Chinese Medicine, 2011, 2, 121-124
doi:10.4236/cm.2011.24020 Published Online December 2011 (http://www.SciRP.org/journal/cm)
121
Determination of Ursolic Acid a Biomarker in Different
Swertia Species through High Performance
Thin Layer Chromatography
Mansi Gupta, Deepa Bisht, Sayyada Khatoon, Sharad Srivastava, Ajay Kumar Singh Rawat
Pharmacognosy and Ethnopharmacology Division, National Botanical Research Institute (CSIR), Lucknow, India
Received January 21, 2011; revised February 10, 2011; accepted April 14, 2011
Abstract
A simple highly precise method has been established for the determination of ursolic acid in three species of
Swertia viz. S. angustifolia, S. paniculata, and S. alata. The assay combines identification and quantification
of the analyte on silica gel 60GF254 HPTLC plates with visualization under UV light and scanning at 510 nm.
Keywords: Ursolic Acid, Swertia Species, HPTLC
1. Introduction
Swertia (family: Gentianaceae) is widely distributed thr-
oughout the temperate regions of Asia, Europe, America,
Africa, and Western Himalayas at altitudes of ca. 1500 -
2400 m from Kashmir to Nepal and China. Around 40
species of Swertia have been found in India and 15 occur
in the Himalayan regions of Uttaranchal, of which, S.
chiraita is well-known for numerous medicinal applica-
tions [1]. Several Swertia species are being used as sub-
stitutes or adulterants of S. chiraita and are known for
their therapeutic potential against fever, hepatitis, chole-
cystitis, pneumonia, osteomyelitis, dysentery, scabies, spa-
sm, pain, and malaria, and used as antidepressant, mu-
tagenic, antipsychotic, tuberculostatic, choleretic, antioxi-
dant, anti-inflammatory, and antidiabetic drugs [2-7].
Swertia is known as a rich source of xanthones, some
species also containing terpenoids and seco-iridoids [2,3,
7]. Previous chemical investigations of the plants have
revealed the presence of the Lupeol [8], Mangiferin [9,
10], β-sitosterol, 3-β-D-glucoside [11] and ursolic acid
[12]. In an earlier study, 1-hydroxy-8-glucosyloxy-3, 5-
dimethoxyxanthone, 1, 8-dihydroxy-3, 7-dimethoxyxa-
nthone, 3-methoxy-1, 5, 8-trihydroxyxanthone and urso-
lic acid have been reported from its aerial parts [7] with
potent antioxidant activity [13], but there has been no re-
port on the quantitative estimation of ursolic acid in these
species.
Hence, in this study we report a simple and highly pre-
cise high-performance thin-layer chromatographic (HP-
TLC) method for the determination of ursolic acid (Fig-
ure 1). An attempt has also been made to validate the
developed method in terms of precision, repeatability,
LOD, LOQ and accuracy. The method is also suitable for
industrial quality control and successful commercial ex-
ploitation of these species.
2. Experimental
2.1. Plant Material and Reagents
The whole plant of three different species of Swertia viz. S.
angustifolia, S. alata and S. paniculata were collected
from Kalamuni (Uttarakhand, India). The specimens were
authenticated and a voucher specimen of each species
was deposited in the Institute’s herbarium (LWG 2224-
37SAL, LWG 222438SAG, and LWG 222439SP, respe-
ctively.
Ursolic acid was from Sigma-Aldrich (Steinheim, Ger-
many); other reagents were from Merck (Germany).
Figure 1. Structure of ursolic acid.

