Qualitative and Quantitative Determination of Various Extracts of Ocimum basilicum L. Leaves

Ocimum basilicum L. (Lamiaceae) commonly known as basil possesses various medicinal activities to treat headache, diarrhoea, kidney malfunctions, diabeties, cancer and several pharmacological activities. These are mainly due to the various chemically diversified constituents present in it. In order to evaluate the chemical composition of basil leaves, the dried leaves were extracted with petroleum ether, chloroform, ethyl acetate, methanol and water by Soxhlet’s extraction. The extracts were screened for the qualitative determination by preliminary phytochemical screening. Quantitative determination was also performed for the total alkaloids, flavonoids, phenols, saponins and tannins. Amongst the extracts, methanol (MeOH) was identified to contain various classes of secondary metabolites, so GCMS analysis was performed on the MeOH extract. The results revealed that chemical constituents claimed for various biological activities were present. also used as ornamental and kitchen herb for the aroma present in it. Essential oils extracted from basil leaves have high aroma and used as herbal flavours in most of the food products 4 . It possesses various pharmacological activities such as antimicrobial, anti-insecticidal, antioxidant, anti-inflammatory, anti-hyperlipidemic, antidiabetic, anticonvulsant, antiplatelet, anti-thrombotic, immune-modulatory, cytotoxicity and anti-cancer. These activities are mainly due to the various chemically diversified constituents present in it 5 – 9 . Alkaloids, flavonoids, phenols, terpenoids are present in basil leaves which have potent medicinal use 10, 11 . This study was designed to evaluate the chemical composition of basil leaves with different solvents such as petroleum ether, chloroform, ethyl acetate, methanol and water. Qualitative and quantitative determination of chemical constituents were evaluated through standard procedures for all the five extracts. which, Methanol (MeOH) extract showed the high percentage yield of different chemical constituents. In order to quantify, MeOH extract was subject to Gas chromatography coupled with Mass spectrometer with suitable working conditions 14 .


Introduction
Ocimum basilicum L. (also called basil) belongs to Lamiaceae, is a culinary herb which is highly spread across Southeast Asia and worldwide. From ancient times, it is widely used to treat various ailments due to its versatile medicinal properties 1,2 . Traditionally, it is used as folk medicine and Uyghur medicine in Turkey; treating pimples, headaches and kidney malfunctions in India; treating aches and pains in Bulgarian folk medicine, Sedative in Spain 3 . It is also used in the treatment of insect stings, snake bites and skin infections externally. It is also used as ornamental and kitchen herb for the aroma present in it. Essential oils extracted from basil leaves have high aroma and used as herbal flavours in most of the food products 4 . It possesses various pharmacological activities such as antimicrobial, anti-insecticidal, antioxidant, anti-inflammatory, anti-hyperlipidemic, antidiabetic, anticonvulsant, antiplatelet, anti-thrombotic,

Extraction
The collected plant leaves were washed, shade dried and ground in a blender with 2 mm dia mesh size. The powdered samples of weight, each 100 g were extracted continuously with 500 ml of petroleum ether, chloroform, ethyl acetate, methanol and aqueous (1:5 w/v) in Soxhlet's apparatus. The process was continued for about 24 hours with a temperature below the boiling point of the solvent. The extracts were collected and filtered through Whatmann no.1 filter paper. Then the filtrate was concentrated in a rotary vacuum evaporator and stored in dark bottles and kept at -20º C till further use 15 .

Qualitative Determination of Chemical
Constituents of Ocimum basilicum Leaf Extracts 16, 17

Sodium Hydroxide Test
To a 2-3 ml of plant extract, 2 ml of 10% sodium hydroxide solution was added to form an intense yellow color which would be turned to colorless in addition of diluted hydrochloric acid, this marks the presence of flavonoids.

Test for Terpenes
Copper Acetate Test To a 3 ml of plant extract, 8-10 drops of copper acetate solution was added. The emerald green color indicates the presence of terpenes.

Molish's Test
To a 2-3 ml of plant extract, a few drops of α-napthol solution was added and shaken well to which a few drops of concentrated sulphuric acid were added along the sides of the test tube. The formation of a violet ring at the junction indicates the presence of carbohydrates.

Tests for Protein
Biuret's Test To a 2-3 ml of plant extract, a few drops of 4 % sodium hydroxide and 1% Copper sulphate solution was added. Appearance of Violet or pink color indicates the presence of proteins.

Millon's Test
To a 3 ml of plant extract, a few drops of million's reagent was added and heated gently. The appearance of reddish brown coloration indicates the presence of proteins.

Spot Test
A small quantity of extract was pressed between two Whatmann No.1 filter papers for about 2 min. The oil stain on the paper indicates the presence of fixed oils.

Saponification Test
To a 2-3 ml of plant extract, 0.5 N alcoholic potassium hydroxide solution along with 2 drops of phenolphthalein was added and heated for about 2 hrs. The formation of soap or partial neutralization of alkali indicates the presence of fixed oils and fats.

Salkowski Tests
To a 2-3 ml of plant extract, 2 ml of chloroform & 2 ml of concentrated sulphuric acid was added and shaken well. The chloroform layer appears red and acid layer shows greenish yellow fluorescence which shows the presence of steroids.

KellarKillani's test
A few ml of plant extracts was dissolved in water with Glacial acetic acid and ferric chloride and concentrated sulphuric acid. The formation of brown ring at the junction indicates the presence of cardiac glycosides.

Ferric Chloride Test
To a 0.5 ml of plant extract, 5 ml of D. H 2 O was added and boiled for 10 min. To the 2 ml of collected filtrate, a few drops of 10% ferric chloride solution were added. Appearance of greenish blue or violet color indicates the presence of a phenolic hydroxyl group.

