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 chromatographicdetermination of its major active constituent, andrographolide.The leaves
of the herb were found to contain the highest amount (2.39%w/w)of andrographolideand 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 chromatography

INTRODUCTION

Andrographis paniculutu Nees (Acanthaceae), popularly known in the Indian system of medicine as
Kalmegh, has been used traditionally for the relief of
general debility, dysentery, dyspepsia and liver disorders (Satyavati and Raina, 1976). The hepatoprotective 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 gravimetric (Sengupta er d.,1948, 1949), titrimetric (Rao,
1962; Talukdar et al., 1968), spectrophotometric
(Gaind et a f . , 1963; Talukdar and Dutta, 1969;

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 andrographolide for the standardization of A . puniculata. and
have compared its sensitivity with that of the spectrophotometric method (Talukdar and Dutta, 1969).
Using the HPLC technique, andrographolide was estimated 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-cumMuseum of the Department of Pharmaceutical Sciences,
Panjab University, Chandigarh.

HOHaC

CH3

1
''

Author t o whom correspondence should he addressed.

09SX-0344/92/030 129-03 $06.50

0 I992 by John Wiley & Sons. Ltd

HPLC analysis. A Waters chromatograph comprising a delivery 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 andrographolide isolated in our laboratory (Handa and Sharrna.
1990a) as an external standard. All analyses were run in
triplicate.
Receioed I October 1991
Accepted (rer>i.ved)9 March I992

ANUPAM SHARMA E T A L

I30

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 i n 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 spectrophotometric method of Talukdar and Dutta (1969). Three separate 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 andrographolide 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 50C (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 1b) and andrographolide was estimated using reference andrographolide as an external standard. The andrographolide 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.
Estimation of andrographolide in various parts of A . puniculatu. Roots, stem, leaves, pericarp and seeds were separated
from A . puniculuru procured from Calcutta, and andrographolide was estimated in these parts following the procedure
described above. Tdbk 2 shows the mean percentage of
andrographolide in different parts of the plant.

.I
0

bmin

Figure 1. Chromatograms of ( a ) reference andrographolide

(peak 1) a n d (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 t o 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 spectrophotometric analysis. The respective regression equations for
HPLC and spectrophotometric assay were: y =
139.8634~-15.1951 (r=0.999) and y=O.O002x0.0017 (r=0.994). Mean coefficients of variation of

Efficiency of HPLC assay. The efficiency of the assay was

determined by estimating the recovery of 5 mg andrographolide added to 1 g of A. puniculufu whole plant (Calcutta
~~~

Table 1. Peak areas and absorbance values obtained from

HPLC and spectrophotometric analysis of reference
andrographolide
HPLC

Spectrophotometry

Amount injected

Meana peak
area

lng)

fIlV x s)

(4

5
10
20
40
80
100

139
400
81 5
1590
31 50
3980

10
15
25
30
40
50
80
100

"n=3.

Amount loaded

Meana absorbance

- 3000 x
= 4000

>

0.000
0.001
0.005
0.007
0.009
0.012
0.019
0.023

.L

2000

1000

0.025

U
A*

0.020

STANDARDIZATION O F A N D R O G R A P H I S PANICULA TA
~

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

Yield of
MeOH ext
I%w/w)

Andrographolide
content
IMeana;tSE i%w/w)l

Confidence
limit
ip = 0.051

6.24
6.33
6.37
6.12

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

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

4.72
3.97
2.17
0.60
0.11

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

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

*
*

'n=3.

peak arealabsorbance from replicate ( n = 3) observations did not exceed O.X6/4.68 (respectively).
Recovery of andrographolide by the HPLC procedure was observed to be around 99% (Table 3) whilst
that reported for the spectrophotometric assay (Talukdar 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-

131

ent sources shows that the andrographolide content is

fairly constant (Table 2). However, different parts of
the plant show wide variations in andrographolide content, with leaves containing the highest (2.39"/0) and
seeds the lowest (0.13%) amounts. It would be pertinent 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, therefore, records a higher content of andrographolide
because of its associated impurities (Talukdar et a f . ,
1968).
The HPLC method of standardization of A. panicufatu described herein is simple, sensitive and precise,
and may be of value in standardizing the raw material
for preparation of formulations containing this plant.
Table 3. Recovery of andrographolide from A. paniculuta
whole plant (Calcutta sample) by HPLC assay
Andrographolide content (mg/g)

Ser'a' No

1
2
3

sample

7.94
8.27
8.15

samples

12.89
13.23
13.08

Recovery

(%I

99.0
99.2
98.6
Mean k SD 98.9 k 0.37

5 m g reference andrographolide added to 1 g of A. paniculata.

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