SCd

Ja

b

c

d

e

a

ARRAA

KCCFHL

1

nooebchctdd

M

(

0d

Journal of Pharmaceutical and Biomedical Analysis 66 (2012) 170– 175

Contents lists available at SciVerse ScienceDirect

Journal of Pharmaceutical and Biomedical Analysis

j ourna l ho me p a ge: www.elsev ier .com/ locate / jpba

imultaneous quantification of coumarins, flavonoids and limonoids in Fructusitri Sarcodactylis by high performance liquid chromatography coupled withiode array detector

un Chua,b, Song-Lin Lia,b,c, Zhi-Qi Yind,∗∗, Wen-Cai Yee, Qing-Wen Zhanga,b,∗

State Key Laboratory of Quality Research in Chinese Medicine (University of Macau), MacaoInstitute of Chinese Medical Sciences, University of Macau, MacaoDepartment of Metabolomics and Key Laboratory of New Drug Delivery System of Chinese Materia Medica, Jiangsu Province Academy of Chinese Medicine, Nanjing 210028, ChinaDepartment of Natural Medicinal Chemistry, China Pharmaceutical University, Nanjing 210009, ChinaInstitute of Traditional Chinese Medicine and Natural Products, Jinan University, Guangzhou 510632, China

r t i c l e i n f o

rticle history:eceived 6 January 2012eceived in revised form 19 March 2012ccepted 19 March 2012vailable online 28 March 2012

eywords:itrus medica var. sarcodactylis

a b s t r a c t

A high performance liquid chromatography coupled with diode array detector (HPLC-DAD) methodwas developed for simultaneous quantification of eleven major bioactive components including sixcoumarins, three flavonoids and two limonoids in Fructus Citri Sarcodactylis. The analysis was performedon a Cosmosil 5 C18-MS-II column (4.6 mm × 250 mm, 5 �m) with water–acetonitrile gradient elution.The method was validated in terms of linearity, sensitivity, precision, stability and accuracy. It was foundthat the calibration curves for all analytes showed good linearity (R2 > 0.9993) within the test ranges. Theoverall limit of detection (LOD) and limit of quantification (LOQ) were less than 3.0 and 10.2 ng. The rela-

oumarinlavonoidPLC-DADimonoid

tive standard deviations (RSDs) for intra- and inter-day repeatability were not more than 4.99% and 4.92%,respectively. The sample was stable for at least 48 h. The spike recoveries of eleven components were95.1–104.9%. The established method was successfully applied to determine eleven components in threesamples from different locations. The results showed that the newly developed HPLC-DAD method waslinear, sensitive, precise and accurate, and could be used for quality control of Fructus Citri Sarcodactylis.

. Introduction

Citrus medica L. var. sarcodactylis (Noot.) Swingle (commonlyamed bergamot) is a native plant of southern Asia and grownn a large scale in China. Fructus Citri Sarcodactylis, the fruitf C. medica var. sarcodactylis is widely used as food ingredi-nt and traditional Chinese medicinal herb [1]. This herb haseen reported having activities of antiasthma [2,3], cytotoxi-ity [4], anti-inflammatory [5,6], immune enhancement [7,8],ypolipemic and hypoglycaemic [9], antioxidant [10,11], antimi-robial [12], and antiobesity [13]. Phytochemical studies showed

hat flavonoids such as 3,5,6-trihydroxy-3′,4′,7-trimethoxyflavone,iosmin, hesperidin and diosmetin [14–16], coumarins such as 5,7-imethoxycoumarin [7,8,17–20], and limonoids such as limonin,

∗ Corresponding author at: Institute of Chinese Medical Sciences, University ofacau, Macao. Tel.: +853 8397 4879; fax: +853 2884 1358.

∗∗ Co-corresponding author. Tel.: +86 25 86185371.E-mail addresses: chyzq2005@126.com (Z.-Q. Yin), qwzhang@umac.mo

Q.-W. Zhang).

731-7085/$ – see front matter © 2012 Elsevier B.V. All rights reserved.oi:10.1016/j.jpba.2012.03.041

© 2012 Elsevier B.V. All rights reserved.

nomilin and limonexic acid [4,21] are major bioactive componentsof Fructus Citri Sarcodactylis.

