Crude ethanolic extraction 5 grams of air dried ground rhizome were macerated and periodically stirred in 50 ml of absolute ethanol for 48 hours. The suspension was filtered by way of Whatman No. 4 filter paper and centrifuged at five,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 ahead of testing along with the solvent was applied as a negative handle. Fractionated solvent extraction 5 grams of air dried ground rhizome have been macerated and periodically stirred in 50 ml of hexane for 48 hours. The suspension was filtered as a result of the filter paper and centrifuged at 5,000 rpm for 15 minutes. The super natant was air dried to acquire 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 hours. The ethyl acetate sus pension was filtered by means of the filter paper, centrifuged at five,000 rpm for 15 minutes, now and air dried to acquire 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 hrs. The methanol suspension was filtered by means of the filter paper, centrifuged at 5,000 rpm for 15 minutes, and air dried to obtain the methanol soluble fraction. Every solvent fraction was reconstituted in an appropri ate car, DMSO or ethanol, before testing. Phenolic extraction Phenolic extraction was carried out by using acidic hy drolysis system with some modifications.
Briefly, two hundred milliliters of 70% methanol were extra to a beaker containing 10 grams of ground rhizome. The mixture was stirred for 2 hours at room temperature after which filtered via the filter paper. The filtrate was evaporated to 60 ml by a rotary evaporator. The remaining filtrate was extra with 50 ml of 2 M NaOH and stirred continuously nearly for 12 hrs at space tempera ture. The mixture was centrifuged at 1,700 g for 20 mi nutes after which filtered by means of the filter paper. The supernatant was repeatedly extracted three times with 80 ml of diethyl ether, in which the aqueous phase was collected as well as diethyl ether phase was discarded. The aqueous phase was adjusted to pH 1. five by 10 M HCl and filtered via the filter paper.
The filtrate was even further extracted by 80 ml of diethyl ether for three times, in which the portion of the diethyl ether was collected. The pooled diethyl ether phase was dehydrated with sodium sulphate anhydrous after which filtered by means of the filter paper. The filtrate was evaporated to 5 ml utilizing a rotary evaporator and ultimately evaporated to dry ness beneath a gentle stream of nitrogen. Determination of complete phenolic material Complete phenolic written content in ethanolic crude extract was established by the Folin Ciocalteu method as described previously. Gallic acid was employed since the conventional and the outcome was calculated as ug Gallic Acid Equivalent per mg dry weight with the extract. HPLC analysis of phenolic rich extract The identification of personal phenolic acids in phenolic rich extract prepared by phenolic extraction as described over was carried out making use of a Waters HPLC technique, determined by matching spectrum and retention occasions of phenolic acid specifications.
The phenolic acid requirements utilized had been 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 procedure consisted of a Waters 600E Multisolvent Delivery method, Waters In Line degasser AF, a Rheodyne injector with sample loop of twenty ul, plus a Waters 2669 photodiode array detector. Empower application was employed for information acquisition. A Waters method column C18 coupled to a guard column was used. The temperature with the column was 25 C as well as the movement fee of mobile phase was 1. 0 ml minute.