Review Article Genus Caulophyllum : An Overview of

Review ArticleGenus Caulophyllum An Overview of Chemistry and Bioactivity

Yong-Gang Xia1 Guo-Yu Li2 Jun Liang1 Bing-You Yang1

Shao-Wa Luuml1 and Hai-Xue Kuang1

1 Key Laboratory of Chinese Materia Medica Heilongjiang University of Chinese MedicineMinistry of Education Harbin 150040 China

2 Pharmaceutical College Harbin Medical University Harbin 150086 China

Correspondence should be addressed to Hai-Xue Kuang hxkuanghotmailcom

Received 21 January 2014 Accepted 2 April 2014 Published 4 May 2014

Academic Editor Yong Jiang

Copyright copy 2014 Yong-Gang Xia et alThis 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

Recently some promising advances have been achieved in understanding the chemistry pharmacology and action mechanismsof constituents from genus Caulophyllum Despite this there is to date no systematic review of those of genus Caulophyllum Thisreview covers naturally occurring alkaloids and saponins and those resulting from synthetic novel taspine derivatives The paperfurther discussed several aspects of this genus including pharmacological propertiesmechanisms of action pharmacokinetics andcell membrane chromatography for activity screening The aim of this paper is to provide a point of reference for pharmaceuticalresearchers to develop new drugs from constituents of Caulophyllum plants

1 Introduction

Caulophyllum is a small genus of perennial herbs in the familyBerberidaceae The genus Caulophyllum is well known for itsdiversity and pharmacological uses in traditional medicinesystem since ancient times All species in this genus are verysimilar [1] C robustum is native to eastern Asia especially inChina while C thalictroides and C giganteum are native toeastern North America It is worth noting that nearly allphytochemical and pharmacological studies on this genus arefocused on C thalictroides and C robustum due to theirimportant medical functions [2]

The roots and rhizomes of C thalictroides (L) Michx(blue cohosh) have been used traditionally by Native Ameri-cans formedicinal purposes [3]The primary function of bluecohosh inmany native communities of North America was toinduce childbirth ease the pain of labor rectify delayed orirregularmenstruation and alleviate heavy bleeding and painduringmenstruation [4] Between 1882 and 1905 blue cohoshwas listed in theUnited States Pharmacopoeia as a labor indu-cer [5] and sold as an herbal supplement that can aid inchildbirth Dietary supplements of blue cohosh are readilyavailable throughout the USA over-the-counter and fromInternet suppliers [6] There is considerable concern about

the safety of blue cohoshwith reports of new born babies hav-ing heart attacks or strokes after the maternal consumptionof blue cohosh to induce labor [7ndash9] There is a heated dis-cussion about using blue cohosh as dietary supplements forwomen [2]

C robustum Maxim is well-known in Hong Mao Qi inChinese which grows widely throughout north-east north-west and south-west China Its roots and rhizomes have beenused as folk medicine to treat external injuries irregular-menses and stomach-ache due to its strong and wide bio-logical activities [10] Modern pharmacological studies havedemonstrated that alkaloids and triterpence saponins areresponsible for its major biological function as an anti-inflammatory [11] analgesic [12] antioxidant [13] antibacte-rial [11] antiacetylcholinesterase [14] and antitumor [15 16]Taspine a lead compound in anticancer agent development[17 18] was firstly screened to possess obvious effect ontumor angiogenesis and human epidermal growth factor rec-eptor by using cell membrane chromatography from the Crobustum [19]

So it is very necessary to deeply explore Caulophyllumplants In the past decades some promising advances havebeen achieved in understanding the chemistry pharmacol-ogy and action mechanisms of constituents from genus

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2014 Article ID 684508 18 pageshttpdxdoiorg1011552014684508

2 Evidence-Based Complementary and Alternative Medicine

Macroporous or (and) ion exchange resins

Vacuum evaporated

Adjust pH 70

Suspension in

pH 90 with

Organic layer

Vacuum evaporated

Vacuum evaporated

Vacuum evaporated Adjust pH 70

Adjust pH 90

Maceration or reflux using methanolor 70 ethanol for extraction

Total extract

Liquid-liquid extraction Column chromatography

Water layer

Water layer

Water layer

Organic layer

Total alkaloidal fraction

Saponin fraction

Neutral fraction

Specific elution

Saponin fraction

Weak base Fr 1

Nonphenolic Fr 2 Phenolic Fr 3

Note ①5 or 01N HCl in H2O②H2O

CHCl3CHCl3

1N NaOH

CHCl3 or EtOAc

CHCl3 or EtOAc

Suspension in ①

Suspension in ① or ②As followeds ① is mentioned

CHCl3 or EtOAc

n-BuOH

Caulophyllum plant materials

n-BuOH layer

CHCl3 residue

NH4OH CHCl3

Scheme 1 Summary of procedures for isolation of alkaloids and saponins from Caulophyllum plants

Caulophyllum From the opinion of safety of using dietarysupplements of blue cohosh a review dealing with quan-titative methods of primary constituents of blue cohosh indietary supplements has been published [2]However to datethere is no systematic reviewof chemistry pharmacology andactionmechanisms of constituents fromgenusCaulophyllum

In this review the different structures of the alkaloidsand saponins in genusCaulophyllum are described includingnaturally occurring constituents and synthetical taspinederivativesThe present review highlighted the chemistry andpharmacological diversity andmechanism of actionThe aimof this paper is to provide a point of reference on Caulo-phyllum plants for pharmaceutical researchers Furthermorevarious perspectives and existing problems for this genus areoffered for consideration

2 Phytochemistry

Phytochemical research carried out on genus Caulophyllumled to the isolation of alkaloids and triterpence saponins and a

few other classes of secondary metabolites A comprehensivesummary of structures and isolation methods of metabolitesclassified by structural types was given in present reviewScheme 1 summarizes the procedures for crude isolation ofalkaloids and triterpene saponins from genus CaulophyllumThe roots and rhizomes of Caulophyllum plants are extractedwithmethanol or 70 ethanol bymaceration [13 20] or reflux[21] and the combined extracts are concentrated in vacuo todryness Then two schemes are available for acquiring thealkaloid and saponin fractions namely liquid-liquid parti-tion and liquid-solid column chromatography methods [21]Liquid-liquid partition is commonly performed for crudeisolation In most cases the residue is suspended in 5 or01 NHCl in water and then partitioned with EtOAc orCHCl

3to remove neutral constituentsThe aqueous layer was

then removed NH4OH was added to make it basic (pH 9)

and the whole was extracted with EtOAc or CHCl3 The

EtOAc or CHCl3soluble part was evaporated to obtain the

total alkaloidal fraction Moreover total alkaloidal fractionwas able to further liquid-liquid partition to afford weak

Evidence-Based Complementary and Alternative Medicine 3

HO

O

HO

O

HO

OO

N

O

O

O

N

O

1 2

6

128

NH

OO

OH

O

N

OH14 15

N

O

OH

NH

O

O

O

13

16

OHHO O

O O

N

17

22

10 H11 AC

R

OOH

R

OON

1819 OH

R

OOH

OH

OO

2021

N

O

HO

O

O

9

7

N

OHO

O

OHH 3

N

HN

N

H

H

4 5

N

O

N

N

O

NH

N

N

HO

N

N

HO

N

O

N

HOR N

N

HO

R1

R1

R2

R2

R3

R3

OCH3

CH3 CH3 CH3CH3 CH3 CH2OH

N+

N+

Figure 1 Chemical structures of alkaloids (1ndash22) from genus Caulophyllum

base (Fr 1) nonphenolic alkaloids (Fr 2) and phenolicalkaloids (Fr 3) [13] The H

2O layer was neutralized with

5HCl and extracted with n-butanolThe combined organiclayers were evaporated to obtain total saponin fraction [20]Column chromatography is also a popular method to enrichtotal alkaloids and saponins from Caulophyllum plants bychoosing optimal macroporous or (and) ion exchange resins[13 21 22]

21 Alkaloids With respect to alkaloid aspects of this genus22 molecules have been isolated and identified from genusCaulophyllum Alkaloid compounds are very important bio-active constituents in genus Caulophyllum Their chemical

structures and sources can be seen in Figure 1 and Table 1These compounds can be divided into several kinds of struc-tural types magnoflorine (1) taspine (2) and boldine (3) arecontributed to aporphine alkaloids Aporphine alkaloids havebeen shown to possess anticancer activity and there is evi-dence that this activity is exerted through induction of apop-tosis inhibiting cell proliferation and inhibiting DNA topoi-somerase [23 24]Magnoflorine (1) a quaternary ammoniumbase is isolated and detectedwith the biggest amounts amongall the alkaloids isolated from genus Caulophyllum 1was alsoisolated from the n-butanol fraction of blue cohosh due to itsstrong water-solubility but it was not active in the rat embryoculture [25] The molecular structure of 2 is characterized byhigh symmetry 4ndash12 are typical quinolizidine alkaloids


Table 1 Chemical structures of alkaloids (1ndash22) from genus Caulophyllum

No Compounds Formula Sourcesa References1 Magnoflorine C20H24NO4

+ Cr Ct [85 86]2 Taspine C20H19NO6 Cr Ct [25 86]3 Boldine C19H21NO4 Cr [86]4 Anagyrine C15H20N2O Cr Ct [20 85 86]5 Sparteine C15H26N2 Cr Ct [25]6 N-methylcytisine C12H16N2O Cr Ct [20 85ndash87]7 Cytisine C11H14N2O Cr [86]8 56-Dehydro-120572-isolupanine C15H22N2O Cr Ct [25 86]9 Lupanine C15H24N2O Cr Ct [25 86]10 Baptifoline or Argentamin C15H20N2O2 Ct [85 86]11 O-acetylbaptifolin C17H22N2O3 Ct [20]12 120572-isolupanine C15H24N2O Cr Ct [25 86]13 Thalictroidine C14H19NO2 Ct [20 25]14 Caulophyllumine A C15H21NO4 Ct [20]15 Caulophyllumine B C14H19NO Ct [20]16 Piperidylacetophenone C15H21NO3 Ct [20]17 Caulophine C19H21NO5 Cr [88]18 Caulophylline A C20H23NO5 Cr [13]19 Caulophylline B C19H21NO5 Cr [13]20 Caulophylline C C20H24NO5 Cr [13]21 Caulophylline D C20H24NO6 Cr [13]22 Caulophylline E C18H17NO4 Cr [13]aCr means C robustum Ct means C thalictroides

Quinolizidine alkaloids have been reported to possess theobvious nematicidal activity [26]

In October 1999 a novel alkaloid thalictroidine (13) withpiperidine-acetophenone conjugate was isolated from therhizomes of C thalictroides using an in vitro rat embryo cul-ture method 13was not teratogenic in the rat embryo cultureat tested concentrations [25] After nine years 13was isolatedagain from C thalictroides together with 14ndash16 [20] 13ndash15piperidine-acetophenone conjugates are rare in the plantkingdom 16 was only reported from Boehmeria genus [27]and is another example of such a type of compound fromnatural sources

In April 2009 a distinct class of alkaloid fluorenonealkaloid (caulophine 17) was firstly reported from the radixof C robustum using cell membrane chromatography as thescreeningmethod 17was identified as 3-(2-(dimethylamino)ethyl)-45-dihydroxy-16-dimethoxy-9H-fluoren-9-one basedon physicochemical and spectroscopic analyses 17 pos-sessed antimyocardial ischemia activity by rat experimentsIt is worth mentioning that a preparative high performanceliquid chromatography method was developed for isolationpurification and enrichment of caulophine (17) [28] Asfollows another four fluorenone alkaloids caulophyllines AndashD (18ndash21) and one dihydroazafluoranthene alkaloid caulo-phylline E (22) were isolated from the roots of C robustum

Fluorenone alkaloid is a newly discovered alkaloid skele-ton in natural products 17ndash21 five new fluorenone alkaloidswere isolated from the same plant suggesting that fluorenonetype alkaloid is another kind ofmetabolites that existed in this

genusCaulophyllum 22 is a novel and rare naturally occurreddihydroazafluoranthene alkaloid there are no reports aboutdihydroazafluoranthene alkaloid isolated from natural prod-ucts except its novel core skeleton first isolated from coal tar[24] 22 has the isoquinoline fragment which is possible to bethe conceivable precursor of different substituted fluorenonealkaloids A hypothetical biosynthetic pathway for 20 wasproposed starting from 22 which undergoes a sequentialnitrogen-related double bond reduction oxidation ring-opening N-methylation and demethoxy process [13]

22 Triterpene Saponins Caulophyllum triterpenes generallyconstitute the main class of secondary metabolites in thegenusCaulophyllum amounting to up to 746of the total dryweight in root and rhizome [29] Until now 32 caulophy-llsaponins were isolated and identified by chemical anddetailed spectroscopic analysis (Table 2) These saponinsgenerally bear one (monodesmosidic) or two (bidesmosidic)carbohydrate chains that are directly attached to the hydroxylgroups in position C-3 for monodesmosidic saponins and topositions C-3 and C-28 in the case of the bidesmosidicsaponins 25 28 29 33 34 37ndash39 and 41ndash44 are bidesmo-sidic triterpenoid saponins with two sugar chains at C-3 andC-28 Others were found to only have one sugar chain at C-3or C-28

Twelve kinds of aglycones have been discovered from thegenus Caulophyllum (Figure 2(a)) [20 21 30] Before 2009only four kinds of sapogenins were discovered from genus


Table 2 Chemical structures of triterpene saponins (23ndash54) from genus Caulophyllum

No Compound names C-3 C-28 Formula Sourcesa References

23 Arararr 3120573-O-AG1 S1 mdash C35H56O7 Ct [21]24 Saponin PE Glc rarr 2Ara rarr 3120573-O-AG1 S2 mdash C41H66O12 Ct [20 21]25 Ciwujianoside A Glc rarr 2Ara rarr 3120573-O-AG1-28-Olarr Glc6 larr Glc4 larr Rha S2 S3 C59H96O26 Ct [20 21]

26 Cauloside A Ara rarr 3120573-O-AG2 S1 mdash C35H56O8 Cr Ct [20 21 30 3137 89]

27 Cauloside C Glc rarr 2Ara rarr 3120573-O-AG2 S2 mdash C41H66O13 Cr Ct [20 21 31 3789]

28 Cauloside D Ara rarr 3120573-O-AG2-28-Olarr Glc6 larr Glc4 larr Rha S1 S3 C53H86O22 Cr Ct [20ndash22 30 90]

29 Cauloside G Glc rarr 2Ara rarr 3120573-O-AG2-28-Olarr Glc6 larr Glc4 larr Rha S2 S3 C59H96O27 Cr Ct [20ndash22 30 91]

30 Cauloside b Rha rarr 2Ara rarr 3120573-O-AG2 S4 mdash C41H66O12 Cr [92]31 Cauloside c Ara rarr 3Rha rarr 2Ara rarr 3120573-O-AG2 S5 mdash C46H74O16 Cr [92]32 AG2-28-Olarr Glc6 larr Glc4 larr Rha mdash S3 C48H78O18 Ct [21]33 Glc rarr 3Ara rarr 3120573-O-AG2-28-Olarr Glc6 larr Glc4 larr Rha S6 S3 C59H96O27 Cr [22 93]34 Ara rarr 3120573-O-AG3-28-Olarr Glc6 larr Glc4 larr Rha S1 S3 C53H86O22 Ct [30]35 Ara rarr 3120573-O-AG3 S1 mdash C35H56O8 Ct Cr [21 31 37]36 Glc rarr 2Ara rarr 3120573-O-AG3 S2 mdash C41H66O13 Ct [21 30]37 Leiyemudanoside C Glc rarr 3Ara rarr 3120573-O-AG3- 28-O larr Glc6 larr Glc4 larr Rha S6 S3 C59H96O27 Cr [36]38 Ara rarr 3120573-O-AG3-28-Olarr Glc6 larr Glc S1 S7 C47H76O19 Ct [21]39 Glc rarr 2Ara rarr 3120573-O-AG3-28-Olarr Glc6 larr Glc4 larr Rha S2 S3 C59H96O27 Cr [30]