M. GUPTA ET AL.
122
2.2. Extraction of Plant Material
Air dried (45˚C - 55˚C) powdered whole plant of the
three different Swertia species (each 2.0 g) were ex-
tracted separately with 3 × 20 ml methanol. Extracts
were evaporated to dryness under vacuum and 10 mg of
residues were re-dissolved in methanol (1 ml).
2.3. Standard Solution
A stock solution (0.1 mg·mL–1) of ursolic acid was pre-
pared by dissolving 2.5 mg ursolic acid standard, accu-
rately weighed, in 25 mL methanol in volumetric flask.
2.3. Chromatography
HPTLC was performed on 10 cm × 10 cm silica gel 60-
GF254 plates (Merck). Samples and ursolic acid standard
of known concentration were applied to the layers as 6
mm-wide bands, 15 mm from the bottom and sides of the
plates by use of a Camag Linomat 5 automated TLC ap-
plicator with the nitrogen flow providing a delivery
speed of 150 nl·s–1 from the application syringe. The pla-
tes were developed to a distance of 80 mm with toluene-
ethyl acetate-formic acid (8:2:0.1) as mobile phase in a
Camag twin-trough glass chamber previously saturated
with mobile phase vapor; temperature was 20˚C. After
removal of plates from chamber, completely dried in air
at room temperature (27˚C) and peak areas for samples
and standard were recorded by densitometry in absorb-
ance/reflection mode at λmax = 510 nm, by means of a
Camag TLC Scanner 3 with WINCATS version 3.2.1
software.
To prepare calibration plot, stock solution (0.1 mg·ml–1)
of ursolic acid was prepared and different volumes of the
solution were analyzed by HPTLC as described above.
Calibration plots were prepared by plotting peak area
against concentration.
2.4. Method Validation
Precision was checked by repeated scanning of the same
spot of (3 µg·spot–1) six times and results were expressed
in terms of relative standard deviation (% RSD). The
repeatability of the method was done by analyzing the
spots of 3 µg standard solutions after application on the
HPTLC plate (n = 5) and expressed in terms of % RSD.
Variability of the method was studied by analyzing ali-
quots of standard solution of (0.4, 0.8, 1.6, 3.2, 6.4 and
8.0 µg·spot–1) on the same day (intra-day precision) and
on different days (inter-day precision) and the results
were expressed in terms of % RSD. The accuracy of the
method was determined by recovery studies. Standard
spiked with three different amounts of (400, 600 and 800
ng) in plant samples were analyzed and the percent re-
covery calculated. Different dilutions of standard solu-
tions in methanol were applied. Limit of detection (LOD)
and limit of quantification (LOQ) were determined on
the basis of signal to noise ratios of 3:1 and 10:1.
3. Results and Discussion
To obtain high resolution and reproducible peaks, mobile
phases of different composition were investigated for
HPTLC analysis. The desired profile was achieved in
toluene: ethyl Acetate: formic acid (8:2:0.1 v/v) at 510
nm. The accuracy of recovery was checked by triplicate
analysis of plant samples spiked with known amount of
ursolic acid at three different concentrations. The aver-
age amounts of the compound in the whole plant of these
species are shown in Table 1. From this table it is clear
that the amount of ursolic acid was maximum in S. an-
gustifolia (0.6475%). Densitometric scans obtained have
similar pattern in extracts of the three Swertia species
along with biomarker ursolic acid. (Figur es 2 and 3).
Table 1. Results from quantitative analysis of Ursolic acid
in different Swertia species.
Species Amount of Ursolic acid* [% dry weight]
Swertia angustifolia 0.6475
Swertia alata 0.3076
Swertia paniculata 0.5510
*Results are mean from triplicate analysis.
Ursolic acid
1 2 3 4
Under visible light
1 = Swertia angustifolia; 2 = Swertia alata; 3 = Swertia paniculata; 4 =
Standard (ursolic acid).
Figure 2. Estimation of ursolic acid.

M. GUPTA ET AL.
123
Figure 3. Densitometric scan (at 510 nm) profile.
We have done qualitative TLC profiling of all the Swer-
tia species along with a parallel run of Oleanolic and
Ursolic acids. In this study we found OA and UA have
almost similar Rf values, however after derivatization
with anisaldihide-sufuric acid, bands of UA appears a
florescent yellow but OA appears as dark bluish-brown
in colour. In the present study we have calculated peak
percentage of UA only (Figure 4).
Precision of scanner was also checked by scanning
same spots five times and coefficient of variance was
calculated. Repeatability of method was also established
by applying 3 g per spot of standard solution five times
and coefficient of variance was calculated. Limit of de-
tection (LOD) and limit of quantification (LOQ) were
also determined. Calibration curve (range 2 - 10 μg) of
ursolic acid was linear (Table 2).
Table 2. Method validation parameters for the estimation of
Ursolic acid by HPTLC densitometry in different Swertia
species.
Parameter Ursolic acid (average values)
Recovery (%) 97.34
Precision (% RSD)
CV (instrument precision) 0.76
CV (method precision) 1.31
Inter-day precision 0.68
Intra-day precision 0.73
Limit of detection 0.86 µg
Limit of quantification 2.62 µg
Specificity Specific
Correlation coefficient 0.986
Linearity range 2 - 10 µg·spot–1
Linear regression coefficient y = 4807.861 + 3224.411
Slope 4.010 ± 0.4694
Y-Intercept 8878 ± 1285
Robustness Robust
Olenolic acid
Ursolic acid
1 2 3 4 5
4
3
2
1
1) Swertia alata; 2) S. angustifolia; 3) S. paniculata; 4) Oleanolic acid (OA);
5) Ursolic acid (UA).
Figure 4. A comparative TLC profile of OA & UA along
with different Swertia species.

M. GUPTA ET AL.
124
Ohe basis of these studies it can be concluded that the
he authors are grateful to Director, NBRI, for providing
] S. Garg, “International Bioscience Monograph,” Today &
ry of In-
hopra, S. L. Nayar and I. C. Chopra, “Glossar
Naithani, “Flora of Chamoli, Botanical Survey of
n
twal and R. S. Bisht, “A Xanthone Glycoside
4. Conclusions
n t
mal
method described above for determination of ursolic acid
as marker compound in different Swertia species is sim-
ple, sensitive, economical, and suitable for rapid screen-
ing of many plant samples. The analysis does not require
special sample pretreatment and as many as fifteen sam-
ples can be analyzed on a single 20 cm × 20 cm TLC
plate. The method can be used for the routine quality
control of Swertia species as well as for the quantifica-
tion of ursolic acid in other plant materials as well as in
compound herbal formulations containing ursolic acid.
5. Acknowledgements
T
all the facilities to conduct this research work and are
also thankful to National Medicinal Plant Board, India
for financial support.
6. References
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