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Chiang Mai J. Sci. 2008; 35(3) Chiang Mai J. Sci. 2008; 35(3) : 521-525 www.science.cmu.ac.th/journal-science/josci.html Short Communications

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High Performance Liquid Chromatographic Analysis of Asiaticoside in Centella asiatica (L.) Urban

Prateek K. Jain and Ram K. Agrawal*

Department of Pharmaceutical Sciences, Dr. H. Gour University, Sagar (M.P.), India. *Author for correspondence; e-mail: [email protected] Received: 3 March 2008 Accepted: 18 April 2008.

ABSTRACT A simple, sensitive high performance liquid chromatographic method was developed for the determination of asiaticoside in Centella asiatica extract. The asiaticoside yielded 1.7 mg/100 mg of the test extract. Calibration graph of asiaticoside showed good linearity in the concentration range of (32 - 1027 g/mL), with a correlation coefficient of 0.999. The average recovery for asiaticoside is 97.5 %. The proposed method is reproducible and statistically validated. The system was successfully used to investigate the presence of the asiaticoside in Centella asiatica plant parts. Keywords: Centella asiatica, asiaticoside, HPLC method.

1. INTRODUCTION

Centella asiatica (Linn) Urban known as Indian pennywort is a prostate, perennial, faintly aromatic herb found wild throughout India [1,2]. Leaves are used as tonic, memory enhancer and also used in the treatment of cataract, eye troubles and in fevers and diarrhea among children. The plant is reported to contain glycosides like brahmosides, indocentelloside, asiaticoside, theankuniside and isotheankuniside [3]. Asiaticoside, a trisaccharide triterpene, has been identified as the most active compound in the plant associated with the healing of wounds and duodenal ulcers, whilst the triterpene saponins are also reported to possess immunomodulatory properties [4]. Asiaticoside is one of the

most active compounds which can serve as a marker [5, 6] and is used for its standardization. A few methods such as gravimetric [7] and column chromatography [8] have been suggested for the quantitative estimation of asiaticoside which are not very precise, sensitive and require multiple step extraction and purification. Literatures reveal that HPLC methods are also available [9, 10-12].

2. MATERIALS AND METHODS

The roots of the Centella asiatica were picked up in the month of February-March from the University Campus of Dr. H.S. Gour Vishwavidyalaya, Sagar (M.P.), collected crude drugs were authenticated from the Botany

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Department, Dr. H.S. Gour Vishwavidyalaya, Sagar (M.P.), India. Reference standard asiaticoside (purity 98.5%) of Fluka Chemie GmbH, Product No. 43191 was taken as standard. 2.1 Chromatographic System Shimadzu high perfor mance liquid chromatographic system equipped with LC10A pump with SPD-M 10Avp Photo diode Array Detector or UV detector in combination with class LC 10A software. 2.2 Chromatographic Conditions Mobile Phase The mobile phase components were filtered through 0.2 m membrane filter before use. Gradient elution was performed using 0.3% orthophosphoric acid (Soluton A) and acetonitrile (Solution B) as detailed in Table 1. Table 1. Gradient elution program.

Time (min) 0.01 5.0 15.0 20.0 23.0 30.0 35.0 40.0 Buffer (A) Acetonitrile (B) (mL) (mL) 95.0 80.0 50.0 20.0 20.0 50.0 80.0 95.0 5 20 50 80 80 50 20 5

2.2.1 Standard Preparation Ten milligrams of asiaticoside reference standard was weighed accurately in to a 10 mL volumetric flask. Dissolved in 5 mL of methanol and then made up to 10 mL with methanol. 2.2.2 Sample Preparation Weighed accurately 100 mg of methanolic extract in a 100 mL volumetric flask and dissolved in small quantity of methanol and made up the volume with methanol. 2.3 Procedure Instrument was adjusted as per conditions prescribed. HPLC analysis of the standard and the samples were carried out using above mentioned protocol and the chromatograms were recorded. The retention time and peak area of the standard and sample were noted. Amount of asiaticoside present in the sample was calculated by comparing the sample peak area with that of the standard. 2.4 Estimation of Asiaticoside The amount of asiaticoside present in the methanolic extract in 20 L is 0.33 g; therefore 100 mg of extract contain 1.7 mg of asiaticoside. 2.5 Method Validation 2.5.1 Calibration Curve Standard asiaticoside solutions concentrations range (32 - 1027 g/mL) were analyzed for studying the linearity and the area count obtained for these solutions. 2.5.2 Limit of Detection and Limit of Quantitation In order to estimate the limit of detection (LOD) and lower limit of quantitation (LLOQ), blank methanol was spotted six times following the same method. The signal to noise ratio (S/N) was determined. LOD was considered as 3:1 and LLOQ as 10:1.

Other conditions are shown below:

Column : Phenomenex-Luna 5 C-18 (2), Size: 250 x 4.60 mm Detector : SPD-M 10Avp Photo diode array detector/UV Detector Wave length : 210 nm Flow rate : 1.8 mL / min. Injection volume : 20 L

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2.5.3 Recovery Studies Accuracy and precision of the method were studied by performing experiments by standard addition technique. Three different levels of standards (0.3 g, 0.6 g and 0.9 g) were added to a previously analyzed sample, each level being repeated in tripicate. The percentage recovery was calculated from amount of drug found.

