Comparative Study On Lipid-Lowering And Anti-Inflammatory Activities Of Gynosteme Pentaphylla In Different Processing Methods

Gynostemma pentaphyllum(Thunb.)Makino is the whole grass of Gynostemma pentaphyllum in Cucurbitaceae.It is famous as ’Southern Ginseng ’ because it contains ginsenosides.It has the effects of clearing heat and detoxifying,relieving cough and removing phlegm,invigorating spleen and replenishing qi,nourishing heart and tranquilizing mind.It can be used as herbal medicine,food and drink.After harvesting,its leaves are often made into tea through procedures such as deactivation,dehydration,rolling and drying.It mainly distributed in Shaanxi and Fujian.The traditional drying method of Gynostemma pentaphyllum is drying in the shade,but modern high temperature deactivation is mostly used.The previous study in our laboratory found that the composition of gypenosides in G.pentaphyllum dried with high temperature deactivation was different from that in shade-dried G.pentaphyllum.Most of the G.pentaphyllum after deactivation were contained trisaccharide saponins and tetrasaccharide saponins,while the proportion of monosaccharide saponins and disaccharide saponins were increased in shade-dried samples.Rare saponins were reported with higher bioavailability,more easily absorbed by the human body,and stronger pharmacological activity.However,the efficacy of different processing methods of Gynostemma pentaphyllum is different and lacks systematic comparison.Therefore,our study intends to compare the differences in chemical components of G.pentaphyllum with different processing methods.Based on this,the pharmacological activities of lipid-lowering and anti-inflammatory were studied,and the mechanism of gypenosides improving NAFLD was elucidated.Mainly experiments and results were as follows:1.Gynostemma samples collectionA total of 20 batches of Fujian G.pentaphyllum samples were collected including 10 batches of high-temperature de-enzyming dried samples and 10 batches of shade-dried samples in the natural environment.2.Comparative study on chemical constituents of G.pentaphyllum by different drying methodsUPLC-Q-TOF-MS was used to characterize the chemical constituents of G.pentaphyllum processed by different methods.A total of 80 saponins were identified,including 12 tetrasaccharide saponins,28 trisaccharide saponins,18 disaccharide saponins,19 monosaccharide saponins and 3 aglycones.In the shade-dried samples,the proportion of tetrasaccharide saponins decreased,the proportion of trisaccharide saponins remained unchanged,and the proportion of disaccharide saponins,monosaccharide saponins and aglycones increased significantly.3.Comparative study on hypoglycemic and lipid-lowering activity of G.pentaph yllum by different drying methods.3.1 Comparison of lipid-lowering activity in vitro based on FXR receptorIn this study,G.pentaphyllum from Fujian Province was selected,and the total saponins(SQGPs,YGGPs)of two different processing methods were isolated and purified by macroporous resin,and two different parts were prepared as monosaccharide disaccharide saponins(GPs1-2).The effects of SQGPs and YGGPs on the expression of Fxr and target genes in Caco-2 cells,MODE-K and mouse primary hepatocytes were determined to investigate the effects of different processing methods on the pharmacological activity of total saponins in vitro.The results showed that YGGPs could significantly increase the expression of Fxr,Fgf15,Osta and Ostβ genes in MODE-K cells and Ibabp gene in Caco-2 cells,and regulate the expression of Fxr and target genes Mrp3,Ntcp,Shp,Cyp7a1 and Ostβ in mouse primary hepatocytes.SQGPs could only significantly increase the expression of Ibabp gene in Caco-2 cells and Fgf15 in MODE-K cells,and the expression of Mrp3,Ntcp and Shp in mouse primary hepatocytes.Although SQGPs also had a certain regulatory effect on gene expression,it was not as strong as YGGPs.The experimental results indicate that there are significant differences in the lipid-lowering activity of gypenosides with different processing methods in vitro.The dried samples have stronger pharmacological activity,which may be due to the role of rare saponins,and it is possible to improve the metabolic phenotype of non-alcoholic fatty liver(NAFLD)mice through FXR.3.2 Comparison of lipid-lowering and hypoglycemic activities in vivo based on NAFLD mouseIn this section,NAFLD mouse model was established by high-sugar,high-fat and high-cholesterol diet,and 100 mg·kg-1 dose of non-tamine and 200mg·kg-1 dose of SQGPs and YGGPs were used as therapeutic drugs for six weeks.By observing the effects of SQGPs and YGGPs on body weight,food intake,liver weight to body weight ratio,adipose tissue weight,serum biochemical indexes,histopathology,glucose tolerance and insulin tolerance in NAFLD mice,the effects of different processing methods on the lipid-lowering and hypoglycemic activity of gypenosides in vivo were explored.The results showed that YGGPs had a stronger lipid-lowering and hypoglycemic effect without affecting food intake.From the second week,YGGPs could significantly inhibit weight gain,reduce the weight ratio of liver weight to body weight and the weight of iWAT and eWAT,reduce the content of TC,TG and ALT in serum and liver,prevent lipid deposition,and have a certain hepatoprotective effect.It can also be seen from pathological tissue sections that YGGPs can