PHYTOCHEMICAL ANALYSIS

Phytochem. Anal. 12, 374–376 (2001)

DOI: 10.1002/pca.606

High-performance Thin Layer Chromatographic

Analysis of Anti-inflammatory Triterpenoids
from Boswellia serrata Roxb.

K. Krohn,1* M. S. Rao,1 N. V. Raman2 and M. Khalilullah2

1
Universität Paderborn, Fachbereich Chemie und Chemietechnik, 33098 Paderborn, Germany
2
Department of Chemistry, College of Engineering, JNT University, Kukatpally, Hyderabad, A.P, 500 072 India

A rapid and simple high-performance thin layer chromatographic (HPTLC) method was developed for the
simultaneous quantitative estimation of the biologically active triterpenoids b-boswellic acid, 3-O-acetyl-b-
boswellic acid, 11-keto-b-boswellic acid and 3-O-acetyl-11-keto-b-boswellic acid from the gum resin of
Boswellia serrata. The assay combines the isolation and separation of boswellic acid derivatives on silica gel
60F254-HPTLC plates with spot visualisation and scanning at 250 nm. Methanol was found to be the most
appropriate solvent for the exhaustive extraction of boswellic acid derivatives. Copyright # 2001 John
Wiley & Sons, Ltd.
Keywords: High-performance thin layer chromatography; triterpene acids; boswellic acids; anti-inflammatory, anti-
leukemia; Boswellia serrata.

INTRODUCTION

The gum resin of Boswellia serrata was used for the

treatment of inflammatory diseases in the traditional
Ayurvedic medicine in India (Kiritikar and Basu, 1935;
Chatterjee and Pal, 1984). A detailed pharmacological
study by Singh and co-workers (Singh and Atal, 1986;
Sharma et al., 1989) established that an alcoholic extract
of the gum displayed marked anti-inflammatory activity
in mice and rats and also inhibited the formation of
leucotrienes in rat peritoneal neutrophils in vitro (Mack
et al., 1990; Ammon et al., 1991). The constituents of
the gum resin were reported to be monoterpenes,
diterpenes and triterpenes (Pardhy and Bhattacharya,
1978a–c).
In this paper the isolation of the pharmacologically
important triterpene acids b-boswellic acid (1), 3-O-
acetyl-b-boswellic acid (2), 11-keto-b-boswellic acid (3)
and 3-O-acetyl-11-keto-b-boswellic acid (4) from B.
serrata is described, and the development of an optimised
high-performance thin layer chromatographic (HPTLC) Reagents and standards. Reagents were purchased
method for the simultaneous analysis of these four from Merck (Darmstadt, Germany). Compounds 1–4
important triterpenoids in gum resin is presented. were isolated from a methanolic extract obtained
from 100 g of oleogum resin of B. serrata. A sample
(45 g) of this extract was dissolved in 2% potassium
hydroxide solution (200 mL) and re-extracted with ethyl
EXPERIMENTAL acetate (5  150 mL). The ethyl acetate extract was
discarded and the aqueous solution was adjusted to pH 6
Plant material. The gum resin of B. serrata was with 2% hydrochloric acid and extracted again with
collected from a tree in a southern region of India. The ethyl acetate (5  150 mL). The combined ethyl acetate
plant material was identified by Dr V. S. Raju (Depart- solution was washed with water, dried over anhydrous
ment of Botany, Kakatiya University, Warangal, India). sodium sulphate and evaporated to dryness to yield 18 g
Voucher specimen reference number: B.248. of residue which consisted of a mixture of boswellic
acids. The residue was subjected to column chroma-
* Correspondence to: K. Krohn, Universität Paderborn, Fachbereich Chemie
tography over silica gel eluted with a gradient of
und Chemietechnik, 33098 Paderborn, Germany. petroleum ether:ethyl acetate to yield, with increasing
Email: kk@chemie.uni-paderborn.de polarity, 3-O-acetyl-b-boswellic acid (2), acetyl-11-keto-