3. RESULTS AND DISCUSSION

A critical analysis of the literatures revealed that this herb finds widespread use in several traditional systems of medicine. The number of analytical reports for the determination of asiaticoside is comparatively small. Only few methods are described in the literature. All of them show major disadvantage, the separation time is extremely long (several hours) or the compounds are not baseline separated and elute more or less with the injection peak. The HPLC analysis of Centella asiatica showed asiaticoside peak at retention time 11.65 min which was comparable with that of standard asiaticoside (retention time, 11.95 min). Figure 1 showed chromatogram of standard asiaticoside and Figure 2 showed

mV 500 400 300 200 100 0 0 5 10 15 20

11.65

chromatogram of methanolic extract of Centella asiatica. The quantification yielded 1.7 mg / 100 mg of the test extract. The estimation of asiaticoside in Centella asiatica showed in Table 2. The signal to noise ratios 3:1 and 10:1 were considered as LOD and LLOQ respectively. The LOD and LLOQ were found to be 50 and 200 ng/spot. Calibration graph, a plot of peak area obtained versus asiaticoside concentration, showed good linearity in the concentration range of 32 - 1027 g/mL, (Y = 2055.5X + 27975), with a correlation coefficient of 0.999. The mean percentage recovery obtained for asiaticoside was 97.5% showed in Table 3.

4. CONCLUSIONS

Standardization of herbal medicines and plant ingredients specially with reference to their active marker is time consuming. We have tried to standardize Centella asiatica using HPLC for its active compound, asiaticoside. The method provides good resolution and separation of asiaticoside from other constituents of Centella asiatica. The proposed HPLC method is rapid, simple and accurate for quantitative monitoring of asiaticoside in Centella asiatica.

1DetA Ch1

25

30

35

40

45

min Figure 1. HPLC chromatogram of standard asiaticoside (128.3 g/mL); HPLC condition see text.

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mV 500

1DetA Ch1

400

300

100

0 0 5 10 15 20 25 30 35 40 45 min

Figure 2. HPLC chromatogram of methanolic extract of Centella asiatica (1 mg/mL); HPLC condition see text. Table 2. Estimation of Asiaticoside in Centella asiatica by HPLC. Replicate 1 2 3 Average Mean S.D., n = 3 Sample Area 811580 796664 799865 802703 7852 Asiaticoside (g) 0.36 0.32 0.32 0.33 0.02

Table 3. Results of Recovery Analysis.

Sample Amount of Amount of Total Total asiaticoside asiaticoside asiaticoside asiaticoside present in added to A taken (A+B) found (g) (g) (g) (g) A B C D % Recovery D/C x100 (Mean)

Centella asiatica Mean

11.95

200

0.33 S.D., n = 3.

0.3 0.6 0.9

0.63 0.93 1.23

0.61 0.92 1.20

0.04 0.01 0.09

96.8 98.2 97.6

6.3 1.1 7.3

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ACKNOWLEDGEMENTS The authors are thankful to Indian Council of Medical Research (ICMR), New Delhi and M.P. Council of Science and Technology (MPCOST) Bhopal, India for providing financial assistance. REFERENCES [1] The Indian Pharmaceutical Codex, Indigenous drugs, New Delhi, Council of Scientific and Industrial Research, Vol. 1, 1953. [2] British Herbal pharmacopoeia, London, British Herbal Medicine association, part 2, 1979. [3] Tiwari N.K., Sharma N.C., Tiwari V., and Singh B.D, Micropropagation of Centella asiatica (L.), A valuable medicinal herb, Plant Cell Tissue and Org. Cult., 2000; 63: 179-185. [4] Plohmann B., Bader G., Streich S., Hilter K., and Franz G., Immunomodulatory effects of triterpenoid saponins, Eur. J. Pharm. Sci., 1994; 2: 12-17. [5] Tyler V., Brady L., Robbers J. Pharmacognosy, 8th Edn, Lea and Febiger, Philadelphia, 1981. [6] Kartnig T., Clinical applications of Centella asiatica (L.) Urb., Herbs, Spices, Medicinal plants, Vol. 3, 1988.

[7] Upadhyay S.C., Khosa, R.L., Sharma D.N., Kumar M., and Chansauria J.P.N., Total glycoside content and antistress activity of Indian and Mauritius Centella asiatica ­ A comparison, Indian Drugs, 1991; 28(8): 388-389. [8] Singh B., and Rastogi R.P., A reinvestigation of the triterpenes of Centella asiatica, Phytochem., 1969; 8: 917-921. [9] Verma R.K., Bhartariya K.G., Gupta M.M., and Kumar S., Reverse phase high performance liquid chromatography of asiaticoside in Centella asiatica, Phytochem. Anal., 1999; 10: 191-193. [10] Chauhan S.K., Singh B., and Agarwal S., A HPLC determination of asiaticoside in Centella asiatica, Indian J. Nat. Prod., 2003; 19 (4): 21-23. [11] Schaneberg B.T., Mikell J.R., Bedir E., and Khan I.A., An improved HPLC method for quantitative determination of six triterpenes in Centella asiatica extracts and commercial products, Pharmazie, 2003; 123-125. [12] Inamdar P.K., Yeole R.D., Ghogare A.B., and De Souza N.J., Determination of biologically active constituents in Centella asiatica, J. Chromatogr. A., 1996; 742:127130.

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