Study On The Liver Protection And Chemical Constituents Of Ethanol Extract Of Angelicae Pubescentis Radix

Angelicae Pubescentis Radix（AP）is the dried root of Angelica pubescens Maxim.F.biserrata Shan et Yuan which was first recored in Shennong Ben Cao Jing the oldest pharmacopeia of traditional Chinese medicine,and is mainly distributed in Sichuan,Hubei,Anhui in China.It has been planted in large scale in Wuxi and Wushan,Chongqing.Modern research shows that AP mainly contains coumarins,volatile oils,organic acids.Coumarin components are the main active components in this herb.AP,one of the important traditional Chinese medicines（TCM）to relieve pain,dispel wind and dampness clinically has a wide range of pharmacological activities such as anti-inflammatory,analgesic,and antioxidant.To our knowledge,there are few research have been conducted to study the liver protective effect of AP.Thus,in the present study,the liver protective effect of AP was evaluated on carbon tetrachloride（CCl₄）induced mice,and then the separation,purification and identification of chemical component study were performed in order to locate the active components.The research may provide theoretical support for the development and utilization of AP in the future.The model of acute liver injury caused by the intraperitoneal injection of CCl₄in mice.The mice were randomly divided into 6 groups:blank group,model group,low/middle/high dose of ethanol extract of AP groups,silybin group.After continuous intragastric administration of assigned drugs for one week,mice except in control group were received0.2%CCl₄by intraperitoneal injection to induce acute liver injury.The pathological change were explored by H&E staining.The levels of ALT,AST,SOD,GSH in serum and TNF-α,IL-1β,IL-6 in liver tissue of mice was detected by ELISA kit.The expression of TLR4,My D88,p-p65,p-IκB,Nrf2,HO-1 proteins were measured by WB.The results showed that the extract relieved the injury state of liver cells,reduced the content of ALT and AST,enhanced the level of GSH and SOD,reduced the content of TNF-α,IL-1βand IL-6,enhanced the expression of Nrf2 and HO-1 protein,reduce the expression of TLR4,My D88,p-p65 and p-IκB protein.As many as 36 compounds including 26 coumarins,5 organic acids,and 3 other components have been isolated and identified from the ethanol extract of AP by the compreshensive application of chromatography and spectroscopy technology including silica gel,Sephadex LH20,semi-preparative HPLC and physicochemical properties and spectroscopic techniques（NMR,IR,UV,CD,HR-ESI-MS,etc.）.The name of the isolated compounds are as follows:Angelol A-3’-β-D-glucose（1）,Angelmanetin（2）,Angelmarin-4’-β-D-glucose（3）,Osthole（4）,Isoimperatorin（5）,Umbelliferone（6）,Columbianetin（7）,Columbianetina cetate（8）,Imperatorin（9）,Bisabolangelone（10）,Columbianadin（11）,Angelol A（12）,Angelica B（13）,Angelica C（14）,Angelica D（15）,Xanthotoxin（16）,Isopimpinellin（17）,Peucedanol（18）,Angelitriol（19）,（E）-suberenol（20）,Meranzin Hydrate（21）,Angelica G（22）,Angelica K（23）,Angelica L（24）,1-（β-D-ribofuranosyl）-l H-1,2,4-triazone（25）,p-hydroxycinnamic acid（26）,Uracil（27）,Columbianetin-β-D-glucopyranoside（28）,Vanillic Acid（29）,Peucedanol（30）,Tortuoside（31）,ligustiphenol（32）,Caffeic Acid（33）,linoleic acid（34）.Among them,compound 1,2 and 3 are new compounds;compounds25,26,31,and 32 are compounds discovered for the first time from the genus;compounds 27,29,and 33 are compounds discovered for the first time from AP.In summary,the ethanol extract of AP may play a hepatoprotective effect by activating the TLR4/My D88/NF-κB signaling pathway and Nrf2/Keap1 pathway,reducing inflam-mation and inhibiting oxidative stress.Three new coumarin glycosides and new angelica components were discovered,which not only enriched the components of AP,but also provided a basis for subsequent experimental research.Whether the new compounds have hepatoprotective activity remains to be further studied...