PHYTOCHEMICAL INVESTIGATION AND STUDY ON ANTIOXIDANT PROPERTIES OF OCIMUM CANUM HYDRO-ALCOHOLIC LEAF EXTRACTS

Natural products are important sources for biologically active drugs 1 . There has been an increasing interest in the medicinal plants as natural products in different parts of the world 2 . Medicinal plants containing high antioxidant properties play an important role in the prevention of various degenerative diseases in the society. The medicinal value of these plants depends on bioactive phytochemical constituent’s action in the human body. Some of the most important bioactive phytochemical constituents include alkaloids, flavonoids, essential oils, tannins and saponins 3 . Phenolics are commonly found in medicinal plants and their biological effects, include antioxidant activity. Due to synthetic antioxidants such as butylated hydroxyl anisole (BHA), butylated hydroxyl toluene (BHT), and tert-butyl hydroquinone (TBHQ), which are widely used in food industry and cosmetic, have been growing concern over the possible carcinogenic effects 4 . Thus interest in natural antioxidant has increased considerably. Nowadays, it is well known that natural antioxidants extracted from herbs and spices have high antioxidant properties and are used in many food applications 5 . Natural antioxidants from p lant sources are potent and safe due to their harmless nature; wild herbs have their antioxidant properties 6. Various plants are proposed to be antioxidantsas their essential oils contain phenylpropanoids in high contents. Essential oils of plant genus Ocimumbelonged to family Lamiaceae. It is collectively called as Basil, is a diverse and rich source of aromat ic essential oil. The species such as Ocimumbasilicum L., O canum Sims., O gratissimum L. and Ocimum sanctum L. are composed of interesting phenyl-propenes e.g. eugenol, methyl eugenol and methyl chavicol. Essential oils of these plants have been broadly used as culinary herbs, as pharmaceutical agents because of their antimicrobial, antiemetic, antid iabetic, antifert ility, antiasthmatic, antistress andanticancer activity 7.


INTRODUCTION
Natural products are important sources for biologically active drugs 1 . There has been an increasing interest in the med icinal plants as natural products in different parts of the world 2 . Medicinal plants containing high antioxidant properties play an important role in the prevention of various degenerative diseases in the society. The medicinal value of these plants depends on bioactive phytochemical constituent's action in the human body. So me of the most important bioactive phytochemical constituents include alkaloids, flavonoids, essential oils, tannins and saponins 3 . Phenolics are commonly found in medicinal plants and their biological effects, include antioxidant activity. Due to synthetic antioxidants such as butylated hydroxyl anisole (BHA), butylated hydroxyl toluene (BHT), and tert-butyl hydroquinone (TBHQ), which are widely used in food industry and cosmetic, have been growing concern over the possible carcinogenic effects 4 . Thus interest in natural antioxidant has increased considerably. Nowadays, it is well known that natural antioxidants extracted fro m herbs and spices have high antio xidant properties and are used in many food applications 5 . Natural antio xidants fro m p lant sources are potent and safe due to their harmless nature; wild herbs have their antioxidant properties 6. Various plants are proposed to be antioxidantsas their essential oils contain phenylpropanoids in high contents. Essential oils of plant genus Ocimumbelonged to family Lamiaceae. It is collectively called as Basil, is a diverse and rich source of aromat ic essential oil. The species such as Ocimumbasilicum L., O canum Sims., O gratissimum L. and Ocimum sanctum L. are co mposed of interesting phenyl-propenes e.g. eugenol, methyl eugenol and methyl chavicol. Essential oils of these plants have been broadly used as culinary herbs, as pharmaceutical agents because of their antimicrobial, antiemetic, antid iabetic, antifert ility, antiasthmatic, antistress andanticancer activity 7.

