| This thesis is composed of four chapters.Chapter 1.Document researchThe chemical composition and pharmacological action of the stems and leaves of S.miltiorrhiza were systematically summarized,which provided scientific evidence for the exploitation and industrialization of the stems and leaves of S.miltiorrhiza.In this paper,the advantages and applications of the non-rodent model organisms—Drosophila melanogaster,Ccaenorhabditis elegans and zebra fish in drug screening and efficacy evaluation were reviewed.For the rodent model based on the modern research,flies,worms and zebrafish in drug screening and evaluation of the advantages and application were summarized,which provides a powerful reference for the establishment and development of non-rodent mode organisms platform.Chapter 2.Evaluation on the anti-aging activity of the stems and leaves of S.miltiorrhiza based on Drosophila and C.elegans1.The content of extract(SM and DJ)and total phenolic acid(SMPA and D JPA)of root and the stems and leaves of S.miltiorrhiza were measured based on UPCL.The result show that the total amount of phenolic acids in root and the aerial parts was similar—SM:13.58%,DJ:12.04%,SMPA:66.31%and DLPA:13.58%.The results showed that the content of phenolic acids in the root as much as that in stems and leaves.2.The anti-aging activity of extracts and total phenolic acids from roots of S.miltiorrhiza and the stems and leaves of S.miltiorrhiza were evaluated by lifespan.There was no significant difference between each groups in food intake,which suggested there was no significant effect on the feeding behavior and body mass of the fruit flies.Results show that compared with the control group,the life span of fruit flies was increased to some extent after treatment of SM,DJ,SMPA and DJPA.It was suggested that the extracts and total phenolic acid of roots of S.miltiorrhiza or the stems and leaves of S.miltiorrhiza had significant anti-aging activity.After the oxidative damage of 6.0 mmol·L-1 PQ or 0.6%SDS,the survival rate of the fruit flies in group M was significantly lower than that in group N(P<0.001).Compared with group M,the survival rates of the fruit flies showed no significant change,suggesting that both the extracts of roots of S.miltiorrhiza or the stems and leaves of S.miltiorrhiza were unable to reverse the lethality caused by 6.0 mmol·L-1 PQ or 0.6%SDS.3.The antioxidant activity and its mechanism of extracts and total phenolic acids from S.miltiorrhiza and the stems and leaves of S.miltiorrhiza were evaluated by weight,SOD and CAT activity,intestinal epithelial cell apoptosis index and number of intestinal stem cells of fruit flies.It was proved that the extracts of S.miltiorrhiza and its aerial parts had significant prolongation of life.The SOD and CAT activity of fruit flies were improved(P<0.05).The root and the stems and leaves of S.miltiorrhiza has antioxidant activity,which may be one of the reasons why flies live longer.There was no significant difference between groups in weight,which suggested extracts and total phenolic acids from S.miltiorrhiza and the stems and leaves of S.miltiorrhiza have no significant effect on the body mass of the fruit flies.The extract of the stems and leaves of S.miltiorrhiza has certain protective effect on the rapid aging model induced by PQ.It was able to reduce the apoptosis rate of intestinal epithelial cells(P<0.05)and reduce the number of intestinal stem cells(P<0.05).It is indicated that the stems and leaves of S.miltiorrhiza have significant anti-aging effect,which may be one of the reasons for its prolonging life.4.Our object is to evaluate anti-aging activity of extracts and total phenolic acids from roots of S.miltiorrhiza and the stems and leaves of S.miltiorrhiza based on C.elegans by lifespan.The results showed that,compared with N group,The average and maximum life span of nematode worms was increased to varying degrees after treatment of SM,DJ,SMPA and DJPA.5.Our object is to evaluate mechanism of antioxidant of extract and total phenolic acids of root and the stems and leaves of S.miltiorrhiza activity based on C.elegans,in terms of active oxygen content,head oscillation,body bending frequency,pharyngeal pump behavior,peroxidase and superoxide dismutase activity and anti-oxidation-related gene expression.Compared with the control group,there was no significant difference in the frequency of head oscillations,pharyngeal pump behavior and the body bends after the treatment of SM or DJ.But the relative expression of sod-1,ctl-2,mtl-1,mtl-2,hsp-16.1 and hsp-16.2 were up-regulated significantly after the treatment of DJ.And the relative expression of sod-1,sod-2,ctl-2 and mtl-1 were up-regulated significantly after the treatment of SM.Compared with the control group,the ROS content in the intestinal tract of the model group was significantly increased(P<0.01).There was no significant difference in the frequency of head oscillations,and pharyngeal pump behavior.The body bends were significantly increased and the CAT and SOD activity decreased significantly(P<0.01)after PQ induction(P<0.05).Compared with model group,nematode