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

This work was supported by Jiangsu Key laboratory for TCM formulae Research,2006'Great Basic Science Research Project of Jiangsu College and University (NO.06KJA36022),2009'Program for New Century Excellent Talents by the Ministry of Education (NCET-09-0163),2009' Program for Excellent Scientific and Technological Innovation Team of Jiangsu Higher Education and Construction Project for Jiangsu Key Laboratory for High Technology Research of TCM Formulae (BM2010576), Construction Project for Jiangsu Engineering Center of Innovative Drug from Blood-conditioning TCM Formulae.This dissertation is divided into four chapters.Chapter 1 was literature research.Section I, a detailed review was given to the research idea and model about Siwu series Decoctions for Treating Primary Dysmenorrhea of Gynecology Blood Stasis Syndrome. Besides, the development of traditional modern research for treating primary dysmenorrheal was also summarized.Section II, the development of modem research on Xiang-Fu-Si-Wu Decoction (XFSWD) for treating primary dysmenorrhea was summarized. Experimental program and technology roadmap were designed based on literature research.Chapter 2 was experimental program and design.In Chapter 3, the cluster analysis was used to analyze the relationship of consisting herbs in Xiang-Fu-Si-Wu Decoction. The method of association rule and classic Apriori algorithm was used to analyze the rule of application TCM for treating primary dysmenorrhea. With hypothesis tests. Radix Angelicae Sinensis, Rhizoma Cyperi and Rhizoma Corydalis were clustered as one group. Radix Angelicae Sinensis and Rhizoma Cyperi as a herb couple was most used in the treatment of dysmenorrhea. Rhizoma Cyperi and Rhizoma Corydalis were used most frequently in the Siwu decoctions.Chapter 4, basic research of bioactive effect and chemical constituents of Xiang-Fu-Si-Wu Decoction.Section I, the evaluation system for effects of Xiang-Fu-Si-Wu Decoction was established.1. Xiang-Fu-Si-Wu Decoction was separated by gradient elution with different concentration ethanol from macroporous adsorptive resins D101 then obtained the different fraction.2. The inhibitory effects of active - guided fraction and its main bioactive components of Xiang-Fu-Si-Wu Decoction on model of non - pregnant mice uterine contraction induced by oxytocin were investigated. Six alkaloids in active fraction XFSW-10 were identified and quantified by HPLC - DAD. The data stated that alkaloids like berberine were main active components in bioactive fraction of Xiang-Fu-Si-Wu Decoction for dysmenorrhea on uterus contraction.3. By lymphocyte proliferation and transformation experiments, XFSWD, XFSW-3 and XFSW-7 showed high effect. Four main compounds including albiflorin, peoniflorin, ferulaic acid, Senkyunolide I in active fraction XFSW-7 were identified and quantified by HPLC - DAD. Albiflorin, peoniflorin, ferulaic acid, Senkyunolide I were the potential active constituents. Xiang-Fu-Si-Wu Decoction and Si-Wu Decoction had the common material basis.4. A model of blood stasis was established by means of giving rats an injection of adrenalin and making it swim in ice-cold water to evaluate the effects of Xiang-Fu-Si-Wu Decoction on hemorheology and ovarian function in rat model of acute blood stasis. XFSWD showed improvements in the indexes of rats in the model of acute blood stasis, prolonged TT, APTT, degraded FIB to normal level and regulated the secretion of E2 and P.5.5-HIAA in hypothalamus of normal and primary dysmenorrheal model rats were rapidly identified using microdialysis coupled with HPLC-ECD. The contents of 5-HIAA increased notably in dysmenorrheal model rats compared to normal control group, which suggested primary dysmenorrhea lead to the increase of 5-HIAA. After treatment with Xiang-Fu-Si-Wu Decoction, the contents of 5-HIAA decreased which showed that Xiang-Fu-Si-Wu Decoction relieve pain syndrome of primary dysmenorrhea through decreasing the metabolism rate of 5-HT in hypothalamus.SectionⅡ, the pharmacological models, including Qi-Zhi Blood Stasis rats model, blood-deficiency mice model, hot-plate test, acetic acid-induced writhing and dysmenorrheal mice model were adopted to evaluating the effects of Xiang-Fu-Si-Wu Decoction and Si-Wu Decoction. The results were listed as follow.1. The results on Qi-Zhi Blood Stasis rats model stated that Xiang-Fu-Si-Wu Decoction showed significant improves the hemorheology of rats in the model of Qi-Zhi blood stasis while Si-Wu Decoction showed low effect on this model.2. Xiang-Fu-Si-Wu Decoction showed low effect on blood-deficiency mice model. Si-Wu Decoction was benefit on accelerating