Volume 20 Issue 4 Article 22

Thin-layer chromatographic separation and validated HPTLC method for

quantification of ursolic acid in various Ocimum species

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Recommended Citation
Rout, K.K.; Singh, R.K.; Barik, D.P.; and Mishra, S.K. (2012) "Thin-layer chromatographic separation and validated
HPTLC method for quantification of ursolic acid in various Ocimum species," Journal of Food and Drug Analysis: Vol.
20 : Iss. 4 , Article 22.
Available at: https://doi.org/10.6227/jfda.2012200416

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Journal of Food and Drug Analysis, Vol. 20, No. 4, 2012, Pages 865-871 doi:10.6227/jfda.2012200416

Thin-Layer Chromatographic Separation and Validated

HPTLC Method for Quantification of Ursolic Acid
in Various Ocimum Species
KEDAR KUMAR ROUT1, RAJESH KUMAR SINGH1, DURGA PRASAD BARIK2 AND
SAGAR KUMAR MISHRA3
1.
Post Graduate Department of Chemistry, North Orissa University, Orissa, India
2.
Department of Botany, School of Life Sciences, Ravenshaw University, Cuttack-753003, Orissa, India
3.
Pharmacognosy and Phytochemistry Division, University Department of Pharmaceutical Sciences, Utkal University, Vani Vihar,
Bhubaneswar-751004, Orissa, India

(Received: October 13, 2011; Accepted: June 13, 2012)

ABSTRACT

An improved thin-layer chromatographic separation and quantitative estimation of ursolic acid in different plant parts of various
Ocimum species have been developed and validated. Excellent separation of the components was achieved on high-performance precoated
TLC plates by using optimized ternary mobile phase consisting of toluene : acetone : formic acid (7.8 : 2.2 : 0.15, v/v/v). Quantification and
densitometric determination were performed after derivatization of the plate with methanol-sulphuric acid reagent in reflection/absorption
mode at 540 nm. The limit of detection and limit of quantification were found to be 20 and 35 ng/spot, respectively. The response of ursolic
acid was linear over the range of 40 to 440 ng/spot with a correlation coefficient of r2=0.9995, indicating good relationship between peak
area and concentration. Recovery values from 98.36 to 100.06% showed excellent accuracy of the method. The method was validated
according to ICH protocol for precision, repeatability and accuracy. Ursolic acid was efficiently separated from the other components by
the proposed method which was very simple, rapid, precise, sensitive and accurate for the quantification of ursolic acid in different plant
parts of Ocimum species. The maximum ursolic acid content was found in the stems.

Key words: HPTLC, Ocimum basilicum, Ocimum sanctum, TLC, ursolic acid

INTRODUCTION toxins(3). In pharmacological prospective, O. sanctum was

reported to have anti-inflammatory, analgesic, antipyretic(4),
Ocimum sanctum L. (Holi Basil) and Ocimum basilicum antioxidant(5), antiulcer(6), antitumour(7), antimutagenic(8),
L. (Sweet Basil) are two important species of genus Ocimum, anticarcinogenic and antifertility activities(9). Leaf powder
which are found in different areas of the world and have enor- was shown to have potent antidiabetic effect(10). The essential
mous medicinal properties(1). Whole plant of O. sanctum have oil of both species was reported as excellent antimicrobial,
been recommended for the treatment of bronchitis, bronchial insecticidal and antioxidant agents(11).
asthma, malaria, diarrhea, dysentery, skin diseases, arthritis, The major chemical constituents reported from O.
chronic fever and insect bite, etc. It has also been reported sanctum are eugenol(12), luteolin and luteolin-7-O-β-D-
to possess antifertility, anticancer, antidiabetic, antifungal, glucuronide, apigenin, orientin(13), ursolic acid(13,14), galu-
antimicrobial, hepatoprotective, cardioprotective, analgesic teolin, vicenin-2(15), vicenin-1(16), and gallic acid(17). Simi-
and adaptogenic properties(2). Similarly, O. basilicum tradi- larly, O. basilicum has been reported to contain a number of
tionally has been used as an antiseptic, preservative, seda- interesting compounds, such as monoterpenoids (carvone,
tive, digestive regulator and diuretic. It has also been recom- cineole, fenchone, geraniol, linalool, myrcene and thujone),
mended for the treatment of headache, cough, infections of sesquiterpenoids (caryophyllene and farnesol), the triterpe-
upper respiratory tract, kidney malfunction and to eliminate noid, ursolic acid and the flavonoid, apigenin(18). Among the
* Author for correspondence. E-mail: kd_rout@yahoo.co.in major constituents of Ocimum, ursolic acid, a pentacyclic
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Journal of Food and Drug Analysis, Vol. 20, No. 4, 2012

