International Food Research Journal 18: 579-582 (2011

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Determination of total phenol, flavonoid and antioxidant activity of edible mushrooms Pleurotus florida and Pleurotus eous

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Mohamed Imran, M., Mohamed Mahroop Raja, M., Abdul Basith, J. and Asarudeen, A.
P.G. Department of Microbiology, Jamal Mohamed College (Autonomous),Tiruchirappalli – 620 020, Tamilnadu, India

Abstract: The antioxidant potential of two commonly used edible mushrooms Pleurotus florida and Pleurotus eous was estimated in this study. The total phenolic and flavonoid contents were determined as these compounds have profound antioxidant properties and antioxidant capacity was tested by phosphomolybdenum assay and metal chelating activity. The total phenolic content and total flavonoid content of the two mushroom extracts revealed that Pleurotus florida showed higher activity in both solvents. The total antioxidant activity was determined by using phosphomolybdenum assay where Pleurotus eous have higher activity. Among the two mushrooms, Pleurotus florida has higher chelating activity against ferrous ion when compared to Pleurotus eous. Thus the study revealed that the consumption of oyster mushrooms may enhance the immune power of our body against diseases due to free radicals. So it can be used as a dietary supplement along with other foods or as a drug itself. Keywords: Pleurotus florida, Pleurotus eous, antioxidant activity, phenol, flavonoid

Introduction Mushrooms are nutritionally functional food and a source of physiologically beneficial and nontoxic medicines. Mushrooms are known for rich proteinacious food. Mushroom consist about 75% proteins, so they are grouped under single cell proteins (SCP). The mushroom does not contain starch and it contain very low amount of sugar. Fat content of mushroom though low, are rich in linolic acid, an essential fatty acid. Mushroom does not contain cholesterol instead; ergo sterol is present which could be converted by the body into vitamin D. There are thousands of different mushroom species and about 700 species have significant pharmacological properties (Jeong et al., 2009). Pleurotus species are commonly called oyster mushrooms and contain about 40 species. Oyster mushrooms now rank second among the important cultivated mushrooms in the world. The oyster mushroom is one of the most suitable fungal organisms for producing protein rich food, from various agro wastes without composting. Like all other mushrooms oyster mushrooms are also rich in protein, vitamin and minerals. They do not contain
*Corresponding author. Email: moim25@gmail.com

cholesterol and fat but contain very little amount of carbohydrates, sugar and hence these mushrooms are safe for heart patients and diabetic patients. Oyster mushrooms are cellulose loving fungi and are growing naturally in the temperate and tropical forest on dead decaying wooden logs or sometimes on dying of deciduous or coniferous woods. It may also grow on decaying organic matter. Oyster mushrooms are rich in vitamins like vitamin C, vitamin B complex particularly Thiamin, Riboflavin, Niacin, Folic acid and vitamin B12 (Lakshmi et al., 2004) An antioxidant is a molecule capable of slowing or preventing the oxidation of other molecules. The free radicals produced by oxidation reactions start chain reactions that damage cells. Antioxidant terminates these chain reactions by removing free radical intermediates and inhibits other oxidation reactions by being oxidized themselves (Sies, 1997). Antioxidants play a major role in the prevention and treatment of a variety of diseases. The proposed mechanism by which antioxidants protects cells from oxidative stress is by scavenging free radicals, chelating catalytic metals and halting lipid per oxidation chain reactions. Antioxidants are also widely used as ingredients in dietary supplements in the hope of maintaining health and preventing
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Mohamed Imran, M., Mohamed Mahroop Raja, M., Abdul Basith, J. and Asarudeen, A.

