WORLD JOURNAL OF PHARMACEUTICAL RESEARCH - WJPR!

S. Gupta et al. World Journal of Pharmaceutical research 

IN-VITRO ANTIOXIDANT AND FREE RADICAL SCAVENGING ACTIVITIES OF 

www.wjpr.net 

OCIMUM SANCTUM 

*Saurabh Gupta 1 , M. N. Sathish Kumar 1 , B. Duraiswamy 2 , Mahavir Chhajed 3 , 

Atika Chhajed 4 

1 Department of Pharmacology, J.S.S. College of Pharmacy, Ootacamund (TN)-643 001 

2 Department of Pharmacognosy, J.S.S. College of Pharmacy, Ootacamund (TN)-643 001 

3 SAFE Institute of Pharmacy, Gram Kanadiya, Indore (MP)-452 016 

4 Devi Ahilya College of Pharmacy, Jaora Compound, Chhawani, Indore (MP)-452010 

Article Received on 

21 January 2012, 

Revised on 017 February 2012, 

Accepted on 27 February 2012 

�� 

�� 

�� 

ABSTRACT 

�� 

�� 

* Saurabh Gupta 

Department of Pharmacology, 

J.S.S. College of Pharmacy, 

Ootacamund - 643 001 

Tamilnadu, India. 

saurabhgupta80@gmail.com, 

satishalya@hotmail.com 

The present study was carried out to evaluate the antioxidant and free 

radical scavenging activity of aqueous ethanolic (1:1) extract of 

Ocimum sanctum (AEOS) in various systems. DPPH radical, nitric 

oxide radical, superoxide anion radical and hydroxyl radical 

scavenging assays were carried out to evaluate the antioxidant 

potential of the extract. The antioxidant activity of aqueous ethanolic 

extract increased in a dose dependent manner. About 50, 100, 200, 

300, 400 and 500 μg/mL of AEOS showed 38.06, 41.45, 44.83, 49.06, 

57.78 and 65.98% inhibition respectively on peroxidation of linoleic 

acid emulsion. Like antioxidant activity, the effect of AEOS on 

reducing power increases in a dose dependent manner, indicating 

some compounds in Ocimum sanctum is both electron donors and 

could react with free radicals to convert them into more stable 

products and to terminate radical chain reactions. In DPPH radical 

scavenging assay the IC50 value of the extract was found to be 

34.21μg/mL where as IC50 value of ascorbic acid was 18.69 μg/mL. 

AEOS was found to inhibit the nitric oxide radicals generated from 

sodium nitroprusside, the IC50 value was found to be 86.91 μg/mL, 

whereas the IC50 value of curcumin was 86.91 μg/mL. Moreover, the 

AEOS was found to scavenge the superoxide generated by 

PMS/NADH-NBT system. The IC50 value of extract was found to be 

78


73.38 μg/mL whereas the IC50 value of curcumin was found to be 24.67 μg/mL. AEOS was 

also found to inhibit the hydroxyl radical generated by the deoxyribose method, the 

concentration of extract needed for 50% inhibition was found to be 42.69 μg/mL. Catechin, 

used as a standard, showing an IC50 value 17.71 μg/mL, which indicates the prooxidant 

activity of AEOS. The total phenolic and flavonoid content of Ocimum sanctum were also 

determined in this study and is found to be 82.02 ± 8.17 mg GAE/g and 74.6±5.1 mg/g 

respectively. The results obtained in the present study indicate that the AEOS can be a 

potential source of natural antioxidant. 

Key words: Ocimum sanctum, antioxidants, DPPH radical, nitric oxide radical, superoxide 

radical, hydroxyl radical 

INTRODUCTION 

Oxidative deterioration of fat components in foods is responsible for the rancid odors and 

flavors which decrease nutritional quality. The addition of antioxidants is required to preserve 

food quality. Synthetic antioxidants such as butylated hydroxytoluene (BHT), butylated 

hydroxyanisole (BHA), tert-butylhydroquinone (TBHQ) and propyl gallate (PG) are widely 

used as antioxidants in the food industry. 

Their safety, however, has been questioned. BHA was shown to be carcinogenic in animal 

experiments. At high doses, BHT may cause internal and external hemorrhaging, which 

contributes to death in some strain of mice and guinea pigs. This effect is due to the ability of 

BHT to reduce vitamin K-depending blood-clotting factor. [1] Therefore, the importance of 

replacing synthetic antioxidants by natural ingredients from oil seeds, herbs and spices and 

other plant materials has increased due to health implications and increased functionality 

which improves solubility in both, oil and water. It is well known that natural antioxidants 

extracted from herbs and spices (rosemary, oregano, thyme, etc.) have high antioxidant 

activity and are used in many food applications. [2-3] A number of studies deal with the 

antioxidant activity of extracts from herbs and spices. [4-7] 

Most of the free radical scavenging potential in herbs and spices is due to the redox properties 

of phenolic compounds which allow them to act as reducing agents, hydrogen donators and 

singlet oxygen quenchers. [8-9] 

The importance of free radicals and reactive oxygen species (ROS) has attracted increasing 

attention. ROS and free radical mediated reactions are involved in degenerative or 

www.wjpr.net 

79


pathological processes such as aging, cancer, rheumatoid arthritis, coronary heart disease and 

