Semisynthesis And Performance Evaluation Of 7-Hydroxyethyl Sophoricoside

The Sophorae Fructus has rich resoures and a variety of pharmacological activities, especially the sophoricoside of it has weak estrogen-like effect and obvious curative effect in treatment of osteoporosis. But because of poor water solubility and low bioavailability, the clinical application of sophoricoside is limited. In order to improve the solubility, this paper first used a selective extraction method to obtain sophoricoside monomer, then used the method of chemical modification to synthesize 7-hydroxyethyl sophoricoside, and last used the method of physical and chemical and animal experiments to study the performance of 7-hydroxyethyl sophoricoside. The main results were as follows:(1) The preparation of sophoricosideThe optimized extraction process with the index of yield and content of sophoricoside, was screened by the single factor and orthogonal experiments. The optimum conditions of sophoricoside extraction process were as follows: 70% ethanol, extraction time 2.5 h, extraction temperature 80℃, liquid-solid ratio 7:1 (mL/g); the yield and content of sophoricoside was 2% and 76% respectively.The purified process used the method of resin purification-solvent recrystallization. Resin screening experiments with the index of the adsorption rate and desorption rate, the results showed that DIOlwas the best among HYP-501, HPD-100, D101, D201, AB-8 five kinds of macroporous resins. The optimum purification parameters of D101 resin were as follows:sample concentration 2 mg/mL, sample flow rate 2 BV/h,80% ethanol elution, elution flow rate 2 BV/h; the purity of intermediate substance reached more than 85%. Then, intermediate substance was crystallined in the ethanol. By measuring the physical, chemical properties and IR,’H-NMR,13C-NMR, the crystallined substance accorded with genistein-4’-O-β-D-glucoside, the purity reached 95.3%by HPLC detection.(2) The synthesis of 7-hydroxyethyl sophoricosideBased on the Williamson reaction theory and column chromatography separation methods, by the single factor experiments, the 7-hydroxyethyl sophoricosides were synthesized with 90% sophoricoside as raw materials, 2-chloride ethanol as hydroxyethylation reagent, purified water as solvent and sodium hydroxide as catalyst. By the response surface experiments, the optimum conditions of the synthesis process of 7-hydroxyethyl sophoricoside were as follows:sodium hydroxide dosage 3.2%(w/v relative to solvent), purified water 1:50 (w/v relative to sophoricoside), synthesis temperature 80℃, synthesis time 4 h. The yield was 43.34%. The crude products were purified by silica gel column chromatography using ethylacetate:methanol=6:1 as elution reagent. Finally the pale yellow solid was obtained by rotating and evaporating the elution whose Rf value was 0.45. By measuring the UV, IR,’H-NMR, 13C-NMR, the pale yellow solid was compared with sophoricoside, it confirmed that the synthetic product was 7-hydroxyethyl sophoricoside.(3) Performance evaluation of 7-hydroxyethyl sophoricosideThe determination of solubility showed that the equilibrium solubility of sophoricoside and 7-hydroxyethyl sophoricoside were 17.74 mg/L and 20.35 mg/L at 37℃; the oil-water partition coefficient were 376.52 and 359.88.The determination of scavenging capacity on free radicals showed that from 2μg/mL to 15 μg/mL, the scavenging capacity of sophoricoside and 7-hydroxyethyl sophoricoside were positively related to their concentrations. When the sample concentration was 15μg/mL, the scavenging capacity of sophoricoside on ·O2-, DPPH,·OH were 25.18%,17.44% and 27.53% respectively and that of 7-dihydroxyethyl sophoricoside were 28.32%,20.32% and 28.98% respectively.The swelling experiments of mice showed that when the dose was 4 mg/(kg·day) by intragastric gavage, the inhibitory rates of sophoricoside on topical edema induced by oxylene on mice ears and formaldehyde on mice foot were 22.98% and 19.61% respectively and that of 7-hydroxyethyl sophoricoside were 26.93% and 22.13% respectively.The experiments of weak estrogen-like effect to mice showed that when the dose was 4 mg/(kg·day) by intragastric gavage, the uterine coefficient and ovarian coefficient of sophoricoside groups were 0.180% and 0.096% respectively and that of 7-hydroxyethyl sophoricoside groups were 0.195% and 0.106% respectively.Thus the conclusion was:the sophoricoside with more than 90% purity were obtained by ethanol reflux extraction and macroporous resin-ethanol recrystallization purification; 7-hydroxyethyl sophoricoside with more than 90% purity were obtained with 2-chloroethanol as alkylating agent and silica gel column chromatography as purified method; by modifying the structure, the equilibrium solubility of 7-hydroxyethyl sophoricoside increased by 14.71%, the oil-water partition coefficient declined by 9.73%, the antioxidant activity, anti-swelling effect and weak estrogen-like effects increased by 16.51%, 17.19% and 10.42%respectively. These results provide an experimental basis for the search for new drugs to treat osteoporosis.