Qualitative and quantitative analysis of major bioactive com-ponents is very important for the quality control of Fructus CitriSarcodactylis. To date, only one or two components, such as 5,7-dimethoxycoumarin [7,8,17–20] and hesperidin [1,20], were usedas chemical markers for the quality control of Fructus Citri Sarco-dactylis. It is well known that herbal medicines exert its activitiesthrough multiple components at multiple targets, and determi-nation of multiple components has also been well accepted forthe quality control of medicinal herbs. However, to the best ofour knowledge, no method has been developed for simultaneousanalysis of coumarins, flavonoids and limonoids in Fructus CitriSarcodactylis.

As a continuous work on the quality research in com-monly used Chinese medicinal herbs, a high performance liquidchromatography coupled with diode array detector (HPLC-DAD)

method was developed for simultaneous quantification of threedifferent types of ingredients, i.e., flavonoids, coumarins andlimonoids, in Fructus Citri Sarcodactylis. Eleven compounds,including isoscopoletin (1), scopoletin (2), 7-hydroxycoumarin

J. Chu et al. / Journal of Pharmaceutical and Biomedical Analysis 66 (2012) 170– 175 171

Table 1Linearity and sensitivity of the assay.

Analyte RT (min) Calibration curve R2 Test range (�g/ml) LODb (ng) LOQc (ng)

1 9.52 y = 34.4729x − 0.3099a 1.0000 0.14–35.00 0.4 1.42 10.26 y = 31.0882x − 0.5423 1.0000 0.16–20.00 0.5 1.63 11.06 y = 43.0959x + 5.6546 0.9998 0.16–40.00 0.5 1.64 12.28 y = 10.7024x − 0.9523 0.9993 0.63–40.00 2.0 6.35 12.97 y = 14.8670x + 5.4972 0.9999 0.39–50.00 0.7 2.16 16.89 y = 36.4464x + 7.1467 0.9998 0.15–38.00 0.5 1.57 30.36 y = 43.2944x + 19.3511 0.9998 0.20–100.00 0.6 2.08 31.39 y = 40.6628x + 10.2006 0.9999 0.12–62.00 0.4 1.29 31.99 y = 19.6955x − 8.6986 0.9997 1.00–16.00 3.0 10.010 33.27 y = 7.1098x + 3.4677 0.9999 0.87–111.70 2.6 8.711 34.96 y = 5.8203x + 3.6074 0.9999 1.02–130.80 3.0 10.2

(53utw

2

2

iPtC

mYpbJ3ptwpM

2

peestmitps4iuEs

a y = peak area and x = concentration (�g/ml).b Limit of detection (S/N = 3).c Limit of quantification (S/N = 10).

3), diosmin (4), hesperidin (5), 6,7-dimethoxycoumarin (6),,7-dimethoxycoumarin (7), bergapten (8), 3,5,6-trihydroxy-′,4′,7-trimethoxyflavone (9), limonin (10) and nomilin (11) weresed as the chemical markers for this analysis. The contents ofhese components in samples collected from different locationsere compared.

. Materials and methods

.1. Materials and chemicals

Fructus Citri Sarcodactylis samples were collected from Pun-ng (GFS-A) and Guangzhou (GFS-B and GFS-C), Guangdongrovince, China. The botanical origin of the materials was authen-icated at the College of Pharmacy, Jinan University, Guangzhou,hina.

Isoscopoletin (1), scopoletin (2), 7-hydroxycoumarin (3), dios-in (4) and 6,7-dimethoxycoumarin (6) were purchased from

uanye Company (Shanghai, China). Hesperidin (5) was sup-lied from Nature Standard Company (Shanghai, China) andergapten (8) was supplied from Zelang Company (Nanjing,iangsu, China). 5,7-dimethoxycoumarin (7) and 3,5,6-trihydroxy-′,4′,7-trimethoxyflavone (9), limonin (10) and nomilin (11) werereviously isolated from this herb in our laboratory refereeing tohe methods reported in the literature [14–16,21,22]. Deionizedater was prepared using a Millipore Milli Q-Plus system (Milli-ore, Bedford, MA, USA). Acetonitrile for HPLC was purchased fromerck (Darmstadt, Germany).