Alzheimer’s disease. Many antioxidants compounds, naturally occurring in plant sources 

have been identified on free radical or active oxygen scavenger. Many synthetic antioxidant 

components have shown toxic or mutagenic effects, which have shifted the attention onto the 

naturally occurring antioxidants. [10] 

Free radicals include super oxide radical (SOR), hydroxyl radical (OH), hydroperoxyl radical 

(HPR), alkoxy radical (AR), peroxyl radical (PR) and nitric oxide radical (NOR). Other 

molecules that act like free radicals are singlet oxygen, hydrogen peroxide (H2O2), and 

hypochlorous acid (HOCl). Collectively free radicals and non -free radicals are called 

oxidants or reactive oxygen species. [11] 

Use of herbal remedies as therapeutic agents is promising and gaining the momentum, which 

constitute important part of traditional medicine system, Ayurveda. Ocimum sanctum is also 

known as Holy Basil in English or Tulsi or Tulasi in Hindi, belongs to the family Lamiaceae 

(Labiatae) and is considered as a sacred plant in Hindu culture. [12] 

The genus Ocimum, of the Family Labiatae, includes at least 60 species and numerous 

varieties. It represents an important source for essential oils and is used in the food, 

perfumery and cosmetic industries. Some Ocimum spp. are used in traditional medicine for 

different applications, especially in many Asian and African countries. The recurring 

polymorphism determines a large number of subspecies, different varieties and forms 

producing essential oils with varying chemical composition. Some present high camphor 

content, others are characterized by citral, geraniol, methyl chavicol, eugenol, thymol, etc. [13- 

16] 

The plants of O. sanctum can belong to the same chemotype, independently of their colour 

(purple or green). The literature describes oils of O. sanctum of different origin that contain 

high amounts of eugenol or methyl eugenol or sesquiterpenes. Eugenol (27–83%) is the main 

component of oils. Some of these oils contain also high amounts of methyl eugenol (3–24%), 

methyl chavicol (10–15%) and/or sesquiterpene hydrocarbons. Among them, ¡-caryophyllene 

is often the main compound. [17] 

In the oil of O. sanctum green, 51 components were identified. These represent about the 

94.2% of the whole oil. Its composition suggests that the oil could be classified as 

eugenol/sesquiterpene type. Eugenol represents 41.7% of the total oil, the sesquiterpene 

www.wjpr.net 

80


hydrocarbons are 39.2% and the total sesquiterpene components are 45.9% of the total oil. 

Among the sesquiterpene hydrocarbons, the main components were ¡-caryophyllene and ¡- 

elemene. [18] The quantitative composition of this oil agrees with some of the literature that 

reports eugenol, ¡-caryophyllene, ¡-elemene and qurcetine as the main components. 

MATERIAL AND METHODS 

Collection and preparation of extract: 

The whole plants were collected in the month of March, 2006, from Salem district, 

Tamilnadu, India. Dr. G. Murthy, Botanist, Botanical Survey of India (BSI), Coimbatore 

authenticated the collected plant; voucher specimen has been preserved in our laboratory 

(BSI/SC/5/23/06-07/Tech) for future reference. The taxonomic identification was carried out 

following Keshavamurthy and Yoganarasimhan [19] and Gamble [20] . 

The collected plant material was washed thoroughly with water to remove any unwanted 

matter. Then dried in shade, grinded to a coarse powder with a mechanical grinder and passed 

through sieve no. 40. Further, it was extracted with cold maceration process. For this, the 

coarse powder material was kept in the mixture of ethanol and distilled water (1:1) for 10 

days with intermittent shaking. The solvent was removed by distillation under reduced 

pressure and resulting semisolid mass was vacuum dried using rotary flash evaporator to get a 

solid residue (yield 4.16% w/w). 

Drugs: 

Ammonium thiocyanate was purchased from E. Merck. Ferrous chloride, ferric chloride, 

polyoxyethylene sorbitan monolaurate (Tween -20), 1,1-diphenyl-2-picryl-hydrazyl (DPPH), 

nicotinamide adenine dinucloetide (NADH), Ethylene Diamine Tetra Acetate (EDTA), 

butylated hydroxy toluene (BHT), butylated hydroxy anisole (BHA), α -tocopherol, ascorbic 

acid, quercetin, catechin, pyrocatechol, nitrobluetetrazolium, thiobarbituric acid, 2-deoxy-2- 

ribose, trichloroacetic acid, phenazine methosulphate, potassium ferricyanide and standard 

qurcetine were purchased from Sigma Chemical Co. Ltd, USA. All other unlabeled chemicals 

and reagents were of analytical grade and were used without further purification. 

Phytochemical screening: 

Phytochemical analysis of the major phytoconstituents of the plant extracts were undertaken 

using standard qualitative methods (color tests and/or TLC). [21] 

www.wjpr.net 

81


Antioxidant studies [22-23] 

Antioxidant activity: 

The antioxidant activity of hydroalcoholic extract was determined according to the 

thiocyanate method. [24] About 10 mg of extract was dissolved in 10 mL distilled water. 

