| Chapter 1 Extraction of total flavonoids in Carya cathayensis leaf and preparation of experimental drugObjective:Extraction and separation of total flavonoids in Carya cathayensis leaf from the leaves of the walnut,determination of the content of total flavonoids,and the preparation of the Lavage liquid.Methods:The preparation of flavonoids in Carya cathayensis leaf.Extracting solution will obtained by the method of reflux extraction,obtained the total flavonoids in Carya cathayensis leaf extract.And then mixed with polyamide,removed the water by reduced pressure distillation.Chromatographed on column and eluted with 30%ethanol.After eluted with 40%ethanol,collected the eluent,removed the water by reduced pressure distillation.Finally,High performance liquid will be used to determine the content of flavonoids in the collected product.Results:Every 200.0g in Hickory leaves can obtained the total flavonoids in Carya cathayensis leaf about 3.0g.The total flavonoids content is 57.3%.Conclusion:The five known flavonoids in the total content are above 50%.And the method has high repeatability,and can be used for the preparation of the following pharmacological experiments.Chapter 2 The improve of the flavonoids in Caryacathayensis leaf to the lipid metabolism disorders in the ovariectomized ratsObjective:To evaluate the improve effect of the flavonoids in Caryacathayensis leaf on lipid metabolism disorders in the ovariectomized rats.Methods:This experiment to establish animal model of rats by removal of healthy mature female bilateral ovaries.One hundred and twenty female rats aged three months,100 ovariectomized,20 which only removed ovarian peripheral fat(sham operation),marked as 1 to 13 groups,ten rats in each group.Group 1,2,3,4,11 and 12 rats were fed with standard fodder and drinking water(normal diet),weighing every 10 days,after 2 months of feeding,then 2,3,4 group feeding the flavonoids in Caryacathayensis leaf.Group 5,7,8,9,10,and 13 rats were fed with high-nutrient and drinking water,weighing every 10 days,after 2 months of feeding,then 8,9,10 group feeding the flavonoids in Caryacathayensis leaf.The effects of the flavonoids in Caryacathayensis leaf on ovariectomized rats were studied by measuring body weight,Lee's index,abdominal fat index,aortic peritoneal fat coefficient,blood lipids,liver lipids and liver and fat slices.Results:1.The weight gain rate of the rats fed with the high-nutrient diet after administration was significantly lower than that before the administration.The Lee's index of each group was slightly different,and there was a decreasing trend in the treatment group.2.The TC and TG levels in serum were detected.The levels of TC and TG in serum were higher than those in the sham-operation group,and there was a decreasing trend in each dose group after administration.The levels of TC and TG in serum of rats fed with high nutrient diet were 2.571mg/mol and 0.719mg/mol,which were significantly higher than those of sham operation group 12.47%and 14.67%,After feeding the flavonoids in Caryacathayensis leaf,each treatment group were significantly decreased;in the low dose group,TC and TG decreased by about 3.9%and 22.53%respectively;the TC and TG in the middle dose group decreased by about 7.2%and 23.92%respectively;the high dose group decreased by 16%and 34.21%respectively,showing a dose-increasing trend.3.Ovariectomized SD rats had severe fat ectopic deposition in the liver tissue,around the aorta and abdomen,and after treatment with flavonoids of hickory leaf,they all improved significantly.1)The levels of TC and TG in the liver of rats fed with common diet were 0.857mg/mol and 3.222mg/mol,which were significantly higher than those of the sham operation group 34.96%,60.86%.,after feedng the flavonoids in Caryacathayensis leaf,each treatment group were significantly decreased,In the low dose group,the TC and TG decreased by about 15.17%and 18.8%respectively.In the middle dose group,TC and TG decreased by about 5.25%and 19.58%respectively;the TC in high dose group did not change significantly,the TG decreased by about 16.33%.The levels of TC and TG in the liver of ovariectomized SD rats were 0.978 mg/mol and 4.225 mg/mol,which were significantly higher than those of the sham-operated rats about 16.99%,42.54%.After feeding the