reverse fatty liver and adipose tissue cell enlargement caused by high-sugar and high-fat diet,improve lipid accumulation in liver and fat,and YGGPs can reduce fasting blood glucose.The AUC values of glucose tolerance test and insulin tolerance test were decreased,and glucose metabolism was improved.Although SQGPs improved these detection indicators,they were not as good as YGGPs.4.Study on lipid-lowering activity mechanism of gypenosidesThis section mainly uses PCR,WB and energy metabolism cage experiments to explore whether gypenosides treat NAFLD by activating FXR.The experimental results show that YGGPs may increase the expression of Fgf15,Ibabp,Ost-α and Ost-β genes by activating intestinal FXR,promote the secretion of intestinal sex hormone FGF15,enter the liver,intestine and systemic circulation,and activate liver FXR,increase the expression of target genes Shp and Fgfr4,inhibit Srebp1,regulate liver lipid metabolism,and reduce the expression of genes related to lipid synthesis and fatty acid absorption.Increase the expression of fatty acid oxidation related genes,reduce the expression of Cyp7a1 and Cyp27a1 genes related to bile acid synthesis,reduce the total amount of bile acids in serum,liver and ileum,increase the total amount of bile acids in feces,promote bile acid circulation,and avoid toxic and side effects caused by bile acid deposition.On the other hand,FGF15 can also activate the expression of ADRB3 in adipose tissue,regulate the lipolysis pathway ADRB3-PKA-HSL,promote the decomposition of white adipose tissue,reduce the excessive accumulation of lipids in the body,and provide raw materials for energy metabolism.The energy metabolism cage experiment showed that YGGPs significantly increased O2 consumption and CO2 exhalation in NAFLD mice,and promoted body heat production.The above experimental results prove that the possible mechanism of YGGPs in improving NAFLD is to promote the secretion of FGF15 in the ileum by activating intestinal FXR,thereby regulating liver glucose and lipid metabolism and energy metabolism.5.Comparative study on anti-inflammatory activity of G.pentaphyllum by different drying methods in Fujian5.1 Comparison of anti-inflammatory activity in vitro based on LPS-induced RAW264.7 cell inflammationIn this section,100 ng·mL-1 LPS was used to induce inflammatory response in RAW264.7 cells,and the anti-inflammatory activities of SQGPs,YGGPs and their differential site GPs1-2 were investigated.The NO release rate was measured at 24 h after LPS modeling for 1 h.It was found that GPs1-2 had the best anti-inflammatory activity,followed by YGGPs and SQGPs.SQGPs significantly inhibited NO release from RAW264.7 cells at 150 μg·mL-1,and the inhibition rate was 13.83%at 200μg·mL-1.YGGPs significantly inhibited NO release at 100 μg·mL-1,and the inhibition rate was 35.97%at 200μg·mL-1.GPs1-2 significantly inhibited NO release at 25μg·mL-1.The inhibition rate was 63.06%at 200 μg·mL-1,and the NO release rate of RAW264.7 cells was almost the same as that of the blank group at 100 μg·mL-1.This shows that the anti-inflammatory components are mainly GPs1-2.5.2 Comparison of anti-inflammatory activity in vivo based on LPS-induced ALI modelThis section focuses on the improvement effect of SQGPs,YGGPs and GPs1-2 on acute lung injury(ALI)animal models.The ALI mouse model was constructed by bronchial spray of 5 mg·kg-1 LPS.After 24 h,dexamethasone and 25 mg·kg-1,50 mg·kg-1,100 mg·kg-1 YGGPs were administered by tail vein.The lung function,protein concentration of inflammatory factors in BALF,pathological section of lung tissue and wet-dry ratio of lung tissue were detected to evaluate the improvement effect of different concentrations of YGGPs on ALI.It was found that the improvement effect was dose-dependent.With the increase of dosage,the improvement of each detection index gradually increased.Because the effect of 50 mg·kg-1 and 100 mg·kg-1 was significantly better than that of 25 mg·kg-1,the dosage of SQGPs and YGGPs was determined to be 50 mg·kg-1.The proportion of GPs1-2 in GPs was low,so 20 mg·kg-1 was selected for tail vein administration after modeling.The results showed that after administration,the Ti,Te and Penh were significantly reduced compared with LPS group,f,TV,EF50,PIF,PEF and Volbal were significantly higher than those in CTRL group.The protein levels of IL1-β,IL-6 and TNFa in BALF were significantly decreased,which alleviated lung inflammation and improved lung inflammation.The improvement effect of SQGPs was worse than that of YGGPs at the same dose.The effect of 50 mg·kg-1 YGGPs was equivalent to that of 20 mg·kg-1 GPs1-2,indicating that there were differences in the anti-inflammatory activity of GPs by different processing methods.The rare saponins produced by the unsterilized sample had stronger anti-inflammatory activity.The results of pharmacological activity evaluation of lipid-lowering and anti-inflammatory activities in vitro and in vivo indicated that the pharmacological activity of traditional shade-dried Gynostemma pentaphyllum was better than that of roller-dried Gynostemma pentaphyllum.The above research results are helpful to scientifically guide the processing technology of Gynostemma pentaphyllum,ensure the production of high-quality Gynostemma pentaphyllum medicinal materials,and provide a demonstration for scientific evaluation of modern processing technology of medicinal materials.