.2. Sample preparation

Pressurized liquid extraction (PLE) is a commonly used sam-le preparation method for plant analysis owing to its highxtraction speed, low extraction solvent assumption and highxtraction efficiency. PLE was performed on a Dionex ASE 200ystem (Dionex Corp., Sunnyvale, CA, USA) under optimized condi-ions. In brief, dried powder of Fructus Citri Sarcodactylis (0.5 g)

ixed with diatomaceous earth at the ratio of 1:1 was putnto an 11 ml stainless steel extraction cell. The conditions ofhe PLE method were: solvent, methanol; temperature, 90 ◦C;article size, 0.125–0.15 mm; static extraction time, 5 min; pres-ure, 1500 psi.; static cycle, 1; number of extraction, 1; and0% of the flush volume. PLE extract solution was transferred

nto a 25 ml volumetric flask which was filled up to its vol-me with the same solvent and filtered through a 0.45 �mconofilter (Agilent Technologies) before injecting into the HPLCystem.

2.3. HPLC analysis

Analysis was performed on an Agilent 1200 HPLC system(Palo Alto, CA, USA), equipped with vacuum degasser, quater-nary gradient pump, autosampler and DAD, connected to anAgilent ChemStation software. A Cosmosil 5 C18-MS-II column(4.6 mm × 250 mm, 5 �m) was used. A binary gradient elutionsystem consisted of water (A) and acetonitrile (B). The gradientprogram was: 0–10 min, 18–23% B; 10–15 min, 23% B; 15–30 min,23–50% B; 30–40 min, 50–100% B; and finally, reconditioning thecolumn with 18% B isocratic for 5 min. The flow-rate was 1.0 ml/minand the column temperature was 25 ◦C. The injection volume was10 �l with needle wash. The UV detection wavelengths were set at210 nm, 254 nm, 280 nm, 320 nm and 340 nm.

2.4. Calibration curves

Methanol stock solution containing isoscopoletin (1), scopoletin(2), 7-hydroxycoumarin (3), diosmin (4), hseperidin (5), 6,7-dimethoxycoumarin (6), 5,7-dimethoxycoumarin (7), bergapten(8), 3,5,6-trihydroxy-3′,4′,7-trimethoxyflavone (9), limonin (10)and nomilin (11) was diluted with methanol to obtain a seriesconcentrations of working solutions, and subjected to HPLC anal-ysis. The calibration curves of compound 1 to compound 11 wereconstructed by plotting the peak areas versus the concentrationsrespectively.

2.5. Sensitivity and stability

The sensitivity study was completed by analyzing the limitof detection (LOD) and limit of quantification (LOQ) which weredetermined at a signal-to-noise (S/N) ratio of about 3 and 10,respectively.

The stability was tested by analyzing the sample of GFS-A andpeak areas of eleven analytes at 0, 2, 4, 8, 12, 24 and 48 h wererecorded and compared.

2.6. Precision and accuracy

Intra- and inter-day variations were chosen to determine theprecision of the developed method. The relative standard deviation(RSD) was taken as a measure of precision. Intra- and inter-dayrepeatability was determined on six replicates within one day andthree consecutive days, respectively.

Spike recovery was used to evaluate the accuracy of the assay.

Known amount of individual standards was added into accuratelyweighed samples (GFS-A, purchased from Puning, Guangdongprovince). The mixtures were extracted and analyzed in triplicateusing the method described in Section 2.2.

172 J. Chu et al. / Journal of Pharmaceutical and Biomedical Analysis 66 (2012) 170– 175

d com

3

3

it

Fig. 1. Structures of eleven investigate

. Results and discussion

.1. Optimization of HPLC conditions

Although the eleven analytes belong to three different types,.e., flavonoids, coumarins and limonoids, those belonging tohe same type have very similar chemical structure (Fig. 1), so

ponents in Fructus Citri Sarcodactylis.

baseline separation of all eleven analytes is a challenge issue forthe development of this method. Different mobile phases andelution programs were tested in order to obtain good resolution.

At last, on a Cosmosil 5 C18-MS-II column (4.6 mm × 250 mm,5 �m), all eleven analytes were eluted with baseline separation(Fig. 2) by using elution program of water (A) and acetonitrile (B).The gradient program is described in Section 2.3.

