell proliferationassay kit was purchased from Promega Roswell Park Memo-rial Institute 1640 (RPMI-1640) and fetal bovine serum (FBS)were purchased from Gibco BRL (Invitrogen USA)

22 Preparation of Decoctions Based on a previous studythe LD

50of aqueous extracts of V nigrum L and RPA after

intragastric administration were 2566 gkg and 160 gkgrespectively [11] In this study the dose of V nigrum L wasfixed at 2566 gkg and the RPA dose varied from 02566 to2566 gkg

The doses ofV nigrum L andRPA in each of 12 groups areshown in Table 1 Each group was extracted using deionizedwater (700mL) for 1 h during microboiling under refluxThe extracts were filtered through three layers of gauzeand the drug extraction was then repeated The filtrateswere combined and were then concentrated to 100mL at60minus70∘C under reduced pressure Samples were then shakencalibrated and stored at 4∘C

All decoctions were centrifuged at 13000 rpm for 10minusing a Heraeus Labofuge 400R refrigerated centrifuge(Thermo Scientific USA) The supernatants were then fil-tered through a 022120583M aqueous microporous membraneand were stored at 4∘C for UPLC-TOF-MS analysis Groupswere prepared in triplicate [12 13]

23 Animals and Experimental Design Seven-week-oldKun-ming (KM) mice (18minus22 g) were obtained from the Experi-mental Animal Center of the Academy of Military MedicalSciences with the certificate of conformity SCXK-(Army)2007-004 Animal subjects were housed at the SPF animalcenter of the Academy of Military Medical Sciences (BeijingChina) according to the regulations of the animal carecommittee Mice were housed at a constant temperature of25 plusmn 1

∘C with 50 plusmn 20 humidity with a 12 h lightdark cycleand 10ndash15 air changes per hour Mice were allowed free accessto water and food during the experimental period

Mice were acclimatized to the facilities and environmentfor 3 days before the experiments Two hundred and fortymice were randomized into 12 groups (AndashL) with 10 miceper group Food was removed from all animals 12 h beforethe experiments Mice received decoctions at 04mL10 g viagavage and were observed for 14 days

Evidence-Based Complementary and Alternative Medicine 3

Table 2 Mice mortality after the intragastric administration of different proportion decoctions of V nigrum L and RPA

Group V nigrum L dose (g) RPA dose (g) Crude drug concentration ratio Female mortality Male mortality Total mortalityA 6415 06415 10 1 100 90 95B 6415 0802 8 1 100 100 100C 6415 1069 6 1 100 60 80D 6415 1604 4 1 100 60 80E 6415 3208 2 1 90 70 80F 6415 6415 1 1 90 100 95G 6415 1283 1 2 50 70 60H 6415 2566 1 4 60 60 60I 6415 3849 1 6 0 20 10J 6415 5132 1 8 0 0 0K 6415 6415 1 10 0 0 0L 6415 0 0 70 70 70

24 UPLC-MS

241 Liquid Chromatography UPLC was performed on aWaters Acquity UPLC system (Waters Milford MA USA)equipped with a binary solvent delivery system an autosam-pler and a photodiode-array detection (PDA) system Chro-matography was performed using a Waters ACQUITYBEHC

18column (100mm times 21mm 17 120583m) [12] The mobile

phase consisted of (A) water containing 01 formic acidand (B) acetonitrile containing 01 formic acid The elutingconditions were as follows isocratic 2 B (0-1min) lineargradients of 2minus5 B (1-2min) 5ndash20 B (2ndash5min) 20ndash30B (5ndash7min) 30ndash33 B (7ndash10min) 33ndash36 B (10ndash13min)36ndash40 B (13ndash17min) 40ndash100 B (17-18min) 100ndash2 B (18-19min) and 2 B (19-20min)The flow rate was 05mLminThe sample chamber was maintained at 4∘C with the columnat 45∘CThe injection volume was 5 120583L [12 14 15]

242 Mass Spectrometry Mass spectrometry was performedusing aWaters SYNAPTmass spectrometer equippedwith anelectrospray ionization (ESI) source Samples were injectedtwice once in positive ESI mode and once in negative ESImode The data acquisition range was 100ndash1500Da The lockspray reference scan frequencywas 20 s with a reference conevoltage of 30V The MS source temperature was set at 100∘Cand the desolvation temperature was 450∘C with a gas flowof 900 LhThe lock mass compound was leucine enkephalin(200 pg120583L) with anmz of 5562771 in the positive ionmodeand 5542615 in negative ion mode The capillary voltageswere set to 29 kV for ESI+ and 3 kV for ESIminus The conevoltage was 40 kV and the collision energies were 6V (trap)and 4V (transfer) with 200mLmin trap gas flow [16]

25 SH-SY5Y Cell Cultures and Cell Proliferation Assay SH-SY5Y cells (ATCC Manassas USA) were cultured in RPMI-1640 supplemented with 10 FBS Cells were maintained at37∘C in an incubator with a saturated humidity atmosphereof 95 air and 5 CO

2 They were cultured in a 96-well plate

and treated with different drug combinations An MTS cellproliferation assay was performed and theOD at 490 nmwasread using a VICTOR X plate reader (Perkin Elmer USA)

26 Data Analysis The UPLC-TOF-MS data of all sampleswere analyzed using MassLynx41 software (Waters Manch-ester UK) and principal component analysis (PCA) wasused for data analysis Pearson correlation coefficients wereused to identify relationships between the study parame-ters and mortality The chemical markers in each groupwere identified using the V nigrum L and RPA chemicaldatabases [12] For data analyses we compared the correlationbetween chemical composition and the acute toxicity of Vnigrum L and RPA between the 12 groups Experimentalvalues are expressed as means plusmn standard deviations (SD)Statistical analyses were performed using two-tailed Studentrsquos119905-tests A value of 119875 lt 001 was considered statisticallysignificant

3 Results and Discussion

31 Mouse Acute Toxicity Mice in the 12 groups wereadministered the decoctions by intragastric administrationDeath occurred 5minus10min after administration Toxicity wasmanifested predominantly as trembling convulsions andspasms The major organs showed no obvious lesions atnecropsy by the naked eye [6 17 18] Mouse mortality isshown in Table 2

Group L (with aV nigrum L dose of the LD502566 gkg)

had a mortality of 70 Groups AminusF were treated with thesame dose of V nigrum L but the groups fed a lower ratioof RPA had a higher mortality than group L Groups GminusKhad a higher proportion of RPA and lower mortality thangroup L suggesting that toxicity was minimized Groups Band F were the most toxic with mortalities of 100 and95 respectively Overall analyses showed that mortalityincreasedwhen the dose ofV nigrum L was less than the doseof RPA Conversely toxicity decreased when the proportionof RPA increased The trends in mortality are shown inFigure 1

Female mouse mortality decreased with increasing pro-portions of RPA whereas males mortality exhibited twopeaks at groups B and F which both had 100 (Figure 2)

4 Evidence-Based Complementary and Alternative Medicine

0

20

40

60

80

100

120

Mor

talit

y ra

te (

)

A B C D E F G H I J K LGroup

Figure 1 Total mortality of mice after the intragastric administra-tion of decoctions containing different proportions of V nigrum Land RPA The groups represent the combinations shown in Table 1

FemaleMale

0

20

40

60

80

100

120

Mor

talit

y ra

te (

)

A B C D E F G H I J K LGroup

Figure 2 Mortality of male and female mice after the intragastricadministration of decoctions containing different proportions of Vnigrum L and RPA The groups represent the combinations shownin Table 1

32 Identification and Quantification of the Chemical Com-ponents of V nigrum L and RPA To explain the cause ofacute toxicity of the combined use of V nigrum L andRPA we compared the chemical composition of the 12groups using a supervised orthogonal partial least squareddiscriminant analysis (OPLS-DA) After Pareto scaling withmean centering the data from both the positive and negativeion modes were displayed as scores plots (Figure 3) Thescores plots clearly revealed that the samples were clustered

minus40

minus20

0

20

40

minus100 0 100

A BC DE FG HI JK L

t[1]

t[2]

Scores comp[1] versus comp[2] colored by sample group

(a)

minus50

0

50

minus200 minus100 0 100 200

A BC DE FG HI JK L

Scores comp[1] versus comp[2] colored by sample group

t[1]

t[2]

(b)

Figure 3 BBThe OPLS-DA score plot of 36 samples obtained usingPareto scaling with mean centering (a) Positive ion mode (b)Negative ion mode The same score plots represent the same groupBoth the positive and negative ion modes showed that the sampleswere clearly clustered into 12 groups Replicates of the 12 groupscould be distinguished easily

into 12 groups replicates of same group were comparable butthe 12 groups could be distinguished easilyThis suggests thatthe changes in chemical compositions of the 12 groups wereconsistent and the experiment was reproducible

UPLC-TOF-MS is a rapid specific and sensitive methodused to identify and quantify individual components Repre-sentative BPI chromatograms of the 36 samples in the positiveand negative mode ESI are shown in Figure 4

The contents of some special alkaloids change signif-icantly in different proportion decoctions To determinewhich constituents contributed to the differences in toxicity

Evidence-Based Complementary and Alternative Medicine 5

F-P-2

95

()

minus5

2481128

4763010

4603062

44632764422959

5363228

4443088

4263004

4103042

5763557

6763667

7163999

108

9

7

3

2

1

465

200 400 600 800 1000 1200 1400 1600 1800

Time

BPI103e5

1 TOF MS ES+

(a)

43909591690150 2550515

5431194

5671754

5251649

4491475

2270695

6311725

6311760

6291927

3292323

10

8

97

3

2

1

4

65

200 400 600 800 1000 1200 1400 1600 1800

Time

BPI430e495

minus5

()

5-2 neg1 TOF MS ESminus

(b)

Figure 4 Representative BPI chromatograms (a) Positive ionmode (b) Negative ionmode Some of identified constituents (1ndash10) are labeledin the BPI chromatogram and the numbers correspond to Tables 3 and 4

observed with the 12 decoctions statistical analyses wereperformed using Pearsonrsquos correlation and the correlationcoefficient (119903) described the degree of linear correlationbetween two variables total mouse mortality and the chem-ical composition of the 12 decoctions The 119903 values werebetween minus1 and +1 119903 gt 0 indicates that two variables werepositively correlated whereas 119903 lt 0 indicates a negativecorrelation The larger the absolute value the higher thecorrelation therefore we defined 119903 = 090ndash100 as extremelyrelated and 119903 = 080ndash089 as highly related We extractedthe relevant chemical component data when 119903 ge 08 andidentified the individual components using the RPA and Vnigrum L chemical composition databases

According to Pearsonrsquos correlation coefficient 131 compo-sitions had an extremely positive relationship (119903 ge 09) withtoxicity However only 14 of these could be identified andall came from V nigrum L In addition 35 components werehighly positively correlated (119903 ge 08) to acute toxicity andthese constituents contributed most of the toxicity identifiedin the 12 decoctions Therefore we hypothesized that thesecomponents might play important roles in the acute toxicity

of this drug combination We identified that these com-poundswere all veratrum alkaloids that were detected in eachsample in various ionized forms The chemical componentswith 119903 ge 08 are shown in Table 3 [7 19ndash24]

3-Veratroylgermine jervine veratramine germanitrinegermidine and germerine were the main chemical compo-nents that were associated with acute toxicity with Pearsonrsquoscorrelation coefficients of 09779 09661 09589 0958109402 and 09393 respectively These compounds are allveratrum alkaloids and the changes in their content areshown in Figure 5The other 29 components that were highlypositively correlated (119903 ge 08) with acute toxicity are shownin Figure 6

Interestingly Pearsonrsquos correlation coefficient identified24 RPA components that were extremely negatively corre-lated (119903 le minus09) with toxicityThe chemical components with119903 le minus09 are shown in Table 4 [25ndash33]

The concentrations of these components increased withincreasing amounts of RPA Paeoniflorin sulfonate ben-zoyloxy paeoniflorin galloyl paeoniflorin paeonilacto-neBpaeonilacto-neC and tetragalloyglucose were the major RPA

6 Evidence-Based Complementary and Alternative Medicine

Table 3 The chemical components that were highly positively correlated with toxicity (119903 ge 08)

Number 119903 tR (min) Assigned identity Molecularformula

Mean measuredmass (Da)

Theoretical exactmass (Da)

Mass accuracy(ppm)

1 09779 1298 3-Veratroylgermine C36H53NO11 6763667 6758061 018052 09661 1239 Jervine C27H39NO3 4263004 4256035 236243 09589 1261 Veretramine C27H39NO2 4103042 4096041 297514 09581 1343 Germanitrine C39H59NO11 7184169 7178859 250155 09402 1220 Germidine C34H53NO10 6363412 6357853 057556 09393 1340 Germerine C37H59NO11 7163999 6938645 034087 09390 1291 3-Angeloylzygadenine C32H49NO8 5763557 5757334 127088 09311 1035 11-Deoxojervine C27H41NO2 4123189 4116199 64392

9 09240 157 11205733120572-Dihydroxy-5120573-jervanin-12-en-11-one C27H41NO4 4443088 4436187 24040

10 09237 696 Jervinone C27H37NO3 4242860 4235876 2014011 09182 1125 Neogermbudine C37H59NO12 7323971 7098639 4916912 09143 425 Germine C27H43NO8 5103059 5096322 15549

13 09131 418 NeogermineVeramanine C27H43NO5 4623213 4616340 14522

14 09034 1158 Veraline B C27H45NO3 4323465 4316511 2964615 08978 1116 Verdine C27H41NO5 4603056 4596181 1524916 08903 1163 Stenophylline A C36H51NO11 6743474 6737902 9865517 08870 1035 Germitrine C39H61NO12 7364278 7359011 0828818 08805 914 Polydatin C20H22O8 4290937 3903839 3484919 08738 1081 Cervdine C32H49NO9 5923498 5917328 20608

20 08675 1234 3-Veratrum acylprotoveratrine C36H51NO12 6903482 6897896 10609

21 08671 1289 Zygadenitic acid120575-lactone-16-angelate C32H47NO8 5743408 5737175 48884

22 08652 1232 Maackinine C39H59NO11 7184171 7178859 06797

23 08641 1238Rubijervine rubivirineetioline epirubijervine

IsorubijervineC27H43NO2 4143357 4136358 36827

24 08639 1049 Solanidine C27H43NO3 4303308 4296352 3049425 08492 1057 Pseudojervine C32H49NO8 5883525 5877441 1971026 08444 1181 Neojerminalanine C39H61NO13 7524244 7519005 3001427 08379 843 Zygacine C29H45NO8 5363223 5356695 0000028 08358 1320 Neoverataline B C27H42N2O9 5563260 5386304 407929 08201 1280 3-Veratroylzygadenine C36H51NO10 6963187 6577908 5346930 08176 583 Zygadenine C27H43NO7 4943110 4936328 1543431 08176 054 120574-Aminobutyric acid C4H9NO2 2460978 1031198 1612132 08160 1273 Veratrosine C33H49NO7 6163528 5717447 6832933 08128 1322 Stenophylline A C37H55NO10 6743901 6738333 05430

34 08119 13347-Acetyl-15-

methylbutyryl-3-veratroylgermine

C43H61NO13 8383791 7999433 13104

35 08017 1213 Vanilloylzygadenine C35H49NO10 6443444 6437643 15156

components that were identified with Pearsonrsquos correlationcoefficients of minus09811 minus09799 minus09793 minus09737 minus09736and minus09734 respectively The changes in content of the 24components that were highly negatively correlated (119903 le minus09)with acute toxicity are shown in Figure 7

33 Cell ProliferationAssay To further validate the toxicity ofthe combination ofV nigrumL andRPAonneurons we usedveratridine and paeoniflorin the major pharmacologicallyactive components of V nigrum L and peony for in vitroexperimentsThe effect of these two chemical components on

Evidence-Based Complementary and Alternative Medicine 7

Table 4 The chemical components that were highly negatively correlated with toxicity (119903 le minus09)

Number 119903 tR (min) Assigned identity Molecularformula

Mean measuredmass (Da)

Theoretical exactmass (Da)

Mass accuracy(ppm)

1 minus09811 495 Paeoniflorinsulfonate C23H28O13S 5431194 5445256 337142 minus09799 1220 Benzoyloxy paeoniflorin C30H32O13 5991785 6001843 030463 minus09793 977 Galloyl paeoniflorin C30H32O15 6311725 6321741 038564 minus09737 1479 Paeonilacto-ne B C17H18O6 3171750 3181103 392095 minus09736 056 Paeonilacto-ne C C10H12O4 1950496 1960736 531926 minus09734 809 Tetragalloyglucose C34H28O22 7871008 7885729 566077 minus09728 230 Vanillic acid C7H6O5 1690161 1701195 374718 minus09716 1391 Desbenzoyl paeoniflorin C16H24O10 3751868 3761369 445059 minus09708 1352 Benzoyl paeoniflorin C30H32O12 5831848 5845679 06258

10 minus09684 415 6-O-120573-D-Glucopyranosyllactinolide C16H26O9 3611537 3623722 93435

11 minus09670 1037 Paeoniflorin albiflorin R1albiflorin mudanpioside I C23H28O11 5191261 4804618 15284

12 minus09655 1167 Gallic acid C9H10O2 1510750 1501745 5959013 minus09640 493 Paeonol C9H10O3 1650545 1661739 4067614 minus09625 1167 Lactiflorin C23H26O10 4801872 4624465 0444915 minus09609 667 Isomaltopaeoniflorin C29H38O16 6432221 6426024 2656916 minus09589 852 Albiflorin C23H27O11 9573118 4794539 9372317 minus09582 679 11015840-O-Benzoylsucrose C19H26O12 4641757 4464025 2366818 minus09567 1057 Pentagalloylglucose C41H32O26 9391166 9406772 6629219 minus09562 802 Oxypaeoniflorin C23H28O12 5141934 4964612 1899220 minus09544 1158 Ethyl gallate C9H10O5 2210433 1981727 3244521 minus09478 1011 Eugeniin C41H30O26 9371010 9386613 6708622 minus09395 585 Paeonilactinone C9H14O2 1530904 1542063 7575023 minus09352 113 Lactinolide C10H16O4 2011111 2002316 7890724 minus09173 1373 Palbinone C22H30O4 3572047 3584712 52115

3-Veratroylgermine250

200

150

100

50

0A B C D E F G H I J K L

Ion

inte

nsity

Sample group

Ion

inte

nsity

Sample group

0010203040506070809 Veratramine

A B C D E F G H I J K L0

02

04

06

08

1

12

14

Ion

inte

nsity

A B C D E F G H I J K LSample group

Jervine

A B C D E F G H I J K L

Ion

inte

nsity

Sample group

020406080100120140160180200

Grmidine

A B C D E F G H I J K L

Germanitrine

Ion

inte

nsity

Sample group

0

02

04

06

08

1

12

14

A B C D E F G H I J K L

Ion

inte

nsity

Sample group

0

01

02

03

04

05

06 Germerine

minus01

Figure 5 Ion intensity trend plots for 3-veratroylgermine jervine veratramine germanitrine germidine and germerine which were highlypositively correlated (119903 ge 09393) with toxicity

8 Evidence-Based Complementary and Alternative Medicine

0100200300400500600700800

A B C D E F G H I J K L A B C D E F G H I J K L

Ion

inte

nsity

Ion

inte

nsity

Ion

inte

nsity

Ion

inte

nsity

Ion

inte

nsity

A B C D E F G H I J K L

A B C D E F G H I J K L

A B C D E F G H I J K LSample groupSample group

Sample group A B C D E F G H I J K LSample group

Sample groupA B C D E F G H I J K L

Sample group

3-Angeloylzygadenine

0

01

02

03

04

05

06

07

Ion

inte

nsity

Ion

inte

nsity

11-Deoxojervine

0

50

100

150

200

250

300

350

Sample group

Ion

inte

nsity

Sample group

A B C D E F G H I J K L0

01

02

03

04

05

06 Jervinone

0

05

1

15

2

25

3 Neogermbudine

0020406081

121416182 Germine

0051

152

253

35 Veraline B

0

1

2

3

4

5

6 Verdine

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

1

2

3

4

5

6 GermitrineIo

n in

tens

ity

A B C D E F G H I J K LSample group

0

05

1

15

25

2

Polydatin

minus05

minus05

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

1

2

3

4

5

6

Ion

inte

nsity

A B C D E F G H I J K LSample group

0051

152

253

Stenophylline A

minus05

35

Neogermineveramanine

jervanin-12-en-11-one

0

2

4

6

8

10

12

14

A B C D E F G H I J K L

Ion

inte

nsity

Sample group

Cervdine

A B C D E F G H I J K L

Ion

inte

nsity

Sample group

0

02

04

06

08

1

12 Maackinine

Ion

inte

nsity

A B C D E F G H I J K LSample group

0123456789 Rubijervine

A B C D E F G H I J K L

Ion

inte

nsity

Sample group

0020406081

12141618 3-Veratrum acyl protoveratrine

Ion

inte

nsity

A B C D E F G H I J K LSample group

0051

152

253

354

Solanidine

minus05

A B C D E F G H I JK L

Ion

inte

nsity

Sample group

0020406081

121416182

Zygadenitic acid 120575-Lactone-16-angelate

11205733a-Dihydroxy-5120573-

(a)

Figure 6 Continued

Evidence-Based Complementary and Alternative Medicine 9

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

20

40

60

80

100

120 Pseudojervine

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

02

04

06

08

1

12

14 Neojerminalanine

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

02

04

06

08

1

12 Zygacine

Ion

inte

nsity

A B C D E F G H I J K LSample group

012345678 Zygadenine

Ion

inte

nsity

A B C D E F G H I J K LSample group

000501015020250303504045 Neoverataline B

minus005

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

01

02

03

04

05

06 3-Veratroylzygadenine

minus01

0

05

1

15

2

25 Vanilloylzygadenine

A B C D E F G H I J K LSample group

Ion

inte

nsity

A B C D E F G H I J K LSample group

Ion

inte

nsity

0

2

4

6

8

10

12 Veratrosine

A B C D E F G H I J K LSample group

Ion

inte

nsity

0

2

4

6

8

10

12 Stenophylline A

A B C D E F G H I J K LSample group

Ion

inte

nsity

0020406081

1214 120574-Aminobutyric acid

minus02

A B C D E F G H I J K LSample group

Ion

inte

nsity

0

02

04

06

08

1

12 7-Acetyl-15-methylbutyryl-3-veratroylgermine

(b)

Figure 6 Ion intensity trend plots for the remaining 29 chemical components that were highly positively correlated (119903 ge 08) with toxicity

the viability of SH-SY5Y cells was tested using MTS assaysAs shown in Figures 8(a) and 8(b) veratridine had an LD

50

of 450 whereas paeoniflorin exerted no significant toxicityat doses of 0ndash2000120583M Therefore the dose of veratridinewas fixed at 400120583M and the RPA dose was varied from50 to 2000120583M SH-SY5Y cell cytotoxicity was increasedwhen the concentration of paeoniflorin was decreased inthe combination (Figure 8(c)) Therefore paeoniflorin couldoffset the cytotoxicity caused by veratridine in SH-SY5Y cells[25]

4 Discussion

The eighteen incompatible medicaments (Shi Ba Fan) is awell-known traditional Chinese medicine (TCM) theory It

basically listed eighteen pairs of TCM herbal medicine thatare not to be used in combination as they may cause fatalconsequences This principal has been used as guidancefor general TCM practices for hundreds of years with littlechemical comprehension In this work for the first time weused the UPLC-TOFMS coupled with multivariate statisticalanalysis in an effort to gain scientific understanding for thecauses of the incompatibility

We previously reported that the LD50of aqueous extracts

of V nigrum L and RPA were 2566 gkg and 160 gkgrespectively after intragastric administration [11] V nigrumL extract was severely toxic whereas RPA had minimaltoxicity Although the amount of V nigrum L in each groupwas unchanged the mortality changed significantly withaltered ratios of V nigrum L to RPA When the doses of

10 Evidence-Based Complementary and Alternative Medicine

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

1

2

3

4

5

6 Tetragalloyglucose

Ion

inte

nsity

A B C D E F G H I J K LSample group

0020406081

121416182 Vanillic acid

Ion

inte

nsity

A B C D E F G H I J K LSample group

minus2

0

2

4

6

8

10

12 Benzoylpaeoniflorin

000501015020250303504

A B C D E F G H I J K L

Ion

inte

nsity

Sample groupminus005

Paeoniflorin sulfonate

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

05

1

15

2 Benzoyloxypaeoniflorin

minus05

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

05

1

15

2 Gallic acid

minus05

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

05

1

15

2

253 Galloylpaeoniflorin

minus05

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

005

01

015

02

025

03

035 Paeonilacto-neC

Ion

inte

nsity

A B C D E F G H I J K LSample group

0123456789 Paeonilacto-neB

minus1

Ion

inte

nsity

A B C D E F G H I J K LSample group

Vanillic acid

0

1

2

3

4

5

6

7

minus1

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

01

02

03

04

05

06

07

minus01

6-O-120573-D-Glucopyranosyllactinolide

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

05

1

15

2

25

3

35

minus05

Paeoniflorinalbiflorin R1albiflorinmudanpioside I

A B C D E F G H I J K L

Ion

inte

nsity

Sample group

Paeonol

0

02

04

06

08

1

12

minus02

Ion

inte

nsity

A B C D E F G H I J K LSample group

01020304050607080 Pentagalloylglucose

minus10

Ion

inte

nsity

A B C D E F G H I J K LSample group

00102030405060708 Lactiflorin

minus01

Ion

inte

nsity

A BC D E F G H I J K LSample group

0020406081

121416 Isomaltopaeoniflorin

minus02

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

05

1

15

2

25

3

minus05

Albiflorin

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

05

1

15

2

25 1998400-O-Benzoylsucrose

(a)

Figure 7 Continued

Evidence-Based Complementary and Alternative Medicine 11

Ion

inte

nsity

Ion

inte

nsity

Ion

inte

nsity

A B C D E F G H I J K LSample group A B C D E F G H I J K L

Sample group

A B C D E F G H I J K LSample group

00102030405060708 Oxypaeoniflorin

001020304050607 Eugeniin

Ion

inte

nsity

A B C D E F G H I J K LSample group

0020406081

12141618 Paeonia lactinone

00102030405060708091 Ethyl gallate

minus01

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

01

02

03

04

05

Lactinolide06

minus01

0

5

10

15

20

25 Palbinone

Ion

inte

nsity

A B C D E F G H I J K LSample group

minus5

minus01

(b)

Figure 7 Ion intensity trend plots for the chemical components that were highly negatively correlated with toxicity (119903 ge 09)

OD

val

ue

25 50 100 200 400 800

lowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

mdashVeratridine (120583M)

10

12

08

06

04

02

0

(a)

OD

val

ue

02

04

06

08

12

Paeoniflorin (120583M) 100 500 1500 2000mdash 1000

10

00

(b)

OD

val

ue

Paeoniflorin (120583M)

mdashmdash

Veratridine (120583M) 400 400 400 400 400 400 400 400 400 400

50 100 250 500 750 1000 1250 1500 1750 2000

02

04

06

08

12

10

00

lowastlowastlowast

lowastlowast

lowastlowast lowastlowast

lowastlowast

lowast

(c)

Figure 8 SH-SY5Y cell viability SH-SY5Y cells were cultured with (a) veratridine (25ndash800120583M) (b) paeoniflorin (50minus2000120583M) and (c)400 120583M veratridine in combination with 50minus2000120583M paeoniflorin for 24 h Cell viability was then assessed using MTS assays Data arepresented as mean plusmn SD Means of three independent experiments Asterisks indicate a statistically significant difference compared withuntreated cells lowast119875 lt 005 lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001 versus controL

12 Evidence-Based Complementary and Alternative Medicine

V nigrum L were equal to or higher than RPA particularlyratios of 8 1 and 1 1 mouse mortality increased significantlyTherefore components of V nigrum L were the majorcausative factors of toxicity In addition RPA modulated thetoxicity ofV nigrum LTherefore clinical applications shouldbe monitored carefully

Mice died 5minus10min after drug administration Toxicitymanifested predominantly as trembling convulsions andspasms but the major organs showed no obvious lesionsat necropsy by the naked eye Based on the toxic reactionsand the time of death in the current study and accord-ing to the guidelines for Chinese natural medicine acutetoxicity testing we speculate that the toxicity was causedpredominantly by suppression of the central nervous systemand that V nigrum L was the major cause of death Vnigrum L water extracts exert toxic effects on the digestivetract mucosa nucleus nervi vagi and central nervous system[6 17 18] Pharmacological studies of veratrum alkaloidshave been performed previously and reported that thesealkaloids stimulate the central nervous system and inhibit thebrain This results in spasms convulsions coma drowsinessand other symptoms of consciousness in animals which isconsistent with the symptoms of themice in the current study[6 17]

Veratrum alkaloid is the toxic component of V nigrum LWe previously investigated the differently expressed genes inSH-SY5Y cells treated with veratridine using cDNAmicroar-rays [34] Data revealed that veratridine treatment alteredthe expression of MMP caused Ca2+ concentration over-load increased reactive oxygen species production enhancedLDH release damaged the cell membrane reduced SH-SY5Y cell viability and induced apoptosis Under oxidizingconditions the MAPK signaling pathway is activated whichincreases apoptosisThis study illustrates the neurotoxicity ofthe combination of V nigrum L and reveals the molecularmechanism behind the neurotoxic effects of V nigrum L(data not shown)

The amount of V nigrum L in each group was the samethe dose of RPA is in ascending order and the chemicalcomponents of RPA are also increased but the mortalityof each group changed significantly with the increase ofRPA so we conclude that higher amount of RPA can haveprotection effect which can offset the toxicity of combinationof V nigrum L and RPA And pharmacological studies havedemonstrated that RPA exerts a wide range of pharmacolog-ical activities including analgesic sedative anticonvulsantand antispasmodic effects on the central nervous systemRPA could also inhibit writhing reactions and antagonizepentylenetetrazol-induced convulsions The most importantactive ingredient in RPA is paeoniflorin which has significantneuroprotective effects [26ndash34]

In this study mouse acute toxicity experiments revealedthat the mortality of mice changed significantly when thedose of V nigrum L was higher than or equal to that ofRPA particularly at ratios of V nigrum L RPA of 8 1and 1 1 UPLC-Q-TOFMS with automated data analysisused to identify and quantify the specific chemical compo-nents According to Pearsonrsquos correlation coefficient most of

the chemical components that were positively correlated withtoxicity were from V nigrum L We conclude that the majorveratrum alkaloids of V nigrum L caused acute toxicity anddeath in mice When the doses of V nigrum L were higherthan or equal to RPA the main chemical components of Vnigrum L and the toxicity of the decoctions were increasedThis suggests that V nigrum L and RPA have opposingroles Additional experiments revealed that the cytotoxicityof veratridine in SH-SY5Y cells was decreasedwith increasingconcentrations of paeoniflorin Therefore the clinical use ofthese agents should be considered carefully This study alsoprovides information to help improve the incompatibilitytheory of TCM and introduces novel ideas for further studieson the development and application of TCM

When preparing V nigrum L and RPA decoctions alower amount of RPA contributes to the dissolution of vera-trum alkaloids Conversely higher amounts of RPA can exertneuroprotective effects which can offset the toxicity causedby the combination of V nigrum L and RPA Under theseconditions dissolution is the most important factor for Vnigrum L toxicity The results of cell-based experiments alsodemonstrated that paeoniflorin could offset the neurotoxiceffects of veratridine when treated in combination whichexcluded the factor of dissolution in preparation

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors are grateful for financial support from NationalNature Science Foundation of China (Grant no 81073149)National Basic Research Program of China (Grant nos2011CB505304 and 2012CB518402) and Natural ScienceFoundation of Beijing (Grant no 7112110)

References

[1] H Guo W Li X Y Wang and G W Fan ldquoAdvance of researchon toxic attenuation by compatibility of traditional Chinesemedicine prescriptionsrdquo Zhongguo Zhongyao Zazhi vol 37 no1 pp 120ndash123 2012

[2] Y Zhang H Hua X Fan C Wang and J Duan ldquoExplorationon eighteen incompatible medicaments of chest pain prescrip-tions based on association rules miningrdquo Zhongguo ZhongyaoZazhi vol 36 no 24 pp 3544ndash3547 2011

[3] C-H Xu P Wang Y Wang et al ldquoPharmacokinetic compar-isons of two different combinations of Shaoyao-Gancao Decoc-tion in rats competing mechanisms between paeoniflorin andglycyrrhetinic acidrdquo Journal of Ethnopharmacology vol 149 no2 pp 443ndash452 2013

[4] D H TianDictionary of Practical Traditional Chinese MedicineII Peoplersquos Medical Beijing China 2002

[5] Y Gaillard and G Pepin ldquoLC-EI-MS determination of ver-atridine and cevadine in two fatal cases of Veratrum albumpoisoningrdquo Journal of Analytical Toxicology vol 25 no 6 pp481ndash485 2001

Evidence-Based Complementary and Alternative Medicine 13

[6] H Li G-Y Gao S-Y Li W-J Zhao Y-T Guo and Z-J Liang ldquoEffects of Veratrum nigrum alkaloids on centralcatecholaminergic neurons of renal hypertensive ratsrdquo ActaPharmacologica Sinica vol 21 no 1 pp 23ndash28 2000

[7] L Wang W Li and Y Liu ldquoHypotensive effect and toxicologyof total alkaloids and veratramine from roots and rhizomesof Veratrum nigrum L in spontaneously hypertensive ratsrdquoPharmazie vol 63 no 8 pp 606ndash610 2008

[8] Y P Jiang Y G Liu and H C Chen ldquoEffect of aqueous extractof Radix Paeoniae Rubra against Carbon Tetrachloride inducedliver fibrosis in ratrdquo Herald of Medicine vol 23 pp 527ndash5292004

[9] D-Y He and S-M Dai ldquoAnti-inflammatory and immunomod-ulatory effects of Paeonia lactiflora Pall a traditional Chineseherbal medicinerdquo Frontiers in Pharmacology 2011

[10] C Feng M Liu X Shi et al ldquoPharmacokinetic properties ofpaeoniflorin albiflorin and oxypaeoniflorin after oral gavage ofextracts of Radix Paeoniae Rubra and Radix Paeoniae Alba inratsrdquo Journal of Ethnopharmacology vol 130 no 2 pp 407ndash4132010

[11] X X Zhang Y G Wang Z C Ma Y Gao and C R XiaoldquoUniform designed research on the compatibility toxicity ofVeratridine and peonyrdquo Zhong Hua Zhong Yi Yao Za Zhi vol28 no 10 pp 2901ndash2904 2013

[12] Q D Liang Y Gao J Ma et al ldquoChemical comparison of driedrehmannia root and prepared rehmannia root by UPLC-TOFMS andHPLC-ELSDwithmultivariate statistical analysisrdquoActaPharmaceutica Sinica B vol 3 no 1 pp 55ndash64 2013

[13] Y Wang S He X Cheng Y Lu Y Zou and Q Zhang ldquoUPLC-Q-TOF-MSMS fingerprinting of traditional Chinese formulaSiJunZiTangrdquo Journal of Pharmaceutical and Biomedical Analy-sis vol 80 pp 24ndash33 2013

[14] L-P Kang K Yu Y Zhao et al ldquoCharacterization of steroidalglycosides from the extract of Paris Polyphylla var Yunnanensisby UPLCQ-TOFMSErdquo Journal of Pharmaceutical and Biomed-ical Analysis vol 62 pp 235ndash249 2012

[15] A Kaufmann P Butcher K Maden and M Widmer ldquoUltra-performance liquid chromatography coupled to time of flightmass spectrometry (UPLC-TOF) a novel tool for multiresiduescreening of veterinary drugs in urinerdquoAnalytica Chimica Actavol 586 no 1-2 pp 13ndash21 2007

[16] S Zhou Z C Ma Q D Liang et al ldquoUPLCQ-TOF-MS basedchemical profiling approach to evaluate chemical compositionof augmentation toxicity in combination of Radix aconiti andPinellia praeparatardquoActa Chimica Sinica vol 70 no 3 pp 284ndash290 2012

[17] N Lin and X Gao ldquoExperimental observation on the absorp-tion of Veratrum nigrum L in the upper digestive tractrdquoZhongguo Zhong Yao Za Zhi vol 17 no 1 pp 43ndash45 1992

[18] Z-Z Wang W-J Zhao X-S Zhang et al ldquoProtection ofVeratrum nigrum L var ussuriense Nakai alkaloids againstischema-reperfusion injury of the rat liverrdquo World Journal ofGastroenterology vol 13 no 4 pp 564ndash571 2007