Various concentrations (50, 100, 200, 300, 400 and 500 μg/mL) of extract was added to 

linoleic acid emulsion (2.5 mL, 0.04 M, pH 7.0) and phosphate buffer (2 mL, 0.04 M, pH 

7.0). The linoleic acid emulsion was prepared by mixing 0.2804 g of linoleic acid, 0.2804 g 

of Tween 20 as emulsifier and 50 mL phosphate buffer and then the mixture was 

homogenized. The final volume was adjusted to 5mL with potassium phosphate buffer (0.04 

M, pH 7.0). Further the mixed samples were incubated at 37 0 C in a glass flask for 60 h to 

accelerate the oxidation process. [34-35] At each 12 h interval, 1 mL of the incubated sample 

was taken and 0.1 mL of FeCl2 (0.02 M) and 0.1 mL of ammonium thiocyanate (30%) were 

added. The amount of peroxide was determined by measuring the absorbance at 500 nm. α- 

Tocopherol was used as the reference compound. To eliminate the solvent effect, the control 

sample, which contains the same amount of solvent added into the linoleic acid emulsion in 

the test sample and reference compound was used. All the data are expressed as mean of 

triplicate determinations. The percentage of inhibition of lipid peroxide generation was 

measured by comparing the absorbance values of control and those of test samples. 

Reductive ability: 

The reducing power of the extract was determined according to the method of Oyaizu. [24] 10 

mg of extract in 1 mL of distilled water was mixed with 2.5 mL of phosphate buffer (0.2 M, 

pH 6.6) and 2.5 mL of potassium ferricyanide [K3Fe(CN)6] (1%). The mixture was incubated 

at 50 0 C for 20 min. A portion (2.5 mL) of trichloroacetic acid (10%) was added to the 

mixture, which was then centrifuged at 3000 g for 10 min. The 2.5 mL of upper layer of the 

solution was mixed with 2.5 mL of distilled water and 0.5 mL of FeCl3 (0.1%) and the 

absorbance was measured at 700 nm. BHT was used as reference compound. All the analyses 

were performed in triplicate and the results were averaged. Increased absorbance of the 

reaction mixture indicated increasing reducing power. 

DPPH radical scavenging activity: 

The free radical scavenging activity of the hydroalcoholic extract of Ocimum sanctum was 

measured by 1,1-diphenyl-2-picryl-hydrazil (DPPH) using the method of Blois; a method 

based on the reduction of a methanolic solution of the coloured DPPH radical. [25-26] Used as a 

reagent, DPPH evidently offers a convenient and accurate method for titrating 0.1 mM 

www.wjpr.net 

82


solution of DPPH in methanol was prepared and 1 mL of this solution was added to 3 mL of 

the extract suspension in water at different concentrations (20, 40, 60, 80 and 100 μg). After 

30 minutes of incubation, absorbance was measured at 517 nm. Ascorbic acid was used as 

reference material. Lower absorbance of the reaction mixture indicated higher free radical 

scavenging activity. All the tests were performed in triplicate and the results averaged. The 

percentage reduction in absorbance was calculated from the initial and final absorbance of 

each solution. [27-28] The percentage inhibition was calculated by comparing the absorbance 

values of control and samples. Percentage scavenging of DPPH radical was calculated using 

the formula, 

www.wjpr.net 

% Scavenging of DPPH = 

Nitric oxide radical scavenging effect: 

[Absorbance of Control- Absorbance of Test] × 100 

[Absorbance of Control] 

Nitric oxide generated from sodium nitroprusside in aqueous solution at physiological pH 

interacts with oxygen to produce nitrite ions which were measured by the Griess reaction. [29- 

30] Scavenger of nitric oxide compete with oxygen leading to reduced production of nitric 

oxide. 

[31] The reaction mixture (3 mL) containing sodium nitroprusside (10 mM) in 

phosphate buffered saline (PBS) and the extract in different concentrations (20, 40, 60, 80 

and 100 µg) were incubated at 25 o C for 150 min. At every 30 min interval, 0.5 mL of the 

incubated sample was removed and 0.5 mL of Griess reagent (1% sulpha nilamide, 0.1% 

naphthylethylene diamine dihydrochloride in 2% H3PO4) was added. The absorbance of the 

chromophore formed was measured at 546 nm. All the analyses were performed in triplicate 

and the results were averaged. The percentage inhibition of nitric oxide generated was 

measured by comparing the absorbance values of control and test. Curcumin was used as a 

reference compound. 

Superoxide anion radical scavenging effect: 

Measurement of superoxide anion scavenging activity of the hydroalcoholic extract of 

Ocimum sanctum was done based on the method described by Nishimiki et.al. [32] and slightly 

modified. About 1 mL of nitroblue tetrazolium (NBT) solution (156 μM NBT in 100 mM 

phosphate buffer, pH 7.4), 1 mL of NADH solution (468 μM in 100 mM phosphate buffer, 

pH 7.4) and 0.1 mL of sample solution of hydroalcoholic extract of Ocimum sanctum (20, 40, 

60, 80 and 100 μg) in distilled water were mixed and the reaction started by adding 100 μl of 

phenazine methosulphate (PMS) solution (60 μM PMS in 100 mM phosphate buffer, pH 7.4). 

83


The reaction mixture was incubated at 25 0 C for 5 min and the absorbance at 560 nm was 

measured against blank samples. Decreased absorbance of the reaction mixture indicated 

increased superoxide anion scavenging activity. Curcumin was used as reference compound. 

All the experiments were performed in triplicate and the results were averaged. The 

percentage of inhibition was determined by comparing the results of control and test samples. 

