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High-Performance Thin-Layer
Chromatographic analysis of hepatoprotective
diterpenoids from...

Article in Phytochemical Analysis · January 2000

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 PHYTOCHEMICAL ANALYSIS
Phytochem. Anal. 11, 34–36 (2000)

High-Performance Thin-Layer
Chromatographic Analysis of Hepatoprotective
Diterpenoids from Andrographis paniculata

Sudhanshu Saxena, Dharam C. Jain,* Madan M. Gupta, Rajendra S. Bhakuni,

Hari O. Mishra and Ram P. Sharma
Central Institute of Medicinal and Aromatic Plants (CIMAP), CIMAP Post Office, Lucknow 226015, U.P., India

A rapid and simple high-performance thin-layer chromatographic (HPTLC) method has been developed
for the simultaneous quantitative estimation of the biologically active diterpenoids, 14-deoxy-11,12-
didehydroandrographolide, andrographolide, neoandrographolide and andrographiside in Andrographis
paniculata. The assay combines the isolation and separation of andrographolide derivatives on silica gel 60
F254 HPTLC plates with spot visualization and scanning at 540 nm. Methanol was found to be the most
appropriate solvent for the exhaustive extraction of andrographolide derivatives. Copyright # 2000 John
Wiley & Sons, Ltd.
Keywords: andrographolide; diterpenoids; hepatoprotective; anti-inflammatory; Andrographis paniculata; Acanthaceae.

INTRODUCTION HPTLC method for the simultaneous analysis of these

four important diterpenoids in plant samples.
Andrographis paniculata (Burm : f) Nees (Acanthaceae),
commonly known as Kalmegh, is widely used in the
traditional system of Indian medicine in the treatment of EXPERIMENTAL
hepatitis. The drug is mainly recognized for its
diterpenoid andrographolide (2) and related compounds
Plant material. Leaves of A. paniculata were collected
(Sharma et al., 1992; Tang and Eisenbrand, 1992). The
from the experimental farm of the Central Institute of
plant is reported to possess protective activity against
Medicinal and Aromatic Plants (CIMAP, Lucknow,
various liver disorders (Choudhury et al., 1987; Handa
India) and a voucher specimen has been deposited in
and Sharma, 1990; Sharma et al., 1991; Rana and
the herbarium of this Institute.
Avadhoot, 1991; Saraswat et al., 1995). Andrographolide
and related compounds were investigated for their
pharmacological properties and all showed varying Chemicals. Reagents and HPTLC plates used were from
degrees of anti-pyretic, anti-malarial and anti-inflamma- Merck (Darmstadt, Germany). Compounds 1–4 were
tory activity (Deng et al., 1982; Mishra et al., 1992; isolated from a methanolic extract obtained from 500 g of
Madav et al., 1995). During our crop improvement leaves of A. paniculata. A sample (18 g) of this extract
programme, a quick, sensitive and accurate analytical was subjected to column chromatography on silica gel
method was required for the analysis of large numbers of (Merck) eluted with mixtures of chloroform and
plant samples for 2 and its derivatives. Although a few methanol as indicated below.
methods (Gaind et al., 1963; Talukadar and Dutta, 1969; 14-Deoxy-11,12-didehydroandrographolide (1) was
Zhu et al., 1984; Sharma et al., 1992) have been applied eluted in fractions 16 and 17 from the silica gel column
for the quantitative determination of the major compound eluted with chloroform:methanol (97:3) as a white
2, many of these procedures are time consuming and lack amorphous powder and crystallized with an ethyl
precision. Moreover all methods are for only the one acetate:methanol mixture; melting point, 196–197°C;
diterpenoid, andrographolide. C20H28O4; MS, [M ‡ H]‡ 333; 13C-NMR (deuterated
In this paper is presented the isolation of the pyridine) d (ppm), 38.7 (C1), 28.9 (C2), 80.2 (C3), 43.3
pharmacologically important diterpenoids viz 14-deoxy- (C4), 55.6 (C5), 24.7 (C6), 37.4 (C7), 149.3 (C8), 61.9
11,12-didehydroandrographolide (1), andrographolide (C9), 39.5 (C10), 134.2 (C11), 122.0 (C12), 129.0 (C13),
(2), neoandrographolide (3) and andrographiside (4) 145.3 (C14), 70.6 (C15), 174.5 (C16), 108.0 (C17), 23.7
from A. paniculata, and the development of an optimised (C18), 64.2 (C19) and 16.0 (C20).
Andrographolide (2) was eluted in fractions 23–25
from the silica gel column eluted with chloroform:metha-
nol (92:8) and crystallized in methanol; melting point,
* Correspondence to: D. C. Jain, Central Institute of Medicinal and Aromatic
229–230°C; C20H30O5; MS, [M ‡ H]‡ 351; 13C-NMR
Plants (CIMAP), CIMAP Post Office, Lucknow 226015, U.P., India. (deuterated pyridine) d (ppm), 37.5 (C1), 29.1 (C2), 80.0
E-mail: root@cimap.ernet.in (C3), 43.4 (C4), 55.5 (C5), 24.5 (C6), 38.3 (C7), 148.0

