Bioactive Components And Formulation Rule Of Si-Wu Series Decoctions For Treating Primary Dysmenorrhea Of Gynecology Blood Stasis Syndrome

This dissertation is divided into five chapters and the contents are summarized as follows:Chapter 1.Literature research,thoughts and designThe relevant literatures of THSW were summarized including the clinical application,the pharmacological effects,components basis and metabolic characteristics.On this basis,research ideas were summarized and experimental program and technology roadmap were designed.The literature research,thoughts and design were provided too much reference for the following research.Chapter 2.Chemical fractions activity evaluation and THSW components basis Section 1 Samples preparation of THSWFor the simulation of the usage of traditional Chinese medicine,water was used to extract THSW.Macroporous resin was used to separate the water extraction of THSW,15 fractions named as THSW-1?THSW-15 were obtained for the following research.Section 2 Activity evaluation on disease and syndrome related models of THSWPart 1 Effects on blood deficiency and blood stasis syndrome model in rats of THSWA simple and stable blood deficiency and blood stasis syndrome model was set up.Acetylphenylhydrazine(APH)and cyclophosphamide(CP)were used to build this model.The different dosages of THSW had varying degrees of improvement.Part 2 Effects on primary dysmenorrhea model in mice of THSW and their different fractionsA primary dysmenorrhea model in mice was established by oxytocin(OT)and estrogen(E2)Volatile oil,water extract,alcohol precipitation deposition part,alcohol precipitation supernatant part and 30%to 50%alcohol elution fractions could decrease mice's writhing times.Mechanisms were related to Ca2+and NO level in uterine tissue.Part 3 Effects on analgesic experiments in mice of THSW and their different fractionsHot-plate test and acetic acid-induced writhing movements in mice models were set up.Water extract,alcohol precipitation deposition part,alcohol precipitation supernatant part and 30%to 40%alcohol elution fractions could increase pain threshold values and decrease mice's writhing times.Part 4 Effects on the isolated mice uterus model of THSW and their different fractionsOT induced contraction of isolated uterine tissue model in vitro.Volatile oil,water extract,alcohol precipitation supernatant part and 30%alcohol elution fraction showed significantly effects on uterine contraction frequency,amplitude,and the role of muscle tension.Section 3 Effects on nerve-endocrine-immune-the blood circulatory system related aspects of THSWPart 1 Effects on neurotransmitters in rats brain tissue and serum of THSWThe primary dysmenorrhea model and acute blood stasis model in rats were built.Contents of serotonin(5-HT),?-endorphin(?-EP)and norepinephrine(NA)in rats brain tissue and serum were detected.THSW could regulate their contents.Part 2 Effects on estrogenic activity of THSWEstrogenic activity of THSW was detected.If E2 in vivo was in the low concentration,THSW could have estrogenic activity.If E2 in vivo was in the high concentration,THSW could have anti-estrogenic activity.Part 3 Effects on LPS-stimulated rat peritoneal macrophages to release NO of THSW and their different fractionsTHSW could inhibit ipopolysaccharide(LPS)to stimulate rat macrophages to release NO production.The inhibition rate:20%to 40%alcohol elution fractions were less than 30%,50%to 80%alcohol elution fractions were more than 30%and less than 50%and 95%alcohol elution fraction was more than 60%.Part 4 Effects on splenic lymphocyte proliferation of THSW and their different fractionsWater extract,alcohol precipitation deposition part,alcohol precipitation supernatant part,water insoluble part and 0%to 40%alcohol elution fractions could significantly promote spleen lymphocyte proliferation.50%to 80%alcohol elution fractions could significantly inhibit spleen lymphocyte proliferation.Part 5 Effects on thrombin activity of THSW and their different fractionsThe optimized method of thrombin time(TT)had been established.Alcohol precipitation supernatant part,water insoluble part and 40%to 70%alcohol elution fractions could significantly inhibit thrombin activity.Part 6 Effects on inhibiting ADP-induced platelet aggregation of THSW and their different fractionsVolatile oil,water extract,alcohol precipitation deposition part,alcohol precipitation supernatant part and 20%to 30%alcohol elution fractions could significantly inhibit ADP-induced platelet aggregation.Part 7 Effects on antioxidant in in vitro of THSW and their different fractionsThe antioxidant effects were evaluated by the application of five antioxidant experiments in vitro.Water extract,alcohol precipitation supernatant part,and 10%to 30%alcohol elution fractions showed significantly antioxidant effects.Section 4 Components basis of THSWPart 1 UHPLC-DAD-TOF-MS qualitative analysis of components basis in THSW16 components were identified according to standards and another 16 components were identified according to literature research.Part 2 UHPLC-DAD-TOF-MS qualitative analysis of components basis of different fractions in THSWAromatic acids were mainly distributed in alcohol precipitation deposition part,alcohol precipitation supernatant part and 0%to 80%alcohol elution fractions.Glycosides components were mainly distributed in alcohol precipitation deposition part,alcohol precipitation supernatant part and 10%to 40%alcohol elution fractions.Phthalides components were mainly distributed in alcohol precipitation supernatant part and 30%to 40%alcohol elution fractions.Part 3 HPLC-DAD quantitative analysis of components basis in THSWA HPLC-DAD detection method was established for the simultaneous determination of 11 components in THSW:gallic acid,amygdalin,chlorogenic acid,caffeic acid,hydroxysafflor yellow A(HYSA),paeoniflorin,coumaric acid,ferulic acid,benzoic acid,senkyunolide I and senkyunolide H.Part 4 HPLC-DAD quantitative analysis of components basis of different fractions in THSWA HPLC-DAD detection method was established for the simultaneous determination of 6 components in the effective 