Extraction And Analysis Of Flavonoids From Chrysanthemum And Its Mechanism Of Reducing Blood Lipid

As a typical representative of medicinal and edible plant,chrysanthemum has the effects of clearing heat,detoxifying and brightening eyes.It is widely used in the traditional Chinese medicine and food industry.Modern pharmacological studies have shown that chrysanthemum has a variety of physiological activities such as antioxidation,anti-inflammatory,anti-tumour,cardiovascular protection,hypoglycaemic and hypolipidemic,etc.As the main functional component of chrysanthemum,chrysanthemum flavonoids have a variety of biological activities,including anti-inflammatory,anti-cancer,antioxidant and free radical scavenging activities,which have become a hot research topic in recent years.Nevertheless,flavonoids have been less studied in terms of their hypolipidemic effects and their mechanisms.Therefore,based on our previous researches,the ultrasonic extraction process of chrysanthemum flavonoids was optimized by response surface analysis,the chemical components of chrysanthemum flavonoids were qualitatively analyzed by UPLC-Triple-TOF/MS,and the lipid-lowering effects of chrysanthemum flavonoids,luteolin and luteoloside were studied by hyperlipidemia animal model and steatosis Hep G2 cell model.Based on the regulation of lipid metabolism related enzymes,the mechanism of reducing blood lipid was preliminarily studied.Using gene chip technology,the molecular mechanism of reducing blood lipid of chrysanthemum flavonoids,luteolin and luteoloside(two monomers of Chrysanthemum flavonoids)was obtained from the perspective of the whole genome,and verified by RT-PCR,which provided a scientific basis for the in-depth study and utilization of flavonoids.The main research work is as follows:1.Optimization of ultrasonic extraction process and qualitative analysis of Flavonoids from Chrysanthemum.According to the results of single cause experiment,the concentration of ethanol(A),ultrasonic extraction time(B)and liquid-solid ratio(C)were selected as the investigation factors of response surface optimization.Response surface analysis was used to optimize the extraction process of chrysanthemum flavonoids.A quadratic multinomial mathematical model for the ultrasonic extraction process of chrysanthemum flavones was established as follows:Y=5.69+0.58×A+0.13×B-0.021×C-0.35×AB+0.077×AC-0.13×BC-0.44×A~2-0.85×B~2-0.31×C~2.The model was highly significant and the fitting was considered excellent.The optimized extraction parameters were ethanol concentration of 77%,ultrasonic time of 25 min and liquid-solid ratio of 31:1(m L/g).Under these conditions,the extraction rate of chrysanthemum flavonoids can reach 5.88%,and the validation value is 5.92%,which is basically in line with the prediction model.Using UPLC-Triple-TOF/MS technology and combined with Scinder and Reaxy database,32 flavonoids in chrysanthemum were identified and speculated,including 26 flavonoids,4 dihydroflavonoids and 2 flavonols,and the functional components of chrysanthemum flavonoids were identified.2.Study on the hypolipidemic effect of chrysanthemum flavonoids.A hyperlipidemic SD rat model was established by D12492 high-fat diet,and the model SD rats were further randomly divided into five groups:model group,chrysanthemum flavonoid group,luteolin group,Luteoloside group and simvastatin group(positive control group).The normal control group was fed with D12450B diet,and the rest groups were fed with D12492 high-fat diet.The intervention period was 6 weeks.At the end of the experiment,liver,adipose tissue,blood and fecal samples were weighed and collected for backup,and relevant indexes such as body weight,organ coefficient,lipid quadruple,fecal lipid content were measured,and Histopathological analysis was carried out on the liver,and adipose tissue.The results showed that the liver wet weight,liver coefficient,total fat and body fat rate in the chrysanthemum flavonoid,luteolin and Luteoloside groups were remarkablely lower than those in the model group;the TC,TG and LDL-C levels in the chrysanthemum flavonoid,luteolin and Luteoloside groups were remarkablely lower than those in the model group,while the HDL-C levels were not significantly different;the Apo-A1 level in the chrysanthemum flavonoid group was obviously higher than that in the model group,while the Apo-B level The fecal levels of TC and TG in the chrysanthemum flavonoid,luteolin and Luteoloside groups were obviously higher than those in the model group.Histopathological analysis showed that the degree of liver lipid degeneration and liver steatosis scores in the chrysanthemum flavonoid,luteolin and Luteoloside groups were obviously lower than those in the model group.The cross-sectional