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Simultaneous Determinationof Senecionine, Senlciphylline,and Senecionine N-Oxide inGynura segetum by RP-HPLCMing Yang a b , Jian-Yu Li a , Xian-Yi Li a , Long-BoCong a b , Jun Zhu c , Huan Xie a , Hai-Long Yuan a &Xiao-He Xiao aa Institute of People's Liberation Army of ChineseMedicine , Beijing, Chinab Key Laboratory of Modern Preparation ofTraditional Chinese Army, Ministry of Education,Jiangxi University of Traditional Chinese Army ,Nanchang, Chinac Hangzhou Vocational and Technical College,Chemical Engineering Department , Hangzhou, ChinaPublished online: 10 Aug 2009.

To cite this article: Ming Yang , Jian-Yu Li , Xian-Yi Li , Long-Bo Cong , Jun Zhu ,Huan Xie , Hai-Long Yuan & Xiao-He Xiao (2009) Simultaneous Determination ofSenecionine, Senlciphylline, and Senecionine N-Oxide in Gynura segetum by RP-HPLC,Analytical Letters, 42:12, 1820-1830, DOI: 10.1080/00032710903060693

To link to this article: http://dx.doi.org/10.1080/00032710903060693

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CHROMATOGRAPHY

Simultaneous Determination of Senecionine,Senlciphylline, and Senecionine N-Oxide

in Gynura segetum by RP-HPLC

Ming Yang,1,2 Jian-Yu Li,1 Xian-Yi Li,1 Long-Bo Cong,1,2 Jun Zhu,3

Huan Xie,1 Hai-Long Yuan,1 and Xiao-He Xiao1

1Institute of People’s Liberation Army of Chinese Medicine, Beijing, China2Key Laboratory of Modern Preparation of Traditional Chinese Army,Ministry of Education, Jiangxi University of Traditional Chinese Army,

Nanchang, China3Hangzhou Vocational and Technical College, Chemical Engineering

Department, Hangzhou, China

Abstract: A rapid reversed-phase high-performance liquid chromatographic(RP-HPLC) method was established for simultaneous determination of senecio-nine, senlciphylline, and senecionine N-oxide in a famous traditional Chinesemedicine, Gynura segetum, which has been commonly used for hemostasis. TheHPLC assay was performed on a Kromasil KR100-5 C18 column(25 cm� 4.6mm, 5 mm) with mobile phase composed of acetonitrile and 0.2%phosphoric acid–triethylamine within 40min. The detection wavelength was220 nm. All the compounds showed good linearity (r2> 0.9997). The methodwas reproducible with intra- and interday variation less than 2.82%. The recoveryof the assay was in the range of 96.55–103.88%. The method was successfullyapplied to the quantification of three constituents in 15 Gynura segetum samples

Received 18 December 2008; accepted 4 May 2009.The authors appreciate the financial support of the Natural Science Fund of

Beijing, China, under Grant No. 7072076.Address correspondence to Hai-Long Yuan and Xiao-He Xiao, 302 Hospital of

People’s Liberation Army, Institute of People’s Liberation Army of Chinese Medi-cine, Beijing 100039, China. E-mail: yhlpharm@126.com; pharmacy302@126.com

Analytical Letters, 42: 1820–1830, 2009Copyright # Taylor & Francis Group, LLCISSN: 0003-2719 print=1532-236X onlineDOI: 10.1080/00032710903060693

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collected from different metropolis. The results indicated that the developed assaycould be considered as a suitable quality-control method for Gynura segetum.

Keywords: Gynura segetum, RP-HPLC, senecionine, senecionine N-oxide,senlciphylline

INTRODUCTION

Gynura segetum is the dry root tuber of compositae plant Gynura segetum(Lour.) Merr. (GS), recorded in the drug standard of Ministry of PublicHealth of the People’s Republic of China (Chinese crude drug). It is awell-known Chinese medicine, named ‘‘Ju-San-Qi’’ in Chinese, and isdistributed extensively in southeast Asia (Committee of Pharmacopeia ofPeople’s Republic of China 1992; Committee of Flora of Chinese Academyof Science 1999). In traditional Chinese medicine, the main uses of GSinclude treatment of hematischesis, analgesia, and detumescence bydetoxification. The reports of modern study of pharmacology indicatethat GS can assuredly stop bleeding (Liu, Pang, and Wang 1982, 1985;Chen and Liu 1985; Zhang et al. 1988), so it has the potential forexploitation.