Received 1 July 2000

Copyright # 2001 John Wiley & Sons, Ltd. Revised 30 January 2001
Accepted 7 February 2001
 DETERMINATION OF BOSWELLIC ACID DERIVATIVES BY HPTLC
Figure 1. HPTLC chromatograms of boswellic acids present in
the resin of Boswellia serrata. Trace St shows the standards
b-boswellic acid (1), 3-O-acetyl-b-boswellic acid (2), 11-keto-b-
boswellic acid (3) and 3-O-acteyl-11-keto-b-boswellic acid (4),
whilst trace Sm is from a gum resin sample.
b-boswellic acid (4), b-boswellic acid (1), and 11-keto-b-
boswellic acid (3). The isolated boswellic acids were
identified by comparison of their mass and IR spectral
data, [a]D values and melting points with those reported
in the literature (Pardhy and Bhattacharya, 1978a).
Extraction of oleogum resin for HPTLC analysis.
Lumps or granules (1.0 g) of gum resin from B. serrata
were extracted with solvents (3  25 mL each) of varying
polarity (chloroform, ethyl acetate, methanol and petro-
leum ether) in order to determine the most efficient
extraction method for boswellic acid and its derivatives.
Extracts were concentrated under vacuum, redissolved in
methanol, filtered, re-concentrated and evaporated to
5 mL prior to HPTLC analysis.
Chromatographic conditions. Chromatography was
performed on pre-activated (110°C) silica gel 60F254
HPTLC plates (10  10 cm; 0.25 mm layer thickness;
Merck). Samples and standard compounds were applied
to the layers as 8 mm wide bands, positioned 10 mm from
the bottom of the plate, using a Camag (Mutten,
Switzerland) Linomat IV automated TLC applicator with
nitrogen flow providing delivery from the syringe at a
speed of 10 mL/s. These parameters proved critical and
were maintained for all analyses performed.
Detection and quantification of compounds 1–4. TLC
plate development was performed using a Camag twin-
trough glass tank which had been pre-saturated with
mobile phase for ca. 2 h. Solvent was allowed to run up
the plate to a height of 8 cm. TLC analyses were made
under laboratory conditions of 20  5°C and 50%
Table 1. Linear regression equations and Rf values for boswellic acids 1–4 analysed by HPLTC
Compound Rf value
b-Boswellic acid (1) 0.42
3-O-Acetyl-b-boswellic acid (2) 0.52
11-Keto-b-boswellicacid (3) 0.46
3-O-Acetyl-11-keto-b-boswellic acid (4) 0.59
Copyright # 2001 John Wiley & Sons, Ltd.
375
Table 2. Inter- and intra-day precisions of the determina-
tion of b-boswellic acid by HPTLC
Concentration Intra-day Inter-day
(mg/spot) precisiona RSD% precisiona RSD%
5 1.85 2.95
10 1.09 1.85
20 0.72 1.06
a
n = 3.
relative humidity. The composition of the mobile phase
was optimised by testing different solvent compositions
of varying polarities. A mixture of hexane:acetone (7:3,
v/v) was employed in the optimised method.
After development, the layers were dried and the com-
ponents were visualised by UV irradiation at 250 and
365 nm. Compounds 1–4 were quantified on the plate
using a Camag model-3 TLC scanner equipped with
Camag CATS 4 software. The slit width was set to
8  0.4 mm and the scan wavelength was 250 nm. In order
to calibrate the method, stock solutions (1.0 mg/mL) of
compounds 1–4 were prepared in methanol and various
amounts of these solutions (containing 1–25 mg of
standard) were analysed by HPTLC exactly as described
above and calibration curves constructed.
The precision of the instrumentation was checked by
repeated scanning of the same spots of boswellic acids
(10 mg/spot) seven times each, and the coefficient of
variation (CV) calculated. The repeatability of the
method was tested by replicate scanning (n = 3) of
standard solutions of boswellic acids (10 mg/spot) after
application to a TLC plate. The variability of the method
was studied by analysing aliquots of different concentra-
tions of standard solutions of boswellic acids (1, 5, 10, 20
and 25 mg) on the same day (intra-day precision) and on
different days (inter-day precision) and the relative
