| This dissertation included five chapters.Chapter one,Literature ReviewA detailed review was performed to the medicinal resources of Salvia miltiorrhiza Bge.（SM）including the herbal textual research,variety and distribution of resources,chemical constituents and pharmacological activities,as well as the growth and harvest pattern of SM.Resource chemical constituents and the pharmacological activities on aerial parts of SM in recent years were summarized and analyzed,the value and utilization ways of the resources were also put forward in order to provide basis and reference for the systematic utilization and industrialization development of SM resources.Research progress on genomics,proteomics,and metabonomics in recent years were carried out to summarize the biosynthesis pathway,key enzymes genes,regulation mechanism and bioconversion and utilization of phenolic acids in SM,which laid scientific foundation for the efficient production and comprehensive utilization of phenolic acids resources in SM.Chapter two,Study on the chemical constituents of Salvia miltiorrhiza 1.The sensitive and fast ultra high performance liquid chromatography-triple quadrupole mass spectrometer（UPLC-TQ/MS）method was developed for simultaneous quantitation of the salvianolic acids,flavonoids,tanshinones and triterpenes in various parts of SM during different growth period.The results indicated that content changing trend of salvianolic acids in roots of SM showed"M" shape,and the content of salvianolic acids reached the highest in the spring seedling stage（April to May）and the vigorous growth period of aerial parts（July to August）,which as high as 6.598%.As for the tanshinones in roots of SM,it reached the highest of 0.716%at the turning green stage of next year.The chemical components of the aerial parts of SM were various and abundant,which can be taken into consideration of the new source of salvianolic acids.As a matter of fact,determination about the suitable harvest season of the aerial parts of SM should be evaluated according to the actual demand,supposing that the salvianolic acid as the main evaluation index,July and August as the optimum harvest time of the aerial parts of SM was recommended.2.The high performance liquid chromatography coupled with an evaporative light scattering detector（HPLC-ELSD）method was established for the detection analysis of four saccharides in various parts of SM during different growth period,mainly for fructose,glucose,sucrose and stachyose.The results revealed that in roots of SM,it contains a decent amount of stachyoses,and the content reached maximum at withering period（29.97%）.Content of monosaccharides（fructose and glucose）in stems showed the highest in wilting and seedling stage,disaccharide（sucrose）and oligosaccharide（stachyose）accumulate the maximum in the vigorous growth period of the aerial parts in July.In leaves of SM,monosaccharides（fructose and glucose）accumulate the maximum at May,with the contents of 3.73%and 4.62%,and content of disaccharide（sucrose）and oligosaccharide（stachyose）reached highest in July,at which period the photosynthesis was the strongest.Content of monosaccharides（fructose and glucose）in the flowers were the highest at the middle of May（full-bloom stage）,with the fructose of 6.91%and glucose of 5.20%,and content of disaccharide（sucrose）and oligosaccharide（stachyose）attained the highest at the mid to end of June（fructescence）.3.The UPLC method combined with similarity evaluation system for chromatographic fingerprint of TCM（2004A）was used for establishing the reference fingerprint and similarity evaluation,and the principal component analysis（PCA）and clustering analysis was carried out by SPSS 19.0.The characteristic fingerprint between SM and SMSL was established and compared,and the contents of seven kinds of salvianolic acids（danshensu,protocatechuic aldehyde,caffeic acid,rosmarinic acid,lithospermic acid,salvianolic acid B and salvianolic acid A）were simultaneously determined.Results showed that fingerprint of water extracts of SM and SMSL have a certain similarity,which up to 0.981,and the salvianolic acids were identified to be common components.Content determination showed that significant difference existed between different batches of SM and SMSL,while the species composition of the salvianolic acids was consistent.4.The fingerprint of alcohol extracts of S.miltiorrhiza stems and leaves（SMSL）was established by UPLC,Similarity evaluation,cluster analysis（CA）,and principal component analysis（PCA）were used to deal with the experimental data,specific chromatogram of SMSL was obtained,and 13 common peaks were identified.Similarities of the samples were 0.823～0.997,the results of CA and PCA were