hematopoiesis recovery.3. Xiang-Fu-Si-Wu Decoction showed higher effect on hot-plate test, acetic acid-induced writhing and dysmenorrheal mice model than Si-Wu Decoction. Analgesic effect was the main bioactivity of Xiang-Fu-Si-Wu Decoction.SectionⅢ, The chemical components in Xiang-Fu-Si-Wu Decoction and its medicine material were studied.1. A new cerebroside, 1-O-(β-D-glucopyranosyloxy)-(2S,3R,4E,8Z)-2-[(2'R)-2'-hydroxylignoceranoyl-amino]-4,8-tetradecene-3-diol was isolated from the 60% EtOH extract of traditional Chinese medical plant Cyperus rotundas L.. Its structure was determined on basis of spectroscopic data. This new compound showed anti-proliferation effect on vascular smooth muscle cells (VSMCs).2. The method of UHPLC-DAD-ESI-MS was adopted to screen and analyze the potential active components in Xiang-Fu-Si-Wu Decoction.34 compounds including aromatic acid, phthalide, alkaloid, terpenes, sugar etc. were identified.3.10 compounds including gallic acid, chlorogenic acid, albiflorin, caffeic acid, paeoniflorin, tetrahydrocolumbamine, protopine, fumalic acid, tetrahydropalmatine and bererine in active fraction of Xiang-Fu-Si-Wu Decoction were simultaneous analyze.4. The polysaccharide content of Xiang-Fu-Si-Wu Decoction ethanol precipitate fraction was analyzed by phenol/carbazole - sulfuric acid method.In Chapter 5, the mechanism and metabolism research of Xiang-Fu-Si-Wu Decoction on human and model animal.1. The mechanism of Xiang-Fu-Si-Wu Decoction for treating primary dysmenorrhea was the result of control on neural/endocrine/immune/blood system and direct role in target organ.2. Plasma metabolic profiling of normal and dysmenorrheal syndrome rats after oral administration Xiang-Fu-Si-Wu Decoction by UHPLC-TOF-MS and multivariate statistical analysis.3. UHPLC-Q-TOF-MS with automated data analysis software (MetaboLynxTM) for fast analysis of the metabolic profile of alkaloids and aromatic acid in Xiang-Fu-Si-Wu Decoction ethanol supernatant fraction. Rat plasma, urine and bile samples collected 1.5 h,0 - 12 h and 0 - 4h after oral administration of Xiang-Fu-Si-Wu Decoction ethanol supernatant fraction were analyzed. The post-acquisition data were processed using MetaboLynx. A total of 45 metabolites were identified in rat biofluid samples compared with blank samples. The major metabolic pathways included Hydroxylation, methylation, Sulfate conjugation etc. Through the online ESI-MSE analysis and automated data analysis (MetaboLynxTM), a total of 15 constituents have been identified or tentatively characterized from the nine tested samples after oral administration of XFSW-10. Six parent compounds and nine metabolites were identified in blood and eight organs. Methylates/ demethylates sulfation, glucuronidation, sulfation and glucuronidation conjugates were the major metabolic pathways of alkaloids in XFSW-10 in vivo.In Chapter 6, formulation Laws of Xiang-Fu-Si-Wu Decoction for Treating Primary Dysmenorrhea of Qi stagnation Blood Stasis Syndrome.Section I:1. Cluster analysis was adopted to analyze the relationship of consisting herbs, and analyze the relationship between bioactivities and active components. The results stated that experimental data consisted with theory of TCM.2. The bio-active contribution of main herb couple in Xiang-Fu-Si-Wu Decoction (XFSWD) was evaluated by using the forward-reverse biological activity test. Four animal models, including hot-plate test, acetic acid-induced writhing, dysmenorrheal mice model, and blood-deficiency mice model, were adopted to test the effect of every sample on different models. Cyperi rhizoma - Corydalis rhizoma (CRCR) have great analgesic effect. When CRCR are removed from XFSWD, its analgesic activity was lowered, even disappeared. While the analgesic effect of Cyperi rhizoma - Angelicae sinensis radix (CRASR) was weaken than CRCR, and When CRASR was removed from XFSWD, the effect was not diminished clearly, some are even increased. In XFSWD, the enrich-blood effect of CRASR are very weak, CRCR played important role in its analgesia effect. HPLC-PDA method was used to analysis the difference of the content of aromatic acid and alkaloids in herb couple of Xiang-Fu-Si-Wu Decoction. By comparing the area of response peak, the result showed that, the content of alkaloids were higher in herb couple of Cyperi rhizoma and Corydalis rhizoma, this herb couple inhibited stripping of aromatic acid. However, herb couple of Angelicae sinensis radix and Cyperi rhizoma promoted stripping of aromatic acid.SectionⅡ, Si-Wu series Decoctions for treating primary dysmenorrhea of gynecology blood stasis syndrome had common and particular formulation laws by clinical research.