flavonoids in Caryacathayensisleaf,the liver lipid levels decreased significantly:in the low dose group,TC,TG decreased by about 19.12%and 16%respectively;the TC and TG of middle dose group decreased by about 6.33%and 30.01%respectively;the TC and TG in high dose group decreased by about 14.51%and 33.4%respectively.2)In order to further observe the liver fat,we to observe the liver tissue section HE staining,found that the rat's liver cells have a lot of fat droplets and inflammatory cells,after the treatment,hepatitis sex cells infiltrating in rats had improved significantly,the number of liver fat droplets in cells decreased significantly.3)We found that adipocytes of ovariectomized rats were larger and denser.The ratsabdominal fat cells diameter decreased to varying degrees after treated with the flavonoids in Caryacathayensis leaf.The fat ratio around the aorta of the rats was measured.4)After the ovariectomy,the aortic arch fat percentage of the model group was 0.117,which was similar to that of the sham-operated group.The aortic arch fat percentage of the model group decreased significantly after the administration of high-nutrient diet.The aortic arch fat ratio 0.1851,compared with sham operation group was increased 37.17%,after feed the flavonoids in Caryacathayensis leaf,Aortic arch fat decreased significantly:low dose group decreased by about 15.3%;middle dose group decreased by about 24%;high dose group decreased by about 32%.After the ovariectomy,the abdominal fat ratio of the model group was 1.5389,which was similar to that of the sham-operated group.The fat content of the aortic arch was decreased.The abdominal fat ratio of the model group fed with high-nutrient feed was 3.1266,which was 40.66%higher than that of the sham operation group.The middle-dose group decreased about 27.91%;high dose group decreased about 39.43%.Summary:1.The flavonoids in Caryacathayensis leaf has certain inhibitory effect on growth of body weight of ovariectomized rats.2.The flavonoids in Caryacathayensis leaf had no significant effect on Lee's index of ovariectomized rats,but could significantly decrease abdominal fat ratio and aortic peri-fat coefficient of ovariectomized rats.It indicated that the flavonoids in Caryacathayensis leaf can reduce the deposition of ectopic fat in ovariectomized rats.3.Ovariectomized rats fed normal diet had little effect on serum lipids,but significantly increased the accumulation of liver fat;high-nutrient diet fed rats after ovariectomy on blood lipids increased liver fat are very obvious.The flavonoids in Carya cathayensis leaf can significantly decrease the concentration of liver fat and serum fat,also can adjust to the function of TC?TG metabolic disorder in rats.4.Total flavonoids of Hickory leaf have obvious alleviation and repair effect on liver tissue lesion of ovariectomized high nutrition diet rats.5.The flavonoids in Caryacathayensis leaf can inhibit adipocyte fat accumulation and growth to a certain extent.Chapter 3 Mechanism of total flavonoids in Carya cathayensis leaf on the Improvement of Lipid Metabolism Disorder in Ovariectomized RatsSection1:The enzyme activity regulation of the flavonoids in Caryacathayensisleaf on the related to lipid metabolism of ovariectomized rats.Objective:To evaluate the activity effect of the flavonoids in Caryacathayensisleaf on the related enzymesactivity to lipid metabolism and the mechanism of lipid metabolism disorderin ovariectomized rats.Methods:Preparation of 10%rat liver homogenateof ovariectomized rats,we tested malate dehydrogenase(MDH),fatty acid synthase(FAS),hepatic lipase(HL),lipoprotein lipase(LPL),total lipase(TL),succinodehydrogenase(SDH)and hormone sensitive lipase(HSL)activity by Enzyme-linked immunosorbent assay(ELISA)and chemicals to evaluate the effect of theflavonoids in Caryacathayensisleaf on the regulatorymechanism of lipid metabolism disorderin ovariectomized rats.SPASS16 software analysis was used in results,and data was expessed in x±s.Single-factor analysis of varience was used for comparing the difference among the groups.Those with homogeneous variance received Least-Significant Difference(LSD),while those with non-homogeneous variance received Dunntt 3.Results:1.Rrats fed with normal diet and high nutritional feed showed similar results:FAS concentrations