Studies On The Chemical Constituents And Bioactivities Of Fraxinus Mandshurica And Morus Australis Roots

This thesis elaborated the studies on the chemical constituents and biological activities(such as anti-obesity,anti-diabetes and liver protection etc.)of Fraxinus mandshurica Rupr.and Morus australis Poir.,hoping to provide references for the full utilization of these two plant resources.(?)F.mandshurica,one of the major species of Fraxinus genus,is the main afforestation tree in northeast China.Currently,there are few reports on the chemical constituents and biological activities of F.mandshurica.Therefore,this study firstly investigated the phytochemical compositions and biological activities of F.mandshurica and main results are as follows:1.Through extensive chromatographic and spectral analysis,40 compounds were isolated and identified from the F.mandshurica seeds,including 19 secoiridoids compounds,they are GI3;nuzhenide;ligstroside;oleoside-11-methyl ester;oleuricine A;nicotiflorine;jaspolyanoside;oleopolynuzhenide A;safghanoside G;excelside B;isooleonuezhenide;lucidumoside A;safghanoside A;jaspolyoleoside B;10-hydroxoleoside-7,11-dimethyl ester;GI5;oleoside-7,11-dimethyl ester;isolignstroside;10-hydroxyligstroside;2 phenethanol glycoside compounds,they are salidroside and osmanthuside H;2 coumarins,they are pimpinellin and fraxinol;1 lignan compound 8-hydroxypinoresinol;3 triterpenoids,they are3?,28-dihydroxyoleana-11,13(18)-diene;ursolic acid;sclerosteroid J;2 sterol compounds,they are?-sitosterol and?-daucosterin;2 phenolic acid compounds,they are gallic acid and caffeic acid;2 fatty acid lactone compounds,they are AL072 and monolinolenin;3flavonoid compounds,they are cymaroside;luteolin;quercetin;4 other compounds,they are tyrosol;D-aromadendrane-4?,10?-diol;1-(4-hydroxy-3-methoxy)-phenyl-1,2,3-propanetriol and saccharide.In addition,GC-MS was used to analyze the fatty acid composition of the oil components in F.mandshurica seeds.A total of 18 fatty acid compounds were identified,they are(Z,Z)-9,12-octadecadienoic acid;(E)-9-octadecenoic acid;hexadecanoic acid;methyl stearate;(Z)-11-octadecenoic acid;18-methylnonadecanoic acid;tetracosanoic acid;cis-11-eicosenoic acid;docosanoic acid;cis-10-Heptadecenoic acid;(Z)-9-hexadecenoic acid;14-methyl-hexadecanoic acid;tricosanoic acid;(Z)-9-octadecenoic acid;heneicosanoic acid;2-octyl-cyclopropaneoctanoic acid;cis-10-nonadecenoic acid;11-methyl-octadecanoic acid,of which(Z,Z)-9,12-octadecadienoic acid has the highest relative content in its fatty acid compounds,reaching 64.37%.2.Through extensive chromatographic and spectral analysis,21 compounds were isolated and identified from the F.mandshurica leaves,including 5 coumarin compounds,they are fraxetin;esculetin;dimethylfraxetin;esculin,fraxin;3 secoiridoid compounds,they are oleuropein;nuzhenide;ligstroside;7 flavonoids compounds,they are kaempferol;quercetin,dihydroquercetin;catechin;isoquercitrin;quercitrin;rutin;2 phenolic acid compounds,they are chlorogenic acid and cinnamic acid;4 triterpene and sterol compounds,they are?-sitosterol;3-oxooleanolic acid;belulin and belulic acid.3.A HPLC-DAD method was developed and validated for the simultaneous characterization and quantification of 19 secoiridoids in the extract of F.mandshurica seeds.The highest content of secoiridoid compound in F.mandshurica seed extract is nuzhenide and the lowest content compound is isolignstroside.And the content of total secoiridoids is181.35 mg/g.The verification results showed that all standard calibration curves exhibited good linearity(R²>0.9991)within the test ranges.The relative standard deviation values for intraday and interday precision,stability and repeatability were<5%.The recoveries were in the range of 97.52-102.28%.The proposed method could serve as a prerequisite for quality control of materials or products of F.mandshurica seeds.4.Inhibitory activities on?-glucosidase,acetylcholinesterase,tyrosinase and pancrelipase of 27 major components from F.mandshurica seeds were screened in vitro.Results showed that most secoiridoids possessed significant?-glucosidase and pancrelipase inhibitory activities,which means they can be good sources of?