Crude ethanolic extraction 5 grams of air dried ground rhizome were macerated and periodically stirred in 50 ml of absolute ethanol for 48 hrs. The suspension was filtered as a result of Whatman No. four filter paper and centrifuged at 5,000 rpm Inhibitors,Modulators,Libraries for 15 minutes. The supernatant was air dried to yield an ethanolic crude extract. The residue was reconstituted in dimethyl sulfoxide or ethanol in advance of testing and also the solvent was used as a unfavorable manage. Fractionated solvent extraction Five grams of air dried ground rhizome have been macerated and periodically stirred in 50 ml of hexane for 48 hours. The suspension was filtered via the filter paper and centrifuged at 5,000 rpm for 15 minutes. The super natant was air dried to get the hexane soluble frac tion.
The precipitate remaining from hexane extraction was dispersed, macerated and periodically stirred in 50 mL of ethyl acetate for 48 hrs. The ethyl acetate sus pension was filtered by the filter paper, centrifuged at 5,000 rpm for 15 minutes, definitely and air dried to get the ethyl acetate soluble fraction. The precipitate remaining from ethyl acetate extraction was dispersed, macerated and periodically stirred in 50 ml of methanol for 48 hours. The methanol suspension was filtered through the filter paper, centrifuged at five,000 rpm for 15 minutes, and air dried to acquire the methanol soluble fraction. Each and every solvent fraction was reconstituted in an appropri ate vehicle, DMSO or ethanol, in advance of testing. Phenolic extraction Phenolic extraction was carried out through the use of acidic hy drolysis method with some modifications.
Briefly, two hundred milliliters of 70% methanol have been extra to a beaker containing 10 grams of ground rhizome. The mixture was stirred for 2 hrs at area temperature and then filtered as a result of the filter paper. The filtrate was evaporated to 60 ml by a rotary evaporator. The remaining filtrate was added with 50 ml of two M NaOH and stirred continuously those for twelve hrs at space tempera ture. The mixture was centrifuged at 1,700 g for twenty mi nutes and after that filtered through the filter paper. The supernatant was repeatedly extracted 3 times with 80 ml of diethyl ether, in which the aqueous phase was collected as well as the diethyl ether phase was discarded. The aqueous phase was adjusted to pH 1. 5 by ten M HCl and filtered by the filter paper.
The filtrate was additional extracted by 80 ml of diethyl ether for three times, during which the portion from the diethyl ether was collected. The pooled diethyl ether phase was dehydrated with sodium sulphate anhydrous after which filtered via the filter paper. The filtrate was evaporated to five ml using a rotary evaporator and lastly evaporated to dry ness under a gentle stream of nitrogen. Determination of total phenolic written content Total phenolic content material in ethanolic crude extract was established by the Folin Ciocalteu strategy as described previously. Gallic acid was utilised because the typical and the result was calculated as ug Gallic Acid Equivalent per mg dry bodyweight on the extract. HPLC examination of phenolic rich extract The identification of personal phenolic acids in phenolic rich extract ready by phenolic extraction as described above was carried out making use of a Waters HPLC procedure, dependant on matching spectrum and retention instances of phenolic acid requirements.
The phenolic acid standards utilized were gallic acid, protocatechuic acid, p hydroxybenzoic acid, vanillic acid, caffeic acid, syringic acid, m hydroxy benzaldehyde, p coumaric acid, ferulic acid, and sinapinic acid. The HPLC program consisted of a Waters 600E Multisolvent Delivery procedure, Waters In Line degasser AF, a Rheodyne injector with sample loop of 20 ul, and also a Waters 2669 photodiode array detector. Empower software program was made use of for data acquisition. A Waters method column C18 coupled to a guard column was employed. The temperature from the column was 25 C plus the flow fee of mobile phase was one. 0 ml minute.