Screening And Preparation Of Anti-inflammatory Active Components From Fermented Forsythia Suspensa Leaves

The leaves of Forsythia suspensa are the leaves of Forsythia suspensa（Thunb.）Vah L,a plant of the Oleaceae family.They have the effects of clearing heat,detoxifying,protecting liver,and lowering lipid.Originally recorded in the Compendium of Materia Medica,"Stems and leaves are bitter,smooth,non-toxic,and can treat heat accumulation in the heart and lungs".In Shanxi,Henan,and other places of China,there were traditional habit of picking forsythia leaves and making them into tea for health care.Studies at home and abroad have shown that the main chemical components of the leaves of Forsythia suspensa are terpenes,lignans,flavonoids,and phenylethanol glycosides,which have pharmacological effects such as anti-inflammatory,antibacterial,and liver protection.The previous research by the team found that natural fermentation of forsythia suspensa leaves can efficiently convert lignan glycosides such as forsythin into glycosides such as forsythin.Moreover,compared with the leaves of Forsythia suspensa,the chemical composition of fermented Forsythia suspensa leaves has undergone significant changes and has better anti-inflammatory activity.Further,the ethanol extract from fermented forsythia suspensa leaves was preliminarily separated and screened for activity using a macroporous resin column.It was found that the site rich in lignan glycosides such as forsythia suspensa had good anti-inflammatory activity.Therefore,it is necessary to study the preparation process of the anti-inflammatory active fraction from fermented forsythia suspensa leaves.In this paper,fermented forsythia suspensa leaves were used as the research object to further screen its anti-inflammatory active sites;The preparation process of anti-inflammatory active sites was studied,and pilot scale experiments were conducted to verify the process parameters;According to the content of the extract standard in the"Chinese Pharmacopoeia",a preliminary quality standard for the extract of the effective part was established,and a preliminary stability study was conducted;Finally,using network pharmacology and molecular docking technology to preliminarily explore its anti-inflammatory mechanism.The main research contents are as follows:1.Screening of anti-inflammatory active sitesUsing fermented forsythia suspensa leaves as raw materials,after extraction and separation,80%ethanol crude extract,40%ethanol elution site,and 90%ethanol elution site were obtained.Using the LPS induced macrophage anti-inflammatory screening model,the anti-inflammatory effects of the 80%ethanol crude extract,40%ethanol eluting site,and 90%ethanol eluting site of fermented forsythia suspensa leaves were evaluated.The IC₅₀ value of the crude extract was 116.4μg/mL,the IC₅₀ value of 40%of the extract was128.5μg/mL,and the IC₅₀ value of 90%of the extract was 53.99μg/mL.The results showed that the 90%ethanol elution site rich in lignan aglycones such as forsythin had good anti-inflammatory effects.2.Study on the preparation process of anti-inflammatory active fraction rich in lignan aglyconeFermented Forsythia suspensa leaves 80%ethanol extract,concentrated under reduced pressure to recover ethanol until the alcohol content in the extract is about 40%.Then,D101macroporous adsorption resin was used as a purification and separation material,and column chromatography was used for separation.40%and 90%ethanol were used as eluents,followed by sequential elution,combined with the extract,and concentrated for drying.Using the loss rate,recovery rate,and content of four lignan aglycones,such as Phillygenin,Pinoresinol,Epipinoresinol and Pinoresinol monomethyl ether as evaluation indicators,column chromatographic separation parameters such as sample loading concentration,sample loading amount,column diameter to height ratio,and amount of eluting solvent were investigated and selected to determine the process conditions for column separation of anti-inflammatory active sites.Preparation process parameters determined through research（4.5×22.5cm）is:the alcohol content of the sample is about40%,and the sample concentration（equivalent to the amount containing fermented forsythia leaves）is 200 mg/mL;When the column diameter to height ratio is 1:5,the sample loading amount is 0.7BV（column volume）;The elution conditions for column chromatography are as follows:elute 5 BV with 40%ethanol first,and then elute 14 BV with 90%ethanol,with a flow rate of 5 BV/h.Under the above conditions,conduct three pilot scale up parallel validation experiments（8.5×42.5cm）。The results showed that using the above preparation and separation process,the total recovery rate of the four lignan aglycones was about 74.3%,and the total content of the four lignan aglycones in the extract was about 53.54%.3.Study on Quality Control Standards and Preliminary Stability of Extracts from Anti-inflammatory Active PartsReferring to the research content on the quality standards of relevant traditional Chinese medicine extracts contained in the"Chinese Pharmacopoeia",a preliminary study was conducted on the quality standards of the extracts from the anti-inflammatory active sites of fermented forsythia suspensa leaves.Establish control standards for the properties,inspection,and identification of the extract;HPLC characteristic chromatograms of the extracts and four methods for the determination of lignan aglycones were established;Draft preliminary quality standards have been drafted.Finally,the factors affecting the stability of the extract were preliminarily investigated through an influencing factor test.The results showed that the properties of the extract were relatively stable,and factors such as high temperature,high humidity,and light did not affect the active site extract.4.Preliminary study on anti-inflammatory mechanism of extracts from anti-inflammatory active sitesUsing the Siwws target prediction database,201 component targets were searched for active ingredients such as Phillygenin,Pinoresinol,Epipinoresinol and Pinoresinol monomethyl ether.Using the disease database,1038 inflammation related targets were obtained.After the intersection of component targets and disease targets,a disease component target network is constructed,and the key components of Phillygenin,Pinoresinol,Epipinoresinol and Pinoresinol monomethyl ether are selected and ranked through the Degree value.The PPI network screened five core targets,including CASP3,ERBB2,TLR4,HIF1A,and MTOR.GO enrichment analysis and KEGG pathway analysis indicate that the therapeutic effects of active sites on inflammation mainly involve biological processes such as regulation of inflammatory reactions,inflammatory reactions,responses to bacterial derived molecules,leukocyte activation,phosphatidylinositol 3-kinase complexes IA,and signal pathways such as PI3K-Akt signaling pathway,chemokine signaling pathway,Th17 cell differentiation pathway,NF-kappa B signaling pathway,and MAPK signaling pathway.Molecular docking results showed that the binding of rosin,Phillygenin,Pinoresinol,and Pinoresinol monomethyl ether to inflammation related targets（CASP3,ERBB2,TLR4,HIF1A,and MTOR）was good,indicating that the above three components may be effective components for the treatment of inflammation in the active site.