Tulsi (Ocimum sanctum), excellent source of phytochemicals

Ocimum sanctum also known as Tulsi or Holybasil is an aromatic plant and it belongs to the family Lamiaceae. It is widely used as medicine to cure various ailments. The objective of the study was to analyse different phytochemical components of tulsi leaf. The dried powder of Tulsi (50g) was placed in the thimble of Soxhlet apparatus and the experiment was done separately for methanol, ethanol and distilled water. The percentage yield was 8%w/w,7%w/w, and 5%w/w respectively. The study reveals that various secondary metabolites such as carbohydrate, tannin, flavonoids, saponins, glycoside, terpenoid, fatty acids and phenol are present in tulsi leaf extract. From the quantitative analysis it was found that high amount of phenols are present in Tulsi leaf ranging from 1.6 to 7.6 percentages. Consequently the amount of alkaloid and flavonoids ranged from 0.91 to 1.28 and 1.56 to 2.24 percentages respectively. From the GC-MS analysis of methanolic extract three compounds were identified as major constituents viz., Eugenol , Benzene, 1, 2-dimethoxy4(2propenyl), α Farnesene and Cyclohexane, 1, 2, 4triethenyl. Thesephyto-chemicals are known to possess antiseptic, analgesic, anti-inflammatory, antimicrobial, antistress, immunomodulatory, hypoglycemic, hypotensive and antioxidant properties. Hence it is more beneficial to use tulsi asan herbal medicine as compare to chemically synthesized drug. Keywords— Ocimum sanctum, phytochemical,


INTRODUCTION
The plant kingdom is an excellent source of potential drugs and in the recent years there has been an increasing awareness about the importance of medicinal plants.
Medicinal plants are rich source of different types of medicines and produce various bioactive molecules. Herbal plant extracts are very useful and are the major sources of medicine which play vital role in controlling various types of pathogens (Doss, 2009) and as growth promoters. These are the cheaper source for therapeutics and viable solution for various pathogens.The medicinal plants extract have now emerged as a good alternative as they are rich in a wide variety of secondary metabolites such as tannins, phenolics, alkaloids and flavonoids etc which enhances growth, innate immune response and disease resistance against pathogenic bacteria in human as well as in different organisms (Edoga et al., 2005). About 80% of individuals from developed countries use various medicinal plants as traditional medicines as anticancer drugs (Dewick, 1996), antimicrobial drugs (Phillipson, 1996), antifungal and in various proposes. The medicinal plants are rich sources of secondary metabolites which are chemically and taxonomically extremely diverse compounds with obscure function. A large number of phytochemicals are widely uses in human therapy, agriculture, veterinary, various scientific researches and in different areas (Vasu et al., 2009) along with inhibitory effects on all types of microorganisms in vitro (Cowan, 1999). Ocimum sanctum L. commonly known as holy basil (Tulsi) is an herbaceous perennial, belongs to family Lamiaecae and is considered as one of the most important source of medicine and drugs with many secondarymetabolites and essential oils recommended fortreatment of malaria, diarrhoea, bronchial asthma,dysentery, bronchitis, skin diseases, arthritis, painfuleye disease, chronic fever and eye diseases etc 5,6. Inaddition, Ocimum sanctum also showsanticancerous, antifungal, antimicrobial, antifertility,hepatoprotective, antispasmodic, cardio protective,antiemetic, antidiabitic, analgesic, adaptogenic, anddiaphoretic properties6-9.The pharmacological studies reported in the present research confirm the therapeutic value of O. Sanctum. Therefore, the present study looks into the extraction and preliminary phytochemical analysis of O. Sanctum leaves.

II.
MATERIALS AND METHODS Collection of plant material: Leaves of Ocimum sanctum L. (tulsi) were collected from different sites of Dibrugarh District, Assam, washed with sterile water and dried in shades. Then the samples were powered in mechanical grinder. Aqueous, methanol and ethanol extract: The dried tulsi (50g) powder was placed in the thimble of Soxhlet apparatus and 500-700 ml of distilled water, methanol and ethanol was used for extraction procedure and the experiment was done separately for all the two solvents and distilled water. The extraction was continued till clear solvent or water was seen in the thimble. The extract was concentrated using rotary evaporator. Then the extract was dried in a digital water bath till a dark green residue was obtained. The percentage yield of the extract was calculated using the following formula: Final weight of the dried extract Initial weight of the powder All the three extracts were kept in separate vials in the refrigerator till further use. Qualitative phytochemical analysis: The extract was tested following standard biochemical methods as described below. Molisch's test: 2ml of Molisch's reagent was added to 0.5 ml of crude extract and the mixture was shaken properly. After that, 2ml of concentrated H2SO4 was poured carefully along the side of the test tube. Appearance of a violet ring at the interface indicated the presence of carbohydrate. Iodine test: 2ml of iodine solution was mixed with 0.5 to 1 ml of crude extract. A dark blue or purple coloration indicated the presence of the carbohydrate. Test for phenol: 2 ml of alcohol and 2-3 drops of ferric chloride solution was added to 1 ml of crude extract, bluegreen or black coloration indicated the presence of phenols Test for tannin: 1 ml of distilled water and 2-3 drops of ferric chloride solution was added to 0.5 ml of crude extract. A black coluration indicated the presence of tannin. Test for flavonoi ds Shinoda test: Crude extract was mixed with small amount of magnesium and concentrated HCl was added drop wise. Appearance of pink scarlet colour after few minutes indicated the presence of flavonoids. Alkaline reagent test: 0.5 ml of crude extract was mixed with 2ml of 2% solution of NaOH. An intense yellow colour was formed which turned colourless on addition of few drops of diluted acid which indicated the presence of flavonoids. Test for saponins: 1ml of crude extract was mixed with 5ml of distilled water in a test tube and it was shaken vigorously. The formation of stable foam was taken as an indication for the presence of saponins.