40 Cauloside B Ara rarr 3120573-O-AG4 S1 mdash C35H56O9 Cr Ct [20ndash22 3031 37 94]

41 Leonticin D Ara rarr 3120573-O-AG4-28-Olarr Glc6 larr Glc4 larr Rha S1 S3 C53H86O23 Ct [21 22 30]42 Cauloside H Glc rarr 2Ara rarr 3120573-O-AG4-28-Olarr Glc6 larr Glc4 larr Rha S2 S3 C59H96O28 Ct [20]43 Leiyemudanoside A Ara rarr 3120573-O-AG4-28-Olarr Glc6 larr Glc S1 S7 C47H76O19 Cr [36]44 Leiyemudanoside B Glc rarr 3Ara rarr 3120573-O-AG4-28-Olarr Glc6 larr Glc4 larr Rha S6 S3 C59H96O28 Cr [36]45 Glc rarr 2Ara rarr 3120573-O-AG4 S2 mdash C41H66O14 Ct [21]46 Ara rarr 3120573-O-AG4-28-Olarr Glc S1 S8 C41H66O14 Ct [21]47 Ara rarr 3120573-O-AG5 S1 mdash C35H54O8 Ct [21]48 Ara rarr 3120573-O-AG6 S1 mdash C35H54O9 Ct [21]49 Glc rarr 2Ara rarr 3120573-O-AG7 S2 mdash C41H64O14 Ct [21]50 Ara rarr 3120573-O-AG8 S1 mdash C35H54O10 Ct [21]51 Ara rarr 3120573-O-AG9 S1 mdash C35H54O9 Ct [21]52 Glc rarr 2Ara rarr 3120573-O-AG10 S2 mdash C41H68O13 Ct [21]53 Glc rarr 2Ara rarr 3120573-O-AG11 S2 mdash C41H66O12 Ct [21]54 Ara rarr 3120573-O-AG12 S1 mdash C35H58O8 Cr [31]aCr means C robustum Ct means C thalictroides

Caulophyllum namely oleanolic acid (AG1) hederagenin(AG2) echinocystic acid (AG3) and caulophyllogenin (AG4)HoweverMa et al reported 47ndash53with abnormal sapogeninsAG5 to AG11 from blue cohosh for the first time As follows54 bearing sapogenin erythrodiol was discovered from genusCaulophyllum in 2012 [31]These aglycones are closely relatedoxygenated pentacyclic triterpenoidal structures that can be

distinguished only by the positions and numbers of the dou-ble bonds in rings C and D and oxygenation patterns in posi-tions C-16 C-23 and C-28 A possible biosynthetic pathwayofCaulophyllum sapogenins can be hypothesized as shown inScheme 2Thefirst is that 23-oxidosqualene is cyclized to thepentacyclic oleanane-type triterpenoid backbone 120573-amyrinby plant oxidosqualene cyclases 120573-amyrin synthase [32 33]


HO

COOH

HO

COOH

OHHO

COOH

OH

HO

COOH

OH

OH CHOHO

COOH

OH

CHOHO

COOH

HOOH

O O O

HO

OH

OH

O O O

HO

COOH

OH

OH

HO

COOHH

OH

HO

H

O

O

H

HO

OH

OH

A B

C D

E

1

3

12

16 28

23

O

AG1 AG2AG3

AG4 AG5 AG6

AG7 AG8 AG9

AG10AG11 AG12

CH2OH

(a)

O

OO

O

OO

OH

O

OO

O

OHO

HO

HOHOHO

HOHOHO

HO

HO

HOHO

HOHO

HOHO

HO HOHO HO

HOHO

HO

HO

OHOHOHOH

OH

OH

OH

OH

OH

OHOH

OH

OH

OHOH

OHOH

OHOH

OH

OHOHO

O

OO

OO

OOO

O

OO O

S1 S2 S3S4

S5 S6 S7S8

(b)

Figure 2 (a) Chemical structures of aglycones (AG) of saponins (b) linkage mode of sugar moieties of saponins from genus Caulophyllum

The 120573-amyrin experienced hydroxylation at C-28 to produceerythrodiol (AG12) Erythrodiol is further oxidized at the C-28 position by a single cytochrome P450 enzyme to yieldoleanolic acid (AG1) [34] The aglycones AG1ndashAG9 may bederived from the common skeleton of oleanolic acid as

precursors that firstly experience selective oxidation at C-23or C-16 or both of C-23 and C-16 to afford hederagenin(AG2) echinocystic acid (AG3) and caulophyllogenin (AG4)respectively Hederagenin may selectively involve a complexprocess such as dehydrogenization oxidation lactonization


O

23-oxidosqualeneHO HO

OH

HO

OH

O

HO

OH

O

OHHO

OH

O

OH

HO

OH

O

OH

OH

HO

OH

O

HO

HO

OH

O

OH

HO

HO

OH

O

OH

HOOH

O O O

HOOH

O O O

HO

OH

O

OH

OH

OH

OHOH

HOOH

O OOH

HOOH

O O

AG1

AG2

AG3 AG4

AG5 AG6

AG7AG8

AG9

AG12120573-Amyrin

Scheme 2 Hypothesized biosynthetic pathway for oleanane aglycones from genus Caulophyllum A series of step-by-step actions from 23-oxidosqualene to 120573-amyrin erythrodiol oleanolic acid and other aglycones are assumed

dehydration and lactone ring hydrolysis to form diverseaglycones in genus Caulophyllum Though the intermediate(312057312120572-dihydroxy-olean-28-oic acid 120574-lactone) has beenartificially synthesized from oleanolic acid (Supplemen-tary Scheme 1 in Supplementary Material available onlineat httpdxdoiorg1011552014684508) [35] this type of

biosynthetic pathway in plants also needs to be furtherconfirmed

The according carbohydrate chains are composed mainlyof arabinose rhamnose and glucose moieties After acidhydrolysis gas chromatography analysis revealed the pres-ence of glucose arabinose and rhamnose through comparing


OH

O

55

RO

R56 H57 Glc

HO 59

HO

58

HO60

Figure 3 Chemical structures of other compounds (55ndash60) from genus Caulophyllum

with derivatives obtained by the same method of standardmonosaccharides [36] For their linkage mode of sugarmoieties it showed the presence ofmany linked types of sugarmoieties including terminal glucose 16-linked glucose ter-minal rhamnose 12-linked rhamnose terminal arabinose12-linked arabinose 13-linked arabinose and 123-linkedarabinose [20 21 30 36] (Figure 2(b)) Diverse aglyconesmonosaccharide residues and diverse linkage mode of sugarmoieties are possible to form diverse structures of triterpenesaponins from genus Caulophyllum

23 Other Compounds Other minor compounds are presentin this genus such as fatty acids and sterols [37 38] Thesecompounds were identified as palmitic acid (55) 120572-spinasterol (56) 120572-spinasterol-120573-D-glucopyranoside (57)stigmasterol (58) lupeol (59) and cholesterol (60) (Figure 3)Polysaccharides are present in this genus The extractionprocess and antioxidant activity of polysaccharides have beenstudied [39 40] The optimal ultrasound-assisted extractionconditions of polysaccharides from C robusutm were extr-action temperature 65∘C time 70min ratio of liquid to solid20 and extracting power of 70W

3 Synthetic Taspine Derivatives

Zhang et al designed and synthesized four novel ring-openedtarget compounds (11015840ndash41015840) by structure-based drug designThis design includes two pathways cleavage of the CndashCbond of diphenyl and ester bond of ring B and ring D(Scheme 3(A)) Targeted compounds 11015840 and 21015840 were synthe-sized by the route outlined in Scheme 3(B) Isovanillin (51015840)was used as the starting material which was firstly oxidizedto afford isovanillic acid (61015840) The methyl ester 71015840 was pre-pared to avoid side-reactions of the carboxylate group Then

refluxing of 71015840 with prenylbromide in the presence of K2CO3

in anhydrous acetone afforded 81015840 A solution of 81015840 in NN-dimethylaniline was heated to reflux to give 91015840 Prenyl groupwas moved into the paraposition of hydroxyl in Claisen rear-rangement processThe next stepwas the coupling of 91015840 to thecarboxyl of 101015840 by an ester bond with DCC and DMAP Theoxidation of 111015840 produced aldehyde 121015840 which reacted withdimethylamine followed by reduction to give 131015840 At last ben-zyl deprotection of 131015840 with palladium-carbon inMeOHgave11015840 The 31015840 and 41015840 were synthesized in the same way from iso-vanillin [17]

Synthetic endeavors into cleavage of the CndashC bondand ester bond of rings B D and E have been studied(Scheme 4(A)) Initially six target biphenyl derivatives (191015840ndash241015840) were successfully synthesized by general routes des-cribed in Scheme 4(B) employing a classical symmetricalUllmann reaction [18] Isovanillin (51015840) was also used for thestartingmaterial which was required for seven steps to afford191015840 by bromination benzylation oxidation substitutionreaction Ullmann reaction and catalytic hydrogenation 191015840is an important intermediate to synthesize the following tar-geted compounds During the synthesis of unsymmetricalbiphenyl (221015840) a novel symmetrical biphenyl derivative (311015840)was surprisingly isolated as a byproduct [41] which exhibitedpotent anticancer activity to attract increasing attention Tofurther investigate this finding researchers aimed to enhancethe structural complexity and diversity of 221015840 by generatingnovel biphenyls (Figure 4) [41] As a result eighteen sym-metrical biphenyls derivatives (311015840ndash481015840) were firstly prepared[42] Following these He et al used 201015840 as the identifyinggroup and synthesized another two novel taspine diphenylderivatives 491015840 and 501015840 which were made by introducingcoumarin groups into the structure of 201015840 [43] Meanwhilederivatives 511015840 and 521015840 were obtained via similar procedures(Scheme 4(B)) [44]


O

OO

N

O

O

OA

B D

C

Taspine 2

COOOH

O

R

O

O

O

O

OHO

OOC

N

O

O

OHO

OHO

a b

OHO

c

OO

COOd

OHO

COO

+

OBnO

eCOOOBn

O

O

O

O

fCOOOBn

O

O

O O

gCOOOBn

O

O

O

O N

h

i

BnO

O

COO

j

BnO

O

OHO

+e

O

OHO

OOC

O

O

f O

OHO

OOC

O

O

O

O

OBnO

OOC

N

O

Og h

(A)

(B)

D

B

O

H

O

OH

O

O

O

H

O

OH

O

O

1998400

1998400

R = N(CH3)23998400 R = morpholine4998400R = 3-Cl-4-F-aniline

5998400 6998400 7998400

7998400

8998400 9998400

9998400

9998400

10998400

11998400 12998400 13998400

1499840015998400 16998400

16998400

17998400 18998400

OCH3

2998400

2998400

Scheme 3 (A) Design of ring-opened target compounds 11015840ndash41015840 (B) preparation of target compounds 11015840ndash41015840 Reagents and conditions (a)NaOH KOH H

2O 84 (b) CH

3OH H

2SO4 90 (c) prenyl bromide K

2CO3 acetone 92 (d) N N-dimethylaniline N

2 reflux 71 (e)

anhydrous THF DCC DMAP 78 (f) OsO4 NaIO

4 acetoneH

2Ot-BuOH 32 (g) dimethylamine Morpholine3-Cl-4-F-aniline THF

CH2Cl2 NaBH(OAc)

3 25ndash40 (h) H

2 PdC 97 (i) BnCl K

2CO3 EtOH 95 (j) NaOHH

2O CH

3OH 93 [17]

4 Bioactivity

41 Antibacterial Activity Earlier biological studies showedthat caulosidesAndashDandG (264027ndash29) have antimicrobialactivity [45] Recently triterpene compounds isolated fromCrobustum showed microorganism inhibitory activities to the

test fungi and bacteria Moreover compound 35 and caulo-side B (40) had notable inhibiting microorganism activitiesto bacteria with minimal inhibitory concentration (MIC) of39 120583gmL [11] Ethanol extract and its five subfractions of Crobustum showed high antibacterial activity against Staphy-lococcus aureus Staphylococcus aureus (clinic bacterial) and


O

OO

N

O

O

O

Taspine 2

CONHHNOC

O

O

(A)

E

A

B

C

DB D E

HN

OF

Cl

N

ON

HNN

OOH

O

Ha

OOH

O

H b

Br OOBn

O

H c

Br OOBn

O

OH d

Br OOBn

O

Cl e

Br

OOBn

O

NH f

Br

O

O

OBnCONHBnO

HNOCg h

(B)

or

oror By products

ij

R1 = H R2 = HR1 =W R2 = HR1 = H R2 = YR1 =W R2 = YR1 =W R2 = XR1 =W R2 = Z

OR2

R1O

W =

X =

Y =

Z =

5199840052998400 49998400 + 50998400

32998400ndash4899840031998400

20998400

20998400

3099840029998400

19998400

23998400

19998400

21998400

21998400

22998400

22998400

23998400

24998400

24998400

25998400 26998400 27998400 289984005998400

Scheme 4 (A) Design of ring-opened target compounds 191015840ndash241015840 (B) preparation of target compounds (191015840ndash241015840) Reagents and Conditions(a) Fe NaOAc AcOH Br

2 81 (b) BnCl K

2CO3 95 (c) NaH

2PO4 NaClO

2 30 H

2O2 93 (d) SOCl

2 DMF(cat) CH

2Cl2 96 (e)

CH2Cl2 30 CH

3NH2 85 (f) Cu DMF 72 (g) H

2 PdC 97 (h) K

2CO3 DMF (26 64 25 76) (i) K

2CO3 EtOH (27 72 28

59 29 54) [18 42ndash44]

Bacillus subtilis and the diameters of the biggest inhibitionzone were 2003mm 2352mm and 2077mm The MICs ofthese were 031ndash063mgmL [46]

42 Anti-Inflammatory and Analgesic Effects The anti-inflammatory and analgesic effects of ethanol extract chlo-roform extract and n-butyl alcohol extract from C robustumwere observed by several animal experiments Among the dif-ferent organic extracts the action of alcohol extractwas betterthan other organic extracts [12] Cauloside A (26) and caulo-side C (27) had anti-inflammatory and analgesic activities atdose dependency and the analgesic effect was themost signif-icant when compounds were injected for 30min [11] Fromthe points of structure-activity relationship of the saponinscauloside C (27) with disaccharide has more potent analgesiceffect than cauloside A (26) with monosaccharide Oppo-sitely cauloside A (26) has more potent anti-inflammatoryactivity than cauloside C (27)The anti-inflammatory activityof taspine hydrochloride has been demonstrated by using thecarrageenan-induced pedal edemamethod the cotton pellet-induced granuloma method and the adjuvant polyarthritismodel [47]

