| With the rapid development of traditional Chinese medicine and natural medicine resources,the cost of traditional Chinese medicine resources is greatly depleted,and "non-medicinal parts"such as plant roots and the above-ground stems and leaves,as well as "stumps" and broken tissues generated during the processing of medicinal herbs production areas are wasted.This has not only become a thorny issue for the development of the Chinese medicine industry,but also brought tremendous pressure on the ecological environment,limiting the development of the resource economy industry chain.The dry root and rhizome of Salvia miltiorrhiza Bge.is a traditional bulk medicinal material and is widely used.However,a large number of the above-ground stems and leaves of S.miltiorrhiza were discarded during the harvest process,which caused serious waste of resources and environmental pressure.The stem and leaves of S.miltiorrhiza contains phenolic acids,flavonoids and their glycosides,triterpenoids,polysaccharides and their glycosides,steroids,coumarins,a mino acids,proteins and volatile components and other components.The content of salvianolic acids is about 1 times than that in the root,the content of total flavonoid is more than 4 times than that in the root.Therefore,it is an effective way to develop the resource value of stem and leaf of S.miltiorrhiza by taking full advantage of the abundant resource components in it.Based on the previous research,we take the total phenolic acids in the stems and leaves of S.miltiorrhiza as the research object.And we aim to study its protective effect on multiple organ damage in diabetes,and explores its possible biological mechanisms.Our study is in order to provide the scientific basis for the development and utilization of stem and leaf of S.miltiorrhiza,and the improvement of resource utilization efficiency and benefits.The work of this paper is important to realize the industrialization of stem and leaf of S.miltiorrhiza and extend the industrial chain of Salvia,miltiorrhiza resource.This paper is divided into four chapters to carry out relevant research work:1 Literature researchThis chapter systematically studies the chemical composition and the pharmacological activities of the non-traditional medicinal parts of S.miltiorrhiza,as the stems and leaves.Simultaneously,we explored the multi-channel,multi-level resource value discovery and resource utilization model of it on the basis of the "three major utilization strategies" and "three types of resource models".We aimed to provide the basis for achieving the development and improving the utilization of stem and leaf of S.miltiorrhiza.2 Experimental research2.1 Evaluation of the value of total phenolic acids in stems and leaves of S.miltiorrhiza in the protection of multiple organ damage in diabetes2.1.1 Preparation of different chemical sites in stems and leaves of S.miltiorrhizaIn the context of industrial production,industrial ethanol was selected as the extraction solvent.With the content of total phenolic acids,total flavonoids and total triterpenoids as index,the optimum extraction process of stems and leaves from S.miltiorrhiza was evaluated.The column chromatography and extraction technology were used to purify different chemical sites in stems and leaves of S.miltiorrhiza.The optimum condition for the sample preparation was as follows:60%ethanol was used for reflux extraction of stems and leaves of S.miltiorrhiza(the ratio of solid to liquid was 1:10;extracted for twice;1.5 hours for each time).Alcohol extract from stem and leaf of S.miltiorrhiza(DJ)was obtained by concentrating the total extraction to dryness.AB-8 was selected to purify the various chemical components.Finally,total phenolic acid was obtained by eluting with 5 BV of 20%ethanol solution;total flavonoid was obtained by eluting with 5 BV 40%ethanol.Under these conditions,purity of phenolic acids could reach 45.86%,and the purity is not up to expectations.So,the extraction technology was combined to achieve the high purity extract.After the combination,the purity of total phenolic acids in stems and leaves of Salvia miltiorrhiza(JF)could reach 87.21%and the purity of flavonoid could reach 1.19%.2.1.2 Study on the protective effect of total phenolic acid in stems and leaves of S.miltiorrhiza on multiple organ damage in diabetesThe oxidative stress and high glucose injuried models were induced with hydrogen peroxide(H2O2)and high glucose.The cell damage model combined with type Ⅱ(T2DM)diabetic mice were used to evaluate the protective effect of JF on multiple organ damage in diabetes both in vitro and in vivo.We aimed to select the organs which JF had the best protective effect,and provid a basis for the development value of JF.