ABS TRACT
M any herbal remedies individually or in combination have been recommended in various medical expositions for the cure of different diseases. Free radicals are implicated for many diseases including diabetes mellitus, arthritis, cancer, aging, etc. In the treatment of these diseases, antioxidant therapy has gained utmost importance. Currently there has been an increased interest globally to identify antioxidant compound that are pharmacologically potent and have low or no side effects. As plants are source of natural antioxidants, much attention has been gain to plants. The quest for natural antioxidants for dietary, cosmetic and pharmaceutical uses has become a major industrial and scientific research challenges over the last two decades. A variety of free radical scavenging antioxidants exists within the body in which many of them are derived from dietary sources like fruits, vegetables and teas. TheOcimumcanum, commonly known as 'Kala Tulsi', has been recognized in different system of traditional medicines for the treatment of different diseases and ailments of human beings. In this study, Antioxidant activity of the hydro-alcoholic extract of Ocimumcanumwas determined by various antioxidant assays. In all the testing, a significant correlation existed between concentrations of the extract and percentage inhibition of free radicals. These findings suggest that the hydro-alcoholic extracts are able to scavenge free radicals, by either hydrogen or electron donating mechanisms, and can therefore act as primary antioxidants. The antioxidant property may be related to the phenols and flavonoids present in the extracts. These results clearly indicate that Ocimumcanumcan be effectively used against free radical mediated diseases. Key words:Ocimumcanum, phytochemical constituents, traditional uses and pharmacological properties, antioxidant activity purposes. O. canum is used specially fo r treating various types of diseases and lowering blood glucose and also treats cold, fever, parasitic infestations on the body and inflammat ion of joints and headaches 8 . Essential oil fro m the leaves of O. canum possesses antibacterial and insecticidal properties 9. It is used in ritual as an incense as well to protect the home and welcome newborns into the world. It is an unusual and very useful addition to the med icinal garden. The hairy leaves and decorative flowers are very aromatic and form a lush mound about 2 feet in height. This annual plant grows well in fu ll sun, welldrained soil and plenty of heat. The plant branches from the base and has an angled stems and oval pubescent leaves. Its leaves are tiny and fuzzy and have violet or white flowers, having a sweet scent res embling that of the clove. The leaves of the Ocimum canum are opposite and toothed. It is irregular and occurs in crowded whorls. The Ocimum canum has a small corolla. These plants have intense floral-fru ity aro mas.The oil of the Oci mum canum is composed of Linalool. The seeds may provide fiber or reduce constipation.

Plant collection
Leaves

Extraction
The dried leaves were coarsely powdered and extracted with a mixture of methanol: water (7:3, v/v) by a Soxhlet apparatus at 50°C. The solvent was completely removed and obtained dried crude ext ract which was used for investigation. Further the extracts were subjected for the phytochemical study as well as pharmaco logical screening.

Phytochemical screening
Phytochemical screenings were perfo rmed using standard procedures. 10

2) Test for carbohydrates and reducing sugar:
The small quantities of the filtrate will be dissolved in 4ml of distilled water and filtered. The filtrate will be subjected to a) Molisch's test: A small portion of the filtrate will be treated with Molisch's reagent and sulphuric acid. The ext ract will be treated with barfoed's reagent and heated. Appearance of reddish orange colour precipitate indicates the presence of nonreducing sugars.

3) Test for steroids:
Libermannburchard's test: The ext ract will be treated with 3ml of acet ic anhydride, few drops of glacial acet ic acid followed by a drop of concentrated sulphuric acid. Appearance of bluish green colour indicates the presence of steroids.

4) Test for proteins:
a) Biuret test: The extract will be treated with copper sulphate solution, followed by addition of sodium hydroxide solution; appearance of violet colour indicates the presence of proteins.

b) Millon's test:
The extract will be treated with Millon's reagent; appearance of pink colour indicates the presence of proteins.

5) Test for tannins:
The ext ract will be treated with 10% lead acetate solution; appearance of white precipitate indicates the presence of tannins.

6) Test for phenolic compounds:
a) The ext ract will be treated with neutral ferric ch loride solution; appearance of violet colour indicates the presence of phenolic co mpounds.
b) The extract will be treated with 10% sodium ch loride solution; appearance of cream co lour ind icates the presence of phenolic co mpounds.