Hydroxyl radical scavenging effect: 

Hydroxyl radical scavenging activity was measured by studying the competition between 

deoxyribose and test compounds for hydroxyl radical generated by Fe3 + -Ascorbate-EDTA- 

H2O2 system (Fenton reaction) according to the method of Kunchandy and Rao. 

hydroxyl radicals attack deoxyribose that eventually results in TBARS formation. 

www.wjpr.net 

[33] The 

The reaction mixture contained in a final volume of 1.0 mL, 100 μl of 2-deoxy-2-ribose (28 

mM in KH2PO4-K2HPO4 buffer, pH 7.4), 500µl solutions of various concentrations of 

hydroalcoholic extract (20, 40, 60, 80 and 100 μg) in KH2PO4-KOH buffer (20 mM, pH 7.4), 

200 µl of 1.04 mM EDTA and 200 µM FeCl3 (1:1 v/v), 100 µl of 1.0 mM H2O2 and 100 µl of 

1.0 mM ascorbic acid was incubated at 37 0 C for 1 h. The free radical damage imposed on the 

substrate, deoxyribose was measured as TBARS by the method of Ohkawa et al. [34] 1.0 mL 

of thiobarbituric acid (1%) and 1.0 mL of trichloroacetic acid (2.8%) were added to the test 

tubes and the incubation was continued at 100 0 C for further 20 min. After cooling, 

absorbance was measured at 532 nm against control containing deoxyribose and buffer. 

Catechin was used as a positive control. All the experiments were performed in triplicate and 

the results were averaged. The percentage inhibition was determined by comparing the results 

of the test compounds and control. 

Total Phenolic and Flavonoid content: 

A precisely weighed amount of whole plant material of Ocimum sanctum were homogenized 

for 5 min and extracted with 1% HCl in methanol (MeOH) (2 ml/g). The extracts were 

shaked and allowed to stand for 1.5 h at room temperature. The extracts were filtered through 

Whatman filter paper and the filtrates were taken to a final volume with distilled water. 

Extractions were carried out in triplicate. The extracts obtained were used to determine total 

phenolic and flavonoid content. 

The total phenolic contents of Ocimum sanctum were determined using the method Singleton. 

[35] Briefly, the appropriate extract dilution was oxidized with the Folin–Ciocalteu reagent 

84


and the reaction was neutralized with sodium carbonate. The absorbance of the resulting blue 

color was measured at 700 nm after 30 min. Quantification was done on the basis of a 

standard curve of gallic acid. Results were expressed as mg gallic acid equivalents (GAE). 

The total flavonoid content in the plant material was determined by the methodology of 

Chang. [33] Quercetin was used as a reference for the calibration curve. The absorbance of the 

reaction mixture was measured at 415 nm. Results were expressed as mg quercetin 

equivalents (QE). Data are reported as means ± standard deviation (SD) for at least three 

replicates. 

Statistical analysis: 

Statistical analysis of difference between groups was evaluated by one-way ANOVA 

followed by student t test. The values P < 0.05 were regarded as significant and the values P 

< 0.01 were considered as highly significant. 

RESULTS AND DISCUSSION 

Phytochemical investigation: 

It was found that hydroalcoholic extract contained carbohydrate, proteins and amino acids, 

saponin, phenolic compounds and tannins. 

Antioxidant activity: 

The most commonly used method for determining antioxidant activity is to measure the 

inhibitory degree of autoxidation of linoleic acid. [24] The results were shown in figure I. The 

different concentration of the hydroalcoholic extract at 50, 100, 200, 300, 400 and 500 µg/mL 

showed antioxidant activities in a dose dependent manner and had 38.06, 41.45, 44.83, 49.06, 

57.78 and 65.98% inhibition respectively on lipid peroxidation of linoleic acid system. At the 

same time α-tocopherol at the same concentration showed 43.47, 52.92, 67.53, 78.71, 83.23 

and 91.09% inhibition respectively. 

Reductive ability: 

The antioxidant activity has been reported to be concomitant with the development of 

reducing power. [36] The reducing power of extract might be due to its hydrogen donating 

ability, as described by Shimada et al. [37] The reducing power of each concentration of 

extract and BHT is shown in figure II. The reducing power increased as the extract 

concentration increased, indicating some compounds in Ocimum sanctum is both electron 

www.wjpr.net 

85


donors and could react with free radicals to convert them into more stable products and to 

terminate radical chain reactions. For the measurements of the reductive ability, we 

investigated the Fe3 + -Fe2 + , transformation in the presence of the each extract using the 

method of Oyaizu. [38] All the amounts of extract showed higher activities than control and 

these differences were statistically highly significant (P< 0.01). 

A bsorbance at 700 nm 

www.wjpr.net 

1.0 

0.8 

0.6 

0.4 

0.2 

Fig II: Red u ctive a b ility o f BH T a n d d ifferen t d o ses o f O cim u m sa n ctu m 

0.0 

0 20 40 60 80 100 

Concentration (microgram/ml) 

DPPH radical scavenging effect: 

DPPH scavenging activity has been used by various researchers as a quick and reliable 

parameter to assess the in vitro antioxidant activity of crude plant extracts. [39-40] In DPPH test 

the ability of a compound to act as donor for hydrogen atoms or electrons was measured 

spectrophotometrically. The scavenging activities of DPPH exerted by each extract as well as 

ascorbic acid were summarized in figure III. The hydroalcoholic extract at the concentration 

of 20, 40, 60, 80 and 100 µg/mL exhibited 44.93, 51.50, 58.66, 66.49, and 77.24% inhibition 

respectively, where standard drug ascorbic acid at the same concentration exhibited 53.47, 

69.02, 81.53, 88.71 and 96.23% inhibition respectively. The IC50 (the concentration required 

to inhibit a radical formation by 50%) of extract was found to be 34.21μg/mL whereas the 

IC50 value of standard ascorbic acid was found to be 18.69 μg/mL. In the present 

investigation Ocimum sanctum at different doses demonstrated significant DPPH scavenging 

activity indicating their abilities to act as radical scavengers. 