triterpenic acid is common to both species and one of the dichloromethane-methanol (1 : 1) in a Soxhlet extractor for
most important bio-active molecule. It is well known to 30 h under hot condition. The dichloromethane-methanol
possess many important pharmacological properties, such extract solution was filtered and concentrated under vacuum
as antimicrobial, antifeedant, hepatoprotective, antiinflam- to solid mass. Three gram of the extract was chromatograhed
matory, antiulcer, hypolipidemic, antiatherosclerotic and over silica gel (100-200 mesh) packed in a glass column and
anticancer activities(19-22). It has also been reported to inhibit eluted with solvents of increasing polarity from chloroform
viral infections of herpes simplex virus and human immu- to a mixture of chloroform-methanol (8%). Furthermore,
nodeficiency virus(23,24). Recently, it has shown marked chromatographic purification of the fractions, obtained by
anti-tumor effects and exhibited cytotoxic activity towards elution of the crude column with 2 to 4% methanol in chlo-
many cancer cell lines(22,25,26). Because of its interesting roform, with a mobile phase of n-hexane: ethyl acetate (4:
biological activities, specialist in the field of pharmacology 1) and recrytalization from chloroform-methanol yielded a
and medicinal chemistry are focusing their increasing interest white amorphous powder (75 mg), m.p. 259°C. TLC: Rf: 0.61
towards this molecule. (methanol-chloroform; 5 : 95, v/v); 1H NMR (DMSO-d6, 400
The literature search revealed two reports available for MHz): δ 5.14 (t, 1H, J = 13.2; 3.4 Hz, H-12), 3.19 (m, 1H,
the quantification of ursolic acid in O. sanctum leaves and J = 5.4 Hz, H-3), 2.20 (d, 1H, J = 11.2 Hz, H-18), 1.92 (dd,
their related formulations(27,28). But there is no literature 1H, J = 13.2 Hz, H-11), 1.56 (m, 1H, H-11), 1.52 (m, 1H,
regarding the quantification for other plant parts of Ocimum H-1), 1.45 (m, each 1H, H-18, H-21), 1.34 (m, 1H, αH-21),
species. Therefore, it was felt necessary to develop a HPTLC 1.30 (m, each 1H, H-6, H-7), 1.05 (s, 3H, H-23), 1.00 (s, 3H,
method for the determination of this important bioactive H-27), 0.98 (s, 3H, H-26), 0.91 (s, 3H, H-24), 0.88 (d, 3H,
molecule in various parts of Ocimum species. In this view, the J = 8.2 Hz, H-30), 0.78 (d, 3H, J = 6.4 Hz, H-29), 0.63 (s,
present study describes the development of a simple, rapid, 3H, H-25); 13C NMR (DMSO, 400 MHz): δ 178.65 (C28),
and validated HPTLC method for the estimation of ursolic 138.54 (C13), 124.95 (C12), 77.20 (C3), 55.15 (C5), 52.73
acid by using an efficient mobile phase in different parts of (C18), 47.39 (C9), 47.19 (C17), 42.00 (C14), 40.50 (C8),
O. sanctum (green & purple) and O. basilicum with excellent 39.25 (C4), 38.87 (C19), 38.74 (C20), 38.61 (C1), 36.89
separation of matrix. (C10), 36.88 (C22), 33.21 (C7), 30.76 (C21), 28.62 (C23),
27.90 (C15), 27.35 (C2), 24.18 (C16), 23.73 (C27), 23.22
(C11), 21.45 (C30), 18.37 (C6), 17.38 (C26), 17.27 (C29),
MATERIALS AND METHODS 16.43 (C24), 15.59 (C25); EI-MS 70 eV (m/z, relative inten-
sity %): 456 (M+, 6), 438 (18), 411 (12), 248 (100), 189 (24)
I. Instrumentation and 175 (27). The obtained spectral data of ursolic acid were
further confirmed by comparing the values with published
The HPTLC system (Camag, Muttenz, Switzerland) literature(29).
consisted of a TLC scanner III with winCATS software
(version 1.4.2), a Limomat 5 autosprayer fitted with 100 μL III. Sample Preparation
syringe and connected to a nitrogen cylinder, a twin trough
chamber (20×10 cm), a plate heater, a derivatization chamber, An accurately weighed powder samples (1 g each) of
and a documentation unit Reproastar 3. Acme brand silica gel leaf, stem and flower of O. sanctum (white & purple varieties)
(100-200 mesh) was used for column chromatography. NMR and O. basilicum were extracted with dichloromethane-meth-
data were recoded on a Jeol 400 MHz spectrometer with anol (1 : 1) in a Soxhlet extractor for 14 h after initial defat-
operating frequency of 399.65 Hz at 294.8 K. The sample ting with n-hexane. Extracts were concentrated and filtered
containing a solution of 10 mg in 1 mL of DMSO-d6 was through Whatman No.1 filter paper under vacuum. Finally,
measured using TMS as internal standard. The mass spectrum they were transferred to 25 mL volumetric flask and made the
(EI-MS) was recorded at 70 eV electron ionization on a Jeol volume with methanol.
D-300 spectrometer.
IV. Standard Stock Solution
II. Materials and Chemicals
A standard stock solution of pure ursolic acid was
The plant materials were collected locally from a Silvi- prepared by dissolving 2 mg in 100 mL methanol (20 ng/μL).
culture farm in Bhubaneswar, Orissa, India and the identity The solution was further diluted with methanol to make the
was confirmed by taxonomist. All the reagents used were of working solutions for the sensitivity study. The above stock
analytical grade and were obtained from the S. D. Fine Chem. solution was also used for the validation and calibration
Ltd. (Mumbai, India) and HPTLC plates were from Merck experiments.
KGaA (Darmstadt, Germany). TLC plates were pre-run with
methanol and activated at 65°C for 40 min prior to chroma- V. Chromatography and HPTLC Scanning
tography. The ursolic acid was isolated from the dichloro-
methane-methanol extract of leaves. After initial defatting of Chromatography was performed on prewashed and
the leaf powder with n-hexane, the marc was extracted with preactivated 20×10 cm aluminum-backed HPTLC plates
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Journal of Food and Drug Analysis, Vol. 20, No. 4, 2012

precoated with silica gel 60 F254 of 0.2 mm layer thickness. II. Method Validation
All the sample and standard solutions were applied on the
plate as 6 mm band located 10 mm from bottom and 11 mm Validation of the developed HPTLC method was carried
band gap using a Camag Linomat 5 applicator mounted with a out as per the International Conference on Harmonization
100-μL syringe, under a continuous drying stream of nitrogen (ICH) guidelines (CPMP/ICH/381/95, CPMP/ICH/281/95)
gas at a constant application rate of 150 nL/s. The standard for specificity, sensitivity, linearity, accuracy, precision,
solution of 4 ng/μL was applied in different volumes whose repeatability, and robustness(30).
concentrations ranged 8-80 ng on the TLC plate for determi-
nation of Limit of Detection (LOD) and Limit of Quantifica-
tion (LOQ). For linearity study, the same stock solution was Track 12, ID: Standard6
used and a series of standard solutions equivalent to 40, 120, 500
AU
200, 280, 360, and 440 ng/spot were applied to the TLC plate. 450
Similarly, various concentrations of sample solutions were
400
applied to the TLC plate in order to get the concentration of
350
ursolic acid in the calibrated range.
The applied HPTLC plates containing sample and 300
ursolic acid
standard spots were developed up to 77 mm in a Camag 250
twin-trough glass chamber (ascending mode of the devel- 200
opment under the laboratory conditions of 25 ± 3°C and 150
61 ± 4% of relative humidity) that has been presaturated with
100
the mobile phase, toluene : acetone : formic acid (7.8 : 2.2 :
0.15, v/v/v) for 4 min. After development, the plate was dried 50

with a stream of hot air and the densitometry scanning was 0

-0.15 0.05 0.25 0.45 0.65 Rf 0.85
performed by using a Camag TLC Scanner 3 with winCATS
software (version 1.4.2) in absorption-reflection scan mode Figure 1. A symmetrical HPTLC chromatogram of standard ursolic
acid (440 ng/spot, Rf = 0.53; Mobile phase = toluene : acetone : formic
after derivatization of the plates with methanolic-sulphuric
acid, 7.8 : 2.2 : 0.15, v/v/v).
acid reagent (5%). The marker compound, ursolic acid was
quantified by its maximum absorbance at 540 nm. The slit
dimension of the scanner was set at 5.0×0.45 mm (micro)
with 100 μm per step data resolution and 20 mm/s scanning
(A) (B) (C)
speed. Concentration of the compound was determined from
the scan intensity of diffusely reflected light using linear cali-
bration via peak areas.

RESULTS AND DISCUSSION

I. Development of Optimum Mobile Phase

In HPTLC analysis, the mobile phase plays a crucial role

for the exact measurement of analytes. A solvent system that
would give dense and compact spots with appropriate and
significantly different Rf value was highly desired. In view of
this, a number of mobile phase have been tested and toluene
: acetone : formic acid (7.8 : 2.2 : 0.15, v/v/v) was found to
give good separation of ursolic acid from its matrix with an
Rf value of 0.53 and the solvent migration distance of 77 mm
with chamber saturation time of 5 min. The optimum mobile
phase enabled satisfactory separation of the components
of the mixtures investigated with dense, compact, and well
shaped spots (Figure 1). Comparative thin layer chromato-
grams have been developed and compared with the earlier Figure 2. Comparative TLC obtained after derivatization with meha-
reports of Verma et al.(27) (toluene : ethyl acetate : acetic acid, nolic-sulphuric reagent. T1-ursolic acid standard, T2-O. sanctum leaf
30 : 3 : 1, v/v/v) and Anandjiwala et al.(28) (toluene : ethyl extract, (A) toluene : acetone : formic acid (7.8 : 2.2 : 0.15, v/v/v); (B)
acetate : formic acid, 35 : 15 : 1, v/v/v) which is presented toluene : ethyl acetate : acetic acid (30 : 3 : 1, v/v/v); (C) toluene : ethyl
in Figure 2. acetate : formic acid (35 : 15 : 1, v/v/v).
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Journal of Food and Drug Analysis, Vol. 20, No. 4, 2012

(I) Sensitivity precision and was found as 0.60 (Table 1).

Intra-day precision of the method within a laboratory
Sensitivity of the method was tested with respect to over a short period of time by the same operator using the
LOD and LOQ of ursolic acid. Series of concentrations of same equipment was evaluated for ursolic acid at three
ursolic acid in the range of 8-80 ng was applied on the TLC different concentrations (200, 280 and 360 ng/spot) over
plate and analyzed to determine LOD and LOQ. Under the entire calibration range for six time (n = 6) on the same day,
experimental conditions employed, LOD and LOQ were whereas inter-day precision was checked at the same concen-
determined to be 10 and 35 ng/spot, respectively (Table 1). tration level over entire calibration range for six time (n =
6) on the consecutive days. In both cases, the coefficient of
(II) Linearity and Calibration Curve variation of peak area of spots was used to evaluate method
precision and the results obtained are shown in Table 2. In
The linearity was tested at six concentration levels i.e. all the case, % RSD values were less than 2% confirming the
40, 120, 200, 280, 360 and 440 ng/spot of the ursolic acid. A precision of the method.
calibration plot was constructed by plotting peak area against
the concentration (ng/spot) with the help of the win-CATS (IV) Accuracy
software. The linear regression data showed a good linear
relationship over a concentration range of 40-440 ng/spot and The accuracy of the analytical method was determined
was defined by the linear equation y = 9.767x+272.512; the from recovery experiments, which were performed by spiking
slope, intercept, and correlation co-efficient were also deter- a known amount of ursolic acid at 3 different levels with 3
mined. The correlation co-efficient of the calibration plot replicates for each level and the coefficient of variation (CV)
was 0.9995 (Table 1), indicating a good linear relationship was calculated. Satisfactory recoveries ranging from 98.36
between peak area and concentration (Figure 3). to 100.06% with an average value of 99.10% for ursolic
acid was obtained by the proposed method. The results are
(III) Precision presented in Table 3. Small CV (< 1.46%) indicated that the
method provides sufficient accuracy.
Instrumental precision was studied by scanning six
bands of ursolic acid at a concentration of 300 ng/spot by the
proposed method. The coefficient of variation of measure-
ment of the peak area was taken to evaluate the system Substance: ursolic acid @ 540 nm Regression mode: Linear
Y = 27.512 + 9.767 X r=0.99951 sdv = 1.95%
5000
AU
4500
Table 1. Method validation data for estimation of ursolic acid
4000
Validation parameters Values 3500
Instrument precision, CV (%), n = 6 0.60 3000
Repeatability of application, CV (%), n = 6 1.04 2500
Limit of detection (ng/spot) 10 2000

Limit of quantification (ng/spot) 35 1500

Correlation coefficient (r) 0.9995 1000

Linearity range (ng/spot) 40-440 500

Number of data points 6 0

0.00 50.00 100.00 150.00 200.00 250.00 300.00 350.00 400.00 450.00
RSD of slope 0.55 ng
Figure 3. Linear calibration plot for ursolic acid (r = 0.99951, sdv =
RSD of intercept 1.32
1.95%), ×; standard ursolic acid levels, +; ursolic acid present in
Specificity Specific sample.

Table 2. Intra- and inter-day precision study of HPTLC methoda

Concentration Intra-day precision Inter-day precision

(ng/spot) Mean area S.D. % R.S.D. Mean area S.D. % R.S.D.
200 2277.65 6.91 0.31 2227.53 23.46 1.05
280 3067.42 16.80 0.55 3022.31 29.50 0.98
360 3766.92 21.70 0.58 3714.63 40.42 1.09
a
n=6
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Journal of Food and Drug Analysis, Vol. 20, No. 4, 2012

Table 3. Results from the recovery study of ursolic acid by the proposed HPTLC method

Amount of Amount of ursolic Amount of ursolic Total amount Total amount C.V Recovery Average recovery
sample (mL) acid present (ng) acid added (ng) (ng) found (ng)a (n = 3) (%) (%)
15 153.12 0 153.12 150.61 1.46 98.36
15 153.12 80 233.12 233.25 0.88 100.06
15 153.12 160 313.12 309.38 1.10 98.80 99.10
15 153.12 240 393.12 389.94 0.91 99.19
a
Each value is the mean of three analyses.

Spectra comparsion
100.0 100.0
[AU] [AU]
Standard Sample
80.0 80.0
70.0 70.0
60.0 60.0
50.0 50.0
40.0 40.0
30.0 30.0
20.0 20.0
10.0 10.0
0.0 0.0
400.0 450.0 500.0 550.0 600.0 650.0 700.0 [nm] 800.0
Figure 4. Matching the identity of ursolic acid in standard and sample
track by comparison of UV-VIS overlay spectra at 540 nm.

Spectra comparsion: Purity

100.0 100.0
Standard Sample
[AU] [AU]
80.0 80.0
70.0 70.0
60.0 60.0
50.0 50.0
40.0 40.0
30.0 30.0 Figure 6. Thin layer chromatogram after derivatization with methanol-
sulphuric reagent. T1-oleanolic acid standard, T2-O. sanctum (white),
20.0 20.0
T3-O, sanctum (purple) and T4-O. basilicum leaf extracts.
10.0 10.0
0.0 0.0
400.0 450.0 500.0 550.0 600.0 650.0 700.0 [nm] 800.0
Figure 5. Comparison of HPTLC UV-VIS spectra obtained at 540 nm
for purity test of ursolic acid present in sample and standard tracks. absorbance of wavelengths with an experimental correlation
limit of 0.99900; it was found that r (start, middle) = 0.999989
and r (middle, end) = 0.999959 (Figure 5).
The specificity of the method was also tested with respect
(V) Specificity to oleanolic acid present in the plant extracts. It was found
that oleanolic acid present in the plant extract was detected as
The specificity of the developed method was confirmed minor component or present in less quantity. Ursolic acid had
by comparing retardation factor (Rf) value of ursolic acid no interference from other extraneous components and well
standard with the sample. Furthermore, the identity of the resolved from them, indicating the method is specific only for
ursolic acid peak was confirmed by comparing the UV-Visible ursolic acid (Figure 6).
absorption spectrum of the peak from the standard with the
corresponding peak from the sample; they were found to be (VI) Robustness
superimposable (Figure 4). The peak purity test was also
conducted for the separated ursolic acid and was confirmed The robustness of the method was tested by small
by comparison of UV-VIS absorption spectra recorded deliberate changes in the composition of mobile phase and
from start to middle and middle to end at their maximum analysis of ursolic acid at 200 ng. It was found that there
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Journal of Food and Drug Analysis, Vol. 20, No. 4, 2012

Table 4. Robustness of method (n = 3)

Amount of ursolic acid detected

Mobile phase composition Rf value Amount of ursolic acid spotted (ng) % RSD
(ng, mean ± SD)

Toluene : acetone : formic acid

0.53 200 201.03 ± 2.13 1.06
(7.8 : 2.2 : 0.15, v/v/v)

Toluene : acetone : formic acid

0.52 200 199.90 ± 3.48 1.74
(8 : 2 : 0.15, v/v/v)

was no significant change in the Rf of the compounds and Table 5. Ursolic acid content in different plant parts of Ocimum
low value of % RSD, thus confirming the robustness of the species
method (Table 4). Ursolic acid content (%)
Plant species Plant parts
(Meana ± SD)
III. Quantification of Ursolic Acid Leaf 0.321 ± 0.024
Ocimum sanctum
Stem 0.565 ± 0.027
The developed method was applied to the determina- (white)
tion of ursolic acid in different plant parts of three Ocimum Flower 0.211 ± 0.032
species. Six replicates for each determination were made Leaf 0.392 ± 0.039
Ocimum sanctum
and the contents of ursolic acid were presented in Table 5. Stem 0.577 ± 0.026
(purple)
The low SD values indicated the suitability of the proposed Flower 0.226 ± 0.027
method for routine analysis of this important bioactive Leaf 0.062 ± 0.023
molecule.
Ocimum basilicum Stem 0.078 ± 0.014
Flower 0.049 ± 0.034
a
CONCLUSIONS n=6

The developed HPTLC method provides a simple,

precise, and accurate analytical method for the identification antioxidant activities of Ocimum species: Ocimum basi-
and exact quantification of ursolic acid in different parts of licum and Ocimum sanctum. J. Cell and Tissue Res. 10:
Ocimum species. A good separation of analyte was achieved 2145-2150.
using toluene : acetone : formic acid (7.8 : 2.2 : 0.15, v/v/v) 2. Prakash, P. and Gupta, N. 2005. Therapeutic uses of
as a mobile phase on precoated silica-gel 60 F254 plates. The Ocimum sanctum Linn (Tulsi) with a note on eugenol
method was successfully validated as per the ICH guidelines and its pharmacological actions: a short review. Indian J.
and statistical data proved that the developed HPTLC method Physiol. Pharmacol. 49: 125-131.
may be used as a tool for routine analysis of this bioactive 3. Zhang, J. W., Li, S. K. and Wu, W. J. 2009. The main
marker. It is evident from the analysis that the stems of chemical composition and in vitro antifungal activity of
Ocimum species accumulate a significant level of ursolic acid the essential oils of Ocimum basilicum Linn. var. pilosum
than the leaves and flower, which was not reported earlier. (Willd.) Benth. Molecules 14: 273-278.
Moreover, the Ocimum species may be used as an alternative 4. Godhwani, S., Godhwani, J. L. and Vyas, D. S. 1987.
source for this important bioactive molecule. Ocimum sanctum: an experimental study evaluating its
anti-inflammatory, analgesic and antipyretic activity in
animals. J. Ethnopharmacol. 21: 153-163.
ACKNOWLEDGMENTS 5. Bhattacharya, S. K., Bhattacharya, A., Das, K., Muru-
ganandam, A. V. and Sairam, K. 2001. Further investi-
The authors wish to express their thanks to Dr. P. K. Satp- gations on the antioxidant activity of Ocimum sanctum
athy, Head, Department of Chemistry, North Orissa Univer- using different paradigms of oxidative stress in rats. J.
sity for providing the necessary facilities for performing this Nat. Rem. 1: 6-16.
research work and also to the scientific officer, Dr. Acharya, 6. Bhargava, K. P. and Singh, N. 1981. Anti-stress
Drug Testing Laboratory, Bhubaneswar for his valuable activity of Ocimum sanctum Linn. Indian J. Med. Res.
support. 73: 443-451.
7. Annapurani, S. and Priya, R. 1999. Antimutagenic, anti-
tumourogenic and antigenotoxic effects of polyphenol
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