Pharmaceutical Biology

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A Rapid Method for Isolation of Andrographolide from

Andrographis Paniculata Nees (Kalmegh)

M. Rajani, Neeta Shrivastava & M.N. Ravishankara

To cite this article: M. Rajani, Neeta Shrivastava & M.N. Ravishankara (2000) A Rapid
Method for Isolation of Andrographolide from Andrographis Paniculata Nees (Kalmegh),
Pharmaceutical Biology, 38:3, 204-209, DOI: 10.1076/1388-0209(200007)3831-SFT204

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2000, Vol. 38, No. 3, pp. 204–209 © Swets & Zeitlinger

A RAPID METHOD FOR ISOLATION OF ANDROGRAPHOLIDE FROM

ANDROGRAPHIS PANICULATA NEES (KALMEGH)

M. Rajani*, Neeta Shrivastava and M.N. Ravishankara

Pharmacognosy and Phytochemistry Department, BV Patel Pharmaceutical Education & Research Development
(PERD) Centre, Thaltej, Ahmedabad – 380 054, India

ABSTRACT Andrographolide, neoandrographolide and

kalmeghnin present in the plant have been reported to
A simple and rapid method for isolation of andro- be the active principles (Handa et al., 1986; Choudhury
grapholide from the leaves of Andrographis paniculata
et al., 1987). Some of the other chemical constituents
is reported. This involves extraction of the leaf powder
by cold maceration in a 1:1 mixture of dichloro- include andrographanin, andrograpanoside, 14-deoxy-
methane and methanol and isolation of andro- 12-methoxyandrographolide, and deoxyandro-
grapholide directly from the resulting extract by grapholide (Fujita et al., 1984). The leaf extract, as well
recrystallisation. The identity of the compound was as andrographolide, have been shown to protect against
confirmed through IR, UV, mass and melting point, and alcohol and CCl4-induced hepatotoxicity and CCl4-
co-chromatography with a reference standard on TLC.
The purity of the compound was confirmed by TLC,
induced microsomal lipid peroxidation (Choudhury et
UV absorption spectrum, HPLC and differential scan- al., 1987; Chander et al., 1995). Andrographolide has
ning calorimetry, the latter of which gave the melting also been reported to have activity against Plasmodium
point of andrographolide as 235.3°C. berghei NK 65 (Misra et al., 1992).
We report a simple method for isolation of andro-
grapholide from the leaves of A. paniculata and details
INTRODUCTION of its characterization and purity check by IR, UV,
LCMS, HPTLC, HPLC and DSC.
Andrographis paniculata (Acanthaceae), commonly
known as kalmegh, is widely used in Indian systems of
medicine as a stomachic, tonic, antipyretic, alterative, MATERIALS AND METHODS
anthelmintic, febrifuge and cholagogue; for liver disor-
ders, general debility and colic pains (Nadkarni, 1954; Plant Material
Bentley & Trimen, 1983; Gupta et al., 1990; Aminud- Fresh leaf material of A. paniculata was obtained from
din et al., 1997). The leaf forms an ingredient of many a local medicinal plant farm in the month of November
patented Indian herbal proprietary preparations (e.g., and authenticated. The leaf material was air-dried
Kalmeghasava and Kalmeghnamay Haub) for the under shade for a day and then in a hot-air oven below
treatment of liver ailments (Handa et al., 1986; 60°C. It was powdered to 40 mesh and stored in an air-
Chaudhri, 1996; Evans, 1996). tight container at 15–20°C until further use.