[19] V Christov B Mikhova A Ivanova et al ldquoSteroidal alka-loids of Veratrum lobelianum Bernh and Veratrum nigrum LrdquoZeitschrift fur Naturforschung C Journal of Biosciences vol 65no 3-4 pp 195ndash200 2010

[20] V Christov BMikhova andD Selenge ldquo(-)-Veranigrine a newsteroidal alkaloid fromVeratrum nigrum Lrdquo Fitoterapia vol 80no 1 pp 25ndash27 2009

[21] Y Cong J-H Wang R Wang Y-M Zeng C-D Liu and XLi ldquoA study on the chemical constituents of Veratrum nigrum

L processed by rice vinegarrdquo Journal of Asian Natural ProductsResearch vol 10 no 7-8 pp 619ndash624 2008

[22] H-L Li J Tang R-H Liu C Zhang and W-D Zhang ldquoTwonew flavanone glycosides from Veratrum nigrum Lrdquo NaturalProduct Research vol 23 no 2 pp 122ndash126 2009

[23] Y Cong Y-B Zhou J Chen Y-M Zeng and J-H WangldquoAlkaloid profiling of crude and processed Veratrum nigrum Lthrough simultaneous determination of ten steroidal alkaloidsby HPLC-ELSDrdquo Journal of Pharmaceutical and BiomedicalAnalysis vol 48 no 3 pp 573ndash578 2008

[24] L-S Wang D-Q Zhao H-M Tao and Y-H Liu ldquoDetermi-nation of vetatramine in Veratrum nigrum by HPLC-ELSDrdquoZhongguo Zhongyao Zazhi vol 33 no 7 pp 791ndash792 2008

[25] Q Wang H-Z Guo C-H Huo et al ldquoChemical constituentsin root of Paeonia lactiflorardquo Chinese Traditional and HerbalDrugs vol 38 no 7 pp 972ndash976 2007

[26] J-J TanQ-C Zhao L Yang Z-P Shang Z-QDu andMYanldquoChemical constituents in roots of Paeonia lactiflorardquo ChineseTraditional and Herbal Drugs vol 41 no 8 pp 1245ndash1248 2010

[27] Q Zhang Z Z Wang W Xiao et al ldquoUPLC characteristicchromatographic profile of Paeoniae Radix Albardquo ZhongguoZhongyao Zazhi vol 36 no 6 pp 712ndash714 2011

[28] X R Gao and Y Tian ldquoActive principles of Paeonia lactifloraPallrdquo Chinese Journal of New Drugs vol 15 pp 416ndash418 2006

[29] N-C Luo W Ding J Wu et al ldquoUPLC-Q-TOFMS coupledwith multivariate statistical analysis as a powerful techniquefor rapidly exploring potential chemicalmarkers to differentiatebetween radix paeoniae alba and radix paeoniae rubrardquoNaturalProduct Communications vol 8 no 4 pp 487ndash491 2013

[30] W K Zhao Y Guo T Yasuhiro and K Tohru ldquoIsolation andstructure determination of echinuline from Veratrum nigrumL var ussuriense Nakairdquo Zhongguo Zhong Yao Za Zhi vol 16no 7 pp 425ndash426 1991

[31] W Zhao Y Guo T Yasuhirb and T Kikuchi ldquoIsolation andstructure determination of aurantiamide acetate fromVeratrumnigrum Lvarussuriense Nakairdquo Zhongguo Zhongyao Zazhi vol23 no 1 p 41 1998

[32] L Yang X H Xia Q Zhu and X P Tan ldquoStudy on therule of influence of two purification methods on the chemicalcompositions in aqueous solution of paeoniae radix albardquoZhong Yao Cai vol 36 pp 118ndash121 2012

[33] X Y Zang and X Li ldquoDevelopment of the study on chemicalconstituentsof Paeonia lact if lora Pallrdquo Journal of ShenyangPharmaceutical University vol 19 pp 70ndash73 2002

[34] Y-L Wang Y-G Wang Q-D Liang et al ldquoUsing cDNAmicroarray to screen differently expressed genes in SH-SY5Ytreated by veratridinerdquo Chinese Pharmacological Bulletin vol29 no 5 pp 643ndash647 2013

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

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BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

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PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

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Diabetes ResearchJournal of

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Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Evidence-Based Complementary and Alternative Medicine 3

Table 2 Mice mortality after the intragastric administration of different proportion decoctions of V nigrum L and RPA

Group V nigrum L dose (g) RPA dose (g) Crude drug concentration ratio Female mortality Male mortality Total mortalityA 6415 06415 10 1 100 90 95B 6415 0802 8 1 100 100 100C 6415 1069 6 1 100 60 80D 6415 1604 4 1 100 60 80E 6415 3208 2 1 90 70 80F 6415 6415 1 1 90 100 95G 6415 1283 1 2 50 70 60H 6415 2566 1 4 60 60 60I 6415 3849 1 6 0 20 10J 6415 5132 1 8 0 0 0K 6415 6415 1 10 0 0 0L 6415 0 0 70 70 70

24 UPLC-MS

241 Liquid Chromatography UPLC was performed on aWaters Acquity UPLC system (Waters Milford MA USA)equipped with a binary solvent delivery system an autosam-pler and a photodiode-array detection (PDA) system Chro-matography was performed using a Waters ACQUITYBEHC

18column (100mm times 21mm 17 120583m) [12] The mobile

phase consisted of (A) water containing 01 formic acidand (B) acetonitrile containing 01 formic acid The elutingconditions were as follows isocratic 2 B (0-1min) lineargradients of 2minus5 B (1-2min) 5ndash20 B (2ndash5min) 20ndash30B (5ndash7min) 30ndash33 B (7ndash10min) 33ndash36 B (10ndash13min)36ndash40 B (13ndash17min) 40ndash100 B (17-18min) 100ndash2 B (18-19min) and 2 B (19-20min)The flow rate was 05mLminThe sample chamber was maintained at 4∘C with the columnat 45∘CThe injection volume was 5 120583L [12 14 15]

242 Mass Spectrometry Mass spectrometry was performedusing aWaters SYNAPTmass spectrometer equippedwith anelectrospray ionization (ESI) source Samples were injectedtwice once in positive ESI mode and once in negative ESImode The data acquisition range was 100ndash1500Da The lockspray reference scan frequencywas 20 s with a reference conevoltage of 30V The MS source temperature was set at 100∘Cand the desolvation temperature was 450∘C with a gas flowof 900 LhThe lock mass compound was leucine enkephalin(200 pg120583L) with anmz of 5562771 in the positive ionmodeand 5542615 in negative ion mode The capillary voltageswere set to 29 kV for ESI+ and 3 kV for ESIminus The conevoltage was 40 kV and the collision energies were 6V (trap)and 4V (transfer) with 200mLmin trap gas flow [16]

25 SH-SY5Y Cell Cultures and Cell Proliferation Assay SH-SY5Y cells (ATCC Manassas USA) were cultured in RPMI-1640 supplemented with 10 FBS Cells were maintained at37∘C in an incubator with a saturated humidity atmosphereof 95 air and 5 CO

2 They were cultured in a 96-well plate

and treated with different drug combinations An MTS cellproliferation assay was performed and theOD at 490 nmwasread using a VICTOR X plate reader (Perkin Elmer USA)

26 Data Analysis The UPLC-TOF-MS data of all sampleswere analyzed using MassLynx41 software (Waters Manch-ester UK) and principal component analysis (PCA) wasused for data analysis Pearson correlation coefficients wereused to identify relationships between the study parame-ters and mortality The chemical markers in each groupwere identified using the V nigrum L and RPA chemicaldatabases [12] For data analyses we compared the correlationbetween chemical composition and the acute toxicity of Vnigrum L and RPA between the 12 groups Experimentalvalues are expressed as means plusmn standard deviations (SD)Statistical analyses were performed using two-tailed Studentrsquos119905-tests A value of 119875 lt 001 was considered statisticallysignificant

3 Results and Discussion

31 Mouse Acute Toxicity Mice in the 12 groups wereadministered the decoctions by intragastric administrationDeath occurred 5minus10min after administration Toxicity wasmanifested predominantly as trembling convulsions andspasms The major organs showed no obvious lesions atnecropsy by the naked eye [6 17 18] Mouse mortality isshown in Table 2

Group L (with aV nigrum L dose of the LD502566 gkg)

had a mortality of 70 Groups AminusF were treated with thesame dose of V nigrum L but the groups fed a lower ratioof RPA had a higher mortality than group L Groups GminusKhad a higher proportion of RPA and lower mortality thangroup L suggesting that toxicity was minimized Groups Band F were the most toxic with mortalities of 100 and95 respectively Overall analyses showed that mortalityincreasedwhen the dose ofV nigrum L was less than the doseof RPA Conversely toxicity decreased when the proportionof RPA increased The trends in mortality are shown inFigure 1

Female mouse mortality decreased with increasing pro-portions of RPA whereas males mortality exhibited twopeaks at groups B and F which both had 100 (Figure 2)

4 Evidence-Based Complementary and Alternative Medicine

0

20

40

60

80

100

120

Mor

talit

y ra

te (

)

A B C D E F G H I J K LGroup

Figure 1 Total mortality of mice after the intragastric administra-tion of decoctions containing different proportions of V nigrum Land RPA The groups represent the combinations shown in Table 1

FemaleMale

0

20

40

60

80

100

120

Mor

talit

y ra

te (

)

A B C D E F G H I J K LGroup

Figure 2 Mortality of male and female mice after the intragastricadministration of decoctions containing different proportions of Vnigrum L and RPA The groups represent the combinations shownin Table 1

32 Identification and Quantification of the Chemical Com-ponents of V nigrum L and RPA To explain the cause ofacute toxicity of the combined use of V nigrum L andRPA we compared the chemical composition of the 12groups using a supervised orthogonal partial least squareddiscriminant analysis (OPLS-DA) After Pareto scaling withmean centering the data from both the positive and negativeion modes were displayed as scores plots (Figure 3) Thescores plots clearly revealed that the samples were clustered

minus40

minus20

0

20

40

minus100 0 100

A BC DE FG HI JK L

t[1]

t[2]

Scores comp[1] versus comp[2] colored by sample group

(a)

minus50

0

50

minus200 minus100 0 100 200

A BC DE FG HI JK L

Scores comp[1] versus comp[2] colored by sample group

t[1]

t[2]

(b)

Figure 3 BBThe OPLS-DA score plot of 36 samples obtained usingPareto scaling with mean centering (a) Positive ion mode (b)Negative ion mode The same score plots represent the same groupBoth the positive and negative ion modes showed that the sampleswere clearly clustered into 12 groups Replicates of the 12 groupscould be distinguished easily

into 12 groups replicates of same group were comparable butthe 12 groups could be distinguished easilyThis suggests thatthe changes in chemical compositions of the 12 groups wereconsistent and the experiment was reproducible

UPLC-TOF-MS is a rapid specific and sensitive methodused to identify and quantify individual components Repre-sentative BPI chromatograms of the 36 samples in the positiveand negative mode ESI are shown in Figure 4

The contents of some special alkaloids change signif-icantly in different proportion decoctions To determinewhich constituents contributed to the differences in toxicity

Evidence-Based Complementary and Alternative Medicine 5

F-P-2

95

()

minus5

2481128

4763010

4603062

44632764422959

5363228

4443088

4263004

4103042

5763557

6763667

7163999

108

9

7

3

2

1

465

200 400 600 800 1000 1200 1400 1600 1800

Time

BPI103e5

1 TOF MS ES+

(a)

43909591690150 2550515

5431194

5671754

5251649

4491475

2270695

6311725

6311760

6291927

3292323

10

8

97

3

2

1

4

65

200 400 600 800 1000 1200 1400 1600 1800

Time

BPI430e495

minus5

()

5-2 neg1 TOF MS ESminus

(b)

Figure 4 Representative BPI chromatograms (a) Positive ionmode (b) Negative ionmode Some of identified constituents (1ndash10) are labeledin the BPI chromatogram and the numbers correspond to Tables 3 and 4

observed with the 12 decoctions statistical analyses wereperformed using Pearsonrsquos correlation and the correlationcoefficient (119903) described the degree of linear correlationbetween two variables total mouse mortality and the chem-ical composition of the 12 decoctions The 119903 values werebetween minus1 and +1 119903 gt 0 indicates that two variables werepositively correlated whereas 119903 lt 0 indicates a negativecorrelation The larger the absolute value the higher thecorrelation therefore we defined 119903 = 090ndash100 as extremelyrelated and 119903 = 080ndash089 as highly related We extractedthe relevant chemical component data when 119903 ge 08 andidentified the individual components using the RPA and Vnigrum L chemical composition databases

According to Pearsonrsquos correlation coefficient 131 compo-sitions had an extremely positive relationship (119903 ge 09) withtoxicity However only 14 of these could be identified andall came from V nigrum L In addition 35 components werehighly positively correlated (119903 ge 08) to acute toxicity andthese constituents contributed most of the toxicity identifiedin the 12 decoctions Therefore we hypothesized that thesecomponents might play important roles in the acute toxicity

of this drug combination We identified that these com-poundswere all veratrum alkaloids that were detected in eachsample in various ionized forms The chemical componentswith 119903 ge 08 are shown in Table 3 [7 19ndash24]

3-Veratroylgermine jervine veratramine germanitrinegermidine and germerine were the main chemical compo-nents that were associated with acute toxicity with Pearsonrsquoscorrelation coefficients of 09779 09661 09589 0958109402 and 09393 respectively These compounds are allveratrum alkaloids and the changes in their content areshown in Figure 5The other 29 components that were highlypositively correlated (119903 ge 08) with acute toxicity are shownin Figure 6

Interestingly Pearsonrsquos correlation coefficient identified24 RPA components that were extremely negatively corre-lated (119903 le minus09) with toxicityThe chemical components with119903 le minus09 are shown in Table 4 [25ndash33]

The concentrations of these components increased withincreasing amounts of RPA Paeoniflorin sulfonate ben-zoyloxy paeoniflorin galloyl paeoniflorin paeonilacto-neBpaeonilacto-neC and tetragalloyglucose were the major RPA

6 Evidence-Based Complementary and Alternative Medicine

Table 3 The chemical components that were highly positively correlated with toxicity (119903 ge 08)

Number 119903 tR (min) Assigned identity Molecularformula

Mean measuredmass (Da)

Theoretical exactmass (Da)

Mass accuracy(ppm)

1 09779 1298 3-Veratroylgermine C36H53NO11 6763667 6758061 018052 09661 1239 Jervine C27H39NO3 4263004 4256035 236243 09589 1261 Veretramine C27H39NO2 4103042 4096041 297514 09581 1343 Germanitrine C39H59NO11 7184169 7178859 250155 09402 1220 Germidine C34H53NO10 6363412 6357853 057556 09393 1340 Germerine C37H59NO11 7163999 6938645 034087 09390 1291 3-Angeloylzygadenine C32H49NO8 5763557 5757334 127088 09311 1035 11-Deoxojervine C27H41NO2 4123189 4116199 64392

9 09240 157 11205733120572-Dihydroxy-5120573-jervanin-12-en-11-one C27H41NO4 4443088 4436187 24040

10 09237 696 Jervinone C27H37NO3 4242860 4235876 2014011 09182 1125 Neogermbudine C37H59NO12 7323971 7098639 4916912 09143 425 Germine C27H43NO8 5103059 5096322 15549

13 09131 418 NeogermineVeramanine C27H43NO5 4623213 4616340 14522

14 09034 1158 Veraline B C27H45NO3 4323465 4316511 2964615 08978 1116 Verdine C27H41NO5 4603056 4596181 1524916 08903 1163 Stenophylline A C36H51NO11 6743474 6737902 9865517 08870 1035 Germitrine C39H61NO12 7364278 7359011 0828818 08805 914 Polydatin C20H22O8 4290937 3903839 3484919 08738 1081 Cervdine C32H49NO9 5923498 5917328 20608

20 08675 1234 3-Veratrum acylprotoveratrine C36H51NO12 6903482 6897896 10609

21 08671 1289 Zygadenitic acid120575-lactone-16-angelate C32H47NO8 5743408 5737175 48884

22 08652 1232 Maackinine C39H59NO11 7184171 7178859 06797

23 08641 1238Rubijervine rubivirineetioline epirubijervine

IsorubijervineC27H43NO2 4143357 4136358 36827

24 08639 1049 Solanidine C27H43NO3 4303308 4296352 3049425 08492 1057 Pseudojervine C32H49NO8 5883525 5877441 1971026 08444 1181 Neojerminalanine C39H61NO13 7524244 7519005 3001427 08379 843 Zygacine C29H45NO8 5363223 5356695 0000028 08358 1320 Neoverataline B C27H42N2O9 5563260 5386304 407929 08201 1280 3-Veratroylzygadenine C36H51NO10 6963187 6577908 5346930 08176 583 Zygadenine C27H43NO7 4943110 4936328 1543431 08176 054 120574-Aminobutyric acid C4H9NO2 2460978 1031198 1612132 08160 1273 Veratrosine C33H49NO7 6163528 5717447 6832933 08128 1322 Stenophylline A C37H55NO10 6743901 6738333 05430

34 08119 13347-Acetyl-15-

methylbutyryl-3-veratroylgermine

C43H61NO13 8383791 7999433 13104

35 08017 1213 Vanilloylzygadenine C35H49NO10 6443444 6437643 15156

components that were identified with Pearsonrsquos correlationcoefficients of minus09811 minus09799 minus09793 minus09737 minus09736and minus09734 respectively The changes in content of the 24components that were highly negatively correlated (119903 le minus09)with acute toxicity are shown in Figure 7

33 Cell ProliferationAssay To further validate the toxicity ofthe combination ofV nigrumL andRPAonneurons we usedveratridine and paeoniflorin the major pharmacologicallyactive components of V nigrum L and peony for in vitroexperimentsThe effect of these two chemical components on

Evidence-Based Complementary and Alternative Medicine 7

Table 4 The chemical components that were highly negatively correlated with toxicity (119903 le minus09)

Number 119903 tR (min) Assigned identity Molecularformula

Mean measuredmass (Da)

Theoretical exactmass (Da)

Mass accuracy(ppm)

1 minus09811 495 Paeoniflorinsulfonate C23H28O13S 5431194 5445256 337142 minus09799 1220 Benzoyloxy paeoniflorin C30H32O13 5991785 6001843 030463 minus09793 977 Galloyl paeoniflorin C30H32O15 6311725 6321741 038564 minus09737 1479 Paeonilacto-ne B C17H18O6 3171750 3181103 392095 minus09736 056 Paeonilacto-ne C C10H12O4 1950496 1960736 531926 minus09734 809 Tetragalloyglucose C34H28O22 7871008 7885729 566077 minus09728 230 Vanillic acid C7H6O5 1690161 1701195 374718 minus09716 1391 Desbenzoyl paeoniflorin C16H24O10 3751868 3761369 445059 minus09708 1352 Benzoyl paeoniflorin C30H32O12 5831848 5845679 06258

10 minus09684 415 6-O-120573-D-Glucopyranosyllactinolide C16H26O9 3611537 3623722 93435

11 minus09670 1037 Paeoniflorin albiflorin R1albiflorin mudanpioside I C23H28O11 5191261 4804618 15284

12 minus09655 1167 Gallic acid C9H10O2 1510750 1501745 5959013 minus09640 493 Paeonol C9H10O3 1650545 1661739 4067614 minus09625 1167 Lactiflorin C23H26O10 4801872 4624465 0444915 minus09609 667 Isomaltopaeoniflorin C29H38O16 6432221 6426024 2656916 minus09589 852 Albiflorin C23H27O11 9573118 4794539 9372317 minus09582 679 11015840-O-Benzoylsucrose C19H26O12 4641757 4464025 2366818 minus09567 1057 Pentagalloylglucose C41H32O26 9391166 9406772 6629219 minus09562 802 Oxypaeoniflorin C23H28O12 5141934 4964612 1899220 minus09544 1158 Ethyl gallate C9H10O5 2210433 1981727 3244521 minus09478 1011 Eugeniin C41H30O26 9371010 9386613 6708622 minus09395 585 Paeonilactinone C9H14O2 1530904 1542063 7575023 minus09352 113 Lactinolide C10H16O4 2011111 2002316 7890724 minus09173 1373 Palbinone C22H30O4 3572047 3584712 52115

3-Veratroylgermine250

200

150

100

50

0A B C D E F G H I J K L

Ion

inte

nsity

Sample group

Ion

inte

nsity

Sample group

0010203040506070809 Veratramine

A B C D E F G H I J K L0

02

04

06

08

1

12

14

Ion

inte

nsity

A B C D E F G H I J K LSample group

Jervine

A B C D E F G H I J K L

Ion

inte

nsity

Sample group

020406080100120140160180200

Grmidine

A B C D E F G H I J K L

Germanitrine

Ion

inte

nsity

Sample group

0

02

04

06

08

1

12

14

A B C D E F G H I J K L

Ion

inte

nsity

Sample group

0

01

02

03

04

05

06 Germerine

minus01

Figure 5 Ion intensity trend plots for 3-veratroylgermine jervine veratramine germanitrine germidine and germerine which were highlypositively correlated (119903 ge 09393) with toxicity

8 Evidence-Based Complementary and Alternative Medicine

0100200300400500600700800

A B C D E F G H I J K L A B C D E F G H I J K L

Ion

inte

nsity

Ion

inte

nsity

Ion

inte

nsity

Ion

inte

nsity

Ion

inte

nsity

A B C D E F G H I J K L

A B C D E F G H I J K L

A B C D E F G H I J K LSample groupSample group

Sample group A B C D E F G H I J K LSample group

Sample groupA B C D E F G H I J K L

Sample group

3-Angeloylzygadenine

0

01

02

03

04

05

06

07

Ion

inte

nsity

Ion

inte

nsity

11-Deoxojervine

0

50

100

150

200

250

300

350

Sample group

Ion

inte

nsity

Sample group

A B C D E F G H I J K L0

01

02

03

04

05

06 Jervinone

0

05

1

15

2

25

3 Neogermbudine

0020406081

121416182 Germine

0051

152

253

35 Veraline B

0

1

2

3

4

5

6 Verdine

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

1

2

3

4

5

6 GermitrineIo

n in

tens

ity

A B C D E F G H I J K LSample group

0

05

1

15

25

2

Polydatin

minus05

minus05

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

1

2

3

4

5

6

Ion

inte

nsity

A B C D E F G H I J K LSample group

0051

152

253

Stenophylline A

minus05

35

Neogermineveramanine

jervanin-12-en-11-one

0

2

4

6

8

10

12

14

A B C D E F G H I J K L

Ion

inte

nsity

Sample group

Cervdine

A B C D E F G H I J K L

Ion

inte

nsity

Sample group

0

02

04

06

08

1

12 Maackinine

Ion

inte

nsity

A B C D E F G H I J K LSample group

0123456789 Rubijervine

A B C D E F G H I J K L

Ion

inte

nsity

Sample group

0020406081

12141618 3-Veratrum acyl protoveratrine

Ion

inte

nsity

A B C D E F G H I J K LSample group

0051

152

253

354

Solanidine

minus05

A B C D E F G H I JK L

Ion

inte

nsity

Sample group

0020406081

121416182

Zygadenitic acid 120575-Lactone-16-angelate

11205733a-Dihydroxy-5120573-

(a)

Figure 6 Continued

Evidence-Based Complementary and Alternative Medicine 9

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

20

40

60

80

100

120 Pseudojervine

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

02

04

06

08

1

12

14 Neojerminalanine

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

02

04

06

08

1

12 Zygacine

Ion

inte

nsity

A B C D E F G H I J K LSample group

012345678 Zygadenine

Ion

inte

nsity

A B C D E F G H I J K LSample group

000501015020250303504045 Neoverataline B

minus005

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

01

02

03

04

05

06 3-Veratroylzygadenine

minus01

0

05

1

15

2

25 Vanilloylzygadenine

A B C D E F G H I J K LSample group

Ion

inte

nsity

A B C D E F G H I J K LSample group

Ion

inte

nsity

0

2

4

6

8

10

12 Veratrosine

A B C D E F G H I J K LSample group

Ion

inte

nsity

0

2

4

6

8

10

12 Stenophylline A

A B C D E F G H I J K LSample group

Ion

inte

nsity

0020406081

1214 120574-Aminobutyric acid

minus02

A B C D E F G H I J K LSample group

Ion

inte

nsity

0

02

04

06

08

1

12 7-Acetyl-15-methylbutyryl-3-veratroylgermine

(b)

Figure 6 Ion intensity trend plots for the remaining 29 chemical components that were highly positively correlated (119903 ge 08) with toxicity

the viability of SH-SY5Y cells was tested using MTS assaysAs shown in Figures 8(a) and 8(b) veratridine had an LD

50

of 450 whereas paeoniflorin exerted no significant toxicityat doses of 0ndash2000120583M Therefore the dose of veratridinewas fixed at 400120583M and the RPA dose was varied from50 to 2000120583M SH-SY5Y cell cytotoxicity was increasedwhen the concentration of paeoniflorin was decreased inthe combination (Figure 8(c)) Therefore paeoniflorin couldoffset the cytotoxicity caused by veratridine in SH-SY5Y cells[25]

4 Discussion

The eighteen incompatible medicaments (Shi Ba Fan) is awell-known traditional Chinese medicine (TCM) theory It

basically listed eighteen pairs of TCM herbal medicine thatare not to be used in combination as they may cause fatalconsequences This principal has been used as guidancefor general TCM practices for hundreds of years with littlechemical comprehension In this work for the first time weused the UPLC-TOFMS coupled with multivariate statisticalanalysis in an effort to gain scientific understanding for thecauses of the incompatibility

We previously reported that the LD50of aqueous extracts

of V nigrum L and RPA were 2566 gkg and 160 gkgrespectively after intragastric administration [11] V nigrumL extract was severely toxic whereas RPA had minimaltoxicity Although the amount of V nigrum L in each groupwas unchanged the mortality changed significantly withaltered ratios of V nigrum L to RPA When the doses of

10 Evidence-Based Complementary and Alternative Medicine

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

1

2

3

4

5

6 Tetragalloyglucose

Ion

inte

nsity

A B C D E F G H I J K LSample group

0020406081

121416182 Vanillic acid

Ion

inte

nsity

A B C D E F G H I J K LSample group

minus2

0

2

4

6

8

10

12 Benzoylpaeoniflorin

000501015020250303504

A B C D E F G H I J K L

Ion

inte

nsity

Sample groupminus005

Paeoniflorin sulfonate

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

05

1

15

2 Benzoyloxypaeoniflorin

minus05

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

05

1

15

2 Gallic acid

minus05

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

05

1

15

2

253 Galloylpaeoniflorin

minus05

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

005

01

015

02

025

03

035 Paeonilacto-neC

Ion

inte

nsity

A B C D E F G H I J K LSample group

0123456789 Paeonilacto-neB

minus1

Ion

inte

nsity

A B C D E F G H I J K LSample group

Vanillic acid

0

1

2

3

4

5

6

7

minus1

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

01

02

03

04

05

06

07

minus01

6-O-120573-D-Glucopyranosyllactinolide

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

05

1

15

2

25

3

35

minus05

Paeoniflorinalbiflorin R1albiflorinmudanpioside I

A B C D E F G H I J K L

Ion

inte

nsity

Sample group

Paeonol

0

02

04

06

08

1

12

minus02

Ion

inte

nsity

A B C D E F G H I J K LSample group

01020304050607080 Pentagalloylglucose

minus10

Ion

inte

nsity

A B C D E F G H I J K LSample group

00102030405060708 Lactiflorin

minus01

Ion

inte

nsity

A BC D E F G H I J K LSample group

0020406081

121416 Isomaltopaeoniflorin

minus02

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

05

1

15

2

25

3

minus05

Albiflorin

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

05

1

15

2

25 1998400-O-Benzoylsucrose

(a)

Figure 7 Continued

Evidence-Based Complementary and Alternative Medicine 11

Ion

inte

nsity

Ion

inte

nsity

Ion

inte

nsity

A B C D E F G H I J K LSample group A B C D E F G H I J K L

Sample group

A B C D E F G H I J K LSample group

00102030405060708 Oxypaeoniflorin

001020304050607 Eugeniin

Ion

inte

nsity

A B C D E F G H I J K LSample group

0020406081

12141618 Paeonia lactinone

00102030405060708091 Ethyl gallate

minus01

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

01

02

03

04

05

Lactinolide06

minus01

0

5

10

15

20

25 Palbinone

Ion

inte

nsity

A B C D E F G H I J K LSample group

minus5

minus01

(b)

Figure 7 Ion intensity trend plots for the chemical components that were highly negatively correlated with toxicity (119903 ge 09)

OD

val

ue

25 50 100 200 400 800

lowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

mdashVeratridine (120583M)

10

12

08

06

04

02

0

(a)

OD

val

ue

02

04

06

08

12

Paeoniflorin (120583M) 100 500 1500 2000mdash 1000

10

00

(b)

OD

val

ue

Paeoniflorin (120583M)

mdashmdash

Veratridine (120583M) 400 400 400 400 400 400 400 400 400 400

50 100 250 500 750 1000 1250 1500 1750 2000

02

04

06

08

12

10

00

lowastlowastlowast

lowastlowast

lowastlowast lowastlowast

lowastlowast

lowast

(c)

Figure 8 SH-SY5Y cell viability SH-SY5Y cells were cultured with (a) veratridine (25ndash800120583M) (b) paeoniflorin (50minus2000120583M) and (c)400 120583M veratridine in combination with 50minus2000120583M paeoniflorin for 24 h Cell viability was then assessed using MTS assays Data arepresented as mean plusmn SD Means of three independent experiments Asterisks indicate a statistically significant difference compared withuntreated cells lowast119875 lt 005 lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001 versus controL

12 Evidence-Based Complementary and Alternative Medicine

V nigrum L were equal to or higher than RPA particularlyratios of 8 1 and 1 1 mouse mortality increased significantlyTherefore components of V nigrum L were the majorcausative factors of toxicity In addition RPA modulated thetoxicity ofV nigrum LTherefore clinical applications shouldbe monitored carefully

Mice died 5minus10min after drug administration Toxicitymanifested predominantly as trembling convulsions andspasms but the major organs showed no obvious lesionsat necropsy by the naked eye Based on the toxic reactionsand the time of death in the current study and accord-ing to the guidelines for Chinese natural medicine acutetoxicity testing we speculate that the toxicity was causedpredominantly by suppression of the central nervous systemand that V nigrum L was the major cause of death Vnigrum L water extracts exert toxic effects on the digestivetract mucosa nucleus nervi vagi and central nervous system[6 17 18] Pharmacological studies of veratrum alkaloidshave been performed previously and reported that thesealkaloids stimulate the central nervous system and inhibit thebrain This results in spasms convulsions coma drowsinessand other symptoms of consciousness in animals which isconsistent with the symptoms of themice in the current study[6 17]

Veratrum alkaloid is the toxic component of V nigrum LWe previously investigated the differently expressed genes inSH-SY5Y cells treated with veratridine using cDNAmicroar-rays [34] Data revealed that veratridine treatment alteredthe expression of MMP caused Ca2+ concentration over-load increased reactive oxygen species production enhancedLDH release damaged the cell membrane reduced SH-SY5Y cell viability and induced apoptosis Under oxidizingconditions the MAPK signaling pathway is activated whichincreases apoptosisThis study illustrates the neurotoxicity ofthe combination of V nigrum L and reveals the molecularmechanism behind the neurotoxic effects of V nigrum L(data not shown)

The amount of V nigrum L in each group was the samethe dose of RPA is in ascending order and the chemicalcomponents of RPA are also increased but the mortalityof each group changed significantly with the increase ofRPA so we conclude that higher amount of RPA can haveprotection effect which can offset the toxicity of combinationof V nigrum L and RPA And pharmacological studies havedemonstrated that RPA exerts a wide range of pharmacolog-ical activities including analgesic sedative anticonvulsantand antispasmodic effects on the central nervous systemRPA could also inhibit writhing reactions and antagonizepentylenetetrazol-induced convulsions The most importantactive ingredient in RPA is paeoniflorin which has significantneuroprotective effects [26ndash34]

In this study mouse acute toxicity experiments revealedthat the mortality of mice changed significantly when thedose of V nigrum L was higher than or equal to that ofRPA particularly at ratios of V nigrum L RPA of 8 1and 1 1 UPLC-Q-TOFMS with automated data analysisused to identify and quantify the specific chemical compo-nents According to Pearsonrsquos correlation coefficient most of

the chemical components that were positively correlated withtoxicity were from V nigrum L We conclude that the majorveratrum alkaloids of V nigrum L caused acute toxicity anddeath in mice When the doses of V nigrum L were higherthan or equal to RPA the main chemical components of Vnigrum L and the toxicity of the decoctions were increasedThis suggests that V nigrum L and RPA have opposingroles Additional experiments revealed that the cytotoxicityof veratridine in SH-SY5Y cells was decreasedwith increasingconcentrations of paeoniflorin Therefore the clinical use ofthese agents should be considered carefully This study alsoprovides information to help improve the incompatibilitytheory of TCM and introduces novel ideas for further studieson the development and application of TCM

When preparing V nigrum L and RPA decoctions alower amount of RPA contributes to the dissolution of vera-trum alkaloids Conversely higher amounts of RPA can exertneuroprotective effects which can offset the toxicity causedby the combination of V nigrum L and RPA Under theseconditions dissolution is the most important factor for Vnigrum L toxicity The results of cell-based experiments alsodemonstrated that paeoniflorin could offset the neurotoxiceffects of veratridine when treated in combination whichexcluded the factor of dissolution in preparation

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors are grateful for financial support from NationalNature Science Foundation of China (Grant no 81073149)National Basic Research Program of China (Grant nos2011CB505304 and 2012CB518402) and Natural ScienceFoundation of Beijing (Grant no 7112110)

References

[1] H Guo W Li X Y Wang and G W Fan ldquoAdvance of researchon toxic attenuation by compatibility of traditional Chinesemedicine prescriptionsrdquo Zhongguo Zhongyao Zazhi vol 37 no1 pp 120ndash123 2012

[2] Y Zhang H Hua X Fan C Wang and J Duan ldquoExplorationon eighteen incompatible medicaments of chest pain prescrip-tions based on association rules miningrdquo Zhongguo ZhongyaoZazhi vol 36 no 24 pp 3544ndash3547 2011

[3] C-H Xu P Wang Y Wang et al ldquoPharmacokinetic compar-isons of two different combinations of Shaoyao-Gancao Decoc-tion in rats competing mechanisms between paeoniflorin andglycyrrhetinic acidrdquo Journal of Ethnopharmacology vol 149 no2 pp 443ndash452 2013

[4] D H TianDictionary of Practical Traditional Chinese MedicineII Peoplersquos Medical Beijing China 2002

[5] Y Gaillard and G Pepin ldquoLC-EI-MS determination of ver-atridine and cevadine in two fatal cases of Veratrum albumpoisoningrdquo Journal of Analytical Toxicology vol 25 no 6 pp481ndash485 2001

Evidence-Based Complementary and Alternative Medicine 13

[6] H Li G-Y Gao S-Y Li W-J Zhao Y-T Guo and Z-J Liang ldquoEffects of Veratrum nigrum alkaloids on centralcatecholaminergic neurons of renal hypertensive ratsrdquo ActaPharmacologica Sinica vol 21 no 1 pp 23ndash28 2000

[7] L Wang W Li and Y Liu ldquoHypotensive effect and toxicologyof total alkaloids and veratramine from roots and rhizomesof Veratrum nigrum L in spontaneously hypertensive ratsrdquoPharmazie vol 63 no 8 pp 606ndash610 2008

[8] Y P Jiang Y G Liu and H C Chen ldquoEffect of aqueous extractof Radix Paeoniae Rubra against Carbon Tetrachloride inducedliver fibrosis in ratrdquo Herald of Medicine vol 23 pp 527ndash5292004

[9] D-Y He and S-M Dai ldquoAnti-inflammatory and immunomod-ulatory effects of Paeonia lactiflora Pall a traditional Chineseherbal medicinerdquo Frontiers in Pharmacology 2011

[10] C Feng M Liu X Shi et al ldquoPharmacokinetic properties ofpaeoniflorin albiflorin and oxypaeoniflorin after oral gavage ofextracts of Radix Paeoniae Rubra and Radix Paeoniae Alba inratsrdquo Journal of Ethnopharmacology vol 130 no 2 pp 407ndash4132010

[11] X X Zhang Y G Wang Z C Ma Y Gao and C R XiaoldquoUniform designed research on the compatibility toxicity ofVeratridine and peonyrdquo Zhong Hua Zhong Yi Yao Za Zhi vol28 no 10 pp 2901ndash2904 2013