(1)CardiovascularThe MTT results showed that JF had better protective effects on H2O2 and high glucose-induced HUVEC and H9C2 cells than total phenolic acids in root and rhizome of Salvia miltiorrhiza Bge(GF).The blood glucose and glycosylated protein levels increased,body weight decreased significantly after diabetic modeling.With the treatment,the blood glucose level of the ad ministration groups decreased compared to the model group.JF was most effective.In addition,DJ and JF not only reduced blood glucose,but also reduced the body weight in different degrees and improved the body condition of the T2DM mice.After modeling,the TG,T-CHO,LDL-C and HDL-C in serum of the T2DM mice were increased significantly,the activity of SOD decreased significantly,the activity of LDH and CK increased significantly.JF significantly relieved the serum oxidative damage and the atherosclerotic dyslipidemia in T2DM mice.JF had better effects on oxidative damage,hyperglycemia,and hyperlipidemia than GF.T2DM mice had imbalance of platelet diastolic and contractile,microcirculatory disturbances and endothelial dysfunction.High doses of JF significantly reduced the ratio of TXB2 to 6-keto-PGF1α,indicating that JF is superior to GF in relieving endothelial dysfunction.Histopathological sections of heart tissue showed that after being treated by high-fat diet combined with STZ injection,slight fibrosis in the myocardium,along with a large amount of lipid accumulation were observed in the T2DM mice,and the function of left heart decreased.DJ can prevent diabetes-induced myocardial remodeling,heart chamber expansion,and the decline of systolic function.The effect of GF on improvement of cardiac function and myocardial tissue damage in diabetic heart injury mice is superior to JF.In summary,JF have slightly less protective effects on diabetic heart damages than that of GF,but its regulation effects on diabetic vascular endothelial injury and endothelial dysfunction is better than roots.(2)Digestive organsJF had o BViously protective effect on H2O2 and high glucose-damaged IEC-6 and HISMC,even at low doses.JF could effectively alleviate the pathological changes caused by diabetes.The gastro-intestinal hormone disorder in T2DM mice,the concentration of motilin(MTL)and Gastrin(Gas)in the serum were significantly increased,and the concentrations of vasoactive intestinal peptide(VIP),glucagon-like peptide 1(GLP-1)and substance(SP)were significantly reduced.DJ and JF had an effect on improving the concentration of various gastrointestinal hormones.Moreover,JF was better than GF on improving the concentration of various gastrointestinal hormones.The serum alanine a minotransferase and aspartate a minotransferase activity of T2DM mice were increased significantly,which means liver function was impaired.Compared with the model group,the high and low doses of JF and the high doses of GF improved liver function significantly.As there was no o BVious pathological change in the stomach tissue,we have not studied about the gastric injury.(3)OthersIn vitro cell experiments showed that the protective effect of JF on hydrogen peroxide and high glucose damaged kidney cells HBZY-1 and HK-2 was superior to GF.But,the protective effect of JF on PC 12 cells daused by H2O2 and HSC cells damaged by high glucose damaged was lower than GF.Compared with the normal group,animal in model group showed wilting,weight lossing,and urine outputing were significantly increased.With ad ministration,the above physiological conditions were improved to varying degrees.In the model group,the cortex and medullar structure of kidney tissue were clear,glomerular was slightly enlarged and glomerular matrix hyperplasia,mesangial area was expansion.The swelling of renal proximal tubular epithelial cell was moderated,main performance for the volume of cell was increased,lightly stained cytoplasm,minimal hyperchromatism,unclear cavity margins,and no vacuolization.A small amount of inflammatory cell infiltration was observed in each drug-ad ministered group,the pathological changes of mice in the high-dose of DJ and JF was close to the normal.The kidney Index,urine volume and 24-hour urinary protein were increased o BViously after diabetic modeling.JF and DJ could reduce kidney Index,urine volume and 24-hour urinary protein,and the effects of JF were better than GF.STZ combined with high-fat diet could significantly reduce the thymus index,spleen index and the immunity of mice,indicating that the thymus and spleen of diabetic mice had certain toxic and side effects.The immune index of diabetic in mice was significantly increased by JF,DJ and GF.The protective effect on thymus index:GF>DJ>JF;protective effect on spleen index:JF>DJ>GF.Compared with normal mice,there was a small number of islets,irregular morphology,uneven cell distribution in the islets of T2DM mice.The pancreas structure was close to the normal group after the treatment,especially the high-dose group had a particularly significant protective effect on the pancreas of T2DM mice.Serum insulin levels in diabetic mice were significantly increased.Insulin levels in diabetic mice were significantly reduced by high doses of DJ and JF.The effect of JF on reducing insulin levels was better than GF.Studies have shown that JF have protective effects on multiple organ damage