Studies On The Synthesis Of Echinocystic Acid Derivatives And Their Lipases Inhibition Activities

Obesity is a global health problem that is primarily related to people's lifestyle disorders and lack of physical activity.Obesity is associated with a large number of chronic diseases and disabilities,such as hypertension,lipid abnormality,insulin resistance,diabetes and cancer.Lipase is an important target for anti-obesity drugs.It is a key enzyme for the hydrolysis of fat into glycerol and free fatty acids during fat digestion.Inhibition of lipase is one of the important methods for anti-obesity drug discovery.Fructus gleditsiae sinensis is a dry mature fruit of Gleditsia sinensis Lam.It mainly contains saponins,flavonoids,phenolic acids,polysaccharides and other ingredients,and has significant anti-inflammatory,anti-microbial,anti-tumor and other pharmacological effects.Echinocystic acid is one of the representative components of saponins in fructus gleditsiae sinensis.It has the functions of reducing blood lipid,decrease blood pressure and regulation of blood glucose.They have low bioavailability because of their ring system stability in the chemical structure,few the active sites,poor solubility and so on.Echinocystic acid was used as a lead compound to carry out an acetylation reaction at positions C-3 and C-16 in its structure.And then a series of amino acid methyl esters was introduceated at its C-28 position to obtain a series of compounds,in order to improve the in vivo bioavailability of echinocystic acid,thereby increasing its pharmacological activity.In this paper,the echinocystic acid was extracted and purified from the fructus gleditsiae sinensis.Then,the saponin was used as the lead compound,and the lipase was used as the target to carry out the derivative synthesis.The oil-water partition coefficient of each derivative was determined and discussed in depth.The structure-activity relationship of each derivative inhibiting lipase activity.The Auto Dock 4.2 software was used to perform virtual molecular docking of echinocystic acid and its derivatives,providing scientific basis and research ideas for the development of new drugs for the treatment of obesity.Taking the fructus gleditsiae sinensis as the raw material,the total saponin of saponin in the saponin was extract with the concentration of ethanol at 75%,the amount of alcohol as 5 times of the medicinal material and the extraction time of 3 h.The obtained total saponin was further hydrolyzed by 2 mol/L hydrochloric acid in45%ethanol for 3 h,and hydrolyzed to obtain total sapogenin.Next,methanol was used as a dissolving agent,and total sapogenin was separated by ODS column to obtain echinocystic acid.The obtained echinocystic acid content was high and the purity can reach to no less than 98%.A series of derivatives were synthesized by echinocystic acid as a lead compound,and finally 21 new compounds which were not never reported before were synthesized.The oil-water partition coefficient of the echinocystic acid and its derivatives was determined by HPLC.The results showed that the pH value?1.2⁷.4?had no significant effect on the solubility of the compound to be tested and the oil-water partition coefficient.The oil-water partition coefficients of compounds 2¹1 were higher than that of echinocystic acid,while the oil-water partition coefficients of compounds 12²1 were lower than that of echinocystic acid.The oil-water partition coefficients of compounds 13,14,20,and 21 were significantly lower than those of echinocystic acid,so they were highly soluble and more favorable for activity screening experiments.Virtual molecular docking of echinocystic acid and its derivatives was done by Auto Dock 4.2 software.Docking results indicated that each derivative can interact with lipase,and the 28-position substitution enhances the stability of the degree of binding of the complex formed by the combination of the derivative and lipase.A screening method for lipase activity using PNPB as a substrate was established.Lipase inhibitors were assayed for the inhibition of lipase activity by echinocystic acid and its derivatives.The experimental results showed that the derivatives which was synthesized after the protection of the hydroxyl group in the echinocystic acid and then the introduction of the amino acid methyl ester in the carboxyl group had a good lipase-inhibiting activity.The IC50 values of the compounds 15,16,22,23,24were 1.05,1.55,1.25,1.13,1.43?g·mL-1,respectively,which was significantly higher than the positive drug orlistat(IC50=20.85?g·mL^-1)and the lead compound echinocystic acid(IC50=3.11?g·mL^-1).In summary,in this study,21 new compounds were prepared with echinocystic acid as the lead compound,and the systemic lipase inhibition activity of echinocystic acid was studied.The structure-activity relationship was carried out by Auto Dock 4.2software.the study.This paper can provide scientific basis and research ideas for the development of new anti-obesity drugs.