Test for glycosides
Liebermann's test: Crude extract was mixed with each of 2ml of chloroform and 2ml of acetic acid. The mixture was cooled in ice. Carefully concentrated H2SO4 was added. If colour change from violet to blue to green which indicated the presence of steroidal nucleus, i.e., glycone portion of glycoside. Salkowski's test: 2ml of chloroform was mixed with crude extract. Then 2ml of concentrated H2SO4 was added carefully and shaken gently. A reddish brown colour indicated the presence of glycoside. Keller-kilani test:0.5 ml of crude extract was mixed with 2ml of glacial acetic acid containing 2-3 drops of 2% solution of FeCl3. Then 2ml of concentrated H2SO4 was poured into the mixture. A brown ring at the interface indicated the presence of cardiac glycosides. Test for steroid (i) 2ml of chloroform was added to the crude extract of Tulsi. Then 2ml of each of concentrated H2SO4 and acetic acid were added into the mixture. The presence of steroids was indicated by appearance of a greenish coloration in the reaction mixture. (ii) Crude extract was mixed with 2ml of chloroform and gently added concentrated H2SO4. A red colour was seen in the lower layer this indicated the presence of steroids. Test for terpenoids: Crude extract was mixed in 2ml of chloroform and evaporated to dryness. To this, 2ml of concentrated H2SO4 was added and heated for about 2 minutes. Presence of terpenoids was indicated by a greyish colour at the interface. Test for alkaloids: 2ml of 1% HCl was mixed with crude extract and heated gently. After heating, Mayer's And Wagner's reagents were added to the mixture. If precipitate was observed in the reaction mixture which indicated the presence of alkaloids.
Test for anthraquinone: 5ml of chloroform and 5 ml of ammonia solution was added to 0.2 gm of plant extract. Appearance of pink, red or violet colour indicated the presence of anthraquinone.

Oils & Fats:
A small quantity of crude extract was pressed between two filter papers separately. An oily appearance on filter paper indicated the presence of fixed oil and fats.

Test for lactones
Baljet's test:Crude extract was treated with sodium picrate solution. Presence of lactone was observed by appearance of yellow to orange colour in the mixture. Quantitative analysis of phytochemical in the plant extract: Determination of total phenolic contents (Singleton et al., 1999): The amount of total phenol for aqueous, methanol and ethanol extract were determined by Folin -Ciocalteu reagent method. 2.5 ml of 10% Folin-Ciocalteu reagentand 2 ml of 2% Na2Co3 were added to 0.5 ml of plant extract. The mixture was then incubated at room temperature for 30 minutes. Gallic acid was used as standard (1mg/ml). The absorbance of the sample was measured at 765nm. All the tests were done in triplicates and the results were determined from standard curve and were expressed as gallic acid equivalent (mg/g of extracted compound). Determination of alkaloid (Harborne, 1973): 5 g of the sample was taken and 200 ml of 10% acetic acid in ethanol was added to the sample and allowed to stand for 4 hours. Then the solution was filtered and the extract was concentrated on water bath Conc. NH4(OH) was added drop wise and the whole solution was allowed to settle and the precipitate was then washed with dilute ammonium hydroxide and filtered. The residue was dried and weighed and this was the amount of alkaloid present in the plant material Determination of flavonoids (Bohm & Kocipai-Abyazan, 1994): 10 g of plant sample was taken and extracted repeatedly with 100ml 80% methanol. Then the solution was filtered and the filtrate was transferred into an empty crucible and evaporated into dryness over water bath and weighed. The final weight dry weight was amount of flavonoids in the plant sample.

Preparation of stock solution
The extracts were reconstituted in methanol. Methanolic extracts (1 μl) were injected for GC-MS analysis.
The oven temperature was programmedfrom 50°C (for 1 min.), with an increaseof 6 0 C / min to 280 0 C, then ending with aisothermal for 15min at 280°C. Mass spectra weretaken at 70 eV; a 0.5 seconds of scan interval andfragments from 40 to 550 Da. Total GC runningtime was 60 minutes.

