QUALITATIVE PHYTOCHEMICAL SCREENING OF VARIOUS SOLVENT EXTRACTS OF CALOCYBE INDICA, MILKY MUSHROOM

The present study investigates the presence of phytochemical compounds in thecold&hot aqueous extract, ethanol extract and methanol extract of Calocybeindica , milky mushroom. The mushroom spawn was prepared from the procured culture with the white sorghum grains as the substrate and well matured spawn were used for the production of mushrooms. The paddy straw was used as a substrate for the growth of the mushrooms. The matured spawn was inoculated in the processed substrate and maintained with the optimum conditions. The well grown mushrooms were harvested and shade dried. The dried mushrooms were powdered and cold&hot aqueous extract, ethanol extract and methanol extract was prepared and used for the qualitative phytochemical analysis. The phytochemicals such as carbohydrates, proteins, alkaloids, flavonoids, phenols, saponins etc. were screened for their presence and the results shown that the four different extractsof Calocybeindica , milky mushroom possess various phytochemicals at considerable proportion which can be said as a source of phytochemicals for various pharmaceutical applications. Potato dextrose agar medium at room temperature as slants and in pertriplates 14 .


ISSN: 2320-5407
Int. J. Adv. Res. 8(05), 573-577 574 the mushroom extracts are target specific and have been regularly tested in the clinical trials by the scientists and also by the pharmaceutical companies for developing functional foods. Because making such products from the fruit bodies, mycelia or its extracts have pharmacological benefits helping in the early intervention of sub-healthy states in humans which prevents the consequences of life threatening diseases 5 .
Edible mushrooms are cultured in media that usually consist of lignocellulosic agricultural wastes such as crop straw 6 . The organic solid waste remaining after cultivation of edible mushrooms is the spent mushroom substrate (SMS) 7 . SMS can be used as compost, as animal feed, to promote health of animals, and to produce packaging and construction materials, biofuels, and enzymes 8 . Hence, the mushroom cultivation is a value added process to convert these materials and represents one of the most efficient biological ways by which these residues can be recycled 9 .
Calocybeindica is a lignocellulolytic mushroom, which requires a temperature of 30-35°C and relative humidity of 80 to 90 % for good growth. Therefore, it is an ideal candidate for hot weather cultivation when no other mushroom excepting Volvariellaspecies can grow. It has a robust sporophore, attractive colour, sustainable yield, delicious taste, unique texture and excellent shelf life as compared to oyster or button mushroom 10 .In nature, milky white mushrooms are seen grown on humus rich soil in agricultural fields or along the roadside in tropical and subtropical parts of India, especially in the plains of Tamil Nadu (South Indian State) and in Rajasthan (located in the western edge of India) 11 . Milky white mushrooms are highly suitable for commercial production in humid tropical and subtropical regions of the world 12 . The six major constituents of mushrooms are water, proteins, carbohydrates, dietary fiber, fat, and ash 13 .
The present study was designed to analyse the presence of various metabolites in the aqueous extract of Calocybeindica in a qualitative manner.

Mushroom culture:
The culture of Calocybeindicawas procured from Vijaya Mushrooms, Coimbatore. The species was sub cultured and maintained in Potato dextrose agar medium at room temperature as slants and in pertriplates 14 .

Mushroom spawn production 15 :
The mushroom spawn was prepared on white sorghum grain. The mature grain procured from local market was well cleaned and boiled in water for 30 min. The boiled grain was mixed with 2% calcium carbonate. 300g of calcium carbonate mixed grain was filled in polypropylene bags of size 11inch x 5 inch and sterilized for 15 psi for one hour. The sterilized bags were cooled to room temperature and inoculated with the mushroom culture maintained in slants. The culture inoculated bags were kept undisturbed at room temperature for mycelium running. After mycelium running, matured spawn after 30 days were used for preparation of mushroom beds.