Lee et al (2012) assessed the in vitro and in vivo effects ofblue cohosh on lipopolysaccharide (LPS)-induced cytokinesin BV2 cells and mice Several lines of evidence indicate thatblue cohosh treatment suppressed the elevation of LPS-induced iNOS (inducible nitric oxide synthase) expression ina concentration-dependent manner in microglia cells Bluecohosh saponins (caulosides AminusD 26 40 27 28) signifi-cantly suppressed the expression of tumor necrosis factor-120572(TNF-120572) interleukin-1120573 (IL-1120573) and IL-6 In addition bluecohosh extract suppressed the expression of COX (cyclo-oxygenase)-2 iNOS and proinflammatory cytokines inadrenal glands of mice So it is concluded that saponinconstituents of blue cohosh exert anti-inflammatory effectsthrough the inhibition of expression of iNOS and proinflam-matory cytokines [48]

43 Antioxidant Effects CaulophyllineAminusD (18ndash21) affordedthe lower scavenging effects against DPPH (11-diphenyl-2-picrylhydrazyl) radical at test concentration (60 to 1074120583gmL) Caulophylline E (22) showed good scavenging effe-cts against DPPH radical with IC

50(half-inhibition con-

centration) of 121 120583gmL [13] Antioxidant activities of the


O

O

O

OHN

OCNH

O

HN

NH

abcdefghij

Isop aIsop bIsop cIsop dIsop eIsop jDiet aDiet b

F

Cl

Cl

Cl

O

Br

F

N F

F

O

O

OHNOC

O

O

OO O

NH

O

F

Cl

O

OHNOC

CONH

CONH

O

O

OOHN

NH

O

F

Cl

O

O

NH

O

NH2

NHOO

F

ClO

O

HN

OF

Cl

O

HN

OOH

O

NH

O

O

NH

NH

O

O

319984003299840033998400349984003599840036998400379984003899840039998400409984004199840042998400439984004499840045998400469984004799840048998400

a =

b =

c =

d =

e =

CF3

f =

g =

h =

i =

j =

49998400

50998400

51998400

52998400

R1

R1

R1

R2

R2

R2

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

Isop = Isopropyl Diet = Diethyl

Figure 4 Structures of 311015840ndash511015840

polysaccharide fraction ethanol extract and different polarfractions of C robustum were evaluated by DPPH hydroxyland superoxide radical and nitrogen dioxide (NO

2) scaveng-

ing assay [39 49]The results showed that ethanol extract anddifferent polar fractions displayed high antioxidant activitiesThe scavenging activities of polysaccharides from C robus-tum for DPPH hydroxyl and superoxide radical and NO

2

were attributed to 80 96 78 and 851 respectivelyfor the concentrations of 50mgmL Another experimentresearch reported that chloroform partition fraction showedIC50

value of DPPH-free radical-scavenging activity whichwas 794 120583gmL [14]

44 Antiacetylcholinesterase Activity As early as 2006 tasp-ine (2) has been confirmed to be an antiacetylcholinesterase(AChE) inhibitory agent by a bioactivity-guided approachin a Magnolia x soulangiana extract using a microplateenzyme assay with Ellmanrsquos reagent [50] 2 showed a sig-nificantly higher effect on AChE than the positive controlgalantamine and selectively inhibited the enzyme in a long-lasting and concentration-dependent fashion with an IC

50

value of 012 120583gmL It could be suggested that taspine might

be a potential candidate for the development of anti-AD(Alzheimerrsquos disease) treatment

More recentlyC robustum has been confirmed to possesssignificantAChE activitywith inhibition rates (8872plusmn 147)at the concentration of 1 gsdotLminus1 through thin layer chromatog-raphy bioautographic method Furthermore chloroformfractions have shown higher AChE inhibitory capacity soit will be further performed bioguided isolation and purifica-tion to obtain active compounds [14] In addition to taspine inC robustum whether to have other compounds responsiblefor the activity of AChE is worthy of studying further

45 Effect on Atherosclerosis and Myocardial Ischemia It wasfound that the n-butanol fraction ofC robustumwas an effec-tive part and caulophine (17) separated from the part was anactive one in vasodilatation [51 52] The n-butanol fractionmay have protective action on H

2O2injured-human umbil-

ical vein endothelial cell line in vitro and its mechanism ofactionmay be related to the increase of the level of nitric oxide(NO) NOS (nitric oxide synthase) and the expression ofNF-120581B (nuclear factor kappa B) [53]The interaction between


the effective component 17 and the membrane or membranereceptor was reflected in the vascular CMC model whichsuggested that 17 may exert bioactivity in the heart [52]The deeper study demonstrates that 17 is able to protectcardiomyocytes from oxidative and ischemic injury throughan antioxidative mechanism [54] and from caffeine-inducedinjury via calcium antagonism [51]

46 Antitumor Activity and Mechanism of Action The cyto-toxicity (IC

50) of taspine (2) was found to be 039 120583gmL

against KB cells and 017 120583gmL against V-79 cells [55] 2showed antitumor activity on the mouse S180 sarcoma in agood dose-dependent manner [56] The inhibition rates ontumor of taspine at lowmiddle and high concentrationswere3908 4399 and 4860 respectively The microvesseldensity and protein expressing of the vascular endothelialgrowth factor (VEGF) basic fibroblast growth factor (bFGF)Bcl-2 and Bax in the tumor were decreased compared withthe negative controlThe ratio of Bax to Bcl-2 was increased 2has antitumor effect on the S180 sarcoma and themechanismmay be through the way of decreasing the expressing ofthe VEGF bFGF Bcl-2 and Bax and inducing the vascularendothelial cell apoptosis

Zhan et al was to investigate the effect of taspine onthe growth of oestrogen-receptor-positive breast cancerxenografts in vivo and the possible mechanism for thisaction [19] Cell cycle and apoptosis analysis documented thattaspinewas able to change cell cycle and induce cell apoptosisThere was a significant decrease in the expression of estrogenreceptor (ER) and progesterone receptor (PR) both in tumortissue and cells after treatment with taspine At the same timeit also showed a reduction in the expression of mRNA forER and PR in the group treated with taspine These datasuggested that taspine might serve as a promising candidateof ER antagonist in the treatment of oestrogen-independentbreast cancer

Many evidences have shown that taspine could suppresstumor-induced angiogenesis Taspine was able to inhibitchicken chorioallantoic membrane angiogenesis throughinterfering with the proliferation and migration of endothe-lial cells in a dose-dependent manner [57] The exact mech-anism [15] has been further demonstrated suggesting thatVEGF and bFGF secretion were downregulated by taspine inhuman non-small cell lung cancer cell (A549 cell) and humanumbilical vein endothelial cells (HUVECs) confirmed bythe decreased mRNA level of VEGF and Flk-1KDR aftertaspine treatment in HUVECs The molecular mechanismsof taspine on tumor angiogenic inhibition have been furtherstudied in vitro [58] which indicated that taspine significantlyinhibited cell proliferation of HUVECs induced by VEGF165via decreasing Akt and Erk12 activities except decreasingVEGF level Authors assume that taspine can inhibit the pro-liferation of vascular endothelial cells in tumor by regulatingPI3 kinase and MAP kinase signal pathways

Additionally taspine could induce apoptosis of HUVECsin a dose-dependent manner [59] Cell cycle was signifi-cantly stopped at the S phase The morphology of HUVECtreated with taspine showed nuclear karyopycnosis chro-matin agglutination and typical apoptotic body detected by

electronic microscope Taspine has an inhibitory effect ongrowth of HUVECs and can induce its apoptosis by decreas-ing Bcl-2 expression and increasing bax expression Zhanget al continuously investigated the effects of taspine on theproliferation and apoptosis in theA431 cell [60]The cell cyclewas significantly stopped at S phase and nuclear karyopy-knosis chromatin agglutination and typical apoptotic bodieswere found after taspine treatment in A431 cells Therewas a decrease in the expression of Bcl-2 whereas the expres-sion of caspase-3 cleaved caspase-3 CDK2 CDK4 and Baxincreased These data demonstrated that taspine can induceapoptosis by activating caspase-3 expression and upregulat-ing the ratio of BaxBcl-2 in A431 cells

The preliminary biological test demonstrated that deriva-tive 31015840 showed much better inhibitory activities againstCACO-2 (IC

50= 0023 120583gmL) and ECV304 (IC

50=

00012120583gmL) than taspine [17] A deep research demon-strated that most derivatives (11015840ndash41015840 and 191015840ndash241015840) possessed amoderate degree of cytotoxicity against human cancer celllines [18] One of them (31015840) exhibited much better antipro-liferative activity against CACO-2 (IC

50= 234 120583gmL) and

ECV304 (IC50

= 119 120583gmL) cells than taspine did Someof the compounds showed good antiproliferative activityagainst colon (HT29) breast (MCF-7) lung (A549) rectum(CACO-2) skin (A375) hepatoma (7721) and pancreatic(PANC-1) cancers cell lines A continual research demon-strated that derivative 31015840 can inhibit the proliferation of andinduce apoptosis in Caco-2 cells by activating caspase-3caspase-8 and caspase-9 downregulating the expressions ofVEGF and upregulating the ratio of baxbcl-2 [61]

Derivatives (311015840) and (321015840) demonstrated themost potentcytotoxic activity with IC

50values between 142 120583gmL and

223 120583gmL among symmetrical taspine derivatives (311015840ndash481015840)[42] Biphenyls without halogen substitution (341015840 381015840 391015840and 441015840) were much less potent than those containinghalogen Halogen substitution played a critical role in theactivity of biphenyls Derivative 311015840 inhibits tumor growth inxenografted A549 cells in nudemice by inhibiting the growthof neovessels In other words derivative 311015840 is an inhibitor ofangiogenesis which functions by downregulating VEGF [62]Furthermore derivative 311015840 had potential to suppress theadhesion migration and invasion of ZR-75-30 cancer cellsand it could serve as a potential novel therapeutic candidatefor the treatment of metastatic breast cancer [63] Derivative481015840 could inhibit proliferation of lovo cell and tumor growthin a human colon tumor xenografted model of athymic micewhich might be a novel angiogenesis inhibitor that reducesangiogenic responses in vivo and in vitro by blocking VEGFRsignaling pathways [64]

Two novel derivatives (491015840 and 501015840) by introducing differ-ent coumarin fluorescent groups into the basic structure havenot only fluorescence but also the ability to inhibit effects ondifferent breast cancer cell lines which indicates their pos-sible further use as dual functional fluorescence probes intracer analysis Derivative 511015840 inhibits tumor growth and cellproliferation by inhibiting cell migration downregulatingmRNA expression of VEGF and EGF and decreasing angio-genic factor production which deserves further consid-eration as a chemotherapeutic agent [44] All evidences


have demonstrated that the lactone ring B is important foractivity while the lactone ring D can be opened thus retain-ing and even improving the antiproliferative properties oftaspine Halogen substitution could potentially improve theanticancer activity of the biphenyl derivatives [17 18 42]

47 Inhibitory Cytochrome P450 Effects Themethanolic extr-acts of the roots of blue cohosh the alkaloidal fractionand isolated constituents were evaluated for their inhibitionof major drug metabolizing cytochrome P450 (CYP450)enzymes [65] The methanolic extracts did not show anyeffect but the alkaloidal fraction showed a strong inhibition ofCYP2C19 3A4 2D6 and 1A2 (gt80 inhibition at 100 120583gmL)with IC

50values in the range of 2ndash20 120583gmL Among the

isolated alkaloids caulophyllumine B (15) O-acetlybaptifolin(11) anagyrine (4) and lupanine (9) inhibited these enzymesto various extents (IC

50 05ndash151120583gmL) N-methylcytisine

(6) showed weak activity against the CYP3A4 in vitro with32 inhibition at 204120583gmL An equimolar mixture ofalkaloids exhibited amore pronounced inhibitory effect on allfour enzymes as compared to the isolated alkaloids Amongthe saponins caulosides C (27) and D (28) showed 43 and35 inhibition of CYP3A4 at the concentration of 766 and1074 120583gmL respectively Other enzymes were not affectedThis in vitro study indicates that dietary supplements contain-ing blue cohosh may pose a risk of drug-drug interactions iftaken with other drugs or herbs metabolism of whichinvolves CYP450 enzymes

48 Topoisomerase Inhibitor Taspine (2) was found to induceconformational activation of the proapoptotic proteins Bakand Bax mitochondrial cytochrome c release andmitochon-drial membrane permeabilization inHCT116 cells [66] Anal-ysis of the gene expression signature of taspine treated cellssuggested that taspine is a topoisomerase inhibitor Taspinehas a reduced cytotoxic effect on a cell line with a mutatedtopoisomerase II enzyme Interestingly in contrast to the top-oisomerase II inhibitors doxorubicin etoposide and mitox-antrone taspine was cytotoxic to cell lines overexpressing thePgP or MRP drug efflux transporters Taspine induces wide-spread apoptosis in colon carcinoma multicellular spheroidsand that apoptosis is induced in two xenograftmouse modelsin vivo Taspine is a dual topoisomerase inhibitor that iseffective in cells overexpressing drug efflux transporters andinduces wide-spread apoptosis in multicellular spheroids

49 Effect on Wound Healing A patent reported that themethod is useful for preparing wound care compositionwhich comprises C robustum which is useful for relievingpostoperative pain and promoting wound healing and bloodcirculation in wound area [67] Further research showed thattaspine was able to promote early phases of wound healing ina dose-dependent manner with no substantial modificationthereafter Its mechanism of action is probably related to itschemotactic properties on fibroblasts and is not mediatedby changes in extracellular matrix [68] Authors summarizedthat taspine opens a pathway of research for new tools to stim-ulate wound repair in the absence of macrophages thereby

helping to better understand the process of wound healingTaspine also exhibited a dose-related cicatrizant effect and amedian effective dose (ED

50) of 0375mgkg which was

nontoxic to human foreskin fibroblasts at concentrationsbelow 150 ngmL and that had no effect on cell proliferation[69]

410 Toxicity N-methylcytisine (6) exhibited teratogenicactivity in the rat embryo culture (REC) an in vitromethod todetect potential teratogens Anagyrine (4) and 120572-isolupanine(12) were not teratogenic in the REC at tested concentrationsTaspine (2) showed high embryotoxicity but no teratogenicactivity in the REC [25] Wu et al have observed that bluecohosh interrupted medaka embryogenesis and produced anabnormal phenotype which identifies blue cohosh as a potentteratogen Moreover the induction of gata2 mRNA followedby edn1 mRNA by BC indicates that the teratogenic responseof blue cohosh is probably mediated by the Gata2-End1signaling pathway [9] Caulosides B (40) and C (21) werereported to have cytotoxicity to developing sea urchinembryos by changing cell permeability It is well-known thatcytotoxic glycoside causes a disturbance of cell membranepermeability that can cause leakage of important cellularcomponents [70 71]

A new born infant whosemother ingested an herbalmed-ication blue cohosh to promote uterine contractions pre-sented with acute myocardial infarction associated with pro-found congestive heart failure and shock [7] One year laterother similar cases were reported [72]Meanwhile Accordingto a survey of midwives in the United States approximately64 of midwives reported using blue cohosh as a labour-inducing aid Severe multiorgan hypoxic injury may occurRecently a review focused on the toxicity of blue cohosh hasbeen reported [2]