20%alcohol elution fraction:amygdalin,albiflorin,paeoniflorin,benzoic acid,coumaric acid and ferulic acid.A HPLC-DAD detection method was established for the simultaneous determination of 8 components in the effective 30%to 50%alcohol elution fractions:p-hydroxybenzoic acid,paeoniflorin,albiflorin,coumaric acid,ferulic acid,benzoic acid,senkyunolide I and senkyunolide H.Chapter 3.Pharmacokinetics,metabolites and metabolic characteristics of THSW Section 1 Pharmacokinetic study of THSW in a primary dysmenorrhea model in ratsPharmacokinetic characteristics of THSW and alcohol precipitation supernatant part were studied in normal and dysmenorrhea model rats.Pharmacokinetic characteristics were changed because of uterus in severe contraction in dysmenorrhea model rats induced by OT and E2.Section 2 Study on metabolites of THSW on primary dysmenorrhea model ratsUrine,feces and bile samples were analyzed after administration.According to MetabLynx software analysis and literature research,22 metabolites were identified in the urine samples,4 in the feces samples and 18 in the bile samples.Section 3 Evaluation on the effect of THSW on blood deficiency and blood stasis syndrome model rats studied by metabonomics approachMetabolic disturbances rules were studied in blood deficiency and blood stasis syndrome model rats induced by APH and CP.Changes metabolic phenotype by THSW in rat urine were studied.11 important metabolic markers were identified after modeling.7 important metabolic markers were significantly changed before and after modeling,then returned to normal levels after administration.Section 3 Evaluation on the effect of THSW on primary dysmenorrhea model rats studiedby metabonomics approachMetabolic disturbances rules were studied in primary dysmenorrhea model rats induced by OT and E2.7 important metabolic markers have been identified and entered into the database IPA to build the analysis of metabolic pathways.Mechanisms may be associated with the intervention of sex hormones and the regulation of amino acid pathway.Chapter 4.Formulation rules of THSWSection 1 Formulation rules and characteristics of THSWAssociation analysis among structures and characteristics of THSW and discover meaningful a herb pair.Angelicae Sinensis Radix and Carthami Flos could be classified as a class according to the results of cluster analysis based on the Xing-Wei-Guijing of THSW.Section 2 Study on biological effects associated with the components basis based on cluster analysisBiological effects associated with the components basis were studied by cluster analysis.Fractions before the elution of 50%alcohol were the main effective parts,including aromatic acids,monoterpene glycosides and phthalides.Components were the presence of cross in adjacent gradient elution parts.Section 3 The herb pair of Semen Persicae and Carthami FlosPart 1 The bio-active contribution of Semen Persicae(Taoren)and Carthami Flos(Honghua)herb pair in THSWCompare THSW,Taoren-Honghua herb pair(TH)and Si-wu Decoction(SW)on primary dysmenorrhea of gynecology blood stasis syndrome and clarify formulation rules.Compare to SW,THSW had stronger effects on promoting blood circulation through influencing on hemorheology,plasma coagulation because of adding TH herb pair in THSW.Part 2 Effective characteristics of the herb pair of THComplex components were analyzed in the herb pair of TH based on MarkerLynx software.The content of amygdalin or HSYA in the herb pair of TH was higher than it in its own herb.Effects of TH and its main components amygdalin and HSYA on hemorheological disorders of blood stasis in rats were investigated.Section 3 The herb pair of Angelica Sinensis and Carthami FlosPart 1 The bio-active contribution of Angelica Sinensis(Danggui)and Carthami Flos(Honghua)herb pair in THSWCompare Danggui,Honghua and Danggui-Honghua herb pair(GH)in blood-deficiency and blood stasis modle.Complex components were analyzed in the herb pair of GH based onMarkerLynx software.GH exhibited synergetic effects.But the mechanism of GH needs to be further study.Part 2 Effective characteristics of the herb pair of GHComplex components were analyzed in the herb pair of GH based on MarkerLynx software.The content of ferulic acid,chlorogenic acid,coumaric acid or HYSA in the herb pair of GH was higher than it in its own herb.Thrombin time was prolonged and the activity of scavenging DPPH was enhanced compared to the same drug dosage of the single decoction of Danggui or Honghua.Chapter 5.Correlation analysis THSW and SW series decoctionsSection 1 Correlation analysis THSW and SWCompare to SW,THSW not only had the function of SW,but also had stronger effects on promoting blood circulation.Section 2 Study on interaction of components basis in SW series decoctions to related targets and proteinPart 1 Effects on inhibiting thrombin activity of flavonoids components in SW series decoctionsA series of natural flavonoids from SW series decoctions collected and their inhibition of thrombin activity in vitro with the optimized method of thrombin time(TT)had been examined.Structure-activity relationships of flavonoids(SARs)on thrombin would facilitate the design of chemical compounds with higher potency to serve as potential thrombin inhibitors.Part 2 Discovery of estrogen receptor a modulators from natural components in SW series decoctionsThe binding between the estrogen receptor a(ER-a)and a variety of components from SW series decoctions as studied using a stably-transfected human breast cancer cell line(MVLN)and molecular docking.The results showed that some components,such as aromatic acids and flavones in SW series decoctions could be used as selective estrogen receptor modulators and could be selected for further development as potential agents for estrogen related diseases.Part 3 Interaction of aromatic acids from SW series decoctions to Human Serum AlbuminInteraction of aromatic acids from SW series decoctions to human serum albumin(HAS)was investigated by fluorescence spectra and molecular docking.The results showed that the effects of cinnamic acid series of aromatic acid were stronger than benzoic acid series of aromatic acid.