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PH Y TO C H EM IC A L A N A LY SIS ,

VOL. 3, 129-131 (1992)

Standardization

of

the Indian Crude Drug

Kalmegh by High Pressure Liquid

Chromatographic Determination of

Andrographolide

A n u p a m S h a r m a , Kr i s h an

La1

a n d S u k h d ev S. Ha n d a *

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 p opular hep atoprotective Indian herbal drug Kalrnegh

(Andrographis pan icd ata)

can be standar dized by

high pressure liquid chrom atograp hicdetermination of its ma jor active constituent, andro grapho lide. The leaves

of the herb were found to contain the highest amount (2.39 w/w )of andrographolideand the seeds to contain

the low est. The techniq ue was found to be much m ore sensitive and sim ple than the known spectrophotometric

method

Keywords: Standard iza t ion ; A ndrographo l ide ; Androgruplzis

paniculutn;

Kalmegh; high pressure liquid chro-

matography

I N T R OD U C T I ON

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. number of methods, including gravi-

metric Sengupta

er d. 948, 1949), titrimetric Rao,

1962; Talukdar

t al .

1968), spectrophotometric

Gaind

et a f . ,

1963; Talukdar and Dutta, 1969;

HOHaC CH3

1

'' Author t o wh o m 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 .

pan icda ta . The

spectrophotometric method Talukdar and Dut ta,

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 Dut ta, 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.

E X P E R I M E N T A L

Plant material.

A puniculata

(whole plant) was procured

from the markets o f Calcu t ta , Bombay . Dehradun and

Haridwar. Its identity was established by comparison with

reference specimens preserved at the Herbarium-cum-

Museum

of

the Depar tmen t

of

Pharmaceutical Sciences,

Panjab Universi ty, Ch andigarh .

HPLC analysis.

Wate rs chrom atograph compris ing a del i-

very pump 510, a gradient controller 680,

an

in jector U6K,

a

UV detec to r

481

and an in teg ra to r 730 was employed. A

resolve 5 pm spherical silica (3.9 mm X 15cm) 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 de tec to r was opera ted a t 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

992 by John Wiley Sons. Ltd

Receioed

O c t o b e r 1991

Accepted (rer>i.ved) Ma r c h I992

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ANUPAM SHARMA

E T A L

4000

-

3000

x

.L

2

x

>

-

1000

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 spectropho-

tometric method of Talukdar and Dutta (1969). Three separ-

ate silica gel G TLC plates ( 2 0 ~ 2 0 c m ;

.25 m m )

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 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

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 0 2 0

- 0 0 2 5

A

1

1 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 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

Meana peak

Amount injected area Amount loaded Meana absorbance

lng) fIlV x

s) 4

5 139 10 0.000

10 400 15 0.001

20 815 25 0.005

40 1590

30

0.007

80 3150

40

0.009

100 3980 50 0.012

80 0.019

100 0.023

n= 3 .

.I

1

2

3

bm i n

b

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 AN D DISCUSSION

An

attempt has been made to standardize the Indian

crude drug Kalmegh using H P L C 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 H P L C

and

0.50-5.00

mg/mL

in

the case of spectrophoto-

metric analysis.

The

respective regression equa tions

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

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STANDARDIZATION

OF A N D R O G R A P H I S

P A N I C U L A T 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)

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 0.013

0.74

5

0.019

0.69 0.010

0.65 k0.017

2.39

k

0.008

0.44

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).

How ever, different parts of

the plant show w ide 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. Th e method

of

quantitation of andrographolide in

the Indian Pharmacopoeia is gravimetric, which does

not involve purification of andrographolide an d, there-

fore, records a higher content of andrographolide

because of its associated impurities (Talukdar

et a f . ,

1968).

T h e HPLC method

of

standardization of A panicu-

fatu described herein is simp le, sensitive an d 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

o f andrographolide could be quantitated by H P L C

in

contriist

to

30

pg

by the spectrop hotom etric method

(Figure 2).

Analysis o f

A .

pu t1 i c lh ta procured from four difter-

Table 3. Recovery of andrographolide from A .

paniculuta

whole plant (Calcutta sample) by HPLC assay

Recovery

of

andrographolide by the

H P L C

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

Meank

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, deoxyandrographol ide 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 est imation 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

intoxica tion in rats.

Indian J. Med. Res. 928,

284-292.

Indian Pharmacopeia (1966). Kalmegh,

pp.

385-386.

Government of India Press, Calcutta.

Rao, S. V.

(1962).

Estimation of andrographolide in Kalmegh

(Andrographis paniculata). ndian J Pharm. 24, 134.

Satyavati,

G.

V. and Raina, M . 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

b y

TLC. lndian J. Appl. Chem. 32, 18-25.

77-78.