Study On The Correlation Between Transformation Of Flavonoids In Liangxue Hemostatic Medicine And Its Hemostatic Activity

The traditional Chinese medicine has been used for two thousand years since it has been used to stop bleeding. It has been widely used in clinic because of its remarkable effect. "Chao Tang Cun Xing" first recorded in "Jin Kui Yao Lue Fang Lun". Modern thinks carbon medicine not only saves part of function of the original drug, and has hemostatic and other therapeutic effects, can be described as "Cun Xing".Chinese hemostatic drugs commonly used in clinic to treat hemorrhage by cooling blood, warming channel, dissipating blood stasis, adtringents. The bleeding cansed by overheating is more common, so liangxue hemostatic medicine is most widely used. The crude kind of medicine cooling blood, enhance hemostasis after charring. A large number of literature analysis and summary found that such medicines contain flavonoids. Studies have suggested that flavonoids may be the main hemostatic components of Cirsium japonicum Carbonisatus, Platycladi Cacumen Carbonisatus, Pollen Typhae Carbonisatus, Flos Sophorae Carbonisatus and Nelumbinis Folium Carbonisatus. Literature found that the content of flavonoid glycoside decreased in Flos Sophorae Carbonisatus, Platycladi Cacumen Carbonisatus and Pollen Typhae Carbonisatus, and flavonoid aglycones content increased, indicating that flavonoids have been transformed in the process of charring. However, there is no report about the correlation between the transformation and the role of hemostasis. Flos Sophorae, Platycladi Cacumen and Pollen Typhae were Liangxue hemostatic medicine, and the flavonoids content were higher. Moreover, the hemostatic effects were significantly enhanced after they were carboned.In this paper, the hypothesis that certain pharmacological and chemical correlations exist that can justify therapeutic effects attributed to the carbonisatus product of Flos Sophorae, Platycladi Cacumen and Pollen Typhae was proposed. In particular the effect could be attributed to transformation of flavonoids during the heating process in carbonisatus development. Flavonoids constituents in Flos Sophorae, Platycladi Cacumen and Pollen Typhae were first obtained. Then, the flavonoids were prepared using single component heating simulated the carbonization process. The haemostatic activity was evaluated from the whole, in vitro, molecular three pharmacological levels. This paper investigates the correlations between therapeutic hemostatic effects of Flos Sophorae Carbonisatus, Platycladi Cacumen Carbonisatus and Pollen Typhae Carbonisatus and transformation of flavonoids during heating. The paper discovered common hemostatic compounds in the three medicines, preliminarily reveals the internal mechanism of hemostatic effect. This paper is divided into five chapters, the main contents of each chapter and the research results are summarized as follows:Chapter 1. Simulated processing of flavonoids in Flos Sophorae, Platycladi Cacumen and Pollen TyphaeIn order to investigate correlation between transformation of flavonoids in Liangxue hemostatic medicine and its hemostatic activity, Flos Sophorae, Platycladi Cacumen and Pollen Typhae was extracted with 60% ethanol and purified by macroporous adsorption resin to obtain the total flavonoids. The purity of the flavonoids in Flos Sophorae, Platycladi Cacumen and Pollen Typhae was measured by HPLC with 95%,89% and 84% respectively.In order to study transformation rules of flavonoids in the process of heating, the total flavonoids of three drugs were heated simulated the carbonization process. According to China Pharmacopeia defined processing technology method, three medicines were stir-fried under 150℃,200℃,250℃and 300℃, and the time to reach the required degree charcoal determined. It is found that the heating time required in Flos Sophorae and Platycladi Cacumen under 250℃, Pollen Typhae under 200℃ is proper, and required time at other temperatures is too long or too short, does not conform to the actual production, so the simulation temperature of the total flavonoids in Flos Sophorae and Platycladi Cacumen is selected at 250℃, the simulation temperature of the total flavonoids in Pollen Typhae is selected at 200℃. Moreover, the three simulation charring methods (direct heating and blank pieces, diatomite as carrier filling) were compared to investigate the diffierences in the effects of flavonoids transformation. The results showed that the effects of three methods on total flavonoids transformation had no significant difference, taking into account to blank pieces and diatomite as carrier filling simulated carbonization process on the composition of the loss is serious and the operation is complicated. Therefore, direct heating method was used to total flavonoids simulation.Chapter 2. Tracing novel hemostatic compounds from heating products of total flavonoids in Flos Sophorae, Platycladi Cacumen and Pollen Typhae by spectrum-effect relationships and column chromatographyIn this study, the total ion chromatogram fingerprints of heating products of total flavonoids in Flos Sophorae, Platycladi Cacumen and Pollen Typhae were established by high performance liquid chromatography-mass spectroscopy/mass spectroscopy to study the transformation rule of the flavonoids in Flos Sophorae, Platycladi Cacumen and Pollen Typhae in the heating process. The results showed that the content of flavonoids in the three kinds of drugs decreased with the increasing of