Study On The Inhibitory Efffect On Diet-induced Obesity Of The Flavonoids Compounds In Camellia Nitidissima Chi Flowers And Its Mechanism

Obesity is a kind of metabolic disorder disease,which results from an energy imbalance due to long-term excess of energy intake over energy expenditure.The excessive energy is converted into triglyceride and accumulated in abdominal adipose tissue and subcutaneous adipose tissue.Obesity has adverse effects on all physiological functions of the human body and it is a serious threat to public health security.Therefore,effective prevention and treatment of obesity has become an urgent problem.Numerous of chemical synthesis medicines have been used to treat obesity.However,these chemicals have uncontrollable side effects?insomnia,elevated blood pressure and rapid heartbeat,headaches,palpitations,gastrointestinal discomfort,oily stools and steatorrhea?,which are not accepted by the patients.Therefore,the development of new,natural,safe,and highly effective products from plants for the treatment of obesity has gained increasing attentions.Camellia nitidissima Chi is an evergreen shrub,and has been approved as a medicinal and edible plant.To the best of our knowledge,there are a variety of natural flavonoids ingredients with potential anti-obesity effect in Camellia nitidissima Chi flowers.However,there is little information about the systematic and comprehensive research on the effect of Camellia nitidissima Chi flower flavonoids compounds?CNFC?on anti-obesity and the underlying mechanism is still unclear.In this study,the CNFC was obtained by separation and purification,and the main components of CNFC were characterized and analyzed.The effect of CNFC on anti-obesity and the underlying mechanism of CNFC were studied in vitro and in vivo.The main research contents and conclusions are as follows:?1?Structural characterization of main components of CNFCCamellia nitidissima Chi flower flavonoids compounds?CNFC?was obtained with Camellia nitidissima Chi flower as raw material by extraction using organic solvent and purification using macroporous resin.The main components of CNFC were separated by liquid chromatography,manual collected,and characterized by time-flight mass spectrometer.The main components of CNFC were identified as epicatechin or catechins,procyanidins dimer,procyanidins trimer,procyanidin tetramer,procyanidin pentamer,apigenin-6,8-di-C-glycoside,6-C-pentosyl-8-C-hexosyl apigenin,quercetin-3-O-glucosyl-rhamnosyl-glucoside,rutin,quercetin-glucoside,phloretin-3',5'-di-C-?-glucoside,and kaempferol-galactoside.Among them,procyanidin tetramer,procyanidin pentamer,apigenin-6,8-di-C-glycoside and phloretin-3',5'-di-C-?-glucoside were found for the first time in Camellia nitidissima Chi flowers.?2?The inhibitory effect of CNFC on the activities of digestive enzymes in vitroThe inhibitory effect of CNFC on the activities of digestive enzymes and its mechanism were studied with?-amylase,?-glucosidase,pancreatic lipase and pancreatic cholesterol esterase as the research object in vitro.The results showed that CNFC not only interacted with the digestive enzymes??-amylase and?-glucosidase?,but also influenced the digestion of starch by binding to the substrate?starch?.The IC₅₀ of?-amylase,?-glucosidase,pancreatic lipase,pancreatic cholesterol esterase and cholesterol solubility was 300?g/m L,45?g/m L,320?g/m L,200?g/m L and 600?g/m L,respectively.The inhibition types of CNFC on?-amylase,?-glucosidase,pancreatic lipase,pancreatic cholesterol esterase were competitive inhibition,mixed inhibition,non-competitive inhibition and non-competitive inhibition,respectively.The results of fluorescence spectrum and circular dichroism spectrum showed that CNFC lowered the catalytic activity of?-amylase,?-glucosidase,pancreatic lipase and pancreatic cholesterol esterase by changing the microenvironment and spatial structure of these enzymes.?3?The regulating effect of CNFC on obese Sprague Dawley?SD?rats induced by high fat dietThe effects of CNFC on the weight gain and complications caused by obesity were studied using obese rats induced by high fat diet as model.The results showed that the anti-obesity effect of CNFC on obese rats was mainly included the following four ways.Firstly,CNFC decreased the appetite and food intake by significantly?p<0.05?up-regulating the secretion of glucagon peptide 1.Secondly,CNFC decreased the absorption of food and accelerated the excretion of energy substances?total glycerides and total cholesterol?in feces by inhibiting the the activity of digestive enzymes.Thirdly,CNFC changed the storage forms of energy substances by inhibiting the differentiation of preadipocytes.Fourthly,CNFC promoted fat decomposition and fat acid oxidation by significantly up-regulating the expression of lipohydrolase?p<0.05?and the secretion of adiponectin?p<0.05?to achieve the effect of control