PHYTOCHEMICAL SCREENING AND IN-VITRO ANTIOXIDANT AND ANTI-INFLAMMATORY POTENTIAL EVALUATIONS OF METHANOLIC EXTRACTS OF COCOS NUCIFERA (L.) LEAVES

Objective: Cocos nucifera (L.) (Arecaceae) is commonly called the ‘‘coconut tree’’ and is the most naturally widespread fruit plant on Earth. Throughout history, humans have used medicinal plants therapeutically, and minerals, plants, and animals have traditionally been the main sources of drugs. The objective in the present study was to screen the phytochemical profile and pharmacological activities of methanolic extract of coconut leaves. Methods: To investigate pharmacological activities DPPH scavenging assay and HRBC membrane stabilization methods were performed for antioxidant and anti-inflammatory potential respectively. Results: The pharmacological studies revealed that the plant extracts may have significant antioxidant effect which is probably mediated by inhibition of DPPH free radical. The IC50 values by DPPH scavenging assay observed for standard and leaves were 97.29μg/ml and 486.78μg/ml respectively. Thus, this plant extracts have significant antioxidant effect. It also had moderate antiinflammatory activity. The IC50 values for anti-inflammatory activity by standard and coconut leaves were 21.46 μg/ml and 831.21 μg/ml respectively. These findings suggest that Cocos nucifera (L.) may be a possible source for the development of a newantiinflammatory drug. Conclusion: The phytochemical analysis of methanolic extract of coconut leaves showed that they contained significant presence of flavonoids, phenols, saponins, terpenoids and triterpenes. Alkaloids, glycosides and tannins are also moderately present. Quantitative evaluations show significant presence of phenols which was more than tannin content.


INTRODUCTION
Plants, which have one or more of its parts having substances that can be used for treatment of diseases, are called medicinal plants 1 . Medicines derived from plants are widely famous due to their safety, easy availability and low cost 2 . Throughout the ages, humans have relied on nature for their basic needs, for the production of food, shelter, clothing, transportation, fertilizers, flavours, fragrances, and medicines 3 . Plants have formed the basis of sophisticated traditional medicine systems that have been in existence for thousands of years and continue to provide mankind with new remedies. Although some of the therapeutic properties attributed to plants have proven to be erroneous, medicinal plant therapy is based on the empirical findings of hundreds and probably thousands of years of use. The first records, written on clay tablets in cuneiform, are from Mesopotamia and date from about 2 600 BC 4 . Among the substances that were used are oils of Cedrus species (cedar) and Cupressus sempervirens (cypress), Glycyrrhiza glabra (licorice), Commiphora species (myrrh) and Papaver somniferum (poppy juice), all of which are still in use today for the treatment of ailments ranging from coughs and colds to parasitic infections and inflammation. In ancient Egypt, bishop's weed (Ammimajus) was reported to be used to treat vitiligo, a skin condition characterized by a loss of pigmentation 5, 6 . More recently, a drug (methoxypsoralen) has been produced from this plant to treat psoriasis and other skin disorders, as well as T-cell lymphoma 6 . The interest in nature as a source of potential chemotherapeutic agents continues. Natural products and their derivatives represent more than 50% of all the drugs in clinical use in the world today.   10 . C. nucifera has been called the 'tree of life' or 'tree of heaven' because of its value as provider of so many useful products. This species provides food, water, oil, medicine, fibre, timber, and fuel for many people living on islands in the Pacific Ocean 11 .

MATERIALS AND METHODS
In this study, all the chemicals, reagents used here provided from laboratory of Department of Pharmacy, USTC which source from Merck Limited, Mumbai, India and were analytical grade, pure and sorted under optimum storage conditions. Moreover, the drug mixtures and solutions were prepared accurately in standard volumetric flasks about one hour prior to obtain and recording the data. In the alkaline condition phenols ionize completely. When Folin-Ciocalteu's reagent is used in this ionized phenolic solution, the reagent will readily oxidize the phenols. Usual color of Folin-Ciocalteu's reagent is yellow and after the oxidation process the solution becomes blue. The intensity of the color change is measured in a spectrophotometer at 760 nm. The absorbance value will reflect the total phenolic content of the compound 12 . 1mL of saturated sodium carbonate solution (8% w/v in water) was added to the mixture and the volume was made up to 3ml with distilled water. The reaction was kept in the dark for 30min and after centrifuging the absorbance of blue color from different samples was measured at 760 nm 14 .

Total Tannin Content (TTC) determination
Fifty micro liters (µl) of tannins extract for each sample was taken in test tube and volume was made to 1.0 ml with distilled water. Then, 0.5ml Folin Ciocalteu reagent was added and mixed properly. Then 2.5ml 20 per cent sodium carbonate solution was added and mixed it and kept for 40 minutes at room temperature. Optical density was taken at 725nm in spectrophotometer and concentration was estimated 15 . Tannic acid was used as standard and tannin contents were measured as tannic acid equivalent.

Figure 5: Antioxidant activity of standard by DPPH SCV assay Anti-inflammatory activity
Percent inhibition of protein denaturation was calculated as follows 16 : The method of HRBC membrane stabilization was chosen to evaluate anti-inflammatory effect.

Anti oxidant activity
The free radical-scavenging activity of extracts was evaluated with the DPPH assay based on the measurement of the reducing ability of antioxidants toward the DPPH radical 17, 18 .

RESULTS AND DISCUSSION
The following tests were done to find the presence of the active chemical constituents such as alkaloids, flavonoids, glycosides, phenols, saponins, tannins, terpenoids and triterpenes is shown in Table 3. Due to the different chemical compositions present in a Cocos nucifera (L.) are obviously responsible for its different therapeutic and pharmacological activities.   Table 4, it is observed that the degree of membrane stabilization was increased by increase in concentration. That means the drug will give required action at higher concentration. As shown in Figure 2, it is observed that in Comparative % inhibition of protein denaturation, the Cocosnucifera leaves under the study of methanolic extract from 1.56±0.5% to 53.53±0.48% has shown moderate inhibition of protein denaturation at any concentration compared to the standard drug Diclofenac Sodium from 79.51±0.46% to 93.47± 0.19%. It revealed that the plant extracts may have moderate anti-inflammatory effect which is probably mediated by HRBC membrane stabilization. The secondary metabolites such as phenolic compounds and tannins which were found in preliminary phytochemical screening might be responsible for such type of activity. The Antioxidant activity was performed by the method of free radical-scavenging assay. The antioxidant potential of the methanolic extract was determined on the basis of their scavenging activity of the stable 1, 1diphenyl-2-picryl hydrazyl (DPPH) free radical. So, the free radical-scavenging activity of extracts was  Figure 7. So, it revealed that the plant extracts may have significant antioxidant effect which is probably mediated by inhibition of DPPH free radical, which is responsible for oxidation.