CCC 0958–0344/2000/010034–03 $17.50 Received 20 April 1998

Copyright # 2000 John Wiley & Sons, Ltd. Revised 30 June 1998
Accepted 29 July 1998
 DITERPENOIDS FROM ANDROGRAPHIS PANICULATA 35

(C8), 56.5 (C9), 39.3 (C10), 25.1 (C11), 147.0 (C12), 130.2
(C13), 66.1 (C14), 75.3 (C15), 170.7 (C16), 108.8 (C17),
23.8 (C18), 64.2 (C19) and 15.3 (C20).
Neoandrographolide (3) was eluted in fractions 31–35
from the silica gel column eluted with chloroform:metha-
nol (88:12) and crystallized in methanol; melting point,
173–174°C; C26H40O8; MS, [M ‡ H]‡ 481; 13C-NMR
(deuterated pyridine) d (ppm), 39.3 (C1), 19.5 (C2), 36.6
(C3), 40.0 (C4), 56.4 (C5), 24.9 (C6), 38.9 (C7), 148.4
(C8), 56.9 (C9), 38.7 (C10), 22.3 (C11), 25.1 (C12), 134.3
(C13), 145.3 (C14), 70.6 (C15), 174.6 (C16), 107.0 (C17),
28.2 (C18), 72.7 (C19), 15.5 (C20), 105.3 (C1'), 75.3 (C2'), After colour development of the separated constitu-
78.7 (C3'), 72.0 (C4'), 78.2 (C5') and 63.1 (C6'). ents, compounds 1–4 were quantified using a TLC
Andrographiside (4) was eluted in fractions 48–50 scanner (Camag, model 3) equipped with CATS 4
from the silica gel column eluted with chloroform:metha- software (Camag) under the following conditions: slit
nol (85:15) and recrystallized in an ethyl acetate:metha- width 8  0.4 mm; wavelength 540 nm; absorption
nol mixture; melting point, 192–194°C; C26H40O10; MS, reflection scan mode. In order to calibrate the method,
[M ‡ H]‡ 513; 13C-NMR (deuterated pyridine) d (ppm), stock solutions of compounds 1–4 were prepared in
38.0 (C1), 29.2 (C2), 79.2 (C3), 43.7 (C4), 56.7 (C5), 25.1 methanol (1.0 mg/mL each) and various amounts of these
(C6), 38.5 (C7), 147.0 (C8), 55.8 (C9), 39.5 (C10), 25.1 solutions (1–25 mg) were analysed by HPTLC, exactly as
(C11), 148.2 (C12), 130.1 (C13), 66.1 (C14), 75.3 (C15), described above, and calibration curves thus constructed.
170.7 (C16), 108.5 (C17), 24.5 (C18), 72.0 (C19), 14.9
(C20), 105.4 (C1'), 74.9 (C2'), 78.7 (C3'), 71.9 (C4'), 78.4
(C5') and 62.9 (C6').
All of the structures were confirmed by comparison of RESULTS AND DISCUSSION
spectral analysis data with that reported in the literature
(Gupta et al., 1990; Matsuda et al., 1994). Different compositions of the mobile phase for HPTLC
were tested and the desired resolution of compounds 1–4
Extraction of plant material for HPTLC analysis. Air (see Table 1), together with symmetrical and reproduci-
dried (35–50°C) leaves (1.0 g) of A. paniculata were ble peaks, was achieved using chloroform:toluene:metha-
extracted with solvents (3  25 mL each) of varying nol (6:2.5:1.5) as the mobile phase (Fig. 1). The
polarity viz. chloroform, ethyl acetate, methanol and calibration curves for compounds 1–4 were linear in the
ethanol, in order to optimize the extraction method for the range 1–25 mg and are shown in Table 1. For the
most efficient extraction of andrographolide and it examination of recovery rates, known amounts of stock
derivatives. Extracts were concentrated under vacuum, solutions of compounds 1–4 were added to plant extracts
redissolved in methanol, filtered, re-concentrated and of A. paniculata and quantitative analysis replicated three
finally made up to 5 mL volume with methanol prior to times. Values determined were 98%, 96%, 98% and 97%,
HPTLC analysis. respectively, for compounds 1–4. Peak purity tests of all
four compounds were performed by comparing the
Chromatographic conditions. Chromatography was spectra (at 540 nm) of each in both the standard and the
performed on pre-activated (at 110°C) silica gel 60 F254 sample tracks.
HPTLC plates (10  10 cm; 0.25 mm layer thickness). Different solvents of varying polarities were used for
Samples and standard compounds were applied to the the extraction of andrographolide and its derivatives from
layer as 8 mm wide bands, positioned 10 mm from the plant material, and the average of three replicates are
bottom of the plate, using an automated TLC applicator presented in Table 2. Methanol was found to be the most
Linomat IV (Camag, Muttenx, Switzerland) with nitro- appropriate solvent for the maximum extraction of
gen flow providing delivery from the syringe at a speed of andrographolide and its derivatives.
10s/mL. These critical parameters were maintained for all In summary, the HPTLC method for the simultaneous
analyses performed.