[12] Q D Liang Y Gao J Ma et al ldquoChemical comparison of driedrehmannia root and prepared rehmannia root by UPLC-TOFMS andHPLC-ELSDwithmultivariate statistical analysisrdquoActaPharmaceutica Sinica B vol 3 no 1 pp 55ndash64 2013

[13] Y Wang S He X Cheng Y Lu Y Zou and Q Zhang ldquoUPLC-Q-TOF-MSMS fingerprinting of traditional Chinese formulaSiJunZiTangrdquo Journal of Pharmaceutical and Biomedical Analy-sis vol 80 pp 24ndash33 2013

[14] L-P Kang K Yu Y Zhao et al ldquoCharacterization of steroidalglycosides from the extract of Paris Polyphylla var Yunnanensisby UPLCQ-TOFMSErdquo Journal of Pharmaceutical and Biomed-ical Analysis vol 62 pp 235ndash249 2012

[15] A Kaufmann P Butcher K Maden and M Widmer ldquoUltra-performance liquid chromatography coupled to time of flightmass spectrometry (UPLC-TOF) a novel tool for multiresiduescreening of veterinary drugs in urinerdquoAnalytica Chimica Actavol 586 no 1-2 pp 13ndash21 2007

[16] S Zhou Z C Ma Q D Liang et al ldquoUPLCQ-TOF-MS basedchemical profiling approach to evaluate chemical compositionof augmentation toxicity in combination of Radix aconiti andPinellia praeparatardquoActa Chimica Sinica vol 70 no 3 pp 284ndash290 2012

[17] N Lin and X Gao ldquoExperimental observation on the absorp-tion of Veratrum nigrum L in the upper digestive tractrdquoZhongguo Zhong Yao Za Zhi vol 17 no 1 pp 43ndash45 1992

[18] Z-Z Wang W-J Zhao X-S Zhang et al ldquoProtection ofVeratrum nigrum L var ussuriense Nakai alkaloids againstischema-reperfusion injury of the rat liverrdquo World Journal ofGastroenterology vol 13 no 4 pp 564ndash571 2007

[19] V Christov B Mikhova A Ivanova et al ldquoSteroidal alka-loids of Veratrum lobelianum Bernh and Veratrum nigrum LrdquoZeitschrift fur Naturforschung C Journal of Biosciences vol 65no 3-4 pp 195ndash200 2010

[20] V Christov BMikhova andD Selenge ldquo(-)-Veranigrine a newsteroidal alkaloid fromVeratrum nigrum Lrdquo Fitoterapia vol 80no 1 pp 25ndash27 2009

[21] Y Cong J-H Wang R Wang Y-M Zeng C-D Liu and XLi ldquoA study on the chemical constituents of Veratrum nigrum

L processed by rice vinegarrdquo Journal of Asian Natural ProductsResearch vol 10 no 7-8 pp 619ndash624 2008

[22] H-L Li J Tang R-H Liu C Zhang and W-D Zhang ldquoTwonew flavanone glycosides from Veratrum nigrum Lrdquo NaturalProduct Research vol 23 no 2 pp 122ndash126 2009

[23] Y Cong Y-B Zhou J Chen Y-M Zeng and J-H WangldquoAlkaloid profiling of crude and processed Veratrum nigrum Lthrough simultaneous determination of ten steroidal alkaloidsby HPLC-ELSDrdquo Journal of Pharmaceutical and BiomedicalAnalysis vol 48 no 3 pp 573ndash578 2008

[24] L-S Wang D-Q Zhao H-M Tao and Y-H Liu ldquoDetermi-nation of vetatramine in Veratrum nigrum by HPLC-ELSDrdquoZhongguo Zhongyao Zazhi vol 33 no 7 pp 791ndash792 2008

[25] Q Wang H-Z Guo C-H Huo et al ldquoChemical constituentsin root of Paeonia lactiflorardquo Chinese Traditional and HerbalDrugs vol 38 no 7 pp 972ndash976 2007

[26] J-J TanQ-C Zhao L Yang Z-P Shang Z-QDu andMYanldquoChemical constituents in roots of Paeonia lactiflorardquo ChineseTraditional and Herbal Drugs vol 41 no 8 pp 1245ndash1248 2010

[27] Q Zhang Z Z Wang W Xiao et al ldquoUPLC characteristicchromatographic profile of Paeoniae Radix Albardquo ZhongguoZhongyao Zazhi vol 36 no 6 pp 712ndash714 2011

[28] X R Gao and Y Tian ldquoActive principles of Paeonia lactifloraPallrdquo Chinese Journal of New Drugs vol 15 pp 416ndash418 2006

[29] N-C Luo W Ding J Wu et al ldquoUPLC-Q-TOFMS coupledwith multivariate statistical analysis as a powerful techniquefor rapidly exploring potential chemicalmarkers to differentiatebetween radix paeoniae alba and radix paeoniae rubrardquoNaturalProduct Communications vol 8 no 4 pp 487ndash491 2013

[30] W K Zhao Y Guo T Yasuhiro and K Tohru ldquoIsolation andstructure determination of echinuline from Veratrum nigrumL var ussuriense Nakairdquo Zhongguo Zhong Yao Za Zhi vol 16no 7 pp 425ndash426 1991

[31] W Zhao Y Guo T Yasuhirb and T Kikuchi ldquoIsolation andstructure determination of aurantiamide acetate fromVeratrumnigrum Lvarussuriense Nakairdquo Zhongguo Zhongyao Zazhi vol23 no 1 p 41 1998

[32] L Yang X H Xia Q Zhu and X P Tan ldquoStudy on therule of influence of two purification methods on the chemicalcompositions in aqueous solution of paeoniae radix albardquoZhong Yao Cai vol 36 pp 118ndash121 2012

[33] X Y Zang and X Li ldquoDevelopment of the study on chemicalconstituentsof Paeonia lact if lora Pallrdquo Journal of ShenyangPharmaceutical University vol 19 pp 70ndash73 2002

[34] Y-L Wang Y-G Wang Q-D Liang et al ldquoUsing cDNAmicroarray to screen differently expressed genes in SH-SY5Ytreated by veratridinerdquo Chinese Pharmacological Bulletin vol29 no 5 pp 643ndash647 2013

Submit your manuscripts athttpwwwhindawicom

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

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OncologyJournal of

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Oxidative Medicine and Cellular Longevity

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PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Computational and Mathematical Methods in Medicine

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Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

4 Evidence-Based Complementary and Alternative Medicine

0

20

40

60

80

100

120

Mor

talit

y ra

te (

)

A B C D E F G H I J K LGroup

Figure 1 Total mortality of mice after the intragastric administra-tion of decoctions containing different proportions of V nigrum Land RPA The groups represent the combinations shown in Table 1

FemaleMale

0

20

40

60

80

100

120

Mor

talit

y ra

te (

)

A B C D E F G H I J K LGroup

Figure 2 Mortality of male and female mice after the intragastricadministration of decoctions containing different proportions of Vnigrum L and RPA The groups represent the combinations shownin Table 1

32 Identification and Quantification of the Chemical Com-ponents of V nigrum L and RPA To explain the cause ofacute toxicity of the combined use of V nigrum L andRPA we compared the chemical composition of the 12groups using a supervised orthogonal partial least squareddiscriminant analysis (OPLS-DA) After Pareto scaling withmean centering the data from both the positive and negativeion modes were displayed as scores plots (Figure 3) Thescores plots clearly revealed that the samples were clustered

minus40

minus20

0

20

40

minus100 0 100

A BC DE FG HI JK L

t[1]

t[2]

Scores comp[1] versus comp[2] colored by sample group

(a)

minus50

0

50

minus200 minus100 0 100 200

A BC DE FG HI JK L

Scores comp[1] versus comp[2] colored by sample group

t[1]

t[2]

(b)

Figure 3 BBThe OPLS-DA score plot of 36 samples obtained usingPareto scaling with mean centering (a) Positive ion mode (b)Negative ion mode The same score plots represent the same groupBoth the positive and negative ion modes showed that the sampleswere clearly clustered into 12 groups Replicates of the 12 groupscould be distinguished easily

into 12 groups replicates of same group were comparable butthe 12 groups could be distinguished easilyThis suggests thatthe changes in chemical compositions of the 12 groups wereconsistent and the experiment was reproducible

UPLC-TOF-MS is a rapid specific and sensitive methodused to identify and quantify individual components Repre-sentative BPI chromatograms of the 36 samples in the positiveand negative mode ESI are shown in Figure 4

The contents of some special alkaloids change signif-icantly in different proportion decoctions To determinewhich constituents contributed to the differences in toxicity

Evidence-Based Complementary and Alternative Medicine 5

F-P-2

95

()

minus5

2481128

4763010

4603062

44632764422959

5363228

4443088

4263004

4103042

5763557

6763667

7163999

108

9

7

3

2

1

465

200 400 600 800 1000 1200 1400 1600 1800

Time

BPI103e5

1 TOF MS ES+

(a)

43909591690150 2550515

5431194

5671754

5251649

4491475

2270695

6311725

6311760

6291927

3292323

10

8

97

3

2

1

4

65

200 400 600 800 1000 1200 1400 1600 1800

Time

BPI430e495

minus5

()

5-2 neg1 TOF MS ESminus

(b)

Figure 4 Representative BPI chromatograms (a) Positive ionmode (b) Negative ionmode Some of identified constituents (1ndash10) are labeledin the BPI chromatogram and the numbers correspond to Tables 3 and 4

observed with the 12 decoctions statistical analyses wereperformed using Pearsonrsquos correlation and the correlationcoefficient (119903) described the degree of linear correlationbetween two variables total mouse mortality and the chem-ical composition of the 12 decoctions The 119903 values werebetween minus1 and +1 119903 gt 0 indicates that two variables werepositively correlated whereas 119903 lt 0 indicates a negativecorrelation The larger the absolute value the higher thecorrelation therefore we defined 119903 = 090ndash100 as extremelyrelated and 119903 = 080ndash089 as highly related We extractedthe relevant chemical component data when 119903 ge 08 andidentified the individual components using the RPA and Vnigrum L chemical composition databases

According to Pearsonrsquos correlation coefficient 131 compo-sitions had an extremely positive relationship (119903 ge 09) withtoxicity However only 14 of these could be identified andall came from V nigrum L In addition 35 components werehighly positively correlated (119903 ge 08) to acute toxicity andthese constituents contributed most of the toxicity identifiedin the 12 decoctions Therefore we hypothesized that thesecomponents might play important roles in the acute toxicity

of this drug combination We identified that these com-poundswere all veratrum alkaloids that were detected in eachsample in various ionized forms The chemical componentswith 119903 ge 08 are shown in Table 3 [7 19ndash24]

3-Veratroylgermine jervine veratramine germanitrinegermidine and germerine were the main chemical compo-nents that were associated with acute toxicity with Pearsonrsquoscorrelation coefficients of 09779 09661 09589 0958109402 and 09393 respectively These compounds are allveratrum alkaloids and the changes in their content areshown in Figure 5The other 29 components that were highlypositively correlated (119903 ge 08) with acute toxicity are shownin Figure 6

Interestingly Pearsonrsquos correlation coefficient identified24 RPA components that were extremely negatively corre-lated (119903 le minus09) with toxicityThe chemical components with119903 le minus09 are shown in Table 4 [25ndash33]

The concentrations of these components increased withincreasing amounts of RPA Paeoniflorin sulfonate ben-zoyloxy paeoniflorin galloyl paeoniflorin paeonilacto-neBpaeonilacto-neC and tetragalloyglucose were the major RPA

6 Evidence-Based Complementary and Alternative Medicine

Table 3 The chemical components that were highly positively correlated with toxicity (119903 ge 08)

Number 119903 tR (min) Assigned identity Molecularformula

Mean measuredmass (Da)

Theoretical exactmass (Da)

Mass accuracy(ppm)

1 09779 1298 3-Veratroylgermine C36H53NO11 6763667 6758061 018052 09661 1239 Jervine C27H39NO3 4263004 4256035 236243 09589 1261 Veretramine C27H39NO2 4103042 4096041 297514 09581 1343 Germanitrine C39H59NO11 7184169 7178859 250155 09402 1220 Germidine C34H53NO10 6363412 6357853 057556 09393 1340 Germerine C37H59NO11 7163999 6938645 034087 09390 1291 3-Angeloylzygadenine C32H49NO8 5763557 5757334 127088 09311 1035 11-Deoxojervine C27H41NO2 4123189 4116199 64392

9 09240 157 11205733120572-Dihydroxy-5120573-jervanin-12-en-11-one C27H41NO4 4443088 4436187 24040

10 09237 696 Jervinone C27H37NO3 4242860 4235876 2014011 09182 1125 Neogermbudine C37H59NO12 7323971 7098639 4916912 09143 425 Germine C27H43NO8 5103059 5096322 15549

13 09131 418 NeogermineVeramanine C27H43NO5 4623213 4616340 14522

14 09034 1158 Veraline B C27H45NO3 4323465 4316511 2964615 08978 1116 Verdine C27H41NO5 4603056 4596181 1524916 08903 1163 Stenophylline A C36H51NO11 6743474 6737902 9865517 08870 1035 Germitrine C39H61NO12 7364278 7359011 0828818 08805 914 Polydatin C20H22O8 4290937 3903839 3484919 08738 1081 Cervdine C32H49NO9 5923498 5917328 20608

20 08675 1234 3-Veratrum acylprotoveratrine C36H51NO12 6903482 6897896 10609

21 08671 1289 Zygadenitic acid120575-lactone-16-angelate C32H47NO8 5743408 5737175 48884

22 08652 1232 Maackinine C39H59NO11 7184171 7178859 06797

23 08641 1238Rubijervine rubivirineetioline epirubijervine

IsorubijervineC27H43NO2 4143357 4136358 36827

24 08639 1049 Solanidine C27H43NO3 4303308 4296352 3049425 08492 1057 Pseudojervine C32H49NO8 5883525 5877441 1971026 08444 1181 Neojerminalanine C39H61NO13 7524244 7519005 3001427 08379 843 Zygacine C29H45NO8 5363223 5356695 0000028 08358 1320 Neoverataline B C27H42N2O9 5563260 5386304 407929 08201 1280 3-Veratroylzygadenine C36H51NO10 6963187 6577908 5346930 08176 583 Zygadenine C27H43NO7 4943110 4936328 1543431 08176 054 120574-Aminobutyric acid C4H9NO2 2460978 1031198 1612132 08160 1273 Veratrosine C33H49NO7 6163528 5717447 6832933 08128 1322 Stenophylline A C37H55NO10 6743901 6738333 05430

34 08119 13347-Acetyl-15-

methylbutyryl-3-veratroylgermine

C43H61NO13 8383791 7999433 13104

35 08017 1213 Vanilloylzygadenine C35H49NO10 6443444 6437643 15156

components that were identified with Pearsonrsquos correlationcoefficients of minus09811 minus09799 minus09793 minus09737 minus09736and minus09734 respectively The changes in content of the 24components that were highly negatively correlated (119903 le minus09)with acute toxicity are shown in Figure 7

33 Cell ProliferationAssay To further validate the toxicity ofthe combination ofV nigrumL andRPAonneurons we usedveratridine and paeoniflorin the major pharmacologicallyactive components of V nigrum L and peony for in vitroexperimentsThe effect of these two chemical components on

Evidence-Based Complementary and Alternative Medicine 7

Table 4 The chemical components that were highly negatively correlated with toxicity (119903 le minus09)

Number 119903 tR (min) Assigned identity Molecularformula

Mean measuredmass (Da)

Theoretical exactmass (Da)

Mass accuracy(ppm)

1 minus09811 495 Paeoniflorinsulfonate C23H28O13S 5431194 5445256 337142 minus09799 1220 Benzoyloxy paeoniflorin C30H32O13 5991785 6001843 030463 minus09793 977 Galloyl paeoniflorin C30H32O15 6311725 6321741 038564 minus09737 1479 Paeonilacto-ne B C17H18O6 3171750 3181103 392095 minus09736 056 Paeonilacto-ne C C10H12O4 1950496 1960736 531926 minus09734 809 Tetragalloyglucose C34H28O22 7871008 7885729 566077 minus09728 230 Vanillic acid C7H6O5 1690161 1701195 374718 minus09716 1391 Desbenzoyl paeoniflorin C16H24O10 3751868 3761369 445059 minus09708 1352 Benzoyl paeoniflorin C30H32O12 5831848 5845679 06258

10 minus09684 415 6-O-120573-D-Glucopyranosyllactinolide C16H26O9 3611537 3623722 93435

11 minus09670 1037 Paeoniflorin albiflorin R1albiflorin mudanpioside I C23H28O11 5191261 4804618 15284

12 minus09655 1167 Gallic acid C9H10O2 1510750 1501745 5959013 minus09640 493 Paeonol C9H10O3 1650545 1661739 4067614 minus09625 1167 Lactiflorin C23H26O10 4801872 4624465 0444915 minus09609 667 Isomaltopaeoniflorin C29H38O16 6432221 6426024 2656916 minus09589 852 Albiflorin C23H27O11 9573118 4794539 9372317 minus09582 679 11015840-O-Benzoylsucrose C19H26O12 4641757 4464025 2366818 minus09567 1057 Pentagalloylglucose C41H32O26 9391166 9406772 6629219 minus09562 802 Oxypaeoniflorin C23H28O12 5141934 4964612 1899220 minus09544 1158 Ethyl gallate C9H10O5 2210433 1981727 3244521 minus09478 1011 Eugeniin C41H30O26 9371010 9386613 6708622 minus09395 585 Paeonilactinone C9H14O2 1530904 1542063 7575023 minus09352 113 Lactinolide C10H16O4 2011111 2002316 7890724 minus09173 1373 Palbinone C22H30O4 3572047 3584712 52115

3-Veratroylgermine250

200

150

100

50

0A B C D E F G H I J K L

Ion

inte

nsity

Sample group

Ion

inte

nsity

Sample group

0010203040506070809 Veratramine

A B C D E F G H I J K L0

02

04

06

08

1

12

14

Ion

inte

nsity

A B C D E F G H I J K LSample group

Jervine

A B C D E F G H I J K L

Ion

inte

nsity

Sample group

020406080100120140160180200

Grmidine

A B C D E F G H I J K L

Germanitrine

Ion

inte

nsity

Sample group

0

02

04

06

08

1

12

14

A B C D E F G H I J K L

Ion

inte

nsity

Sample group

0

01

02

03

04

05

06 Germerine

minus01

Figure 5 Ion intensity trend plots for 3-veratroylgermine jervine veratramine germanitrine germidine and germerine which were highlypositively correlated (119903 ge 09393) with toxicity

8 Evidence-Based Complementary and Alternative Medicine

0100200300400500600700800

A B C D E F G H I J K L A B C D E F G H I J K L

Ion

inte

nsity

Ion

inte

nsity

Ion

inte

nsity

Ion

inte

nsity

Ion

inte

nsity

A B C D E F G H I J K L

A B C D E F G H I J K L

A B C D E F G H I J K LSample groupSample group

Sample group A B C D E F G H I J K LSample group

Sample groupA B C D E F G H I J K L

Sample group

3-Angeloylzygadenine

0

01

02

03

04

05

06

07

Ion

inte

nsity

Ion

inte

nsity

11-Deoxojervine

0

50

100

150

200

250

300

350

Sample group

Ion

inte

nsity

Sample group

A B C D E F G H I J K L0

01

02

03

04

05

06 Jervinone

0

05

1

15

2

25

3 Neogermbudine

0020406081

121416182 Germine

0051

152

253

35 Veraline B

0

1

2

3

4

5

6 Verdine

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

1

2

3

4

5

6 GermitrineIo

n in

tens

ity

A B C D E F G H I J K LSample group

0

05

1

15

25

2

Polydatin

minus05

minus05

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

1

2

3

4

5

6

Ion

inte

nsity

A B C D E F G H I J K LSample group

0051

152

253

Stenophylline A

minus05

35

Neogermineveramanine

jervanin-12-en-11-one

0

2

4

6

8

10

12

14

A B C D E F G H I J K L

Ion

inte

nsity

Sample group

Cervdine

A B C D E F G H I J K L

Ion

inte

nsity

Sample group

0

02

04

06

08

1

12 Maackinine

Ion

inte

nsity

A B C D E F G H I J K LSample group

0123456789 Rubijervine

A B C D E F G H I J K L

Ion

inte

nsity

Sample group

0020406081

12141618 3-Veratrum acyl protoveratrine

Ion

inte

nsity

A B C D E F G H I J K LSample group

0051

152

253

354

Solanidine

minus05

A B C D E F G H I JK L

Ion

inte

nsity

Sample group

0020406081

121416182

Zygadenitic acid 120575-Lactone-16-angelate

11205733a-Dihydroxy-5120573-

(a)

Figure 6 Continued

Evidence-Based Complementary and Alternative Medicine 9

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

20

40

60

80

100

120 Pseudojervine

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

02

04

06

08

1

12

14 Neojerminalanine

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

02

04

06

08

1

12 Zygacine

Ion

inte

nsity

A B C D E F G H I J K LSample group

012345678 Zygadenine

Ion

inte

nsity

A B C D E F G H I J K LSample group

000501015020250303504045 Neoverataline B

minus005

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

01

02

03

04

05

06 3-Veratroylzygadenine

minus01

0

05

1

15

2

25 Vanilloylzygadenine

A B C D E F G H I J K LSample group

Ion

inte

nsity

A B C D E F G H I J K LSample group

Ion

inte

nsity

0

2

4

6

8

10

12 Veratrosine

A B C D E F G H I J K LSample group

Ion

inte

nsity

0

2

4

6

8

10

12 Stenophylline A

A B C D E F G H I J K LSample group

Ion

inte

nsity

0020406081

1214 120574-Aminobutyric acid

minus02

A B C D E F G H I J K LSample group

Ion

inte

nsity

0

02

04

06

08

1

12 7-Acetyl-15-methylbutyryl-3-veratroylgermine

(b)

Figure 6 Ion intensity trend plots for the remaining 29 chemical components that were highly positively correlated (119903 ge 08) with toxicity

the viability of SH-SY5Y cells was tested using MTS assaysAs shown in Figures 8(a) and 8(b) veratridine had an LD

50

of 450 whereas paeoniflorin exerted no significant toxicityat doses of 0ndash2000120583M Therefore the dose of veratridinewas fixed at 400120583M and the RPA dose was varied from50 to 2000120583M SH-SY5Y cell cytotoxicity was increasedwhen the concentration of paeoniflorin was decreased inthe combination (Figure 8(c)) Therefore paeoniflorin couldoffset the cytotoxicity caused by veratridine in SH-SY5Y cells[25]

4 Discussion

The eighteen incompatible medicaments (Shi Ba Fan) is awell-known traditional Chinese medicine (TCM) theory It

basically listed eighteen pairs of TCM herbal medicine thatare not to be used in combination as they may cause fatalconsequences This principal has been used as guidancefor general TCM practices for hundreds of years with littlechemical comprehension In this work for the first time weused the UPLC-TOFMS coupled with multivariate statisticalanalysis in an effort to gain scientific understanding for thecauses of the incompatibility

We previously reported that the LD50of aqueous extracts

of V nigrum L and RPA were 2566 gkg and 160 gkgrespectively after intragastric administration [11] V nigrumL extract was severely toxic whereas RPA had minimaltoxicity Although the amount of V nigrum L in each groupwas unchanged the mortality changed significantly withaltered ratios of V nigrum L to RPA When the doses of

10 Evidence-Based Complementary and Alternative Medicine

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

1

2

3

4

5

6 Tetragalloyglucose

Ion

inte

nsity

A B C D E F G H I J K LSample group

0020406081

121416182 Vanillic acid

Ion

inte

nsity

A B C D E F G H I J K LSample group

minus2

0

2

4

6

8

10

12 Benzoylpaeoniflorin

000501015020250303504

A B C D E F G H I J K L

Ion

inte

nsity

Sample groupminus005

Paeoniflorin sulfonate

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

05

1

15

2 Benzoyloxypaeoniflorin

minus05

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

05

1

15

2 Gallic acid

minus05

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

05

1

15

2

253 Galloylpaeoniflorin

minus05

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

005

01

015

02

025

03

035 Paeonilacto-neC

Ion

inte

nsity

A B C D E F G H I J K LSample group

0123456789 Paeonilacto-neB

minus1

Ion

inte

nsity

A B C D E F G H I J K LSample group

Vanillic acid

0

1

2

3

4

5

6

7

minus1

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

01

02

03

04

05

06

07

minus01

6-O-120573-D-Glucopyranosyllactinolide

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

05

1

15

2

25

3

35

minus05

Paeoniflorinalbiflorin R1albiflorinmudanpioside I

A B C D E F G H I J K L

Ion

inte

nsity

Sample group

Paeonol

0

02

04

06

08

1

12

minus02

Ion

inte

nsity

A B C D E F G H I J K LSample group

01020304050607080 Pentagalloylglucose

minus10

Ion

inte

nsity

A B C D E F G H I J K LSample group

00102030405060708 Lactiflorin

minus01

Ion

inte

nsity

A BC D E F G H I J K LSample group

0020406081

121416 Isomaltopaeoniflorin

minus02

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

05

1

15

2

25

3

minus05

Albiflorin

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

05

1

15

2

25 1998400-O-Benzoylsucrose

(a)

Figure 7 Continued

Evidence-Based Complementary and Alternative Medicine 11

Ion

inte

nsity

Ion

inte

nsity

Ion

inte

nsity

A B C D E F G H I J K LSample group A B C D E F G H I J K L

Sample group

A B C D E F G H I J K LSample group

00102030405060708 Oxypaeoniflorin

001020304050607 Eugeniin

Ion

inte

nsity

A B C D E F G H I J K LSample group

0020406081

12141618 Paeonia lactinone

00102030405060708091 Ethyl gallate

minus01

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

01

02

03

04

05

Lactinolide06

minus01

0

5

10

15

20

25 Palbinone

Ion

inte

nsity

A B C D E F G H I J K LSample group

minus5

minus01

(b)

Figure 7 Ion intensity trend plots for the chemical components that were highly negatively correlated with toxicity (119903 ge 09)

OD

val

ue

25 50 100 200 400 800

lowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

mdashVeratridine (120583M)

10

12

08

06

04

02

0

(a)

OD

val

ue

02

04

06

08

12

Paeoniflorin (120583M) 100 500 1500 2000mdash 1000

10

00

(b)

OD

val

ue

Paeoniflorin (120583M)

mdashmdash

Veratridine (120583M) 400 400 400 400 400 400 400 400 400 400

50 100 250 500 750 1000 1250 1500 1750 2000

02

04

06

08

12

10

00

lowastlowastlowast

lowastlowast

lowastlowast lowastlowast

lowastlowast

lowast

(c)

Figure 8 SH-SY5Y cell viability SH-SY5Y cells were cultured with (a) veratridine (25ndash800120583M) (b) paeoniflorin (50minus2000120583M) and (c)400 120583M veratridine in combination with 50minus2000120583M paeoniflorin for 24 h Cell viability was then assessed using MTS assays Data arepresented as mean plusmn SD Means of three independent experiments Asterisks indicate a statistically significant difference compared withuntreated cells lowast119875 lt 005 lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001 versus controL

12 Evidence-Based Complementary and Alternative Medicine

V nigrum L were equal to or higher than RPA particularlyratios of 8 1 and 1 1 mouse mortality increased significantlyTherefore components of V nigrum L were the majorcausative factors of toxicity In addition RPA modulated thetoxicity ofV nigrum LTherefore clinical applications shouldbe monitored carefully

Mice died 5minus10min after drug administration Toxicitymanifested predominantly as trembling convulsions andspasms but the major organs showed no obvious lesionsat necropsy by the naked eye Based on the toxic reactionsand the time of death in the current study and accord-ing to the guidelines for Chinese natural medicine acutetoxicity testing we speculate that the toxicity was causedpredominantly by suppression of the central nervous systemand that V nigrum L was the major cause of death Vnigrum L water extracts exert toxic effects on the digestivetract mucosa nucleus nervi vagi and central nervous system[6 17 18] Pharmacological studies of veratrum alkaloidshave been performed previously and reported that thesealkaloids stimulate the central nervous system and inhibit thebrain This results in spasms convulsions coma drowsinessand other symptoms of consciousness in animals which isconsistent with the symptoms of themice in the current study[6 17]

Veratrum alkaloid is the toxic component of V nigrum LWe previously investigated the differently expressed genes inSH-SY5Y cells treated with veratridine using cDNAmicroar-rays [34] Data revealed that veratridine treatment alteredthe expression of MMP caused Ca2+ concentration over-load increased reactive oxygen species production enhancedLDH release damaged the cell membrane reduced SH-SY5Y cell viability and induced apoptosis Under oxidizingconditions the MAPK signaling pathway is activated whichincreases apoptosisThis study illustrates the neurotoxicity ofthe combination of V nigrum L and reveals the molecularmechanism behind the neurotoxic effects of V nigrum L(data not shown)

The amount of V nigrum L in each group was the samethe dose of RPA is in ascending order and the chemicalcomponents of RPA are also increased but the mortalityof each group changed significantly with the increase ofRPA so we conclude that higher amount of RPA can haveprotection effect which can offset the toxicity of combinationof V nigrum L and RPA And pharmacological studies havedemonstrated that RPA exerts a wide range of pharmacolog-ical activities including analgesic sedative anticonvulsantand antispasmodic effects on the central nervous systemRPA could also inhibit writhing reactions and antagonizepentylenetetrazol-induced convulsions The most importantactive ingredient in RPA is paeoniflorin which has significantneuroprotective effects [26ndash34]

In this study mouse acute toxicity experiments revealedthat the mortality of mice changed significantly when thedose of V nigrum L was higher than or equal to that ofRPA particularly at ratios of V nigrum L RPA of 8 1and 1 1 UPLC-Q-TOFMS with automated data analysisused to identify and quantify the specific chemical compo-nents According to Pearsonrsquos correlation coefficient most of

the chemical components that were positively correlated withtoxicity were from V nigrum L We conclude that the majorveratrum alkaloids of V nigrum L caused acute toxicity anddeath in mice When the doses of V nigrum L were higherthan or equal to RPA the main chemical components of Vnigrum L and the toxicity of the decoctions were increasedThis suggests that V nigrum L and RPA have opposingroles Additional experiments revealed that the cytotoxicityof veratridine in SH-SY5Y cells was decreasedwith increasingconcentrations of paeoniflorin Therefore the clinical use ofthese agents should be considered carefully This study alsoprovides information to help improve the incompatibilitytheory of TCM and introduces novel ideas for further studieson the development and application of TCM

When preparing V nigrum L and RPA decoctions alower amount of RPA contributes to the dissolution of vera-trum alkaloids Conversely higher amounts of RPA can exertneuroprotective effects which can offset the toxicity causedby the combination of V nigrum L and RPA Under theseconditions dissolution is the most important factor for Vnigrum L toxicity The results of cell-based experiments alsodemonstrated that paeoniflorin could offset the neurotoxiceffects of veratridine when treated in combination whichexcluded the factor of dissolution in preparation

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors are grateful for financial support from NationalNature Science Foundation of China (Grant no 81073149)National Basic Research Program of China (Grant nos2011CB505304 and 2012CB518402) and Natural ScienceFoundation of Beijing (Grant no 7112110)

References

[1] H Guo W Li X Y Wang and G W Fan ldquoAdvance of researchon toxic attenuation by compatibility of traditional Chinesemedicine prescriptionsrdquo Zhongguo Zhongyao Zazhi vol 37 no1 pp 120ndash123 2012

[2] Y Zhang H Hua X Fan C Wang and J Duan ldquoExplorationon eighteen incompatible medicaments of chest pain prescrip-tions based on association rules miningrdquo Zhongguo ZhongyaoZazhi vol 36 no 24 pp 3544ndash3547 2011

[3] C-H Xu P Wang Y Wang et al ldquoPharmacokinetic compar-isons of two different combinations of Shaoyao-Gancao Decoc-tion in rats competing mechanisms between paeoniflorin andglycyrrhetinic acidrdquo Journal of Ethnopharmacology vol 149 no2 pp 443ndash452 2013

[4] D H TianDictionary of Practical Traditional Chinese MedicineII Peoplersquos Medical Beijing China 2002

[5] Y Gaillard and G Pepin ldquoLC-EI-MS determination of ver-atridine and cevadine in two fatal cases of Veratrum albumpoisoningrdquo Journal of Analytical Toxicology vol 25 no 6 pp481ndash485 2001

Evidence-Based Complementary and Alternative Medicine 13

[6] H Li G-Y Gao S-Y Li W-J Zhao Y-T Guo and Z-J Liang ldquoEffects of Veratrum nigrum alkaloids on centralcatecholaminergic neurons of renal hypertensive ratsrdquo ActaPharmacologica Sinica vol 21 no 1 pp 23ndash28 2000

[7] L Wang W Li and Y Liu ldquoHypotensive effect and toxicologyof total alkaloids and veratramine from roots and rhizomesof Veratrum nigrum L in spontaneously hypertensive ratsrdquoPharmazie vol 63 no 8 pp 606ndash610 2008

[8] Y P Jiang Y G Liu and H C Chen ldquoEffect of aqueous extractof Radix Paeoniae Rubra against Carbon Tetrachloride inducedliver fibrosis in ratrdquo Herald of Medicine vol 23 pp 527ndash5292004

[9] D-Y He and S-M Dai ldquoAnti-inflammatory and immunomod-ulatory effects of Paeonia lactiflora Pall a traditional Chineseherbal medicinerdquo Frontiers in Pharmacology 2011

[10] C Feng M Liu X Shi et al ldquoPharmacokinetic properties ofpaeoniflorin albiflorin and oxypaeoniflorin after oral gavage ofextracts of Radix Paeoniae Rubra and Radix Paeoniae Alba inratsrdquo Journal of Ethnopharmacology vol 130 no 2 pp 407ndash4132010

[11] X X Zhang Y G Wang Z C Ma Y Gao and C R XiaoldquoUniform designed research on the compatibility toxicity ofVeratridine and peonyrdquo Zhong Hua Zhong Yi Yao Za Zhi vol28 no 10 pp 2901ndash2904 2013

[12] Q D Liang Y Gao J Ma et al ldquoChemical comparison of driedrehmannia root and prepared rehmannia root by UPLC-TOFMS andHPLC-ELSDwithmultivariate statistical analysisrdquoActaPharmaceutica Sinica B vol 3 no 1 pp 55ndash64 2013

[13] Y Wang S He X Cheng Y Lu Y Zou and Q Zhang ldquoUPLC-Q-TOF-MSMS fingerprinting of traditional Chinese formulaSiJunZiTangrdquo Journal of Pharmaceutical and Biomedical Analy-sis vol 80 pp 24ndash33 2013

[14] L-P Kang K Yu Y Zhao et al ldquoCharacterization of steroidalglycosides from the extract of Paris Polyphylla var Yunnanensisby UPLCQ-TOFMSErdquo Journal of Pharmaceutical and Biomed-ical Analysis vol 62 pp 235ndash249 2012

[15] A Kaufmann P Butcher K Maden and M Widmer ldquoUltra-performance liquid chromatography coupled to time of flightmass spectrometry (UPLC-TOF) a novel tool for multiresiduescreening of veterinary drugs in urinerdquoAnalytica Chimica Actavol 586 no 1-2 pp 13ndash21 2007

[16] S Zhou Z C Ma Q D Liang et al ldquoUPLCQ-TOF-MS basedchemical profiling approach to evaluate chemical compositionof augmentation toxicity in combination of Radix aconiti andPinellia praeparatardquoActa Chimica Sinica vol 70 no 3 pp 284ndash290 2012

[17] N Lin and X Gao ldquoExperimental observation on the absorp-tion of Veratrum nigrum L in the upper digestive tractrdquoZhongguo Zhong Yao Za Zhi vol 17 no 1 pp 43ndash45 1992

[18] Z-Z Wang W-J Zhao X-S Zhang et al ldquoProtection ofVeratrum nigrum L var ussuriense Nakai alkaloids againstischema-reperfusion injury of the rat liverrdquo World Journal ofGastroenterology vol 13 no 4 pp 564ndash571 2007

[19] V Christov B Mikhova A Ivanova et al ldquoSteroidal alka-loids of Veratrum lobelianum Bernh and Veratrum nigrum LrdquoZeitschrift fur Naturforschung C Journal of Biosciences vol 65no 3-4 pp 195ndash200 2010

[20] V Christov BMikhova andD Selenge ldquo(-)-Veranigrine a newsteroidal alkaloid fromVeratrum nigrum Lrdquo Fitoterapia vol 80no 1 pp 25ndash27 2009