in diabetes,especially for the damage of intestinal tract and kidney.2.1.3 The protective effect of DJ on intestinal injury in type 1 diabetic mice and diabetic nephropathy rats,and intestinal toxicity in normal mice(1)Type 1 diabetesThe layer and mucosa thickness in the diabetic duodenum did not differ from the normal group.Whereas,the intestinal mucosa was altered in the ileum and colon of mice induced by STZ as compared to control group,with mucosa atrophy,intestinal villi shorter,lower number of crypts,distortion and zonal sclerosis of the la mina propria.JF ameliorates the levels of vasoactive intestinal peptide(VIP)and advanced glycation end products(AGEs).The protein expressions of zonula occludens-1(ZO-1)and occluding were reduced significantly in ileum and colon with the induction of STZ.With the treatment of DJ,the intestinal barrier was repaired due to the up-regulated expressions of ZO-1 and Occludin in the ileum and colon DM mice.However,consistent with pathological changes,no significant differences were observed among protein expressions of ZO-1 and Occludin in the duodenum of all groups.Besides,the mRNA expressions of TJP(ZO-1,Occludin,claudin-5)in colon tissue were detected by Q-PCR.The results revealed that the mRNA expressions of TJP(ZO-1,Occludin,claudin-5)were decreased after STZ injury and DJ ad ministration reversed the lower mRNA expressions of TJP significantly.The gut microbiota of each group was determined by 16SrRNA.With the treatment of DJ,the diversity of gut microbiota was increased.The ratio of relative abundances of phylum Firmicutes and Bacteroides was found to be significantly higher in DM animals compared to the control group.In contrast,the load of phylum Deferribacteres was significantly lower in DM group vs.the control group.However,the Firmicutes/B act eroidetes ratio and the relative abundances of Deferribacteres in DM mice were significantly improved upon treatment of DJ.After DJ treatment,the lowered relative abundances of Anaerotruncus coliho minis,Mucispirillum schaedleri and Butyricimonas virosa in DM mice were increased significantly.In addition,the relative abundances of Proteus hauseri and Helicobacter winghamensis were decreased significantly by DJ in high dose or low dose vs.the model group.UniFrac PCoA(principal co-ordinate analysis)of OTUs showed that the samples in the model group were well separated from those in the control group based on the weighted UniFrac distances.(2)Diabetic nephropathy ratsThe gastrointestinal hormones were disordered in rats with diabetic nephropathy,the serum levels of VIP and SP in the serum of rats were significantly decreased,and the concentration of SS was significantly increased in diabetic nephropathy rats.The serum gastrointestinal hormone concentrations in rats were improved by DJ.The results of intestinal pathology in diabetic nephropathy rats showed that the intestinal lesions were mainly concentrated in the proximal rectum,showing atrophy of the intestinal glands and atrophy and thinning of the mucosal layer.After intervention,intestinal damage was improved,especially in the high-dose group.After the ad ministration of intervention,the decreased TJP expression in the intestine of rats with diabetic nephropathy was significantly increased.(3)Normal miceBesides,we detect the enterotoxicity of DJ on normal mice.DJ was effect on controlling the weight and blood sugar of normal mice at 4th week.Besides,the level of VIP in serum,the protein and mRNA expression of ZO-1,Occludin Claudin-5 of ileum or colon tissue was increased but insignificantly with DJ treatment.16S rRNA-based molecular sequencing analysis of intestinal contents revealed that number of Proteobacteria was significantly increased while Tenericutes was significantly decreased in DJ group compared to the control group at phylum level.At genus level,the relative abundances of Odoribacter,Anaeroplasma,Clostridium XlVb,Lactococcus and Pseudomonas were decreased significantly,whereas the relative abundances of Dehalobacterium and Lachnospira were significantly increased by DJ.UniFrac PCoA of OTUs showed that the samples in the DJ group were similar with those in control group.2.2 Study on the protective mechanism of JF on the diabetic intestinal injury2.2.1 Intestinal mechanical barrierAfter DM mice models were induced by high-fat diet combined with STZ,the levels of DAO and Zonulin were significantly higher than those in normal mice in serum of diabetic mice.The high doses of JF significantly reduced the levels of DAO and Zonulin in serum of T2DM mice,indicating that JF can significantly repair the damage of intestinal mucosal barrier in T2DM mice and improve intestinal permeability of model group mice.Compared with control mice,tight junction proteins expression of ZO-1,Occludin,Claudinl,and Claudin5 were down-regulated significantly in T2DM mice.DJ and JF significantly increased the expression of intestinal TJP in T2DM mice,especially the effect of improving protein expression of Occludin,Claudinl,and Claudin5 was