5 Pharmacokinetics

Magnoflorine (1) taspine (2) and caulophine (17) were themain components of genus Caulophyllum Several studieshave been carried out to understand the distribution absorp-tion metabolism and excretion of magnoflorine (1) taspine(2) and caulophine (17) using modern analytical methods

51 Pharmacokinetics of Magnoflorine As far as magnoflo-rine is concerned a new sample-preparation method basedon hollow-fiber liquid-phase microextraction (HFLPME)was developed and successfully used for pharmacoki-netic studies of magnoflorine in rat plasma after intra-venous administration The magnoflorine disappears fromrat plasma in accordance with a two-compartment openmodel The plasma concentration of magnoflorine reached apeak immediately after completion of administration thenbegan to decline Without doubt the chromatographic andHFLPME sample-preparation procedures of magnoflorinewill facilitate the development and validation of other meth-ods of analysis of magnoflorine in other biological matrixes[73]


52 Pharmacokinetics of Taspine Lu et al (2008) preparedtaspine solid lipid nanoparticles (2-SLN) and taspine solidlipid nanoparticles with galactoside (2-G2SLN) separatelyusing the film evaporation extrusion method The pharma-cokinetics and liver target efficiency after IV administrationsof 2-SLN and 2-G2SLN to ICR mice were finally compared[59] The pharmacokinetics and tissue distribution afterintravenous administrations of taspine solution and taspineliposome to ICR mice were compared Incorporation intoliposomes prolonged taspine retention within the systemiccirculation and increased its distribution to the spleen andliver but reduced its distribution to the heart and brain [74]

53 Pharmacokinetics of Caulophine Pharmacokinetic stud-ies have shown that caulophine (17) is easily absorbed afteroral administration but it is eliminated from the body slowlyIn fact 125 h after treating rats treated with caulophine thehighest concentration of caulophine was found in the liverTherefore hepatic metabolism is probably the main route forthe in vivo processing of caulophine [75] Two metabolitesincluding glucuronide conjugate and N-oxide of caulophinewere found in rat urine and feces byHPLC-MSMoreover thesame caulophine glucuronide conjugate was observed in ratliver microsomes system However caulophine glucuronideconjugate was not observed in dog liver microsomes [28]

6 Cell Membrane Chromatography forActivity Screening

Cell membrane chromatography (CMC) is a novel bioaffinitychromatographic technique The CMC combined with highperformance liquid chromatography (HPLC) or HPLCMSwill be of great utility in drug discovery using natural medici-nal herbs as a source of novel compounds In reported studiesthe model of CMC in which cell membrane is enriched withcertain receptors is used as the stationary phase was appliedto screen the target components from medicinal herbs [76ndash78] and to investigate the interactions between drug andreceptor [79 80] This system has been successfully appliedto the screening and identification of active components fromC robustum

A combined A431CMC-HPLC method was developedand was successfully applied to recognize separate andidentify target components ldquotaspinerdquo and ldquocaulophinerdquo fromC robustum [81] A combined A431CMC with onlineHPLCMS was also established for identifying active com-ponents from C robustum acting on human epidermalgrowth factor receptor (EGFR) [77] Retention fractions onA431CMCmodel were captured onto an enrichment columnand the components were directly analyzed by combining a10-port column switcher with an LCMS system for separa-tion and preliminary identification Using sorafenib tosylateas a positive control taspine (2) and caulophine (17) wereidentified as the active molecules which could act on theEGFR Other research results showed that taspine (2) was theactive molecule acting on the tumor vasodilatation [52] andmagnoflorine (1) and caulophine (17) were the active

molecules acting on the human 1205721A-adrenoceptor (1205721AAR)

[82]This system has been also successfully applied to inves-

tigate the interactions between active compounds from Crobustum and receptor A new high-expression vascularendothelial growth factor receptor-2 (VEGFR-2) CMCmethod combined with mathematical treatments was pro-posed for evaluating taspine-receptor interactions [83] Acompetitive binding study was performed and the resultsindicate that there are multiple types of binding sites onVEGFR-2 for taspine (2) Following this Du and coworkersdeveloped another new high-expression EGFRCMCmethodto recognize the ligands acting on EGFR specifically andinvestigate the affinity of gefitiniba novel taspine derivativeHMQ1611 to EGFR [84] It has been proven that the CMCmethod combined zonal elution provides a powerful tech-nique for the characterization of HMQ1611 binding to theEGFR

7 Conclusions and Future Prospects

The present review discusses the chemistry and pharma-cological aspects of the genus Caulophyllum and especiallyprovides a detailed analysis of the literature published sincethe year of 2000 The state of the science on Caulophyllumchemistry and pharmacological activity leaves considerableopportunity for future discoveries

Two new classes of alkaloids piperidine-acetophenoneconjugates (13ndash16) and fluorenone (17ndash22) alkaloids havebeen reported from genus Caulophyllum suggesting thatpiperidine-acetophenone conjugates and fluorenone typealkaloids are another two major kinds of metabolites thatexisted in this genus Caulophyllum In addition to commonaglycones (oleanolic acid hederagenin echinocystic acidand caulophyllogenin) eight other kinds of aglycones havebeen found from Caulophyllum species Diverse aglyconesmonosaccharide residues and linked modes of sugars arepossible to form diverse structures of triterpene saponinsfrom genus Caulophyllum Many new compounds have beenidentified in recent years and we are convinced that moretrace constituents with novel structures will be discoveredwith the development of new technology for isolation andidentification

Currently althoughmany purified compounds have beentested for activity which are 1 2 4 5 6 9 11 15 17 26ndash2935 and40 only 2 (taspine) is performed in-depth study on itsanti-tumor and anti-angiogenic mechanisms and could serveas a lead compound in anticancer agent development Mean-while a class of biphenyl derivatives of taspine was designedand synthesized for screening potential novel anticanceragents Besides 2 caulophine (17) was identified as anotheractive molecule which could act on the EGFR and 120572

1AARby combining 120572

1AARCMC and A431CMC with onlineHPLCMS 17 also merits further research to see its actionof mechanisms On the other hand a number of compoundswith novel structure skeleton such as 13ndash16 18ndash22 50 51 5253 and 54 have previously been isolated but no further testshave been performed It is possible that these compounds are


usually overlooked due to their low abundance in Caulophyl-lum Pharmacokinetic study is also limited for compoundsisolated mainly involving three active alkaloids 1 2 and 17So it is very urgent to develop pharmacokinetic study in vivofor other bioactive compounds in genus Caulophyllum

Pharmacological studies carried out on crude extractsand pure metabolites provided pragmatic documents for itstraditional uses and have revealed that this genus is a valuablesource formedicinally importantmoleculesMany importantbiological activities of this genus have been demonstratedsuch as anti-inflammatory and analgesic effects antioxidanteffects antiacetylcholinesterase activity and antitumor et alThough many promising results were confirmed by animalmodels it should be further investigated by clinical trialsRegarding the constituents contributed to medicinal valuesthe findings indicated that alkaloids and triterpene saponinswere regarded as the major constituents in this genus whilepolysaccharides that occurred in the genus are worthy offurther researching their chemical and pharmacologicalactivities [37] However most of the plant extracts used in theabove bioassaywere notwell characterized and this defect ledto the difficulty to reproduce the reported results To add theavailability of primary experimental data suitable analyticaland standardization protocols of plant materials should bedeveloped since these are the ground work for convincingand reproducible pharmacological studies

The toxicity of Caulophyllum species is not negligiblemainly involving the teratogenic effects and inducing heartfailure and shock by ingesting blue cohosh From the view ofcurrent research results alkaloid fractionsmay be responsibleformajor toxicity However exact individuals are required forfurther research by chemical and pharmacological experi-mentsThe future work should be focused on the relationshipbetween clinical effects and side-effects of Caulophyllumextracts to screen a safe and effective dosage Moreover astrict quality control procedure should be adopted to guaran-tee its quality On the other hand the alkaloids and triterpenesaponins are two major kinds of constituents in blue cohoshwhich are easily divided by chromatography methods [22]The individual pharmacological tests for fractions of alkaloidsand triterpene saponins should be considered according tothe traditional and modern uses of Caulophyllum plantsWhether alkaloids and triterpene fractions can be usedseparately in the future according to each medical functionit may be a good choice for Caulophyllum plants for reducingthe drug interaction and enhancing their efficiency

Abbreviations

AChE AntiacetylcholinesteraseAG AglyconesAR AdrenoceptorAra 120572-L-ArabinopyranoseBfgf Basic fibroblast growth factorCMC Cell membrane chromatographyCOX CyclooxygenaseCYP450 Cytochrome P450DPPH 11-Diphenyl-2-picrylhydrazyl

EGFR Epidermal growth factor receptorER Estrogen receptorGCMS Gas chromatographymass spectrumGlc 120573-D-GlucopyranoseHFLPME Hollow-fiber liquid-phase microextractionHPLC High performance liquid chromatographyHPLCMS High performance liquid

chromatographymass spectrumHUVECs Human umbilical vein endothelial cellsIC50 Half-inhibition concentration

IL InterleukiniNOS Inducible nitric oxide synthaseLPS LipopolysaccharideMIC Minimal inhibitory concentrationNF-120581B Nuclear factor kappa BNO Nitric oxideNO2 Nitrogen dioxide

PR Progesterone receptorREC Rat embryo cultureRha 120572-L-RhamnopyranoseTNF-120572 Tumor necrosis factor-120572TV Tumor vasodilatationVEGF Vascular endothelial growth factorVEGFR-2 Vascular endothelial growth factor

receptor-2

Conflict of Interests

The authors declare that they have no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Yong-Gang Xia and Guo-Yu Li equally contributed to thiswork

Acknowledgments

The authorsrsquo work was financially supported by academicvisitor plans for China Scholarship Council (2011823174) theState Key Creative New Drug Project of 12th Five-year Planof China (2013ZX09102019) the National Natural ScienceFoundation of China (81373929) and New Century ExcellentTalents in Heilongjiang Provincial University

References

[1] Tropicos ldquoMissouri Botanical Gardenrdquo 2014 httpwwwtrop-icosorgNameSearchaspx

[2] J I Rader andR S Pawar ldquoPrimary constituents of blue cohoshquantification in dietary supplements and potential for toxicityrdquoAnalytical and Bioanalytical Chemistry vol 405 no 13 pp4409ndash4417 2013

[3] A Ankli E Reich and M Steiner ldquoRapid high-performancethin-layer chromatographic method for detection of 5 adul-teration of black cohoshwithCimicifuga foetidaC heracleifoliaC dahurica or C americanardquo Journal of AOAC Internationalvol 91 no 6 pp 1257ndash1264 2008


[4] A Chevallier The Encyclopedia of Medicinal Plants DorlingKindersley Limited London UK 1996

[5] J-J Dugoua D Perri D Seely E Mills and G Koren ldquoSafetyand efficacy of blue cohosh (Caulophyllum thalictroides) duringpregnancy and lactationrdquo The Canadian Journal of ClinicalPharmacology vol 15 no 1 pp e66ndashe73 2008

[6] M Ganzera H R Dharmaratne N P Nanayakkara and I AKhan ldquoDetermination of saponins and alkaloids in Caulophyl-lum thalictroides (blue cohosh) by high-performance liquidchromatography and evaporative light scattering detectionrdquoPhytochemical Analysis vol 14 no 1 pp 1ndash7 2003

[7] T K Jones and B M Lawson ldquoProfound neonatal congestiveheart failure caused by maternal consumption of blue cohoshherbal medicationrdquoThe Journal of Pediatrics vol 132 no 3 part1 pp 550ndash552 1998

[8] G M Chan and L S Nelson ldquoMore on blue cohosh and peri-natal strokerdquoTheNew England Journal of Medicine vol 351 no21 pp 2239ndash2241 2004

[9] MWu YHu Z Ali I A Khan A J Verlangeiri andA K Das-mahapatra ldquoTeratogenic effects of blue cohosh (Caulophyllumthalictroides) in Japanese medaka (Oryzias latipes) are probablymediated through GATA2EDN1 signaling pathwayrdquo ChemicalResearch in Toxicology vol 23 no 8 pp 1405ndash1416 2010

[10] Jiangsu New Medica College Zhong Yao Da Ci Dian ShanghaiScience and Technology Press Shanghai China 2009 (Chi-nese)

[11] X F Yang Y M Ma H Xing J J Liu and Y X Kang ldquoStudieson triterpene saponins and their biological activity of Caulo-phyllum robustumrdquo Journal Shaannxi University Science Tech-nology vol 31 no 2 pp 62ndash69 2013 (Chinese)

[12] Y C Yang S Z Chen H Y Yang and X Xiong ldquoExperimentalstudy on anti-inflammatory and analgesic effects of three kindsof organic extracts of Leontice robustumrdquo China PracticeMedicine vol 32 no 2 pp 1ndash3 2007 (Chinese)

[13] X-L Wang B-R Liu C-K Chen J-R Wang and S-S LeeldquoFour new fluorenone alkaloids and one new dihydroazaflu-oranthene alkaloid from Caulophyllum robustum MaximrdquoFitoterapia vol 82 no 6 pp 793ndash797 2011

[14] Y Yang M Liu Y Q Zhang and L Lu ldquoScreening of somemedicinal plants for acetylcholinesterase inhibition and antiox-idant activityrdquoChina Journal Experimental TraditionalMedicineFormular vol 19 no 2 pp 213ndash218 2013 (Chinese)

[15] Y Zhang L He L Meng W Luo and X Xu ldquoSuppression oftumor-induced angiogenesis by taspine isolated from Radix etRhizoma Leonticis and its mechanism of action in vitrordquo CancerLetters vol 262 no 1 pp 103ndash113 2008

[16] Y Zhang L He and Y Zhou ldquoTaspine isolated from Radix etRhizoma Leonticis inhibits growth of human umbilical veinendothelial cell (HUVEC) by inducing its apoptosisrdquo Phy-tomedicine vol 15 no 1-2 pp 112ndash119 2008

[17] J Zhang Y Zhang S Zhang S Wang and L He ldquoDiscovery ofnovel taspine derivatives as antiangiogenic agentsrdquo Bioorganicamp Medicinal Chemistry Letters vol 20 no 2 pp 718ndash721 2010

[18] J Zhang Y Zhang Y Shan N Li W Ma and L He ldquoSyn-thesis and preliminary biological evaluation of novel taspinederivatives as anticancer agentsrdquo European Journal of MedicinalChemistry vol 45 no 7 pp 2798ndash2805 2010

[19] Y Zhan Y Zhang Y ChenNWang L Zheng and LHe ldquoActi-vity of taspine isolated from Radix et Rhizoma Leonticis againstestrogen-receptor-positive breast cancerrdquo Fitoterapia vol 82no 6 pp 896ndash902 2011

[20] Z Ali and I A Khan ldquoAlkaloids and saponins from bluecohoshrdquo Phytochemistry vol 69 no 4 pp 1037ndash1042 2008

[21] Y Matsuo K Watanabe and Y Mimaki ldquoTriterpene glycosidesfrom the underground parts of Caulophyllum thalictroidesrdquoJournal of Natural Products vol 72 no 6 pp 1155ndash1160 2009

[22] G Y Li Y G Xia J Liang Y H Zhang and H X Kuang ldquoSim-ultaneous determination of five triterpenoid saponins in theroot of Caulophyllum robustum Maxim by HPLC-ELSDrdquo inPhytochemicals Occurrence in Nature Health Effects and Anti-oxidant Properties chapter 14 Nova Science New York NYUSA 2013

