1. INTRODUCTION:

Nature has been a source of medicinal agents for thousands of years and an impressive number of modern drugs have been isolated from natural sources, many based on their use in traditional medicine. Various medicinal plants have been used for years in daily life to treat disease all over the world. According to World Health Organization, medicinal plants are the best source to obtain a variety of newer herbal drugs. About 80% of individuals from developed countries use traditional medicine, which has compounds derived from medicinal plants. Therefore, such plants should be investigated to better understand their properties, safety and efficacy¹. Plant-derived substances have recently become of great interest owing to their versatile applications. Medicinal plants are the richest bio-resource of drugs of traditional systems of medicine, modern medicines, nutraceuticals, food supplements, folk medicines, pharmaceutical intermediates and chemical entities for synthetic drugs^2.

Sapindus emarginatus vahl is extensively used in Indian traditional and folklore medicines to cure various human ailments. Traditionally, it is used as anti-inflammatory and antiprurutic. It is used to purify blood. The seed is an intoxicant and the fruit rind has oxytropic action. Its powder is used in nasal insufflations³. It has been suggested that aqueous and ethanolic extracts from plants used in allopathic medicine are potential sources of antiviral, antitumoural and antimicrobial agents^4-6.

The medicinal values of Sapindus emarginatus Vahl includes its traditional usage for treating salivation, epilepsy, cholera, diarrhoea, ulcers, tonsillitis, fever, arthritis, rheumatism, dysentery, emetics, paralysis, convulsions, spasm, pulmonary troubles, and vermifuges etc. However there is no exclusive report on the medicinal values of the nuts of Sapindus emarginatus vahl. Hence the present investigation is designed to screen the phytochemical contents and to evaluate the anti-inflammatory activity of this potential plant.

2. MATERIALS AND METHOD:

2.1 Extract preparation:

The dried pericarps of fruits of Sapindus emarginatus Vahl were collected from local market and were authenticated by Prof. P.Jayaraman, Ph.D, National Institute of Herbal Science (PARC). Aqueous extract was prepared using, 100g of its powder which was mixed in 1 litre of distilled water and boiled for 5hours so that the volume is reduced to 10g of paste.

2.2 Phytochemical screening:

Phytochemical analysis of the aqueous extract was performed according to the methods of Harborne (1998) and Evans, C.W. (1989)^7,8. By this analysis, the presence of several phytochemicals like flavonoids, tannins, saponins, sugars and glycosides were tested.

Test for terpenoids: 5.0ml of the extract is mixed in 2ml of chloroform and concentrated sulphuric acid is added to form a layer. Red brown colour of the interface shows positive test for terpenes.

Test for tannins: 0.5g of dried powdered sample was boiled in 20ml of water in a test tube and filtered. This was followed by few drops of 0.1% ferric chloride. A brownish or blue black colour shows the presence of tannins.

Test for flavanoids: A portion of powdered sample with 10ml of ethyl acetate was heated over a steam bath for 3 mins .The mixture is filtered and 4.0 ml of the filtrate is shaken with 1.0 ml of dilute ammonia solution. Yellow coloration is observed.

Test for saponin: 2.5 g of plant material extracted with boiling water was cooled and shaken vigorously to froth and stand for 15 to 20 min. No froth = -ve, froth<1cm = weakly +ve, froth 1.2cm=+ve, froth >2cm = strongly +ve.

Test for cardiac glycosides: To 5.0 ml of the extract, 2ml of glacial acetic acid containing 1 drop of ferric chloride solution is added. This is followed by addition of 1.0 ml of concentrated sulphuric acid. A brown ring of the interface indicates the deoxy sugar characterstic of cardenolides. A violet ring may appear below the brown ring. While in acetic acid layer, a greenish layer may gradually spread throughout thin layer.

Test for steroids: 2.0ml of acetic anhydride is added to 0.5gm of ethanolic extract of each sample followed by 2ml of sulphuric acid. Color changes from violet to blue or green in some samples.

