Active Constituents And In Vitro Bioactivities Of The Mulberry Branch Bark Ethanolic Extract

In this experiment, we take the branch bark of lake mulberry NO.32 as experimental material, and used ethanol-aqueous solution as the extraction solvent, and got the ethanolic extracts from mulberry branch barker (MBBEE). We isolated and purified the MBBEE by means of organic solvent extraction and macroporous resin adsorption,and detected the contents of total flavonoids, total phenolics, total sugar, total tannic acid, free amino acids of MBBEE and its fractions, we explored their in vitro bioactivities, too. By the organic solvent extraction, we got fractions of petroleum ether, chloroform, ethyl acetate, n-butanol (n-BuOH) and the residue (Res, the remaining water). The n-BuOH and Res were the major fractions, and their inhibition rates (IC₅₀) of the scavenging activity of DPPH radical were 0.16 mg/mL and 0.10 mg/mL respectively. The n-BuOH fraction had the strongest tyrosinase inhibitory activity, the IC₅₀ value was 5.2μg/mL. While the Res fraction had the strongestα-glucosidase inhibitory activity, the IC₅₀ value was 2.5μg/mL. By the macroporous resin adsorption, we got four fractions: D101-P, D101-DW, D101-E30 and D101-E100, their yields were 44%, 34%, 21% and 2% respectively. And we assayed the contents of free saccharides, DNJ, quercetin, rutin and resveratrol in MBBEE and its D101-fractions by HPLC. The contents of free amino acids in D101-P and D101-DW were 14568μg/g, 17902μg/g, and the contents ofγ-Aba were 580μg/g, 518μg/g respectively. The mass ratios of fructose in D101-P and D101-DW were 177.30 mg/g, 129.15 mg/g, and the mass ratios of DNJ which hadα-glucosidase inhibitory activity were 1.9 mg/g, 0.76 mg/g, the contents of quercetin were 46.05 mg/g, 56.03 mg/g respectively. D101-P and D101-DW had strongerα-glucosidase inhibitory activity than MBBEE (the IC₅₀ value was 2.75μg/mL), the IC₅₀ values were 1.7μg/mL, 2.3μg/mL respectively. The highest content of saccharides in D101-E30 was rhamnose, and it was 233.95 mg/g; the contents of quercetin and rutin were 69.52 mg/g,62.19 mg/g, D101-E30 had stronger scavenging activity of DPPH radical and stronger tyrosinase inhibitory activity than MBBEE and other fractions, the IC₅₀ value was 142μg/mL, 0.25μg/mL respectively. We isolated and purified the basis compound from D101-E30 by half-preparative HPLC, by means of LC-MS, UV spectra, fluorescence emission spectra, 1H NMR and 13C NMR, we identified it as Oxyresveratrol-4-O-β-D-glucopyranosyl-3′-O-β-D-glucopyranoside (Mulberroside A), we also tested its in vitro bioactives. The results showed that the scavenging activity of DPPH radical and tyrosinase inhibitory activity mostly from mulberroside A, and its IC₅₀ values were 35.33μmol/L, 0.07 nmol/L respectively.The results show that the MBBEE consists of many bioactive substances including rutin, quercetin, DNJ, resveratrol andγ-Aba etc. About 80% of MBBEE is aqueous phase (D101-P and D101-DW), which has strongα-glucosidase inhibitory activity, while about 20% of MBBEE is alcohol aqueous phase, and it has strong tyrosinase inhibitory activity, and its bases is mulberroside A. MBBEE is not only used widely as hypoglycemic biomedicine for diabetes and its complications, but also has potential applicative worth in senior whitening cosmetics and so on.