For storage, the cells had been frozen at 70 C liquid nitro gen in complete medium supplemented with 5% di methyl sulfoxide as a cryoprotectant. Extraction of P. giganteus fruiting bodies The fresh fruiting bodies had been sliced, weighed and freeze dried for 1 2 days. The freeze dried fruiting bodies were then ground working with a blender. The resulting dried powder was weighed and stored in 4 8 C. Aqueous extraction system was according to Eik et al. Briefly, the freeze dried powder was soaked in distilled water and was left overnight at space temperature and 200 rpm in a shaker. The combine ture was double boiled in water bath for 30 min and fil tered soon after cooling. The resulting aqueous extract was freeze dried and kept at selleck inhibitor 40 C prior to use.
For ethanol extraction, the freeze dried powder was soaked in 95% ethanol at space temperature for three days along with the procedure was repeated three times. The ethanol solvent was evaporated employing a rotary evaporator to offer a brownish vis cous extract. Dietary composition of freeze dried fruiting bodies of P. giganteus Fifty grams sample of P. giganteus fruiting selleck chemicals bodies was sent to Consolidated Laboratory Sdn. Bhd. for nutri tional analysis. Cell viability and cytotoxicity assay Cell viability and proliferation was determined by MTT assay. Around twelve,000 cells per properly were seeded on a 96 properly plate and incubated at 37 C above night inside a humidified setting of 5% CO2 and 95% air. Fresh medium have been then replaced plus the cells had been exposed to 0 to 1000 ug ml of aqueous or ethanolic ex tract of P. giganteus for 48 hours.
Subsequently, 20 ul of sterilized MTT in phosphate buffered saline buffer was spiked into every well and incubated at 37 C for 4 hours. The supernatant was then meticulously removed, and 200 ul of dimethyl sulfoxide was added into each and every nicely to dissolve the MTT formazan in the bottom of the wells. Soon after 15 min, the absorbance at 540 nm with 690 nm as back ground absorbance was measured with an ELISA micro plate reader. The complete development medium was the blank, and cells incubated in medium only devoid of mushroom extracts had been denoted as good handle. Neurite outgrowth stimulation action Neurite outgrowth stimulation assay was according to Eik et al. with some modifications. The cells had been seeded within a 6 nicely plate at an initial density of 5,000 cells per properly in two ml complete development medium with distinctive concentrations of aqueous and ethanolic mushroom extracts. For freeze dried aqueous extract, a stock solu tion of ten mg ml was prepared freshly each time before assay. The stock remedy was then diluted five times in sterile distilled water to last concentrations ranging from 5 a hundred ug ml. For ethanolic extract, ten mg ml of stock option in DMSO was ready freshly.