in the liver of rats after the ovaries were increased,but there was no significant difference compared with the sham operated group.Compared with the OVX group,hickory leaves low,middle and high dose concentration of FAS treatment,after a downward trend,but there was no significant difference.2.Compared with the sham-operated group,the activities of hepatic lipase HL in the ovariectomized model group which fed with normal or high-nutrient diet were 53.25%and 42.85%lower than those in the sham-operated group.The total flavonoids in Carya Cathayensis Leaf had a significant effect on liver hepatic lipase HL in ovariectomized rats fed with normal diet.The activity of hepatic lipase HL in the liver of rats treated with low,medium and high doses was 2.03 times,1.72 times and 1.03 times higher than that of the model group respectively,but the activity of hepatic lipase HL of the rats fed with high nutrient diet was decreased.3.The activity of hepatic lipase LPL in the ovariectomized model group which fed with normal or high-nutrient diet was 41.39%and 71.25%lower than that in the sham-operated group,respectively.The total flavonoids in Carya Cathayensis Leaf had a significant effect on LPL activity.The activity of LPL was 2.61 times,2.61 times and 1.44 times higher than that of the normal diet group,the low and middle dose groups showed very significant difference(P<0.01);the high dose group showed a significant difference(P<0.05).The LPL activity of ovariectomized rats fed with high-nutrient diet was 1.68 times,0.71 times and 1.57 times higher than that of the control group,and low,medium and high doses groups showed significant differences(P<0.05).4.Compared with sham operation group,the activity of total lipase TL in ovariectomized rats fed with normal diet and high nutrient diet was significantly lower(P<0.05).The total flavonoids in Carya Cathayensis Leaf significantly increased the TL activity.The activities of TL in the liver of ovariectomized rats fed with low,middle and high doses of feed were 2.31 times,2.15 times and 1.24 times higher respectively,the low and middle dose groups showed very significant difference(P<0.01);the high dose groups showed significant difference(P<0.05).The TL activity of low,medium and high dose groups increased 64.03%,32.37%and 66.19%,respectively,and the low dose groups showed significant difference(P<0.05).5.SDH activity of liver tissue in high-nutrition ovariectomized group was lower than that in normal diet group.The activity of SDH in the liver was significantly lower than that in the sham-operated group after the ovariectomized rats were fed with high-nutrient diet.The activity of SDH was improved after the total flavonoids in Caryacathayensis leaf.Compared with ovariectomized group,the low,middle and high dose group has increased by 9.8%,29.5%and 43.3%.There were significant differences among the high dose group.6.Compared with the sham-operated group,the MDH activity of the ovariectomized rats fed with normal and high-nutrient diet was 8.21%and 13.53%lower,respectively.The MDH activity in the ovariectomized model groups fed with high-nutrient diet was 23.77%lower than that in the ovariectomized model groups fed with normal diet.The activity of MDH increased in both the normal and high nutrient feeding groups.The MDH activity of ovariectomized rats which treated with low,medium and high dose of total flavonoids in Caryacathayensis leaf and fed with normal diet were increased by 13.62%,5.64%and 3.14%,respectively;the MDH activity of ovariectomized rats fed with high-nutrient diet increased by 23.78%26.42%and 30.44%respectively.The effect of total flavonoids in Caryacathayensis leaf on MDH activity of ovariectomized rats fed with high-nutrient diet was more obvious.Summary:1.The total flavonoids in Caryacathayensis leaf can regulate the activities of lipid-related enzymes in the liver of ovariectomized rats and increase the activity of TL,LPL,HL and so on.It is possible to decrease the lipid Quality synthesis to achieve the purpose of Lipid-lowering,while lipolysis-related enzymes such as HSL do not have much impact.2.It indicated that the total flavonoids in Caryacathayensis leaf could improve the energy metabolism of the liver to reduce excess energy to achieve lipid-lowering purposes.3.The activity of lipidase-related