Nitric oxide radical scavenging effect: 

Nitric oxide (NO) is an important chemical mediator generated by endothelial cells, 

macrophages, neurons, etc. and is involved in the regulation of various physiological 

processes. [41] Excess concentration of NO is associated with several diseases. [42-43] Oxygen 

reacts with the excess nitric oxide to generate nitrite and peroxynitrite anions, which act as 

free radicals. [44-45] In the present study, the extract competes with oxygen to react with nitric 

BH T 

extract 

86


oxide and thus inhibits generation of the anions. Figure IV illustrates the percentage 

inhibition of nitric oxide generation by hydroalcoholic extract of Ocimum sanctum. Curcumin 

was used as a reference compound. The extract at the concentration of 20, 40, 60, 80 and 100 

µg/mL exhibited 22.66, 26.92, 36.44, 46.17 and 57.26% inhibition respectively, where as 

standard at the same concentration exhibited 40.01, 42.67, 50.53, 63.97 and 79.88% 

inhibition respectively. The IC50 value of extract was found to be 86.91 μg/mL whereas the 

IC50 value of curcumin was found to be 58.11 μg/mL. 

Percent Inhibition 

www.wjpr.net 

100 

90 

80 

70 

60 

50 

40 

30 

20 

10 

Fig IV: Percentage inhibition of nitric oxide radical by 

curcumin and different doses of Ocimum sanctum 

0 

0 20 40 60 80 100 

Concentration (microgram/ml) 

Superoxide anion radical scavenging effect: 

Curcumin 

extract 

Superoxides are produced from molecular oxygen due to oxidative enzymes [46 ] of body as 

well as via non enzymatic reaction such as autoxidation by catecholamines. [47] In the present 

study, superoxide radical reduces NBT to a blue colored formation that is measured at 560 

nm. [48] Figure V shows the superoxide scavenging effect of each extact and curcumin on the 

PMS/NADH-NBT system. The decrease of absorbance at 560 nm with antioxidants thus 

indicates the consumption of superoxide anion in the reaction mixture. Ocimum sanctum at 

concentration 20, 40, 60, 80 and 100 μg/mL inhibited the production of superoxide anion 

radicals by 31.62, 38.83, 43.53, 52.61 and 58.86% respectively whereas at same 

concentration standard curcumin showed 45.87, 62.63, 68.09, 74.41 and 81.32% inhibition 

respectively. The IC50 value of extract was found to be 73.38 μg/mL whereas the IC50 value 

of curcumin was found to be 24.67 μg/mL. 

87


www.wjpr.net 

P e rc e n t In h ib itio n 

90 

80 

70 

60 

50 

40 

30 

20 

10 

F i g V : P e r ce n t a g e i n h i b i t i o n o f s u p e r o x i d e r a d i ca l b y 

cu r cu m i n a n d d i f f e r e n t d o s e s o f O cim u m s a n ctu m 

0 

0 20 40 60 80 100 

C onc e ntra tion (m ic rogra m /m l) 

Hydroxyl radical scavenging effect: 

C urc um in 

Hydroxyl radicals are the major active species causing lipid oxidation and enormous 

biological damage. [49-50] The deoxyribose method is a simple assay to determine the rate 

constants for reactions of hydroxyl radicals. [51] Ferric-EDTA was incubated with H2O2 and 

ascorbic acid at pH 7.4. Hydroxyl radicals were formed in free solution and were detected by 

their ability to degrade 2-deoxy-2-ribose in to fragments that on heating with TBA at low pH 

form a pink chromogen. [52-53] Any hydroxyl radical scavenger added to the reaction would 

compete with deoxyribose for the availability of hydroxyl radicals, thus reducing the amount 

of MDA formation. We herein tested the scavenging activity of each extract along with 

positive control catechin. Ocimum sanctum at concentration 20, 40, 60, 80 and 100 μg/mL 

inhibited the production of superoxide anion radicals by 38.06, 49.45, 52.83, 57.06 and 

66.70% while at same concentration catechin showed 57.47, 63.02, 71.53, 77.71 and 83.23% 

respectively (Figure VI). The concentration extract needed for 50% inhibition was found to 

be 42.69 μg/mL. Catechin, used as a standard was highly effective in inhibiting the oxidative 

DNA damage, showing an IC50 value 17.71 μg/mL. 