better than that of the GF.The effect of GF on the Occludin,Claudinl and Claudin5 protein of intestinal tract was not significantly different from that of the model group.Compared with the model group,the expression of ZO-1 was significantly improved in all ad ministration groups.Especially the high and low doses groups of JF can significantly improve the expression of ZO-1 protein in T2DM mice.Moreover,compared with normal mice,the expression of β-catenin,E-cadherin protein in intestinal tissue of T2DM mice was significantly increaseThe results showed that expression level of proteins each administration groups was reversed to the normal group.However,the low dose of the ad ministration group had no significant effect on the expression ofβ-catenin protein.Compared with the control mice,the expression of the tight junction proteins of ZO-1,Ocludin,Claudinl and Claudin5 in T2DM mice was significantly decreased.After ad ministration of low doses of DJ,the mRNA expression of ZO-1,Claudinl and Claudin5 in the intestine of T2DM mice was not significantly improved,while the expression of TJP mRNA in the intestine of mice in the other groups was significantly increased.2.2.2 Enteric neurons and intestinal motilityCompared with normal mice,proteins expression related T2DM mouse enteric nervous system of acetylcholine transferase(CHAT),protein gene product 9.5(PGP9.5),neurotrophic factor(GDNF)and neuronal nitric oxide synthase(nNOS)was increased significantly.These results indicate that the enteric nervous system of diabetic mice is reactively activated.The increase in the number of nerve fibers may be reactive hyperplasia.Increased nerve fibers may secrete excessive neurotransmitters and cytokines and increase the nerve impulse transmission pathway,thereby promoting the intestinal motility and secretion.Increased peristalsis and secretion,and then participate in the production of diabetic intestinal diarrhea.JF and GF can significantly reduce the expression of related proteins,repair the disordered enteric nervous system in diabetic mice,and relieve the symptoms of diabetic diarrhea.Cajal mesenchymal cells are the main expression cells of tyrosine kinase receptor c-Kit.The ligand of c-Kit is hematopoietic stem cell growth factor(SCF),which induces ICC extracellular tyrosine kinase after binding.Then,phosphorylation further regulates multiple signaling pathways between cells.After the mice were induced with diabetes,the stools became sparse and diarrhea.Compared with normal mice,the expression of c-Kit and SCF mRNA in the intestine of DM mice was significantly increased,SCF/c-Kit signaling pathway was activated,intestinal motility was disturbed,and diarrhea was induced.After ad ministration,the DJ and total phenolic acids significantly decreased the expression of c-Kit and SCF protein and mRNA and regulated the intestinal motility.2.2.3 Immune barrierAfter diabetic model was established,the PI3K/Akt/NF-KB p65 signaling pathway was activated in the intestine of mice,which showed that PI3K,NF-κB p65 protein expression,p-IKKa/IKKa and p-Akt/Akt ratios were significantly increased,the phosphorylation levels of IKKa and Akt were increased significantly.After ad ministration,the level of each protein in the pathway decreased,and JF significantly decreased the ratios of p-IKKa/IKKa and p-Akt/Akt even at low doses.The effect is better than that of GF.The protein and mRNA expressions of TNF-a,IL-1β,and IL-6 in the intestine of diabetic mice were significantly increased.After ad ministration,the levels were significantly decreased.DJ and JF significantly reduced the expression of IL-6 and TNF-a protein,but DJ did not significantly down-regulate IL-1β.The results showed that JF can reduce the intestinal inflammatory response in mice by inhibiting the PI3K/Akt/NF-KB p65 signaling pathway,thereby protecting diabetic intestinal damage.2.2.4 Intestinal microbiota and their metabolitesAfter the drug intervention,the diversity of intestinal microbiota in diabetic mice was increased,especially the diversity index of JF.Metastats analysis showed that the abundance of the phylum Bacteroides,Bacteroidetes and Tenericutes in T2DM mice was significantly reduced.The change in the relative abundance of the bacteria was reversed after ad ministration.In particular,JF significantly reduced the ratio of Firmicutes/Bacteroidetes at the phylum level.Based on the level of genus,compared with the blank control group,abundance of Alistipes,Butyricimonas,Akkermansia,Mucispirillum,Lactococcus,Streptococcus,Anaerotruncus,Burkholderia and Ralstonia was decreased,while abundance of Proteus and Odoribacter significantly increased,which indicated that the structure of the intestinal microbiota in mice has changed.DJ and JF significantly increased the abundance of Lactobacillus,Streptococcus、Mucispirillum、Akkermansia、Burkholderia and Butyricimonas,reduced the abundance of Proteus and Odoribacter.Six types of short-chain fatty acids(SCFAs)in fresh feces from each group of mice were measured.The contents of intestinal