According to the literature, alkaloids (including senecionine, seneciphyl-line, sineciphyllinine, and (E)-seneciphylline) are the main constituents ofGS, and they were proven to be responsible for the various biological activ-ities of the herb (Yuan,Gu, andWei 1990; Liang andRoeder 1984;Hua et al.1983). Based on pharmacological tests in vivo and in vitro, we have success-fully screened the active fractions for hemostasis from GS. The results indi-cated that the ethyl acetate extracts of GS have noticeable activity in PT,APTT, and FIB experiments. Further separation and purification of theethyl acetate extracts were performed by silica column chromatographyand Rp-C18 column chromatography to give three compounds, and theywere identified as senecionine (SO), seneciphylline (SP), and senecionineN-oxide (SONO) (see Fig. 1) by direct comparison of their 1H NMR, 13CNMR,mass spectroscopic (MS), ultraviolet (UV), and infrared (IR) spectraldata with those reported in the literature (Yuan, Gu, and Wei 1990; Liangand Roeder 1984; Hua et al. 1983). All of them were confirmed as the effec-tive compounds that have significant activity on blood clotting. Amongthem, SONO was obtained from this plant for the first time.

However, up to the present, no method is available for simultaneousquantification of these compounds in GS. The current study aimed at devel-oping a simple, sensitive, and feasible HPLC method for the simultaneousquantification of SO, SP, and SONO in GS to control the quality of thisimportant Chinese herbal medicine.

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EXPERIMENTAL

Apparatus and Reagents

Samples of GS were obtained from different habitats and authenticatedby Prof. Xiao-he Xiao (Institute of People’s Liberation Army ofChinese Medicine). The standards of SO, SP, and SONO (98.45%,98.87%, and 98.96%, respectively) were isolated from GS by a seriesof chromatographic procedures in our institute. Acetonitrile was ofHPLC grade (Fisher, USA). Phosphoric acid, triethylamine, and othersolvents used for compound isolation were of analytical grade (BeijingChemical Factory, China). Doubly distilled water was prepared from aMillipore water-purification system (Millipore, Milford, MA, USA) andwas filtered through 0.45-mm membranes.

Figure 1. Chemical structures of senecionine, senlciphylline, and senecionineN-oxide.

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Chromatographic Instrument and Conditions

An HP 1100 chromatographic system consisting of a quaternary pump(G1100A QuatPump, Agilent), degasser, diode array detector (G1100ADAD, Agilent), and HP Chemstation Data system (Agilent Technologies,Palo Alto, CA) was used. The injection volume was 20mL. Analysis wasachieved on a Kromasil KR100–5 C18 column (25cm� 4.6mm, 5mm) pro-tected by a Alltima C18 guard column (20� 4mm, 5mm). The mobile phaseis consist of solvent A (acetonitrile) and solvent B (0.2% phosphoric acid–triethylamine, v=v) with a ratio of 90:10. The detection wavelength wasmonitored at 220nm. The flow rate was 1.0ml=min, and sample injectionvolume was 10mL. The column temperature was set at 25�C.

Sample and Calibration Curve Preparation

All the reference compounds were accurately weighed and dissolved inmethanol to give the mixed standard stock solution containing SO(0.3164mg=mL), SP (0.3296mg=mL), and SONO (0.7896mg=mL).Working standard solutions were prepared by diluting the mixedstandard solution with methanol to give six different concentrationswithin the ranges of 7.91–79.10 mg=ml for SO, 8.24–82.40 mg=ml for SP,and 19.74–197.40 mg=ml for SONO for calibration curves. These rangeswere specified as approximately 25–250% of the test concentration ofthe samples, covered the amounts of the analytes of all samples, andprovided suitable level of linearity, accuracy, and precision.