J. Chu et al. / Journal of Pharmaceutical and Biomedical Analysis 66 (2012) 170– 175 173

F .06 �1 : 5,7-d3 .77 �g

pt((ifsuD3

3

iltct3fiiftmwv4

tents of isoscopoletin (1), scopoletin (2), 7-hydroxycoumarin (3),6,7-dimethoxycoumarin (6) are relatively lower, and the contentsof bergapten (8) and 3,5,6-trihydroxy-3′,4′,7-trimethoxyflavone (9)

Table 2Precision test of the assay.

Analyte Intra-day repeatability (n = 6) Inter-day repeatability (n = 6)

Content (�g/g) RSDa (%) Content (�g/g) RSD (%)

1 64.2 ± 1.9 2.99 65.6 ± 1.8 2.722 11.5 ± 0.3 2.23 11.4 ± 0.2 1.563 7.7 ± 0.4 4.56 7.8 ± 0.4 4.924 1214.3 ± 12.2 1.01 1222.0 ± 22.0 1.805 669.8 ± 11.7 1.74 672.4 ± 14.3 2.136 99.1 ± 1.4 1.40 99.5 ± 1.7 1.717 882.1 ± 0.4 0.05 876.7 ± 4.7 0.538 NDb ND ND ND9 ND ND ND ND

ig. 2. HPLC-DAD chromatograms of reference compounds. (A) 1: isoscopoletin, 633.51 �g/ml; 5: hesperidin, 30.36 �g/ml; 6: 6,7-dimethoxycoumarin, 38.79 �g/ml; 7′ ,4′ ,7-trimethoxyflavone, 21.70 �g/ml; 10: limonin, 94.13 �g/ml; 11: nomilin, 152

Considering the different UV �max values of different com-ounds, the UV detection wavelengths were set at 254 nm for 3,5,6-rihydroxy-3′,4′,7-trimethoxyflavone (9), 280 nm for hesperidin5), 320 nm for 5,7-dimethoxycoumarin (7), 7-hydroxycoumarin3) and bergapten (8), 340 nm for 6,7-dimethoxycoumarin (6),soscopoletin (1), scopoletin (2) and diosmin (4), and 210 nmor limonin (10) and nomilin (11). Typical chromatograms arehown in Fig. 2. In order to obtain a distinct chromatogram, wesed the function DAD Timetable. The program changes in theAD wavelength were: 0–31 min, 340 nm; 31–32.5 min, 254 nm;2.5–36 min, 210 nm; 36–40 min, 340 nm.

.2. Validation of the developed method

The method was validated in terms of linearity, sensitiv-ty, precision, stability and accuracy. Table 1 summarizes theinearity, test range, limit of detection (LOD) and limit of quan-ification (LOQ) of the analytes. It was found that the calibrationurves for all analytes showed good linearity (R2 > 0.9993) withinhe test range. The overall LODs and LOQs were less than.0 and 10.2 ng for DAD, indicating that this method is suf-ciently sensitive. The relative standard deviations (RSDs) for

ntra- and inter-day repeatability are shown in Table 2. It wasound that overall intra- and inter-day variations were not morehan 4.99% and 4.92% respectively, suggesting that the developed

ethod was precise. The spike recoveries of eleven componentsere 95.1–104.9%, demonstrating that this method was also

ery accurate. Furthermore, the sample was stable for at least8 h.

11

g/ml; 2: scopoletin, 53.56 �g/ml; 3: 7-hydroxycoumarin, 40.23 �g/ml; 4: diosmin,imethoxycoumarin, 106.47 �g/ml; 8: bergapten, 35.07 �g/ml; 9: 3,5,6-trihydroxy-

/ml and (B) extract of sample GFS-A.

3.3. Sample determination

The established method was applied for the quantification ofthree Fructus Citri Sarcodactylis samples from different collec-tions. The contents of the eleven compounds are shown in Table 3.From Table 3, it was found that diosmin (4), hesperidin (5), 5,7-dimethoxycoumarin (7), limonin (10) and nomilin (11) are fivemajor components of Fructus Citri Sarcodactylis, while the con-

0 223.4 ± 11.4 4.99 227.3 ± 5.8 2.561 789.1 ± 22.2 2.82 801.3 ± 21.1 2.64

a RSD (%) = 100 × S.D./mean.b ND, not detected.

174 J. Chu et al. / Journal of Pharmaceutical and B

Tab

le

3C

onte

nts

of

elev

en

com

pou

nd

s

in

sam

ple

s

from

dif

fere

nt

coll

ecti

ons

(n

=

3,

�g/

g).