area of adipocytes in the chrysanthemum flavonoid,luteolin and Luteoloside groups were obviously smaller than that in the model group;the number of adipocytes in the chrysanthemum flavonoid,luteolin and Luteoloside groups was significantly more than that in the model group under 200×light microscopy and within 25mm~2.3.Study on the hypolipidemic mechanism of chrysanthemum flavonoids based on the regulation of lipid metabolism-related enzymes.Using animal experimental sera and liver tissues in Chapter 2,the activities of lipid synthesis-related enzymes(FAS,HMG-Co A and DGAT),lipid metabolism-related enzymes(FAβO,CYP7A and HL),antioxidant systemic enzymes(GSH-Px,CAT,SOD and MDA)and hepatoprotective enzymes(AST,ALP,ALT)were examined by systematically studying the lipid metabolism-related enzymes.The purpose of this study was to pxplore the hypolipidemic mechanism of chrysanthemum flavonoids from the perspective of regulating lipid metabolism-related enzymes.The research findings showed that the levels of FAS,HMG-Co A and DGAT in the chrysanthemum flavonoids,luteolin and luteoloside groups were significantly lower than those in the model group,while the levels of FAβO,CYP7A and HL were significantly higher than those in the model group.The levels of AST,ALP and ALT(except AST in the luteolin group)in the chrysanthemum flavonoid group,luteolin group and Luteolin glycoside group were significantly lower than those in the model group.4.Study on the lipid-lowering mechanism of chrysanthemum flavonoids based on gene chip technology.The total RNA of liver tissue of SD rats in Chrysanthemum flavonoids group,luteolin group,luteoloside group and high-fat model group were extracted,and the detection of gene chip and the screening of differential genes were carried out.Through GO and KEGG analysis,some differential genes related to lipid metabolism were selected for RT-PCR verification,and the molecular mechanism of chrysanthemum flavonoids,luteolin and Luteoloside in reducing blood lipid was studied from the perspective of gene expression level and overall genome.The screening results of differentially expressed genes indicated that there were 299differentially expressed genes in the model group compared with the normal control group,of which 217 genes were significantly up-regulated and 82 genes were significantly down-regulated;There were 427 differentially expressed genes in Chrysanthemum flavonoids group compared with model group,of which 167 were significantly up-regulated and 260were significantly down-regulated;There were 465 differentially expressed genes in luteolin group compared with model group,of which 123 were significantly up-regulated and 342were significantly down-regulated;There were 420 differentially expressed genes in the luteolin group compared with the model group,of which 160 were significantly up-regulated and 260 were significantly down-regulated.The results of GO function analysis showed that the differential genes in Chrysanthemum flavonoids group and model group were significantly enriched with 121biological processes,9 cell components and 32 molecular functions,a total of 162 functional categories;The differential genes in luteolin group and model group were significantly enriched with 163 biological processes,24 cell components and 55 molecular functions,a total of 242 functional categories;The differential genes between luteoloside group and model group were obviously enriched in 131 biological processes,17 cell components and 37molecular functions,a total of 185 functional categories.KEGG analysis showed that the differential genes in Chrysanthemum flavonoids group and model group were remarkablely enriched in 13 metabolic pathways,including circadian rhythm,cysteine and methionine metabolism,steroid biosynthesis,p53 signal pathway,metabolic pathway,biosynthetic antibiotics,glycine,serine and threonine metabolism,etc;The differential genes between luteolin group and model group were remarkablely enriched in27 metabolic pathways,including glycine,serine and threonine metabolism,circadian rhythm,metabolic pathway,glycerol phospholipid metabolism,steroid hormone biosynthesis,bile secretion,fatty acid elongation,glycerol ester metabolism,unsaturated fatty acid biosynthesis,etc;The differential genes between luteoloside group and model group were remarkablely enriched in 18 metabolic pathways,including circadian rhythm,cysteine and methionine metabolism,PPAR signaling pathway,p53 signaling pathway,metabolic pathway,fatty acid biosynthesis,glycine,serine and threonine metabolism,PI3K Akt signaling pathway,fatty acid metabolism,etc.RT-PCR results showed that compared with the model control group,the expression of SQLE,GCK,GPAM and IDI1 in Chrysanthemum flavonoids group was