enzymes in SD rats fed with high-nutrient diets,after administration of the total flavonoids in Caryacathayensis leaf,was significantly more than that in normal diet-fed SD rats.Section2:Regulation effect of the flavonoids in Carya cathayensis leaf on gene related lipid metabolism in ovariectomized ratsObjective:To evaluate the regulation effect of the total flavonoids in Carya cathayensis leaf on enzyme gene and protein related lipid metabolism in ovariectomized rats.Method:Real-time quantitative PCR was used to detect the mRNA expression of fatty acid synthase(FAS),sterol regulatory element binding protein-lc(SREBP-1c),3-hydroxy-3-methylglutaryl-CoAreductase(HMGCoAR)and peroxisome proliferator activated receptor?(PPAR?),phosphoenolpyruvate carboxykinase(PEPCK),glucose-6-phosphatase(G6Pase)and glucose transporter 2(GLUT 2)in the liver.Results:1.FAS are the key enzyme of endogenous fatty acid synthesis,and their main functions are to synthesize fatty acid.The expression of FAS mRNA of the control group of normal and high nutrient fed groups were both higher than that of sham groups.But,the expression of FAS mRNA in the high nutrient was 4.36 times higher than that in the normal diet group.After treated with total flavonoids in Carya cathayensis leaf,the expression of FAS mRNA with normal nutrient feed group was lower than its control group 36.6%,54.7%,33.3%;the expression of FAS mRNA with high nutrient feed group was lower than its control group 75.9%,85.7%,91,8%.2.The expression of SREBP-1c mRNA in the ovariectomized model group was 41.26%higher than that in the sham-operated group,and the low,middle and high dose groups were 21.7%,41.6%and 46.3%lower than the model group respectively.Compared with the normal diet group,the expression of SREBP-1c mRNA was significantly higher in the high-nutrient diet group than that in the normal diet group,the expression of SREBP-1c mRNA in the high-nutrient diet group was increased by 2.97 times.The expression of SREBP-1c mRNA in the ovariectomized model group was 4.7 times higher than that in the sham-operated group and the low,middle and high dosage groups of the total flavonoids in Caryacathayensis leaf decreased by 65.9%,79.5%,86.7%.The effect of total flavonoids in Caryacathayensis leaf on the expression of SREBP-1c mRNA in the high-nutrient diet group was more obvious.The expression of SREBP-1c protein was decreased by 36.59%,respectively.Compared with the sham-operated group,and the expression of SREBP-lc protein in low,middle and high dose groups were decreased by 29.55%,24.43%,35.70%.The expression of SREBP-lc protein in the high-nutrient diet group was 4.88%,8.51%and 12.49%lower than that in the model group after treated of low,middle and high doses of total flavonoids in Caryacathayensis leaf.3.There was almost no difference between normal and high nutrient feed groups in expression of HMGCoAR mRNA.After treated with total flavonoids in Carya cathayensis leaf,the expression of HMGCoAR mRNA in normal nutrient feed groups was lower than its control group 66.5%and 40.7%in high nutrient fed group.4.The expression of PPAR? mRNA in ovariectomized group was significantly lower than that in other groups,which was 1/10 of that in the sham-operation group.PPARa mRNA in the three dose groups of total flavonoids in Carya cathayensis leaf significantly reversed the decrease(P<0.01),and the PPARa mRNA expression level in the three dose groups of total flavonoids in Carya cathayensis leaf increased by 36.2 times,2.3 times and 9.8 times respectively compared with the model group.Compared with sham-operated group,the expression of PPARa protein in ovariectomized model group had no obvious change.Compared with model group,the contents of PPAR? protein in normal ovariectomized group were 12.91%,37.20%and 77.87%higher than that in sham-operated group,middle and high dose had significant difference(P<0.01).5.The level of PPARamRNA in the high-nutrient diet group was significantly lower than that in the sham-operated group,which was 1/10 of that in the sham-operation groups.The PPARa mRNA in the three dose groups of total flavonoids in Carya cathayensis leaf significantly reversed the decrease(P<0.01).The PPARa mRNA expression levels of total flavonoids in Carya cathayensis leaf total flavonoids were 16 times,30 times and 18 times higher than that of the model group,respectively.The expression of PPARa protein in the group which treated with the total flavonoids in Carya cathayensis leaf was significantly 22.01%lower than that in the sham operation group;the expression of PPARa protein in low,middle and high dose groups were increased by 52.86%,190.71%and 92.86%,respectively,with significant difference(P<0.01).The total flavonoids in Carya cathayensis leaf can oxidize the fatty acids to reduce the fat accumulation in the liver tissue cells by activating the PPARa oxidase ?