The dried powders of GS samples (1.0 g, 100 mesh) were accuratelyweighed, soaked in 25mL methanol solution, and then refluxing extractedat 80�C for 2 h. The resultant mixture was cooled to room temperature,adjusted to the original weight, and filtered through a 0.45-mmmembranebefore HPLC injection. All samples were prepared in triplicate.

RESULTS AND DISCUSSION

Optimization of Extraction Conditions

Sample 2 (Duchang, Jiangxi) was collected for optimization of extractionconditions. To obtain the best extraction efficiency, the extractionmethod, extraction solvent, and extraction time were investigated. Theresults suggested that reflux extraction was more effective than ultrasonicextraction and soxhlet extraction, which allowed extraction of all theconstituents in good yields (see Fig. 2). Hence, the reflux extraction

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was chosen as the preferred method. Ethyl acetate, ethanol, and metha-nol were performed as extraction solvents to analyze the effect of the sol-vent on extraction efficiency. The results showed that methanol was themost suitable extraction solvent. Then 1.0-g samples were extracted with25ml methanol by refluxing for 60, 90, 120, 150, and 180min, respec-tively, to determine optimal extraction time. It was showed that the com-pounds were almost completely extracted within 120min (see Fig. 3).Hence, 120min was chosen as optimal extraction time.

Figure 2. Effect of different sample preparation methods on extraction yield.

Figure 3. Extraction efficiency of different extracts.

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Optimization of Chromatographic Conditions

Different mobile phases, including methanol–water, acetonitrile–water,methanol–water–SDS, acetonitrile–water–SDS, methanol–water–phos-phoric acid, and acetonitrile–water–phosphoric acid, were examined forsatisfactory resolution. The acetonitrile–water–phosphoric acid solventsystem was most effective. It was also found that adding triethylaminein the mobile phase could enhance the resolution and eliminate the peaktailing of the target compounds, so the concentrations of phosphoric acidand triethylamine were examined. As a result, acetonitrile and watercontaining 0.2% phosphoric acid and 0.2% triethylamine was chosen asthe eluting solvent system with desired separation and acceptable tailingfactor within a run time of 40min. Representative chromatograms ofmixture standard (a) and GS sample (b,c) are shown in Figure 4.

From the DAD online spectra results, it was found that all the threecompounds have strong absorption between 200 nm and 240 nm. Whenthe monitoring wavelength was set as 220 nm, sufficient absorption wasachieved. Hence, the detection wavelength was set as 220 nm.

It was also suggested that separation was better when columntemperature was kept at 25�C rather than 20, 30, or 35�C.

Figure 4. HPLC chromatograms of mixture standard (a), GS sample 2 (b), andGS sample 12 (c): 1, SO; 2, SP; and 3, SONO.

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Linearities and Sensitivities of the Method

The calibration curve for each compound was performed with sixdifferent concentrations in triplicate using the same HPLC method. Allcalibration curves were constructed from peak areas of the referencestandards vs. their concentrations. The regression equations of the threemarker compounds were as follows:

SO : y ¼ 10:680xþ 2:768 ðr2 ¼ 0:9998Þ

SP : y ¼ 6:361x� 1:378 ðr2 ¼ 0:9997Þ

SONO : y ¼ 10:590xþ 4:487 ðr2 ¼ 0:9997Þ

As can be seen from these equations, all the three compounds showedgood linearity (r2> 0.9997) under current chromatographic conditions.The detection limits (signal-to-noise ratio¼ 3) of SO, SP, and SONOwere 0.21, 0.85, and 0.52, respectively. The results are listed in Table 1.

Validation of the Assay Method

The method for quantitative analysis of GS was validated with regard toits specificity, precision, accuracy, and stability.

