Study On Antioxidant Activity Of Cercidiphyllum Japonicum Sieb.et Zucc Extract And Anti-tumor Mechanism Of In Human Hepatoma SMMC-7721 Cells

Cercidiphyllum japonicum Sieb.et zucc.also known as the mountain ginkgo,Yunyi tree and Wujun tree,is an ancient rare deciduous tall tree of the Cercidiphyllaceae.It is one of the woody plants of the East Asian Tertiary,and is listed as a national secondary protected wild plant.C.japonicum contains a wide range of chemical constituents.Its roots,stems and leaves can be used for medicinal purposes,especially for bark simmering water.It has a special effect on the treatment of colds and dysentery.At present,the research on C.japonicum mostly focuses on endangered conditions,breeding and reproduction,community structure,genetic diversity and ecological characteristics,and has less biological activity on its secondary metabolites.In this study,the C.japonicum leaves from Yanjinghe Breeding Farm in Wangcang County,Guangyuan City,Sichuan Province were used as experimental materials to extract and analyze the components of secondary metabolites,and to study the in vitro antioxidant activity and on human liver cancer SMMC-7721 cell anti-tumor activity and mechanism of C.japonicum extracts,laying the foundation for further better development and utilization.The research content is as follows:?1?Extraction and component analysis of secondary metabolites of C.japonicum.The secondary metabolites in the leaves of C.japonicum were extracted by water extraction,ultrasonic extraction and steam distillation to obtain C.japonicum aqueous extract,C.japonicum ultrasonic extract and C.japonicum essential oil.The components of C.japonicum aqueous extract and ultrasonic extract were analyzed by ultra performance liquid chromatography and time-of-flight mass spectrometry?UPLC-TOF/MS?.Three flavonoids of C.japonicum aqueous extract were identified by compound fragment analysis,namely Kaempferol,alizarin,isoquercitrin;Three flavonoids of C.japonicum ultrasonic extract were identified by compound fragment analysis,namely naphthol,naringenin,quercetin 3-O-?-D-Glucosin.The components of C.japonicum essential oil were analyzed by gas chromatography-mass spectrometry?GC/MS?,mainly containing eucalyptus,?,?-4-trimethyl-3-cyclohexene-1-methanol,4-methyl-1-?1-A compound such as methylethyl?-3-cyclohexen-1-ol,phthalic acid or isobutyloctyl ester.?2?Study on the antioxidant activity of C.japonicum extract in vitro.Using C.japonicum aqueous extract,ultrasonic extract and C.japonicum essential oil as the experimental materials,the in vitro antioxidant activity of C.japonicum extract was comprehensively evaluated by scavenging hydroxyl radicals,DPPH free radicals,superoxide anion and reducing ferric ions.The results showed that C.japonicum aqueous extract and ultrasonic extract have the ability to scavenge hydroxyl radicals,DPPH free radicals,superoxide anions and reduce ferric ions.The antioxidant effect increased with increasing extract concentration,and the ability to scavenge superoxide anion(IC₅₀ values were 0.083 mg/mL and 0.002 mg/mL,respectively)was stronger than the positive control Vc(IC₅₀ value was 0.238 mg/mL)and the IC₅₀ value of the iron ion reducing power(the IC₅₀ value of the aqueous extract is 0.014 mg/mL;the IC₅₀ value of the ultrasonic extract is 0.001 mg/mL)is relatively small,indicating the total The antioxidant activity is strong,indicating that both have good antioxidant activity in vitro.With the increase of concentration,the effect of C.japonicum essential oil on scavenging three kinds of free radicals was not obvious,and the reducing power was also weak.The difference was not significant at p=0.05 level,and there was no statistical significance,indicating that its antioxidant activity was weak.?3?Anti-tumor activity and mechanism of C.japonicum extract on human hepatoma SMMC-7721 cells.The effect of C.japonicum extract on the viability of human hepatoma SMMC-7721 cells was determined by MTT assay.The results showed that C.japonicum aqueous extract and C.japonicum ultrasonic extract had weak cytotoxicity to human liver cancer SMMC-7721 cells,and the cell inhibition rate did not increase with the increase of concentration.C.japonicum essential oil showed significant cytotoxicity,so C.japonicum essential oil was used for subsequent experiments.After treatment of human hepatoma SMMC-7721 cells with C.japonicum essential oil for 12 h,24 h and48 h,the cell inhibition rate increased with the increase of concentration,and the IC₅₀values were 0.108 mg/mL and 0.104 mg/mL,respectively.0.132 mg/mL.Among them,the IC₅₀ value was the smallest when 24 h was processed,and 24 h was selected as the processing time of the subsequent test.The morphology of SMMC-7721 cells treated with C.japonicum essential oil was further observed by microscopy.As the concentration increased,the cell morphology gradually shrunk and rounded and collapsed in a three-dimensional fusiform shape,showing a flat irregular shape.Flow cytometry was used to detect the effect of C.japonicum essential oil on the cell cycle and apoptosis rate of human hepatoma SMMC-7721.Cell cyclic results indicate that C.japonicum essential oil acts on SMMC-7721 cells,inhibit cell proliferation by blocking part of G2/M phase cells.With the increase of C.japonicum essential oil concentration,the proportion of cells in G2/M phase increased from 7.14%to 11.14%;Cell apoptotic rate results showed that C.japonicum essential oil induced apoptosis of SMMC-7721cells,and the apoptosis rate increased in a dose-dependent manner.When the treatment concentration was 0.6 mg/mL,C.japonicum essential oil significantly promoted apoptosis of human liver cancer SMMC-7721 cells,and the apoptosis rate?early wither+late wither?was 20.58%.The apoptotic cells were further observed by AO/EB staining and transmission electron microscopy.AO/EB staining showed that the number of apoptotic cells showing orange-red or red fluorescence increased with the increase of C.japonicum essential oil concentration.Transmission electron microscopy also observed the scattered distribution of chromatin in the cell core,and many apoptotic features such as vacuoles and apoptotic bodies appeared.The JC-1 mitochondrial membrane potential reagent detection kit detected that C.japonicum essential oil treatment of human liver cancer SMMC-7721 cells,with the increase of essential oil concentration,the mitochondrial membrane potential ratio decreased significantly,and dose-dependent.The effects of C.japonicum essential oil on autophagy of human hepatoma SMMC-7721 cells were detected by transmission electron microscopy and Western Blot.Transmission electron microscopy showed many distinct bilayer membrane structures or autophagosomes enveloping the organelles that needed to be degraded.Western Blot assay showed that the accumulation of LC3-II protein in the cells and the protein level of Beclin-1 were up-regulated.Both of them indicated that C.japonicum essential oil induced higher levels of autophagy in human liver cancer SMMC-7721 cells.In-depth study of the role of autophagy induced by C.japonicum essential oil in apoptosis,after the addition of the autophagy inhibitor chloroquine,the level of Bcl-2 anti-apoptotic protein in SMMC-7721 cells was determined.Compared with the control group,the anti-apoptotic protein of Bcl-2 was down-regulated with the increase of the concentration of essential oil,indicating that autophagy induced by C.japonicum essential oil after treatment of human hepatoma SMMC-7721 cells existed as a protective mechanism.In summary,this study analyzed the composition of C.japonicum extract and determined its antioxidant activity,and elucidated its anti-tumor mechanism.The study found that C.japonicum aqueous extract and ultrasonic extract have good antioxidant activity.C.japonicum essential oil can induce apoptosis and autophagy in human hepatoma SMMC-7721 cells,and promote the apoptosis of SMMC-7721 by inhibiting autophagy.