Introduction:

The physiological and biochemical processes involved in living cell, leads to free radical and reactive oxygen species. The accumulation of free radicals leads to oxidative damage to lipids and DNA. Moreover, it causes various diseases namely diabetes, cancer, hypertension, cardiovascular, necrosis of hepatocytes, etc. The antioxidant compound protects the living cell from free radicals generated by oxidative damage. The secondary metabolites such as flavonoids, polyphenol, glycosides, tannins, coumarins, isoflavonoids etc present in plants impart highly antioxidant activities.

It is scientifically documented that the eating of fruits and vegetables diminishes the prospects of diversified diseases in human. These defense properties of fruits and vegetables are due to presence of flavonoids and polyphenol components. Moreover, the antioxidant components are used as nutritional supplements in food product, to keep body free from diseases^1-4.

Mushrooms are recognized to be medically active in various therapies, namely antitumour, antibacterial, antiviral, haematological and immunomodulating treatments. Additionally, the mushrooms are containing rich source of protein, and it is used as healthy food. Pleurotus ostreatus mushrooms, usually known as oyster mushrooms, cultivate in the wild in tropical, subtropical and temperate regions. P. ostreatus are effortlessly cultivated in artificial method, and this enhances the yield of mushroom. The capability of extract from P. ostreatus to screen leading organs of animals such as the liver, heart, and brain of aged rats against oxidative stress has been reported³. In this study, we planned to evaluate the preliminary phytochemical and in vitro antioxidant activity of P. ostreatus

MATERIALS AND METHODS:

Plant material

The P. ostreatus was collected from the different area of Bastar and Sarjuga district. The collected material was authenticated by Dr. A.P. Singh, Principal Scientist, Department of Agronomy College of Agriculture, Indira Gandhi Krishi Vishwavidyalaya, Raipur (C.G.). The material was dried under shade, powdered mechanically and stored in air tight container.

Preparation of extracts

The powder of the P. ostreatus was extracted successively with petroleum ether, ethyl acetate and ethanol by cold maceration method. The extract was filtered and the resultant extract was distilled in vacuum under reduced pressure in order to remove the solvent completely, and later dried in a desiccator. The extracts of P. ostreatus were kept in air tight container for further study.

Preliminary Phytochemical analysis

Preliminary phytochemical screening was performed to identify secondary metabolites in petroleum ether, ethyl acetate and ethanol extract of P. ostreatus^5,6.

In vitro antioxidant activity

Hydrogen-donating activity

In this study, methanolic solution of DPPH (100 mM, 2.95 ml), 0.05 ml of ethanol extract dissolved in methanol, and added at various concentrations (50-250 µg/ml). Reaction mixture was shaken and after 30 min at room temperature, the absorbance values were measured at 517 nm and converted into percentage of antioxidant activity (% AA). Ascorbic acid was used as standard. The degree of discoloration indicates the scavenging efficacy of the extract, was calculated by the following equation^7,8.

% AA = 100 – {[(Abs_sample – Abs_blank) x 100] / Abs_DPPH}

Superoxide scavenging activity

Superoxide scavenging was carried out by using alkaline Dimethyl sulfoxide (DMSO). Solid potassium superoxide was allowed to stand in contact with dry DMSO for at least 24 h and the solution was filtered immediately before use. Filtrate (200 ml) was added to 2.8ml of an aqueous solution containing nitrobluetetrazolium (56 mM), EDTA (10 mM) and potassium phophate buffer (10 mM, pH 7.4). Sample extract (1 ml) at various concentrations (50-250 µg/ml) in water was added and the absorbance was recorded at 560 nm against a control in which pure DMSO has been added instead of alkaline DMSO^7,8.

Total polyphenol content

Total polyphenol content was determined using colorimetric method. 2.0 ml of the prepared extract was oxidized using Folin - Ciocalteu reagent (400 μl), and sodium carbonate solution (75 g/l) was then added to the reaction mixture to reach a 10.0 ml volume. After 2 h, the suspension was centrifuged for 10 min at 5000 rpm, and absorption was measured at a 760 nm wavelength. The amount was calculated using the gallic acid calibration curve^9,10. The results were expressed as gallic acid equivalent (GAE) mg per 100 ml of the sample.

Statistics analysis

The data were reported as mean values ± standard deviation (SEM). Values representing the concentrations of investigated extract that cause 50% of neutralization/inhibition (IC₅₀) were determined by the linear regression analysis.