7) Test for flavonoids:
a) 5ml of ext ract will be hydrolyzed with 10%sulphuric acid and cooled. Then, it will be extract ing with diethyl ether and divided in to three portions in three separate test tubes. 1ml of d iluted sodium carbonate, 1ml of 0.1N sodium hydroxide, and 1ml of strong ammonia solution will be added to the first, second and third test tubes respectively. In each test tube, development of yellow colour demonstrated the presence of flavonoids.
b) Shinoda's test: The extract will be dissolved in alcohol, to which few magnesiu m turnings will beaded followed by concentrated HCL drop wise and heated, and appearance of magenta colour shows the presence of flavonoids.

8) Test for gums and mucilage:
The extract was treated with 25 ml of absolute alcohol, and filtered. The filtrate will examine for its swelling properties.

9) Test for glycosides:
When a pinch the extract was treated with glacial acetic acid and few drops of ferric chloride solution, followed by the addition of conc. Sulphuric acid, formation of ring at the junction of two liquids indicates the presence of glycosides.

10) Test for saponins:
Foam test About 1 ml of the ext ract was diluted to 20 ml of with distilled water and shaken well in a test tube. The formation of foam in the upper part of test tube indicates the presence of saponins.

11) Test for Triterpenoids:
The substance was warmed with tin and thionyl chloride. Pink colour indicates the presence of triterpenoids.

Determination of antioxi dant acti vity a) DPPH radical scavenging assay 13
To the Methanol solution of DPPH (1 mM ) an equal volume of the extract dissolved in alcohol was added at various concentrations from 250 to 2000 μg/ ml in a final volume of 1.0 ml. An equal amount of alcohol was added to the control. After 20 min, absorbance was recorded at 517 n m. Experiment was performed in trip licate.

b) AB TS radical scavenging assay 13
To the reaction mixtu re containing 0.3 ml of ABTS radical, 1.7 ml phosphate buffer and 0.5 ml ext ract was added at various concentrations from 250 to 2000 μg/ ml. Blank was carried out without drug. Absorbance was recorded at 734 n m. Experiment was performed in triplicate.

c) Nitric oxi de radical scavenging 13
Sodiu m nitroprusside (5μM) in standard phosphate buffer solution was incubated with different concentration of the test extracts dissolved in standard phosphate buffer (0.025M , pH 7.4) and the tubes were incubated at 25 °C for 5 hr. After 5 h, 0.5 ml of incubation solution was removed and diluted with 0.5 ml Griess reagent (prepared by mixing equal volu me of 1% sulphanilamide in 2% phosphoric acid and 0.1% naphthylethylene di-amine dihydrochloride in water). The absorbance of chromophore formed was read at 546 n m. The control experiment was also carried out in similar manner, using distilled water in the place of extracts. The experiment was performed (in triplicate) and % scavenging activity was calculated using the formula :-

− [100/blank absorbance × sample absorbance]
The activity was co mpared with ascorbic acid, which was used as a standard antioxidant.

d) Superoxi de scavenging (NBT reduction assay) 13
Alkaline DM SO was used as a super oxide generating system. To 0.5 ml of different concentrations of the test compound, 1 ml of alkaline DMSO and 0.2 ml of NBT 20 mM in phosphate buffer pH 7.4 was added. The experiment was performed in triplicate.

e) Iron chelating acti vity assay 13
The reaction mixture containing 1 ml O-Phenanthroline, 2 ml Ferric chloride, and 2 ml ext ract at various concentrations ranging from 250 to 2000 μg/ ml in a final volume of 5 ml was incubated for 10 minutes at ambient temperature. The absorbance at 510 n m was recorded. Ascorbic acid was added instead of extract and absorbance obtained was taken as equivalent to 100% reduction of all ferric ions. Blank was carried out without extract. Experiment was performed in triplicate.