[23] C Stevigny C Bailly and J Quetin-Leclercq ldquoCytotoxic andantitumor potentialities of aporphinoid alkaloidsrdquo CurrentMedicinal Chemistry Anti-Cancer Agents vol 5 no 2 pp 173ndash182 2005

[24] S Ponnala and W W Harding ldquoA route to azafluoranthenenatural products through direct arylationrdquo European Journal ofOrganic Chemistry vol 2013 no 6 pp 1107ndash1115 2013

[25] E J Kennelly T J Flynn E P Mazzola et al ldquoDetecting poten-tial teratogenic alkaloids from blue cohosh rhizomes using anin vitro rat embryo culturerdquo Journal of Natural Products vol 62no 10 pp 1385ndash1389 1999

[26] B Zhao ldquoNematicidal activity of quinolizidine alkaloids and thefunctional group pairs in their molecular structurerdquo Journal ofChemical Ecology vol 25 no 10 pp 2205ndash2214 1999

[27] A Al-Shamma S Drake L Guagliardi L Mitscher and JSwayze ldquoAntimicrobial alkaloids from Boehmeria cylindricardquoPhytochemistry vol 21 no 2 pp 485ndash487 1982

[28] B Wen S Wang L Wang and L He ldquoPreparative chromato-graphic isolation of caulophine from Radix caulophylli and itsmetabolism in vivo and in vitrordquo Analytical Letters vol 44 no7 pp 1277ndash1289 2011

[29] S Lv G Li Y Zuo and H Kuang ldquoDetermination the contentof total saponins from Hong Mao Qirdquo Acta Traditional ChineseMedicine vol 33 no 5 pp 14ndash15 2005 (Chinese)

[30] J-W Jhoo S Sang K He et al ldquoCharacterization of thetriterpene saponins of the roots and rhizomes of blue cohosh(Caulophyllum thalictroides)rdquo Journal of Agricultural and FoodChemistry vol 49 no 12 pp 5969ndash5974 2001

[31] Y M Ma H Xing J J Liu and Y X Kang ldquoStudy on thechemical constitutes of C robustum rootrdquo Journal of AnhuiAgriculturaL Science vol 40 no 2 pp 745ndash747 2012 (Chinese)

[32] I Abe ldquoEnzymatic synthesis of cyclic triterpenesrdquo NaturalProduct Reports vol 24 no 6 pp 1311ndash1331 2007

[33] T Kushiro M Shibuya and Y Ebizuka ldquo120573-amyrin synthasemdashcloning of oxidosqualene cyclase that catalyzes the formationof the most popular triterpene among higher plantsrdquo EuropeanJournal of Biochemistry vol 256 no 1 pp 238ndash244 1998

[34] J Pollier andA Goossens ldquoOleanolic acidrdquo Phytochemistry vol77 pp 10ndash15 2012

[35] A Garcıa-Granados P E Lopez E Melguizo A Parra and YSimeo ldquoPartial synthesis of C-ring derivatives from oleanolicand maslinic acids Formation of several triene systems bychemical and photochemical isomerization processesrdquo Tetrahe-dron vol 60 no 7 pp 1491ndash1503 2004

[36] G Li Y Zhang B Yang et al ldquoLeiyemudanosides A-C threenew bidesmosidic triterpenoid saponins from the roots of Cau-lophyllum robustumrdquo Fitoterapia vol 81 no 3 pp 200ndash2042010

[37] F J Dan Z J Cai Y Tian Z Y Guo and L J Chu ldquoChem-ical constituents of Caulophyllum robustum Maximrdquo China


Traditional Patent Medicine vol 33 no 6 pp 1011ndash1014 2011(Chinese)

[38] Y M Ma H Xing J J Liu and Y X Kang ldquoStudy on thechemical constitutes of C robustum rootrdquo Journal of AnhuiAgricultural Science vol 40 no 2 pp 745ndash747 2012 (Chinese)

[39] F JDan Z L Jun Z J Cai andWYang ldquoStudies on antioxidantactivity of Caulophyllum robustum polysaccharidesrdquo JiangsuAgricultural Science vol 38 no 5 pp 398ndash400 2010 (Chinese)

[40] F J Dan Z J Cai G Z Liu and D Z Zhou ldquoStudies onextraction process of polysaccharides from ldquoHong Mao Qirdquo byultrasonic technologyrdquoChinese Journal ofNewDrugs vol 18 no22 pp 2172ndash2175 2009 (Chinese)

[41] D Pizzirani M Roberti S Grimaudo et al ldquoIdentification ofbiphenyl-based hybrid molecules able to decrease the intracel-lular level of Bcl-2 protein in Bcl-2 overexpressing leukemiacellsrdquo Journal of Medicinal Chemistry vol 52 no 21 pp 6936ndash6940 2009

[42] J Zhang Y Zhang X Pan S Wang and L He ldquoSynthesis andcytotoxic evaluation of novel symmetrical taspine derivatives asanticancer agentsrdquo Medicinal Chemistry vol 7 no 4 pp 286ndash294 2011

[43] H He Y Zhan Y Zhang J Zhang and L He ldquoSynthesis ofnovel taspine diphenyl derivatives as fluorescence probes andinhibitors of breast cancer cell proliferationrdquo Luminescence vol27 no 4 pp 310ndash314 2012

[44] H Z He N Wang J Zhang L Zheng and Y M ZhangldquoTas13D inhibits growth of SMMC-7721 cell via suppressionVEGF and EGF expressionrdquo Asian Pacific Journal of CancerPrevention vol 13 no 5 pp 2009ndash2014 2012

[45] M M Anisimov L I Strigina S I Baranova A L Kulrsquoga andN S Chetyrina ldquoThe antimicrobial activity of the triterpeneglycosides of Caulophyllum robustum maximrdquo Antibiotiki vol17 no 9 pp 834ndash837 1972

[46] Z J Cai F J Dan G H Chen H L Li Y P Xiong and D ZZhou ldquoStudy on in vitro antibacterial activity of CaulophyllumrobustumMaximrdquo Journal of Anhui Agricultural Science vol 36no 35 pp 15541ndash15543 2008 (Chinese)

[47] G P Perdue R N Blomster D A Blake and N R FarnsworthldquoSouth American plants II taspine isolation and anti-infla-mmatory activityrdquo Journal of Pharmaceutical Sciences vol 68no 1 pp 124ndash126 1979

[48] Y Lee J C Jung Z Ali I A Khan and S Oh ldquoAnti-infla-mmatory effect of triterpene saponins isolated fromblue cohosh(Caulophyllum thalictroides)rdquo Evidence-Based Complementaryand Alternative Medicine vol 2012 Article ID 798192 8 pages2012

[49] F J Dan W F Yan L J Chu W Y Lu and Z J Cai ldquoStudieson antioxidant activities of ethanol extract and different polarfractions of Caulophyllum robustum Maxmirdquo Science Technol-ogy Food Industry vol 32 no 1 pp 68ndash74 2011 (Chinese)

[50] J M Rollinger D Schuster E Baier E P Ellmerer T Langerand H Stuppner ldquoTaspine bioactivity-guided isolation andmolecular ligand-target insight of a potent acetylcholinesteraseinhibitor from Magnolia x soulangianardquo Journal of NaturalProducts vol 69 no 9 pp 1341ndash1346 2006

[51] K-W Si J-T Liu L-C He et al ldquoEffects of caulophine oncaffeine-induced cellular injury and calcium homeostasis in ratcardiomyocytesrdquo Basic amp Clinical Pharmacology amp Toxicologyvol 107 no 6 pp 976ndash981 2010

[52] K Gao L-C He and G-D Yang ldquoScreening the effectivecomponent of Leontice robustum by cell membrane chromatog-raphyrdquo Chinese Pharmaceutical Journal vol 38 no 1 pp 14ndash162003 (Chinese)

[53] R Lin J-T Liu L-C He W-J Gan and H-D Yang ldquoEffectsof Leontice robustum extract on the expression of NF-120581B andnitroxide production in ECV injured by H

2O2rdquo Chinese Phar-

maceutical Journal vol 39 no 11 pp 826ndash828 2004 (Chinese)[54] K Si J Liu L He et al ldquoCaulophine protects cardiomyocytes

from oxidative and ischemic injuryrdquo Journal of PharmacologicalSciences vol 113 no 4 pp 368ndash377 2010

[55] H Itokawa Y Ichihara M Mochizuki et al ldquoA cytotoxic sub-stance from Sangre de Gradordquo Chemical amp PharmaceuticalBulletin vol 39 no 4 pp 1041ndash1042 1991

[56] Y Zhang L He and H Wang ldquoInhibitory effect of taspine onmouse S180 sarcoma and its mechanismrdquo China Journal ofChinese Materia Medica vol 32 no 10 pp 953ndash956 2007(Chinese)

[57] Y Zhang L He L Meng and W Luo ldquoTaspine isolated fromRadix et Rhizoma Leonticis inhibits proliferation and migrationof endothelial cells as well as chicken chorioallantoicmembraneneovascularisationrdquoVascular Pharmacology vol 48 no 2-3 pp129ndash137 2008

[58] J Zhao L Zhao W Chen L He and X Li ldquoTaspine downreg-ulates VEGF expression and inhibits proliferation of vascularendothelial cells through PI3 kinase and MAP kinase signalingpathwaysrdquo Biomedicine amp Pharmacotherapy vol 62 no 6 pp383ndash389 2008

[59] W Lu L C He C H Wang Y H Li and S Q Zhang ldquoTheuse of solid lipid nanoparticles to target a lipophilic molecule tothe liver after intravenous administration tomicerdquo InternationalJournal of Biological Macromolecules vol 43 no 3 pp 320ndash3242008

[60] Y Zhang Q Jiang N Wang B Dai Y Chen and L HeldquoEffects of taspine on proliferation and apoptosis by regulatingcaspase-3 expression and the ratio of BaxBcl-2 in A431 cellsrdquoPhytotherapy Research vol 25 no 3 pp 357ndash364 2011

[61] Y Zhang J Zhang B Dai N Wang and L He ldquoAnti-pro-liferative and apoptotic effects of the novel taspine derivativetas41 in the Caco-2 cell linerdquo Environmental Toxicology andPharmacology vol 31 no 3 pp 406ndash415 2011

[62] W Lu B Dai W Ma and Y Zhang ldquoA novel taspine analogHMQ1611 inhibits growth of non-small cell lung cancer byinhibiting angiogenesisrdquoOncology letters vol 4 no 5 pp 1109ndash1113 2012

[63] Y Zhan NWang C Liu Y Chen L Zheng and L He ldquoA noveltaspine derivative HMQ1611 suppresses adhesion migrationand invasion of ZR-75-30 human breast cancer cellsrdquo BreastCancer 2012

[64] Y M Zhang B L Dai L Zheng et al ldquoA novel angiogenesisinhibitor impairs lovo cell survival via targeting against humanVEGFR and its signaling pathway of phosphorylationrdquo CellDeath and Disease vol 3 article e406 2012

[65] V L Madgula Z Ali T Smillie I A Khan L A Walker and SI Khan ldquoAlkaloids and saponins as cytochrome P450 inhibitorsfrom blue cohosh (Caulophyllum thalictroides) in an in vitroassayrdquo Planta Medica vol 75 no 4 pp 329ndash332 2009

[66] W Fayad M Fryknas S Brnjic M H Olofsson R Larssonand S Linder ldquoIdentification of a novel topoisomerase inhibitoreffective in cells overexpressing drug efflux transportersrdquo PLoSONE vol 4 no 10 article e7238 2009


[67] S Tian ldquoPreparing wound care composition comprises egcleaning drying and crushing kamuning acutangular Aniso-dus root soap thorn Leontice Caulophyllum robustum and pur-slane and taking tea oil and then filtering and heatingrdquo Patent2012

[68] B H Porras-Reyes W H Lewis J Roman L Simchowitz andT A Mustoe ldquoEnhancement of wound healing by the alkaloidtaspine defining mechanism of actionrdquo Proceedings of theSociety for Experimental Biology and Medicine vol 203 no 1pp 18ndash25 1993

[69] A J Vaisberg M Milla M C Planas et al ldquoTaspine is thecicatrizant principle in Sangre de Grado extracted from Crotonlechlerirdquo Planta Medica vol 55 no 2 pp 140ndash143 1989

[70] D Aminin I Agafonova S Gnedoi L Strigina and M Anisi-mov ldquoThe effect of pH on biological activity of plant cytotoxincauloside Crdquo Comparative Biochemistry and Physiology AMolecular amp Integrative Physiology vol 122 no 1 pp 45ndash511999

[71] M Anisimov E Shentsova and V Shcheglov ldquoMechanism ofcytotoxic action of some triterpene glycosidesrdquo Toxicon vol 16no 3 pp 207ndash218 1978

[72] J Edmunds ldquoBlue cohosh and newborn myocardial infarc-tionrdquo Midwifery Today with International Midwife no 52 pp34ndash35 1999

[73] J Zhou J B Sun P Zheng et al ldquoOrthogonal array design foroptimization of hollow-fiber-based liquid-phase microextrac-tion combined with high-performance liquid chromatographyfor study of the pharmacokinetics of magnoflorine in ratplasmardquo Analytical and Bioanalytical Chemistry vol 403 no 7pp 1951ndash1960 2012

[74] W Lu L C He and X-M Zeng ldquoHPLC method for the phar-macokinetics and tissue distribution of taspine solution andtaspine liposome after intravenous administrations to micerdquoJournal of Pharmaceutical and Biomedical Analysis vol 46 no1 pp 170ndash176 2008

[75] B Wen S Wang and L He ldquoDevelopment and validation ofa solid phase extraction and LC-MS method for the determi-nation of caulophine in rat plasma and tissue application tostudy its pharmacokineticsrdquo Analytical Letters vol 43 no 18pp 2872ndash2882 2010

[76] C Li L He H Dong and J Jin ldquoScreening for theanti-inflammatory activity of fractions and compounds fromAtractylodes macrocephala koidzrdquo Journal of Ethnopharmacol-ogy vol 114 no 2 pp 212ndash217 2007

[77] S Wang M Sun Y Zhang H Du and L He ldquoA new A431

cellmembrane chromatography and online high performance liq-uid chromatographymass spectrometry method for screen-ing epidermal growth factor receptor antagonists from Radixsophorae flavescentisrdquo Journal of Chromatography A vol 1217no 32 pp 5246ndash5252 2010

[78] X Huang L Kong X Li X Chen M Guo and H Zou ldquoStrat-egy for analysis and screening of bioactive compounds in tra-ditional Chinese medicinesrdquo Journal of Chromatography BAnalytical Technologies in the Biomedical and Life Sciences vol812 no 1-2 pp 71ndash84 2004

[79] W Yu B Yuan X Deng L He Z Youyi and H Qide ldquoThepreparation of HEK293 1205721A or HEK293 1205721B cell membranestationary phase and the chromatographic affinity study ofligands of 120572

1adrenoceptorrdquo Analytical Biochemistry vol 339

no 2 pp 198ndash205 2005[80] B X Yuan J Hou G D Yang L M Zhao and L C He ldquoCom-

parison of determination of drug-muscarinic receptor affinity

by cell-membrane chromatography and by radioligand-bind-ing assay with the cerebrum membrane of the ratrdquo Chro-matographia vol 61 no 7-8 pp 381ndash384 2005