-pathway.6.The total flavonoids in Carya cathayensis leaf increased the expression of PEPCK mRNA in ovariectomized rats fed with normal diet.There was a significant difference in low dose group(P<0.01).The total flavonoids in Carya cathayensis leaf were 81.49%higher than that of the model group,and the difference was significant(P<0.01).The expression of G6Pase mRNA decreased by 9.90%in the low dose total flavonoids in Carya cathayensis leaf group,and there was no significant difference.The middle dose and the high dose total flavonoids in Carya cathayensis leaf decreased the expression of G6Pase mRNA and those were lower 1.60%and 39.48%.There was no significant difference in the PEPCK mRNA between the sham-operated group and the model group fed with high-nutrient diet,G6Pase mRNA expression levels were significantly higher than the model group,total flavonoids in Carya cathayensis leaf can significantly reduce the expression of PEPCK and G6Pase in high nutritional feeding rats group(P<0.01).Compared with the model group,the levels of PEPCK mRNA in the three dose groups of total flavonoids in Carya cathayensis leaf decreased by 19.4%,40.9%and 41.2%respectively(P<0.01),G6Pase mRNA expression levels were 46.0 times,47.3 times and 39.7 times lower than those in the model group,respectively(P<0.01).It is important for inhibition of gluconeogenesis and the protection of the liver.7.The expression of GLUT2 mRNA in ovariectomized group was significantly higher than that in sham-operated group,the expression of GLUT2 mRNA was significantly decreased in three dose groups(P<0.01),and decreased by 5.76%,22.34%and 30.25%in the normal diet group,and decreased by 39.79%,38.35%and 56.23%in the high-nutrient diet group(P<0.01).GLUT2 is the main glucose transporter in the liver.When eating and other factors caused by elevated blood glucose levels,GLUT2 in the liver rapidly absorbs too much glucose and reduces the liver's excessive accumulation of glucose.Conclusion:1.The normal and high nutrient diet both induced ovariectomized rats metabolic syndrome,fat deposition in the liver and abnormal expression of the hepatic mRNA genes related to fat and glucose metabolism in rats.2.The total flavonoids in Carya cathayensis leaf can prevent and improve this metabolic disorder and hepatic fat deposition by regulating the expression of these genes.The total flavonoids in Carya cathayensis leaf can reduce the expression of FAS mRNA,HMGCoAR mRNA and SREBP-1c mRNA and protein in ovariectomized rats,and the effect of FASL mRNA on the expression of FASL mRNA in the high-nutrient diet group was more obvious.Maybe it can inhibit the synthesis of fatty acids and the accumulation of lipids by down-regulating FAS mRNA expression.The total flavonoids in Carya cathayensis leaf could promote the oxidation of fatty acid and decrease the accumulation of serum and liver lipid by up-regulating the expression of PPARa mRNA and protein in ovariectomized rats.3.The total flavonoids in Carya cathayensis leaf increased the expression of PEPCK mRNA in ovariectomized rats,which may promote the gluconeogenesis and have a positive effect on the control of blood glucose,and the expression of G6Pase mRNA was down-regulated.The total flavonoids in Carya cathayensis leaf could down-regulate the expression of PEPCK mRNA and G6Pase mRNA in ovariectomized rats,which may inhibit the gluconeogenesis and protect the liver.The total flavonoids in Carya cathayensis leaf could significantly decrease the GLUT2 mRNA expression in the glucose transporter,this have a positive effect in inhibition of liver excessive intake of glucose,reducing liver excessive accumulation of sugar and protecting the liver.Section3:Effect of the flavonoids in Carya cathayensis leaf on