Keywords: Andrographis paniculata Nees., andrographolide,

Extraction and Isolation
Kalmegh. Three samples of leaf powder, 50 g each, were sub-
jected to extraction with 3 different solvents:
• One sample of leaf powder was extracted exhaus-
Address correspondence to: M. Rajani, Pharmacognosy and
Phytochemestry Department, BV Patel Pharmaceutical Edu- tively with a 1:1 mixture of dichloromethane and
cation and Research Development (PERD) Centre, Thaltej, methanol by cold maceration. The extract was fil-
Ahmedadad – 308 054, India. tered and the solvent removed under vacuum
 RAPID METHOD FOR ISOLATION OF ANDROGRAPHOLIDE 205

(extract: 4.8g) – (AP I denotes andrographolide mobile phase containing methanol and water (1:1),
sample isolated from this extract). at a flow rate of 1 ml/min and the eluted compound
• The other two samples were macerated overnight, detected at 223 nm (method given in Anonymous,
one in methanol and the other in 95% alcohol and 1998, with some modifications).
filtered. The marc was packed into a Soxhlet and 4. By recording the melting point of the compound on
extracted with the respective solvents (10 cycles in a differential scanning calorimeter (DSC 7,
each case). The extracts were collected separately PERKIN ELMER)
and the solvents removed under vacuum (MeOH
extract: 5.4 g; 95% alcohol extract: 5.5 g) – (AP II
UV, IR and MS
and AP III denote andrographolide samples iso-
The sample along with the reference standard of
lated from the above methanolic extract and 95%
andrographolide was spotted and developed in a sol-
alcoholic extract, respectively).
vent system containing chloroform:methanol:ethyl
The dark green crystalline mass obtained was acetate (8:1.5:1). The UV absorption spectrum was
washed, separately, with toluene several times until recorded on a CAMAG TLC Scanner 3. UV absorp-
most of the colouring matter was removed from the tion spectrum of the sample and the reference stan-
residue. Then the toluene was completely removed dard, in methanol, were also recorded on UV/VIS
from the residue. The crystalline material left behind spectrophotometer (JASCO model 7850). IR spectra
(yield 1.9–2.0 g) was dissolved in hot methanol and were recorded on a BUCK SCIENTIFIC IR Spec-
cooled in a refrigerator for crystallisation. The process trophotometer (model 500). Atmospheric pressure ion-
was repeated several times until colourless plates of isation with ion spray mass spectra of molecular ions
constant melting point (uncorrected) of 230–231°C were obtained on a PE SCIEX API 165 MS with
(cc. Merck Index, 1989) were obtained (melting point WATERS LC.
was taken in a Melting Point Apparatus, TOSHNI-
WAL, Bombay).
RESULTS AND DISCUSSION
Thin-Layer Chromatography
For TLC experiments, precoated plates of silica gel The yield of crude andrographolide isolated, using the
60F254 (E. Merck) were used and spotting was done on three different solvents, was found to be about the
CAMAG LINOMAT IV Automatic TLC spotter. For same (1.9–2.0 g). Preliminary TLC evaluation of the
purity assessment of the isolated compounds and for three extracts showed andrographolide to be the major
recording UV spectrum of the compound, the plates component. Isolation of this compound in pure form
were scanned on a CAMAG TLC Scanner 3. was achieved by repeated washing of the crystalline
matter off the green colouring material with toluene
Methods Adopted for Testing the Purity of the and repeated recrystallization from methanol and final
Andrographolide Isolated washing of the crystals with cold methanol. The
The purity of the compound isolated was checked by purity of the sample at every stage of recrystallization
the following: was monitored through TLC. Among all the solvent
systems tried, the one containing chloroform:
1. By carrying out TLC in different solvent systems
methanol:ethyl acetate (8:1.5:1) was found to be suit-
(Puri et al., 1993; Chander et al., 1995) and co-
able for both extracts and pure andrographolide (Rf:
chromatography along with reference standard
0.65).
andrographolide (obtained from Regional Research
Laboratory, Jammu-Tawi, India).
Purity of the Andrographolide Isolated
2. By recording chromatogram and UV absorption
The purity of andrographolide isolated was established
spectrum of the compound developed on TLC plate
by the following:
in the solvent system containing chloroform:
methanol:ethyl acetate (8:1.5:1) on a CAMAG 1. TLC of the isolated sample, carried out in different
TLC Scanner 3 solvent systems (some of them reported, Puri et al.,
3. By carrying out HPLC of the isolated compounds 1993; Chander et al., 1995), showed a single spot
on a  Bondapack C-18 (10 m) column (3.9  with its Rf value varying from 0.2 and 0.9 in
300 mm), using a JASCO HPLC system, with a different solvent systems:
206 M. RAJANI ET AL.