-glucosidase and pancrelipase inhibitors,and suggesting that F.mandshurica seeds may have anti-diabetes and anti-obesity potentials.5.The effects of F.mandshurica seeds on high-fat diet-induced obese C57BL/6 mice were studied in vivo.Results showed that F.mandshurica seeds can significantly reduce the body weight of obese mice,reduce the content of LDL,TC and TG in serum and liver,and increase HDL level in serum of obese mice.The hypolipidemic mechanism may be related to the up-regulation of PPAR-?expression and inhibition of LXR-?and SREBP-1c expressions,thereby up-regulating the expression of Apo A5 and improving the activities of HL and LPL in obese mice.And F.mandshurica seeds could reduce the serum ALT and AST levels,increase the SOD,CAT and GSH levels,and reduce the levels of MDA and NO in the liver of obese mice.F.mandshurica seeds could also decrease the levels of inflammatory factors including TNF-?,PGE2 and IL-6.Additionally,F.mandshurica seeds effectively reduced the serum Cre,BUN and BUA contents.Finally,F.mandshurica seeds could modulate the structural alteration of gut microbiota in obese mice,increasing the proportion of anti-obesity gut microbiota(Bacteroidetes,Bacteroidia,S24-7 and Allobaculum),and reducing the proportion of obesogenic gut microbiota(Firmicutes and Dorea).In the free fatty acid-induced Hep G2 cells,F.mandshurica seeds could significantly reduce the content of TG and TC in the cells.6.The effects of F.mandshurica seeds on HFD/STZ-induced diabetes C57BL/6 mice were studied in vivo.Results showed that F.mandshurica seeds could reduce fasting blood glucose,enhance glucose tolerance and insulin sensitivity of diabetic mice.F.mandshurica seeds could also reduce the serum TG and TC levels,decreased the serum ALT and AST levels,increase the activity of SOD and the content of GSH,and reduce the MDA content in the liver of diabetic mice.In addition,F.mandshurica seeds could reduce the content of Cre,BUN and BUA in the serum of diabetic mice.Finally,F.mandshurica seeds could reduce the Firmicute/Bacteroidetes ratio and significantly increase the relative abundance of probiotics Lactobacillaceae and Lactobacillus of diabetic mice.In the L02 cells,19secoiridoids isolated from F.mandshurica seeds can reduce the glucose concentration in the cell culture medium.Among them,Isooleonuezhenide has the strongest activity.(?)M.australis is one of the major Morus species growing in Northern China.In our previous research,ten flavonoids and two stilbenes were identified as major constituents of M.australis roots.In the present study,biological activities of M.australis roots and its constituents were evaluated and main results are as follows:1.Inhibitory activities on?-glucosidase,acetylcholinesterase and tyrosinase of components from M.australis roots were screened in vitro.Results showed that morusingin L,cudraflavone C,Austrone A,morusin and kuwanon H possessed significant?-glucosidase inhibitory activity;cudraflavone C,kuwanon C,mulberrofuran G,mulberrofuran F,moracenin B,kuwanon H and cathafuran B showed significant acetylcholinesterase inhibitory activity;morusingin L,kuwanon C,austrone A,morusin,mulberrofuran G,mulberrofuran F,morcin M and cathafuran B exhibited significant tyrosinase inhibitory activity.2.The antitumor activities of these compounds from M.australis roots were investigated on various tissue-derived cancer cell lines,like human hepatocellular carcinoma cell line Hep3b and Hep G2,breast cancer cell line MCF-7,triple negative breast cancer cell line MDA-MB-231,and adenocarcinomic human alveolar basal epithelial cells A549.Interestingly,A549 cells were more sensitive for these compounds than other cell lines.Among these polyphenols,cudraflavone B,austrone A and morusin showed significant cytotoxicity on A549 cells.Three flavonoids induced apoptosis on A549 cells via intrinsic mitochondria pathway and mediated inhibition of autophagic flux contributed cell death via PI3k/Akt/m TOR pathway.3.The hepatoprotective effect of ethanol-water extract of M.australis roots was studied using an alcohol-intoxicated liver injury model in mice.Results showed that M.australis roots extrat could decrease the levels of serum ALT and AST,TG and MDA,and increase GSH contents in the liver of mice.The hepatoprotective effect of ten flavonoids was evaluated in CCl₄-induced Hep G2 cell lines.All the flavonoids(especially cudraflavone B)showed protection against CCl₄intoxicated Hep G2 cell lines by decreased LDH levels.Also,contents of ten individual flavonoids and total flavonoids of the extract were analyzed by HPLC-DAD method.Results showed that the flavonoid with the highest content is moracenin B(86.51 mg/g),and the total flavonoid content in the extract is 192.69 mg/g.