Identification of Compounds
Interpretation on mass spectrum GC-MS was conducted using the database of department of Chemistry; Dibrugarh University. The mass spectrum of the unknown component was compared with the spectrum of the known components stored in the department of Chemistry library.

III. RESULTS
The yield of residue after Soxhlet extraction and evaporation of 50 gm dried plant leaves in methanol, ethanol and water were as follows: The phytochemicals analysis in Ocimum sanctum (Tulsi) leave extracts in the two solvents and aqueous conditions were summarized in Table 2. Various bioactive molecules were found in Tulsi leaf extract from the phytochemical screening. The amount of extraction is more in case of organic solvent then that of water. From the quantitative analysis it was found that high amount of phenols are present in Tulsi leaf ranging from 1.6 to 7.6 percentages. Consequently the amount of alkaloid and flavonoids ranged from 0.91 to 1.28 and 1.56 to 2.24 percentages respectively.    DISCUSSION Ocimum sanctum has various properties such as antistress, antiseptic, analgesic, anti-inflammatory, antimicrobial, immunomodulatory, hypoglycemic, hypotensive, cardioprotective and antioxidant (Williamson, 2002, Tanwar et al., 2015. Eugenol (1-hydroxy-2-methoxy-4allylbenzene), the active constituents present in O. sanctum have been found to be largely responsible for the therapeutic potentials (Sailaja et al., 2010). This plant has various properties such as antistress, antiseptic, analgesic, anti-inflammatory, antimicrobial, immunomodulatory, hypoglycemic, hypotensive, cardioprotective and antioxidant (Williamson, 2002, Tanwar et al., 2015. Eugenol (1-hydroxy-2-methoxy-4-allylbenzene), the active constituents present in O. sanctum have been found to be largely respons ible for the therapeutic potentials (Sailaja et al., 2010). The study reveals that various secondary metabolites such as carbohydrate, tannin, flavonoids, saponins , glycoside, terpenoid, fatty acids and phenol are present in tulsi leaf extract. Leaves of Ocimum sanctum contain water-soluble phenolic compounds and various other constituents, such as eugenol, methyl eugenol and caryophylllene that may act as an immunostimulant. Saponins act as antihyperlipedemic, hypotensive and cardiodepessive properties (Bairwaet al., 2012). The phytochemical constituents such as alkaloids, steroids, flavanoids, tannins, phenols and several other aromatic compounds of plants serve a defense mechanism against predation by many microorganisms, insects and other herbivore (Bonjar et al., 2004) Glycosides can act as cardiostimulants in cases of cardiac failure (Sood et al., 2005). Tannins have anti diarrheal and haemostasis properties (Asquith et al., 1986).  Table No.1. Benzene, 1, 2-dimethoxy-4-(1propenyl) (Methyl-Isoeugenol) has the property of Antifungal activity (Kurita et al., 1981), Nematicidal activity (Park et al., 2003) and Antifeedant activity (Katsumi, 1987).Eugenol that methanolic extract of Ocimum sanctum has anticancer effect by inhibition of nitric oxide synthesis (Kim et al., 1998). The use of medicinal plants acts as a source of antimicrobial agent also for aquaculture. In Macrognathus pancalus, the extract of O. sanctum was found to enhance the antibody response (Dugenci et al., 2003). The different leaf extracts of Tulsi (Ocimum sanctum), shows antimicrobial activity against three human pathogens Escherichia coli, Staphylococcus aureus and Candida albicans. (Subramanian et al.,2014). Tulsi oil showed significant anti-inflammatory, analgesic, antipyretic and antimicrobial effects. It has also shown memory enhancing, antifertility, anticataract, antithyroid, antiulcer, antidiabetic, antiarthritic, antiamnesic, antihelmenthic, anticataract, hepatoprotective and no tropic activity (Rajesh et al., 2013). Alcoholic extract increased step down latency and acetyl cholinesterase inhibition and so used in the treatment of cognitive disorders. Ocimum sanctum has been widely employed in traditional medicines. Hence phytochemicals from this plant can be used in variety of disorders afflicting ma nkind. The herbs are cheap, available in large quantity around us and they pose no danger to the living organisms, the environment and the consumers and hence greatly helpful for living organisms.

V.
CONCLUSION The presence of various bioactive compounds in the tulsi leaves justifies the uses for various ailments by living population. The results confirm the use of Ocimum sanctum plant as traditional medicinal properties andsuggest that some of the plant extracts possess compounds with antimicrobial properties that can be used asantimicrobial agents in new drugs for the therapy of infectious diseases caused by various pathogens. It is more beneficial to use tulsi asan herbal medicine as compare to chemically synthesized drug.