Mushroom Cultivation Technology 15
The well matured paddy straws were cut into 3-5 cm in length and soaked in water overnight. The soaked paddy straw was washed and sterilized for 45 min in steam and shade dried. The shade dried paddy straw wasfilled in polypropylene bags with the matured mushroom spawn in alternate layers. The filled bags were hanged in the mushroom cultivation chamber for spawn running and growth of mushrooms. The temperature of 25 -30°C and humidity of 80% is maintained in the chamber. After the complete mycelium spreading, the beds were cut into the two equal halves and casing soil prepared was applied on the beds forthickness of 2.5 cm and the beds were placed in the underground chamber for the growth of mushrooms. The temperature in the underground chamber was maintained at a range of 30 -35 °C and a humidity of 80 -85 %. Water is sprayed on the beds at periodic intervals. The well grown mushrooms were harvested and processed for further analysis.

Preparation of the extract:
The shade dried mushrooms were grinded to a coarse fine powder and stored for further use. Varioussolvent extracts such ascold&hot aqueous extract, ethanol extract and methanol extract was prepared by following the standardized procedure and used for the qualitative analysis of the various metabolites present in each solvent extract.

Results and Discussion:-Preparation of mushroom spawn and Mushroom substrate beds:
The mushroom spawn was prepared from the culture of Calocybeindica and used for the making of mushroom substrate beds. The well matured spawn was used as inoculum for the preparation of the beds. The well grown mushrooms were harvested, dried and various solvent extracts were prepared and subjected to phytochemical analysis.

Qualitative phytochemical analysis:
The qualitative phytochemical analysis of the cold&hot aqueous extracts, ethanol and methanol extracts revealed the presence of various metabolites such as alkaloids, flavonoids, saponins, tannins, phenol, anthroquinone, carboxylic acids, coumarines, quinone, thiols, terpenoids, triterpenoids, cardiac glycosides, gums and mucilages, carbohydrates and proteins.   The results recorded in the table.1 showed that most of the phytochemicals screened qualitatively were found to be present in all the four different extracts made. Alkaloids, flavonoids, saponins, tannins, phenols, coumarins, quinones, thiols, terpenoids, cardiac glycosides, proteins and steroids were found to be present in all the four different extracts which showed the abundant presence of the phytochemical in the mushroom. Each test for the phytochemical revealed the less presence, moderate presence and more presence of the compounds which is depicted in the above table. On the whole, the mushroom was found to possess most of the important biochemical metabolites that have therapeutic and pharmaceutic applications. The presence of these phytochemicals can be attributed to the pharmacologic and therapeutic properties of the Calocybeindica.

S.No
Free radicals cause several disorders, including diabetes, and the agents that scavenge free radicals may have great potential in ameliorating these diseases 17 .Phenolics have antioxidative, antidiabetic, anticarcinogenic, antimicrobial, antiallergic, antimutageneic, and antiinflammatory activities 18,19 . Tannins have been documented to retain functional and severehaemostatic characteristics that facilitates the healing of woundand enhance irritated mucus membrane and also impede the development of bacteria 20 . Saponins is very important in the treatment of cough and inthe controlling of soreness of the upper respirational region.Furthermore, plant based saponins serve as heart tonic naturallyand have been documented to prevent diabetics and hinder thegrowth of fungi 21 . The importance of flavonoids lies in the fact that it controls andprevent tissue damage due to the presence of triggered, radical or singlet oxygen species 22 . The coumarins are important because of their biotic characteristics.They possess physiologic functions, and inhibits bacterial andcancer growth. Coumarin together with some of its derivativesand are known probable inhibitors of cell propagation in several carcinoma cell shapes 23 . Controlled intake of cardiac glycosides, helps inarresting cardiac arrhythmia and give strength to a weak heart, thus helping the heart perform more efficiently 24 .

Conclusion:-
The mushrooms were cultivated and analysed for the presence of various phytochemicals. The present study revealed the presence of various phytochemicals in the four different solvent extracts such as cold aqueous, hot aqueous, ethanol and methanol. Based on the results of the qualitative phytochemical analysis, it is inference that, Calocybeindica may harbour several phytochemicals that can be exploited for the various medical and disease treating process through pharmaceutical and therapeutic properties of the compounds.