RESULTS AND DISCUSSION:

Phytochemical study

The preliminary phytochemical screening was done to assess the presence of phytoconstituent in petroleum ether, ethyl acetate and ethanol extract of P. ostreatus. Table 1 exhibited that maximum number of secondary metabolites like alkaloid, glycoside, flavonoids, polyphenol and saponin are present in ethanol extracts. The petroleum ether and ethyl acetate extract contains steroids and alkaloids respectively. The flavonoids and polyphenol component are responsible for antioxidant activity. From phytochemical screening, it was found that the ethanol extracts contain flavonoids and polyphenol compound. Hence this result supports us to continue antioxidant activity with ethanol extracts.

Table 1: Phytochemicals present in various extracts of P. ostreatus

Phytoconstituent	Petroleum ether extract	Ethyl acetate extract	Ethanol extract
Alkaloids	-	₊	₊
Saponins	-	-	₊
Glycosides	-	-	₊
Carbohydrates	-	-	-
Polyphenols	-	-	₊
Flavonoids	-	-	₊
Terpenoids	-	-	-
Steroids	-	-	-

+ = Detected, - = Not detected

Table 2: Free radical scavenging capacity of ethanol extract of P. ostreatus

Concentration (µg/ml)	DPPH Scavenging %
Concentration (µg/ml)	Ethanol Extract	Ascorbic Acid
50	40.25±1.05	94.18±1.19
100	65.34±1.28	-
150	89.15±0.98	-
200	117.29±1.16	-
250	138.57±1.21	-
IC₅₀	69.30	-

Values are mean ± SEM of six determinations

In vitro antioxidant activity

The percentage inhibition of free radicals by the ethanol extract of P. ostreatus for DPPH and superoxide anion radical are depicted in table 2 and table 3 respectively. The ethanol extract strongly neutralized the DPPH radical with the IC₅₀ being 69.30 µg/ml (Fig. 1). This inferred that the extracts contain phytoconstituent have ability to donate hydrogen, and neutralize the odd electrons which is responsible for radical's reactivity. The rates of DPPH scavenging activity of ethanol extracts are probably due to the presence phenolic compounds. The DPPH data supports the finding of preliminary phytochemical study. It also confirms the ethanol extract of P. ostreatus acquire high content of phenolic compound. It inferred that extract can be used for the healing of radical related pathological damage.

Fig. 1: IC₅₀ values of ethanol extract of P. ostreatus

Table 3: Superoxide scavenging capacity of ethanol extract of P. ostreatus

Concentration (µg/ml)	Superoxide Scavenging %
Concentration (µg/ml)	Ethanol Extract	Ascorbic Acid
50	29.14±1.31	85.29±0.95
100	42.53±1.09	-
150	63.74±1.53	-
200	95.27±1.16	-
250	121.62±1.24	-
IC₅₀	107.05	_-

Values are mean ± SEM of six determinations

Fig. 2: IC₅₀ values of ethanol extract of P. ostreatus

Superoxide anion radical is one of the strongest reactive oxygen species among the free radicals that are generated. During superoxide scavenging study it was observed that ethanol extract moderately scavenged the superoxide anion radicals. The ethanol extract of P. ostreatus scavenged superoxide radical with the IC₅₀ values of 107.05 µg/ml (Fig. 2). It indicates the protective effect of ethanol extract on antioxidant enzyme SOD in mitochondria exposed to H₂O₂. The phenolic compound scavenged H₂O₂ by donating electron to H₂O₂, and reducing it to water. The finding implies that the extracts contain phenolic compound, and it imparts chief role in healing of diseases.

The ethanol extract of P. ostreatus was prepared for examination of the total phenolic content. The quantification of total phenolic content in ethanol extract was done with respect to the gallic acid, and result expressed as gallic acid equivalents. The ethanol extract of P. ostreatus exhibited 83.47±1.53 mg/100 gm of total polyphenol content. The polyphenolic compounds are extensively used in nutritional supplement to improve the physiological process of cells. The polyphenolic compounds are well known for its antioxidant properties.

CONCLUSIONS:

The findings of present study affirm that ethanol extract of P. ostreatus contain variety of secondary metabolites. These components can be effectively used to protect body from the free radical generated due to oxidative stress. Hence, P. ostreatus can be used as natural antioxidant; to protect body from various diseases generated by free radicals namely diabetes mellitus, cancer, liver diseases, renal failure and degenerative diseases etc.

ACKNOWLEDGMENT:

Authors wish to thank the AICTE, New Delhi, India for their financial support (File No. 8023/RID/RPS-22//2011-12).

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Received on 18.05.2014 Modified on 30.05.2014

Res. J. Pharmacology & P’dynamics. 6(3): July- Sept. 2014; Page 135-138

Preliminary Phytochemical Screening and In Vitro Antioxidant Properties of the Pleurotus ostreatus extract