weight gain.CNFC significantly decreased?p<0.05?the content of total triglyceride,total cholesterol,low density lipoprotein cholesterin,lipid peroxides malondialdehyde in serum and liver of obese rats induced by high-fat diet.Meanwhile,CNFC significantly decreased?p<0.05?the content of alkaline phosphatase,alanine transaminase,and alanine transaminase in serum of obese rats induced by high-fat diet.In addition,CNFC significantly increased?p<0.05?the content of high-density lipoprotein cholesterin,catalase,glutathione peroxidase,total superoxide dismutase in the serum and liver tissue of obese rats induced by high-fat diet.Furthermore,CNFC restored dyslipidemia and abnormal hepatic lipids,improved oxidative stress environment and liver inflammation,and reduced the damage caused by lipid peroxides and oxidative stress.CNFC also improved glucose intolerance,increased insulin sensitivity by lowering area under the curve and insulin resistance index.?4?The regulation effect of CNFC on gut microbiota in high-fat diet-induced obese ratsThe effect of CNFC on gut microbiota was investigated with the faeces of high-fat diet-induced obese rats as the research object using the method of high-throughput sequencing of the 16s r RNA gene.The results showed that CNFC restored the imbalance of intestinal gut microbiota in high-fat diet-induced obese rats by increasing bacterial alpha diversity and restoring gut microbiota composition.CNFC decreased the abundance of Firmicutes and increased the abundance of Bacteroidetes and Verrucomicrobia at the phylum level.At the family level,CNFC increased the abundance of Lactobacillaceae,Bacteroidaceae and Akkermansiaceae,and decreased the abundance of Lachnospiraceae.Spearman correlation analysis showed that the OTU564,OTU606,OTU489,OTU453;Phyla:Verrucomicrobia,Proteobacteria and Bacteroidetes;Family:Akkermansiaceae,Enterobacteriaceae and Bacteroidetes were negatively correlated with phenotypes and most serum biomarkers.The OTU107,OTU91,and OTU433;Phyla:Firmicutes;Family:Lachnospiracea and Ruminococcaceae were positively correlated with numerous metabolic parameters of the host.?5?The regulating effect of CNFC on the lipids accumulation and differentiation of white adipose tissue and 3T3-L1 preadipocytesTo explore the regulating mechanism of CNFC on obese rats induced by high fat diet,white adipose tissue of obese rat induced by high fat diet and 3T3-L1 preadipocytes were selected as research object.The methods of real-time fluorescent quantitative-PCR and western blot were used to measure the expression of genes and protein related with fat metabolism in rats.The results showed that CNFC can effectively inhibit the lipids accumulation of 3T3-L1 fat cells.After treatment with CNFC?100 g/m L?for 10 days,the content of total triglyceride in 3T3-L1 preadipocytes was significantly decreased by 80±4.1%?p<0.01?.The inhibitory effect of CNFC on the lipids accumulation was mainly in the early?2 days?and late?6 days?of 3T3-L1 preadipocytes differentiation.CNFC regulated the differentiation of 3T3-L1 preadipocytes and fat metabolism of white adipose tissue by activating the AMPK signal pathway.CNFC inhibited the formation of 3T3-L1 fat cells by down-regulating?p<0.05?the expression of the key transcription factor CCAAT/enhancer binding protein?C/EBP??and peroxisome proliferator-activated receptor gamma?PPAR??.CNFC inhibited fat synthesis and delayed cell maturation by down-regulating the expression of transcription factor sterol regulatory element binding transcription factor 1c?SREBP-1c?,which furtherly regulated the expression of fatty acid synthase?FAS?and Acetyl-Co A carboxylase?ACC?.CNFC significantly?p<0.05?up-regulated the expression of adipose triglyceride lipase?ATGL?and carnitine palmitoyltransferase1?CPT1?,which promoted fat decomposition and fat acid oxidation.However,CNFC could not up-regulate the expression of fat hormone sensitive lipase?HSL?.?6?The regulation of CNFC on fat metabolism in liver tissueTo study the inhibitory effect of CNFC on the fat accumulation in liver tissue,the methods of real-time fluorescent quantitative-PCR and western blot were used to measure the expression of genes and protein related with fat metabolism in rat liver tissue.The results showed that CNFC inhibited the fat formation of liver tissues by significantly?p<0.05?down-regulating the expression of transcription factors C/EBP?,C/EBP?,C/EBP?and PPAR?.Meanwhile,CNFC inhibited the fat accumulation in liver by significantly?p<0.05?down-regulating the expression of transcription factor SREBP-1C,ACC and FAS.In addition,CNFC significantly?p<0.05?up-regulated the expression of ATGL,HSL and CPT-1,and promoted fat decomposition and fat acid oxidation in liver tissue.Furthermore,CNFC promoted browning by up-regulating the expression of uncoupling protein 1?UCP1?and proliferator-activated receptor gamma coactivator 1-a?PGC-1a?.