significantly down-regulated,while the expression of MSMO1 was down-regulated,but there was no significant difference;In luteolin group,the expression of CYP7A1 and ACOT4 were up-regulated,the expression of ACSL3,DHCR7 and LPIN1 were significantly down-regulated,and there was no significant difference in the down-regulation of GPAM expression;In luteoloside group,the expression of ACACA and CYP7A1 were up-regulated,the expression of PLTP and LPIN1 were significantly down-regulated,and the expression of ANGPTL4 and Pdgfrb were down-regulated without statistical significance.The results of RT-PCR were agree with those of microarray.5.In vitro lipid-lowering effect and mechanism verification of chrysanthemum flavonoidsTaking human hepatoma cell Hep G2 as the research object,the steatosis cell model was induced by oleic acid.The effects of chrysanthemum flavonoids,luteolin and luteolin on lipid metabolism in steatosis Hep G2 cells were observed respectively,and the lipid-lowering effects of chrysanthemum flavonoids,luteolin and luteoloside were further verified at the cell level.15 genes closely related to lipid metabolism were selected by comprehensive analysis of animal experimental gene chip and verified by RT-PCR at the cell level.The results showed that the contents of TC,TG and FFA in the oleic acid model group were significantly higher than those in the model group.After the intervention of chrysanthemum flavonoids,luteolin and luteoloside,the contents of TC,TG and FFA in the cells decreased significantly.The results of oil red O staining showed that after intervention with chrysanthemum flavonoids,luteolin,luteoloside and simvastatin,the red lipid droplets in steatosis Hep G2 cells decreased significantly.The results of RT-PCR were agree with those of microarray.Conclusion:1.Optimization of ultrasonic extraction process of flavonoids from chrysanthemum.The extraction parameters were ethanol concentration of 77%,ultrasonic time of 25 min and liquid-solid ratio of 31:1(m L/g).Under these conditions,the extraction rate of flavonoids from chrysanthemum can reach 5.88%.UPLC-Triple-TOF/MS technology was used to quickly identify 32 flavonoids in chrysanthemum,including 26 flavonoids,4dihydroflavonoids and 2 flavonols.2.Chrysanthemum flavonoids,luteolin and luteoloside have good lipid-lowering effects and can prevent hyperlipidemia induced by high-fat diet.The preliminary study on the mechanism of reducing blood lipid showed that chrysanthemum flavonoids,luteolin and luteoloside can inhibit the activities of lipid synthesis related enzymes(FAS,HMG-Co A and DGAT)and improve lipid metabolism related enzymes FAβO,CYP7A and HL)activity,increase the level of liver antioxidant,improve the peripheral environment of lipid metabolism,so as to reduce blood lipid.3.The hypolipidemic mechanism of chrysanthemum flavonoids may be through down regulating the expression of lipid metabolism related genes such as sqle,GCK,GPAM,IDI1and MSMO1 in the liver.On the one hand,it participates in biological processes such as cholesterol synthesis and metabolism,sterol biosynthesis,insulin secretion and regulation,lipid homeostasis,triglyceride synthesis and metabolism,fatty acid metabolism,on the other hand,it regulates metabolic pathways such as circadian rhythm,steroid biosynthesis,p53signal pathway and other pathways,so as to play the role of lipid-lowering.The mechanism of luteolin lowering blood lipid may be through regulating the expression of related lipid metabolism genes such as GPAM,CYP7A1,ACSL3,ACOT4,DHCR7 and LPIN1 in the liver.On the one hand,luteolin participates in biological processes such as redox,cholesterol bile acid conversion and cholesterol synthesis,on the other hand,it regulates metabolic pathways such as circadian rhythm,steroid hormone biosynthesis,bile secretion and glyceride metabolism,so as to reduce blood lipid.The lipid-lowering mechanism of luteoloside may be through regulating the expression of related lipid metabolism genes such as ACACA,CYP7A1,PLTP,Lpin1,ANGPTL4 and PDGFRB in the liver.On the one hand,luteolin participates in biological processes such as lipid metabolism process,lipid homeostasis,adipocyte differentiation regulation and cell response to cholesterol,on the other hand,it regulates circadian rhythm,PPAR signal pathway,p53 signal pathway,fatty acid biosynthesis and PI3K-Akt signal pathway,Glycerol metabolism and other pathways,so as to play the role of lipid-lowering.4.The RT-PCR results of animal experiment and cell experiment are consistent with the results of gene chip,which further reveals the credibility of the lipid-lowering mechanism of chrysanthemum flavonoids.5.Luteolin and luteoloside may be the effective components of chrysanthemum flavonoids to reduce blood lipid.