PHYTOCHEMICAL ANALYSIS, VOL. 3, 129-131 (1992)

Standardization of the Indian Crude Drug Kalmegh by High Pressure Liquid Chromatographic Determination of Andrographolide

Anupam Sharma, Krishan La1 and Sukhdev S. Handa* ICMR Ccntrc for Advanced Research on Standardization, Quality Control and Formulation of Traditional ReinedieslNatural Products, Department of Pharmaceutical Sciences. Panjab University, Chandigarh-160014. India

The popular hepatoprotective Indian herbal drug Kalrnegh (Andrographis panicdata) can be standardized by high pressure liquid chromatographic determination of its major active constituent, andrographolide. The leaves of the herb were found to contain the highest amount (2.39%w/w) of andrographolide and the seeds to contain the lowest. The technique was found to be much more sensitive and simple than the known spectrophotometric method

Keywords: Standardization; Andrographolide; Androgruplzis paniculutn; Kalmegh; high pressure liquid chro- matography

INTRODUCTION

Andrographis paniculutu Nees (Acanthaceae), popu- larly known in the Indian system of medicine as Kalmegh, has been used traditionally for the relief of general debility, dysentery, dyspepsia and liver dis- orders (Satyavati and Raina, 1976). The hepatoprotec- tive activity of the plant has been attributed (Handa and Sharma, 1989) to its major diterpenoid lactone andrographolide (1) which significantly neutralizes the hepatotoxic effects of carbon tetrachloride (Handa and Sharma, 1990a), paracetamol, galactosamine (Handa and Sharma, 1990b) and ethanol (Choudhury and Poddar, 1984), and has been shown (Choudhury et d., 1987) to be an inhibitor of hepatic microsomal enzymes in rodents. A number of methods, including gravi- metric (Sengupta er d., 1948, 1949), titrimetric (Rao, 1962; Talukdar et al., 1968), spectrophotometric (Gaind et a f . , 1963; Talukdar and Dutta, 1969;

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1

'' Author t o whom correspondence should he addressed.

Bandhopadhyay et al., 1986) and densitometric (Chen et af., 1986), have been previously reported for the estimation of andrographolide in A . panicdata. The spectrophotometric method (Talukdar and Dutta, 1969), involving isolation of andrographolide from the plant extract by preparative thin layer chromatography (PTLC) followed by its spectrophotometric estimation, though simple and most specific, lacks sensitivity and is tedious. We have developed a high pressure liquid chromatographic (HPLC) method to estimate andro- grapholide for the standardization of A . puniculata. and have compared its sensitivity with that of the spectro- photometric method (Talukdar and Dutta, 1969). Using the HPLC technique, andrographolide was esti- mated in A . paniculata procured from four different commercial sources, and in different parts of the plant.

EXPERIMENTAL

Plant material. A. puniculata (whole plant) was procured from the markets of Calcutta, Bombay. Dehradun and Haridwar. Its identity was established by comparison with reference specimens preserved at the Herbarium-cum- Museum of the Department of Pharmaceutical Sciences, Panjab University, Chandigarh.

HPLC analysis. A Waters chromatograph comprising a deli- very pump 510, a gradient controller 680, an injector U6K, a UV detector 481 and an integrator 730 was employed. A resolve 5 pm spherical silica (3.9 mm X 15 cm) column was used for the isocratic resolution of andrographolide using chloroform: methanol (90: 10) as the mobile phase at a flow rate of 0.7mL/min. The detector was operated at 254nm. Andrographolide was estimated in the plant using androgra- pholide isolated in our laboratory (Handa and Sharrna. 1990a) as an external standard. All analyses were run in triplicate.

09SX-0344/92/030 129-03 $06.50 0 I992 by John Wiley & Sons. Ltd

Receioed I October 1991 Accepted (rer>i.ved) 9 March I992

I30 ANUPAM SHARMA E T A L

4000

- 3000

x .L = 2000 x > - g 1000 U

Comparative sensitivity of the HPLC and spectrophotometric methods. Dilutions containing 0.50. 1.00. 2.00, 4.00, 8.00 and 10.00 pg/mL were prepared from a stock solution (300 pg/ mL) of andrographolide in the mobile phase. An aliquot (10 pL) of each dilution was subjected to HPLC and the area under the peak of andrographolide ( R , = 2.90 min; capacity factor. k ' = 1.6) was recorded (Table 1). Figure la shows a typical chromatogram of pure andrographolide. A standard plot (Figure 2 ) of andrographolide was prepared by plotting the mean peak area calculated from three separate sets of observations against the injected amount of andrographolide.