consistent with similarity evaluation.The establishment of UPLC fingerprint of SMSL and the application of chemical pattern recognition can provide a more comprehensive reference for the quality control of SMSL and the utilization value of non-medicinal parts of SM.Chapter three,Correlation analysis between expression level of key enzyme genes and active components accumulation in different parts of Salvia miltiorrhiza during different growth period1.The expression level of five key enzyme genes（SmPALl,Sm4CLl,SmHPPR,SmRAS and SmC YP98A14）which are related to the salvianolic acid synthesis and the five key enzyme genes（SmAACT,SmDXR,SmGGPPS,SmCPS and SmKSL）which are related to the tanshinone synthesis in various parts of SM during different growth period were investigated by fluorescent quantitative PCR.As to the key enzyme genes that are related to the salvianolic acid synthesis,the relative expression of SmC YP98A14,SmPALl and Sm4CL1 were high,and the SmCYP98A14 as the key enzyme in the downstream pathway of rosmarinic acid biosynthesis,its level reached the highest at flowering stage（May）and the aerial parts stable stage（August）.Relative expression quantity of SmPAL1 in the stems and flowers was highest in May and August,SmHPPR in stems shown low expression,while in leaves it maily express in the late growth stage of aerial parts（July to September）.When it comes to the flowers of SM,general trend of the genes were decreasing first and increasing afterwards,except for the SmHPPR without being detected in April and May.The expression of SmDXR in the root decreased in the whole growth phase,and the expression of SmGGPPS was relatively high in 4～9 months,and reached its peak in September.2.The UPLC-TQ/MS method was used for dynamic change study of the salvianolic acids and tanshinones in various parts of SM during different growth period,combined with qPCR technology for analyzing the expression of key enzyme genes which participate in the biosynthesis of salvianolic acids and tanshinones.Meanwhile,the statistical software was carried out to establish the correlation of gene expression quantity and composition accumulation.The results showed that SmPAL1 as the first key enzyme genes of phenylalanine branch in salvianolic acid synthesis pathway,it was significantly correlated with rosmarinic acid accumulation in S.miltiorrhiza roots（r=0.430）,and it was also correlated with salvianolic acid B and total phenolic acid synthetize.In the stems,the gene express of SmPALl and accumulation of protocatechuic aldehyde,caffeic acid and salvianolic acid A shown significantly negative correlation（P<0.05）,there were only SmHPPR in leaves which shown significantly positive correlation（P<0.01）with lithospermic acid synthesis.SmHPPR and SmCYP98A14 expression in flowers of SM were negatively correlated with danshensu,rosmarinic acid,salvianolic acid B,salvianolic acid A and total phenolic acid accumulation,and it were significant negative correlation to rosmarinic acid and total phenolic acid（P<0.01）.SmDXR,SmGGPPS and SmKSL in the root were positively related to the synthesis and accumulation of miltionone.Chapter four,Evaluation of the promoting blood effects of the stems and leaves of Salvia miltiorrhiza1.Based on the acute blood stasis rat model,and take the water and alcohol extracts of SMSL and SM as objectives,total activating blood circulation and dissipating blood stasis effects of SMSL and SM was evaluated by analyzing hemorheology and the blood coagulation function in acute blood stasis rats,combined with the principal component analysis and multi-attribute comprehensive index methods.It indicated that the group of high-dose alcohol extract of SM showed the best effect of activating blood circulation and dissipating blood stasis,which could obviously improve the abnormality of hemorheology and blood coagulation function in acute blood stasis rats.Moreover,the high-dose water extract of SMSL group also showed good effect,and the total activating blood circulation effect value was next to the low-dose alcohol extract of SM,which confirmed the the pharmacological activity of SMSL for promoting blood circulation and removing blood stasis.In addition,the change of inflammatory factors(TNF-α and IL-1β),endogenous active fector（TXB2 and 6-keto-PGF1α）and inducible nitric oxide synthase（iNOS）levels were observed in order to discuss the mechanism for promoting blood circulation and removing blood stasis effect of SMSL.The results shown that,levels of TNF-α,IL-1β,TXB2 and iNOS elevated in the model group compared with the control group,6-keto-PGF1α levels decreased（P<0.05,0.01）,after drug administration,the TNF-α,IL-1β,and iNOS levels decreased,and close to the normal level Compared