2.3 Anti-inflammatory studies:

Protease inhibitory activity:

The reaction mixtures (2.0ml) contained 0.06mg trypsin, 1.0ml of 25mM tris-HCl buffer (p^H 7.4) and 1.0ml of aqueous solution of aqueous extract (50-250µg/ml). The mixtures were incubated at 37°c for 5 minutes. Then 1.0ml of 0.8% (w/v) casein was added. The mixtures were incubated for an additional 20 minutes. 2.0ml of 70% perchloric acid was added to terminate the reaction. Cloudy suspension was centrifuged. Absorbance of the supernatant was read at 660nm against buffer as blank^9.The percentage of inhibition was calculated. Each experiment was done in triplicate and taken the average.

Inhibition of protein denaturation:

Test solution (1.0ml) containing different concentration (50-250µg/ml) of drug was mixed with 1.0ml of egg albumin solution(1.0mM) and incubated at 27±1°c for 15 min. Denaturation was induced by keeping the reaction mixture at 70°c in a water bath for 10min. After cooling the turbidity was measured spectrophotometrically (ELICO) at 280 nm¹⁰. Percentage inhibition of denaturation was calculated from control where no drug was added. Each experiment was done in triplicate and average was taken.

3. RESULTS AND DISCUSSION:

The results of the phytochemical screening reveal the notable presence of Saponins, Flavonoids, Tanins and other phytochemicals too (Table.1). However Steroids and Alkaloids were absent. Screening of various natural organic compounds and identifying active agents is the need of the hour, because successful prediction of lead molecule and drug like properties at the onset of drug discovery will pay off later in drug development. In our study the present findings suggests that the presence of various phytochemicals in the Sapindus emarginatus, mark it as a potential candidate for further exploration to be an effective therapeutic agent.

Table 1.The phytochemical profile of the aqueous extract of Sapindus emarginatus

Phytochemcials	Presence/Absence
Sugar	+
Tannin	+
Alkaloid	-
Flavonoid	+
Saponin	+
Steroid	-
Cardiac Glycoside	+
Terpenoid	+

The aqueous extract of Sapindus emarginatus Vahl showed dose dependent inhibition of thermal denaturation. The results of the anti protease activity (figure1) and inhibition of protein denaturation is shown in figure2.

Fig.1. Effect of aqueous extract of Sapindus emarginatus on protease inhibition

Fig.2. Inhibition of Protein Denaturation by Sapindus emarginatus

Denaturation of protein is a well documented cause of inflammation and rheumatoid arthritis. Several anti- inflammatory drugs have shown dose dependent ability to inhibit thermally induced protein denaturation. The aqueous extract showed dose dependent inhibition of thermal denaturation. The ability of aqueous extract of Sapindus emarginatus to bring down thermal denaturation of protein is possibly a contributing factor with the mechanism of action.

Enzymes work with the body’s innate mechanisms to promote healing and allow inflammation to progress naturally. They support the production of various cytokines, activate large proteins, such as alpha-2-macroglobulins, and slow the clotting mechanism, which provokes a healthier healing response. Proteolytic enzymes in themselves, however, can also be a cause of inflammation. Extracellular endogenous proteases react with cell-surface receptors in the airways to generate leukocyte infiltration and to amplify the response to allergens. protease-activated receptors (PARs), are 7-transmembrane proteins coupled to G proteins. PARs are widely distributed on the cells of the airways, where they contribute to the inflammation. Aqueous extract of Sapindus emarginatus exhibited significant antiprotease activity and the activity was dose dependant. It may be due to the saponin constituent. This finding justifies the usefulness of Sapindus emarginatus in the treatment of inflammation associated diseases.

4. CONCLUSION:

The nuts of Sapindus emarginatus have been around for a very long time in India. Sapindus emarginatus is traditionally used as a natural and effective treatment for skin complaints including eczema, chronic itching and psoriasis. However, no attempt has been made to evaluate the anti-inflammatory effect of Sapindus emarginatus. The finding of our investigation reveals that the aqueous extract of Sapindus emarginatus showed notable presence of saponins, flavonoids and other phytochemicals. The aqueous extract also showed marked inhibition of thermally induced protein denaturation, and exhibited anti-protease activity that justifies its anti-inflammatory property too. So this plant can be used to discover bioactive natural products that may serve as leads for the development of new pharmaceutical that address hither unmet therapeutic needs.

5. REFERENCES:

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Received on 08.04.2010 Modified on 03.05.2010

Research J. Pharm. and Tech.3 (4): Oct.-Dec.2010; Page 1125-1127