The specificity of the method was confirmed by reliability of thethree marker peaks in the GS sample (sample 2, Duchang, Jiangxi).The standard mixture of the three marker compounds and GS samplewere separated by mobile phases mentioned previously with differentratios. As a result, the retention times of the three marker compoundsin GS sample changed consistently with their corresponding standards,

Table 1. Regression data and limits of detection (LODs) of the three analytes

CompoundRegressionequationa

Correlationcoefficient (r2)

Linear range(mg=ml)

LOD(mg=ml)

SO y¼ 10.680xþ 2.768 0.9998 7.91–79.10 0.21SP y¼ 6.361x – 1.378 0.9997 8.24–82.40 0.85SONO y¼ 10.590xþ 4.487 0.9997 19.74–197.40 0.52

aIn the regression equation y¼ axþ b, x refers to the concentration of thecompound (mg=ml) and y to the peak area. Triplicate assay about the differentconcentration (n¼ 6).

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and each analyzed spectra from GS matched perfectly with thecorresponding standard spectra. The data are listed in Table 2.

The intra- and interday variabilities (expressed as relative standarddeviation, RSD) for each marker compound were measured to evaluatethe precision of the method. The intraday assay was examined on sixindividual samples within 1 day, and the interday test was determinedfor three independent days. The method showed good precision withintra- and interday variabilities of less than 2.82%, as shown in Table 3.

To further evaluate the accuracy of the method, a recovery test wasperformed by adding accurate quantities of the mixed standard solutionto GS sample (sample 2, Duchang, Jiangxi) of known amounts and thenextracting and analyzing them as described. All the samples were tested intriplicate. The accuracy was calculated with the value of detected vs.added amounts. As shown in Table 4, the recovery was in the range of96.55–103.88% with RSDs less than 2.86%, indicating the good accuracyof the method.

The stability of sample solution was tested by analyzing the sample(sample 2, Duchang, Jiangxi) solution in triplicate every 12 h over 3 days.The results of the stability test shown in Table 3 demonstrated that theanalytes in the sample solution were stable over 3 days (RSDs< 2.89%).

Table 3. Precision and stability data for the HPLC method

Precision

Intraday (n¼ 6) Interday (n¼ 6) Stability (n¼ 6)

Compound Mean� SDaRSD(%) Mean� SD

RSD(%) Mean� SD

RSD(%)

SO 0.43� 0.01 1.70 0.43� 0.01 2.82 0.42� 0.01 2.89SP 1.05� 0.02 1.69 1.06� 0.02 2.30 1.06� 0.02 2.31SONO 4.40� 0.03 0.69 4.41� 0.04 0.98 4.42� 0.03 0.77

aData were mg constituents per gram drug.

Table 2. Retention time and purity factor of the three analytes

Purity factor

Compound Retention time (min) Mixed standards Sample 2

SO 15.47 999.796 995.876SP 27.44 999.649 996.672SONO 33.45 999.575 998.719

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Applications of the Method

A total amount of 15 GS samples collected from different places wereprepared as described. The developed analytical method was then appliedto simultaneously determine the three constituents in 15 GS samples. Allthree compounds were detected in herbal samples. Each sample wasdetermined in triplicate, and the content of each analyte was calculatedfrom the corresponding calibration curve.

The contents of the three compounds analyzed are listed in Table 5.From the results, it was easy to note that all the three compounds couldbe detected, and there is a significant variability in the contents of thethree compounds in the 15 samples. The total amounts of the three com-pounds varied from 1.61 to 5.88mg=g, with nearly four-fold variation.Meanwhile, a single constituent was concerned. For example, the contentof SONO in GS varied from 0.74 to 4.40mg=g (5.9-fold variation).Similar situations were also found in SO and SP with 3.6-fold and2.1-fold variations, respectively.

The variation in contents of constituents could certainly lead to thevariation of therapeutic effects; hence, each procedure involved (suchas genetic variation, plant origin, and storage conditions) should bestandardized. Otherwise, as all the three compounds are active ingredi-ents and significant variations in the yield of those compounds in GSof different habitats were observed, it is necessary to quantify all the threecompounds in GS to ensure and improve its therapeutic benefits.