P e rc e n t In h ib itio n 

100 

80 

60 

40 

20 

F ig V I: P e r ce n ta g e in h ib itio n o f h y d r o x il r a d ica l b y 

ca te ch in a n d d iffe r e n t d o s e s o f O cim u m s a n ctu m 

0 

0 20 40 60 80 100 

C onc e ntra tion (m ic rogra m /m l) 

e xtra c t 

C a te c hin 

P e t.e the r 

88


Total Phenolic and Flavonoid content: 

Phenols are very important plant constituents because of their scavenging ability due to their 

hydroxyl groups. [54] The total phenolic content of Ocimum sanctum is 82.02 ± 8.17 mg AE/g 

while the total flavonoid content of Ocimum sanctum extract is 74.6±5.1 mg/g. The phenolic 

compounds may contribute directly to antioxidative action. 

www.wjpr.net 

[55] 

It is suggested that 

polyphenolic compounds have inhibitory effects on mutagenesis and carcinogenesis in 

humans, when ingested up to 1g daily from a diet rich in fruits and vegetables. [36] 

CONCLUSION 

Reactive oxygen species (ROS), from both endogenou s and exogenous sources, may be 

involved in the etiologies of such diverse human diseases as atherosclerosis, ischemic injury, 

cancer, and neurodegenerative diseases, as well as in processes like inflammation and ageing. 

[56-58] There is evidence that indigenous antioxidants may be useful in preventing the 

deleterious consequences of oxidative stress and there is increasing interest in the protective 

biochemical functions of natural antioxidants contained in spices, herbs, and medicinal 

plants. [59-60] Medicinal herbs are known to contain a variety of antioxidants. Each herb 

generally contained different phenolic compounds, and each of these compounds possesses 

differing amounts of antioxidant activity. It is rather difficult to characterize every compound 

and assess or compare their antioxidant activities. The most detailed investigations so far 

were concerned with reactions involving phenolic compounds ranging from polymer 

chemistry to biochemistry and food chemistry. [61] It has been revealed that various phenolic 

antioxidants such as flavonoids, tannins, coumarins, xanthones and more recently 

procyanidins scavenge radicals dose dependently, thus they are viewed as promising 

therapeutic drugs for free radical pathologies. [62] The results of the present study, which 

demonstrate the radical scavenging of different extracts, indicate that the use of Ocimum 

sanctum for the treatment of various neurogenerative and inflammatory diseases, and cancer 

seems quite useful and reasonable. The antioxidant and free radical scavenging activities of 

Ocimum sanctum might be due to the presence phenolic compounds in extracts which is 

confirmed by FCR reagent test. Also, it can be concluded that antioxidant activity of plant 

extracts is not limited to phenolics. Activity may also come from the presence of other 

antioxidant secondary metabolites, such as volatile oils, carotenoids, and vitamins. [63] 

Further studies are in progress in our laboratory to evaluate the in vivo antioxidant potential 

of these extracts in various animal models and phytochemical studies are required to establish 

the types of compounds responsible for the bioactivity of this medicinal plant and to 

89


determine the value of the ethnobotanical approach for the screening of plants as potential 

source of bioactive substances. 

ACKNOWLEDGEMENT 

The authors wish to thank Jagadguru Sri Sri Shivarathri Deshikendra Mahaswamigalavaru of 

Sri Suttur Mutt, Mysore for providing the necessary facilities to carry out this study. 

REFERENCES 

1. Ito N, Hirose M, Fukushima H, Tsuda T, Shirai T, Tatenatsu M. Studies on 

antioxidants: Their carcinogenic and modifying effects on chemical carcinogens. Food 

Chem Toxicol, 1986; 24: 1071-1092. 

2. Hirasa K , Takemasa M. Cooking with spices. In: Spice Science Technology. Marcel 

Dekker, New York. 1998; 68-70. 

3. Nakatani N. Antioxidants from spices and herbs. In F. Shahidi (Eds), Natural 

antioxidants: Chemistry, health effects, and application. AOAC Press, Champaign, 

1997; 64-75. 

4. Cuvelier ME, Berset C, Richard H.(1994). Antioxidant constituents in sage ( Salvia 

officinalis). J Agric Food Chem,1994; 42: 665-669. 

5. Economou KD, Oreopoulou V, Thomopoulos CD. Antioxidative activity of some 

plants of the family Labiatae, J Am Oil Chem Soc, 1991; 68: 109-133. 

6. Kikuzaki H, and Nakatani N. Antioxidant effects of some ginger constituents. J Food 

Sci, 1993; 58(6): 1407-1410. 

7. Lu Y, Yeap Foo L. Antioxidant activities of polyphenols from sage (Salvia officinalis). 

Food Chem, 2001; 75(2): 197-202. 

8. Caragay AB. Cancer preventive foods and ingredients. Food Tech, 1992; 46: 65-68. 

9. Rice-Evans CA, Miller NJ, Paganga G. Antioxidant properties of phenolics compounds. 

Trends Plant Sci. 1997; 4: 304-309. 

10. Fejes S, Blazovics A, Lugasi A, Lemberkovics E, Petri G, Kery A. In vitro antioxidant 

activity of Anthriscus cerefolium L. (H offm) extracts. J Ethnopharmacol, 2000; 69: 

259-265. 

11. Hou WC, Lin RD, Cheng KT. Free radical-scavenging activity of Taiwanese native 

plants. Phytomed, 2003; 10: 170-175. 

12. Khare CP. Indian Medicinal Plants, an Illustrated Dictionary, Springer-Verlag, Berlin, 

2007; 445. 

www.wjpr.net 

90


13. Mondello L, Zappia G, Cotroneo A, Chowdhury JU, Yusuf M, Dugo G. Studies on the 

essential oils bearing plants of bangladesh. part viii.- composition of some ocimum oils 

(Ocimum basilicum L. var. purpurascens; Ocimum sanctum L. green; Ocimum sanctum 

L. purple; Ocimum americanum L., citral type; Ocimum americanum L., camphor type. 

Flavour Frag J, 2002; 17: 335-340. 