[21] Y Cong J-H Wang R Wang Y-M Zeng C-D Liu and XLi ldquoA study on the chemical constituents of Veratrum nigrum

L processed by rice vinegarrdquo Journal of Asian Natural ProductsResearch vol 10 no 7-8 pp 619ndash624 2008

[22] H-L Li J Tang R-H Liu C Zhang and W-D Zhang ldquoTwonew flavanone glycosides from Veratrum nigrum Lrdquo NaturalProduct Research vol 23 no 2 pp 122ndash126 2009

[23] Y Cong Y-B Zhou J Chen Y-M Zeng and J-H WangldquoAlkaloid profiling of crude and processed Veratrum nigrum Lthrough simultaneous determination of ten steroidal alkaloidsby HPLC-ELSDrdquo Journal of Pharmaceutical and BiomedicalAnalysis vol 48 no 3 pp 573ndash578 2008

[24] L-S Wang D-Q Zhao H-M Tao and Y-H Liu ldquoDetermi-nation of vetatramine in Veratrum nigrum by HPLC-ELSDrdquoZhongguo Zhongyao Zazhi vol 33 no 7 pp 791ndash792 2008

[25] Q Wang H-Z Guo C-H Huo et al ldquoChemical constituentsin root of Paeonia lactiflorardquo Chinese Traditional and HerbalDrugs vol 38 no 7 pp 972ndash976 2007

[26] J-J TanQ-C Zhao L Yang Z-P Shang Z-QDu andMYanldquoChemical constituents in roots of Paeonia lactiflorardquo ChineseTraditional and Herbal Drugs vol 41 no 8 pp 1245ndash1248 2010

[27] Q Zhang Z Z Wang W Xiao et al ldquoUPLC characteristicchromatographic profile of Paeoniae Radix Albardquo ZhongguoZhongyao Zazhi vol 36 no 6 pp 712ndash714 2011

[28] X R Gao and Y Tian ldquoActive principles of Paeonia lactifloraPallrdquo Chinese Journal of New Drugs vol 15 pp 416ndash418 2006

[29] N-C Luo W Ding J Wu et al ldquoUPLC-Q-TOFMS coupledwith multivariate statistical analysis as a powerful techniquefor rapidly exploring potential chemicalmarkers to differentiatebetween radix paeoniae alba and radix paeoniae rubrardquoNaturalProduct Communications vol 8 no 4 pp 487ndash491 2013

[30] W K Zhao Y Guo T Yasuhiro and K Tohru ldquoIsolation andstructure determination of echinuline from Veratrum nigrumL var ussuriense Nakairdquo Zhongguo Zhong Yao Za Zhi vol 16no 7 pp 425ndash426 1991

[31] W Zhao Y Guo T Yasuhirb and T Kikuchi ldquoIsolation andstructure determination of aurantiamide acetate fromVeratrumnigrum Lvarussuriense Nakairdquo Zhongguo Zhongyao Zazhi vol23 no 1 p 41 1998

[32] L Yang X H Xia Q Zhu and X P Tan ldquoStudy on therule of influence of two purification methods on the chemicalcompositions in aqueous solution of paeoniae radix albardquoZhong Yao Cai vol 36 pp 118ndash121 2012

[33] X Y Zang and X Li ldquoDevelopment of the study on chemicalconstituentsof Paeonia lact if lora Pallrdquo Journal of ShenyangPharmaceutical University vol 19 pp 70ndash73 2002

[34] Y-L Wang Y-G Wang Q-D Liang et al ldquoUsing cDNAmicroarray to screen differently expressed genes in SH-SY5Ytreated by veratridinerdquo Chinese Pharmacological Bulletin vol29 no 5 pp 643ndash647 2013

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Evidence-Based Complementary and Alternative Medicine 5

F-P-2

95

()

minus5

2481128

4763010

4603062

44632764422959

5363228

4443088

4263004

4103042

5763557

6763667

7163999

108

9

7

3

2

1

465

200 400 600 800 1000 1200 1400 1600 1800

Time

BPI103e5

1 TOF MS ES+

(a)

43909591690150 2550515

5431194

5671754

5251649

4491475

2270695

6311725

6311760

6291927

3292323

10

8

97

3

2

1

4

65

200 400 600 800 1000 1200 1400 1600 1800

Time

BPI430e495

minus5

()

5-2 neg1 TOF MS ESminus

(b)

Figure 4 Representative BPI chromatograms (a) Positive ionmode (b) Negative ionmode Some of identified constituents (1ndash10) are labeledin the BPI chromatogram and the numbers correspond to Tables 3 and 4

observed with the 12 decoctions statistical analyses wereperformed using Pearsonrsquos correlation and the correlationcoefficient (119903) described the degree of linear correlationbetween two variables total mouse mortality and the chem-ical composition of the 12 decoctions The 119903 values werebetween minus1 and +1 119903 gt 0 indicates that two variables werepositively correlated whereas 119903 lt 0 indicates a negativecorrelation The larger the absolute value the higher thecorrelation therefore we defined 119903 = 090ndash100 as extremelyrelated and 119903 = 080ndash089 as highly related We extractedthe relevant chemical component data when 119903 ge 08 andidentified the individual components using the RPA and Vnigrum L chemical composition databases

According to Pearsonrsquos correlation coefficient 131 compo-sitions had an extremely positive relationship (119903 ge 09) withtoxicity However only 14 of these could be identified andall came from V nigrum L In addition 35 components werehighly positively correlated (119903 ge 08) to acute toxicity andthese constituents contributed most of the toxicity identifiedin the 12 decoctions Therefore we hypothesized that thesecomponents might play important roles in the acute toxicity

of this drug combination We identified that these com-poundswere all veratrum alkaloids that were detected in eachsample in various ionized forms The chemical componentswith 119903 ge 08 are shown in Table 3 [7 19ndash24]

3-Veratroylgermine jervine veratramine germanitrinegermidine and germerine were the main chemical compo-nents that were associated with acute toxicity with Pearsonrsquoscorrelation coefficients of 09779 09661 09589 0958109402 and 09393 respectively These compounds are allveratrum alkaloids and the changes in their content areshown in Figure 5The other 29 components that were highlypositively correlated (119903 ge 08) with acute toxicity are shownin Figure 6

Interestingly Pearsonrsquos correlation coefficient identified24 RPA components that were extremely negatively corre-lated (119903 le minus09) with toxicityThe chemical components with119903 le minus09 are shown in Table 4 [25ndash33]

The concentrations of these components increased withincreasing amounts of RPA Paeoniflorin sulfonate ben-zoyloxy paeoniflorin galloyl paeoniflorin paeonilacto-neBpaeonilacto-neC and tetragalloyglucose were the major RPA

6 Evidence-Based Complementary and Alternative Medicine

Table 3 The chemical components that were highly positively correlated with toxicity (119903 ge 08)

Number 119903 tR (min) Assigned identity Molecularformula

Mean measuredmass (Da)

Theoretical exactmass (Da)

Mass accuracy(ppm)

1 09779 1298 3-Veratroylgermine C36H53NO11 6763667 6758061 018052 09661 1239 Jervine C27H39NO3 4263004 4256035 236243 09589 1261 Veretramine C27H39NO2 4103042 4096041 297514 09581 1343 Germanitrine C39H59NO11 7184169 7178859 250155 09402 1220 Germidine C34H53NO10 6363412 6357853 057556 09393 1340 Germerine C37H59NO11 7163999 6938645 034087 09390 1291 3-Angeloylzygadenine C32H49NO8 5763557 5757334 127088 09311 1035 11-Deoxojervine C27H41NO2 4123189 4116199 64392

9 09240 157 11205733120572-Dihydroxy-5120573-jervanin-12-en-11-one C27H41NO4 4443088 4436187 24040

10 09237 696 Jervinone C27H37NO3 4242860 4235876 2014011 09182 1125 Neogermbudine C37H59NO12 7323971 7098639 4916912 09143 425 Germine C27H43NO8 5103059 5096322 15549

13 09131 418 NeogermineVeramanine C27H43NO5 4623213 4616340 14522

14 09034 1158 Veraline B C27H45NO3 4323465 4316511 2964615 08978 1116 Verdine C27H41NO5 4603056 4596181 1524916 08903 1163 Stenophylline A C36H51NO11 6743474 6737902 9865517 08870 1035 Germitrine C39H61NO12 7364278 7359011 0828818 08805 914 Polydatin C20H22O8 4290937 3903839 3484919 08738 1081 Cervdine C32H49NO9 5923498 5917328 20608

20 08675 1234 3-Veratrum acylprotoveratrine C36H51NO12 6903482 6897896 10609

21 08671 1289 Zygadenitic acid120575-lactone-16-angelate C32H47NO8 5743408 5737175 48884

22 08652 1232 Maackinine C39H59NO11 7184171 7178859 06797

23 08641 1238Rubijervine rubivirineetioline epirubijervine

IsorubijervineC27H43NO2 4143357 4136358 36827

24 08639 1049 Solanidine C27H43NO3 4303308 4296352 3049425 08492 1057 Pseudojervine C32H49NO8 5883525 5877441 1971026 08444 1181 Neojerminalanine C39H61NO13 7524244 7519005 3001427 08379 843 Zygacine C29H45NO8 5363223 5356695 0000028 08358 1320 Neoverataline B C27H42N2O9 5563260 5386304 407929 08201 1280 3-Veratroylzygadenine C36H51NO10 6963187 6577908 5346930 08176 583 Zygadenine C27H43NO7 4943110 4936328 1543431 08176 054 120574-Aminobutyric acid C4H9NO2 2460978 1031198 1612132 08160 1273 Veratrosine C33H49NO7 6163528 5717447 6832933 08128 1322 Stenophylline A C37H55NO10 6743901 6738333 05430

34 08119 13347-Acetyl-15-

methylbutyryl-3-veratroylgermine

C43H61NO13 8383791 7999433 13104

35 08017 1213 Vanilloylzygadenine C35H49NO10 6443444 6437643 15156

components that were identified with Pearsonrsquos correlationcoefficients of minus09811 minus09799 minus09793 minus09737 minus09736and minus09734 respectively The changes in content of the 24components that were highly negatively correlated (119903 le minus09)with acute toxicity are shown in Figure 7

33 Cell ProliferationAssay To further validate the toxicity ofthe combination ofV nigrumL andRPAonneurons we usedveratridine and paeoniflorin the major pharmacologicallyactive components of V nigrum L and peony for in vitroexperimentsThe effect of these two chemical components on

Evidence-Based Complementary and Alternative Medicine 7

Table 4 The chemical components that were highly negatively correlated with toxicity (119903 le minus09)

Number 119903 tR (min) Assigned identity Molecularformula

Mean measuredmass (Da)

Theoretical exactmass (Da)

Mass accuracy(ppm)

1 minus09811 495 Paeoniflorinsulfonate C23H28O13S 5431194 5445256 337142 minus09799 1220 Benzoyloxy paeoniflorin C30H32O13 5991785 6001843 030463 minus09793 977 Galloyl paeoniflorin C30H32O15 6311725 6321741 038564 minus09737 1479 Paeonilacto-ne B C17H18O6 3171750 3181103 392095 minus09736 056 Paeonilacto-ne C C10H12O4 1950496 1960736 531926 minus09734 809 Tetragalloyglucose C34H28O22 7871008 7885729 566077 minus09728 230 Vanillic acid C7H6O5 1690161 1701195 374718 minus09716 1391 Desbenzoyl paeoniflorin C16H24O10 3751868 3761369 445059 minus09708 1352 Benzoyl paeoniflorin C30H32O12 5831848 5845679 06258

10 minus09684 415 6-O-120573-D-Glucopyranosyllactinolide C16H26O9 3611537 3623722 93435

11 minus09670 1037 Paeoniflorin albiflorin R1albiflorin mudanpioside I C23H28O11 5191261 4804618 15284

12 minus09655 1167 Gallic acid C9H10O2 1510750 1501745 5959013 minus09640 493 Paeonol C9H10O3 1650545 1661739 4067614 minus09625 1167 Lactiflorin C23H26O10 4801872 4624465 0444915 minus09609 667 Isomaltopaeoniflorin C29H38O16 6432221 6426024 2656916 minus09589 852 Albiflorin C23H27O11 9573118 4794539 9372317 minus09582 679 11015840-O-Benzoylsucrose C19H26O12 4641757 4464025 2366818 minus09567 1057 Pentagalloylglucose C41H32O26 9391166 9406772 6629219 minus09562 802 Oxypaeoniflorin C23H28O12 5141934 4964612 1899220 minus09544 1158 Ethyl gallate C9H10O5 2210433 1981727 3244521 minus09478 1011 Eugeniin C41H30O26 9371010 9386613 6708622 minus09395 585 Paeonilactinone C9H14O2 1530904 1542063 7575023 minus09352 113 Lactinolide C10H16O4 2011111 2002316 7890724 minus09173 1373 Palbinone C22H30O4 3572047 3584712 52115

3-Veratroylgermine250

200

150

100

50

0A B C D E F G H I J K L

Ion

inte

nsity

Sample group

Ion

inte

nsity

Sample group

0010203040506070809 Veratramine

A B C D E F G H I J K L0

02

04

06

08

1

12

14

Ion

inte

nsity

A B C D E F G H I J K LSample group

Jervine

A B C D E F G H I J K L

Ion

inte

nsity

Sample group

020406080100120140160180200

Grmidine

A B C D E F G H I J K L

Germanitrine

Ion

inte

nsity

Sample group

0

02

04

06

08

1

12

14

A B C D E F G H I J K L

Ion

inte

nsity

Sample group

0

01

02

03

04

05

06 Germerine

minus01

Figure 5 Ion intensity trend plots for 3-veratroylgermine jervine veratramine germanitrine germidine and germerine which were highlypositively correlated (119903 ge 09393) with toxicity

8 Evidence-Based Complementary and Alternative Medicine

0100200300400500600700800

A B C D E F G H I J K L A B C D E F G H I J K L

Ion

inte

nsity

Ion

inte

nsity

Ion

inte

nsity

Ion

inte

nsity

Ion

inte

nsity

A B C D E F G H I J K L

A B C D E F G H I J K L

A B C D E F G H I J K LSample groupSample group

Sample group A B C D E F G H I J K LSample group

Sample groupA B C D E F G H I J K L

Sample group

3-Angeloylzygadenine

0

01

02

03

04

05

06

07

Ion

inte

nsity

Ion

inte

nsity

11-Deoxojervine

0

50

100

150

200

250

300

350

Sample group

Ion

inte

nsity

Sample group

A B C D E F G H I J K L0

01

02

03

04

05

06 Jervinone

0

05

1

15

2

25

3 Neogermbudine

0020406081

121416182 Germine

0051

152

253

35 Veraline B

0

1

2

3

4

5

6 Verdine

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

1

2

3

4

5

6 GermitrineIo

n in

tens

ity

A B C D E F G H I J K LSample group

0

05

1

15

25

2

Polydatin

minus05

minus05

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

1

2

3

4

5

6

Ion

inte

nsity

A B C D E F G H I J K LSample group

0051

152

253

Stenophylline A

minus05

35

Neogermineveramanine

jervanin-12-en-11-one

0

2

4

6

8

10

12

14

A B C D E F G H I J K L

Ion

inte

nsity

Sample group

Cervdine

A B C D E F G H I J K L

Ion

inte

nsity

Sample group

0

02

04

06

08

1

12 Maackinine

Ion

inte

nsity

A B C D E F G H I J K LSample group

0123456789 Rubijervine

A B C D E F G H I J K L

Ion

inte

nsity

Sample group

0020406081

12141618 3-Veratrum acyl protoveratrine

Ion

inte

nsity

A B C D E F G H I J K LSample group

0051

152

253

354

Solanidine

minus05

A B C D E F G H I JK L

Ion

inte

nsity

Sample group

0020406081

121416182

Zygadenitic acid 120575-Lactone-16-angelate

11205733a-Dihydroxy-5120573-

(a)

Figure 6 Continued

Evidence-Based Complementary and Alternative Medicine 9

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

20

40

60

80

100

120 Pseudojervine

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

02

04

06

08

1

12

14 Neojerminalanine

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

02

04

06

08

1

12 Zygacine

Ion

inte

nsity

A B C D E F G H I J K LSample group

012345678 Zygadenine

Ion

inte

nsity

A B C D E F G H I J K LSample group

000501015020250303504045 Neoverataline B

minus005

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

01

02

03

04

05

06 3-Veratroylzygadenine

minus01

0

05

1

15

2

25 Vanilloylzygadenine

A B C D E F G H I J K LSample group

Ion

inte

nsity

A B C D E F G H I J K LSample group

Ion

inte

nsity

0

2

4

6

8

10

12 Veratrosine

A B C D E F G H I J K LSample group

Ion

inte

nsity

0

2

4

6

8

10

12 Stenophylline A

A B C D E F G H I J K LSample group

Ion

inte

nsity

0020406081

1214 120574-Aminobutyric acid

minus02

A B C D E F G H I J K LSample group

Ion

inte

nsity

0

02

04

06

08

1

12 7-Acetyl-15-methylbutyryl-3-veratroylgermine

(b)

Figure 6 Ion intensity trend plots for the remaining 29 chemical components that were highly positively correlated (119903 ge 08) with toxicity

the viability of SH-SY5Y cells was tested using MTS assaysAs shown in Figures 8(a) and 8(b) veratridine had an LD

50

of 450 whereas paeoniflorin exerted no significant toxicityat doses of 0ndash2000120583M Therefore the dose of veratridinewas fixed at 400120583M and the RPA dose was varied from50 to 2000120583M SH-SY5Y cell cytotoxicity was increasedwhen the concentration of paeoniflorin was decreased inthe combination (Figure 8(c)) Therefore paeoniflorin couldoffset the cytotoxicity caused by veratridine in SH-SY5Y cells[25]

4 Discussion

The eighteen incompatible medicaments (Shi Ba Fan) is awell-known traditional Chinese medicine (TCM) theory It

basically listed eighteen pairs of TCM herbal medicine thatare not to be used in combination as they may cause fatalconsequences This principal has been used as guidancefor general TCM practices for hundreds of years with littlechemical comprehension In this work for the first time weused the UPLC-TOFMS coupled with multivariate statisticalanalysis in an effort to gain scientific understanding for thecauses of the incompatibility

We previously reported that the LD50of aqueous extracts

of V nigrum L and RPA were 2566 gkg and 160 gkgrespectively after intragastric administration [11] V nigrumL extract was severely toxic whereas RPA had minimaltoxicity Although the amount of V nigrum L in each groupwas unchanged the mortality changed significantly withaltered ratios of V nigrum L to RPA When the doses of

10 Evidence-Based Complementary and Alternative Medicine

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

1

2

3

4

5

6 Tetragalloyglucose

Ion

inte

nsity

A B C D E F G H I J K LSample group

0020406081

121416182 Vanillic acid

Ion

inte

nsity

A B C D E F G H I J K LSample group

minus2

0

2

4

6

8

10

12 Benzoylpaeoniflorin

000501015020250303504

A B C D E F G H I J K L

Ion

inte

nsity

Sample groupminus005

Paeoniflorin sulfonate

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

05

1

15

2 Benzoyloxypaeoniflorin

minus05

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

05

1

15

2 Gallic acid

minus05

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

05

1

15

2

253 Galloylpaeoniflorin

minus05

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

005

01

015

02

025

03

035 Paeonilacto-neC

Ion

inte

nsity

A B C D E F G H I J K LSample group

0123456789 Paeonilacto-neB

minus1

Ion

inte

nsity

A B C D E F G H I J K LSample group

Vanillic acid

0

1

2

3

4

5

6

7

minus1

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

01

02

03

04

05

06

07

minus01

6-O-120573-D-Glucopyranosyllactinolide

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

05

1

15

2

25

3

35

minus05

Paeoniflorinalbiflorin R1albiflorinmudanpioside I

A B C D E F G H I J K L

Ion

inte

nsity

Sample group

Paeonol

0

02

04

06

08

1

12

minus02

Ion

inte

nsity

A B C D E F G H I J K LSample group

01020304050607080 Pentagalloylglucose

minus10

Ion

inte

nsity

A B C D E F G H I J K LSample group

00102030405060708 Lactiflorin

minus01

Ion

inte

nsity

A BC D E F G H I J K LSample group

0020406081

121416 Isomaltopaeoniflorin

minus02

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

05

1

15

2

25

3

minus05

Albiflorin

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

05

1

15

2

25 1998400-O-Benzoylsucrose

(a)

Figure 7 Continued

Evidence-Based Complementary and Alternative Medicine 11

Ion

inte

nsity

Ion

inte

nsity

Ion

inte

nsity

A B C D E F G H I J K LSample group A B C D E F G H I J K L

Sample group

A B C D E F G H I J K LSample group

00102030405060708 Oxypaeoniflorin

001020304050607 Eugeniin

Ion

inte

nsity

A B C D E F G H I J K LSample group

0020406081

12141618 Paeonia lactinone

00102030405060708091 Ethyl gallate

minus01

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

01

02

03

04

05

Lactinolide06

minus01

0

5

10

15

20

25 Palbinone

Ion

inte

nsity

A B C D E F G H I J K LSample group

minus5

minus01

(b)

Figure 7 Ion intensity trend plots for the chemical components that were highly negatively correlated with toxicity (119903 ge 09)

OD

val

ue

25 50 100 200 400 800

lowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

mdashVeratridine (120583M)

10

12

08

06

04

02

0

(a)

OD

val

ue

02

04

06

08

12

Paeoniflorin (120583M) 100 500 1500 2000mdash 1000

10

00

(b)

OD

val

ue

Paeoniflorin (120583M)

mdashmdash

Veratridine (120583M) 400 400 400 400 400 400 400 400 400 400

50 100 250 500 750 1000 1250 1500 1750 2000

02

04

06

08

12

10

00

lowastlowastlowast

lowastlowast

lowastlowast lowastlowast

lowastlowast

lowast

(c)

Figure 8 SH-SY5Y cell viability SH-SY5Y cells were cultured with (a) veratridine (25ndash800120583M) (b) paeoniflorin (50minus2000120583M) and (c)400 120583M veratridine in combination with 50minus2000120583M paeoniflorin for 24 h Cell viability was then assessed using MTS assays Data arepresented as mean plusmn SD Means of three independent experiments Asterisks indicate a statistically significant difference compared withuntreated cells lowast119875 lt 005 lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001 versus controL

12 Evidence-Based Complementary and Alternative Medicine

V nigrum L were equal to or higher than RPA particularlyratios of 8 1 and 1 1 mouse mortality increased significantlyTherefore components of V nigrum L were the majorcausative factors of toxicity In addition RPA modulated thetoxicity ofV nigrum LTherefore clinical applications shouldbe monitored carefully

Mice died 5minus10min after drug administration Toxicitymanifested predominantly as trembling convulsions andspasms but the major organs showed no obvious lesionsat necropsy by the naked eye Based on the toxic reactionsand the time of death in the current study and accord-ing to the guidelines for Chinese natural medicine acutetoxicity testing we speculate that the toxicity was causedpredominantly by suppression of the central nervous systemand that V nigrum L was the major cause of death Vnigrum L water extracts exert toxic effects on the digestivetract mucosa nucleus nervi vagi and central nervous system[6 17 18] Pharmacological studies of veratrum alkaloidshave been performed previously and reported that thesealkaloids stimulate the central nervous system and inhibit thebrain This results in spasms convulsions coma drowsinessand other symptoms of consciousness in animals which isconsistent with the symptoms of themice in the current study[6 17]

Veratrum alkaloid is the toxic component of V nigrum LWe previously investigated the differently expressed genes inSH-SY5Y cells treated with veratridine using cDNAmicroar-rays [34] Data revealed that veratridine treatment alteredthe expression of MMP caused Ca2+ concentration over-load increased reactive oxygen species production enhancedLDH release damaged the cell membrane reduced SH-SY5Y cell viability and induced apoptosis Under oxidizingconditions the MAPK signaling pathway is activated whichincreases apoptosisThis study illustrates the neurotoxicity ofthe combination of V nigrum L and reveals the molecularmechanism behind the neurotoxic effects of V nigrum L(data not shown)

The amount of V nigrum L in each group was the samethe dose of RPA is in ascending order and the chemicalcomponents of RPA are also increased but the mortalityof each group changed significantly with the increase ofRPA so we conclude that higher amount of RPA can haveprotection effect which can offset the toxicity of combinationof V nigrum L and RPA And pharmacological studies havedemonstrated that RPA exerts a wide range of pharmacolog-ical activities including analgesic sedative anticonvulsantand antispasmodic effects on the central nervous systemRPA could also inhibit writhing reactions and antagonizepentylenetetrazol-induced convulsions The most importantactive ingredient in RPA is paeoniflorin which has significantneuroprotective effects [26ndash34]

In this study mouse acute toxicity experiments revealedthat the mortality of mice changed significantly when thedose of V nigrum L was higher than or equal to that ofRPA particularly at ratios of V nigrum L RPA of 8 1and 1 1 UPLC-Q-TOFMS with automated data analysisused to identify and quantify the specific chemical compo-nents According to Pearsonrsquos correlation coefficient most of

the chemical components that were positively correlated withtoxicity were from V nigrum L We conclude that the majorveratrum alkaloids of V nigrum L caused acute toxicity anddeath in mice When the doses of V nigrum L were higherthan or equal to RPA the main chemical components of Vnigrum L and the toxicity of the decoctions were increasedThis suggests that V nigrum L and RPA have opposingroles Additional experiments revealed that the cytotoxicityof veratridine in SH-SY5Y cells was decreasedwith increasingconcentrations of paeoniflorin Therefore the clinical use ofthese agents should be considered carefully This study alsoprovides information to help improve the incompatibilitytheory of TCM and introduces novel ideas for further studieson the development and application of TCM

When preparing V nigrum L and RPA decoctions alower amount of RPA contributes to the dissolution of vera-trum alkaloids Conversely higher amounts of RPA can exertneuroprotective effects which can offset the toxicity causedby the combination of V nigrum L and RPA Under theseconditions dissolution is the most important factor for Vnigrum L toxicity The results of cell-based experiments alsodemonstrated that paeoniflorin could offset the neurotoxiceffects of veratridine when treated in combination whichexcluded the factor of dissolution in preparation

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors are grateful for financial support from NationalNature Science Foundation of China (Grant no 81073149)National Basic Research Program of China (Grant nos2011CB505304 and 2012CB518402) and Natural ScienceFoundation of Beijing (Grant no 7112110)

References

[1] H Guo W Li X Y Wang and G W Fan ldquoAdvance of researchon toxic attenuation by compatibility of traditional Chinesemedicine prescriptionsrdquo Zhongguo Zhongyao Zazhi vol 37 no1 pp 120ndash123 2012

[2] Y Zhang H Hua X Fan C Wang and J Duan ldquoExplorationon eighteen incompatible medicaments of chest pain prescrip-tions based on association rules miningrdquo Zhongguo ZhongyaoZazhi vol 36 no 24 pp 3544ndash3547 2011

[3] C-H Xu P Wang Y Wang et al ldquoPharmacokinetic compar-isons of two different combinations of Shaoyao-Gancao Decoc-tion in rats competing mechanisms between paeoniflorin andglycyrrhetinic acidrdquo Journal of Ethnopharmacology vol 149 no2 pp 443ndash452 2013

[4] D H TianDictionary of Practical Traditional Chinese MedicineII Peoplersquos Medical Beijing China 2002

[5] Y Gaillard and G Pepin ldquoLC-EI-MS determination of ver-atridine and cevadine in two fatal cases of Veratrum albumpoisoningrdquo Journal of Analytical Toxicology vol 25 no 6 pp481ndash485 2001

Evidence-Based Complementary and Alternative Medicine 13

[6] H Li G-Y Gao S-Y Li W-J Zhao Y-T Guo and Z-J Liang ldquoEffects of Veratrum nigrum alkaloids on centralcatecholaminergic neurons of renal hypertensive ratsrdquo ActaPharmacologica Sinica vol 21 no 1 pp 23ndash28 2000

[7] L Wang W Li and Y Liu ldquoHypotensive effect and toxicologyof total alkaloids and veratramine from roots and rhizomesof Veratrum nigrum L in spontaneously hypertensive ratsrdquoPharmazie vol 63 no 8 pp 606ndash610 2008

[8] Y P Jiang Y G Liu and H C Chen ldquoEffect of aqueous extractof Radix Paeoniae Rubra against Carbon Tetrachloride inducedliver fibrosis in ratrdquo Herald of Medicine vol 23 pp 527ndash5292004

[9] D-Y He and S-M Dai ldquoAnti-inflammatory and immunomod-ulatory effects of Paeonia lactiflora Pall a traditional Chineseherbal medicinerdquo Frontiers in Pharmacology 2011

[10] C Feng M Liu X Shi et al ldquoPharmacokinetic properties ofpaeoniflorin albiflorin and oxypaeoniflorin after oral gavage ofextracts of Radix Paeoniae Rubra and Radix Paeoniae Alba inratsrdquo Journal of Ethnopharmacology vol 130 no 2 pp 407ndash4132010

[11] X X Zhang Y G Wang Z C Ma Y Gao and C R XiaoldquoUniform designed research on the compatibility toxicity ofVeratridine and peonyrdquo Zhong Hua Zhong Yi Yao Za Zhi vol28 no 10 pp 2901ndash2904 2013

[12] Q D Liang Y Gao J Ma et al ldquoChemical comparison of driedrehmannia root and prepared rehmannia root by UPLC-TOFMS andHPLC-ELSDwithmultivariate statistical analysisrdquoActaPharmaceutica Sinica B vol 3 no 1 pp 55ndash64 2013

[13] Y Wang S He X Cheng Y Lu Y Zou and Q Zhang ldquoUPLC-Q-TOF-MSMS fingerprinting of traditional Chinese formulaSiJunZiTangrdquo Journal of Pharmaceutical and Biomedical Analy-sis vol 80 pp 24ndash33 2013

[14] L-P Kang K Yu Y Zhao et al ldquoCharacterization of steroidalglycosides from the extract of Paris Polyphylla var Yunnanensisby UPLCQ-TOFMSErdquo Journal of Pharmaceutical and Biomed-ical Analysis vol 62 pp 235ndash249 2012

[15] A Kaufmann P Butcher K Maden and M Widmer ldquoUltra-performance liquid chromatography coupled to time of flightmass spectrometry (UPLC-TOF) a novel tool for multiresiduescreening of veterinary drugs in urinerdquoAnalytica Chimica Actavol 586 no 1-2 pp 13ndash21 2007

[16] S Zhou Z C Ma Q D Liang et al ldquoUPLCQ-TOF-MS basedchemical profiling approach to evaluate chemical compositionof augmentation toxicity in combination of Radix aconiti andPinellia praeparatardquoActa Chimica Sinica vol 70 no 3 pp 284ndash290 2012

[17] N Lin and X Gao ldquoExperimental observation on the absorp-tion of Veratrum nigrum L in the upper digestive tractrdquoZhongguo Zhong Yao Za Zhi vol 17 no 1 pp 43ndash45 1992

[18] Z-Z Wang W-J Zhao X-S Zhang et al ldquoProtection ofVeratrum nigrum L var ussuriense Nakai alkaloids againstischema-reperfusion injury of the rat liverrdquo World Journal ofGastroenterology vol 13 no 4 pp 564ndash571 2007

[19] V Christov B Mikhova A Ivanova et al ldquoSteroidal alka-loids of Veratrum lobelianum Bernh and Veratrum nigrum LrdquoZeitschrift fur Naturforschung C Journal of Biosciences vol 65no 3-4 pp 195ndash200 2010

[20] V Christov BMikhova andD Selenge ldquo(-)-Veranigrine a newsteroidal alkaloid fromVeratrum nigrum Lrdquo Fitoterapia vol 80no 1 pp 25ndash27 2009

[21] Y Cong J-H Wang R Wang Y-M Zeng C-D Liu and XLi ldquoA study on the chemical constituents of Veratrum nigrum

L processed by rice vinegarrdquo Journal of Asian Natural ProductsResearch vol 10 no 7-8 pp 619ndash624 2008

[22] H-L Li J Tang R-H Liu C Zhang and W-D Zhang ldquoTwonew flavanone glycosides from Veratrum nigrum Lrdquo NaturalProduct Research vol 23 no 2 pp 122ndash126 2009

[23] Y Cong Y-B Zhou J Chen Y-M Zeng and J-H WangldquoAlkaloid profiling of crude and processed Veratrum nigrum Lthrough simultaneous determination of ten steroidal alkaloidsby HPLC-ELSDrdquo Journal of Pharmaceutical and BiomedicalAnalysis vol 48 no 3 pp 573ndash578 2008

[24] L-S Wang D-Q Zhao H-M Tao and Y-H Liu ldquoDetermi-nation of vetatramine in Veratrum nigrum by HPLC-ELSDrdquoZhongguo Zhongyao Zazhi vol 33 no 7 pp 791ndash792 2008

[25] Q Wang H-Z Guo C-H Huo et al ldquoChemical constituentsin root of Paeonia lactiflorardquo Chinese Traditional and HerbalDrugs vol 38 no 7 pp 972ndash976 2007

[26] J-J TanQ-C Zhao L Yang Z-P Shang Z-QDu andMYanldquoChemical constituents in roots of Paeonia lactiflorardquo ChineseTraditional and Herbal Drugs vol 41 no 8 pp 1245ndash1248 2010

[27] Q Zhang Z Z Wang W Xiao et al ldquoUPLC characteristicchromatographic profile of Paeoniae Radix Albardquo ZhongguoZhongyao Zazhi vol 36 no 6 pp 712ndash714 2011

[28] X R Gao and Y Tian ldquoActive principles of Paeonia lactifloraPallrdquo Chinese Journal of New Drugs vol 15 pp 416ndash418 2006

[29] N-C Luo W Ding J Wu et al ldquoUPLC-Q-TOFMS coupledwith multivariate statistical analysis as a powerful techniquefor rapidly exploring potential chemicalmarkers to differentiatebetween radix paeoniae alba and radix paeoniae rubrardquoNaturalProduct Communications vol 8 no 4 pp 487ndash491 2013

[30] W K Zhao Y Guo T Yasuhiro and K Tohru ldquoIsolation andstructure determination of echinuline from Veratrum nigrumL var ussuriense Nakairdquo Zhongguo Zhong Yao Za Zhi vol 16no 7 pp 425ndash426 1991

[31] W Zhao Y Guo T Yasuhirb and T Kikuchi ldquoIsolation andstructure determination of aurantiamide acetate fromVeratrumnigrum Lvarussuriense Nakairdquo Zhongguo Zhongyao Zazhi vol23 no 1 p 41 1998

[32] L Yang X H Xia Q Zhu and X P Tan ldquoStudy on therule of influence of two purification methods on the chemicalcompositions in aqueous solution of paeoniae radix albardquoZhong Yao Cai vol 36 pp 118ndash121 2012

[33] X Y Zang and X Li ldquoDevelopment of the study on chemicalconstituentsof Paeonia lact if lora Pallrdquo Journal of ShenyangPharmaceutical University vol 19 pp 70ndash73 2002

[34] Y-L Wang Y-G Wang Q-D Liang et al ldquoUsing cDNAmicroarray to screen differently expressed genes in SH-SY5Ytreated by veratridinerdquo Chinese Pharmacological Bulletin vol29 no 5 pp 643ndash647 2013

Submit your manuscripts athttpwwwhindawicom

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

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OncologyJournal of

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Oxidative Medicine and Cellular Longevity

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PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

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Computational and Mathematical Methods in Medicine

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Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

6 Evidence-Based Complementary and Alternative Medicine

Table 3 The chemical components that were highly positively correlated with toxicity (119903 ge 08)

Number 119903 tR (min) Assigned identity Molecularformula

Mean measuredmass (Da)

Theoretical exactmass (Da)

Mass accuracy(ppm)

1 09779 1298 3-Veratroylgermine C36H53NO11 6763667 6758061 018052 09661 1239 Jervine C27H39NO3 4263004 4256035 236243 09589 1261 Veretramine C27H39NO2 4103042 4096041 297514 09581 1343 Germanitrine C39H59NO11 7184169 7178859 250155 09402 1220 Germidine C34H53NO10 6363412 6357853 057556 09393 1340 Germerine C37H59NO11 7163999 6938645 034087 09390 1291 3-Angeloylzygadenine C32H49NO8 5763557 5757334 127088 09311 1035 11-Deoxojervine C27H41NO2 4123189 4116199 64392

9 09240 157 11205733120572-Dihydroxy-5120573-jervanin-12-en-11-one C27H41NO4 4443088 4436187 24040

10 09237 696 Jervinone C27H37NO3 4242860 4235876 2014011 09182 1125 Neogermbudine C37H59NO12 7323971 7098639 4916912 09143 425 Germine C27H43NO8 5103059 5096322 15549