1

2

3

4

5

6

7

8

9

10

11

GFS

-A

58.8

±

0.4

12.7

±

0.1

7.5

±

0.4

1223

.5

±

1.2

666.

8

±

2.0

100.

5

±

0.2

897.

7

±

0.6

–a–

236.

9

±

5.6

801.

2

±

4.3

GFS

-B34

.2

±

0.3

15.5

±

0.2

13.4

±

0.1

1204

.8

±

1.4

801.

6

±

18.8

35.8

±

0.3

779.

0

±

7.3

1.7

±

0.0

–

973.

1

±

1.8

3199

.9

±

27.5

GFS

-C20

.6

±

0.2

36.8

±

1.5

12.7

±

0.1

462.

3

±

7.7

243.

4

±

4.4

16.0

±

0.1

1206

.6

±

11.7

–

51.3

±

0.5

546.

2

±

2.1

274.

0

±

2.7

a–,

beyo

nd

LOQ

.

[

[

[

iomedical Analysis 66 (2012) 170– 175

were too low to be quantified in some samples. It should benoticed that although the content of 5,7-dimethoxycoumarin (7)was consistent among three samples analyzed, the contents ofother components, in particular that of nomilin (11) were markedlydifferent among three samples, ranging from 274.0 to 3199.9 �g/gfor nomilin (11), suggesting that although all these samples werecollected from the genuine locations of Guangdong Province ofChina, the quality difference still existed.

4. Conclusion

An HPLC-DAD method was developed for the quantifica-tion of eleven major components in Fructus Citri Sarcodactylis.Compared with previously reported methods, the newly estab-lished method could simultaneously determine three differenttypes of major components, including six coumarins (isoscopo-letin, scopoletin, 7-hydroxycoumarin, 6,7-dimethoxycoumarin,5,7-dimethoxycoumarin and bergapten), three flavonoids (dios-min, hesperidin, and 3,5,6-trihydroxy-3′,4′,7-trimethoxyflavone)and two limonoids (limonin and nomilin) in Fructus Citri Sar-codactylis. Furthermore, different wavelengths were chosen toensure the high sensitivity of the assay, so that the establishedmethod could be successfully applied for the “holistic” quality con-trol of Fructus Citri Sarcodactylis.

Acknowledgments

We are grateful to Mr. Leon Lai from our institute for histechnical assistance. The research was supported by grants fromMacau Science and Technology Development Fund (013/2008/A1to Dr. Zhang and SKL/QRCM/EST/11-Y1/ICMS for state key lab),the Team Project of the Natural Science Foundation of Guangdong(8351063201000003 to Dr. Ye), and the Research Starting Fund(RC1101) and the Open Fund (No. 2011NDDCM02003) to Dr. Li fromJiangsu Province Academy of Chinese Medicine.

References

[1] E. Committee, Pharmacopoeia of the People’s Republic of China, China MedicalScience Press, Beijing, 2010, p. 167.

[2] H.P. Yin, S.M. Xu, Effect of ethyl acetate extract from bergamot in asthmaticmouse model, Health Res. 29 (2009) 92–94.

[3] H.P. Yin, G.-H. Lin, L.-P. Yu, H. Yuan, Experimental study on antitussive andexpectorant effects of ethyl acetate extract from bergamot, J. Hangzhou Teach.Coll. (Med. Ed.) 27 (2009) 377–379.

[4] Y.Y. Chan, T.S. Wu, Y.H. Kuo, Chemical constituents and cytotoxicity from thestem bark of Citrus medica, Heterocycles 78 (2009) 1309–1316.

[5] J.Y. Wang, C.C. Shi, W.P. Zhu, W.H. Huang, Tests on anti-inflammation and acutetoxicity of Jinhua finger citron volatile oil, Res. Pract. Chin. Med. 18 (2004)46–48.

[6] Y.Y. Chan, C.H. Li, Y.C. Shen, T.S. Wu, Anti-inflammatory principles from thestem and root barks of Citrus medica, Chem. Pharm. Bull. 58 (2010) 61–65.

[7] L. Huang, Z.Y. Kuang, M. Zhang, Effect of CMPS on cytokine of mice inducedby cyclophosphamide, Mod. J. Integr. Tradit. Chin. Western Med. 9 (2000)871–872.