14. Pauli A. Anticandidal low molecular compounds from higher plants with special 

reference to compounds from essential oils. Med Res Rev, 2006; 26(2):223-68. 

15. Peach K, Tracey MV. Modern Methods of Plant Analysis, Vol. I, Springler verlag, 

Berlin, 1955; 367. 

16. Woisky R, Salatino R. Analysis of propolis: some parameters and procedures for 

chemical quality control. J Agric Res, 1998; 37: 99-105. 

17. Bozin B, Mimica Dukic N, Simin N. Characterization of the volatile composition of 

essential oils of some lamiaceae spices and the antimicrobial and antioxidant activities 

of the entire oils. J Agric Food Chem, 2006; 54: 1822-1828. 

18. Haftman E. History of chromatography, A laboratory hand book of chromatographic 

and elecrotophoretic methods, 3 rd Edn., Van Nostrand Reinhold, NewYork. 1975; 1-15. 

19. Keshavamurthy KR, Yoganarasimhan SN. Flora of Coorg (Kodagu). Karnataka. 

Bangalore: Vimsat Publishers, 1990; 353. 

20. Gamble, J.S. Flora of the presidency of Madras, Botanical. Survey of India, Calcutta. 

1967; Vol-I: 167. 

21. Harborne JB. Phytochemical method- a guide to modern technique of plant analysis, 2 nd 

Edn. Chapman and Hall, New York, 1984; p.85. 

22. Yerra R, Senthil Kumar GP, Gupta M, Mazumdar UK. Studies on in vitro antioxidant 

activities of methanol extract of Mucuna pruriens (Fabaceae) seeds. European Bull 

Drug Res, 2005; 13: 31-38. 

23. Ricci D, Fratemale D, Giamperi L, Bucchini A, Epifano F, Burini G, Curini M. 

Chemical composition, antimicrobial and antioxidant activity of the essential oil of 

Teucrium marum (Lamiaceae). J Ethnopharmacol, 2005; 98: 195-200. 

24. Mitsuda H, Yuasumoto K, Iwami K. Antioxidation action of indole compounds during 

the autoxidation of linoleic acid. Eiyo to Shokuryo, 1996; 19: 210-14. 

25. Blois MS. Antioxidant determination by the use of a stable free radical. Nature, 1958; 

29: 1199-1200. 

26. Cao G, Sofic E, Prior RL. Antioxidant and prooxidant behavior of flavonoids: structure- 

activity relationships. Free Rad Biol Med, 1997; 22: 749-60. 

www.wjpr.net 

91


27. Shishoo CJ, Shah SA, Rathod IS, Patel SG. Quality assurance of chyavanprash through 

determination of free radical scavenging activity. Indian J Pharm Sci, 1998; 60(3): 179- 

181. 

28. Shishoo CJ, Ravikumar T, Jain KS, Rathod IS, Gandhi TP, Satia MC. Synthesis of 

novel 1,2-(un) substituted-3-amino-5-aryl-6-arylaminopyrazolo [3,4-d]pyrimidin- 

4(5H)-ones and their biological activities. Indian J. Chem, 1999; 38: 1075-1085. 

29. Green LC, Wagner DA, Glogowski J, Skipper PL, Wishnok JK, Tannenbaum SR, 

Analysis of nitrate, nitrite, and [15N] nitrate in biological fluids. Anal Biochem, 1982; 

126: 131–36. 

30. Marcocci L, Maguire JJ, Droy-Lefaix MT, Packer L. The nitric oxide scavenging 

property of Ginkgo biloba extract EGb 761. Biochem Biophys Res Comm, 1994; 201: 

748-55. 

31. Mondal S, Chakraborty G, Gupta M , Muzumdar U. In vitro antioxidant activity of 

Diospyros malabarica kostel bark. Indian J Exp Biol, 2006; 44: 39-44. 

32. Nishimiki M, Rao NA, Yagi K. The occurrence of superoxide anion in the reaction of 

reduced phenazine methosulfate and molecular oxygen. Biochem Biophys Res Comm, 

1972; 46: 849-853. 

33. Kunchandy E, Rao MNA. Oxygen radical scavenching activity of curcumin. Intern J 

Pharmacog, 1990; 58: 237-240. 

34. Ohkawa H, Ohishi N, Yagi K. Assay for lipid peroxides in animal tissues by 

thiobarbituric acid reaction. Anal Biochem, 1979; 95: 351-358. 

35. Singleton VL, Orthofer R, Lamuela-Raventos RM, Analysis of total phenols and other 

oxidation substrates and antioxidants by means of Folin-Ciocalteu Reagent. Methods in 

Enzymology, 1999; 299: 152-178. 

36. Tanaka M, Kuei CW, Nagashima Y, Taguchi T. Application of antioxidative maillrad 

reaction products from histidine and glucose to sardine products. Nippon Suisan 

Gakkaishi, 1998; 54: 1409-1414. 

37. Shimada K, Fujukawa K, Yahara K, Nakamura T. Antioxidative properties of xanthan 

on the autoxidation of soybean oil in cyclodextrin emulsion. J Agric Food Chem, 1992; 

40(6): 945-948. 

