Improving The Content Of Lignan Glycosides In Forsythia Suspensa Leaves By Biotransformation And The Quality Standard

Forsythia suspensa leaves are the leaves of Forsythia suspensa(Thunb.)Vahl,cool in nature,bitter in taste,have the effects of clearing heat,detoxifying the body,lowering fire and opening the bowels.F.suspensa leaves contain compounds such as lignans,phenylethanoid glycosides and flavonoids,of which lignans are one of the main pharmacodynamic components.Studies have shown that lignan aglycones have better pharmacological activity than lignan glycosides in liver protection,anti-inflammatory and anti-tumor aspects.Previous research by our group has found that F.suspensa leaves may contain enzymes that convert phillyrin into phillygenin.The lignan aglycones such as phillygenin,pinoresinol and matairesinol were increased by natural fermentation of F.suspensa stems.In order to improve the content of lignan aglycones in F.suspensa leaves and develop the medicinal value,the technological conditions for improving the content of lignan aglycones in F.suspensa leaves by biotransformation method were explored,the quality control standards of F.suspensa leaves and fermented F.suspensa leaves were established,and the changes of biological activity before and after F.suspensa leaves fermentation were compared.The details are as follows :1 A HPLC method was established for determination of six components of forsythoside A,phillyrin,phillygenin,pinoresinol,epipinoresinol,and pinoresinol monomethyl ether.The method investigated the linear relationship and validated the methodology.The results showed that the precision,repeatability and stability of the method were within the specified range,and the accuracy of method recovery was high.In addition,the contents of impurities,moisture,total ash and alcohol-soluble extract in 17 batches of F.suspensa leaves were investigated,supplemented phillygenin as a characteristic component for content determination.The quality control standard of F.suspensa leaves was established.The impurities,moisture and total ash should not exceed3.0%,9.0% and 8.0%,and the alcohol-soluble extract should not less than 28.0%.In the content determination,it was tentatively determined that the content of forsythoside A was not less than 6.0%,phillyrin was not less than 3.0%,and phillygenin was not less than0.1%.The characteristic chromatography of F.suspensa leaves was established,and four characteristic peaks were selected,which were forsythiaside A,rutin,phillyrin and phillygenin.2 In order to investigate the reasons for biotransformation of lignan glycosides into lignan glycosides in F.suspensa leaves,the transformation by endogenous crude enzyme solution of F.suspensa leaves was investigated.The results showed that the enzyme solution could hydrolyze lignan glycosides.According to the content of lignan aglycones,the optimum fermentation process of fresh or dried F.suspensa leaves was investigated.Studies have shown that the best fermentation conditions for fresh leaves were fermentation temperature 55℃,fermentation humidity 55% and fermentation time 50 minutes.The moisture content of dried F.suspensa was 60%,the fermentation temperature was 50℃,the fermentation humidity was 75%,and the fermentation time was 50 minutes.The chromatograms showed that the contents of four lignan aglycones,including phillygenin,pinoresinol,epipinoresinol,and pinoresinol monomethyl ether were significantly increased after fermentation.3 To establish the quality standard of fermented F.suspensa leaves,ten batches of fermented F.suspensa leaves were identified by TLC,impurities,moisture,total ash,alcohol-soluble extract and content determination.When petroleum ether: acetone = 7:6dichloromethane: methanol: formic acid = 7:3: 0.1 was developed twice,the separation effect was good and it could be used for the identification of fermented F.suspensa leaves.It is tentatively determined that the impurities,moisture and total ash of fermented F.suspensa leaves shall not exceed 9.0%,and the alcohol-soluble leachables shall not exceed25.0%,phillygenin shall not be less than 2.6%,pinoresinol shall not be less than 0.6%,epipinoresinol shall not be less than 0.2%,and the pinoresinol monomethyl ether shall not be less than 0.1%.4 The extraction conditions and chromatographic conditions of fermented F.suspensa leaves were optimized,and the HPLC characteristic chromatograms of fermented F.suspensa leaves were established.Forsythiaside A,rutin,phillyrin,phillygenin,pinoresinol,epipinoresinol,and pinoresinol monomethyl ether were selected as characteristic peaks of fermented F.suspensa leaves.The method has good precision,reproducibility and stability,and is suitable for the characteristic chromatograms of fermented F.suspensa leaves.5 The activity changes of F.suspensa leaves before and after fermentation were compared in lower blood sugar,liver protection and anti-inflammatory aspects.The inhibitory effects of F.suspensa leaves and fermented F.suspensa leaves on α-glucosidase and α-amylase,the LPS-induced NO in the supernatant of RAW264.7 cells;and the protective effects on APAP-induced drug-induced liver injury in HepG2 cells were investigated,respectively.Studies have shown that fermentation could increase the inhibitory effect of F.suspensa leaves on α-glucosidase and α-amylase,effectively inhibit NO produced by LPS-induced RAW 264.7 cells,and alleviate APAP-induced hepatotoxicity.