13 09131 418 NeogermineVeramanine C27H43NO5 4623213 4616340 14522

14 09034 1158 Veraline B C27H45NO3 4323465 4316511 2964615 08978 1116 Verdine C27H41NO5 4603056 4596181 1524916 08903 1163 Stenophylline A C36H51NO11 6743474 6737902 9865517 08870 1035 Germitrine C39H61NO12 7364278 7359011 0828818 08805 914 Polydatin C20H22O8 4290937 3903839 3484919 08738 1081 Cervdine C32H49NO9 5923498 5917328 20608

20 08675 1234 3-Veratrum acylprotoveratrine C36H51NO12 6903482 6897896 10609

21 08671 1289 Zygadenitic acid120575-lactone-16-angelate C32H47NO8 5743408 5737175 48884

22 08652 1232 Maackinine C39H59NO11 7184171 7178859 06797

23 08641 1238Rubijervine rubivirineetioline epirubijervine

IsorubijervineC27H43NO2 4143357 4136358 36827

24 08639 1049 Solanidine C27H43NO3 4303308 4296352 3049425 08492 1057 Pseudojervine C32H49NO8 5883525 5877441 1971026 08444 1181 Neojerminalanine C39H61NO13 7524244 7519005 3001427 08379 843 Zygacine C29H45NO8 5363223 5356695 0000028 08358 1320 Neoverataline B C27H42N2O9 5563260 5386304 407929 08201 1280 3-Veratroylzygadenine C36H51NO10 6963187 6577908 5346930 08176 583 Zygadenine C27H43NO7 4943110 4936328 1543431 08176 054 120574-Aminobutyric acid C4H9NO2 2460978 1031198 1612132 08160 1273 Veratrosine C33H49NO7 6163528 5717447 6832933 08128 1322 Stenophylline A C37H55NO10 6743901 6738333 05430

34 08119 13347-Acetyl-15-

methylbutyryl-3-veratroylgermine

C43H61NO13 8383791 7999433 13104

35 08017 1213 Vanilloylzygadenine C35H49NO10 6443444 6437643 15156

components that were identified with Pearsonrsquos correlationcoefficients of minus09811 minus09799 minus09793 minus09737 minus09736and minus09734 respectively The changes in content of the 24components that were highly negatively correlated (119903 le minus09)with acute toxicity are shown in Figure 7

33 Cell ProliferationAssay To further validate the toxicity ofthe combination ofV nigrumL andRPAonneurons we usedveratridine and paeoniflorin the major pharmacologicallyactive components of V nigrum L and peony for in vitroexperimentsThe effect of these two chemical components on

Evidence-Based Complementary and Alternative Medicine 7

Table 4 The chemical components that were highly negatively correlated with toxicity (119903 le minus09)

Number 119903 tR (min) Assigned identity Molecularformula

Mean measuredmass (Da)

Theoretical exactmass (Da)

Mass accuracy(ppm)

1 minus09811 495 Paeoniflorinsulfonate C23H28O13S 5431194 5445256 337142 minus09799 1220 Benzoyloxy paeoniflorin C30H32O13 5991785 6001843 030463 minus09793 977 Galloyl paeoniflorin C30H32O15 6311725 6321741 038564 minus09737 1479 Paeonilacto-ne B C17H18O6 3171750 3181103 392095 minus09736 056 Paeonilacto-ne C C10H12O4 1950496 1960736 531926 minus09734 809 Tetragalloyglucose C34H28O22 7871008 7885729 566077 minus09728 230 Vanillic acid C7H6O5 1690161 1701195 374718 minus09716 1391 Desbenzoyl paeoniflorin C16H24O10 3751868 3761369 445059 minus09708 1352 Benzoyl paeoniflorin C30H32O12 5831848 5845679 06258

10 minus09684 415 6-O-120573-D-Glucopyranosyllactinolide C16H26O9 3611537 3623722 93435

11 minus09670 1037 Paeoniflorin albiflorin R1albiflorin mudanpioside I C23H28O11 5191261 4804618 15284

12 minus09655 1167 Gallic acid C9H10O2 1510750 1501745 5959013 minus09640 493 Paeonol C9H10O3 1650545 1661739 4067614 minus09625 1167 Lactiflorin C23H26O10 4801872 4624465 0444915 minus09609 667 Isomaltopaeoniflorin C29H38O16 6432221 6426024 2656916 minus09589 852 Albiflorin C23H27O11 9573118 4794539 9372317 minus09582 679 11015840-O-Benzoylsucrose C19H26O12 4641757 4464025 2366818 minus09567 1057 Pentagalloylglucose C41H32O26 9391166 9406772 6629219 minus09562 802 Oxypaeoniflorin C23H28O12 5141934 4964612 1899220 minus09544 1158 Ethyl gallate C9H10O5 2210433 1981727 3244521 minus09478 1011 Eugeniin C41H30O26 9371010 9386613 6708622 minus09395 585 Paeonilactinone C9H14O2 1530904 1542063 7575023 minus09352 113 Lactinolide C10H16O4 2011111 2002316 7890724 minus09173 1373 Palbinone C22H30O4 3572047 3584712 52115

3-Veratroylgermine250

200

150

100

50

0A B C D E F G H I J K L

Ion

inte

nsity

Sample group

Ion

inte

nsity

Sample group

0010203040506070809 Veratramine

A B C D E F G H I J K L0

02

04

06

08

1

12

14

Ion

inte

nsity

A B C D E F G H I J K LSample group

Jervine

A B C D E F G H I J K L

Ion

inte

nsity

Sample group

020406080100120140160180200

Grmidine

A B C D E F G H I J K L

Germanitrine

Ion

inte

nsity

Sample group

0

02

04

06

08

1

12

14

A B C D E F G H I J K L

Ion

inte

nsity

Sample group

0

01

02

03

04

05

06 Germerine

minus01

Figure 5 Ion intensity trend plots for 3-veratroylgermine jervine veratramine germanitrine germidine and germerine which were highlypositively correlated (119903 ge 09393) with toxicity

8 Evidence-Based Complementary and Alternative Medicine

0100200300400500600700800

A B C D E F G H I J K L A B C D E F G H I J K L

Ion

inte

nsity

Ion

inte

nsity

Ion

inte

nsity

Ion

inte

nsity

Ion

inte

nsity

A B C D E F G H I J K L

A B C D E F G H I J K L

A B C D E F G H I J K LSample groupSample group

Sample group A B C D E F G H I J K LSample group

Sample groupA B C D E F G H I J K L

Sample group

3-Angeloylzygadenine

0

01

02

03

04

05

06

07

Ion

inte

nsity

Ion

inte

nsity

11-Deoxojervine

0

50

100

150

200

250

300

350

Sample group

Ion

inte

nsity

Sample group

A B C D E F G H I J K L0

01

02

03

04

05

06 Jervinone

0

05

1

15

2

25

3 Neogermbudine

0020406081

121416182 Germine

0051

152

253

35 Veraline B

0

1

2

3

4

5

6 Verdine

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

1

2

3

4

5

6 GermitrineIo

n in

tens

ity

A B C D E F G H I J K LSample group

0

05

1

15

25

2

Polydatin

minus05

minus05

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

1

2

3

4

5

6

Ion

inte

nsity

A B C D E F G H I J K LSample group

0051

152

253

Stenophylline A

minus05

35

Neogermineveramanine

jervanin-12-en-11-one

0

2

4

6

8

10

12

14

A B C D E F G H I J K L

Ion

inte

nsity

Sample group

Cervdine

A B C D E F G H I J K L

Ion

inte

nsity

Sample group

0

02

04

06

08

1

12 Maackinine

Ion

inte

nsity

A B C D E F G H I J K LSample group

0123456789 Rubijervine

A B C D E F G H I J K L

Ion

inte

nsity

Sample group

0020406081

12141618 3-Veratrum acyl protoveratrine

Ion

inte

nsity

A B C D E F G H I J K LSample group

0051

152

253

354

Solanidine

minus05

A B C D E F G H I JK L

Ion

inte

nsity

Sample group

0020406081

121416182

Zygadenitic acid 120575-Lactone-16-angelate

11205733a-Dihydroxy-5120573-

(a)

Figure 6 Continued

Evidence-Based Complementary and Alternative Medicine 9

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

20

40

60

80

100

120 Pseudojervine

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

02

04

06

08

1

12

14 Neojerminalanine

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

02

04

06

08

1

12 Zygacine

Ion

inte

nsity

A B C D E F G H I J K LSample group

012345678 Zygadenine

Ion

inte

nsity

A B C D E F G H I J K LSample group

000501015020250303504045 Neoverataline B

minus005

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

01

02

03

04

05

06 3-Veratroylzygadenine

minus01

0

05

1

15

2

25 Vanilloylzygadenine

A B C D E F G H I J K LSample group

Ion

inte

nsity

A B C D E F G H I J K LSample group

Ion

inte

nsity

0

2

4

6

8

10

12 Veratrosine

A B C D E F G H I J K LSample group

Ion

inte

nsity

0

2

4

6

8

10

12 Stenophylline A

A B C D E F G H I J K LSample group

Ion

inte

nsity

0020406081

1214 120574-Aminobutyric acid

minus02

A B C D E F G H I J K LSample group

Ion

inte

nsity

0

02

04

06

08

1

12 7-Acetyl-15-methylbutyryl-3-veratroylgermine

(b)

Figure 6 Ion intensity trend plots for the remaining 29 chemical components that were highly positively correlated (119903 ge 08) with toxicity

the viability of SH-SY5Y cells was tested using MTS assaysAs shown in Figures 8(a) and 8(b) veratridine had an LD

50

of 450 whereas paeoniflorin exerted no significant toxicityat doses of 0ndash2000120583M Therefore the dose of veratridinewas fixed at 400120583M and the RPA dose was varied from50 to 2000120583M SH-SY5Y cell cytotoxicity was increasedwhen the concentration of paeoniflorin was decreased inthe combination (Figure 8(c)) Therefore paeoniflorin couldoffset the cytotoxicity caused by veratridine in SH-SY5Y cells[25]

4 Discussion

The eighteen incompatible medicaments (Shi Ba Fan) is awell-known traditional Chinese medicine (TCM) theory It

basically listed eighteen pairs of TCM herbal medicine thatare not to be used in combination as they may cause fatalconsequences This principal has been used as guidancefor general TCM practices for hundreds of years with littlechemical comprehension In this work for the first time weused the UPLC-TOFMS coupled with multivariate statisticalanalysis in an effort to gain scientific understanding for thecauses of the incompatibility

We previously reported that the LD50of aqueous extracts

of V nigrum L and RPA were 2566 gkg and 160 gkgrespectively after intragastric administration [11] V nigrumL extract was severely toxic whereas RPA had minimaltoxicity Although the amount of V nigrum L in each groupwas unchanged the mortality changed significantly withaltered ratios of V nigrum L to RPA When the doses of

10 Evidence-Based Complementary and Alternative Medicine

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

1

2

3

4

5

6 Tetragalloyglucose

Ion

inte

nsity

A B C D E F G H I J K LSample group

0020406081

121416182 Vanillic acid

Ion

inte

nsity

A B C D E F G H I J K LSample group

minus2

0

2

4

6

8

10

12 Benzoylpaeoniflorin

000501015020250303504

A B C D E F G H I J K L

Ion

inte

nsity

Sample groupminus005

Paeoniflorin sulfonate

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

05

1

15

2 Benzoyloxypaeoniflorin

minus05

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

05

1

15

2 Gallic acid

minus05

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

05

1

15

2

253 Galloylpaeoniflorin

minus05

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

005

01

015

02

025

03

035 Paeonilacto-neC

Ion

inte

nsity

A B C D E F G H I J K LSample group

0123456789 Paeonilacto-neB

minus1

Ion

inte

nsity

A B C D E F G H I J K LSample group

Vanillic acid

0

1

2

3

4

5

6

7

minus1

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

01

02

03

04

05

06

07

minus01

6-O-120573-D-Glucopyranosyllactinolide

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

05

1

15

2

25

3

35

minus05

Paeoniflorinalbiflorin R1albiflorinmudanpioside I

A B C D E F G H I J K L

Ion

inte

nsity

Sample group

Paeonol

0

02

04

06

08

1

12

minus02

Ion

inte

nsity

A B C D E F G H I J K LSample group

01020304050607080 Pentagalloylglucose

minus10

Ion

inte

nsity

A B C D E F G H I J K LSample group

00102030405060708 Lactiflorin

minus01

Ion

inte

nsity

A BC D E F G H I J K LSample group

0020406081

121416 Isomaltopaeoniflorin

minus02

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

05

1

15

2

25

3

minus05

Albiflorin

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

05

1

15

2

25 1998400-O-Benzoylsucrose

(a)

Figure 7 Continued

Evidence-Based Complementary and Alternative Medicine 11

Ion

inte

nsity

Ion

inte

nsity

Ion

inte

nsity

A B C D E F G H I J K LSample group A B C D E F G H I J K L

Sample group

A B C D E F G H I J K LSample group

00102030405060708 Oxypaeoniflorin

001020304050607 Eugeniin

Ion

inte

nsity

A B C D E F G H I J K LSample group

0020406081

12141618 Paeonia lactinone

00102030405060708091 Ethyl gallate

minus01

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

01

02

03

04

05

Lactinolide06

minus01

0

5

10

15

20

25 Palbinone

Ion

inte

nsity

A B C D E F G H I J K LSample group

minus5

minus01

(b)

Figure 7 Ion intensity trend plots for the chemical components that were highly negatively correlated with toxicity (119903 ge 09)

OD

val

ue

25 50 100 200 400 800

lowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

mdashVeratridine (120583M)

10

12

08

06

04

02

0

(a)

OD

val

ue

02

04

06

08

12

Paeoniflorin (120583M) 100 500 1500 2000mdash 1000

10

00

(b)

OD

val

ue

Paeoniflorin (120583M)

mdashmdash

Veratridine (120583M) 400 400 400 400 400 400 400 400 400 400

50 100 250 500 750 1000 1250 1500 1750 2000

02

04

06

08

12

10

00

lowastlowastlowast

lowastlowast

lowastlowast lowastlowast

lowastlowast

lowast

(c)

Figure 8 SH-SY5Y cell viability SH-SY5Y cells were cultured with (a) veratridine (25ndash800120583M) (b) paeoniflorin (50minus2000120583M) and (c)400 120583M veratridine in combination with 50minus2000120583M paeoniflorin for 24 h Cell viability was then assessed using MTS assays Data arepresented as mean plusmn SD Means of three independent experiments Asterisks indicate a statistically significant difference compared withuntreated cells lowast119875 lt 005 lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001 versus controL

12 Evidence-Based Complementary and Alternative Medicine

V nigrum L were equal to or higher than RPA particularlyratios of 8 1 and 1 1 mouse mortality increased significantlyTherefore components of V nigrum L were the majorcausative factors of toxicity In addition RPA modulated thetoxicity ofV nigrum LTherefore clinical applications shouldbe monitored carefully

Mice died 5minus10min after drug administration Toxicitymanifested predominantly as trembling convulsions andspasms but the major organs showed no obvious lesionsat necropsy by the naked eye Based on the toxic reactionsand the time of death in the current study and accord-ing to the guidelines for Chinese natural medicine acutetoxicity testing we speculate that the toxicity was causedpredominantly by suppression of the central nervous systemand that V nigrum L was the major cause of death Vnigrum L water extracts exert toxic effects on the digestivetract mucosa nucleus nervi vagi and central nervous system[6 17 18] Pharmacological studies of veratrum alkaloidshave been performed previously and reported that thesealkaloids stimulate the central nervous system and inhibit thebrain This results in spasms convulsions coma drowsinessand other symptoms of consciousness in animals which isconsistent with the symptoms of themice in the current study[6 17]

Veratrum alkaloid is the toxic component of V nigrum LWe previously investigated the differently expressed genes inSH-SY5Y cells treated with veratridine using cDNAmicroar-rays [34] Data revealed that veratridine treatment alteredthe expression of MMP caused Ca2+ concentration over-load increased reactive oxygen species production enhancedLDH release damaged the cell membrane reduced SH-SY5Y cell viability and induced apoptosis Under oxidizingconditions the MAPK signaling pathway is activated whichincreases apoptosisThis study illustrates the neurotoxicity ofthe combination of V nigrum L and reveals the molecularmechanism behind the neurotoxic effects of V nigrum L(data not shown)

The amount of V nigrum L in each group was the samethe dose of RPA is in ascending order and the chemicalcomponents of RPA are also increased but the mortalityof each group changed significantly with the increase ofRPA so we conclude that higher amount of RPA can haveprotection effect which can offset the toxicity of combinationof V nigrum L and RPA And pharmacological studies havedemonstrated that RPA exerts a wide range of pharmacolog-ical activities including analgesic sedative anticonvulsantand antispasmodic effects on the central nervous systemRPA could also inhibit writhing reactions and antagonizepentylenetetrazol-induced convulsions The most importantactive ingredient in RPA is paeoniflorin which has significantneuroprotective effects [26ndash34]

In this study mouse acute toxicity experiments revealedthat the mortality of mice changed significantly when thedose of V nigrum L was higher than or equal to that ofRPA particularly at ratios of V nigrum L RPA of 8 1and 1 1 UPLC-Q-TOFMS with automated data analysisused to identify and quantify the specific chemical compo-nents According to Pearsonrsquos correlation coefficient most of

the chemical components that were positively correlated withtoxicity were from V nigrum L We conclude that the majorveratrum alkaloids of V nigrum L caused acute toxicity anddeath in mice When the doses of V nigrum L were higherthan or equal to RPA the main chemical components of Vnigrum L and the toxicity of the decoctions were increasedThis suggests that V nigrum L and RPA have opposingroles Additional experiments revealed that the cytotoxicityof veratridine in SH-SY5Y cells was decreasedwith increasingconcentrations of paeoniflorin Therefore the clinical use ofthese agents should be considered carefully This study alsoprovides information to help improve the incompatibilitytheory of TCM and introduces novel ideas for further studieson the development and application of TCM

When preparing V nigrum L and RPA decoctions alower amount of RPA contributes to the dissolution of vera-trum alkaloids Conversely higher amounts of RPA can exertneuroprotective effects which can offset the toxicity causedby the combination of V nigrum L and RPA Under theseconditions dissolution is the most important factor for Vnigrum L toxicity The results of cell-based experiments alsodemonstrated that paeoniflorin could offset the neurotoxiceffects of veratridine when treated in combination whichexcluded the factor of dissolution in preparation

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors are grateful for financial support from NationalNature Science Foundation of China (Grant no 81073149)National Basic Research Program of China (Grant nos2011CB505304 and 2012CB518402) and Natural ScienceFoundation of Beijing (Grant no 7112110)

References

[1] H Guo W Li X Y Wang and G W Fan ldquoAdvance of researchon toxic attenuation by compatibility of traditional Chinesemedicine prescriptionsrdquo Zhongguo Zhongyao Zazhi vol 37 no1 pp 120ndash123 2012

[2] Y Zhang H Hua X Fan C Wang and J Duan ldquoExplorationon eighteen incompatible medicaments of chest pain prescrip-tions based on association rules miningrdquo Zhongguo ZhongyaoZazhi vol 36 no 24 pp 3544ndash3547 2011

[3] C-H Xu P Wang Y Wang et al ldquoPharmacokinetic compar-isons of two different combinations of Shaoyao-Gancao Decoc-tion in rats competing mechanisms between paeoniflorin andglycyrrhetinic acidrdquo Journal of Ethnopharmacology vol 149 no2 pp 443ndash452 2013

[4] D H TianDictionary of Practical Traditional Chinese MedicineII Peoplersquos Medical Beijing China 2002

[5] Y Gaillard and G Pepin ldquoLC-EI-MS determination of ver-atridine and cevadine in two fatal cases of Veratrum albumpoisoningrdquo Journal of Analytical Toxicology vol 25 no 6 pp481ndash485 2001

Evidence-Based Complementary and Alternative Medicine 13

[6] H Li G-Y Gao S-Y Li W-J Zhao Y-T Guo and Z-J Liang ldquoEffects of Veratrum nigrum alkaloids on centralcatecholaminergic neurons of renal hypertensive ratsrdquo ActaPharmacologica Sinica vol 21 no 1 pp 23ndash28 2000

[7] L Wang W Li and Y Liu ldquoHypotensive effect and toxicologyof total alkaloids and veratramine from roots and rhizomesof Veratrum nigrum L in spontaneously hypertensive ratsrdquoPharmazie vol 63 no 8 pp 606ndash610 2008

[8] Y P Jiang Y G Liu and H C Chen ldquoEffect of aqueous extractof Radix Paeoniae Rubra against Carbon Tetrachloride inducedliver fibrosis in ratrdquo Herald of Medicine vol 23 pp 527ndash5292004

[9] D-Y He and S-M Dai ldquoAnti-inflammatory and immunomod-ulatory effects of Paeonia lactiflora Pall a traditional Chineseherbal medicinerdquo Frontiers in Pharmacology 2011

[10] C Feng M Liu X Shi et al ldquoPharmacokinetic properties ofpaeoniflorin albiflorin and oxypaeoniflorin after oral gavage ofextracts of Radix Paeoniae Rubra and Radix Paeoniae Alba inratsrdquo Journal of Ethnopharmacology vol 130 no 2 pp 407ndash4132010

[11] X X Zhang Y G Wang Z C Ma Y Gao and C R XiaoldquoUniform designed research on the compatibility toxicity ofVeratridine and peonyrdquo Zhong Hua Zhong Yi Yao Za Zhi vol28 no 10 pp 2901ndash2904 2013

[12] Q D Liang Y Gao J Ma et al ldquoChemical comparison of driedrehmannia root and prepared rehmannia root by UPLC-TOFMS andHPLC-ELSDwithmultivariate statistical analysisrdquoActaPharmaceutica Sinica B vol 3 no 1 pp 55ndash64 2013

[13] Y Wang S He X Cheng Y Lu Y Zou and Q Zhang ldquoUPLC-Q-TOF-MSMS fingerprinting of traditional Chinese formulaSiJunZiTangrdquo Journal of Pharmaceutical and Biomedical Analy-sis vol 80 pp 24ndash33 2013

[14] L-P Kang K Yu Y Zhao et al ldquoCharacterization of steroidalglycosides from the extract of Paris Polyphylla var Yunnanensisby UPLCQ-TOFMSErdquo Journal of Pharmaceutical and Biomed-ical Analysis vol 62 pp 235ndash249 2012

[15] A Kaufmann P Butcher K Maden and M Widmer ldquoUltra-performance liquid chromatography coupled to time of flightmass spectrometry (UPLC-TOF) a novel tool for multiresiduescreening of veterinary drugs in urinerdquoAnalytica Chimica Actavol 586 no 1-2 pp 13ndash21 2007

[16] S Zhou Z C Ma Q D Liang et al ldquoUPLCQ-TOF-MS basedchemical profiling approach to evaluate chemical compositionof augmentation toxicity in combination of Radix aconiti andPinellia praeparatardquoActa Chimica Sinica vol 70 no 3 pp 284ndash290 2012

[17] N Lin and X Gao ldquoExperimental observation on the absorp-tion of Veratrum nigrum L in the upper digestive tractrdquoZhongguo Zhong Yao Za Zhi vol 17 no 1 pp 43ndash45 1992

[18] Z-Z Wang W-J Zhao X-S Zhang et al ldquoProtection ofVeratrum nigrum L var ussuriense Nakai alkaloids againstischema-reperfusion injury of the rat liverrdquo World Journal ofGastroenterology vol 13 no 4 pp 564ndash571 2007

[19] V Christov B Mikhova A Ivanova et al ldquoSteroidal alka-loids of Veratrum lobelianum Bernh and Veratrum nigrum LrdquoZeitschrift fur Naturforschung C Journal of Biosciences vol 65no 3-4 pp 195ndash200 2010

[20] V Christov BMikhova andD Selenge ldquo(-)-Veranigrine a newsteroidal alkaloid fromVeratrum nigrum Lrdquo Fitoterapia vol 80no 1 pp 25ndash27 2009

[21] Y Cong J-H Wang R Wang Y-M Zeng C-D Liu and XLi ldquoA study on the chemical constituents of Veratrum nigrum

L processed by rice vinegarrdquo Journal of Asian Natural ProductsResearch vol 10 no 7-8 pp 619ndash624 2008

[22] H-L Li J Tang R-H Liu C Zhang and W-D Zhang ldquoTwonew flavanone glycosides from Veratrum nigrum Lrdquo NaturalProduct Research vol 23 no 2 pp 122ndash126 2009

[23] Y Cong Y-B Zhou J Chen Y-M Zeng and J-H WangldquoAlkaloid profiling of crude and processed Veratrum nigrum Lthrough simultaneous determination of ten steroidal alkaloidsby HPLC-ELSDrdquo Journal of Pharmaceutical and BiomedicalAnalysis vol 48 no 3 pp 573ndash578 2008

[24] L-S Wang D-Q Zhao H-M Tao and Y-H Liu ldquoDetermi-nation of vetatramine in Veratrum nigrum by HPLC-ELSDrdquoZhongguo Zhongyao Zazhi vol 33 no 7 pp 791ndash792 2008

[25] Q Wang H-Z Guo C-H Huo et al ldquoChemical constituentsin root of Paeonia lactiflorardquo Chinese Traditional and HerbalDrugs vol 38 no 7 pp 972ndash976 2007

[26] J-J TanQ-C Zhao L Yang Z-P Shang Z-QDu andMYanldquoChemical constituents in roots of Paeonia lactiflorardquo ChineseTraditional and Herbal Drugs vol 41 no 8 pp 1245ndash1248 2010

[27] Q Zhang Z Z Wang W Xiao et al ldquoUPLC characteristicchromatographic profile of Paeoniae Radix Albardquo ZhongguoZhongyao Zazhi vol 36 no 6 pp 712ndash714 2011

[28] X R Gao and Y Tian ldquoActive principles of Paeonia lactifloraPallrdquo Chinese Journal of New Drugs vol 15 pp 416ndash418 2006

[29] N-C Luo W Ding J Wu et al ldquoUPLC-Q-TOFMS coupledwith multivariate statistical analysis as a powerful techniquefor rapidly exploring potential chemicalmarkers to differentiatebetween radix paeoniae alba and radix paeoniae rubrardquoNaturalProduct Communications vol 8 no 4 pp 487ndash491 2013

[30] W K Zhao Y Guo T Yasuhiro and K Tohru ldquoIsolation andstructure determination of echinuline from Veratrum nigrumL var ussuriense Nakairdquo Zhongguo Zhong Yao Za Zhi vol 16no 7 pp 425ndash426 1991

[31] W Zhao Y Guo T Yasuhirb and T Kikuchi ldquoIsolation andstructure determination of aurantiamide acetate fromVeratrumnigrum Lvarussuriense Nakairdquo Zhongguo Zhongyao Zazhi vol23 no 1 p 41 1998

[32] L Yang X H Xia Q Zhu and X P Tan ldquoStudy on therule of influence of two purification methods on the chemicalcompositions in aqueous solution of paeoniae radix albardquoZhong Yao Cai vol 36 pp 118ndash121 2012

[33] X Y Zang and X Li ldquoDevelopment of the study on chemicalconstituentsof Paeonia lact if lora Pallrdquo Journal of ShenyangPharmaceutical University vol 19 pp 70ndash73 2002

[34] Y-L Wang Y-G Wang Q-D Liang et al ldquoUsing cDNAmicroarray to screen differently expressed genes in SH-SY5Ytreated by veratridinerdquo Chinese Pharmacological Bulletin vol29 no 5 pp 643ndash647 2013

Submit your manuscripts athttpwwwhindawicom

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

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OncologyJournal of

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Oxidative Medicine and Cellular Longevity

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PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

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Diabetes ResearchJournal of

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Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Evidence-Based Complementary and Alternative Medicine 7

Table 4 The chemical components that were highly negatively correlated with toxicity (119903 le minus09)

Number 119903 tR (min) Assigned identity Molecularformula

Mean measuredmass (Da)

Theoretical exactmass (Da)

Mass accuracy(ppm)

1 minus09811 495 Paeoniflorinsulfonate C23H28O13S 5431194 5445256 337142 minus09799 1220 Benzoyloxy paeoniflorin C30H32O13 5991785 6001843 030463 minus09793 977 Galloyl paeoniflorin C30H32O15 6311725 6321741 038564 minus09737 1479 Paeonilacto-ne B C17H18O6 3171750 3181103 392095 minus09736 056 Paeonilacto-ne C C10H12O4 1950496 1960736 531926 minus09734 809 Tetragalloyglucose C34H28O22 7871008 7885729 566077 minus09728 230 Vanillic acid C7H6O5 1690161 1701195 374718 minus09716 1391 Desbenzoyl paeoniflorin C16H24O10 3751868 3761369 445059 minus09708 1352 Benzoyl paeoniflorin C30H32O12 5831848 5845679 06258

10 minus09684 415 6-O-120573-D-Glucopyranosyllactinolide C16H26O9 3611537 3623722 93435

11 minus09670 1037 Paeoniflorin albiflorin R1albiflorin mudanpioside I C23H28O11 5191261 4804618 15284

12 minus09655 1167 Gallic acid C9H10O2 1510750 1501745 5959013 minus09640 493 Paeonol C9H10O3 1650545 1661739 4067614 minus09625 1167 Lactiflorin C23H26O10 4801872 4624465 0444915 minus09609 667 Isomaltopaeoniflorin C29H38O16 6432221 6426024 2656916 minus09589 852 Albiflorin C23H27O11 9573118 4794539 9372317 minus09582 679 11015840-O-Benzoylsucrose C19H26O12 4641757 4464025 2366818 minus09567 1057 Pentagalloylglucose C41H32O26 9391166 9406772 6629219 minus09562 802 Oxypaeoniflorin C23H28O12 5141934 4964612 1899220 minus09544 1158 Ethyl gallate C9H10O5 2210433 1981727 3244521 minus09478 1011 Eugeniin C41H30O26 9371010 9386613 6708622 minus09395 585 Paeonilactinone C9H14O2 1530904 1542063 7575023 minus09352 113 Lactinolide C10H16O4 2011111 2002316 7890724 minus09173 1373 Palbinone C22H30O4 3572047 3584712 52115

3-Veratroylgermine250

200

150

100

50

0A B C D E F G H I J K L

Ion

inte

nsity

Sample group

Ion

inte

nsity

Sample group

0010203040506070809 Veratramine

A B C D E F G H I J K L0

02

04

06

08

1

12

14

Ion

inte

nsity

A B C D E F G H I J K LSample group

Jervine

A B C D E F G H I J K L

Ion

inte

nsity

Sample group

020406080100120140160180200

Grmidine

A B C D E F G H I J K L

Germanitrine

Ion

inte

nsity

Sample group

0

02

04

06

08

1

12

14

A B C D E F G H I J K L

Ion

inte

nsity

Sample group

0

01

02

03

04

05

06 Germerine

minus01

Figure 5 Ion intensity trend plots for 3-veratroylgermine jervine veratramine germanitrine germidine and germerine which were highlypositively correlated (119903 ge 09393) with toxicity

8 Evidence-Based Complementary and Alternative Medicine

0100200300400500600700800

A B C D E F G H I J K L A B C D E F G H I J K L

Ion

inte

nsity

Ion

inte

nsity

Ion

inte

nsity

Ion

inte

nsity

Ion

inte

nsity

A B C D E F G H I J K L

A B C D E F G H I J K L

A B C D E F G H I J K LSample groupSample group

Sample group A B C D E F G H I J K LSample group

Sample groupA B C D E F G H I J K L

Sample group

3-Angeloylzygadenine

0

01

02

03

04

05

06

07

Ion

inte

nsity

Ion

inte

nsity

11-Deoxojervine

0

50

100

150

200

250

300

350

Sample group

Ion

inte

nsity

Sample group

A B C D E F G H I J K L0

01

02

03

04

05

06 Jervinone

0

05

1

15

2

25

3 Neogermbudine

0020406081

121416182 Germine

0051

152

253

35 Veraline B

0

1

2

3

4

5

6 Verdine

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

1

2

3

4

5

6 GermitrineIo

n in

tens

ity

A B C D E F G H I J K LSample group

0

05

1

15

25

2

Polydatin

minus05

minus05

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

1

2

3

4

5

6

Ion

inte

nsity

A B C D E F G H I J K LSample group

0051

152

253

Stenophylline A

minus05

35

Neogermineveramanine

jervanin-12-en-11-one

0

2

4

6

8

10

12

14

A B C D E F G H I J K L

Ion

inte

nsity

Sample group

Cervdine

A B C D E F G H I J K L

Ion

inte

nsity

Sample group

0

02

04

06

08

1

12 Maackinine

Ion

inte

nsity

A B C D E F G H I J K LSample group

0123456789 Rubijervine

A B C D E F G H I J K L

Ion

inte

nsity

Sample group

0020406081

12141618 3-Veratrum acyl protoveratrine

Ion

inte

nsity

A B C D E F G H I J K LSample group

0051

152

253

354

Solanidine

minus05

A B C D E F G H I JK L

Ion

inte

nsity

Sample group

0020406081

121416182

Zygadenitic acid 120575-Lactone-16-angelate

11205733a-Dihydroxy-5120573-

(a)

Figure 6 Continued

Evidence-Based Complementary and Alternative Medicine 9

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

20

40

60

80

100

120 Pseudojervine

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

02

04

06

08

1

12

14 Neojerminalanine

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

02

04

06

08

1

12 Zygacine

Ion

inte

nsity

A B C D E F G H I J K LSample group

012345678 Zygadenine

Ion

inte

nsity

A B C D E F G H I J K LSample group

000501015020250303504045 Neoverataline B

minus005

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

01

02

03

04

05

06 3-Veratroylzygadenine

minus01

0

05

1

15

2

25 Vanilloylzygadenine

A B C D E F G H I J K LSample group

Ion

inte

nsity

A B C D E F G H I J K LSample group

Ion

inte

nsity

0

2

4

6

8

10

12 Veratrosine

A B C D E F G H I J K LSample group

Ion

inte

nsity

0

2

4

6

8

10

12 Stenophylline A

A B C D E F G H I J K LSample group

Ion

inte

nsity

0020406081

1214 120574-Aminobutyric acid

minus02

A B C D E F G H I J K LSample group

Ion

inte

nsity

0

02

04

06

08

1

12 7-Acetyl-15-methylbutyryl-3-veratroylgermine

(b)

Figure 6 Ion intensity trend plots for the remaining 29 chemical components that were highly positively correlated (119903 ge 08) with toxicity

the viability of SH-SY5Y cells was tested using MTS assaysAs shown in Figures 8(a) and 8(b) veratridine had an LD

50

of 450 whereas paeoniflorin exerted no significant toxicityat doses of 0ndash2000120583M Therefore the dose of veratridinewas fixed at 400120583M and the RPA dose was varied from50 to 2000120583M SH-SY5Y cell cytotoxicity was increasedwhen the concentration of paeoniflorin was decreased inthe combination (Figure 8(c)) Therefore paeoniflorin couldoffset the cytotoxicity caused by veratridine in SH-SY5Y cells[25]

4 Discussion

The eighteen incompatible medicaments (Shi Ba Fan) is awell-known traditional Chinese medicine (TCM) theory It

basically listed eighteen pairs of TCM herbal medicine thatare not to be used in combination as they may cause fatalconsequences This principal has been used as guidancefor general TCM practices for hundreds of years with littlechemical comprehension In this work for the first time weused the UPLC-TOFMS coupled with multivariate statisticalanalysis in an effort to gain scientific understanding for thecauses of the incompatibility

We previously reported that the LD50of aqueous extracts

of V nigrum L and RPA were 2566 gkg and 160 gkgrespectively after intragastric administration [11] V nigrumL extract was severely toxic whereas RPA had minimaltoxicity Although the amount of V nigrum L in each groupwas unchanged the mortality changed significantly withaltered ratios of V nigrum L to RPA When the doses of