with the model group,each drug administration group could decrease the content of TXB2（P<0.05,0.01）,increased 6-keto-PGF1α,which suggesting that the mechanism of promoting blood circulation and removing blood stasis by SMSL and SM may by the means of inhibiting the inflammatory factor secretion,protecting vascular endothelial function and so on,which provide scientific basis for the development and utilization of SMSL.2.To observe the effects on rabbit coagulation function of water and alcohol extract of SMSL in July（SY-7 and CY-7）,SMSL in December(SY-12 and CY-12)and SM（SG and CG）,the evaluation index of plasma prothrombin time（PT）,thrombin time（TT）,activated partial thromboplastin time（APTT）and fibrinogen（FIB）were detected in vitro experiment.The results showed that anticoagulant effect of alcohol extract of SMSL and SM was stronger than the water extract.The most effective index was TT,and the sequence was CG>CY-7>CY-12,compared with the blank group,there was significant difference（P<0.01）,the three extracts can not prolonged the prothrombin time（PT）.As to the activated partial thromboplastin time（APTT）,the effect sequence was CY-7>CG>CY-12,and in the FIB evaluation,CG>CY-7>CY-12.Besides,there was no significant difference on the in vitro anti-coagulation effect between CY-7 and CG.3.The antioxidant capacity of water and alcohol extracts of SMSL（harvest in July and December）and SM was investigated on the basis of ability to scavenge DPPH radical,ABTS radical and reduce Fe3+,and meanwhile the UPLC-TQ/MS method was applied for qualitative and quantitative analysis of the chemical components.The results showed that SMSL as the non-traditional medicinal parts in S.miltiorrhiza,it possessed strong antioxidant ability in vitro,and the antioxidant and free radical scavenging ability of SY-7 was the strongest,the EC50 of DPPH and ABTS radical scavenging were 19.14 μg·mL-1 and 9.24 μg·mL-1.In extracts it was rich of water-soluble salvianolic acids,mainly for danshensu,caflfeic acid,rosmarinic acid and salvianolic acid B,the antioxidant activity of these four main monomers were evaluated as well,results showed that activity of caffeic acid was the best,with the EC50 of DPPH and ABTS radical scavenging were 4.76μg·mL-1 and 2.65 μg·mL-1.Chapter five,Protective effects of the stems and leaves of Salvia miltiorrhiza and Salvia miltiorrhiza on vascular endothelial function1.The human umbilical vein endothelial cells（HUVECs）were cultured in vitro to observe the protective effect of water and alcohol extracts of SMSL and SM on HUVECs injury induced by H2O2,and discuss the possible mechanism The HUVECs was modeled by 6.06 mmol·L-1 H2O2 injured for 3 h and Vc as positive control,the effect of cell viability on water and alcohol extracts of SMSL and SM,positive control Vc and the representative salvianolic acids,flavonoids and tanshinones included rosmarinic acid,salvianolic acid B,rutin,isoquercitrin and cryptotanshinone was measured by using MTT method,the supernatant was extracted and the levels of GSH-Px,CAT,NO and ET-lwere measured by corresponding kits.Results showed that alcohol extracts of SMSL（CJ）and SM（CG）,water extracts of SMSL（SJ）and SM（SG）had protective effect on HUVECs damage induced by H2O2 in a certain concentration,and the best was CJ with the protection rate of 46.01%at the concentration of 50 μg·mL-1.Compared to the model group,contents of GSH-Px,CAT and NO in the supernatant of the high,middle and low dose groups were increased,and the secretion of ET-1 was decreased（P<0.05,0.01）,which showed a dose-effect relationship.2.Choose the HUVECs in vitro for research objective,the oxidative injury model of HUVECs was established by high glucose,and the cells were treated with different concentration of water and alcohol extracts of SMSL and SM,positive control amino guanidine and the representative salvianolic acids,flavonoids and tanshinones included rosmarinic acid,salvianolic acid B,rutin,isoquercitrin and cryptotanshinone.The survival rate of HUVECs in each group was measured by using MTT method,contents of NO,ICAM-1 and TNF-α were determined by kits.Results showed that the oxidative stress of HUVECs in model group was significantly enhanced compared with the control group,and the alcohol extracts of SMSL（CJ）possessed the strongest protection effect,with the protection rate of 67.23%at concentration of 50 μg·mL-1.After administration,all the indexes were returned to normal level,which revealed that water and alcohol extracts of SMSL and SM could reduce the level of ICAM-1 and TNF-a,and increase the content of NO,so as to improve the damage of oxidative stress in HUVECs induced by high glucose. |
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