Dilutions containing 0.50, 0.75. 1.25, 1.50, 2.00, 2.50. 4.00 and 5.00mg/mL were prepared from a stock solution (10.00 mg/mL) of andrographolide in 95% ethanol. and 20 pL of each dilution was analysed following the spectropho- tometric method of Talukdar and Dutta (1969). Three separ- ate silica gel G TLC plates ( 2 0 ~ 2 0 c m ; 0.25 mm) were spotted and developed using chloroform: benzene : ethanol (7:1.5: 1.5) as the solvent system. UV absorbance of andro- grapholide isolated from each loading was recorded at 223 nm (Milton Roy Spectronic 1201). The mean absorbance values (7'able 1) were plotted against the amount of andrographolide loaded onto the TLC plates (Figure 2).

Estimation of andrographolide in A. puniculutu by HPLC. A. puniculufu (whole plant), procured from each of the four sources. was dried to constant weight at 50°C (24 h), and 10 g of the dried sample was subjected to exhaustive extraction ( 5 h) with methanol in a Soxhlet apparatus. The methanol extracts were evaporated to dryness under reduced pressure and dissolved separately in the mobile phase (50 pg/mL). Aliquots (10 pL) of each solution were subjected to HPLC (Figure 1 b) and andrographolide was estimated using refer- ence andrographolide as an external standard. The androgra- pholide content was calculated in the test material by linear regression. The mean percentage of andrographolide in the samples calculated from three separate observations is shown in Table 2.

- - - - 0.020

- 0.025

A* I 1 1 I 1

Estimation of andrographolide in various parts of A . punicu- latu. Roots, stem, leaves, pericarp and seeds were separated from A . puniculuru procured from Calcutta, and androgra- pholide was estimated in these parts following the procedure described above. Tdbk 2 shows the mean percentage of andrographolide in different parts of the plant.

Efficiency of HPLC assay. The efficiency of the assay was determined by estimating the recovery of 5 mg andrographo- lide added to 1 g of A. puniculufu whole plant (Calcutta

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Table 1 . Peak areas and absorbance values obtained from HPLC and spectrophotometric analysis of reference andrographolide

HPLC Spectrophotometry -

Meana peak Amount injected area Amount loaded Meana absorbance

lng) fIlV x s) (4 5 139 10 0.000

10 400 15 0.001 20 81 5 25 0.005 40 1590 30 0.007 80 31 50 40 0.009

100 3980 50 0.012 80 0.019

100 0.023 "n=3.

.I 0 1 2 3 bmin

b Figure 1. Chromatograms of (a ) reference andrographolide (peak 1) and (b) a methanol extract of A. panicdata

sample) by the method described above and comparing the content of andrographolide with that of an unspiked control sample of the plant. Table 3 shows the recovery percentage o f andrographolide.

RESULTS AND DISCUSSION

An attempt has been made to standardize the Indian crude drug Kalmegh using HPLC for quantitation of the major bioactive constituent, andrographolide. A calibration curve was prepared for andrographolide over the range 0.5-10.0yglmL in the case of HPLC and 0.50-5.00 mg/mL in the case of spectrophoto- metric analysis. The respective regression equations for HPLC and spectrophotometric assay were: y = 139.8634~- 15.1951 (r=0.999) and y=O.O002x- 0.0017 (r=0.994). Mean coefficients of variation of

STANDARDIZATION OF ANDROGRAPHIS PANICULA TA 131

~

Table 2. Estimation of andrographolide in A. paniculata by HPLC

Test sample

A. paniculata Whole plant (source)

Calcutta Bombay Dehradun Haridwar

Calcutta sample Leaves Roots Stem Pericarp Seeds

' n = 3 .

Yield of MeOH ext I% w/w)

6.24 6.33 6.37 6.12

4.72 3.97 2.17 0.60 0.11

Andrographolide content

IMeana;tSE i%w/w)l

0.81 f 0.013 0.74 5 0.019 0.69 f 0.010 0.65 k 0.017

2.39 k 0.008 0.44 f 0.01 1 0.20 * 0.019 0.18 t 0.009 0.13 * 0.013

Confidence limit

ip = 0.051

0.75-0.87 0.66-0.82 0.65-0.73 0.58-0.72

2.36-2.42 0.39-0.49 0.12-0.28 0.14-0.18 0.07-0.19

ent sources shows that the andrographolide content is fairly constant (Table 2). However, different parts of the plant show wide variations in andrographolide con- tent, with leaves containing the highest (2.39"/0) and seeds the lowest (0.13%) amounts. It would be perti- nent to mention here that although the Indian Pharmacopoeia (1966) specifies a minimum of 1% w/w of andrographolide in Kalmegh, its actual content is less. The method of quantitation of andrographolide in the Indian Pharmacopoeia is gravimetric, which does not involve purification of andrographolide and, there- fore, records a higher content of andrographolide because of its associated impurities (Talukdar et a f . , 1968).