Fig. 1. TLC chromatogram of andrographolide isolated (AP I, APII and AP III) and reference standard (R. Std).

Solvent system Rf of Andrographolide standard andrographolide. The UV and IR spectra of

Chloroform:methanol:ethyl acetate (8:1.5:1) 0.65 the isolated sample and the reference standards were
Chloroform:methanol (9:1) 0.9 superimposable.
Chloroform:ethyl acetate (6:4) 0.2
Chloroform:acetone:formic acid (7.5:1.65:0.85) 0.8
Spectral Data
2. UV absorption spectrum, recorded (on CAMAG
UV: max  232 nm (CAMAG TLC Scanner 3), max
TLC Scanner 3) at start, middle and end positions
in MeOH  222 nm (JASCO UV/VIS spectro-
of the band for purity of the sample, completely
photometer).
over lapped, and gave an absorption maxima (max)
IR: 3340–3200 (OH), 1725 (lactone), 1667 (double
at 232 nm (Fig. 2). Further, the TLC chromatogram
bond), 909 (external CH2).
also showed a single peak (Fig. 1).
LCMS: Mobile Phase: MeOH  2 mM NH4OAc;
3. The HPLC analysis of the isolated compound gave
351 (M  H), 368 (M  NH4), 373 (M  Na)
a single peak (Fig. 3) with a retention time of 4.8
(cc. Mol. wt. 350.46, Merck Index, 1989).
min, as with the standard andrographolide.
The above spectral data of the isolated sample and
4. The analysis by DSC gave a single sharp peak with
the reference standard confirm the identification of the
a melting point of 235.3°C and it is comparable
isolated compound as andrographolide (IR comparable
with that of the standard compound (Fig. 4). To the
with the reported data of Fujita et al., 1984). The
best of our knowledge, this is the first report on the
reported activities of andrographolide, as antihepato-
accurate melting point recorded for andro-
toxic, analgesic, antipyretic, antiulcerogenic, anti-
grapholide on DSC (cc with the m.p. of
inflammatory and immunostimulant agent (Puri et al.,
230–231°C obtained on melting point apparatus,
1993; Chander et al., 1995; Saraswat et al., 1995;
TOSHNIWAL).
Madav et al., 1995, 1996), justify the development of
The isolated sample of andrographolide was char- this simple and rapid method for its extraction and iso-
acterised by its spectral data (UV, IR and mass) and lation. Since in Ayurveda texts it has been recom-
found that it matched well with that of the reference mended to collect the plant material in the months of
 RAPID METHOD FOR ISOLATION OF ANDROGRAPHOLIDE 207

Fig. 2. Absorption spectra of andrographolide isolated (AP I, APII and AP III) and reference standard (R. Std), in the UV range, taken on a
CAMAG TLC Scanner 3.

Fig. 3. HPLC of andrographolide isolated (AP I, APII and AP III) and reference standard (R. Std).
208 M. RAJANI ET AL.

Fig. 4. DSC curve for andrographolide isolated (AP I) and reference standard (R. Std).

October-November, we collected the sample in Novem- ACKNOWLEDGEMENTS

ber and obtained a high yield of andrographolide.
The authors thank the Director, BV Patel PERD Center for
facilities, Dr. Kamala K Vasu, Department of Medicinal
Chemistry, BV Patel PERD Centre, for help in interpreting
CONCLUSION
IR and mass spectra, and Ms. Swati Guttiker and Mr. Ajay
Pillai for their help.
Of the three solvents used for the extraction of A. pan-
iculata leaf, the mixture of dichloromethane and
methanol (1:1) was found to be comparable in its
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