f) Hydroxyl Radical Scavenging Acti vity (Deoxyri bose degradati on assay)
The scavenging capacity for hydroxyl radical was Where, A 0 was the absorbance of the control (blan k) and A 1 was the absorbance in the presence different concentrations of the extract g) Total reducti on capability 15 The Fe 3+ reducing power of the extract was determined by the method of Oyaizu et al., with a slight modification. Different concentrations (250-2000 µg/ mL) of extract (0.5 mL) were mixed with 0.5-mL Phosphate buffer (pH 6.6) and 0.5-mL 0.1% potassium hex-cyanoferrate, followed by incubation at 50°C in water bath for 20 min. After incubation, 0.5-mL 10% TCA was added to terminate the reaction. The upper portion of the solution (1 mL) was mixed with 1 mL of d istilled water and 0.1 mL 0.01% FeCl 3 solution was added. The reaction mixtu re was left for 10 min at room temperature and the absorbance was measured at 700 n m against appropriate blank solution. All tests were performed three times. A higher absorbance of the reaction mixture indicated greater reducing power. Ascorbic acid was used as a positive control. . The reaction mixture was then vortexed and incubated at room temperature for 30 min. The absorbance of ferric-xy lenol orange complex was measured at 560 n m. All tests were carried out three times and sodium pyruvate was used as the reference co mpound

Statistical anal ysis 17
The experimental results were expressed as mean ± SEM of three replicates. Where applicable, the data were subjected to one way analysis of variance (ANOVA ) and two way analysis of variance (ANOVA ). All these analysis was done by Graph Pad Pris m So ftware program (version 5). P values < 0.05 were regarded as significant.

Phytochemical screening
Phytochemical screening of the extracts revealed the presence of carbohydrates, flavonoids and tannins

Antioxi dant assays DPPH radical scavenging assay
The proton radical scavenging action is known to be one of the various mechanisms fo r measuring antio xidant activity.
The DPPH test provides informat ion on their activity of the test compounds with a stable free radical. Th is assay determines the scavenging of stable radical species of DPPH by antio xidants. The degree of reduction in absorbance measurement by Ocimumcanumis indicative of the radical scavenging (antio xidant) power of the plant. The study showed that the hydro-alcoholic extract have the proton-donating ability and can serve as free radical inhibitors or scavenger, acting possibly as primary antioxidant. Glycosides --4 Phtyosterols --5 Fixed oils --6 Saponins --7 Tannins + -8 Protein and amino acids --9 Gu ms and mucilage --10 Flavonoids + + 11 Terpenoids + + + = presence, -= absence. Reducing power assay measures the electron-donating capacity of an antioxidant. The reduction of the ferric ion (Fe 3+ ) to ferrous ion (Fe 2+ ) is measured by the intensity of the resultant blue-green solution which absorbs at 700 n m, and an increased absorbance is indicative of higher reducing power.The reducing power of the extract increased progressively over the concentration range studied. Extract solutions at 1000µg/ mlhad co mparable reducing power to Ascorbic acid at 250 µg/ ml. These findings suggest that the Ocimumcanumhydro-alcoholic extract is capable of donating electrons, and could therefore react with free radicals or terminate chain reactions.

Scavenging Acti vity of Hydrogen Peroxi de 18
The scavenging of hydrogen peroxide by the standard (ascorbic acid) and extract after incubation for 10 minutes increased with increased concentration.Ocimumcanumhydro-alcoholic extracts exhibited higher hydrogen peroxide scavenging activity than ascorbic acids at similar concentrations.While hydrogen peroxide itself is not very reactive, it can generate the highly reactive hydroxyl rad ical ( OH) through the Fenton reaction(Equation The scavenging of hydrogen peroxide by phenolic compounds has been attributed to their electron-donating ability. The hydro-alcoholic OC ext racts have high electron-donating abilit ies, and 68.21 ± 0.35scavenging was achieved with concentrations of hydro-alcoholic extracts at 2000 µg/ ml. In comparison, the hydrogen peroxide scavenging activity of ascorbic acid at 2000 µg/ mlwere found to be 98.79 ± 0.28