The HPLC method of standardization of A. panicu- fatu described herein is simple, sensitive and precise, and may be of value in standardizing the raw material for preparation of formulations containing this plant.

peak arealabsorbance from replicate ( n = 3) obser- vations did not exceed O.X6/4.68 (respectively).

cedure was observed to be around 99% (Table 3) whilst that reported for the spectrophotometric assay (Taluk- Ser'a' No sample samples

dar and Dutta, 1969) was about 94'Yo. As little as I 0 ng of andrographolide could be quantitated by HPLC in contriist to 30 pg by the spectrophotometric method (Figure 2).

Analysis o f A . put1iclhta procured from four difter-

Table 3. Recovery of andrographolide from A. paniculuta whole plant (Calcutta sample) by HPLC assay

Recovery of andrographolide by the HPLC pro- Andrographolide content (mg/g)

Recovery ( % I 1 7.94 12.89 99.0 2 8.27 13.23 99.2 3 8.15 13.08 98.6

Mean k SD 98.9 k 0.37

a 5 mg reference andrographolide added to 1 g of A. paniculata.

REFERENCES

Bandhopadhyay, K., Datta, S. K. and Sukul, N. C. (1986). A spectrophotometric estimation of andrographolide and examination of nematicidal activity of the crude extract of Andrographis paniculata. lndian Drugs 23, 510-512.

Chen, D., Shao, A,, Cai, Y. and Wang, G. (1986). Determination of andrographolide, deoxyandrographolide and neoandrogra- pholide in Andrographis paniculata (Burm. F. Nees). Yaowu Fenxi Zazhi 6, 232-234.

Choudhary, B. R. and Poddar, M. K. (1984). Andrographolide and Kalmegh (Andrographis paniculata) extract effect on rat liver and serum transaminases. lRCS Med. Sci. 12, 466- 467.

Choudhary, B. R.. Hague, S. J. and Poddar, M. K. (1987). In vivo and in vitro effects of Kalmeg h (Andrographis paniculata) extract and andrographolide on hepatic microsomal meta- bolising enzymes. Planta Med. 135-140.

Gaind, K. N.. Dar, R. N. and Kaul, R. N. (1963). Spectrophotometric estimation of andrographolide in Kalmegh. lndian J. Pharm. 25, 225-226.

Handa, S. S. and Sharma, A. (1989). Evidence of hepatoprotec- tive activity of Andrographis paniculata Nees due to diter- pene lactone andrographolide. In Research and Development of Indigenous Drugs (Dandiya, P. C. and Vohora, S. B., eds.), pp. 147-151. lnsitute of History of Medicine and Medical Research, New Delhi.

Handa, S. S. and Sharma, A. (1990a). Hepatoprotective activity of andrographolide from Andrographis panicuiata against carbon tetrachloride. Indian J. Med. Res. 928. 276-283.

Handa, S. S. and Sharma, A. (1990b). Hepatoprotective activity of andrographolide against galactosamine and paracetamol intoxication in rats. Indian J. Med. Res. 928, 284-292.

Indian Pharmacopeia (1 966). Kalmegh, pp. 385-386. Government of India Press, Calcutta.

Rao, S. V. (1962). Estimation of andrographolide in Kalmegh (Andrographis paniculata). lndian J. Pharm. 24, 134.

Satyavati, G. V. and Raina, M. K. (eds.) (1976). Medicinal Plants of India, Vol. 1, pp. 64-67. Indian Council of Medical Research, New Delhi.

Sengupta, S. B., Dutta, A. P. and Banerjee, S. (1948). Studies in the specification of Indian medicinal plants. II. Andrographis paniculata. lndian J. Pharm. 10, 70-72.

Sengupta, S. B., Banerjee, S. and Chakravarti, D. (1949). Studies in the specification of Indian plants. V. Estimation of andro- grapholide in Andrographis paniculata. lndian J. Pharm. 11,

Talukdar, P. B., Banerjee, S., Chatterjee, P. K. and Dutta, H. K. (1968). Estimation of andrographolide in Andrographis paniculata. lndian J. Chem. 6, 359-363.

Talukdar, P. 6. and Dutta, A. K. (1969). Quantitative estimation of andrographolide by TLC. lndian J. Appl. Chem. 32, 18-25.

77-78.