Detection and quantification of compounds 1–4. The

development of the TLC layer was performed using a
Camag twin trough glass tank which had been pre-
saturated with the mobile phase of chloroform:toluene:
methanol (6:2.5:1.5) for ca 2 h, and the solvent front was
allowed to run to a height of 8 cm. The composition of the
mobile phase was optimized by testing different solvent
compositions of varying polarities. TLC analyses were
made under laboratory conditions of 20  5°C and 50%
relative humidity. After development, the layer was dried
and the components visualized by immersing the plate in
a freshly prepared mixture of glacial acetic acid:concen-
trated sulphuric acid:anisaldehyde (100:2:1) using an Figure 1. Scan (at 540 nm) of an HPTLC layer showing the
separation of 14-deoxy-11,12-didehydroandrographolide (1),
automatic immersion device (Camag) followed by andrographolide (2), neoandrographolide (3) and androgra-
heating of the layer at 110°C for 15 min with the aid of phiside (4) in standard (St) and sample (Sm) tracks. (For
a Camag TLC plate heater. chromatographic protocol see the Experimental section.)

# 2000 John Wiley & Sons, Ltd. Phytochem. Anal. 11: 34–36 (2000)
36 S. SAXENA ET AL.

Table 1. Linear regressions and Rf values for andrographolide and its derivatives
Compound Rf value Regression equation r

14-Deoxy-11,12-didehydroandrographolide (1) 0.67 Y = 631.9 X ‡ 104.4 0.995

Andrographolide (2) 0.57 Y = 1197.6 X ‡ 233.2 0.997
Neoandrographolide (3) 0.42 Y = 1812.0 X ‡ 328.0 0.995
Andrographiside (4) 0.15 Y = 1551.6 X ‡ 310.8 0.995

Different solvents of varying polarity have been applied

Table 2. Effect of the extracting solvent on the determina- for the extraction, and methanol was found suitable for
tion of different andrographolide derivatives in
Andrographis paniculata the most efficient extraction of these andrographolide
derivatives. The method is particularly suitable for the
Solvent used for Content of constituent (%)a
plant extraction 1 2 3 4 analysis of a large number of plant samples for the
improvement of A. paniculata drug for these major and
Chloroform 1.47 2.52 0.56 Ð
Ethyl acetate 1.03 1.91 0.35 Ð
biologically important diterpenoids. Results of the
Methanol 1.71 2.66 0.62 0.09 analytical programme will be presented elsewhere.
Ethanol 0.81 1.77 0.28 0.04
a
Average (n = 3) expressed on a dry weight basis.

Acknowledgements
analysis of compounds 1–4 from A. paniculata, reported The authors are thankful to the Director of CIMAP for his constant
here for the first time, is simple, rapid and accurate. encouragement.

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