10 Evidence-Based Complementary and Alternative Medicine

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

1

2

3

4

5

6 Tetragalloyglucose

Ion

inte

nsity

A B C D E F G H I J K LSample group

0020406081

121416182 Vanillic acid

Ion

inte

nsity

A B C D E F G H I J K LSample group

minus2

0

2

4

6

8

10

12 Benzoylpaeoniflorin

000501015020250303504

A B C D E F G H I J K L

Ion

inte

nsity

Sample groupminus005

Paeoniflorin sulfonate

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

05

1

15

2 Benzoyloxypaeoniflorin

minus05

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

05

1

15

2 Gallic acid

minus05

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

05

1

15

2

253 Galloylpaeoniflorin

minus05

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

005

01

015

02

025

03

035 Paeonilacto-neC

Ion

inte

nsity

A B C D E F G H I J K LSample group

0123456789 Paeonilacto-neB

minus1

Ion

inte

nsity

A B C D E F G H I J K LSample group

Vanillic acid

0

1

2

3

4

5

6

7

minus1

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

01

02

03

04

05

06

07

minus01

6-O-120573-D-Glucopyranosyllactinolide

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

05

1

15

2

25

3

35

minus05

Paeoniflorinalbiflorin R1albiflorinmudanpioside I

A B C D E F G H I J K L

Ion

inte

nsity

Sample group

Paeonol

0

02

04

06

08

1

12

minus02

Ion

inte

nsity

A B C D E F G H I J K LSample group

01020304050607080 Pentagalloylglucose

minus10

Ion

inte

nsity

A B C D E F G H I J K LSample group

00102030405060708 Lactiflorin

minus01

Ion

inte

nsity

A BC D E F G H I J K LSample group

0020406081

121416 Isomaltopaeoniflorin

minus02

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

05

1

15

2

25

3

minus05

Albiflorin

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

05

1

15

2

25 1998400-O-Benzoylsucrose

(a)

Figure 7 Continued

Evidence-Based Complementary and Alternative Medicine 11

Ion

inte

nsity

Ion

inte

nsity

Ion

inte

nsity

A B C D E F G H I J K LSample group A B C D E F G H I J K L

Sample group

A B C D E F G H I J K LSample group

00102030405060708 Oxypaeoniflorin

001020304050607 Eugeniin

Ion

inte

nsity

A B C D E F G H I J K LSample group

0020406081

12141618 Paeonia lactinone

00102030405060708091 Ethyl gallate

minus01

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

01

02

03

04

05

Lactinolide06

minus01

0

5

10

15

20

25 Palbinone

Ion

inte

nsity

A B C D E F G H I J K LSample group

minus5

minus01

(b)

Figure 7 Ion intensity trend plots for the chemical components that were highly negatively correlated with toxicity (119903 ge 09)

OD

val

ue

25 50 100 200 400 800

lowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

mdashVeratridine (120583M)

10

12

08

06

04

02

0

(a)

OD

val

ue

02

04

06

08

12

Paeoniflorin (120583M) 100 500 1500 2000mdash 1000

10

00

(b)

OD

val

ue

Paeoniflorin (120583M)

mdashmdash

Veratridine (120583M) 400 400 400 400 400 400 400 400 400 400

50 100 250 500 750 1000 1250 1500 1750 2000

02

04

06

08

12

10

00

lowastlowastlowast

lowastlowast

lowastlowast lowastlowast

lowastlowast

lowast

(c)

Figure 8 SH-SY5Y cell viability SH-SY5Y cells were cultured with (a) veratridine (25ndash800120583M) (b) paeoniflorin (50minus2000120583M) and (c)400 120583M veratridine in combination with 50minus2000120583M paeoniflorin for 24 h Cell viability was then assessed using MTS assays Data arepresented as mean plusmn SD Means of three independent experiments Asterisks indicate a statistically significant difference compared withuntreated cells lowast119875 lt 005 lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001 versus controL

12 Evidence-Based Complementary and Alternative Medicine

V nigrum L were equal to or higher than RPA particularlyratios of 8 1 and 1 1 mouse mortality increased significantlyTherefore components of V nigrum L were the majorcausative factors of toxicity In addition RPA modulated thetoxicity ofV nigrum LTherefore clinical applications shouldbe monitored carefully

Mice died 5minus10min after drug administration Toxicitymanifested predominantly as trembling convulsions andspasms but the major organs showed no obvious lesionsat necropsy by the naked eye Based on the toxic reactionsand the time of death in the current study and accord-ing to the guidelines for Chinese natural medicine acutetoxicity testing we speculate that the toxicity was causedpredominantly by suppression of the central nervous systemand that V nigrum L was the major cause of death Vnigrum L water extracts exert toxic effects on the digestivetract mucosa nucleus nervi vagi and central nervous system[6 17 18] Pharmacological studies of veratrum alkaloidshave been performed previously and reported that thesealkaloids stimulate the central nervous system and inhibit thebrain This results in spasms convulsions coma drowsinessand other symptoms of consciousness in animals which isconsistent with the symptoms of themice in the current study[6 17]

Veratrum alkaloid is the toxic component of V nigrum LWe previously investigated the differently expressed genes inSH-SY5Y cells treated with veratridine using cDNAmicroar-rays [34] Data revealed that veratridine treatment alteredthe expression of MMP caused Ca2+ concentration over-load increased reactive oxygen species production enhancedLDH release damaged the cell membrane reduced SH-SY5Y cell viability and induced apoptosis Under oxidizingconditions the MAPK signaling pathway is activated whichincreases apoptosisThis study illustrates the neurotoxicity ofthe combination of V nigrum L and reveals the molecularmechanism behind the neurotoxic effects of V nigrum L(data not shown)

The amount of V nigrum L in each group was the samethe dose of RPA is in ascending order and the chemicalcomponents of RPA are also increased but the mortalityof each group changed significantly with the increase ofRPA so we conclude that higher amount of RPA can haveprotection effect which can offset the toxicity of combinationof V nigrum L and RPA And pharmacological studies havedemonstrated that RPA exerts a wide range of pharmacolog-ical activities including analgesic sedative anticonvulsantand antispasmodic effects on the central nervous systemRPA could also inhibit writhing reactions and antagonizepentylenetetrazol-induced convulsions The most importantactive ingredient in RPA is paeoniflorin which has significantneuroprotective effects [26ndash34]

In this study mouse acute toxicity experiments revealedthat the mortality of mice changed significantly when thedose of V nigrum L was higher than or equal to that ofRPA particularly at ratios of V nigrum L RPA of 8 1and 1 1 UPLC-Q-TOFMS with automated data analysisused to identify and quantify the specific chemical compo-nents According to Pearsonrsquos correlation coefficient most of

the chemical components that were positively correlated withtoxicity were from V nigrum L We conclude that the majorveratrum alkaloids of V nigrum L caused acute toxicity anddeath in mice When the doses of V nigrum L were higherthan or equal to RPA the main chemical components of Vnigrum L and the toxicity of the decoctions were increasedThis suggests that V nigrum L and RPA have opposingroles Additional experiments revealed that the cytotoxicityof veratridine in SH-SY5Y cells was decreasedwith increasingconcentrations of paeoniflorin Therefore the clinical use ofthese agents should be considered carefully This study alsoprovides information to help improve the incompatibilitytheory of TCM and introduces novel ideas for further studieson the development and application of TCM

When preparing V nigrum L and RPA decoctions alower amount of RPA contributes to the dissolution of vera-trum alkaloids Conversely higher amounts of RPA can exertneuroprotective effects which can offset the toxicity causedby the combination of V nigrum L and RPA Under theseconditions dissolution is the most important factor for Vnigrum L toxicity The results of cell-based experiments alsodemonstrated that paeoniflorin could offset the neurotoxiceffects of veratridine when treated in combination whichexcluded the factor of dissolution in preparation

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors are grateful for financial support from NationalNature Science Foundation of China (Grant no 81073149)National Basic Research Program of China (Grant nos2011CB505304 and 2012CB518402) and Natural ScienceFoundation of Beijing (Grant no 7112110)

References

[1] H Guo W Li X Y Wang and G W Fan ldquoAdvance of researchon toxic attenuation by compatibility of traditional Chinesemedicine prescriptionsrdquo Zhongguo Zhongyao Zazhi vol 37 no1 pp 120ndash123 2012

[2] Y Zhang H Hua X Fan C Wang and J Duan ldquoExplorationon eighteen incompatible medicaments of chest pain prescrip-tions based on association rules miningrdquo Zhongguo ZhongyaoZazhi vol 36 no 24 pp 3544ndash3547 2011

[3] C-H Xu P Wang Y Wang et al ldquoPharmacokinetic compar-isons of two different combinations of Shaoyao-Gancao Decoc-tion in rats competing mechanisms between paeoniflorin andglycyrrhetinic acidrdquo Journal of Ethnopharmacology vol 149 no2 pp 443ndash452 2013

[4] D H TianDictionary of Practical Traditional Chinese MedicineII Peoplersquos Medical Beijing China 2002

[5] Y Gaillard and G Pepin ldquoLC-EI-MS determination of ver-atridine and cevadine in two fatal cases of Veratrum albumpoisoningrdquo Journal of Analytical Toxicology vol 25 no 6 pp481ndash485 2001

Evidence-Based Complementary and Alternative Medicine 13

[6] H Li G-Y Gao S-Y Li W-J Zhao Y-T Guo and Z-J Liang ldquoEffects of Veratrum nigrum alkaloids on centralcatecholaminergic neurons of renal hypertensive ratsrdquo ActaPharmacologica Sinica vol 21 no 1 pp 23ndash28 2000

[7] L Wang W Li and Y Liu ldquoHypotensive effect and toxicologyof total alkaloids and veratramine from roots and rhizomesof Veratrum nigrum L in spontaneously hypertensive ratsrdquoPharmazie vol 63 no 8 pp 606ndash610 2008

[8] Y P Jiang Y G Liu and H C Chen ldquoEffect of aqueous extractof Radix Paeoniae Rubra against Carbon Tetrachloride inducedliver fibrosis in ratrdquo Herald of Medicine vol 23 pp 527ndash5292004

[9] D-Y He and S-M Dai ldquoAnti-inflammatory and immunomod-ulatory effects of Paeonia lactiflora Pall a traditional Chineseherbal medicinerdquo Frontiers in Pharmacology 2011

[10] C Feng M Liu X Shi et al ldquoPharmacokinetic properties ofpaeoniflorin albiflorin and oxypaeoniflorin after oral gavage ofextracts of Radix Paeoniae Rubra and Radix Paeoniae Alba inratsrdquo Journal of Ethnopharmacology vol 130 no 2 pp 407ndash4132010

[11] X X Zhang Y G Wang Z C Ma Y Gao and C R XiaoldquoUniform designed research on the compatibility toxicity ofVeratridine and peonyrdquo Zhong Hua Zhong Yi Yao Za Zhi vol28 no 10 pp 2901ndash2904 2013

[12] Q D Liang Y Gao J Ma et al ldquoChemical comparison of driedrehmannia root and prepared rehmannia root by UPLC-TOFMS andHPLC-ELSDwithmultivariate statistical analysisrdquoActaPharmaceutica Sinica B vol 3 no 1 pp 55ndash64 2013

[13] Y Wang S He X Cheng Y Lu Y Zou and Q Zhang ldquoUPLC-Q-TOF-MSMS fingerprinting of traditional Chinese formulaSiJunZiTangrdquo Journal of Pharmaceutical and Biomedical Analy-sis vol 80 pp 24ndash33 2013

[14] L-P Kang K Yu Y Zhao et al ldquoCharacterization of steroidalglycosides from the extract of Paris Polyphylla var Yunnanensisby UPLCQ-TOFMSErdquo Journal of Pharmaceutical and Biomed-ical Analysis vol 62 pp 235ndash249 2012

[15] A Kaufmann P Butcher K Maden and M Widmer ldquoUltra-performance liquid chromatography coupled to time of flightmass spectrometry (UPLC-TOF) a novel tool for multiresiduescreening of veterinary drugs in urinerdquoAnalytica Chimica Actavol 586 no 1-2 pp 13ndash21 2007

[16] S Zhou Z C Ma Q D Liang et al ldquoUPLCQ-TOF-MS basedchemical profiling approach to evaluate chemical compositionof augmentation toxicity in combination of Radix aconiti andPinellia praeparatardquoActa Chimica Sinica vol 70 no 3 pp 284ndash290 2012

[17] N Lin and X Gao ldquoExperimental observation on the absorp-tion of Veratrum nigrum L in the upper digestive tractrdquoZhongguo Zhong Yao Za Zhi vol 17 no 1 pp 43ndash45 1992

[18] Z-Z Wang W-J Zhao X-S Zhang et al ldquoProtection ofVeratrum nigrum L var ussuriense Nakai alkaloids againstischema-reperfusion injury of the rat liverrdquo World Journal ofGastroenterology vol 13 no 4 pp 564ndash571 2007

[19] V Christov B Mikhova A Ivanova et al ldquoSteroidal alka-loids of Veratrum lobelianum Bernh and Veratrum nigrum LrdquoZeitschrift fur Naturforschung C Journal of Biosciences vol 65no 3-4 pp 195ndash200 2010

[20] V Christov BMikhova andD Selenge ldquo(-)-Veranigrine a newsteroidal alkaloid fromVeratrum nigrum Lrdquo Fitoterapia vol 80no 1 pp 25ndash27 2009

[21] Y Cong J-H Wang R Wang Y-M Zeng C-D Liu and XLi ldquoA study on the chemical constituents of Veratrum nigrum

L processed by rice vinegarrdquo Journal of Asian Natural ProductsResearch vol 10 no 7-8 pp 619ndash624 2008

[22] H-L Li J Tang R-H Liu C Zhang and W-D Zhang ldquoTwonew flavanone glycosides from Veratrum nigrum Lrdquo NaturalProduct Research vol 23 no 2 pp 122ndash126 2009

[23] Y Cong Y-B Zhou J Chen Y-M Zeng and J-H WangldquoAlkaloid profiling of crude and processed Veratrum nigrum Lthrough simultaneous determination of ten steroidal alkaloidsby HPLC-ELSDrdquo Journal of Pharmaceutical and BiomedicalAnalysis vol 48 no 3 pp 573ndash578 2008

[24] L-S Wang D-Q Zhao H-M Tao and Y-H Liu ldquoDetermi-nation of vetatramine in Veratrum nigrum by HPLC-ELSDrdquoZhongguo Zhongyao Zazhi vol 33 no 7 pp 791ndash792 2008

[25] Q Wang H-Z Guo C-H Huo et al ldquoChemical constituentsin root of Paeonia lactiflorardquo Chinese Traditional and HerbalDrugs vol 38 no 7 pp 972ndash976 2007

[26] J-J TanQ-C Zhao L Yang Z-P Shang Z-QDu andMYanldquoChemical constituents in roots of Paeonia lactiflorardquo ChineseTraditional and Herbal Drugs vol 41 no 8 pp 1245ndash1248 2010

[27] Q Zhang Z Z Wang W Xiao et al ldquoUPLC characteristicchromatographic profile of Paeoniae Radix Albardquo ZhongguoZhongyao Zazhi vol 36 no 6 pp 712ndash714 2011

[28] X R Gao and Y Tian ldquoActive principles of Paeonia lactifloraPallrdquo Chinese Journal of New Drugs vol 15 pp 416ndash418 2006

[29] N-C Luo W Ding J Wu et al ldquoUPLC-Q-TOFMS coupledwith multivariate statistical analysis as a powerful techniquefor rapidly exploring potential chemicalmarkers to differentiatebetween radix paeoniae alba and radix paeoniae rubrardquoNaturalProduct Communications vol 8 no 4 pp 487ndash491 2013

[30] W K Zhao Y Guo T Yasuhiro and K Tohru ldquoIsolation andstructure determination of echinuline from Veratrum nigrumL var ussuriense Nakairdquo Zhongguo Zhong Yao Za Zhi vol 16no 7 pp 425ndash426 1991

[31] W Zhao Y Guo T Yasuhirb and T Kikuchi ldquoIsolation andstructure determination of aurantiamide acetate fromVeratrumnigrum Lvarussuriense Nakairdquo Zhongguo Zhongyao Zazhi vol23 no 1 p 41 1998

[32] L Yang X H Xia Q Zhu and X P Tan ldquoStudy on therule of influence of two purification methods on the chemicalcompositions in aqueous solution of paeoniae radix albardquoZhong Yao Cai vol 36 pp 118ndash121 2012

[33] X Y Zang and X Li ldquoDevelopment of the study on chemicalconstituentsof Paeonia lact if lora Pallrdquo Journal of ShenyangPharmaceutical University vol 19 pp 70ndash73 2002

[34] Y-L Wang Y-G Wang Q-D Liang et al ldquoUsing cDNAmicroarray to screen differently expressed genes in SH-SY5Ytreated by veratridinerdquo Chinese Pharmacological Bulletin vol29 no 5 pp 643ndash647 2013

Submit your manuscripts athttpwwwhindawicom

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

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Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

8 Evidence-Based Complementary and Alternative Medicine

0100200300400500600700800

A B C D E F G H I J K L A B C D E F G H I J K L

Ion

inte

nsity

Ion

inte

nsity

Ion

inte

nsity

Ion

inte

nsity

Ion

inte

nsity

A B C D E F G H I J K L

A B C D E F G H I J K L

A B C D E F G H I J K LSample groupSample group

Sample group A B C D E F G H I J K LSample group

Sample groupA B C D E F G H I J K L

Sample group

3-Angeloylzygadenine

0

01

02

03

04

05

06

07

Ion

inte

nsity

Ion

inte

nsity

11-Deoxojervine

0

50

100

150

200

250

300

350

Sample group

Ion

inte

nsity

Sample group

A B C D E F G H I J K L0

01

02

03

04

05

06 Jervinone

0

05

1

15

2

25

3 Neogermbudine

0020406081

121416182 Germine

0051

152

253

35 Veraline B

0

1

2

3

4

5

6 Verdine

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

1

2

3

4

5

6 GermitrineIo

n in

tens

ity

A B C D E F G H I J K LSample group

0

05

1

15

25

2

Polydatin

minus05

minus05

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

1

2

3

4

5

6

Ion

inte

nsity

A B C D E F G H I J K LSample group

0051

152

253

Stenophylline A

minus05

35

Neogermineveramanine

jervanin-12-en-11-one

0

2

4

6

8

10

12

14

A B C D E F G H I J K L

Ion

inte

nsity

Sample group

Cervdine

A B C D E F G H I J K L

Ion

inte

nsity

Sample group

0

02

04

06

08

1

12 Maackinine

Ion

inte

nsity

A B C D E F G H I J K LSample group

0123456789 Rubijervine

A B C D E F G H I J K L

Ion

inte

nsity

Sample group

0020406081

12141618 3-Veratrum acyl protoveratrine

Ion

inte

nsity

A B C D E F G H I J K LSample group

0051

152

253

354

Solanidine

minus05

A B C D E F G H I JK L

Ion

inte

nsity

Sample group

0020406081

121416182

Zygadenitic acid 120575-Lactone-16-angelate

11205733a-Dihydroxy-5120573-

(a)

Figure 6 Continued

Evidence-Based Complementary and Alternative Medicine 9

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

20

40

60

80

100

120 Pseudojervine

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

02

04

06

08

1

12

14 Neojerminalanine

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

02

04

06

08

1

12 Zygacine

Ion

inte

nsity

A B C D E F G H I J K LSample group

012345678 Zygadenine

Ion

inte

nsity

A B C D E F G H I J K LSample group

000501015020250303504045 Neoverataline B

minus005

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

01

02

03

04

05

06 3-Veratroylzygadenine

minus01

0

05

1

15

2

25 Vanilloylzygadenine

A B C D E F G H I J K LSample group

Ion

inte

nsity

A B C D E F G H I J K LSample group

Ion

inte

nsity

0

2

4

6

8

10

12 Veratrosine

A B C D E F G H I J K LSample group

Ion

inte

nsity

0

2

4

6

8

10

12 Stenophylline A

A B C D E F G H I J K LSample group

Ion

inte

nsity

0020406081

1214 120574-Aminobutyric acid

minus02

A B C D E F G H I J K LSample group

Ion

inte

nsity

0

02

04

06

08

1

12 7-Acetyl-15-methylbutyryl-3-veratroylgermine

(b)

Figure 6 Ion intensity trend plots for the remaining 29 chemical components that were highly positively correlated (119903 ge 08) with toxicity

the viability of SH-SY5Y cells was tested using MTS assaysAs shown in Figures 8(a) and 8(b) veratridine had an LD

50

of 450 whereas paeoniflorin exerted no significant toxicityat doses of 0ndash2000120583M Therefore the dose of veratridinewas fixed at 400120583M and the RPA dose was varied from50 to 2000120583M SH-SY5Y cell cytotoxicity was increasedwhen the concentration of paeoniflorin was decreased inthe combination (Figure 8(c)) Therefore paeoniflorin couldoffset the cytotoxicity caused by veratridine in SH-SY5Y cells[25]

4 Discussion

The eighteen incompatible medicaments (Shi Ba Fan) is awell-known traditional Chinese medicine (TCM) theory It

basically listed eighteen pairs of TCM herbal medicine thatare not to be used in combination as they may cause fatalconsequences This principal has been used as guidancefor general TCM practices for hundreds of years with littlechemical comprehension In this work for the first time weused the UPLC-TOFMS coupled with multivariate statisticalanalysis in an effort to gain scientific understanding for thecauses of the incompatibility

We previously reported that the LD50of aqueous extracts

of V nigrum L and RPA were 2566 gkg and 160 gkgrespectively after intragastric administration [11] V nigrumL extract was severely toxic whereas RPA had minimaltoxicity Although the amount of V nigrum L in each groupwas unchanged the mortality changed significantly withaltered ratios of V nigrum L to RPA When the doses of

10 Evidence-Based Complementary and Alternative Medicine

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

1

2

3

4

5

6 Tetragalloyglucose

Ion

inte

nsity

A B C D E F G H I J K LSample group

0020406081

121416182 Vanillic acid

Ion

inte

nsity

A B C D E F G H I J K LSample group

minus2

0

2

4

6

8

10

12 Benzoylpaeoniflorin

000501015020250303504

A B C D E F G H I J K L

Ion

inte

nsity

Sample groupminus005

Paeoniflorin sulfonate

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

05

1

15

2 Benzoyloxypaeoniflorin

minus05

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

05

1

15

2 Gallic acid

minus05

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

05

1

15

2

253 Galloylpaeoniflorin

minus05

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

005

01

015

02

025

03

035 Paeonilacto-neC

Ion

inte

nsity

A B C D E F G H I J K LSample group

0123456789 Paeonilacto-neB

minus1

Ion

inte

nsity

A B C D E F G H I J K LSample group

Vanillic acid

0

1

2

3

4

5

6

7

minus1

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

01

02

03

04

05

06

07

minus01

6-O-120573-D-Glucopyranosyllactinolide

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

05

1

15

2

25

3

35

minus05

Paeoniflorinalbiflorin R1albiflorinmudanpioside I

A B C D E F G H I J K L

Ion

inte

nsity

Sample group

Paeonol

0

02

04

06

08

1

12

minus02

Ion

inte

nsity

A B C D E F G H I J K LSample group

01020304050607080 Pentagalloylglucose

minus10

Ion

inte

nsity

A B C D E F G H I J K LSample group

00102030405060708 Lactiflorin

minus01

Ion

inte

nsity

A BC D E F G H I J K LSample group

0020406081

121416 Isomaltopaeoniflorin

minus02

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

05

1

15

2

25

3

minus05

Albiflorin

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

05

1

15

2

25 1998400-O-Benzoylsucrose

(a)

Figure 7 Continued

Evidence-Based Complementary and Alternative Medicine 11

Ion

inte

nsity

Ion

inte

nsity

Ion

inte

nsity

A B C D E F G H I J K LSample group A B C D E F G H I J K L

Sample group

A B C D E F G H I J K LSample group

00102030405060708 Oxypaeoniflorin

001020304050607 Eugeniin

Ion

inte

nsity

A B C D E F G H I J K LSample group

0020406081

12141618 Paeonia lactinone

00102030405060708091 Ethyl gallate

minus01

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

01

02

03

04

05

Lactinolide06

minus01

0

5

10

15

20

25 Palbinone

Ion

inte

nsity

A B C D E F G H I J K LSample group

minus5

minus01

(b)

Figure 7 Ion intensity trend plots for the chemical components that were highly negatively correlated with toxicity (119903 ge 09)

OD

val

ue

25 50 100 200 400 800

lowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

mdashVeratridine (120583M)

10

12

08

06

04

02

0

(a)

OD

val

ue

02

04

06

08

12

Paeoniflorin (120583M) 100 500 1500 2000mdash 1000

10

00

(b)

OD

val

ue

Paeoniflorin (120583M)

mdashmdash

Veratridine (120583M) 400 400 400 400 400 400 400 400 400 400

50 100 250 500 750 1000 1250 1500 1750 2000

02

04

06

08

12

10

00

lowastlowastlowast

lowastlowast

lowastlowast lowastlowast

lowastlowast

lowast

(c)

Figure 8 SH-SY5Y cell viability SH-SY5Y cells were cultured with (a) veratridine (25ndash800120583M) (b) paeoniflorin (50minus2000120583M) and (c)400 120583M veratridine in combination with 50minus2000120583M paeoniflorin for 24 h Cell viability was then assessed using MTS assays Data arepresented as mean plusmn SD Means of three independent experiments Asterisks indicate a statistically significant difference compared withuntreated cells lowast119875 lt 005 lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001 versus controL

12 Evidence-Based Complementary and Alternative Medicine

V nigrum L were equal to or higher than RPA particularlyratios of 8 1 and 1 1 mouse mortality increased significantlyTherefore components of V nigrum L were the majorcausative factors of toxicity In addition RPA modulated thetoxicity ofV nigrum LTherefore clinical applications shouldbe monitored carefully

Mice died 5minus10min after drug administration Toxicitymanifested predominantly as trembling convulsions andspasms but the major organs showed no obvious lesionsat necropsy by the naked eye Based on the toxic reactionsand the time of death in the current study and accord-ing to the guidelines for Chinese natural medicine acutetoxicity testing we speculate that the toxicity was causedpredominantly by suppression of the central nervous systemand that V nigrum L was the major cause of death Vnigrum L water extracts exert toxic effects on the digestivetract mucosa nucleus nervi vagi and central nervous system[6 17 18] Pharmacological studies of veratrum alkaloidshave been performed previously and reported that thesealkaloids stimulate the central nervous system and inhibit thebrain This results in spasms convulsions coma drowsinessand other symptoms of consciousness in animals which isconsistent with the symptoms of themice in the current study[6 17]

Veratrum alkaloid is the toxic component of V nigrum LWe previously investigated the differently expressed genes inSH-SY5Y cells treated with veratridine using cDNAmicroar-rays [34] Data revealed that veratridine treatment alteredthe expression of MMP caused Ca2+ concentration over-load increased reactive oxygen species production enhancedLDH release damaged the cell membrane reduced SH-SY5Y cell viability and induced apoptosis Under oxidizingconditions the MAPK signaling pathway is activated whichincreases apoptosisThis study illustrates the neurotoxicity ofthe combination of V nigrum L and reveals the molecularmechanism behind the neurotoxic effects of V nigrum L(data not shown)

The amount of V nigrum L in each group was the samethe dose of RPA is in ascending order and the chemicalcomponents of RPA are also increased but the mortalityof each group changed significantly with the increase ofRPA so we conclude that higher amount of RPA can haveprotection effect which can offset the toxicity of combinationof V nigrum L and RPA And pharmacological studies havedemonstrated that RPA exerts a wide range of pharmacolog-ical activities including analgesic sedative anticonvulsantand antispasmodic effects on the central nervous systemRPA could also inhibit writhing reactions and antagonizepentylenetetrazol-induced convulsions The most importantactive ingredient in RPA is paeoniflorin which has significantneuroprotective effects [26ndash34]

In this study mouse acute toxicity experiments revealedthat the mortality of mice changed significantly when thedose of V nigrum L was higher than or equal to that ofRPA particularly at ratios of V nigrum L RPA of 8 1and 1 1 UPLC-Q-TOFMS with automated data analysisused to identify and quantify the specific chemical compo-nents According to Pearsonrsquos correlation coefficient most of

the chemical components that were positively correlated withtoxicity were from V nigrum L We conclude that the majorveratrum alkaloids of V nigrum L caused acute toxicity anddeath in mice When the doses of V nigrum L were higherthan or equal to RPA the main chemical components of Vnigrum L and the toxicity of the decoctions were increasedThis suggests that V nigrum L and RPA have opposingroles Additional experiments revealed that the cytotoxicityof veratridine in SH-SY5Y cells was decreasedwith increasingconcentrations of paeoniflorin Therefore the clinical use ofthese agents should be considered carefully This study alsoprovides information to help improve the incompatibilitytheory of TCM and introduces novel ideas for further studieson the development and application of TCM

When preparing V nigrum L and RPA decoctions alower amount of RPA contributes to the dissolution of vera-trum alkaloids Conversely higher amounts of RPA can exertneuroprotective effects which can offset the toxicity causedby the combination of V nigrum L and RPA Under theseconditions dissolution is the most important factor for Vnigrum L toxicity The results of cell-based experiments alsodemonstrated that paeoniflorin could offset the neurotoxiceffects of veratridine when treated in combination whichexcluded the factor of dissolution in preparation

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors are grateful for financial support from NationalNature Science Foundation of China (Grant no 81073149)National Basic Research Program of China (Grant nos2011CB505304 and 2012CB518402) and Natural ScienceFoundation of Beijing (Grant no 7112110)

References

[1] H Guo W Li X Y Wang and G W Fan ldquoAdvance of researchon toxic attenuation by compatibility of traditional Chinesemedicine prescriptionsrdquo Zhongguo Zhongyao Zazhi vol 37 no1 pp 120ndash123 2012

[2] Y Zhang H Hua X Fan C Wang and J Duan ldquoExplorationon eighteen incompatible medicaments of chest pain prescrip-tions based on association rules miningrdquo Zhongguo ZhongyaoZazhi vol 36 no 24 pp 3544ndash3547 2011

[3] C-H Xu P Wang Y Wang et al ldquoPharmacokinetic compar-isons of two different combinations of Shaoyao-Gancao Decoc-tion in rats competing mechanisms between paeoniflorin andglycyrrhetinic acidrdquo Journal of Ethnopharmacology vol 149 no2 pp 443ndash452 2013

[4] D H TianDictionary of Practical Traditional Chinese MedicineII Peoplersquos Medical Beijing China 2002

[5] Y Gaillard and G Pepin ldquoLC-EI-MS determination of ver-atridine and cevadine in two fatal cases of Veratrum albumpoisoningrdquo Journal of Analytical Toxicology vol 25 no 6 pp481ndash485 2001

Evidence-Based Complementary and Alternative Medicine 13

[6] H Li G-Y Gao S-Y Li W-J Zhao Y-T Guo and Z-J Liang ldquoEffects of Veratrum nigrum alkaloids on centralcatecholaminergic neurons of renal hypertensive ratsrdquo ActaPharmacologica Sinica vol 21 no 1 pp 23ndash28 2000

[7] L Wang W Li and Y Liu ldquoHypotensive effect and toxicologyof total alkaloids and veratramine from roots and rhizomesof Veratrum nigrum L in spontaneously hypertensive ratsrdquoPharmazie vol 63 no 8 pp 606ndash610 2008

[8] Y P Jiang Y G Liu and H C Chen ldquoEffect of aqueous extractof Radix Paeoniae Rubra against Carbon Tetrachloride inducedliver fibrosis in ratrdquo Herald of Medicine vol 23 pp 527ndash5292004

[9] D-Y He and S-M Dai ldquoAnti-inflammatory and immunomod-ulatory effects of Paeonia lactiflora Pall a traditional Chineseherbal medicinerdquo Frontiers in Pharmacology 2011

[10] C Feng M Liu X Shi et al ldquoPharmacokinetic properties ofpaeoniflorin albiflorin and oxypaeoniflorin after oral gavage ofextracts of Radix Paeoniae Rubra and Radix Paeoniae Alba inratsrdquo Journal of Ethnopharmacology vol 130 no 2 pp 407ndash4132010

[11] X X Zhang Y G Wang Z C Ma Y Gao and C R XiaoldquoUniform designed research on the compatibility toxicity ofVeratridine and peonyrdquo Zhong Hua Zhong Yi Yao Za Zhi vol28 no 10 pp 2901ndash2904 2013

[12] Q D Liang Y Gao J Ma et al ldquoChemical comparison of driedrehmannia root and prepared rehmannia root by UPLC-TOFMS andHPLC-ELSDwithmultivariate statistical analysisrdquoActaPharmaceutica Sinica B vol 3 no 1 pp 55ndash64 2013

[13] Y Wang S He X Cheng Y Lu Y Zou and Q Zhang ldquoUPLC-Q-TOF-MSMS fingerprinting of traditional Chinese formulaSiJunZiTangrdquo Journal of Pharmaceutical and Biomedical Analy-sis vol 80 pp 24ndash33 2013

[14] L-P Kang K Yu Y Zhao et al ldquoCharacterization of steroidalglycosides from the extract of Paris Polyphylla var Yunnanensisby UPLCQ-TOFMSErdquo Journal of Pharmaceutical and Biomed-ical Analysis vol 62 pp 235ndash249 2012

[15] A Kaufmann P Butcher K Maden and M Widmer ldquoUltra-performance liquid chromatography coupled to time of flightmass spectrometry (UPLC-TOF) a novel tool for multiresiduescreening of veterinary drugs in urinerdquoAnalytica Chimica Actavol 586 no 1-2 pp 13ndash21 2007

[16] S Zhou Z C Ma Q D Liang et al ldquoUPLCQ-TOF-MS basedchemical profiling approach to evaluate chemical compositionof augmentation toxicity in combination of Radix aconiti andPinellia praeparatardquoActa Chimica Sinica vol 70 no 3 pp 284ndash290 2012

[17] N Lin and X Gao ldquoExperimental observation on the absorp-tion of Veratrum nigrum L in the upper digestive tractrdquoZhongguo Zhong Yao Za Zhi vol 17 no 1 pp 43ndash45 1992

[18] Z-Z Wang W-J Zhao X-S Zhang et al ldquoProtection ofVeratrum nigrum L var ussuriense Nakai alkaloids againstischema-reperfusion injury of the rat liverrdquo World Journal ofGastroenterology vol 13 no 4 pp 564ndash571 2007

[19] V Christov B Mikhova A Ivanova et al ldquoSteroidal alka-loids of Veratrum lobelianum Bernh and Veratrum nigrum LrdquoZeitschrift fur Naturforschung C Journal of Biosciences vol 65no 3-4 pp 195ndash200 2010

[20] V Christov BMikhova andD Selenge ldquo(-)-Veranigrine a newsteroidal alkaloid fromVeratrum nigrum Lrdquo Fitoterapia vol 80no 1 pp 25ndash27 2009

[21] Y Cong J-H Wang R Wang Y-M Zeng C-D Liu and XLi ldquoA study on the chemical constituents of Veratrum nigrum

L processed by rice vinegarrdquo Journal of Asian Natural ProductsResearch vol 10 no 7-8 pp 619ndash624 2008

[22] H-L Li J Tang R-H Liu C Zhang and W-D Zhang ldquoTwonew flavanone glycosides from Veratrum nigrum Lrdquo NaturalProduct Research vol 23 no 2 pp 122ndash126 2009

[23] Y Cong Y-B Zhou J Chen Y-M Zeng and J-H WangldquoAlkaloid profiling of crude and processed Veratrum nigrum Lthrough simultaneous determination of ten steroidal alkaloidsby HPLC-ELSDrdquo Journal of Pharmaceutical and BiomedicalAnalysis vol 48 no 3 pp 573ndash578 2008

[24] L-S Wang D-Q Zhao H-M Tao and Y-H Liu ldquoDetermi-nation of vetatramine in Veratrum nigrum by HPLC-ELSDrdquoZhongguo Zhongyao Zazhi vol 33 no 7 pp 791ndash792 2008

[25] Q Wang H-Z Guo C-H Huo et al ldquoChemical constituentsin root of Paeonia lactiflorardquo Chinese Traditional and HerbalDrugs vol 38 no 7 pp 972ndash976 2007

[26] J-J TanQ-C Zhao L Yang Z-P Shang Z-QDu andMYanldquoChemical constituents in roots of Paeonia lactiflorardquo ChineseTraditional and Herbal Drugs vol 41 no 8 pp 1245ndash1248 2010

[27] Q Zhang Z Z Wang W Xiao et al ldquoUPLC characteristicchromatographic profile of Paeoniae Radix Albardquo ZhongguoZhongyao Zazhi vol 36 no 6 pp 712ndash714 2011

[28] X R Gao and Y Tian ldquoActive principles of Paeonia lactifloraPallrdquo Chinese Journal of New Drugs vol 15 pp 416ndash418 2006

[29] N-C Luo W Ding J Wu et al ldquoUPLC-Q-TOFMS coupledwith multivariate statistical analysis as a powerful techniquefor rapidly exploring potential chemicalmarkers to differentiatebetween radix paeoniae alba and radix paeoniae rubrardquoNaturalProduct Communications vol 8 no 4 pp 487ndash491 2013