[81] X Hou S Wang J Hou and L He ldquoEstablishment of A431 cellmembrane chromatography-RPLCmethod for screening targetcomponents from Radix Caulophyllirdquo Journal of SeparationScience vol 34 no 5 pp 508ndash513 2011

[82] L Wang J Ren M Sun and S Wang ldquoA combined cellmembrane chromatography and online HPLCMS method forscreening compounds from Radix Caulophylli acting on thehuman 1205721A-adrenoceptorrdquo Journal of Pharmaceutical and Bio-medical Analysis vol 51 no 5 pp 1032ndash1036 2010

[83] H Du S Wang J Ren N Lv and L He ldquoRevealing multi-binding sites for taspine to VEGFR-2 by cell membranechromatography zonal elutionrdquo Journal of Chromatography BAnalytical Technologies in the Biomedical and Life Sciences vol887-888 pp 67ndash72 2012

[84] H Du N Lv S Wang and L He ldquoRapid characterization ofa novel taspine derivative-HMQ1611 binding to EGFR by a cellmembrane chromatographymethodrdquoCombinatorial Chemistryamp High Throughput Screening vol 16 no 4 pp 324ndash329 2013

[85] M S Flom R W Doskotch and J L Beal ldquoIsolation and char-acterization of alkaloids from Caulophyllum thalictroidesrdquo Jour-nal of Pharmaceutical Sciences vol 56 no 11 pp 1515ndash1517 1967

[86] Y Li Z Hu and L He ldquoAn approach to develop binarychromatographic fingerprints of the total alkaloids from Caulo-phyllum robustum by high performance liquid chromatogra-phydiode array detector and gas chromatographymass spec-trometryrdquo Journal of Pharmaceutical and Biomedical Analysisvol 43 no 5 pp 1667ndash1672 2007

[87] F B Power and A H Salway ldquoThe constituents of the rhizomeand roots ofCaulophyllum thalictroidesrdquo Journal of the ChemicalSociety vol 103 pp 191ndash209 1913

[88] S Wang B Wen N Wang J Liu and L He ldquoFluorenone alka-loid fromCaulophyllum robustumMaximwith anti-myocardialischemia activityrdquoArchives of Pharmacal Research vol 32 no 4pp 521ndash526 2009

[89] M Nagasawa S Urayama T Satake and T Murakarni ldquoNewtriterpenoid saponins in the rhizome and roots ofCaulophyllumrobustumMaximrdquo Yakagaku Zasshi vol 88 no 2 pp 321ndash3241968

[90] L I Strigina N S Chetyrina V V Isakov Y N Elkin A KDzizenko and G B Elyakov ldquoCauloside D a new triterpenoidglycoside from Caulophyllum robustum maxim identificationof cauloside Ardquo Phytochemistry vol 14 no 7 pp 1583ndash15861975

[91] L I Strigina N S Chetyrina and V V Isakov ldquoCauloside Ga new triterpene glycoside from Caulophyllum robustum iden-tification of cauloside Crdquo Khimiya Prirodnykh Soedinenii no 5pp 619ndash623 1976

[92] N S Chetyrina and A I Kalinovskii ldquoTriterpene glycosidesof Cauliphyllum robustum The structures of caulosides B andCrdquo Chemistry of Natural Compounds vol 15 no 2 pp 146ndash1481979

[93] C Wegner M Hamburger O Kunert and E Haslinger ldquoTen-sioactive compounds from the aquatic plant Ranunculus flui-tans L (Ranunculaceae)rdquo Helvetica Chimica Acta vol 83 no 7pp 1454ndash1464 2000

[94] L I Strigina N S Chetyrina V V Isakov A K DzizenkoandG B Elyakov ldquoCaulophyllogenin a novel triterpenoid fromroots of Caulophyllum robustumrdquo Phytochemistry vol 13 no 2pp 479ndash480 1974

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


Macroporous or (and) ion exchange resins

Vacuum evaporated

Adjust pH 70

Suspension in

pH 90 with

Organic layer

Vacuum evaporated

Vacuum evaporated

Vacuum evaporated Adjust pH 70

Adjust pH 90

Maceration or reflux using methanolor 70 ethanol for extraction

Total extract

Liquid-liquid extraction Column chromatography

Water layer

Water layer

Water layer

Organic layer

Total alkaloidal fraction

Saponin fraction

Neutral fraction

Specific elution

Saponin fraction

Weak base Fr 1

Nonphenolic Fr 2 Phenolic Fr 3

Note ①5 or 01N HCl in H2O②H2O

CHCl3CHCl3

1N NaOH

CHCl3 or EtOAc

CHCl3 or EtOAc

Suspension in ①

Suspension in ① or ②As followeds ① is mentioned

CHCl3 or EtOAc

n-BuOH

Caulophyllum plant materials

n-BuOH layer

CHCl3 residue

NH4OH CHCl3

Scheme 1 Summary of procedures for isolation of alkaloids and saponins from Caulophyllum plants

Caulophyllum From the opinion of safety of using dietarysupplements of blue cohosh a review dealing with quan-titative methods of primary constituents of blue cohosh indietary supplements has been published [2]However to datethere is no systematic reviewof chemistry pharmacology andactionmechanisms of constituents fromgenusCaulophyllum

In this review the different structures of the alkaloidsand saponins in genusCaulophyllum are described includingnaturally occurring constituents and synthetical taspinederivativesThe present review highlighted the chemistry andpharmacological diversity andmechanism of actionThe aimof this paper is to provide a point of reference on Caulo-phyllum plants for pharmaceutical researchers Furthermorevarious perspectives and existing problems for this genus areoffered for consideration

2 Phytochemistry

Phytochemical research carried out on genus Caulophyllumled to the isolation of alkaloids and triterpence saponins and a

few other classes of secondary metabolites A comprehensivesummary of structures and isolation methods of metabolitesclassified by structural types was given in present reviewScheme 1 summarizes the procedures for crude isolation ofalkaloids and triterpene saponins from genus CaulophyllumThe roots and rhizomes of Caulophyllum plants are extractedwithmethanol or 70 ethanol bymaceration [13 20] or reflux[21] and the combined extracts are concentrated in vacuo todryness Then two schemes are available for acquiring thealkaloid and saponin fractions namely liquid-liquid parti-tion and liquid-solid column chromatography methods [21]Liquid-liquid partition is commonly performed for crudeisolation In most cases the residue is suspended in 5 or01 NHCl in water and then partitioned with EtOAc orCHCl

3to remove neutral constituentsThe aqueous layer was

then removed NH4OH was added to make it basic (pH 9)

and the whole was extracted with EtOAc or CHCl3 The

EtOAc or CHCl3soluble part was evaporated to obtain the

total alkaloidal fraction Moreover total alkaloidal fractionwas able to further liquid-liquid partition to afford weak


HO

O

HO

O

HO

OO

N

O

O

O

N

O

1 2

6

128

NH

OO

OH

O

N

OH14 15

N

O

OH

NH

O

O

O

13

16

OHHO O

O O

N

17

22

10 H11 AC

R

OOH

R

OON

1819 OH

R

OOH

OH

OO

2021

N

O

HO

O

O

9

7

N

OHO

O

OHH 3

N

HN

N

H

H

4 5

N

O

N

N

O

NH

N

N

HO

N

N

HO

N

O

N

HOR N

N

HO

R1

R1

R2

R2

R3

R3

OCH3


N+

N+
































HO

COOH

HO

COOH

OHHO

COOH

OH

HO

COOH

OH

OH CHOHO

COOH

OH

CHOHO

COOH

HOOH

O O O

HO

OH

OH

O O O

HO

COOH

OH

OH

HO

COOHH

OH

HO

H

O

O

H

HO

OH

OH

A B

C D

E

1

3

12

16 28

23

O

AG1 AG2AG3

AG4 AG5 AG6

AG7 AG8 AG9

AG10AG11 AG12

CH2OH

(a)

O

OO

O

OO

OH

O

OO

O

OHO

HO

HOHOHO

HOHOHO

HO

HO

HOHO

HOHO

HOHO

HO HOHO HO

HOHO

HO

HO

OHOHOHOH

OH

OH

OH

OH

OH

OHOH

OH

OH

OHOH

OHOH

OHOH

OH

OHOHO

O

OO

OO

OOO

O

OO O

S1 S2 S3S4

S5 S6 S7S8

(b)





O


OH

HO

OH

O

HO

OH

O

OHHO

OH

O

OH

HO

OH

O

OH

OH

HO

OH

O

HO

HO

OH

O

OH

HO

HO

OH

O

OH

HOOH

O O O

HOOH

O O O

HO

OH

O

OH

OH

OH

OHOH

HOOH

O OOH

HOOH

O O

AG1

AG2

AG3 AG4

AG5 AG6

AG7AG8

AG9

AG12120573-Amyrin






OH

O

55

RO

R56 H57 Glc

HO 59

HO

58

HO60










O

OO

N

O

O

OA

B D

C

Taspine 2

COOOH

O

R

O

O

O

O

OHO

OOC

N

O

O

OHO

OHO

a b

OHO

c

OO

COOd

OHO

COO

+

OBnO

eCOOOBn

O

O

O

O

fCOOOBn

O

O

O O

gCOOOBn

O

O

O

O N

h

i

BnO

O

COO

j

BnO

O

OHO

+e

O

OHO

OOC

O

O

f O

OHO

OOC

O

O

O

O

OBnO

OOC

N

O

Og h

(A)

(B)

D

B

O

H

O

OH

O

O

O

H

O

OH

O

O

1998400

1998400


5998400 6998400 7998400

7998400

8998400 9998400

9998400

9998400

10998400

11998400 12998400 13998400

1499840015998400 16998400

16998400

17998400 18998400

OCH3

2998400

2998400


2O 84 (b) CH

3OH H



2 reflux 71 (e)


4 acetoneH


CH2Cl2 NaBH(OAc)

3 25ndash40 (h) H

2 PdC 97 (i) BnCl K


2O CH

3OH 93 [17]

4 Bioactivity




O

OO

N

O

O

O

Taspine 2

CONHHNOC

O

O

(A)

E

A

B

C

DB D E

HN

OF

Cl

N

ON

HNN

OOH

O

Ha

OOH

O

H b

Br OOBn

O

H c

Br OOBn

O

OH d

Br OOBn

O

Cl e

Br

OOBn

O

NH f

Br

O

O

OBnCONHBnO

HNOCg h

(B)

or

oror By products

ij


OR2

R1O

W =

X =

Y =

Z =

5199840052998400 49998400 + 50998400

32998400ndash4899840031998400

20998400

20998400

3099840029998400

19998400

23998400

19998400

21998400

21998400

22998400

22998400

23998400

24998400

24998400

25998400 26998400 27998400 289984005998400


2 81 (b) BnCl K

2CO3 95 (c) NaH

2PO4 NaClO

2 30 H

2O2 93 (d) SOCl

2 DMF(cat) CH

2Cl2 96 (e)

CH2Cl2 30 CH

3NH2 85 (f) Cu DMF 72 (g) H

2 PdC 97 (h) K

2CO3 DMF (26 64 25 76) (i) K

2CO3 EtOH (27 72 28

59 29 54) [18 42ndash44]








O

O

O

OHN

OCNH

O

HN

NH

abcdefghij


F

Cl

Cl

Cl

O

Br

F

N F

F

O

O

OHNOC

O

O

OO O

NH

O

F

Cl

O

OHNOC

CONH

CONH

O

O

OOHN

NH

O

F

Cl

O

O

NH

O

NH2

NHOO

F

ClO

O

HN

OF

Cl

O

HN

OOH

O

NH

O

O

NH

NH

O

O

319984003299840033998400349984003599840036998400379984003899840039998400409984004199840042998400439984004499840045998400469984004799840048998400

a =

b =

c =

d =

e =

CF3

f =

g =

h =

i =

j =

49998400

50998400

51998400

52998400

R1

R1

R1

R2

R2

R2

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3




2) scaveng-


2




50

















50= 0023 120583gmL) and ECV304 (IC

50=


50= 234 120583gmL) and

ECV304 (IC50




















5 Pharmacokinetics






















Abbreviations






receptor-2





Acknowledgments


References














































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















HO

O

HO

O

HO

OO

N

O

O

O

N

O

1 2

6

128

NH

OO

OH

O

N

OH14 15

N

O

OH

NH

O

O

O

13

16

OHHO O

O O

N

17

22

10 H11 AC

R

OOH

R

OON

1819 OH

R

OOH

OH

OO

2021

N

O

HO

O

O

9

7

N

OHO

O

OHH 3

N

HN

N

H

H

4 5

N

O

N

N

O

NH

N

N

HO

N

N

HO

N

O

N

HOR N

N

HO

R1

R1

R2

R2

R3

R3

OCH3


N+

N+
































HO

COOH

HO

COOH

OHHO

COOH

OH

HO

COOH

OH

OH CHOHO

COOH

OH

CHOHO

COOH

HOOH

O O O

HO

OH

OH

O O O

HO

COOH

OH

OH

HO

COOHH

OH

HO

H

O

O

H

HO

OH

OH

A B

C D

E

1

3

12

16 28

23

O

AG1 AG2AG3

AG4 AG5 AG6

AG7 AG8 AG9

AG10AG11 AG12

CH2OH

(a)

O

OO

O

OO

OH

O

OO

O

OHO

HO

HOHOHO

HOHOHO

HO

HO

HOHO

HOHO

HOHO

HO HOHO HO

HOHO

HO

HO

OHOHOHOH

OH

OH

OH

OH

OH

OHOH

OH

OH

OHOH

OHOH

OHOH

OH

OHOHO

O

OO

OO

OOO

O

OO O

S1 S2 S3S4

S5 S6 S7S8

(b)





O


OH

HO

OH

O

HO

OH

O

OHHO

OH

O

OH

HO

OH

O

OH

OH

HO

OH

O

HO

HO

OH

O

OH

HO

HO

OH

O

OH

HOOH

O O O

HOOH

O O O

HO

OH

O

OH

OH

OH

OHOH

HOOH

O OOH

HOOH

O O

AG1

AG2

AG3 AG4

AG5 AG6

AG7AG8

AG9

AG12120573-Amyrin






OH

O

55

RO

R56 H57 Glc

HO 59

HO

58

HO60










O

OO

N

O

O

OA

B D

C

Taspine 2

COOOH

O

R

O

O

O

O

OHO

OOC

N

O

O

OHO

OHO

a b

OHO

c

OO

COOd

OHO

COO

+

OBnO

eCOOOBn

O

O

O

O

fCOOOBn

O

O

O O

gCOOOBn

O

O

O

O N

h

i

BnO

O

COO

j

BnO

O

OHO

+e

O

OHO

OOC

O

O

f O

OHO

OOC

O

O

O

O

OBnO

OOC

N

O

Og h

(A)

(B)

D

B

O

H

O

OH

O

O

O

H

O

OH

O

O

1998400

1998400


5998400 6998400 7998400

7998400

8998400 9998400

9998400

9998400

10998400

11998400 12998400 13998400

1499840015998400 16998400

16998400

17998400 18998400

OCH3

2998400

2998400


2O 84 (b) CH

3OH H



2 reflux 71 (e)


4 acetoneH


CH2Cl2 NaBH(OAc)

3 25ndash40 (h) H

2 PdC 97 (i) BnCl K


2O CH

3OH 93 [17]

4 Bioactivity




O

OO

N

O

O

O

Taspine 2

CONHHNOC

O

O

(A)

