Study On Camellia Petelotii（Merrill） Sealy Leaves Aqueous Ethanol Extract Against CCl₄-induced Acute Liver Injury In SD Rats And Its Medium-polar Chemical Components Reference Substances

Camellia petelotii（Merrill）sealy,belongs to the Theaceae family,is a national primary protected plant,which was mainly distributed in Guangxi province and Vietnam.The wild C.petelotii is extremely rare and narrowly distributed,and it is a rare and precious plant in the world.Now C.petelotii has been successfully cultivated in Fangchenggang,Guangxi,China.C.petelotii is known as"Oriental Magic Tea","Panda in the Botanical World",and"Queen of the Tea Clan",not only because of its beauty,but also because of its high medicinal value.As C.petelotii leaves are used for both medicine and food,its extracts can treat sore throat,bloating,edema,unfavorable urination,sores,diarrhea,dysentery,etc.Modern pharmacology studies showed that C.petelotii shows pharmacological activities such as anti-inflammatory,anti-oxidation,inhibition of hepatocellular carcinoma proliferation,removal of advanced glycation end products and regulation of blood lipids.According to reports,some chemical components have been isolated and identified from C.petelotii,mainly including saponins,flavonoids,phenols,alkaloids and other compounds.Objectives:The purpose of this study was to explore the protective effect of C.petelotii leaves on acute liver injury（ALI）and its polar chemical constituents reference substances,so as to providing a pharmacological,chemical basis,and reference substances for the research and development of C.petelotii leaves.Methods:1.10%aqueous ethanol solution was used to extract the C.petelotii leaves.The filtrates were evaporated using a rotary evaporator.Trace solvent was removed by lyophilization with a freeze-dryer to obtain the dry C.petelotii leaves aqueous ethanol extract（CPE）,and the phenol and flavonoid content of C.petelotii was determined by ultraviolet spectrophotometry.2.Sixty male SD rats were randomly divided into 6 groups（n=10）as follows:control group and negative group:0.5%CMC-Na（5 ml/kg/d）;positive group:thiopronin（20 ml/kg/d）;CPE groups:CPE（40,80,and160 ml/kg/d）.All the groups were treated once a day for 7 consecutive days,and 1 h after the last administration on 7^thday,control group was administered olive oil intraperitoneally,and other groups were administered CCl₄[2 ml/kg,dissolved in olive oil（50%,v/v）]intraperitoneally to induce ALI.The animals were fasted for 14 h but allowed free access to water.The rats were sacrificed,and the livers and serum were collected.Hematoxylin&Hosin（H&E）staining was used for histological examination to observe the liver pathological changes of the rats in each group;the contents of aspartate transaminase（AST）,alanine aminotransfease（ALT）and malondialdehyde（MDA）in serum and liver tissues,and superoxide dismutase（SOD）and glutataione（GSH）levels in liver tissues were detected using commercial kits;Using enzyme-linked immunosorbent assay（ELISA）kit to analyze the content of reactive oxygen species（ROS）in rat serum and the protein level of TNF-α,IL-1β,IL-6;Real-time quantitative PCR detecting system（QPCR）analyzed the m RNAs levels of nuclear factor（erythroid-derived 2）-like-2factor（Nrf2）,heme oxygenase-1（HO-1）,TNF-α,IL-1βand IL-6;Western blotting（WB）analyzed protein levels of p65,p-p65,p-IκBα,Nrf2,HO-1 in liver tissue;Immunohistochemistry（HIC）was used to observe the expression of p65 in liver cells.3.The 70%aqueous ethanol extract of C.petelotiileaves was separated and purified by various chromatographic methods,and its structures were identified by physical and chemical properties,¹H-NMR,¹³C-NMR,ESI-MS and compared with reference standards.Results:1.The content of phenols and flavonoids was determined as 34.474±1.026 and 15.228±0.422 mg/g crude drug in CPE,respectively.2.Pretreatment with CPE（80 and 160 mg/kg）attenuated the histological changes caused by CCl₄,including necrosis,infiltration of inflammatory cells,steatosis;decreased ALT,AST,and MDA levels in serum and liver tissues（p<0.05）.Moreover,CPE reduced the concentrations of ROS,TNF-α,IL-1β,and IL-6（p<0.05）,and blocked the nuclear translocation of p65,and up-regulated the expression p-p65,p-IκBα（p<0.05）.Pretreatment with CPE increased the levels of HO-1,SOD,and GHS through up-regulating Nrf2.3.Seven compounds were isolated and identified from 70%aqueous ethanol extract of C.petelotii leaves.The structures of these compounds include one sterol compoundβ-sitosterol（1）,one coumarin compound umbelliferone（2）,four flavonoid compounds quercetin（3）,kaempferol（4）,（-）-epigallocatechin（5）,（-）-epicatechin)（6）,one phenolic acid compound chlorogenic acid（7）.Among these compounds,phenolic compounds such as flavonoids and phenolic acids can be used as reference products,providing reference products for establishing quality standards for C.petelotii leaves.Conclusion:CPE can play an obvious protective effect on ALI,which may be related to the rich content of phenols and total flavonoids.Sevene compounds were isolated and identified from CPE.The results of this experiment provide pre-scientific experimental basis and medium-polarity chemical reference for the scientific development and utilization of C.petelotii leaves as a liver-protecting food or medicine.