[30] W K Zhao Y Guo T Yasuhiro and K Tohru ldquoIsolation andstructure determination of echinuline from Veratrum nigrumL var ussuriense Nakairdquo Zhongguo Zhong Yao Za Zhi vol 16no 7 pp 425ndash426 1991

[31] W Zhao Y Guo T Yasuhirb and T Kikuchi ldquoIsolation andstructure determination of aurantiamide acetate fromVeratrumnigrum Lvarussuriense Nakairdquo Zhongguo Zhongyao Zazhi vol23 no 1 p 41 1998

[32] L Yang X H Xia Q Zhu and X P Tan ldquoStudy on therule of influence of two purification methods on the chemicalcompositions in aqueous solution of paeoniae radix albardquoZhong Yao Cai vol 36 pp 118ndash121 2012

[33] X Y Zang and X Li ldquoDevelopment of the study on chemicalconstituentsof Paeonia lact if lora Pallrdquo Journal of ShenyangPharmaceutical University vol 19 pp 70ndash73 2002

[34] Y-L Wang Y-G Wang Q-D Liang et al ldquoUsing cDNAmicroarray to screen differently expressed genes in SH-SY5Ytreated by veratridinerdquo Chinese Pharmacological Bulletin vol29 no 5 pp 643ndash647 2013

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Evidence-Based Complementary and Alternative Medicine 9

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

20

40

60

80

100

120 Pseudojervine

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

02

04

06

08

1

12

14 Neojerminalanine

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

02

04

06

08

1

12 Zygacine

Ion

inte

nsity

A B C D E F G H I J K LSample group

012345678 Zygadenine

Ion

inte

nsity

A B C D E F G H I J K LSample group

000501015020250303504045 Neoverataline B

minus005

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

01

02

03

04

05

06 3-Veratroylzygadenine

minus01

0

05

1

15

2

25 Vanilloylzygadenine

A B C D E F G H I J K LSample group

Ion

inte

nsity

A B C D E F G H I J K LSample group

Ion

inte

nsity

0

2

4

6

8

10

12 Veratrosine

A B C D E F G H I J K LSample group

Ion

inte

nsity

0

2

4

6

8

10

12 Stenophylline A

A B C D E F G H I J K LSample group

Ion

inte

nsity

0020406081

1214 120574-Aminobutyric acid

minus02

A B C D E F G H I J K LSample group

Ion

inte

nsity

0

02

04

06

08

1

12 7-Acetyl-15-methylbutyryl-3-veratroylgermine

(b)

Figure 6 Ion intensity trend plots for the remaining 29 chemical components that were highly positively correlated (119903 ge 08) with toxicity

the viability of SH-SY5Y cells was tested using MTS assaysAs shown in Figures 8(a) and 8(b) veratridine had an LD

50

of 450 whereas paeoniflorin exerted no significant toxicityat doses of 0ndash2000120583M Therefore the dose of veratridinewas fixed at 400120583M and the RPA dose was varied from50 to 2000120583M SH-SY5Y cell cytotoxicity was increasedwhen the concentration of paeoniflorin was decreased inthe combination (Figure 8(c)) Therefore paeoniflorin couldoffset the cytotoxicity caused by veratridine in SH-SY5Y cells[25]

4 Discussion

The eighteen incompatible medicaments (Shi Ba Fan) is awell-known traditional Chinese medicine (TCM) theory It

basically listed eighteen pairs of TCM herbal medicine thatare not to be used in combination as they may cause fatalconsequences This principal has been used as guidancefor general TCM practices for hundreds of years with littlechemical comprehension In this work for the first time weused the UPLC-TOFMS coupled with multivariate statisticalanalysis in an effort to gain scientific understanding for thecauses of the incompatibility

We previously reported that the LD50of aqueous extracts

of V nigrum L and RPA were 2566 gkg and 160 gkgrespectively after intragastric administration [11] V nigrumL extract was severely toxic whereas RPA had minimaltoxicity Although the amount of V nigrum L in each groupwas unchanged the mortality changed significantly withaltered ratios of V nigrum L to RPA When the doses of

10 Evidence-Based Complementary and Alternative Medicine

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

1

2

3

4

5

6 Tetragalloyglucose

Ion

inte

nsity

A B C D E F G H I J K LSample group

0020406081

121416182 Vanillic acid

Ion

inte

nsity

A B C D E F G H I J K LSample group

minus2

0

2

4

6

8

10

12 Benzoylpaeoniflorin

000501015020250303504

A B C D E F G H I J K L

Ion

inte

nsity

Sample groupminus005

Paeoniflorin sulfonate

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

05

1

15

2 Benzoyloxypaeoniflorin

minus05

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

05

1

15

2 Gallic acid

minus05

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

05

1

15

2

253 Galloylpaeoniflorin

minus05

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

005

01

015

02

025

03

035 Paeonilacto-neC

Ion

inte

nsity

A B C D E F G H I J K LSample group

0123456789 Paeonilacto-neB

minus1

Ion

inte

nsity

A B C D E F G H I J K LSample group

Vanillic acid

0

1

2

3

4

5

6

7

minus1

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

01

02

03

04

05

06

07

minus01

6-O-120573-D-Glucopyranosyllactinolide

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

05

1

15

2

25

3

35

minus05

Paeoniflorinalbiflorin R1albiflorinmudanpioside I

A B C D E F G H I J K L

Ion

inte

nsity

Sample group

Paeonol

0

02

04

06

08

1

12

minus02

Ion

inte

nsity

A B C D E F G H I J K LSample group

01020304050607080 Pentagalloylglucose

minus10

Ion

inte

nsity

A B C D E F G H I J K LSample group

00102030405060708 Lactiflorin

minus01

Ion

inte

nsity

A BC D E F G H I J K LSample group

0020406081

121416 Isomaltopaeoniflorin

minus02

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

05

1

15

2

25

3

minus05

Albiflorin

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

05

1

15

2

25 1998400-O-Benzoylsucrose

(a)

Figure 7 Continued

Evidence-Based Complementary and Alternative Medicine 11

Ion

inte

nsity

Ion

inte

nsity

Ion

inte

nsity

A B C D E F G H I J K LSample group A B C D E F G H I J K L

Sample group

A B C D E F G H I J K LSample group

00102030405060708 Oxypaeoniflorin

001020304050607 Eugeniin

Ion

inte

nsity

A B C D E F G H I J K LSample group

0020406081

12141618 Paeonia lactinone

00102030405060708091 Ethyl gallate

minus01

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

01

02

03

04

05

Lactinolide06

minus01

0

5

10

15

20

25 Palbinone

Ion

inte

nsity

A B C D E F G H I J K LSample group

minus5

minus01

(b)

Figure 7 Ion intensity trend plots for the chemical components that were highly negatively correlated with toxicity (119903 ge 09)

OD

val

ue

25 50 100 200 400 800

lowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

mdashVeratridine (120583M)

10

12

08

06

04

02

0

(a)

OD

val

ue

02

04

06

08

12

Paeoniflorin (120583M) 100 500 1500 2000mdash 1000

10

00

(b)

OD

val

ue

Paeoniflorin (120583M)

mdashmdash

Veratridine (120583M) 400 400 400 400 400 400 400 400 400 400

50 100 250 500 750 1000 1250 1500 1750 2000

02

04

06

08

12

10

00

lowastlowastlowast

lowastlowast

lowastlowast lowastlowast

lowastlowast

lowast

(c)

Figure 8 SH-SY5Y cell viability SH-SY5Y cells were cultured with (a) veratridine (25ndash800120583M) (b) paeoniflorin (50minus2000120583M) and (c)400 120583M veratridine in combination with 50minus2000120583M paeoniflorin for 24 h Cell viability was then assessed using MTS assays Data arepresented as mean plusmn SD Means of three independent experiments Asterisks indicate a statistically significant difference compared withuntreated cells lowast119875 lt 005 lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001 versus controL

12 Evidence-Based Complementary and Alternative Medicine

V nigrum L were equal to or higher than RPA particularlyratios of 8 1 and 1 1 mouse mortality increased significantlyTherefore components of V nigrum L were the majorcausative factors of toxicity In addition RPA modulated thetoxicity ofV nigrum LTherefore clinical applications shouldbe monitored carefully

Mice died 5minus10min after drug administration Toxicitymanifested predominantly as trembling convulsions andspasms but the major organs showed no obvious lesionsat necropsy by the naked eye Based on the toxic reactionsand the time of death in the current study and accord-ing to the guidelines for Chinese natural medicine acutetoxicity testing we speculate that the toxicity was causedpredominantly by suppression of the central nervous systemand that V nigrum L was the major cause of death Vnigrum L water extracts exert toxic effects on the digestivetract mucosa nucleus nervi vagi and central nervous system[6 17 18] Pharmacological studies of veratrum alkaloidshave been performed previously and reported that thesealkaloids stimulate the central nervous system and inhibit thebrain This results in spasms convulsions coma drowsinessand other symptoms of consciousness in animals which isconsistent with the symptoms of themice in the current study[6 17]

Veratrum alkaloid is the toxic component of V nigrum LWe previously investigated the differently expressed genes inSH-SY5Y cells treated with veratridine using cDNAmicroar-rays [34] Data revealed that veratridine treatment alteredthe expression of MMP caused Ca2+ concentration over-load increased reactive oxygen species production enhancedLDH release damaged the cell membrane reduced SH-SY5Y cell viability and induced apoptosis Under oxidizingconditions the MAPK signaling pathway is activated whichincreases apoptosisThis study illustrates the neurotoxicity ofthe combination of V nigrum L and reveals the molecularmechanism behind the neurotoxic effects of V nigrum L(data not shown)

The amount of V nigrum L in each group was the samethe dose of RPA is in ascending order and the chemicalcomponents of RPA are also increased but the mortalityof each group changed significantly with the increase ofRPA so we conclude that higher amount of RPA can haveprotection effect which can offset the toxicity of combinationof V nigrum L and RPA And pharmacological studies havedemonstrated that RPA exerts a wide range of pharmacolog-ical activities including analgesic sedative anticonvulsantand antispasmodic effects on the central nervous systemRPA could also inhibit writhing reactions and antagonizepentylenetetrazol-induced convulsions The most importantactive ingredient in RPA is paeoniflorin which has significantneuroprotective effects [26ndash34]

In this study mouse acute toxicity experiments revealedthat the mortality of mice changed significantly when thedose of V nigrum L was higher than or equal to that ofRPA particularly at ratios of V nigrum L RPA of 8 1and 1 1 UPLC-Q-TOFMS with automated data analysisused to identify and quantify the specific chemical compo-nents According to Pearsonrsquos correlation coefficient most of

the chemical components that were positively correlated withtoxicity were from V nigrum L We conclude that the majorveratrum alkaloids of V nigrum L caused acute toxicity anddeath in mice When the doses of V nigrum L were higherthan or equal to RPA the main chemical components of Vnigrum L and the toxicity of the decoctions were increasedThis suggests that V nigrum L and RPA have opposingroles Additional experiments revealed that the cytotoxicityof veratridine in SH-SY5Y cells was decreasedwith increasingconcentrations of paeoniflorin Therefore the clinical use ofthese agents should be considered carefully This study alsoprovides information to help improve the incompatibilitytheory of TCM and introduces novel ideas for further studieson the development and application of TCM

When preparing V nigrum L and RPA decoctions alower amount of RPA contributes to the dissolution of vera-trum alkaloids Conversely higher amounts of RPA can exertneuroprotective effects which can offset the toxicity causedby the combination of V nigrum L and RPA Under theseconditions dissolution is the most important factor for Vnigrum L toxicity The results of cell-based experiments alsodemonstrated that paeoniflorin could offset the neurotoxiceffects of veratridine when treated in combination whichexcluded the factor of dissolution in preparation

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors are grateful for financial support from NationalNature Science Foundation of China (Grant no 81073149)National Basic Research Program of China (Grant nos2011CB505304 and 2012CB518402) and Natural ScienceFoundation of Beijing (Grant no 7112110)

References

[1] H Guo W Li X Y Wang and G W Fan ldquoAdvance of researchon toxic attenuation by compatibility of traditional Chinesemedicine prescriptionsrdquo Zhongguo Zhongyao Zazhi vol 37 no1 pp 120ndash123 2012

[2] Y Zhang H Hua X Fan C Wang and J Duan ldquoExplorationon eighteen incompatible medicaments of chest pain prescrip-tions based on association rules miningrdquo Zhongguo ZhongyaoZazhi vol 36 no 24 pp 3544ndash3547 2011

[3] C-H Xu P Wang Y Wang et al ldquoPharmacokinetic compar-isons of two different combinations of Shaoyao-Gancao Decoc-tion in rats competing mechanisms between paeoniflorin andglycyrrhetinic acidrdquo Journal of Ethnopharmacology vol 149 no2 pp 443ndash452 2013

[4] D H TianDictionary of Practical Traditional Chinese MedicineII Peoplersquos Medical Beijing China 2002

[5] Y Gaillard and G Pepin ldquoLC-EI-MS determination of ver-atridine and cevadine in two fatal cases of Veratrum albumpoisoningrdquo Journal of Analytical Toxicology vol 25 no 6 pp481ndash485 2001

Evidence-Based Complementary and Alternative Medicine 13

[6] H Li G-Y Gao S-Y Li W-J Zhao Y-T Guo and Z-J Liang ldquoEffects of Veratrum nigrum alkaloids on centralcatecholaminergic neurons of renal hypertensive ratsrdquo ActaPharmacologica Sinica vol 21 no 1 pp 23ndash28 2000

[7] L Wang W Li and Y Liu ldquoHypotensive effect and toxicologyof total alkaloids and veratramine from roots and rhizomesof Veratrum nigrum L in spontaneously hypertensive ratsrdquoPharmazie vol 63 no 8 pp 606ndash610 2008

[8] Y P Jiang Y G Liu and H C Chen ldquoEffect of aqueous extractof Radix Paeoniae Rubra against Carbon Tetrachloride inducedliver fibrosis in ratrdquo Herald of Medicine vol 23 pp 527ndash5292004

[9] D-Y He and S-M Dai ldquoAnti-inflammatory and immunomod-ulatory effects of Paeonia lactiflora Pall a traditional Chineseherbal medicinerdquo Frontiers in Pharmacology 2011

[10] C Feng M Liu X Shi et al ldquoPharmacokinetic properties ofpaeoniflorin albiflorin and oxypaeoniflorin after oral gavage ofextracts of Radix Paeoniae Rubra and Radix Paeoniae Alba inratsrdquo Journal of Ethnopharmacology vol 130 no 2 pp 407ndash4132010

[11] X X Zhang Y G Wang Z C Ma Y Gao and C R XiaoldquoUniform designed research on the compatibility toxicity ofVeratridine and peonyrdquo Zhong Hua Zhong Yi Yao Za Zhi vol28 no 10 pp 2901ndash2904 2013

[12] Q D Liang Y Gao J Ma et al ldquoChemical comparison of driedrehmannia root and prepared rehmannia root by UPLC-TOFMS andHPLC-ELSDwithmultivariate statistical analysisrdquoActaPharmaceutica Sinica B vol 3 no 1 pp 55ndash64 2013

[13] Y Wang S He X Cheng Y Lu Y Zou and Q Zhang ldquoUPLC-Q-TOF-MSMS fingerprinting of traditional Chinese formulaSiJunZiTangrdquo Journal of Pharmaceutical and Biomedical Analy-sis vol 80 pp 24ndash33 2013

[14] L-P Kang K Yu Y Zhao et al ldquoCharacterization of steroidalglycosides from the extract of Paris Polyphylla var Yunnanensisby UPLCQ-TOFMSErdquo Journal of Pharmaceutical and Biomed-ical Analysis vol 62 pp 235ndash249 2012

[15] A Kaufmann P Butcher K Maden and M Widmer ldquoUltra-performance liquid chromatography coupled to time of flightmass spectrometry (UPLC-TOF) a novel tool for multiresiduescreening of veterinary drugs in urinerdquoAnalytica Chimica Actavol 586 no 1-2 pp 13ndash21 2007

[16] S Zhou Z C Ma Q D Liang et al ldquoUPLCQ-TOF-MS basedchemical profiling approach to evaluate chemical compositionof augmentation toxicity in combination of Radix aconiti andPinellia praeparatardquoActa Chimica Sinica vol 70 no 3 pp 284ndash290 2012

[17] N Lin and X Gao ldquoExperimental observation on the absorp-tion of Veratrum nigrum L in the upper digestive tractrdquoZhongguo Zhong Yao Za Zhi vol 17 no 1 pp 43ndash45 1992

[18] Z-Z Wang W-J Zhao X-S Zhang et al ldquoProtection ofVeratrum nigrum L var ussuriense Nakai alkaloids againstischema-reperfusion injury of the rat liverrdquo World Journal ofGastroenterology vol 13 no 4 pp 564ndash571 2007

[19] V Christov B Mikhova A Ivanova et al ldquoSteroidal alka-loids of Veratrum lobelianum Bernh and Veratrum nigrum LrdquoZeitschrift fur Naturforschung C Journal of Biosciences vol 65no 3-4 pp 195ndash200 2010

[20] V Christov BMikhova andD Selenge ldquo(-)-Veranigrine a newsteroidal alkaloid fromVeratrum nigrum Lrdquo Fitoterapia vol 80no 1 pp 25ndash27 2009

[21] Y Cong J-H Wang R Wang Y-M Zeng C-D Liu and XLi ldquoA study on the chemical constituents of Veratrum nigrum

L processed by rice vinegarrdquo Journal of Asian Natural ProductsResearch vol 10 no 7-8 pp 619ndash624 2008

[22] H-L Li J Tang R-H Liu C Zhang and W-D Zhang ldquoTwonew flavanone glycosides from Veratrum nigrum Lrdquo NaturalProduct Research vol 23 no 2 pp 122ndash126 2009

[23] Y Cong Y-B Zhou J Chen Y-M Zeng and J-H WangldquoAlkaloid profiling of crude and processed Veratrum nigrum Lthrough simultaneous determination of ten steroidal alkaloidsby HPLC-ELSDrdquo Journal of Pharmaceutical and BiomedicalAnalysis vol 48 no 3 pp 573ndash578 2008

[24] L-S Wang D-Q Zhao H-M Tao and Y-H Liu ldquoDetermi-nation of vetatramine in Veratrum nigrum by HPLC-ELSDrdquoZhongguo Zhongyao Zazhi vol 33 no 7 pp 791ndash792 2008

[25] Q Wang H-Z Guo C-H Huo et al ldquoChemical constituentsin root of Paeonia lactiflorardquo Chinese Traditional and HerbalDrugs vol 38 no 7 pp 972ndash976 2007

[26] J-J TanQ-C Zhao L Yang Z-P Shang Z-QDu andMYanldquoChemical constituents in roots of Paeonia lactiflorardquo ChineseTraditional and Herbal Drugs vol 41 no 8 pp 1245ndash1248 2010

[27] Q Zhang Z Z Wang W Xiao et al ldquoUPLC characteristicchromatographic profile of Paeoniae Radix Albardquo ZhongguoZhongyao Zazhi vol 36 no 6 pp 712ndash714 2011

[28] X R Gao and Y Tian ldquoActive principles of Paeonia lactifloraPallrdquo Chinese Journal of New Drugs vol 15 pp 416ndash418 2006

[29] N-C Luo W Ding J Wu et al ldquoUPLC-Q-TOFMS coupledwith multivariate statistical analysis as a powerful techniquefor rapidly exploring potential chemicalmarkers to differentiatebetween radix paeoniae alba and radix paeoniae rubrardquoNaturalProduct Communications vol 8 no 4 pp 487ndash491 2013

[30] W K Zhao Y Guo T Yasuhiro and K Tohru ldquoIsolation andstructure determination of echinuline from Veratrum nigrumL var ussuriense Nakairdquo Zhongguo Zhong Yao Za Zhi vol 16no 7 pp 425ndash426 1991

[31] W Zhao Y Guo T Yasuhirb and T Kikuchi ldquoIsolation andstructure determination of aurantiamide acetate fromVeratrumnigrum Lvarussuriense Nakairdquo Zhongguo Zhongyao Zazhi vol23 no 1 p 41 1998

[32] L Yang X H Xia Q Zhu and X P Tan ldquoStudy on therule of influence of two purification methods on the chemicalcompositions in aqueous solution of paeoniae radix albardquoZhong Yao Cai vol 36 pp 118ndash121 2012

[33] X Y Zang and X Li ldquoDevelopment of the study on chemicalconstituentsof Paeonia lact if lora Pallrdquo Journal of ShenyangPharmaceutical University vol 19 pp 70ndash73 2002

[34] Y-L Wang Y-G Wang Q-D Liang et al ldquoUsing cDNAmicroarray to screen differently expressed genes in SH-SY5Ytreated by veratridinerdquo Chinese Pharmacological Bulletin vol29 no 5 pp 643ndash647 2013

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

10 Evidence-Based Complementary and Alternative Medicine

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

1

2

3

4

5

6 Tetragalloyglucose

Ion

inte

nsity

A B C D E F G H I J K LSample group

0020406081

121416182 Vanillic acid

Ion

inte

nsity

A B C D E F G H I J K LSample group

minus2

0

2

4

6

8

10

12 Benzoylpaeoniflorin

000501015020250303504

A B C D E F G H I J K L

Ion

inte

nsity

Sample groupminus005

Paeoniflorin sulfonate

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

05

1

15

2 Benzoyloxypaeoniflorin

minus05

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

05

1

15

2 Gallic acid

minus05

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

05

1

15

2

253 Galloylpaeoniflorin

minus05

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

005

01

015

02

025

03

035 Paeonilacto-neC

Ion

inte

nsity

A B C D E F G H I J K LSample group

0123456789 Paeonilacto-neB

minus1

Ion

inte

nsity

A B C D E F G H I J K LSample group

Vanillic acid

0

1

2

3

4

5

6

7

minus1

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

01

02

03

04

05

06

07

minus01

6-O-120573-D-Glucopyranosyllactinolide

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

05

1

15

2

25

3

35

minus05

Paeoniflorinalbiflorin R1albiflorinmudanpioside I

A B C D E F G H I J K L

Ion

inte

nsity

Sample group

Paeonol

0

02

04

06

08

1

12

minus02

Ion

inte

nsity

A B C D E F G H I J K LSample group

01020304050607080 Pentagalloylglucose

minus10

Ion

inte

nsity

A B C D E F G H I J K LSample group

00102030405060708 Lactiflorin

minus01

Ion

inte

nsity

A BC D E F G H I J K LSample group

0020406081

121416 Isomaltopaeoniflorin

minus02

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

05

1

15

2

25

3

minus05

Albiflorin

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

05

1

15

2

25 1998400-O-Benzoylsucrose

(a)

Figure 7 Continued

Evidence-Based Complementary and Alternative Medicine 11

Ion

inte

nsity

Ion

inte

nsity

Ion

inte

nsity

A B C D E F G H I J K LSample group A B C D E F G H I J K L

Sample group

A B C D E F G H I J K LSample group

00102030405060708 Oxypaeoniflorin

001020304050607 Eugeniin

Ion

inte

nsity

A B C D E F G H I J K LSample group

0020406081

12141618 Paeonia lactinone

00102030405060708091 Ethyl gallate

minus01

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

01

02

03

04

05

Lactinolide06

minus01

0

5

10

15

20

25 Palbinone

Ion

inte

nsity

A B C D E F G H I J K LSample group

minus5

minus01

(b)

Figure 7 Ion intensity trend plots for the chemical components that were highly negatively correlated with toxicity (119903 ge 09)

OD

val

ue

25 50 100 200 400 800

lowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

mdashVeratridine (120583M)

10

12

08

06

04

02

0

(a)

OD

val

ue

02

04

06

08

12

Paeoniflorin (120583M) 100 500 1500 2000mdash 1000

10

00

(b)

OD

val

ue

Paeoniflorin (120583M)

mdashmdash

Veratridine (120583M) 400 400 400 400 400 400 400 400 400 400

50 100 250 500 750 1000 1250 1500 1750 2000

02

04

06

08

12

10

00

lowastlowastlowast

lowastlowast

lowastlowast lowastlowast

lowastlowast

lowast

(c)

Figure 8 SH-SY5Y cell viability SH-SY5Y cells were cultured with (a) veratridine (25ndash800120583M) (b) paeoniflorin (50minus2000120583M) and (c)400 120583M veratridine in combination with 50minus2000120583M paeoniflorin for 24 h Cell viability was then assessed using MTS assays Data arepresented as mean plusmn SD Means of three independent experiments Asterisks indicate a statistically significant difference compared withuntreated cells lowast119875 lt 005 lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001 versus controL

12 Evidence-Based Complementary and Alternative Medicine

V nigrum L were equal to or higher than RPA particularlyratios of 8 1 and 1 1 mouse mortality increased significantlyTherefore components of V nigrum L were the majorcausative factors of toxicity In addition RPA modulated thetoxicity ofV nigrum LTherefore clinical applications shouldbe monitored carefully

Mice died 5minus10min after drug administration Toxicitymanifested predominantly as trembling convulsions andspasms but the major organs showed no obvious lesionsat necropsy by the naked eye Based on the toxic reactionsand the time of death in the current study and accord-ing to the guidelines for Chinese natural medicine acutetoxicity testing we speculate that the toxicity was causedpredominantly by suppression of the central nervous systemand that V nigrum L was the major cause of death Vnigrum L water extracts exert toxic effects on the digestivetract mucosa nucleus nervi vagi and central nervous system[6 17 18] Pharmacological studies of veratrum alkaloidshave been performed previously and reported that thesealkaloids stimulate the central nervous system and inhibit thebrain This results in spasms convulsions coma drowsinessand other symptoms of consciousness in animals which isconsistent with the symptoms of themice in the current study[6 17]

Veratrum alkaloid is the toxic component of V nigrum LWe previously investigated the differently expressed genes inSH-SY5Y cells treated with veratridine using cDNAmicroar-rays [34] Data revealed that veratridine treatment alteredthe expression of MMP caused Ca2+ concentration over-load increased reactive oxygen species production enhancedLDH release damaged the cell membrane reduced SH-SY5Y cell viability and induced apoptosis Under oxidizingconditions the MAPK signaling pathway is activated whichincreases apoptosisThis study illustrates the neurotoxicity ofthe combination of V nigrum L and reveals the molecularmechanism behind the neurotoxic effects of V nigrum L(data not shown)

The amount of V nigrum L in each group was the samethe dose of RPA is in ascending order and the chemicalcomponents of RPA are also increased but the mortalityof each group changed significantly with the increase ofRPA so we conclude that higher amount of RPA can haveprotection effect which can offset the toxicity of combinationof V nigrum L and RPA And pharmacological studies havedemonstrated that RPA exerts a wide range of pharmacolog-ical activities including analgesic sedative anticonvulsantand antispasmodic effects on the central nervous systemRPA could also inhibit writhing reactions and antagonizepentylenetetrazol-induced convulsions The most importantactive ingredient in RPA is paeoniflorin which has significantneuroprotective effects [26ndash34]

In this study mouse acute toxicity experiments revealedthat the mortality of mice changed significantly when thedose of V nigrum L was higher than or equal to that ofRPA particularly at ratios of V nigrum L RPA of 8 1and 1 1 UPLC-Q-TOFMS with automated data analysisused to identify and quantify the specific chemical compo-nents According to Pearsonrsquos correlation coefficient most of

the chemical components that were positively correlated withtoxicity were from V nigrum L We conclude that the majorveratrum alkaloids of V nigrum L caused acute toxicity anddeath in mice When the doses of V nigrum L were higherthan or equal to RPA the main chemical components of Vnigrum L and the toxicity of the decoctions were increasedThis suggests that V nigrum L and RPA have opposingroles Additional experiments revealed that the cytotoxicityof veratridine in SH-SY5Y cells was decreasedwith increasingconcentrations of paeoniflorin Therefore the clinical use ofthese agents should be considered carefully This study alsoprovides information to help improve the incompatibilitytheory of TCM and introduces novel ideas for further studieson the development and application of TCM

When preparing V nigrum L and RPA decoctions alower amount of RPA contributes to the dissolution of vera-trum alkaloids Conversely higher amounts of RPA can exertneuroprotective effects which can offset the toxicity causedby the combination of V nigrum L and RPA Under theseconditions dissolution is the most important factor for Vnigrum L toxicity The results of cell-based experiments alsodemonstrated that paeoniflorin could offset the neurotoxiceffects of veratridine when treated in combination whichexcluded the factor of dissolution in preparation

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors are grateful for financial support from NationalNature Science Foundation of China (Grant no 81073149)National Basic Research Program of China (Grant nos2011CB505304 and 2012CB518402) and Natural ScienceFoundation of Beijing (Grant no 7112110)

References

[1] H Guo W Li X Y Wang and G W Fan ldquoAdvance of researchon toxic attenuation by compatibility of traditional Chinesemedicine prescriptionsrdquo Zhongguo Zhongyao Zazhi vol 37 no1 pp 120ndash123 2012

[2] Y Zhang H Hua X Fan C Wang and J Duan ldquoExplorationon eighteen incompatible medicaments of chest pain prescrip-tions based on association rules miningrdquo Zhongguo ZhongyaoZazhi vol 36 no 24 pp 3544ndash3547 2011

[3] C-H Xu P Wang Y Wang et al ldquoPharmacokinetic compar-isons of two different combinations of Shaoyao-Gancao Decoc-tion in rats competing mechanisms between paeoniflorin andglycyrrhetinic acidrdquo Journal of Ethnopharmacology vol 149 no2 pp 443ndash452 2013

[4] D H TianDictionary of Practical Traditional Chinese MedicineII Peoplersquos Medical Beijing China 2002

[5] Y Gaillard and G Pepin ldquoLC-EI-MS determination of ver-atridine and cevadine in two fatal cases of Veratrum albumpoisoningrdquo Journal of Analytical Toxicology vol 25 no 6 pp481ndash485 2001

Evidence-Based Complementary and Alternative Medicine 13

[6] H Li G-Y Gao S-Y Li W-J Zhao Y-T Guo and Z-J Liang ldquoEffects of Veratrum nigrum alkaloids on centralcatecholaminergic neurons of renal hypertensive ratsrdquo ActaPharmacologica Sinica vol 21 no 1 pp 23ndash28 2000

[7] L Wang W Li and Y Liu ldquoHypotensive effect and toxicologyof total alkaloids and veratramine from roots and rhizomesof Veratrum nigrum L in spontaneously hypertensive ratsrdquoPharmazie vol 63 no 8 pp 606ndash610 2008

[8] Y P Jiang Y G Liu and H C Chen ldquoEffect of aqueous extractof Radix Paeoniae Rubra against Carbon Tetrachloride inducedliver fibrosis in ratrdquo Herald of Medicine vol 23 pp 527ndash5292004

[9] D-Y He and S-M Dai ldquoAnti-inflammatory and immunomod-ulatory effects of Paeonia lactiflora Pall a traditional Chineseherbal medicinerdquo Frontiers in Pharmacology 2011

[10] C Feng M Liu X Shi et al ldquoPharmacokinetic properties ofpaeoniflorin albiflorin and oxypaeoniflorin after oral gavage ofextracts of Radix Paeoniae Rubra and Radix Paeoniae Alba inratsrdquo Journal of Ethnopharmacology vol 130 no 2 pp 407ndash4132010

[11] X X Zhang Y G Wang Z C Ma Y Gao and C R XiaoldquoUniform designed research on the compatibility toxicity ofVeratridine and peonyrdquo Zhong Hua Zhong Yi Yao Za Zhi vol28 no 10 pp 2901ndash2904 2013

[12] Q D Liang Y Gao J Ma et al ldquoChemical comparison of driedrehmannia root and prepared rehmannia root by UPLC-TOFMS andHPLC-ELSDwithmultivariate statistical analysisrdquoActaPharmaceutica Sinica B vol 3 no 1 pp 55ndash64 2013

[13] Y Wang S He X Cheng Y Lu Y Zou and Q Zhang ldquoUPLC-Q-TOF-MSMS fingerprinting of traditional Chinese formulaSiJunZiTangrdquo Journal of Pharmaceutical and Biomedical Analy-sis vol 80 pp 24ndash33 2013

[14] L-P Kang K Yu Y Zhao et al ldquoCharacterization of steroidalglycosides from the extract of Paris Polyphylla var Yunnanensisby UPLCQ-TOFMSErdquo Journal of Pharmaceutical and Biomed-ical Analysis vol 62 pp 235ndash249 2012

[15] A Kaufmann P Butcher K Maden and M Widmer ldquoUltra-performance liquid chromatography coupled to time of flightmass spectrometry (UPLC-TOF) a novel tool for multiresiduescreening of veterinary drugs in urinerdquoAnalytica Chimica Actavol 586 no 1-2 pp 13ndash21 2007

[16] S Zhou Z C Ma Q D Liang et al ldquoUPLCQ-TOF-MS basedchemical profiling approach to evaluate chemical compositionof augmentation toxicity in combination of Radix aconiti andPinellia praeparatardquoActa Chimica Sinica vol 70 no 3 pp 284ndash290 2012

[17] N Lin and X Gao ldquoExperimental observation on the absorp-tion of Veratrum nigrum L in the upper digestive tractrdquoZhongguo Zhong Yao Za Zhi vol 17 no 1 pp 43ndash45 1992

[18] Z-Z Wang W-J Zhao X-S Zhang et al ldquoProtection ofVeratrum nigrum L var ussuriense Nakai alkaloids againstischema-reperfusion injury of the rat liverrdquo World Journal ofGastroenterology vol 13 no 4 pp 564ndash571 2007

[19] V Christov B Mikhova A Ivanova et al ldquoSteroidal alka-loids of Veratrum lobelianum Bernh and Veratrum nigrum LrdquoZeitschrift fur Naturforschung C Journal of Biosciences vol 65no 3-4 pp 195ndash200 2010

[20] V Christov BMikhova andD Selenge ldquo(-)-Veranigrine a newsteroidal alkaloid fromVeratrum nigrum Lrdquo Fitoterapia vol 80no 1 pp 25ndash27 2009

[21] Y Cong J-H Wang R Wang Y-M Zeng C-D Liu and XLi ldquoA study on the chemical constituents of Veratrum nigrum

L processed by rice vinegarrdquo Journal of Asian Natural ProductsResearch vol 10 no 7-8 pp 619ndash624 2008

[22] H-L Li J Tang R-H Liu C Zhang and W-D Zhang ldquoTwonew flavanone glycosides from Veratrum nigrum Lrdquo NaturalProduct Research vol 23 no 2 pp 122ndash126 2009

[23] Y Cong Y-B Zhou J Chen Y-M Zeng and J-H WangldquoAlkaloid profiling of crude and processed Veratrum nigrum Lthrough simultaneous determination of ten steroidal alkaloidsby HPLC-ELSDrdquo Journal of Pharmaceutical and BiomedicalAnalysis vol 48 no 3 pp 573ndash578 2008

[24] L-S Wang D-Q Zhao H-M Tao and Y-H Liu ldquoDetermi-nation of vetatramine in Veratrum nigrum by HPLC-ELSDrdquoZhongguo Zhongyao Zazhi vol 33 no 7 pp 791ndash792 2008

[25] Q Wang H-Z Guo C-H Huo et al ldquoChemical constituentsin root of Paeonia lactiflorardquo Chinese Traditional and HerbalDrugs vol 38 no 7 pp 972ndash976 2007

[26] J-J TanQ-C Zhao L Yang Z-P Shang Z-QDu andMYanldquoChemical constituents in roots of Paeonia lactiflorardquo ChineseTraditional and Herbal Drugs vol 41 no 8 pp 1245ndash1248 2010

[27] Q Zhang Z Z Wang W Xiao et al ldquoUPLC characteristicchromatographic profile of Paeoniae Radix Albardquo ZhongguoZhongyao Zazhi vol 36 no 6 pp 712ndash714 2011

[28] X R Gao and Y Tian ldquoActive principles of Paeonia lactifloraPallrdquo Chinese Journal of New Drugs vol 15 pp 416ndash418 2006

[29] N-C Luo W Ding J Wu et al ldquoUPLC-Q-TOFMS coupledwith multivariate statistical analysis as a powerful techniquefor rapidly exploring potential chemicalmarkers to differentiatebetween radix paeoniae alba and radix paeoniae rubrardquoNaturalProduct Communications vol 8 no 4 pp 487ndash491 2013

[30] W K Zhao Y Guo T Yasuhiro and K Tohru ldquoIsolation andstructure determination of echinuline from Veratrum nigrumL var ussuriense Nakairdquo Zhongguo Zhong Yao Za Zhi vol 16no 7 pp 425ndash426 1991