intestinal ROS levels and the body bends have been significantly reduced(P<0.05).And CAT activity significantly increased relatively after the treatment of SM or DJ.It was showed that the stem leaf extract has antioxidant activity,and it may be associated with the expression of sod-1,ctl-2 and mtl-1 which related with oxidative stress.Chapter 3 Study on the biological activity of the stems and leaves of S.miltiorrhiza in the Drosophila with metabolic disorders.1.The objective is to evaluate the effect of stems and leaves of S.miltiorrhiza(DJ)on a Drosophila model of diet-induced insulin resistance by food intake,body weight,glucose,triglyceride,protein and pupa time.Compared with group N,the level of glucose and triglyceride of flies in group M were significantly increased(P<0.05).Compared with group M,the glucose level of flies in group GLZ,DJ-M and DJ-H were significantly decreased(P<0.05 or P<0.01).Compared with the M group,the TG level of insects in group GLZ and DJ-H were significantly decreased(P<0.05 or P<0.01).2.To explore the mechanism of activity of stems and leaves of S.miltiorrhiza(DJ)based on Drosophila model of diet-induced insulin resistance by related expression of genes which take part in oxidative stress and blood sugar regulation.The results showed that compared with group N,the expression of dFOXO,Ilp5 and 1(2)efl in the group M were significantly down-regulated,and upd2 was significantly up-regulated.Compared with group M,the expression of dFOXO,l(2)efl were significantly raised in groups GZL.The expression of Ilp5 were significantly up-regulated in group SM,and the expression of srl and upd2 significantly down-regulated.he expression of Ilp5 and l(2)efl rised significantly up-regulated in DJ group,and the expression upd2 were significantly decreased.Compared with the group M,the metabolic level of the flies was adjusted after the DJ intervention,and 17 potential biomarkers and 4 main metabolic pathways were analyzed.It suggested that DJ in fruit flies may regulate the expression of dFOXO,Ilp5,1(2)efl and upd2,affect the metabolism of histidine,glycerol phospholipid metabolism,pentose and glucuronic acid salt interactions and glyceride metabolism and other metabolic pathways to regulate glucolipid metabolism of flies back to normal.Chapter 4 Study on the value evaluation of stems and leaves of S.miltiorrhiza based on zebrafish model1.With 25 ?mol·L-1 prednisone dragon induced osteoporosis zebrafish model is set up.Zebra fish head bone dyeing area and the optical density value as an index.15 ?g mL-1 Disodium ethylphosphonate for positive drugs to explore the stems and leaves of S.miltiorrhiza total phenolic acids(12.5,25,50,100,200 ?g mL-1)effect on zebrafish model of osteoporosis.The results showed that compared with the control group,the head bone staining area and staining light density were significantly decreased in the model group(P<0.01).Compared with model group,positive drug group and the stems and leaves of S.miltiorrhiza total phenolic acids(100 ?g·mL-1 group)can make zebrafish head bone dyeing area increased significantly(P<0.05),positive drug group and the stems and leaves of S.miltiorrhiza total phenolic acids(50,100 and 200 ?g·mL-1 group)of dyeing optical density value increased significantly(P<0.01 or P<0.05).It showed that the total phenolic acid of the aerial parts of S.miltiorrhiza had remarkable anti-osteoporosis activity.2.By using phenylhydrazine to induce zebrafish thrombosis model,the blood circulation and blood stasis of the stems and leaves of S.miltiorrhiza were evaluated by taking aspirin as the positive drug.The results showed that compared with the control group,the tail vein of zebrafish in the model group formed a significant thrombosis,and the staining strength of the heart erythrocyte decreased significantly.Compared with model group,positive drug group and extract of the stems and leaves of S.miltiorrhiza 50?200 ?g·mL-1 set of heart red staining intensity and antithrombotic efficiency significantly increased(P<0.05 or P<0.001)and S.miltiorrhiza stem leaf extract EC50 is 57.17 g·mL-1.The extract of the stems and leaves of S.miltiorrhiza can effectively inhibit thrombosis of the model of zebrafish.The vascular defect model of zebra fish was induced by the VEGF receptor tyrosine kinase inhibitor ?,and the vascular integrity index of the internode was evaluated to evaluate the angiogenesis of the stems and leaves of S.miltiorrhiza.The results showed that compared with the control group,zebrafish had significantly lower internode vascular index,and the expression of kdrl,flt-1,vWF and VEGF-A were significantly down-regulated.Compared with the model group,the stems and leaves of S.miltiorrhiza can significantly increase the number of internode vessels(P<0.05 or P<0.01),and the expression of kdrl,flt-1,vWF and VEGF-A were significantly up-regulated.It suggested that the extract of the stems and leaves of S.miltiorrhiza has significant pro-angiogenic activities,and it concerned with the expression of kdrl,flt-1,vWF and VEGF-A whitch regulating angiogenesis. |
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