E

A

B

C

DB D E

HN

OF

Cl

N

ON

HNN

OOH

O

Ha

OOH

O

H b

Br OOBn

O

H c

Br OOBn

O

OH d

Br OOBn

O

Cl e

Br

OOBn

O

NH f

Br

O

O

OBnCONHBnO

HNOCg h

(B)

or

oror By products

ij


OR2

R1O

W =

X =

Y =

Z =

5199840052998400 49998400 + 50998400

32998400ndash4899840031998400

20998400

20998400

3099840029998400

19998400

23998400

19998400

21998400

21998400

22998400

22998400

23998400

24998400

24998400

25998400 26998400 27998400 289984005998400


2 81 (b) BnCl K

2CO3 95 (c) NaH

2PO4 NaClO

2 30 H

2O2 93 (d) SOCl

2 DMF(cat) CH

2Cl2 96 (e)

CH2Cl2 30 CH

3NH2 85 (f) Cu DMF 72 (g) H

2 PdC 97 (h) K

2CO3 DMF (26 64 25 76) (i) K

2CO3 EtOH (27 72 28

59 29 54) [18 42ndash44]








O

O

O

OHN

OCNH

O

HN

NH

abcdefghij


F

Cl

Cl

Cl

O

Br

F

N F

F

O

O

OHNOC

O

O

OO O

NH

O

F

Cl

O

OHNOC

CONH

CONH

O

O

OOHN

NH

O

F

Cl

O

O

NH

O

NH2

NHOO

F

ClO

O

HN

OF

Cl

O

HN

OOH

O

NH

O

O

NH

NH

O

O

319984003299840033998400349984003599840036998400379984003899840039998400409984004199840042998400439984004499840045998400469984004799840048998400

a =

b =

c =

d =

e =

CF3

f =

g =

h =

i =

j =

49998400

50998400

51998400

52998400

R1

R1

R1

R2

R2

R2

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3




2) scaveng-


2




50

















50= 0023 120583gmL) and ECV304 (IC

50=


50= 234 120583gmL) and

ECV304 (IC50




















5 Pharmacokinetics






















Abbreviations






receptor-2





Acknowledgments


References














































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of









































HO

COOH

HO

COOH

OHHO

COOH

OH

HO

COOH

OH

OH CHOHO

COOH

OH

CHOHO

COOH

HOOH

O O O

HO

OH

OH

O O O

HO

COOH

OH

OH

HO

COOHH

OH

HO

H

O

O

H

HO

OH

OH

A B

C D

E

1

3

12

16 28

23

O

AG1 AG2AG3

AG4 AG5 AG6

AG7 AG8 AG9

AG10AG11 AG12

CH2OH

(a)

O

OO

O

OO

OH

O

OO

O

OHO

HO

HOHOHO

HOHOHO

HO

HO

HOHO

HOHO

HOHO

HO HOHO HO

HOHO

HO

HO

OHOHOHOH

OH

OH

OH

OH

OH

OHOH

OH

OH

OHOH

OHOH

OHOH

OH

OHOHO

O

OO

OO

OOO

O

OO O

S1 S2 S3S4

S5 S6 S7S8

(b)





O


OH

HO

OH

O

HO

OH

O

OHHO

OH

O

OH

HO

OH

O

OH

OH

HO

OH

O

HO

HO

OH

O

OH

HO

HO

OH

O

OH

HOOH

O O O

HOOH

O O O

HO

OH

O

OH

OH

OH

OHOH

HOOH

O OOH

HOOH

O O

AG1

AG2

AG3 AG4

AG5 AG6

AG7AG8

AG9

AG12120573-Amyrin






OH

O

55

RO

R56 H57 Glc

HO 59

HO

58

HO60










O

OO

N

O

O

OA

B D

C

Taspine 2

COOOH

O

R

O

O

O

O

OHO

OOC

N

O

O

OHO

OHO

a b

OHO

c

OO

COOd

OHO

COO

+

OBnO

eCOOOBn

O

O

O

O

fCOOOBn

O

O

O O

gCOOOBn

O

O

O

O N

h

i

BnO

O

COO

j

BnO

O

OHO

+e

O

OHO

OOC

O

O

f O

OHO

OOC

O

O

O

O

OBnO

OOC

N

O

Og h

(A)

(B)

D

B

O

H

O

OH

O

O

O

H

O

OH

O

O

1998400

1998400


5998400 6998400 7998400

7998400

8998400 9998400

9998400

9998400

10998400

11998400 12998400 13998400

1499840015998400 16998400

16998400

17998400 18998400

OCH3

2998400

2998400


2O 84 (b) CH

3OH H



2 reflux 71 (e)


4 acetoneH


CH2Cl2 NaBH(OAc)

3 25ndash40 (h) H

2 PdC 97 (i) BnCl K


2O CH

3OH 93 [17]

4 Bioactivity




O

OO

N

O

O

O

Taspine 2

CONHHNOC

O

O

(A)

E

A

B

C

DB D E

HN

OF

Cl

N

ON

HNN

OOH

O

Ha

OOH

O

H b

Br OOBn

O

H c

Br OOBn

O

OH d

Br OOBn

O

Cl e

Br

OOBn

O

NH f

Br

O

O

OBnCONHBnO

HNOCg h

(B)

or

oror By products

ij


OR2

R1O

W =

X =

Y =

Z =

5199840052998400 49998400 + 50998400

32998400ndash4899840031998400

20998400

20998400

3099840029998400

19998400

23998400

19998400

21998400

21998400

22998400

22998400

23998400

24998400

24998400

25998400 26998400 27998400 289984005998400


2 81 (b) BnCl K

2CO3 95 (c) NaH

2PO4 NaClO

2 30 H

2O2 93 (d) SOCl

2 DMF(cat) CH

2Cl2 96 (e)

CH2Cl2 30 CH

3NH2 85 (f) Cu DMF 72 (g) H

2 PdC 97 (h) K

2CO3 DMF (26 64 25 76) (i) K

2CO3 EtOH (27 72 28

59 29 54) [18 42ndash44]








O

O

O

OHN

OCNH

O

HN

NH

abcdefghij


F

Cl

Cl

Cl

O

Br

F

N F

F

O

O

OHNOC

O

O

OO O

NH

O

F

Cl

O

OHNOC

CONH

CONH

O

O

OOHN

NH

O

F

Cl

O

O

NH

O

NH2

NHOO

F

ClO

O

HN

OF

Cl

O

HN

OOH

O

NH

O

O

NH

NH

O

O

319984003299840033998400349984003599840036998400379984003899840039998400409984004199840042998400439984004499840045998400469984004799840048998400

a =

b =

c =

d =

e =

CF3

f =

g =

h =

i =

j =

49998400

50998400

51998400

52998400

R1

R1

R1

R2

R2

R2

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3




2) scaveng-


2




50

















50= 0023 120583gmL) and ECV304 (IC

50=


50= 234 120583gmL) and

ECV304 (IC50




















5 Pharmacokinetics






















Abbreviations






receptor-2





Acknowledgments


References














































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






























HO

COOH

HO

COOH

OHHO

COOH

OH

HO

COOH

OH

OH CHOHO

COOH

OH

CHOHO

COOH

HOOH

O O O

HO

OH

OH

O O O

HO

COOH

OH

OH

HO

COOHH

OH

HO

H

O

O

H

HO

OH

OH

A B

C D

E

1

3

12

16 28

23

O

AG1 AG2AG3

AG4 AG5 AG6

AG7 AG8 AG9

AG10AG11 AG12

CH2OH

(a)

O

OO

O

OO

OH

O

OO

O

OHO

HO

HOHOHO

HOHOHO

HO

HO

HOHO

HOHO

HOHO

HO HOHO HO

HOHO

HO

HO

OHOHOHOH

OH

OH

OH

OH

OH

OHOH

OH

OH

OHOH

OHOH

OHOH

OH

OHOHO

O

OO

OO

OOO

O

OO O

S1 S2 S3S4

S5 S6 S7S8

(b)





O


OH

HO

OH

O

HO

OH

O

OHHO

OH

O

OH

HO

OH

O

OH

OH

HO

OH

O

HO

HO

OH

O

OH

HO

HO

OH

O

OH

HOOH

O O O

HOOH

O O O

HO

OH

O

OH

OH

OH

OHOH

HOOH

O OOH

HOOH

O O

AG1

AG2

AG3 AG4

AG5 AG6

AG7AG8

AG9

AG12120573-Amyrin






OH

O

55

RO

R56 H57 Glc

HO 59

HO

58

HO60










O

OO

N

O

O

OA

B D

C

Taspine 2

COOOH

O

R

O

O

O

O

OHO

OOC

N

O

O

OHO

OHO

a b

OHO

c

OO

COOd

OHO

COO

+

OBnO

eCOOOBn

O

O

O

O

fCOOOBn

O

O

O O

gCOOOBn

O

O

O

O N

h

i

BnO

O

COO

j

BnO

O

OHO

+e

O

OHO

OOC

O

O

f O

OHO

OOC

O

O

O

O

OBnO

OOC

N

O

Og h

(A)

(B)

D

B

O

H

O

OH

O

O

O

H

O

OH

O

O

1998400

1998400


5998400 6998400 7998400

7998400

8998400 9998400

9998400

9998400

10998400

11998400 12998400 13998400

1499840015998400 16998400

16998400

17998400 18998400

OCH3

2998400

2998400


2O 84 (b) CH

3OH H



2 reflux 71 (e)


4 acetoneH


CH2Cl2 NaBH(OAc)

3 25ndash40 (h) H

2 PdC 97 (i) BnCl K


2O CH

3OH 93 [17]

4 Bioactivity




O

OO

N

O

O

O

Taspine 2

CONHHNOC

O

O

(A)

E

A

B

C

DB D E

HN

OF

Cl

N

ON

HNN

OOH

O

Ha

OOH

O

H b

Br OOBn

O

H c

Br OOBn

O

OH d

Br OOBn

O

Cl e

Br

OOBn

O

NH f

Br

O

O

OBnCONHBnO

HNOCg h

(B)

or

oror By products

ij


OR2

R1O

W =

X =

Y =

Z =

5199840052998400 49998400 + 50998400

32998400ndash4899840031998400

20998400

20998400

3099840029998400

19998400

23998400

19998400

21998400

21998400

22998400

22998400

23998400

24998400

24998400

25998400 26998400 27998400 289984005998400


2 81 (b) BnCl K

2CO3 95 (c) NaH

2PO4 NaClO

2 30 H

2O2 93 (d) SOCl

2 DMF(cat) CH

2Cl2 96 (e)

CH2Cl2 30 CH

3NH2 85 (f) Cu DMF 72 (g) H

2 PdC 97 (h) K

2CO3 DMF (26 64 25 76) (i) K

2CO3 EtOH (27 72 28

59 29 54) [18 42ndash44]








O

O

O

OHN

OCNH

O

HN

NH

abcdefghij


F

Cl

Cl

Cl

O

Br

F

N F

F

O

O

OHNOC

O

O

OO O

NH

O

F

Cl

O

OHNOC

CONH

CONH

O

O

OOHN

NH

O

F

Cl

O

O

NH

O

NH2

NHOO

F

ClO

O

HN

OF

Cl

O

HN

OOH

O

NH

O

O

NH

NH

O

O

319984003299840033998400349984003599840036998400379984003899840039998400409984004199840042998400439984004499840045998400469984004799840048998400

a =

b =

c =

d =

e =

CF3

f =

g =

h =

i =

j =

49998400

50998400

51998400

52998400

R1

R1

R1

R2

R2

R2

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3




2) scaveng-


2




50

















50= 0023 120583gmL) and ECV304 (IC

50=


50= 234 120583gmL) and

ECV304 (IC50




















5 Pharmacokinetics






















Abbreviations






receptor-2





Acknowledgments


References














































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















HO

COOH

HO

COOH

OHHO

COOH

OH

HO

COOH

OH

OH CHOHO

COOH

OH

CHOHO

COOH

HOOH

O O O

HO

OH

OH

O O O

HO

COOH

OH

OH

HO

COOHH

OH

HO

H

O

O

H

HO

OH

OH

A B

C D

E

1

3

12

16 28

23

O

AG1 AG2AG3

AG4 AG5 AG6

AG7 AG8 AG9

AG10AG11 AG12

CH2OH

(a)

O

OO

O

OO

OH

O

OO

O

OHO

HO

HOHOHO

HOHOHO

HO

HO

HOHO

HOHO

HOHO

HO HOHO HO

HOHO

HO

HO

OHOHOHOH

OH

OH

OH

OH

OH

OHOH

OH

OH

OHOH

OHOH

OHOH

OH

OHOHO

O

OO

OO

OOO

O

OO O

S1 S2 S3S4

S5 S6 S7S8

(b)





O


OH

HO

OH

O

HO

OH

O

OHHO

OH

O

OH

HO

OH

O

OH

OH

HO

OH

O

HO

HO

OH

O

OH

HO

HO

OH

O

OH

HOOH

O O O

HOOH

O O O

HO

OH

O

OH

OH

OH

OHOH

HOOH

O OOH

HOOH

O O

AG1

AG2

AG3 AG4

AG5 AG6

AG7AG8

AG9

AG12120573-Amyrin






OH

O

55

RO

R56 H57 Glc

HO 59

HO

58

HO60










O

OO

N

O

O

OA

B D

C

Taspine 2

COOOH

O

R

O

O

O

O

OHO

OOC

N

O

O

OHO

OHO

a b

OHO

c

OO

COOd

OHO

COO

+

OBnO

eCOOOBn

O

O

O

O

fCOOOBn

O

O

O O

gCOOOBn

O

O

O

O N

h

i

BnO

O

COO

j

BnO

O

OHO

+e

O

OHO

OOC

O

O

f O

OHO

OOC

O

O

O

O

OBnO

OOC

N

O

Og h

(A)

(B)

D

B

O

H

O

OH

O

O

O

H

O

OH

O

O

1998400

1998400


5998400 6998400 7998400

7998400

8998400 9998400

9998400

9998400

10998400

11998400 12998400 13998400

1499840015998400 16998400

16998400

17998400 18998400

OCH3

2998400

2998400


2O 84 (b) CH

3OH H



2 reflux 71 (e)


4 acetoneH


CH2Cl2 NaBH(OAc)

3 25ndash40 (h) H

2 PdC 97 (i) BnCl K


2O CH

3OH 93 [17]

4 Bioactivity




O

OO

N

O

O

O

Taspine 2

CONHHNOC

O

O

(A)

E

A

B

C

DB D E

HN

OF

Cl

N

ON

HNN

OOH

O

Ha

OOH

O

H b

Br OOBn

O

H c

Br OOBn

O

OH d

Br OOBn

O

Cl e

Br

OOBn

O

NH f

Br

O

O

OBnCONHBnO

HNOCg h

(B)

or

oror By products

ij


OR2

R1O

W =

X =

Y =

Z =

5199840052998400 49998400 + 50998400

32998400ndash4899840031998400

20998400

20998400

3099840029998400

19998400

23998400

19998400

21998400

21998400

22998400

22998400

23998400

24998400

24998400

25998400 26998400 27998400 289984005998400


2 81 (b) BnCl K

2CO3 95 (c) NaH

2PO4 NaClO

2 30 H

2O2 93 (d) SOCl

2 DMF(cat) CH

2Cl2 96 (e)

CH2Cl2 30 CH

3NH2 85 (f) Cu DMF 72 (g) H

2 PdC 97 (h) K

2CO3 DMF (26 64 25 76) (i) K

2CO3 EtOH (27 72 28

59 29 54) [18 42ndash44]