[31] W Zhao Y Guo T Yasuhirb and T Kikuchi ldquoIsolation andstructure determination of aurantiamide acetate fromVeratrumnigrum Lvarussuriense Nakairdquo Zhongguo Zhongyao Zazhi vol23 no 1 p 41 1998

[32] L Yang X H Xia Q Zhu and X P Tan ldquoStudy on therule of influence of two purification methods on the chemicalcompositions in aqueous solution of paeoniae radix albardquoZhong Yao Cai vol 36 pp 118ndash121 2012

[33] X Y Zang and X Li ldquoDevelopment of the study on chemicalconstituentsof Paeonia lact if lora Pallrdquo Journal of ShenyangPharmaceutical University vol 19 pp 70ndash73 2002

[34] Y-L Wang Y-G Wang Q-D Liang et al ldquoUsing cDNAmicroarray to screen differently expressed genes in SH-SY5Ytreated by veratridinerdquo Chinese Pharmacological Bulletin vol29 no 5 pp 643ndash647 2013

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Evidence-Based Complementary and Alternative Medicine 11

Ion

inte

nsity

Ion

inte

nsity

Ion

inte

nsity

A B C D E F G H I J K LSample group A B C D E F G H I J K L

Sample group

A B C D E F G H I J K LSample group

00102030405060708 Oxypaeoniflorin

001020304050607 Eugeniin

Ion

inte

nsity

A B C D E F G H I J K LSample group

0020406081

12141618 Paeonia lactinone

00102030405060708091 Ethyl gallate

minus01

Ion

inte

nsity

A B C D E F G H I J K LSample group

0

01

02

03

04

05

Lactinolide06

minus01

0

5

10

15

20

25 Palbinone

Ion

inte

nsity

A B C D E F G H I J K LSample group

minus5

minus01

(b)

Figure 7 Ion intensity trend plots for the chemical components that were highly negatively correlated with toxicity (119903 ge 09)

OD

val

ue

25 50 100 200 400 800

lowast

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

mdashVeratridine (120583M)

10

12

08

06

04

02

0

(a)

OD

val

ue

02

04

06

08

12

Paeoniflorin (120583M) 100 500 1500 2000mdash 1000

10

00

(b)

OD

val

ue

Paeoniflorin (120583M)

mdashmdash

Veratridine (120583M) 400 400 400 400 400 400 400 400 400 400

50 100 250 500 750 1000 1250 1500 1750 2000

02

04

06

08

12

10

00

lowastlowastlowast

lowastlowast

lowastlowast lowastlowast

lowastlowast

lowast

(c)

Figure 8 SH-SY5Y cell viability SH-SY5Y cells were cultured with (a) veratridine (25ndash800120583M) (b) paeoniflorin (50minus2000120583M) and (c)400 120583M veratridine in combination with 50minus2000120583M paeoniflorin for 24 h Cell viability was then assessed using MTS assays Data arepresented as mean plusmn SD Means of three independent experiments Asterisks indicate a statistically significant difference compared withuntreated cells lowast119875 lt 005 lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001 versus controL

12 Evidence-Based Complementary and Alternative Medicine

V nigrum L were equal to or higher than RPA particularlyratios of 8 1 and 1 1 mouse mortality increased significantlyTherefore components of V nigrum L were the majorcausative factors of toxicity In addition RPA modulated thetoxicity ofV nigrum LTherefore clinical applications shouldbe monitored carefully

Mice died 5minus10min after drug administration Toxicitymanifested predominantly as trembling convulsions andspasms but the major organs showed no obvious lesionsat necropsy by the naked eye Based on the toxic reactionsand the time of death in the current study and accord-ing to the guidelines for Chinese natural medicine acutetoxicity testing we speculate that the toxicity was causedpredominantly by suppression of the central nervous systemand that V nigrum L was the major cause of death Vnigrum L water extracts exert toxic effects on the digestivetract mucosa nucleus nervi vagi and central nervous system[6 17 18] Pharmacological studies of veratrum alkaloidshave been performed previously and reported that thesealkaloids stimulate the central nervous system and inhibit thebrain This results in spasms convulsions coma drowsinessand other symptoms of consciousness in animals which isconsistent with the symptoms of themice in the current study[6 17]

Veratrum alkaloid is the toxic component of V nigrum LWe previously investigated the differently expressed genes inSH-SY5Y cells treated with veratridine using cDNAmicroar-rays [34] Data revealed that veratridine treatment alteredthe expression of MMP caused Ca2+ concentration over-load increased reactive oxygen species production enhancedLDH release damaged the cell membrane reduced SH-SY5Y cell viability and induced apoptosis Under oxidizingconditions the MAPK signaling pathway is activated whichincreases apoptosisThis study illustrates the neurotoxicity ofthe combination of V nigrum L and reveals the molecularmechanism behind the neurotoxic effects of V nigrum L(data not shown)

The amount of V nigrum L in each group was the samethe dose of RPA is in ascending order and the chemicalcomponents of RPA are also increased but the mortalityof each group changed significantly with the increase ofRPA so we conclude that higher amount of RPA can haveprotection effect which can offset the toxicity of combinationof V nigrum L and RPA And pharmacological studies havedemonstrated that RPA exerts a wide range of pharmacolog-ical activities including analgesic sedative anticonvulsantand antispasmodic effects on the central nervous systemRPA could also inhibit writhing reactions and antagonizepentylenetetrazol-induced convulsions The most importantactive ingredient in RPA is paeoniflorin which has significantneuroprotective effects [26ndash34]

In this study mouse acute toxicity experiments revealedthat the mortality of mice changed significantly when thedose of V nigrum L was higher than or equal to that ofRPA particularly at ratios of V nigrum L RPA of 8 1and 1 1 UPLC-Q-TOFMS with automated data analysisused to identify and quantify the specific chemical compo-nents According to Pearsonrsquos correlation coefficient most of

the chemical components that were positively correlated withtoxicity were from V nigrum L We conclude that the majorveratrum alkaloids of V nigrum L caused acute toxicity anddeath in mice When the doses of V nigrum L were higherthan or equal to RPA the main chemical components of Vnigrum L and the toxicity of the decoctions were increasedThis suggests that V nigrum L and RPA have opposingroles Additional experiments revealed that the cytotoxicityof veratridine in SH-SY5Y cells was decreasedwith increasingconcentrations of paeoniflorin Therefore the clinical use ofthese agents should be considered carefully This study alsoprovides information to help improve the incompatibilitytheory of TCM and introduces novel ideas for further studieson the development and application of TCM

When preparing V nigrum L and RPA decoctions alower amount of RPA contributes to the dissolution of vera-trum alkaloids Conversely higher amounts of RPA can exertneuroprotective effects which can offset the toxicity causedby the combination of V nigrum L and RPA Under theseconditions dissolution is the most important factor for Vnigrum L toxicity The results of cell-based experiments alsodemonstrated that paeoniflorin could offset the neurotoxiceffects of veratridine when treated in combination whichexcluded the factor of dissolution in preparation

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors are grateful for financial support from NationalNature Science Foundation of China (Grant no 81073149)National Basic Research Program of China (Grant nos2011CB505304 and 2012CB518402) and Natural ScienceFoundation of Beijing (Grant no 7112110)

References

[1] H Guo W Li X Y Wang and G W Fan ldquoAdvance of researchon toxic attenuation by compatibility of traditional Chinesemedicine prescriptionsrdquo Zhongguo Zhongyao Zazhi vol 37 no1 pp 120ndash123 2012

[2] Y Zhang H Hua X Fan C Wang and J Duan ldquoExplorationon eighteen incompatible medicaments of chest pain prescrip-tions based on association rules miningrdquo Zhongguo ZhongyaoZazhi vol 36 no 24 pp 3544ndash3547 2011

[3] C-H Xu P Wang Y Wang et al ldquoPharmacokinetic compar-isons of two different combinations of Shaoyao-Gancao Decoc-tion in rats competing mechanisms between paeoniflorin andglycyrrhetinic acidrdquo Journal of Ethnopharmacology vol 149 no2 pp 443ndash452 2013

[4] D H TianDictionary of Practical Traditional Chinese MedicineII Peoplersquos Medical Beijing China 2002

[5] Y Gaillard and G Pepin ldquoLC-EI-MS determination of ver-atridine and cevadine in two fatal cases of Veratrum albumpoisoningrdquo Journal of Analytical Toxicology vol 25 no 6 pp481ndash485 2001

Evidence-Based Complementary and Alternative Medicine 13

[6] H Li G-Y Gao S-Y Li W-J Zhao Y-T Guo and Z-J Liang ldquoEffects of Veratrum nigrum alkaloids on centralcatecholaminergic neurons of renal hypertensive ratsrdquo ActaPharmacologica Sinica vol 21 no 1 pp 23ndash28 2000

[7] L Wang W Li and Y Liu ldquoHypotensive effect and toxicologyof total alkaloids and veratramine from roots and rhizomesof Veratrum nigrum L in spontaneously hypertensive ratsrdquoPharmazie vol 63 no 8 pp 606ndash610 2008

[8] Y P Jiang Y G Liu and H C Chen ldquoEffect of aqueous extractof Radix Paeoniae Rubra against Carbon Tetrachloride inducedliver fibrosis in ratrdquo Herald of Medicine vol 23 pp 527ndash5292004

[9] D-Y He and S-M Dai ldquoAnti-inflammatory and immunomod-ulatory effects of Paeonia lactiflora Pall a traditional Chineseherbal medicinerdquo Frontiers in Pharmacology 2011

[10] C Feng M Liu X Shi et al ldquoPharmacokinetic properties ofpaeoniflorin albiflorin and oxypaeoniflorin after oral gavage ofextracts of Radix Paeoniae Rubra and Radix Paeoniae Alba inratsrdquo Journal of Ethnopharmacology vol 130 no 2 pp 407ndash4132010

[11] X X Zhang Y G Wang Z C Ma Y Gao and C R XiaoldquoUniform designed research on the compatibility toxicity ofVeratridine and peonyrdquo Zhong Hua Zhong Yi Yao Za Zhi vol28 no 10 pp 2901ndash2904 2013

[12] Q D Liang Y Gao J Ma et al ldquoChemical comparison of driedrehmannia root and prepared rehmannia root by UPLC-TOFMS andHPLC-ELSDwithmultivariate statistical analysisrdquoActaPharmaceutica Sinica B vol 3 no 1 pp 55ndash64 2013

[13] Y Wang S He X Cheng Y Lu Y Zou and Q Zhang ldquoUPLC-Q-TOF-MSMS fingerprinting of traditional Chinese formulaSiJunZiTangrdquo Journal of Pharmaceutical and Biomedical Analy-sis vol 80 pp 24ndash33 2013

[14] L-P Kang K Yu Y Zhao et al ldquoCharacterization of steroidalglycosides from the extract of Paris Polyphylla var Yunnanensisby UPLCQ-TOFMSErdquo Journal of Pharmaceutical and Biomed-ical Analysis vol 62 pp 235ndash249 2012

[15] A Kaufmann P Butcher K Maden and M Widmer ldquoUltra-performance liquid chromatography coupled to time of flightmass spectrometry (UPLC-TOF) a novel tool for multiresiduescreening of veterinary drugs in urinerdquoAnalytica Chimica Actavol 586 no 1-2 pp 13ndash21 2007

[16] S Zhou Z C Ma Q D Liang et al ldquoUPLCQ-TOF-MS basedchemical profiling approach to evaluate chemical compositionof augmentation toxicity in combination of Radix aconiti andPinellia praeparatardquoActa Chimica Sinica vol 70 no 3 pp 284ndash290 2012

[17] N Lin and X Gao ldquoExperimental observation on the absorp-tion of Veratrum nigrum L in the upper digestive tractrdquoZhongguo Zhong Yao Za Zhi vol 17 no 1 pp 43ndash45 1992

[18] Z-Z Wang W-J Zhao X-S Zhang et al ldquoProtection ofVeratrum nigrum L var ussuriense Nakai alkaloids againstischema-reperfusion injury of the rat liverrdquo World Journal ofGastroenterology vol 13 no 4 pp 564ndash571 2007

[19] V Christov B Mikhova A Ivanova et al ldquoSteroidal alka-loids of Veratrum lobelianum Bernh and Veratrum nigrum LrdquoZeitschrift fur Naturforschung C Journal of Biosciences vol 65no 3-4 pp 195ndash200 2010

[20] V Christov BMikhova andD Selenge ldquo(-)-Veranigrine a newsteroidal alkaloid fromVeratrum nigrum Lrdquo Fitoterapia vol 80no 1 pp 25ndash27 2009

[21] Y Cong J-H Wang R Wang Y-M Zeng C-D Liu and XLi ldquoA study on the chemical constituents of Veratrum nigrum

L processed by rice vinegarrdquo Journal of Asian Natural ProductsResearch vol 10 no 7-8 pp 619ndash624 2008

[22] H-L Li J Tang R-H Liu C Zhang and W-D Zhang ldquoTwonew flavanone glycosides from Veratrum nigrum Lrdquo NaturalProduct Research vol 23 no 2 pp 122ndash126 2009

[23] Y Cong Y-B Zhou J Chen Y-M Zeng and J-H WangldquoAlkaloid profiling of crude and processed Veratrum nigrum Lthrough simultaneous determination of ten steroidal alkaloidsby HPLC-ELSDrdquo Journal of Pharmaceutical and BiomedicalAnalysis vol 48 no 3 pp 573ndash578 2008

[24] L-S Wang D-Q Zhao H-M Tao and Y-H Liu ldquoDetermi-nation of vetatramine in Veratrum nigrum by HPLC-ELSDrdquoZhongguo Zhongyao Zazhi vol 33 no 7 pp 791ndash792 2008

[25] Q Wang H-Z Guo C-H Huo et al ldquoChemical constituentsin root of Paeonia lactiflorardquo Chinese Traditional and HerbalDrugs vol 38 no 7 pp 972ndash976 2007

[26] J-J TanQ-C Zhao L Yang Z-P Shang Z-QDu andMYanldquoChemical constituents in roots of Paeonia lactiflorardquo ChineseTraditional and Herbal Drugs vol 41 no 8 pp 1245ndash1248 2010

[27] Q Zhang Z Z Wang W Xiao et al ldquoUPLC characteristicchromatographic profile of Paeoniae Radix Albardquo ZhongguoZhongyao Zazhi vol 36 no 6 pp 712ndash714 2011

[28] X R Gao and Y Tian ldquoActive principles of Paeonia lactifloraPallrdquo Chinese Journal of New Drugs vol 15 pp 416ndash418 2006

[29] N-C Luo W Ding J Wu et al ldquoUPLC-Q-TOFMS coupledwith multivariate statistical analysis as a powerful techniquefor rapidly exploring potential chemicalmarkers to differentiatebetween radix paeoniae alba and radix paeoniae rubrardquoNaturalProduct Communications vol 8 no 4 pp 487ndash491 2013

[30] W K Zhao Y Guo T Yasuhiro and K Tohru ldquoIsolation andstructure determination of echinuline from Veratrum nigrumL var ussuriense Nakairdquo Zhongguo Zhong Yao Za Zhi vol 16no 7 pp 425ndash426 1991

[31] W Zhao Y Guo T Yasuhirb and T Kikuchi ldquoIsolation andstructure determination of aurantiamide acetate fromVeratrumnigrum Lvarussuriense Nakairdquo Zhongguo Zhongyao Zazhi vol23 no 1 p 41 1998

[32] L Yang X H Xia Q Zhu and X P Tan ldquoStudy on therule of influence of two purification methods on the chemicalcompositions in aqueous solution of paeoniae radix albardquoZhong Yao Cai vol 36 pp 118ndash121 2012

[33] X Y Zang and X Li ldquoDevelopment of the study on chemicalconstituentsof Paeonia lact if lora Pallrdquo Journal of ShenyangPharmaceutical University vol 19 pp 70ndash73 2002

[34] Y-L Wang Y-G Wang Q-D Liang et al ldquoUsing cDNAmicroarray to screen differently expressed genes in SH-SY5Ytreated by veratridinerdquo Chinese Pharmacological Bulletin vol29 no 5 pp 643ndash647 2013

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

12 Evidence-Based Complementary and Alternative Medicine

V nigrum L were equal to or higher than RPA particularlyratios of 8 1 and 1 1 mouse mortality increased significantlyTherefore components of V nigrum L were the majorcausative factors of toxicity In addition RPA modulated thetoxicity ofV nigrum LTherefore clinical applications shouldbe monitored carefully

Mice died 5minus10min after drug administration Toxicitymanifested predominantly as trembling convulsions andspasms but the major organs showed no obvious lesionsat necropsy by the naked eye Based on the toxic reactionsand the time of death in the current study and accord-ing to the guidelines for Chinese natural medicine acutetoxicity testing we speculate that the toxicity was causedpredominantly by suppression of the central nervous systemand that V nigrum L was the major cause of death Vnigrum L water extracts exert toxic effects on the digestivetract mucosa nucleus nervi vagi and central nervous system[6 17 18] Pharmacological studies of veratrum alkaloidshave been performed previously and reported that thesealkaloids stimulate the central nervous system and inhibit thebrain This results in spasms convulsions coma drowsinessand other symptoms of consciousness in animals which isconsistent with the symptoms of themice in the current study[6 17]

Veratrum alkaloid is the toxic component of V nigrum LWe previously investigated the differently expressed genes inSH-SY5Y cells treated with veratridine using cDNAmicroar-rays [34] Data revealed that veratridine treatment alteredthe expression of MMP caused Ca2+ concentration over-load increased reactive oxygen species production enhancedLDH release damaged the cell membrane reduced SH-SY5Y cell viability and induced apoptosis Under oxidizingconditions the MAPK signaling pathway is activated whichincreases apoptosisThis study illustrates the neurotoxicity ofthe combination of V nigrum L and reveals the molecularmechanism behind the neurotoxic effects of V nigrum L(data not shown)

The amount of V nigrum L in each group was the samethe dose of RPA is in ascending order and the chemicalcomponents of RPA are also increased but the mortalityof each group changed significantly with the increase ofRPA so we conclude that higher amount of RPA can haveprotection effect which can offset the toxicity of combinationof V nigrum L and RPA And pharmacological studies havedemonstrated that RPA exerts a wide range of pharmacolog-ical activities including analgesic sedative anticonvulsantand antispasmodic effects on the central nervous systemRPA could also inhibit writhing reactions and antagonizepentylenetetrazol-induced convulsions The most importantactive ingredient in RPA is paeoniflorin which has significantneuroprotective effects [26ndash34]

In this study mouse acute toxicity experiments revealedthat the mortality of mice changed significantly when thedose of V nigrum L was higher than or equal to that ofRPA particularly at ratios of V nigrum L RPA of 8 1and 1 1 UPLC-Q-TOFMS with automated data analysisused to identify and quantify the specific chemical compo-nents According to Pearsonrsquos correlation coefficient most of

the chemical components that were positively correlated withtoxicity were from V nigrum L We conclude that the majorveratrum alkaloids of V nigrum L caused acute toxicity anddeath in mice When the doses of V nigrum L were higherthan or equal to RPA the main chemical components of Vnigrum L and the toxicity of the decoctions were increasedThis suggests that V nigrum L and RPA have opposingroles Additional experiments revealed that the cytotoxicityof veratridine in SH-SY5Y cells was decreasedwith increasingconcentrations of paeoniflorin Therefore the clinical use ofthese agents should be considered carefully This study alsoprovides information to help improve the incompatibilitytheory of TCM and introduces novel ideas for further studieson the development and application of TCM

When preparing V nigrum L and RPA decoctions alower amount of RPA contributes to the dissolution of vera-trum alkaloids Conversely higher amounts of RPA can exertneuroprotective effects which can offset the toxicity causedby the combination of V nigrum L and RPA Under theseconditions dissolution is the most important factor for Vnigrum L toxicity The results of cell-based experiments alsodemonstrated that paeoniflorin could offset the neurotoxiceffects of veratridine when treated in combination whichexcluded the factor of dissolution in preparation

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors are grateful for financial support from NationalNature Science Foundation of China (Grant no 81073149)National Basic Research Program of China (Grant nos2011CB505304 and 2012CB518402) and Natural ScienceFoundation of Beijing (Grant no 7112110)

References

[1] H Guo W Li X Y Wang and G W Fan ldquoAdvance of researchon toxic attenuation by compatibility of traditional Chinesemedicine prescriptionsrdquo Zhongguo Zhongyao Zazhi vol 37 no1 pp 120ndash123 2012

[2] Y Zhang H Hua X Fan C Wang and J Duan ldquoExplorationon eighteen incompatible medicaments of chest pain prescrip-tions based on association rules miningrdquo Zhongguo ZhongyaoZazhi vol 36 no 24 pp 3544ndash3547 2011

[3] C-H Xu P Wang Y Wang et al ldquoPharmacokinetic compar-isons of two different combinations of Shaoyao-Gancao Decoc-tion in rats competing mechanisms between paeoniflorin andglycyrrhetinic acidrdquo Journal of Ethnopharmacology vol 149 no2 pp 443ndash452 2013

[4] D H TianDictionary of Practical Traditional Chinese MedicineII Peoplersquos Medical Beijing China 2002

[5] Y Gaillard and G Pepin ldquoLC-EI-MS determination of ver-atridine and cevadine in two fatal cases of Veratrum albumpoisoningrdquo Journal of Analytical Toxicology vol 25 no 6 pp481ndash485 2001

Evidence-Based Complementary and Alternative Medicine 13

[6] H Li G-Y Gao S-Y Li W-J Zhao Y-T Guo and Z-J Liang ldquoEffects of Veratrum nigrum alkaloids on centralcatecholaminergic neurons of renal hypertensive ratsrdquo ActaPharmacologica Sinica vol 21 no 1 pp 23ndash28 2000

[7] L Wang W Li and Y Liu ldquoHypotensive effect and toxicologyof total alkaloids and veratramine from roots and rhizomesof Veratrum nigrum L in spontaneously hypertensive ratsrdquoPharmazie vol 63 no 8 pp 606ndash610 2008

[8] Y P Jiang Y G Liu and H C Chen ldquoEffect of aqueous extractof Radix Paeoniae Rubra against Carbon Tetrachloride inducedliver fibrosis in ratrdquo Herald of Medicine vol 23 pp 527ndash5292004

[9] D-Y He and S-M Dai ldquoAnti-inflammatory and immunomod-ulatory effects of Paeonia lactiflora Pall a traditional Chineseherbal medicinerdquo Frontiers in Pharmacology 2011

[10] C Feng M Liu X Shi et al ldquoPharmacokinetic properties ofpaeoniflorin albiflorin and oxypaeoniflorin after oral gavage ofextracts of Radix Paeoniae Rubra and Radix Paeoniae Alba inratsrdquo Journal of Ethnopharmacology vol 130 no 2 pp 407ndash4132010

[11] X X Zhang Y G Wang Z C Ma Y Gao and C R XiaoldquoUniform designed research on the compatibility toxicity ofVeratridine and peonyrdquo Zhong Hua Zhong Yi Yao Za Zhi vol28 no 10 pp 2901ndash2904 2013

[12] Q D Liang Y Gao J Ma et al ldquoChemical comparison of driedrehmannia root and prepared rehmannia root by UPLC-TOFMS andHPLC-ELSDwithmultivariate statistical analysisrdquoActaPharmaceutica Sinica B vol 3 no 1 pp 55ndash64 2013

[13] Y Wang S He X Cheng Y Lu Y Zou and Q Zhang ldquoUPLC-Q-TOF-MSMS fingerprinting of traditional Chinese formulaSiJunZiTangrdquo Journal of Pharmaceutical and Biomedical Analy-sis vol 80 pp 24ndash33 2013

[14] L-P Kang K Yu Y Zhao et al ldquoCharacterization of steroidalglycosides from the extract of Paris Polyphylla var Yunnanensisby UPLCQ-TOFMSErdquo Journal of Pharmaceutical and Biomed-ical Analysis vol 62 pp 235ndash249 2012

[15] A Kaufmann P Butcher K Maden and M Widmer ldquoUltra-performance liquid chromatography coupled to time of flightmass spectrometry (UPLC-TOF) a novel tool for multiresiduescreening of veterinary drugs in urinerdquoAnalytica Chimica Actavol 586 no 1-2 pp 13ndash21 2007

[16] S Zhou Z C Ma Q D Liang et al ldquoUPLCQ-TOF-MS basedchemical profiling approach to evaluate chemical compositionof augmentation toxicity in combination of Radix aconiti andPinellia praeparatardquoActa Chimica Sinica vol 70 no 3 pp 284ndash290 2012

[17] N Lin and X Gao ldquoExperimental observation on the absorp-tion of Veratrum nigrum L in the upper digestive tractrdquoZhongguo Zhong Yao Za Zhi vol 17 no 1 pp 43ndash45 1992

[18] Z-Z Wang W-J Zhao X-S Zhang et al ldquoProtection ofVeratrum nigrum L var ussuriense Nakai alkaloids againstischema-reperfusion injury of the rat liverrdquo World Journal ofGastroenterology vol 13 no 4 pp 564ndash571 2007

[19] V Christov B Mikhova A Ivanova et al ldquoSteroidal alka-loids of Veratrum lobelianum Bernh and Veratrum nigrum LrdquoZeitschrift fur Naturforschung C Journal of Biosciences vol 65no 3-4 pp 195ndash200 2010

[20] V Christov BMikhova andD Selenge ldquo(-)-Veranigrine a newsteroidal alkaloid fromVeratrum nigrum Lrdquo Fitoterapia vol 80no 1 pp 25ndash27 2009

[21] Y Cong J-H Wang R Wang Y-M Zeng C-D Liu and XLi ldquoA study on the chemical constituents of Veratrum nigrum

L processed by rice vinegarrdquo Journal of Asian Natural ProductsResearch vol 10 no 7-8 pp 619ndash624 2008

[22] H-L Li J Tang R-H Liu C Zhang and W-D Zhang ldquoTwonew flavanone glycosides from Veratrum nigrum Lrdquo NaturalProduct Research vol 23 no 2 pp 122ndash126 2009

[23] Y Cong Y-B Zhou J Chen Y-M Zeng and J-H WangldquoAlkaloid profiling of crude and processed Veratrum nigrum Lthrough simultaneous determination of ten steroidal alkaloidsby HPLC-ELSDrdquo Journal of Pharmaceutical and BiomedicalAnalysis vol 48 no 3 pp 573ndash578 2008

[24] L-S Wang D-Q Zhao H-M Tao and Y-H Liu ldquoDetermi-nation of vetatramine in Veratrum nigrum by HPLC-ELSDrdquoZhongguo Zhongyao Zazhi vol 33 no 7 pp 791ndash792 2008

[25] Q Wang H-Z Guo C-H Huo et al ldquoChemical constituentsin root of Paeonia lactiflorardquo Chinese Traditional and HerbalDrugs vol 38 no 7 pp 972ndash976 2007

[26] J-J TanQ-C Zhao L Yang Z-P Shang Z-QDu andMYanldquoChemical constituents in roots of Paeonia lactiflorardquo ChineseTraditional and Herbal Drugs vol 41 no 8 pp 1245ndash1248 2010

[27] Q Zhang Z Z Wang W Xiao et al ldquoUPLC characteristicchromatographic profile of Paeoniae Radix Albardquo ZhongguoZhongyao Zazhi vol 36 no 6 pp 712ndash714 2011

[28] X R Gao and Y Tian ldquoActive principles of Paeonia lactifloraPallrdquo Chinese Journal of New Drugs vol 15 pp 416ndash418 2006

[29] N-C Luo W Ding J Wu et al ldquoUPLC-Q-TOFMS coupledwith multivariate statistical analysis as a powerful techniquefor rapidly exploring potential chemicalmarkers to differentiatebetween radix paeoniae alba and radix paeoniae rubrardquoNaturalProduct Communications vol 8 no 4 pp 487ndash491 2013

[30] W K Zhao Y Guo T Yasuhiro and K Tohru ldquoIsolation andstructure determination of echinuline from Veratrum nigrumL var ussuriense Nakairdquo Zhongguo Zhong Yao Za Zhi vol 16no 7 pp 425ndash426 1991

[31] W Zhao Y Guo T Yasuhirb and T Kikuchi ldquoIsolation andstructure determination of aurantiamide acetate fromVeratrumnigrum Lvarussuriense Nakairdquo Zhongguo Zhongyao Zazhi vol23 no 1 p 41 1998

[32] L Yang X H Xia Q Zhu and X P Tan ldquoStudy on therule of influence of two purification methods on the chemicalcompositions in aqueous solution of paeoniae radix albardquoZhong Yao Cai vol 36 pp 118ndash121 2012

[33] X Y Zang and X Li ldquoDevelopment of the study on chemicalconstituentsof Paeonia lact if lora Pallrdquo Journal of ShenyangPharmaceutical University vol 19 pp 70ndash73 2002

[34] Y-L Wang Y-G Wang Q-D Liang et al ldquoUsing cDNAmicroarray to screen differently expressed genes in SH-SY5Ytreated by veratridinerdquo Chinese Pharmacological Bulletin vol29 no 5 pp 643ndash647 2013

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Evidence-Based Complementary and Alternative Medicine 13

[6] H Li G-Y Gao S-Y Li W-J Zhao Y-T Guo and Z-J Liang ldquoEffects of Veratrum nigrum alkaloids on centralcatecholaminergic neurons of renal hypertensive ratsrdquo ActaPharmacologica Sinica vol 21 no 1 pp 23ndash28 2000

[7] L Wang W Li and Y Liu ldquoHypotensive effect and toxicologyof total alkaloids and veratramine from roots and rhizomesof Veratrum nigrum L in spontaneously hypertensive ratsrdquoPharmazie vol 63 no 8 pp 606ndash610 2008

[8] Y P Jiang Y G Liu and H C Chen ldquoEffect of aqueous extractof Radix Paeoniae Rubra against Carbon Tetrachloride inducedliver fibrosis in ratrdquo Herald of Medicine vol 23 pp 527ndash5292004

[9] D-Y He and S-M Dai ldquoAnti-inflammatory and immunomod-ulatory effects of Paeonia lactiflora Pall a traditional Chineseherbal medicinerdquo Frontiers in Pharmacology 2011

[10] C Feng M Liu X Shi et al ldquoPharmacokinetic properties ofpaeoniflorin albiflorin and oxypaeoniflorin after oral gavage ofextracts of Radix Paeoniae Rubra and Radix Paeoniae Alba inratsrdquo Journal of Ethnopharmacology vol 130 no 2 pp 407ndash4132010

[11] X X Zhang Y G Wang Z C Ma Y Gao and C R XiaoldquoUniform designed research on the compatibility toxicity ofVeratridine and peonyrdquo Zhong Hua Zhong Yi Yao Za Zhi vol28 no 10 pp 2901ndash2904 2013

[12] Q D Liang Y Gao J Ma et al ldquoChemical comparison of driedrehmannia root and prepared rehmannia root by UPLC-TOFMS andHPLC-ELSDwithmultivariate statistical analysisrdquoActaPharmaceutica Sinica B vol 3 no 1 pp 55ndash64 2013

[13] Y Wang S He X Cheng Y Lu Y Zou and Q Zhang ldquoUPLC-Q-TOF-MSMS fingerprinting of traditional Chinese formulaSiJunZiTangrdquo Journal of Pharmaceutical and Biomedical Analy-sis vol 80 pp 24ndash33 2013

[14] L-P Kang K Yu Y Zhao et al ldquoCharacterization of steroidalglycosides from the extract of Paris Polyphylla var Yunnanensisby UPLCQ-TOFMSErdquo Journal of Pharmaceutical and Biomed-ical Analysis vol 62 pp 235ndash249 2012

[15] A Kaufmann P Butcher K Maden and M Widmer ldquoUltra-performance liquid chromatography coupled to time of flightmass spectrometry (UPLC-TOF) a novel tool for multiresiduescreening of veterinary drugs in urinerdquoAnalytica Chimica Actavol 586 no 1-2 pp 13ndash21 2007

[16] S Zhou Z C Ma Q D Liang et al ldquoUPLCQ-TOF-MS basedchemical profiling approach to evaluate chemical compositionof augmentation toxicity in combination of Radix aconiti andPinellia praeparatardquoActa Chimica Sinica vol 70 no 3 pp 284ndash290 2012

[17] N Lin and X Gao ldquoExperimental observation on the absorp-tion of Veratrum nigrum L in the upper digestive tractrdquoZhongguo Zhong Yao Za Zhi vol 17 no 1 pp 43ndash45 1992

[18] Z-Z Wang W-J Zhao X-S Zhang et al ldquoProtection ofVeratrum nigrum L var ussuriense Nakai alkaloids againstischema-reperfusion injury of the rat liverrdquo World Journal ofGastroenterology vol 13 no 4 pp 564ndash571 2007

[19] V Christov B Mikhova A Ivanova et al ldquoSteroidal alka-loids of Veratrum lobelianum Bernh and Veratrum nigrum LrdquoZeitschrift fur Naturforschung C Journal of Biosciences vol 65no 3-4 pp 195ndash200 2010

[20] V Christov BMikhova andD Selenge ldquo(-)-Veranigrine a newsteroidal alkaloid fromVeratrum nigrum Lrdquo Fitoterapia vol 80no 1 pp 25ndash27 2009

[21] Y Cong J-H Wang R Wang Y-M Zeng C-D Liu and XLi ldquoA study on the chemical constituents of Veratrum nigrum

L processed by rice vinegarrdquo Journal of Asian Natural ProductsResearch vol 10 no 7-8 pp 619ndash624 2008

[22] H-L Li J Tang R-H Liu C Zhang and W-D Zhang ldquoTwonew flavanone glycosides from Veratrum nigrum Lrdquo NaturalProduct Research vol 23 no 2 pp 122ndash126 2009

[23] Y Cong Y-B Zhou J Chen Y-M Zeng and J-H WangldquoAlkaloid profiling of crude and processed Veratrum nigrum Lthrough simultaneous determination of ten steroidal alkaloidsby HPLC-ELSDrdquo Journal of Pharmaceutical and BiomedicalAnalysis vol 48 no 3 pp 573ndash578 2008

[24] L-S Wang D-Q Zhao H-M Tao and Y-H Liu ldquoDetermi-nation of vetatramine in Veratrum nigrum by HPLC-ELSDrdquoZhongguo Zhongyao Zazhi vol 33 no 7 pp 791ndash792 2008

[25] Q Wang H-Z Guo C-H Huo et al ldquoChemical constituentsin root of Paeonia lactiflorardquo Chinese Traditional and HerbalDrugs vol 38 no 7 pp 972ndash976 2007

[26] J-J TanQ-C Zhao L Yang Z-P Shang Z-QDu andMYanldquoChemical constituents in roots of Paeonia lactiflorardquo ChineseTraditional and Herbal Drugs vol 41 no 8 pp 1245ndash1248 2010

[27] Q Zhang Z Z Wang W Xiao et al ldquoUPLC characteristicchromatographic profile of Paeoniae Radix Albardquo ZhongguoZhongyao Zazhi vol 36 no 6 pp 712ndash714 2011

[28] X R Gao and Y Tian ldquoActive principles of Paeonia lactifloraPallrdquo Chinese Journal of New Drugs vol 15 pp 416ndash418 2006

[29] N-C Luo W Ding J Wu et al ldquoUPLC-Q-TOFMS coupledwith multivariate statistical analysis as a powerful techniquefor rapidly exploring potential chemicalmarkers to differentiatebetween radix paeoniae alba and radix paeoniae rubrardquoNaturalProduct Communications vol 8 no 4 pp 487ndash491 2013

[30] W K Zhao Y Guo T Yasuhiro and K Tohru ldquoIsolation andstructure determination of echinuline from Veratrum nigrumL var ussuriense Nakairdquo Zhongguo Zhong Yao Za Zhi vol 16no 7 pp 425ndash426 1991

[31] W Zhao Y Guo T Yasuhirb and T Kikuchi ldquoIsolation andstructure determination of aurantiamide acetate fromVeratrumnigrum Lvarussuriense Nakairdquo Zhongguo Zhongyao Zazhi vol23 no 1 p 41 1998

[32] L Yang X H Xia Q Zhu and X P Tan ldquoStudy on therule of influence of two purification methods on the chemicalcompositions in aqueous solution of paeoniae radix albardquoZhong Yao Cai vol 36 pp 118ndash121 2012

[33] X Y Zang and X Li ldquoDevelopment of the study on chemicalconstituentsof Paeonia lact if lora Pallrdquo Journal of ShenyangPharmaceutical University vol 19 pp 70ndash73 2002

[34] Y-L Wang Y-G Wang Q-D Liang et al ldquoUsing cDNAmicroarray to screen differently expressed genes in SH-SY5Ytreated by veratridinerdquo Chinese Pharmacological Bulletin vol29 no 5 pp 643ndash647 2013

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom