American Journal of Molecular Biology, 2013, 3, 183-186 AJMB
http://dx.doi.org/10.4236/ajmb.2013.34024 Published Online October 2013 (http://www.scirp.org/journal/ajmb/)
Comparative study on some selected species of
Ocimum genus on free radical scavenging activity and
hepatoprotective activity against CCl4 induced
intoxication in rats
Myron Green1, Rajeswara Rao Pragada2*, Sambasivarao Ethadi2, Bettaiya Rajanna1
1Alcorn State Unive rsity, Mississippi, USA
2AU College of Pharmaceutical Sciences, Andhra University, Visakhapatnam, India
Received 9 July 2013; revised 10 August 2013; accepted 6 September 2013
Copyright © 2013 Myron Green et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
In this study, we compared the in vitro antioxidant
property among the selected Ocimum species (O.
sanctum, O. americanum, O. basilicum and O. gratis-
simum) and hepatoprotective activities of their ex-
tracts against CCl4 induced intoxication. The results
suggested that the four species of Ocimum genus showed
variability in Phenolic content and in vitro antioxi-
dant activity against DPPH, superoxide and hydroxyl
radicals in the following manner: O. sanctum > O.
americanum > O. basilicum > O. gratissimum respec-
tively. Based on serum AST, ALT, ALP and T. Bil
levels, the alcoholic extracts of Ocimum species showed
a significant dose dependent (250 mg and 500 mg and
750 mg/kg p.o.) and a protective effect against CCl4
induced hepatotoxicity in albino rats. The results fur-
ther revealed that the potential hepatoprotective ac-
tivity of Ocimum sanctum among t he Ocimum species.
Keywords: Ocimum sanctum; Ocimum americanum;
Ocimum basilicum; Ocimum gratissimum; Antioxidant
Activity; Carbon Tetrachloride; Hepatotoxicity
1. INTRODUCTION
The liver is the most important organ concerned with the
biochemical activities in the human body. It has great
capacity to detoxicate substances and synthesize useful
principles [1]. A major cause of these disorders is due to
exposure to different environmental pollutants, viruses,
and xenobiotics e.g., paracetamol, carbon tetrachloride,
thioacetamide, alcohol, etc. Reactive oxygen species are
implicated in liver damage [2] through covalent binding
and lipid peroxidation and have been shown to augment
fibrosis as seen from increased collagen synthesis [3].
Scavenging of free radicals by antioxidants could reduce
the fibrosis process in the tissues [4].
Ocimum sanctum (Family: Lamiaceae) is globally
known for its immense medicinal properties, especially
in India, and is popularly known as Tulsi. It is a holy plant
common to most Indian households. Ocimum sanctum is
known to possess anti-inflammatory, antimicrobial, anti-
diabetic, hepatoprotective, cardioprotective, antioxidant,
adaptogenic, anticancer and radioprotective activities.
There are other species in the same genus (Ocimum) and
its influence on hepatoprotective activity is not es-
tablished scientifically. Hence, the selected four species
O. sanctum, O. americanum, O. basilicum and O. gratis-
simum were subjected to the estimation of total phenolic
content, antioxidant activity and evaluated for hepato-
protective activity.
2. MATERIALS & METHODS
2.1. Plant Material and Extraction
The palnts O. sanctum, O. ameri can um, O. basilicum and
O. gratissimum were collected in Visakhapatnam region,
A.P, India and authenticity of the plants were confirmed
by Taxonomist Prof. M. Venkaiah, Department of Botany,
Andhra University, Visakhapatnam. Freshly collected
whole plants were dried under shade and were made into
coarse powder. 400 gms of O. sanctum, O. americanum,
O. basilicum an d O. gratissimum were macerated in 2.5
lts of 95% v/v ethanol. The alcoholic extracts thus ob-
tained were dried under reduced pressure at a room tem-
*Corresponding a uthor.

M. Green et al. / American Journal of Molecular Biology 3 (2013) 183-186 184
perature not exceeding 40˚C to get the crude extracts.
2.2. Chemicals
1,1-diphenyl-2-picrylhydrazyl (DPPH), 2-Deoxyribose,
Nitroblue tetrazolium, Silymarin purchased from Sigma
Chemicals Co. (St. Louis, MO, USA), Folin-Ciocalteau
reagent, Riboflavin, Hydrogen peroxide (H2O2), Carbon
tetrachloride (CCl4) from Loba Chemie Pvt Ltd. (Mum-
bai, India). AST, ALT, ALP and Total bilirubin assay kits
from Span diagnostics Pvt Ltd. Gujarat, India. All other
chemicals and reagents used were of analytical grade.
2.3. Animals
Adult Wistar rats (National Institute of Nutrition, Hy-
derabad, India) of either sex weighing 200 - 250 gms
were used in this study. The animals were maintained
under standard laboratory conditions at an ambient tem-
perature of 25˚C ± 2˚C having 50% ± 5% relative hu-
midity with 12 h light and dark cycle. The rats were ac-
climatized to the laboratory conditions for one week
prior to experimentation and provided with standard rat
pellet diet (Rayans biotech Pvt Ltd., Hyderabad, India)
and water ad libitum. The use and care of animals in the
experimental protocol has been approved by the local In-
stitutional Animal Ethics Committee (Regd. No. 516/01
/A/CPCSEA) following the guidelines of the committee
for the Purpose of Control and Supervision of Experi-
ments on animals (CPCSEA), Government of India.
2.4. Total Phenolic Content
Total phenolic co ntent of the extracts was determined by
using Folin-Ciocalteau reagent method [5]. Folin-Cio-
calteau colorimetry is based on a chemical reduction of
the reagent with a mixture of tungsten and molybdenum
oxides. The products of the metal oxide reduction have a
blue colour with absorption maximum at 765 nm. By us-
ing standard Gallic acid calibration curve, measured the
concentration of phenolic content in Gallic acid total
equivalents using units mg/gm.
2.5. Superoxide Radical Scavenging Activity
Superoxide scavenging activity of the plant extract was
determined by McCord and Fridovich method, 1969 [6]
which depends on light induced superoxide generation
by riboflavin and the corresponding reduction of Ni-
troblue tetrazolium was measured spectrophotometrically
at 560 nm.
2.6. Hydroxyl Radical Scavenging Activity
Hydroxyl radical scavenging activity was measured by
studying the competition between deoxyribose and the
extracts for hydroxyl radicals generated from the Fe2+/
EDTA/H2O2 system (fenton reaction). The hydroxyl
radical attacks deoxyribose, which eventually results in
the formation of thiobarbituric acid reacting substances
(TBARS) [ 7 ].
2.7. DPPH Radical Scavenging Activity
The scavenging activity for DPPH free radicals was
measured according to the procedure described by Braca
et al., 2003 [8]. In DPPH assay, method is based on the
reduction of alcoholic DPPH solution (dark blue in col-
our) in the presence of a hydrogen donating antioxidant
converted to the non radical form of yellow colored di-
phenyl-picrylhydrazine.
2.8. CCl4 Induced Intoxicated Rat Model
The animals were divided into 15 groups, each consisting
of 6 animals. The standard and test group animals were
treated with 100 mg/kg dose of Silymarin and 250, 500,
750 mg/kg dose of alcoholic extracts of selected plant
drugs for 5 days. On the 6th day, 1 h after treatment with
standard and test doses, the animals were intoxicated
with CCl4 in liquid paraffin (1:1 v/v, 1 ml of CCl4/kg,
p.o.). Serum was separated by centrifugation at 37˚C and
used for estimation of various biochemical parameters.
Biochemical parameters like serum enzymes, Aspartate
transaminase (AST) and Alanine transaminase (ALT)
were estimated by Reitman and Frankel, 1957 method
[9], Serum alkaline phosphatase (ALP) by King and
Armstrong, 1980 method [10], Serum Total bilirubin
(T.Bil) by Jendrassik and Grof, 1938 method [11] by
using commercial reagent kits in Aut oanalyzer (RM4000,
Biochemical systems International, Italy).
2.9. Statistical Analysis
Results of the biochemical estimations are reported as
Mean ± S.E.M. Significance was tested by One-Way
analysis of variance (ANOVA) followed by Dunnett’s
post-hoc test using Graphpad Prism-5 softw are.
3. RESULTS
The selected four species of Ocimum genus showed
variability in phenolic content in the following manner:
O. americanum (70.0 µg/gm) > O. sanctum (56.2 µg/gm)
> O. gratissimum (52.0 µg/gm) > O. basilicum (38.0
µg/gm). The alcoholic extracts of selected plants and
Ascorbic acid showed antioxidant activity with IC50
value on DPPH radical in the following manner: Ascor-
bic acid (60.24 ± 0.5 µg/ml) > O. sanctum (85.5 ± 0.7
µg/ml) > O. americanum (175.25 ± 1.5 µg/ml) > O.
basilicum (220.30 ± 1.6 µg/ml) > O. gratissimum
(230.35 ± 2.1 µg/ml) , respectively (Table 1).
Similarly the IC50 values of superoxide radical and

M. Green et al. / American Journal of Molecular Biology 3 (2013) 183-186 185
Table 1. Total Phenolic content of alcoholic extracts of O.
sanctum, O. americanum, O. basilicum and O. gratissimum.
Name of the alcoholic extract GAE(Gallic acid equivalent) mg/gm
O. sanctum 56.2 ± 1.2
O. americanum 70.0 ± 2.2
O. basilicum 38.0 ± 2.1
O. gratissimum 52.0 ± 2.3
Each value represents as mean ± S.E.M (n = 3).
hydroxyl radicals are 80.24 ± 0.4 µg/ml (Ascorbic acid)
> 225.23 ± 1.5 (O. sanctum) > 300.90 ± 2.1 µg/ml (O.
americanum) > 350.33 ± 1.4 (O. basilicum) > 360.35 ±
1.3 µg/ml (O. gratissimum) and 190.20 ± 0.5 µg/ml
(Ascorbic acid) > 140 ± 1.3 µg/ml (O. sanctum) > 200.90
± 2.2 µg/ml (O. americanum) > 285.31 ± 1.2 µg/ml (O.
basilicum) > 400.50 ± 1.3 µg/ml (O. gratissimum) re-
spectively (Table 2).
Based on ALT levels, the percentage protecti on offer ed
by the selected plant drugs at a dose of 750 mg/kg against
CCl4 is as follows: O. sanctum (83.72%), O. americanum
(73.71%), O. basilicum (62.48%) and O. gratissimum
(58.69%) respectively. The standard drug Silymarin
produced 87.04% protecti o n ( Table 3).
4. DISCUSSION
The genus Ocimum consists of more than 30 species and
the plants belonging to the genus contains numerous
phytochemicals including phenolics. The selected four
plant extracts O. sanctum, O. americanum, O. basilicum
and O. gratissimum contains phenolics ranging from
38.00 to 70.00 mg/gm gallic acid equivalents (GAE).
Mostly the phenolics are possessing antioxidant activity
and the plant extracts also showed good antioxidant ac-
tivity. A mong the four extracts O. sanctum showed b etter
antioxidant activity.
Hepatotoxicity produced by CCl4 seems to be medi-
ated by reactive metabolite trichloro methyl free radical
(.CCl3) formed by the hemolytic cleavage of CCl4 or
even by more reactive species trichloro methyl peroxy
free radical (Cl3COO.) formed by the reaction of .CCl3
with O2 caused membrane damage of hepatocytes and
resulted in centrilobular necrosis. The four plant extracts
showed dose dependent hepatoprotective activity against
CCl4 induced hepatotoxicity and maximum percent pro-
tection was observed at a dose of 750 mg/kg. Among th e
four extracts Ocimum sanctum showed better activity and
the results are comparable with that of the standard drug
Silymarin. The mechanism of action may be due to its
free radical scavenging (antioxidant) activity. Further the
study revealed that it is not the quantity b ut the quality of
phenolic compounds is important for showing good
Table 2. In vitro 50% inhibition concentration (IC50) of alco-
holic extracts of O. sanctum, O. americanum, O. basilicum, O.
gratissimum and Ascorbic acid on free radicals scavenging
activity.
Quantity of various extracts (µg)
Free radicals, reactive oxygen species
Extracts/Compounds
Superoxide
Radical Hydroxyl Radical DPPH Radical
Alc. extract of
O. sanctum 225.23 ± 1.5 140.0 ± 0.5 85.5 ± 0.7
Alc. extract of
O. americanum 300.90 ± 2.1 200.90 ± 2. 2 175.25 ± 1.5
Alc. extract of
O. basilicum 350.33 ± 1.4 285.31 ± 1. 2 220.30 ± 1.6
Alc. extract of
O. gratissimum 360.35 ± 1.3 400.50 ± 1.3 230.35 ± 2.1
Ascorbic acid 80.24 ± 0.4 190.20 ± 0. 5 60.24 ± 0.5
Each value represents as mean ± S.E.M (n = 3).
hepatoprotective activity and th e same was proved by the
good antioxidant activity of Ocimum sanctum.
5. CONCLUSIONS
The selected four species showed variability in phenolic
content ranging from 38.00 to 70.00 mg/gm GAE. The
selected plant extracts showed free radical scavenging
activity in a dose dependent manner. Among the selected
plants, Oc imum sa nctum showed better free radical scav-
enging activity and the results are comparable with that
of the standard drug Ascorbic acid. The four plant ex-
tracts showed hepatoprotective activity against CCl4 in-
duced intoxication. The study revealed the hepatoprotec-
tive activity of O. americanum for the first time and the
results are comparable with that of Ocimum sanctum.