SCFAs in the model group were significantly lower than those in the normal group.After treatment,the content of SCFAs in feces of treatment groups increased to varying degrees.The change trends of propionic acid,butyric acid and isovaleric acid content were most o BVious,but the increase of acetic acid and isobutyric acid content was not significant.The acetate acid content was increased significantly only in the high dose of GF,the isobutyric acid content was only significantly increased at high doses of DJ.The effect of JF on reversing the content of propionic acid and butyrate in the fecal of T2DM mice was significant.The content of propionic acid and butyric acid were similar to those in the blank group,indicating that the improvement of JF on improving intestinal damage may through increasing the content of short-chain fatty acids in feces of T2DM mice.2.3 The metabolomics study of JF in regulating diabetic organ damagesDJ,JF and GF can regulate 20 endogenous markers of diabetes mellitus with multiple organ damage,10 in plasma and 10 in urine.JF reversed differential metabolites in serum and urine of diabetic mice.Cortolone-3-glucuronide,15(S)-HPETE,leukotriene A4 levels in groups were reduced,and 2-Methyl-1-hydroxypropyl-ThPP,3-A minoisobutanoic acid,LysoPC(20:4(8Z,11Z,14Z,17Z),Cholic acid glucuronide and Thromboxane were increased.Four metabolic pathways such as pentose and glucuronate interconversions,arachidonic acid metabolismhave,methane metabolism and Taurine and hypotaurine metabolism also found to be improved.After ad ministration of JF,there were 19 differential metabolites in the colon tissue of diabetic mice.Among them,the content of imidazole-4-acetaldehyde,phenylpyruvic acid,deoxyribose,cytidine triphosphate,AICAR,Nicotinate D-ribonucleoside,Nervonic acid,LysoPC(16:0),D-Xylono-1,5-lactone,Chenodeoxycholic acid,6-Mercaptopurine ribonucleoside triphosphate and PC(16:1(9Z)/14:1(9Z)were significantly decreased,while the level of Ceramide(d18:1/12:0),11,12,15-THETA,13-OxoODE,17a-Hydroxypregnenolone,LysoPC(16:0)and Ceramide(d18:1/18:0)were increased.8 differential metabolites identified in kidney tissue,including gamma-A minobutyric acid,norepinephrine,PA and LysoPC(18:3(9Z,12Z,15Z))were lowered,and the level of epinephrine was increased.7 differential metabolites was found in the liver,including ceramide(d 18:1/12:0),phosphorylcholine,normetanephrine,glycosylceramide(d18:1/12:0)levels were decreased,arachidonic acid levels were increased.Analysis of metabolic pathways revealed that JF regulates abnormal sphingolipid metabolism,phenylalanine metabolism and glycerophospholipid metabolism pathways in the colon tissues;regulates starch and sucrose metabolism pathway in heart tissue;sphingolipid metabolism,selenoa mino acid metabolism,arginine and proline,alanine,aspartate and glutamate metabolism and tyrosine metabolism pathways in kidney tissues.Also regulates sphingolipid metabolism pathway in liver tissue.2.4 Study on molecular mechanism prediction of stems and leaves from S.miltiorrhiza for the treatment of diabetes mellitus and its multiple organ complications based on the network pharmacologyNine main compounds in stems and leaves of S.miltiorrhiza were slected as research object,and network pharmacology research methods was used to study the correlation among multicomponent,multi-target,signal pathway of diabetes and its multiple organ complications.In order to provide a basis for further clarifying the molecular mechanism of stems and leaves from S.miltiorrhiza in treating diabetes mellitus and its multiple organ complications.The network pharmacological analysis showed the interaction network of 9 compounds/110 protein targets and 29 signal pathways.It showed that Insulin,MAPK,and Adherens junction signal pathways were ranked ahead of each other in the network(the number of targets acting on the pathway is greater than or equal to 8)and these pathways are the hub of the network.The result can suggest that these three pathways may be the main pathways for stems and leaves of S.miltiorrhiza to protect against diabetes and its complications.And the mechanisms may be related to the regulation of glucose and lipid metabolism,improvement of vascular permeability and anti-inflammatory.Several a mino acid metabolic pathways,which was related,echoes the metabolomics study on the regulation of diabetes mellitus organ damage of stems and leaves from Salvia miltiorrhiza.In addition,it showed multiple a mino acid metabolic pathways,which echoes the metabolomics research on the regulation of multiple organ damage in diabetes.The research results of our topic showed that to develop a protective agent of JF in diabetic intestinal lesions is a feasible approach for the utilization of DJ.The protective mechanism we studied provides basis for the further development of JF.It is of great significance for realizing the comprehensive development and utilization of DJ and improving the efficiency and efficiency of its resource utilization. |
PDF Full Text Request