O

O

O

OHN

OCNH

O

HN

NH

abcdefghij


F

Cl

Cl

Cl

O

Br

F

N F

F

O

O

OHNOC

O

O

OO O

NH

O

F

Cl

O

OHNOC

CONH

CONH

O

O

OOHN

NH

O

F

Cl

O

O

NH

O

NH2

NHOO

F

ClO

O

HN

OF

Cl

O

HN

OOH

O

NH

O

O

NH

NH

O

O

319984003299840033998400349984003599840036998400379984003899840039998400409984004199840042998400439984004499840045998400469984004799840048998400

a =

b =

c =

d =

e =

CF3

f =

g =

h =

i =

j =

49998400

50998400

51998400

52998400

R1

R1

R1

R2

R2

R2

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3




2) scaveng-


2




50

















50= 0023 120583gmL) and ECV304 (IC

50=


50= 234 120583gmL) and

ECV304 (IC50




















5 Pharmacokinetics






















Abbreviations






receptor-2





Acknowledgments


References














































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















O


OH

HO

OH

O

HO

OH

O

OHHO

OH

O

OH

HO

OH

O

OH

OH

HO

OH

O

HO

HO

OH

O

OH

HO

HO

OH

O

OH

HOOH

O O O

HOOH

O O O

HO

OH

O

OH

OH

OH

OHOH

HOOH

O OOH

HOOH

O O

AG1

AG2

AG3 AG4

AG5 AG6

AG7AG8

AG9

AG12120573-Amyrin






OH

O

55

RO

R56 H57 Glc

HO 59

HO

58

HO60










O

OO

N

O

O

OA

B D

C

Taspine 2

COOOH

O

R

O

O

O

O

OHO

OOC

N

O

O

OHO

OHO

a b

OHO

c

OO

COOd

OHO

COO

+

OBnO

eCOOOBn

O

O

O

O

fCOOOBn

O

O

O O

gCOOOBn

O

O

O

O N

h

i

BnO

O

COO

j

BnO

O

OHO

+e

O

OHO

OOC

O

O

f O

OHO

OOC

O

O

O

O

OBnO

OOC

N

O

Og h

(A)

(B)

D

B

O

H

O

OH

O

O

O

H

O

OH

O

O

1998400

1998400


5998400 6998400 7998400

7998400

8998400 9998400

9998400

9998400

10998400

11998400 12998400 13998400

1499840015998400 16998400

16998400

17998400 18998400

OCH3

2998400

2998400


2O 84 (b) CH

3OH H



2 reflux 71 (e)


4 acetoneH


CH2Cl2 NaBH(OAc)

3 25ndash40 (h) H

2 PdC 97 (i) BnCl K


2O CH

3OH 93 [17]

4 Bioactivity




O

OO

N

O

O

O

Taspine 2

CONHHNOC

O

O

(A)

E

A

B

C

DB D E

HN

OF

Cl

N

ON

HNN

OOH

O

Ha

OOH

O

H b

Br OOBn

O

H c

Br OOBn

O

OH d

Br OOBn

O

Cl e

Br

OOBn

O

NH f

Br

O

O

OBnCONHBnO

HNOCg h

(B)

or

oror By products

ij


OR2

R1O

W =

X =

Y =

Z =

5199840052998400 49998400 + 50998400

32998400ndash4899840031998400

20998400

20998400

3099840029998400

19998400

23998400

19998400

21998400

21998400

22998400

22998400

23998400

24998400

24998400

25998400 26998400 27998400 289984005998400


2 81 (b) BnCl K

2CO3 95 (c) NaH

2PO4 NaClO

2 30 H

2O2 93 (d) SOCl

2 DMF(cat) CH

2Cl2 96 (e)

CH2Cl2 30 CH

3NH2 85 (f) Cu DMF 72 (g) H

2 PdC 97 (h) K

2CO3 DMF (26 64 25 76) (i) K

2CO3 EtOH (27 72 28

59 29 54) [18 42ndash44]








O

O

O

OHN

OCNH

O

HN

NH

abcdefghij


F

Cl

Cl

Cl

O

Br

F

N F

F

O

O

OHNOC

O

O

OO O

NH

O

F

Cl

O

OHNOC

CONH

CONH

O

O

OOHN

NH

O

F

Cl

O

O

NH

O

NH2

NHOO

F

ClO

O

HN

OF

Cl

O

HN

OOH

O

NH

O

O

NH

NH

O

O

319984003299840033998400349984003599840036998400379984003899840039998400409984004199840042998400439984004499840045998400469984004799840048998400

a =

b =

c =

d =

e =

CF3

f =

g =

h =

i =

j =

49998400

50998400

51998400

52998400

R1

R1

R1

R2

R2

R2

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3




2) scaveng-


2




50

















50= 0023 120583gmL) and ECV304 (IC

50=


50= 234 120583gmL) and

ECV304 (IC50




















5 Pharmacokinetics






















Abbreviations






receptor-2





Acknowledgments


References














































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















OH

O

55

RO

R56 H57 Glc

HO 59

HO

58

HO60










O

OO

N

O

O

OA

B D

C

Taspine 2

COOOH

O

R

O

O

O

O

OHO

OOC

N

O

O

OHO

OHO

a b

OHO

c

OO

COOd

OHO

COO

+

OBnO

eCOOOBn

O

O

O

O

fCOOOBn

O

O

O O

gCOOOBn

O

O

O

O N

h

i

BnO

O

COO

j

BnO

O

OHO

+e

O

OHO

OOC

O

O

f O

OHO

OOC

O

O

O

O

OBnO

OOC

N

O

Og h

(A)

(B)

D

B

O

H

O

OH

O

O

O

H

O

OH

O

O

1998400

1998400


5998400 6998400 7998400

7998400

8998400 9998400

9998400

9998400

10998400

11998400 12998400 13998400

1499840015998400 16998400

16998400

17998400 18998400

OCH3

2998400

2998400


2O 84 (b) CH

3OH H



2 reflux 71 (e)


4 acetoneH


CH2Cl2 NaBH(OAc)

3 25ndash40 (h) H

2 PdC 97 (i) BnCl K


2O CH

3OH 93 [17]

4 Bioactivity




O

OO

N

O

O

O

Taspine 2

CONHHNOC

O

O

(A)

E

A

B

C

DB D E

HN

OF

Cl

N

ON

HNN

OOH

O

Ha

OOH

O

H b

Br OOBn

O

H c

Br OOBn

O

OH d

Br OOBn

O

Cl e

Br

OOBn

O

NH f

Br

O

O

OBnCONHBnO

HNOCg h

(B)

or

oror By products

ij


OR2

R1O

W =

X =

Y =

Z =

5199840052998400 49998400 + 50998400

32998400ndash4899840031998400

20998400

20998400

3099840029998400

19998400

23998400

19998400

21998400

21998400

22998400

22998400

23998400

24998400

24998400

25998400 26998400 27998400 289984005998400


2 81 (b) BnCl K

2CO3 95 (c) NaH

2PO4 NaClO

2 30 H

2O2 93 (d) SOCl

2 DMF(cat) CH

2Cl2 96 (e)

CH2Cl2 30 CH

3NH2 85 (f) Cu DMF 72 (g) H

2 PdC 97 (h) K

2CO3 DMF (26 64 25 76) (i) K

2CO3 EtOH (27 72 28

59 29 54) [18 42ndash44]








O

O

O

OHN

OCNH

O

HN

NH

abcdefghij


F

Cl

Cl

Cl

O

Br

F

N F

F

O

O

OHNOC

O

O

OO O

NH

O

F

Cl

O

OHNOC

CONH

CONH

O

O

OOHN

NH

O

F

Cl

O

O

NH

O

NH2

NHOO

F

ClO

O

HN

OF

Cl

O

HN

OOH

O

NH

O

O

NH

NH

O

O

319984003299840033998400349984003599840036998400379984003899840039998400409984004199840042998400439984004499840045998400469984004799840048998400

a =

b =

c =

d =

e =

CF3

f =

g =

h =

i =

j =

49998400

50998400

51998400

52998400

R1

R1

R1

R2

R2

R2

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3




2) scaveng-


2




50

















50= 0023 120583gmL) and ECV304 (IC

50=


50= 234 120583gmL) and

ECV304 (IC50




















5 Pharmacokinetics






















Abbreviations






receptor-2





Acknowledgments


References














































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















O

OO

N

O

O

OA

B D

C

Taspine 2

COOOH

O

R

O

O

O

O

OHO

OOC

N

O

O

OHO

OHO

a b

OHO

c

OO

COOd

OHO

COO

+

OBnO

eCOOOBn

O

O

O

O

fCOOOBn

O

O

O O

gCOOOBn

O

O

O

O N

h

i

BnO

O

COO

j

BnO

O

OHO

+e

O

OHO

OOC

O

O

f O

OHO

OOC

O

O

O

O

OBnO

OOC

N

O

Og h

(A)

(B)

D

B

O

H

O

OH

O

O

O

H

O

OH

O

O

1998400

1998400


5998400 6998400 7998400

7998400

8998400 9998400

9998400

9998400

10998400

11998400 12998400 13998400

1499840015998400 16998400

16998400

17998400 18998400

OCH3

2998400

2998400


2O 84 (b) CH

3OH H



2 reflux 71 (e)


4 acetoneH


CH2Cl2 NaBH(OAc)

3 25ndash40 (h) H

2 PdC 97 (i) BnCl K


2O CH

3OH 93 [17]

4 Bioactivity




O

OO

N

O

O

O

Taspine 2

CONHHNOC

O

O

(A)

E

A

B

C

DB D E

HN

OF

Cl

N

ON

HNN

OOH

O

Ha

OOH

O

H b

Br OOBn

O

H c

Br OOBn

O

OH d

Br OOBn

O

Cl e

Br

OOBn

O

NH f

Br

O

O

OBnCONHBnO

HNOCg h

(B)

or

oror By products

ij


OR2

R1O

W =

X =

Y =

Z =

5199840052998400 49998400 + 50998400

32998400ndash4899840031998400

20998400

20998400

3099840029998400

19998400

23998400

19998400

21998400

21998400

22998400

22998400

23998400

24998400

24998400

25998400 26998400 27998400 289984005998400


2 81 (b) BnCl K

2CO3 95 (c) NaH

2PO4 NaClO

2 30 H

2O2 93 (d) SOCl

2 DMF(cat) CH

2Cl2 96 (e)

CH2Cl2 30 CH

3NH2 85 (f) Cu DMF 72 (g) H

2 PdC 97 (h) K

2CO3 DMF (26 64 25 76) (i) K

2CO3 EtOH (27 72 28

59 29 54) [18 42ndash44]








O

O

O

OHN

OCNH

O

HN

NH

abcdefghij


F

Cl

Cl

Cl

O

Br

F

N F

F

O

O

OHNOC

O

O

OO O

NH

O

F

Cl

O

OHNOC

CONH

CONH

O

O

OOHN

NH

O

F

Cl

O

O

NH

O

NH2

NHOO

F

ClO

O

HN

OF

Cl

O

HN

OOH

O

NH

O

O

NH

NH

O

O

319984003299840033998400349984003599840036998400379984003899840039998400409984004199840042998400439984004499840045998400469984004799840048998400

a =

b =

c =

d =

e =

CF3

f =

g =

h =

i =

j =

49998400

50998400

51998400

52998400

R1

R1

R1

R2

R2

R2

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3




2) scaveng-


2




50

















50= 0023 120583gmL) and ECV304 (IC

50=


50= 234 120583gmL) and

ECV304 (IC50




















5 Pharmacokinetics






















Abbreviations






receptor-2





Acknowledgments


References














































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















O

OO

N

O

O

O

Taspine 2

CONHHNOC

O

O

(A)

E

A

B

C

DB D E

HN

OF

Cl

N

ON

HNN

OOH

O

Ha

OOH

O

H b

Br OOBn

O

H c

Br OOBn

O

OH d

Br OOBn

O

Cl e

Br

OOBn

O

NH f

Br

O

O

OBnCONHBnO

HNOCg h

(B)

or

oror By products

ij


OR2

R1O

W =

X =

Y =

Z =

5199840052998400 49998400 + 50998400

32998400ndash4899840031998400

20998400

20998400

3099840029998400

19998400

23998400

19998400

21998400

21998400

22998400

22998400

23998400

24998400

24998400

25998400 26998400 27998400 289984005998400


2 81 (b) BnCl K

2CO3 95 (c) NaH

2PO4 NaClO

2 30 H

2O2 93 (d) SOCl

2 DMF(cat) CH

2Cl2 96 (e)

CH2Cl2 30 CH

3NH2 85 (f) Cu DMF 72 (g) H

2 PdC 97 (h) K

2CO3 DMF (26 64 25 76) (i) K

2CO3 EtOH (27 72 28

59 29 54) [18 42ndash44]








O

O

O

OHN

OCNH

O

HN

NH

abcdefghij


F

Cl

Cl

Cl

O

Br

F

N F

F

O

O

OHNOC

O

O

OO O

NH

O

F

Cl

O

OHNOC

CONH

CONH

O

O

OOHN

NH

O

F

Cl

O

O

NH

O

NH2

NHOO

F

ClO

O

HN

OF

Cl

O

HN

OOH

O

NH

O

O

NH

NH

O

O

319984003299840033998400349984003599840036998400379984003899840039998400409984004199840042998400439984004499840045998400469984004799840048998400

a =

b =

c =

d =

e =

CF3

f =

g =

h =

i =

j =

49998400

50998400

51998400

52998400

R1

R1

R1

R2

R2

R2

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3




2) scaveng-


2




50

















50= 0023 120583gmL) and ECV304 (IC

50=


50= 234 120583gmL) and

ECV304 (IC50




















5 Pharmacokinetics






















Abbreviations






receptor-2





Acknowledgments


References














































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















O

O

O

OHN

OCNH

O

HN

NH

abcdefghij


F

Cl

Cl

Cl

O

Br

F

N F

F

O

O

OHNOC

O

O

OO O

NH

O

F

Cl

O

OHNOC

CONH

CONH

O

O

OOHN

NH

O

F

Cl

O

O

NH

O

NH2

NHOO

F

ClO

O

HN

OF

Cl

O

HN

OOH

O

NH

O

O

NH

NH

O

O

319984003299840033998400349984003599840036998400379984003899840039998400409984004199840042998400439984004499840045998400469984004799840048998400

a =

b =

c =

d =

e =

CF3

f =

g =

h =

i =

j =

49998400

50998400

51998400

52998400

R1

R1

R1

R2

R2

R2

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3

CH3




2) scaveng-


2




50

















50= 0023 120583gmL) and ECV304 (IC

50=


50= 234 120583gmL) and

ECV304 (IC50




















5 Pharmacokinetics






















Abbreviations






receptor-2





Acknowledgments


References














































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


























50= 0023 120583gmL) and ECV304 (IC

50=


50= 234 120583gmL) and

ECV304 (IC50




















5 Pharmacokinetics






















Abbreviations






receptor-2





Acknowledgments


References














































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






























5 Pharmacokinetics






















Abbreviations






receptor-2





Acknowledgments


References














































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



































Abbreviations






receptor-2





Acknowledgments


References














































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















Abbreviations






receptor-2





Acknowledgments


References














































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


























































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of























































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Documents

Review Article Genus Caulophyllum : An Overview of