standard deviation (RSD) values were calculated.
RESULTS AND DISCUSSION
Different compositions of the mobile phase for HPTLC
were tested and the desired resolution of compounds 1–4
(see Fig. 1), together with symmetrical and reproducible
peaks, was achieved using hexane:acetone (7:3, v/v) as
the mobile phase. The calibration curves for compounds
1–4 were linear over the range 1–25 mg and are presented
in Table 1, whilst the inter- and intra-day precisions of
the determination are given in Table 2. The recovery
rates, determined by adding known amounts of stock
solutions of each of the compounds to an extract of B.
Regression equation Correlation coef®cient
y = 631.9 x ‡ 104.4 0.999
y = 1197.6 x ‡ 233.2 1.000
y = 1812.0 x ‡ 328.0 1.000
y = 1551.6 x ‡ 310.8 0.999
Phytochem. Anal. 12: 374–376 (2001)
376
Table 3. Effect of the extracting solvent on the determination of boswellic acids 1–4 in Boswellia serrata
Solvent used for
plant extraction 1 2
Chloroform 2.52  0.0036
Methanol 2.66  0.0012
Petroleum ether 1.91  0.0023
Ethyl acetate 1.77  0.0015
a
Mean value (n = 3) expressed on a dry weight basis.
b
ND = none detected.
serrata followed by replicate (n = 3) quantitative ana-
lyses, were determined to be 97, 98, 96 and 98%,
respectively, for compounds 1–4. The purities of the
peaks corresponding to each of the four compounds were
confirmed by the demonstration that the UV absorption
spectra recorded on the CAMAG TLC scanner at the
start, middle and end position of each band were
completely superimposable.
Different solvents of varying polarities were used for
the extraction of the boswellic acids from the gum resin
of B. serrata, and the average of three replicates is
presented in Table 3. Methanol was found to be the most
appropriate solvent for the optimal extraction of
boswellic acids.
REFERENCES
Ammon HPT, Mack J, Singh GB, Safayhi H. 1991. Protection
of boswellic acids against galactosamine/endotoxin-
induced hepatitis in mice. Planta Med 57: 203±207.
Chatterjee GK, Pal SD. 1984. Anti-in¯ammatory agents from
Indian medicinal plants. Ind Drugs 21: 431.
Kirtikar KR, Basu BD. 1935. The anti-in¯ammatory action of
Indian medicinal plants. Ind Med Plants 1: 521±529.
Mack T, Ammon HPT, Safayhi H. 1990. Protection of bos-
wellic acids against galactosamine/endotoxin-induced
hepatitis in mice. Biochem Pharmac 41: 156±157.
Pardhy RS, Bhattacharya SC. 1978a. Structure of servatol, a
new diterpene cembranoid alcohol from Boswellia
serrata. Ind J Chem 16B: 171±173.
Copyright # 2001 John Wiley & Sons, Ltd.
K. KROHN ET AL.
Contenta of triterpene acid (%)
3 4
0.56  0.0021 1.47  0.0018 0.04  0.0022
0.62  0.0037 1.71  0.0031 0.09  0.0013
1.03  0.0014 0.33  0.0023 NDb
0.81  0.0025 0.28  0.0026 0.05  0.0036
In summary, the HPTLC method for the simultaneous
analysis of compounds 1–4 from B. serrata, reported here
for the first time, is simple, rapid and accurate. Different
solvents of varying polarity were used for the extraction
of the gum, but methanol was found to be most suitable
for the extraction of the boswellic acids.
Acknowledgements
The authors are grateful to Ram Reddy (Principal) and M. S. N. Raju
(Head, Department of Chemistry) at the JNTU College of Engineering,
Kukatpally, Hyderabad for their kind cooperation.
Pardhy RS, Bhattacharya SC. 1978b. Tetracyclic triterpene
acids from the resin of Boswellia serrata Roxb. Ind J
Chem 16B: 174±175.
Pardhy RS, Bhattacharya SC. 1978c. b-Boswellic acid, acetyl-
b-boswellic, acid-11-keto-b-boswellic acid and 11-keto-b-
boswellic acid from the resin of Boswellia serrata Roxb.
Ind J Chem 16B: 176±178.
Sharma ML, Bani S, Singh GB, 1989. Anti-arthritic activity of
boswellic acids in bovine serum albumin-induced arthri-
tis. Ind J Immunopharmac 11: 647±652.
Singh GB, Atal CK. 1986. Chemistry of some biologically
active Indian medicinal plants. Agents Actions 18: 407±
411.
Phytochem. Anal. 12: 374–376 (2001)