diseases (Nikolova et al., 2007). Antioxidants used as food additives which provide protection against oxidative degradation of foods by free radicals. Antioxidants are classified as natural or synthetic. Examples of natural antioxidants are tocopherol and ascorbic acid while examples of synthetic antioxidants are Butylated Hydroxy Toluene (BHT), Butylated Hydroxy Anisole (BHA) and Ethoxyquin (Hilton, 1989). Materials and Methods Collection and preparation of spawns The spawns of oyster mushrooms Pleurotus florida and Pleurotus eous were collected and subcultured on potato dextrose agar plates for mycelial growth. The sorghum seeds were boiled and mixed with 20% by weight of calcium carbonate to prevent excess moisture and then the contents were bottled, autoclaved at 1210C, 15lbs for 15 min. The sterilized bottles were then cooled, inoculated with mycelial discs and kept for incubation at 23 – 280C for 20 days. After incubation, the mother spawns were subcultured in sorghum seeds as above to produce spawn ready for cultivation. Cultivation and harvesting of oyster mushrooms Steam sterilized paddy straw was used as a substrate for the cultivation. The substrate was filled in the bags at a height of 5 inches followed by addition of spawns on the sides as three to four layers. The substrate was compressed and made air tight. The spawned bags were incubated in a dark room for 18 days. During this period of incubation, the mycelium growth appears in white colour during which bags were transferred to the cropping room .Relative humidity was maintained between 85-90% by spraying water on the side walls and floors of the cropping room daily. After 15 -20 days of cultivation, the fruiting bodies developed and the mushrooms were harvested on maturity. Determination of total phenolic content Gallic acid in various concentrations (0-250 mg/l) was prepared in methanol: water (50:50 v/v) as standard. 0.5 ml of mushroom extracts was taken in test tubes and 5 ml of Folins-ciocalteau reagent and 4 ml of aqueous sodium carbonate were added to the tubes. The tubes were kept at room temperature for 15 min. The optical density was measured at 745 nm using spectrophotometer.

Determination of total flavonoid content Quercetin in various concentrations (12.5-100 μg/ml) was prepared in methanol. 0.5 ml of each methanol extracts (1:10) were taken in test tubes and 1.5 ml methanol, 0.1ml of 10% aluminium chloride, 0.1 ml of 1 M potassium acetate and 2.8 ml distilled water were added separately to each tubes. All the tubes were incubated at room temperature for 30 min optical density was measured at 415 nm by using spectrophotometer. Determination of total antioxidant activity by phosphomolybdenum assay Different concentrations of Butylated Hydroxy Toluene (BHT) (25-125 µg/ml) in distilled water were prepared. 0.3 ml of mushroom extracts were taken in test tubes. The reagent solution was prepared by mixing 10 ml of 0.6 M sulphuric acid, 10 ml of 28 mM sodium phosphate and 10 ml of 4 mM ammonium molybdate into a beaker and 3 ml reagent solution was added to all the tubes. 0.3 ml of methanol served as blank. All the tubes were incubated at 950C for 90 min. The tubes were cooled to room temperature and the optical density was measured at 695 nm using spectrophotometer. Results and Discussion The oyster mushrooms Pleurotus florida and Pleurotus eous were cultivated and the fruiting bodies were collected for the preparation of methanolic extracts. By using rotor evaporator 15 gm of extracts in powder form were obtained. The extracts were diluted in methanol at a concentration of 200 mg/ml and screened for its antioxidant properties. Several tests were carried out to determine the antioxidant capacity. The main tests include determination of total phenolic content, determination of total flavonoid content, and determination of total antioxidant capacity by phosphomolybdenum assay. The total phenolic content of Pleurotus florida and Pleurotus eous were estimated by the Folincioclteau method as 3.125 mg GAE/gm of dry extract and 2.725 mg GAE/gm of dry extract. The content of total phenols was higher for P. florida than that for P. eous extract. The total phenolic content per 1 g of dry extract was higher than that reported for garlic extract (0.98 mg GAE per gram of extract), (Bozin et al., 2008), but much lower than reported for content of total phenols per gram of asparagus extract (26.95 mg GAE/g extract) ( Zekovic et al., 2006). The final results were expressed in terms of gallic acid equivalents per gram of extracts (Table 1