38. Oyaizu M. Studies on products of browning reaction: antioxidant activities of products 

of browning reaction prepared from glucosamine. Japanese J Nut, 1986; 44: 307-15. 

39. Navarro MC, Montilla MP, Martin A, Jimenez J, Utrilla MP. Free radical scavenger and 

antihepatotoxic activity of Rosmarinus tomentosus. Planta Med, 1992; 59: 312-314. 

www.wjpr.net 

92


40. Thabrew MI, Hughes RD, Mc Farlane IG. Antioxidant activity of Osbeckla aspera. 

Phytother Res, 1998; 12: 288-290. 

41. Lata H, Ahuja GK. Role of free radicals in health and disease. Ind J Physiol Allied Sci, 

2003; 57: 124-128. 

42. Ialenti S, Moncada M, Di Rosa. Modulation of adjuvant arthritis by endogenous nitric 

oxide. Br J Pharmacol,1993; 110: 701-705. 

43. Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature, 1993; 

3: 62, 801. 

44. Cotran RS, Kumar V, Collins T. Robbin’s Pathological Basis of Diseases, 6 th Edn., 

Thomson Press, Noida, 1999; 1-43. 

45. Sainani GS, Manika JS, Sainani RG. Oxidative stress: a key factor in pathogenesis of 

chronic diseases. Med Update, 1997; 1: 1. 

46. El Naggar EB, Bartosikova L, Zemlicka M, Svajdlenka E, Rabiskova M, Strnadova V, 

Necas J. Antidiabetic effect of Cleome droserifloria aerial parts on lipid peroxidation- 

induced oxidative stress in diabetic rats. Acta Vet Brno, 2005; 3: 347. 

47. Hemmani T, Parihar MS. Reactive oxygen species and oxidative DNA damage. Ind J 

Physiol Pharmacol, 1998; 42: 440-444. 

48. Khanam S, Shivprasad HN, Kashama D. In vitro antioxidant screening models: A 

review. Ind J Pharm Edu, 2004; 38: 180-194. 

49. Aruoma OI, Kaur H, Halliwell B. Oxygen free radicals and human diseases. J Royal 

Soc Health, 1991; 111: 172-177. 

50. Milic B, Djilas SM, Canadanovic-Brunet JM. Antioxidative activity of phenolic 

compounds on the metal-ion breakdown of lipid peroxidation system. Food Chem, 

1998; 61: 443-447. 

51. Halliwell B, Gutteridge JMC. In: Handbook of methods for oxygen radical research, 

Greenwald RA, Eds., CRC Press, Boca Raton, FL, 1985; 177-180. 

52. Larson RA. Naturally occurring antioxidants. Lewis Publishers, New York, 1997; 1- 

195. 

53. Aruoma OI, Laughton MJ, Halliwell B. Carnosine, homocarnosine and anserine; could 

they act as antioxidants in-vivo? Biochem J, 1989; 264: 863-869. 

54. Hatano T, Edamatsu R, Hiramatsu M, Mori A, Fujita Y, Yasuhara A. Effects of the 

interaction of tannins with co-existing substances. Chem Pharm Bull, 1989; 37: 2016– 

2021. 

www.wjpr.net 

93


55. Duh PD, Tu YY, Yen GC. Antioxidant acitvity of aqueous extract of harn jyur 

(Chrysanthemum morifolium Ramat). Lebensmittel-Wissenschaft und Technologie, 

1999; 32: 269-277. 

56. Halliwell B, Gutteridge JMC. Free Radical in Biology and Medicine, 3 rd Edn., 

Clarendon Press, Oxford, 1993; 22-81. 

57. Good PF, Werner P, Hsu A. Evaluation of neuronal oxidative damage in Alzheimer’s 

disease. Am J Pathol, 1996; 149: 21-28. 

58. Gassen M, Youdim MB, The potential role of iron chelators in the treatment of 

Parkinson’s disease and related neurological disorders. Pharmacol Toxicol, 1997; 80: 

159-166. 

59. Osawa T, Katsuzaki H, Kumon H. Protective role of phenolics antioxidants in plant 

against oxidative damage. In; Frontiers of active oxygen species in biology and 

medicine, Asada K and Yoshikava T, Eds., 1994; 333-336. 

60. Noda Y, Anzai Kmori A, Kohono M. Hydroxyl and superoxide anion radical 

scavenging activities of natural source antioxidants using the computerized JES-FR30 

ESR spectrometer system. Bioch Mol Biol Inter, 1997; 42: 35-44. 

61. Bravo L. Polyphenols: chemistry, dietary sources, metabolism and nutritional 

significance. Nutr Rev, 1998; 56: 317-333. 

62. Lee S, Suh S, Kim S. Protective effects of the green tea polyphenol (-)-epigallocatechin 

gallate against hippocampal neuronal damage after transient global ischemia in gerbils. 

Neurosci Lett, 2000; 287: 191-194. 

63. Javanmardi J, Stushnoff C, Locke E, Vivanco JM. Antioxidant activity and total 

phenolic content of Iranian Ocimum accessions. Food Chem, 2003; 83; 547–550. 

www.wjpr.net 

94

WORLD JOURNAL OF PHARMACEUTICAL RESEARCH - WJPR!

You also want an ePaper? Increase the reach of your titles

Delete template?

Save as template?