regulation of visceral fat function in ovariectomized ratsObjective:In the second chapter,we found that the risk factors in ovariectomized rats such as the high levels of blood glucose,the ectopic fat accumulation and the largen of adipocytes.The dysfunction of visceral fat might lead to the occurrence of lipid metabolism disorder.Whether the shape and the morphology has changed of visceral fat,especially abdominal fat and the aorta fat.Whether the total flavonoids in Carya cathayensis leaf regulate fat metabolism by improving fat function.Therefore,we observe the effect of ovariectomy and the total flavonoids in Carya cathayensis leaf supplementation on visceral fat adipose tissue function in this chapter.Method:Lep,LepR,Adiponectin levels in serum were tested by Enzyme-linked immunosorbent assay(ELISA);Adiponectin mRNA,LEP MRNA and LEPR mRNA;PPA?mRNA of intra-abdominal adipose tissue were tested by Real-time PCR;PPA?VEGF C/EBP? protein of intra-abdominal adipose tissue was tested by Western Blot.Results:1.The effect of ovariectomy and the flavonoids in Carya cathayensis leaf supplementation on serum leptin,adiponectin:The results of Enzyme-linked immunosorbent assay(ELISA)showed that the flavonoids in Carya cathayensis leaf was able to raise the serum adiponectin levels in adipose tissue of normal diet rats.However,there was no significant difference of adiponectin levels.And there was no significant difference of serum adiponectin levels in adipose tissue of high nutrition diet rats.2.The results of Real-time PCR showed that the flavonoids was able to increase expression the levels of the adiponectin mRNA in adipose tissue of normal diet rats.Comparing with model group,ovariectomized rats had higher 25.86%,81.79%,66.23%mRNA adiponectin levels in adipose tissue.The adiponectin mRNA level in adipose tissue of high nutrition diet rats was similar to the model group,and that was same as adiponectin levels.3.The serum leptin levels in ovariectomized rats fed with high nutrition feed were significantly higher than the sham group rats.The serum leptin levels in adipose tissue were significantly lower than the normal diet rats.The flavonoids was able to increase the serum leptin levels in adipose tissue of normal diet rats(*P<0.05,??P<0.01).However,there was no significant difference of the expression levels of serum leptin mRNA in adipose tissue.The flavonoids was able to reduce the serum leptin levels in adipose tissue of high nutrition diet rats(*P<0.05).Comparing with model group,ovariectomized rats had low 18.43%,75.71%,26.56%serum leptin mRNA levels in adipose tissue of high nutrition diet rats.The flavonoids was able to reduce the expression levels of serum leptin mRNA in adipose tissue.And it can improve the LEP resistance,increase the sensitivity of LEP,and reduce the body weight by LEP.There was no significant difference of LEPR levels in adipose tissue.4.Ovariectomized rats had lower PPARy mRNA levels in adipose tissue.There was significant difference of PPAR? mRNA levels in high nutrition diet rats.And ovariectomized rats had 52.73%,50.30%,12.64%lower PPAR? mRNA levels in adipose tissue comparing with model group.The flavonoids could increase the expression of PPARy levels in adipose tissue to inhibit fat precursor cells to differentiate into adipocytes.Summary:1.The cardiometabolic risk factors excerbated in ovariectomized rats such as serum levels of LEP and adiponectin,the ectopic fat accumulation and the largen of adipocyte.2.The flavonoids was able to increase the expression levels of the adiponectin mRNA and the serum adiponectin in ovariectomized rats.And there was no significant difference of serum adiponectin levels in high nutrition diet rats,and that was same as adiponectin levels.3.The serum leptin levels in adipose tissue were significantly lower than the normal diet rats.The flavonoids were able to reduce the serum leptin and leptin mRNA levels in ovariectomized rats.And it can improve the LEP resistance,increase the sensitivity of LEP,and reduce the body weight by LEP.There was no significant difference of LEPR levels in adipose tissue.4.The flavonoids could reduce the expression of PPARy levels in adipose tissue to inhibit fat precursor cells to differentiate into adipocytes. |
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