International Food Research Journal 18: 579-582

Determination of total phenol, flavonoid and antioxidant activity of edible mushrooms Pleurotus florida and Pleurotus eous

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and Figure 1). The total flavonoid content of the selected mushrooms were estimated by using aluminium chloride colorimetric technique in terms of quercetin equivalents as 4.4 mg of quercetin equivalent per gm of extract, and 3.0 mg of quercetin equivalent per gram of extract for P. florida and P. eous respectively. The compounds such as flavonoids, which hold hydroxyls groups, are responsible for the radical scavenging activity in the plants (Das and Pereira, 1990; Younes, 1981). The methanolic extract of white mushroom P. florida shows more flavovoid content than that of pink mushroom P. eous. It has been acknowledged that flavonoids show significant antioxidant action on human health and fitness. The flavonoids act through scavenging or chelating process (Cook and Samman, 1996; Kessler et al., 2003) (Table 2 and Figure 2).

The antioxidant activity of the methanol extracts of mushrooms were measured spectrophotometrically through phosphomolybdenum assay and metal chelating activity. The total antioxidant capacity of both the mushroom extracts was estimated by phosphomolybdenum assay with the help of Butylated Hydroxy Toluene (BHT) as standard. Finally the total antioxidant capacities of the mushrooms were expressed in terms of BHT equivalents. The standard graph was plotted and the results were compared (Table 3 and Figure 3).The total antioxidant activity of the methanol extracts were found to be 220 µg equivalent of Butylated Hydroxy Toluene/gm of P. florida and 540 µg equivalent of Butylated Hydroxy Toluene/gm of P. eous in phosphomolybdenum assay. Here in this test only the pink mushroom P. eous shows more total antioxidant capacity than the white mushroom, P. florida (Aziz et al., 2007).

Table 1. Determination of total phenolic content Conc. of Galic acid ( µg/ml) Mean OD (745 nm) 20 0.214 40 0.374 60 0.705 80 0.967 100 1.322 109 1.330 120 1.404 125 1.550 140 1.673 Sample: Pleurotus florida 1.546 Pleurotus eous 1.331
The phenolic content of P. florida = 125 µg GAE/40mg dry mass of extract The phenolic content of P. eous = 109 µg GAE/40mg dry mass of extract Figure 1. Estimation of total phenolics (allic acid equivalents) using methanolic extracts of P. florida and P. eous

Table 2. Determination of total flavonoid content Conc. of Quercetin standard (µg/ml) 12.5 25 37.5 50 62.5 75 87.6 100 Sample: Pleurotus florida Pleurotus eous Mean OD (415 nm) 0.065 0.120 0.215 0.300 0.350 0.425 0.490 0.545 0.241 0.170
Figure 2. Estimation of total flavonoids (Quercetin equivalents) using methanolic extracts of P.florida and P. eous

Total flavanoid content of P. florida = 44 µg QE/10 mg dry mass of extract Total flavanoid content of P. eous = 30 µg QE/10 mg dry mass of extract

Table 3. Determination of total antioxidant capacity by phosphomolybdenum assay Conc. of BHT (µg/ml) 25 50 75 100 125 Pleurotus florida Pleurotus eous Mean OD (695 nm) 0.35 0.65 0.95 1.45 1.75 0.56 1.51

Sample:

Total antioxidant activity of P.florida = 44 µg BHTE/200 mg dry mass of extract Total antioxidant activity of P.eous = 108 µg BHTE/200 mg dry mass of extract Figure 3. Total antioxidant activity of methanolic extracts of P. florida and P. eous

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Acknowledgement We thank the Department of Microbiology, Jamal Mohamed College (Autonomous), Tiruchirappalli – 20, Tamilnadu, India for supporting us to carry out this work in a successful manner. References
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International Food Research Journal 18: 579-582