Based on ALT levels among the four extracts, Ocimum
sanctum showed better activity (83.78 per c ent production)
and the results are comparable with that of the standard
drug Silymarin (87.04 percent protection). As per the
superoxide and hydroxyl radical scavenging activity, the
selected extracts showed good correlation between IC50
value and percentage of hepatoprotection. Overall the
study revealed the selected four plants possessing hepato-
protective activity and showed scopes for biomass utility.
6. ACKNOWLEDGEMENTS
The present work was carried out at Andhra University, Visakhapatnam,
India in Minority International Research Training (MHIRT) pro-
gramme-2010 awarded (NIH/NCMHD/MHIRT# 9T37 MD 001532) to
lcorn State University for their support and encouragement. The A

M. Green et al. / American Journal of Molecular Biology 3 (2013) 183-186 186
Table 3. Effect of alcoholic extracts of Ocimu m species on Serum parameters in rats intoxicated with CCl4.
Treatment Dose (p.o.) AST (IU/L) ALT (IU/L) ALP (IU/L) T.BIL (mg/dL)
Vehicle control 1 ml/kg 87.87 ± 2.6 45.33 ± 1.5 135.45 ± 4.9 0.10 ± 0.01
CCl4 1 ml/kg 247.48 ± 4.8 197.81 ± 5.0 363.51 ± 6.2 0.22 ± 0.01
Silymarin 50 mg/kg 112.03 ± 0.9a ( 8 4.46) 65.09 ± 3.2a (87. 04) 196.42 ± 2.2a (73. 27) 0.13 ± 0.01a (75.00)
250 mg/kg 164.52 ± 2.3a (51.98) 120.76 ± 2.2a (50.53) 243.23 ± 2.8a (52.74) 0.16 ± 0.01a (50.00)
500 mg/kg 126.42 ± 1.7a (75.85) 76.26 ± 3.6a (79. 72) 221.81 ± 2.8a (62. 13) 0.15 ± 0.01a (58.33)
Alc. ext of
O. sanctum
750 mg/kg 110.18 ± 1.4a (86.02) 70.06 ± 1.2a (83.78) 175.46 ± 1.4a (82.46) 0.13 ± 0.02a (75.00)
250 mg/kg 171.30 ± 2.3a (47.73) 130.68 ± 2.4a (44.03) 260.48±1.8a (45.18) 0.17 ± 0.01a (41.67)
500 mg/kg 144.10 ± 2.9a (64.77) 101.16 ± 3.2a (63.39) 250.25 ± 4.5a (49.66) 0.16 ± 0.02a (50.00)
Alc. ext of
O. americanum
750 mg/kg 131.19 ± 1.0a (72.86) 85.41 ± 1.7a (73.71) 197.33±1.0a (72.87) 0.14 ± 0. 02a (66.67)
250 mg/kg 185.08 ± 1.6a (39.10) 141.42 ± 1.5a (36.98) 276.42 ± 3.0a (38.19) 0.18 ± 0.01a (33.33)
500 mg/kg 155.79 ± 2.3a (57.45) 112.80 ± 1 .0a (55.75) 259.09 ± 4.5a (45.79) 0.16 ± 0.02a (50.00 )
Alc. ext of
O. basilicum
750 mg/kg 136.13 ± 1.0a (69.76) 102.54 ± 1.3a (62.48) 210.42 ± 1.1a (67.13) 0.15 ± 0.02a (58.33)
250 mg/kg 189.01 ± 2.1a (36.63) 150.24 ± 1.7a (31.20) 281.93 ±2.4a (35.77) 0.18 ± 0.01a (33.33)
500 mg/kg 152.94 ± 3.3a (59.23) 117.29 ± 2 .6a (52.81) 261.13 ± 6.7a (44.89) 0.17 ± 0.01a (41.67 )
Alc. ext of
O. gratissimum
750 mg/kg 148.40 ± 1.2a (62.08) 108.32 ± 1.2a (58. 69) 240.10 ± 1.0a (54.11) 0.14 ± 0.01a (66.67)
aP < 0.0001. All groups were compared with CCl4 group. Values are mean ± S.E.M., n = 6 animals per group. Values in the parenthesis indicate percent protec-
tion in individual biochemical parameters from their elevated values caused by the hepatoprotection. The percentage of the protection is calculated as 100 ×
(values of CCl 4 control—values of sample)/(values of CCl4 control—values of control). O. sanctum—Ocimum sanctum; O. americanum—Ocimum americanum;
O. basilicum—Ocimum basilicum; O. gratissimum—Ocimum gratissimum.
authors are also thankful to National Institute of Health (NIH), Mary-
land, USA for their financial assistance.
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