[8] L. Huang, Z.Y. Kuang, M. Zhang, Y. Li, Effect of Fructus Citri Sarcodactylispolysaccharide on IL-6 in macrophages of cyclophosphamide-treated mice, J.Guangzhou Univ. Tradit. Chin. Med. 17 (2000) 58–59.

[9] V. Mollace, I. Sacco, E. Janda, C. Malara, D. Ventrice, C. Colica, V. Visalli, S.Muscoli, S. Ragusa, C. Muscoli, D. Rotiroti, F. Romeo, Hypolipemic and hypo-glycaemic activity of bergamot polyphenols: from animal models to humanstudies, Fitoterapia 82 (2011) 309–316.

10] A. Wei, T. Shibamoto, Antioxidant/lipoxygenase inhibitory activities and chem-ical compositions of selected essential oils, J. Agric. Food Chem. 58 (2010)7218–7225.

11] D. Ramful, T. Bahorun, E. Bourdon, E. Tarnus, O. Aruoma, Bioactive phenolicsand antioxidant propensity of flavedo extracts of Mauritian citrus fruits: poten-tial prophylactic ingredients for functional foods application, Toxicology 278

(2010) 75–87.

12] G. Mandalari, R.N. Bennett, G. Bisignano, D. Trombetta, A. Saija, C.B. Faulds, M.J.Gasson, A. Narbad, Antimicrobial activity of flavonoids extracted from berg-amot (Citrus bergamia Risso) peel, a byproduct of the essential oil industry, J.Appl. Microbiol. 103 (2007) 2056–2064.

and B

[

[

[

[

[

[

[

[

J. Chu et al. / Journal of Pharmaceutical

13] C.H. Peng, Y.B. Ker, C.F. Weng, C.C. Peng, C.N. Huang, L.Y. Lin, R.Y. Peng, Insulinsecretagogue bioactivity of finger citron fruit (Citrus medica L. var. sarcodactylisHort, Rutaceae), J. Agric. Food Chem. 57 (2009) 8812–8819.

14] H.Y. He, L.Q. Ling, Chemical studies of a Chinese traditional drug fingered citron,Citrus medica L. var sarcodactylis (Noot.) Swingle, Acta Pharm. Sin. 20 (1985)433–435.

15] Y.K. Nogata, K. Sakamoto, H. Siratsuchi, T. Ishii, M. Yano, H. Ohta, Flavonoidcomposition of fruit tissues of citrus species, Biosci. Biotechnol. Biochem. 70(2006) 178–192.

16] F. Yin, L. Cheng, F.C. Lou, Studies on the constituents of Citrus medica L. var.sarcodactylis (Noot.) Swingle, Chin. J. Nat. Med. 2 (2004) 149–151.

17] L.W. Lin, L. Jiang, D.Q. Hao, G.D. Zheng, X. Yang, Determination of 5,7-dimethoxycoumarin in Fructus Citri Sarcodactylis from Guangdong at differentcollecting times, Res. Pract. Chin. Med. 22 (2009) 15–17.

[

[

iomedical Analysis 66 (2012) 170– 175 175

18] Y.H. Gao, Y.M. Diao, X.S. Peng, H.H. Xu, Determination of 5,7-dimethoxycoumarin in Fructus Citri Sarcodactylis by HPLC, Tradit. Chin.Drug Res. Clin. Pharmacol. 14 (2003) 250–251.

19] R.F. Zhang, Y.H. Gao, H.H. Cui, S.L. Liang, Fingerprints of Fructus Citri Sar-codactylis from Guangdong by HPLC, Chin. Tradit. Herb. Drugs 38 (2007)1075–1077.

20] L.W. Lin, L. Jiang, D.Q. Hao, G.D. Zheng, X. Yang, Q. Wang, J.Y. Zhang, HPLC fin-gerprint of Fructus Citri Sarcodactylis from Guangdong province, Chin. Tradit.Patent Med. 31 (2009) 1805–1808.

21] H.Y. He, L.Q. Ling, G.P. Shi, N. Zhang, Q.M. Mao, Studies on the chemical con-stituents of Chinese traditional drug fingered citron, China J. Chin. Mater. Med.13 (1988) 352–354.

22] F. Yin, F.C. Lou, Studies on the constituents of Citrus medica L. var. sarcodactylis,Chin. Pharm. J. 39 (2004) 20–21.