Lead Acetate Test
To a 2-3 ml of plant extract, 3 ml of lead acetate solution was added. The occurrence of white precipitate indicates the presence of tannins and phenols.

Foam Test
To a few quantity of the plant sample, respective amount of water was added and shaken vigorously for about 10 min. The observation of persistent stable foam indicates the presence of saponins.

Determination of Total Alkaloids
1 mg/ml equivalent of plant sample was dissolved in a dimethyl sulfoxide and 1 ml of 2 N hydrochloric acid was added and the mixture was filtered. The filtrate was transferred to a separating funnel, 5 ml of bromocresol green solution and 5 ml of phosphate buffer were added. The mixture was shaken vigorously with 1, 2, 3 and 4 ml chloroform and collected in a 10ml volumetric flask and diluted to the volume with chloroform. The absorbance for test and standard solutions were determined against the reagent blank at 470 nm with an UV-Vis spectrophotometer. A set of reference standard solutions of atropine (20, 40, 60, 80 and 100 μg/ml) were prepared in the same manner as described earlier 16 .

Determination of Total flavonoids
Total flavonoids were determined by the aluminium chloride colorimetric assay. The reaction mixture consists of 1 ml of extract and 4 ml of distilled water was subjected to the 0.3 ml of 5 % sodium nitrite solution. After 5 minutes, 0.3 ml of 10 % aluminium chloride was mixed. After 5 minutes, 2 ml of 1M Sodium hydroxide was treated and diluted to 10 ml with distilled water. The absorbance for test and standard solutions were determined against the reagent blank at 510 nm with an UV-Vis spectrophotometer. A set of reference standard solutions of quercetin (20, 40, 60, 80 and 100 μg/ml) were prepared in the same manner as described earlier 18 .

Determination of Total Tannins
The total tannins were determined by Folin-Ciocalteu method. About 0.1 ml of the sample was subjected to a 7.5 ml of distilled water and 0.5 ml of Folin-Ciocalteu phenol reagent and 1 ml of 35 % Na 2 CO 3 solution and diluted to 10 ml with distilled water. The mixture was shaken well and kept at room temperature for 30 min. Absorbance for test and standard solutions were measured against the blank at 725 nm with an UV-Vis spectrophotometer. A set of reference standard solutions of Gallic acid (20, 40, 60, 80 and 100 μg/ ml) were prepared in the same manner as described earlier 20 .

Determination of Total Phenolics
The total phenolics was determined by Folin-Ciocalteu assay method. About 1 ml of extract was added with 9 ml of distilled water & 1 ml of Folin-Ciocalteu phenol reagent and shaken well. After 5minutes, 10 ml of 7 % Na 2 CO 3 solution was treated to the mixture and the volume were made up to 25 ml. Incubated for 90 min at room temperature and the absorbance for test and standard solutions were determined against the reagent blank at 550 nm with an UV-Vis spectrophotometer. A set of standard solutions of Gallic acid (20,40,40,60,80 and 100 μg/ml) were prepared in the same manner as described earlier 21 .

GC-MS Analysis
The identification and quantification of basil chemical constituents were evaluated by Gas chromatography coupled with Mass spectrometry QP2010 plus, Shimadzu, Japan equipped with RTX-5 MS GC capillary column (5% diphenyl/ 95% dimethyl polysiloxane) of 0.5 µm dia and 30 m length. GC working conditions: The temperature was kept between 40-290º C with a gradual increase of 8º C/min. Column oven and injection temperatures were set at 100º C and 270º C respectively. Injection mode was set as split with a ratio of 20; Helium was used as carrier gas  Table 1 represents the presence of basil chemical constituents in various extracts such as petroleum ether, chloroform; ethyl acetate; methanol; aqueous. PE and EA extracts showed various class of secondary metabolites than CF extract but lesser that MeOH and aq extracts. Alkaloids, flavonoids, steroids, cardiac glycosides and phenolics were present in almost all the extracts. Comparatively, MeOH extract showed various chemical constituents of basil that the other extracts. Table 2 represents the quantitative determination of chemical constituents of Ocimum basilicum L leaf extracts in respect to total alkaloids, flavonoids, phenols, tannins and saponins content. Alkaloids, flavonoids, phenols and saponins were highly present in MeOH but saponin content was low. Comparatively, MeOH extracts showed more quantity of chemical constituents amongst all the extracts, so it was taken to the GCMS quantification. The values here are mentioned in percentage by converting mg/standard gram equivalent. Figure 1 and Table 3    of methanol leaf extract of basil. The peaks in the chromatogram were compared with the Wiley and NIST database of spectrum installed with GCMS library. The results revealed the presence of chemical composition with area % and m/z ratio with respect to their retention times. Each component of the composition possesses unique retention times which vary in peak areas. The most abundant components in the methanol leaf extract were found to be eugenol, germacrene, β-elemene, gurjunene and menthol with higher peak area percentage. Mostly terpene alcohols and hydrocarbons were detected, and sesquiterpene also with considerable concentrations.

Conclusion
Ocimum basilicum L. leaf extracts were tested for the presence of medicinally important chemical constituents. The qualitative and quantitative determination of chemical constituents of basil leaf extracts were evaluated. GCMS analysis reveals the exact composition with m/z ratio in respect to retention time. The results revealed that the methanol (MeOH) is the most suitable solvent to extract the basil leaf essential oil with high yield of chemical constituents. Further investigation will be helpful in the isolation and characterization of these bio-active compounds and by exploring their pharmacological activities will lead to the development of new therapeutic drugs.