Table 4. Recoveries of the three constituents in GS (n¼ 3)

Compound Added (mg) Detected (mg)a Recovery (%)b RSD (%)c

SO 0.48 0.46� 0.01 97.56 1.260.59 0.62� 0.01 103.88 1.710.71 0.70� 0.01 97.58 1.01

SP 0.49 0.48� 0.01 96.55 2.570.62 0.60� 0.02 97.22 2.860.74 0.76� 0.02 102.33 2.27

SONO 1.18 1.21� 0.02 101.79 1.751.48 1.50� 0.04 101.41 2.781.78 1.76� 0.04 98.83 2.38

aCalculated by subtracting the total amount after spiking from the amount inthe herb before spiking. Data were means of three experiments.

bCalculated as detected amount=added amount� 100%. Data were means ofthree experiments.

cRSD (%)¼ (SD=mean)� 100.

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CONCLUSIONS

A simple, rapid, and accurate method was developed for simultaneousdetermination of three bioactive alkaloids in GS for the first time. Thisdocuments that the verified HPLC method can be successively appliedto quantification of SO, SP, and SONO in GS with good precision,accuracy, and repeatability.

REFERENCES

Chen, X. S., and Z. X. Liu. 1985. The pharmacological study of Gynura segetum–?local anesthetic action Pharmacol. Clin. Chin. Mat. Med. 10: 95.

Committee of Flora of Chinese Academy of Sciences. 1999. The Plant Index ofChina. Beijing: Science Press.

Committee of Pharmacopeia of People’s Republic of China. 1992. Drug Standardof Ministry of Public Healthy of PRC, vol. 21. Beijing: Chemical Industry Press.

Hua, Z. Q., X. J. Xu, X. C. Wei, S. R. Tang. 1983. Molecular and crystalstructure of a pyrrolizidine alkaloid from Gynura segetum (lour.) Merr. ActaScicent. Natural. Uni. Pekinesis 4: 89.

Liang, X. T., and E. Roeder. 1984. Senecionine from Gynura segetum. Planta.Med. 4: 362.

Table 5. Contents of the three constituents in different GS samples (n¼ 3)

Content (mg=g)

No. Origina SO SP SONO Total

1 Nanchang, Jiangxi 0.41 0.97 3.63 5.012 Duchang, Jiangxi 0.43 1.05 4.40 5.883 Jiujiang, Jiangxi 0.38 1.12 3.54 5.044 Guangzhou, Guangdong 0.47 1.14 1.36 2.975 Zhuhai, Guangdong 0.42 1.10 1.38 2.906 Chengdu, Sichuan 0.34 0.52 1.20 2.067 Emei, Sichuan 0.31 0.43 1.13 1.878 Jintan, Jiangsu 0.32 0.44 1.08 1.849 Nanjing, Jiangsu 0.36 0.38 1.11 1.8510 Wenzhou, Zhejiang 0.28 0.35 0.98 1.6111 Hangzhou, Zhejiang 0.32 0.45 1.26 2.0312 Anguo, Hebei 0.60 0.64 0.74 1.9813 Kunming, Yuannan 0.41 0.32 1.13 1.8614 Guizhou, Guangxi 0.43 0.43 1.25 2.1115 Nepal 0.42 0.65 1.40 2.47

aSamples 1–14 were collected in China and sample 15 was collected in Nepal.

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Liu, H. Z. J. Pang, Z. L. Wang, G. H. Ying, and S. R. Li. 1982. Studies of theeffects of Gynura segetum and panax no toginseng on the ultrastructure ofplatelets in Guinea-Pigs. Acta Pharm. Sinica 11: 801.

Liu, H. Z., Z. J. Pang, and Z. L. Wang. 1985. Study on the hemostatic effect ofGynura segetum (lour.) MERR. Chin. J. Hospital Pharm. 7: 292.

Yuan, S. Q., G. M. Gu, and T. T. Wei. 1990. Studies on the alkaloids of Gynurasegetum (lour.) MERR. Acta Pharm. Sin. 3: 191.

Zhang, M. L., W. B. Liu, X. Y. Li, and X. D. Li. 1988. The extraction of thealkaloids of Gynura segetum and the comparison on the pharmacologicaleffects of its analogues. Jilin J. Trad. Chin. Med. 14: 35.

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