heating time, the content of flavonoid aglycones increased first and then decreased, and then heated to a certain period of time to produce a new chromatographic peak.The whole animal experiment was used to study the different effects of products of total flavonoids in different heating time. The rat tail bleeding time (BT), four items of blood coagulation (APTT, PT, TT, FIB) and euglobulin lysis time (ELT) and ADP, thrombin, platelet aggregation induced by collagen was choosed as index. The results showed that heating products of total flavonoids could reduce the BT, APTT, PT and collagen induced platelet aggregation to plays a role in hemostasis, no significant effect on other indexes; the hemostatic effect was first increased and then decreased, the products of total flavonoids in Flos Sophorae, Platycladi Cacumen and Pollen Typhae was heated for 10 min,12 min and 25 min showed the strongest hemostatic effect.On the basis of the component analysis and hemostatic activity of the research results, the canonical correlation analysis was used to establish the spectrum effect relationship to track with hemostatic activity peaks. Results showed that peak 8 and peak 11 that genetated from heating products of flavonoids in Sophora japonica and peak 10 and peak 12 that genetated from heating products of flavonoids in Platycladi Cacumen were correlated most closely, suggesting that these correlation peaks may be the main active ingredients of hemostasis. Furthermore, the predicted active peaks in fingerprints were isolated by column chromatography and their structures were identified by spectroscopy. Based on the analysis of the structure, three new compounds were identified, which were named huaicarbon A, huaicarbon B, cecarbon, respectively. Total flavonoids of Flos Sophorae heating product track is huaicarbon A/B and heating of Platycladus orientalis and total flavonoids in the product tracking is huaicarbon B and cecarbon, pollen Typhae flavonoids heating products also generate huaicarbon A/B. The hemostatic activities the three compounds were investigated in vitro by measuring the platelet aggregation induced by various agonists and coagulation parameters (APTT, PT). The results showed that the three compounds promoted collagen-induced platelet aggregation and decreased APTT and PT in a dose-dependent manner. These results agreed well with the prediction. Huaicarbon B was traced from the heating products of the total flavonoids from three kinds of medicine, which is a common hemostatic component.Chapter 3. The content and hemestatic activity of compound huaicarbon A/B, cecarbon in Flos Sophorae Carbonisatus, Platycladi Cacumen Carbonisatus and Pollen Typhae CarbonisatusThe contents of huaicarbon A/B, cecarbon in Flos Sophorae Carbonisatus, Platycladi Cacumen Carbonisatus and Pollen Typhae Carbonisatus were determined by HPLC-PDA. It was found that the hemostatic compounds huaicarbon A/B, cecarbon could be detected from three laboratory-made products. But there is great difference in the content of flavonoids in the same drug batches of different processed products. The contents of laboratory-made products that have strict control of processing were relatively stable, however the contents of commercially available products were different greatly, some batches do not even contain the three components. It was showed that the processing of charcoal is not controllable will directly affect the composition of processed products.In order to evaluate the hemostatic effect of A/B huaicarbon in vivo, these two compounds were investigated whether are the material basis of the hemostatic effect of the drug. Rats were gavaged to huaicarbon the A/B and three drug carbon product powder. BT, APTT, PT, TT and ADP, thrombin and collagen induced platelet aggregation were choosed as index to research the hemostatic activity of huaicarbon A/B and Flos Sophorae Carbonisatus, Platycladi Cacumen Carbonisatus. The results showed that Flos Sophorae Carbonisatus, Platycladi Cacumen Carbonisatus and Pollen Typhae Carbonisatus and huaicarbon A/B shortened BT, APTT, PT, TT and promoted three induction agents induced platelet aggregation in different degree. The hemostatic effect of commercially available carbon products that do not contain huaicarbon A/B and cecarbon was significantly weaker than that of laboratory-made products. The experimental results not only verified the hemostatic effect of huaicarbon A/B in vivo, illustrated charring processing technology can directly affect the carbon products in vivo, and confirmed that the the scientific theory of "Chao Tang Zhi Xue" and "Chao Tang Cun Xing". In order to compare the different hemostatic effect of Flos Sophorae Carbonisatus and huaicarbon A/B, the dosage of huaicarbon A/B was based on the calculation of its content in Flos Sophorae Carbonisatus 3 group, found that huaicarbon the A/B could shorten BT, APTT, PT and could promote collagen, thrombin induced platelet aggregation. Compared to Flos Sophorae Carbonisatus 3 group, BT, APTT, PT, collagen induced platelet aggregation were no significant differences, which showed that huaicarbon A/B was the primary hemostatic components of Flos Sophorae Carbonisatus; however, TT, ADP and thrombin induced platelet aggregation was significant difference, indicating that there exist in other hemostatic components.Chapter 4. Transformation of quercetin and rhamnose into huaicarbon A/B by heatingQuercetin and rhamnose were the precursor materials for huaicarbon A/B. The mixture of quercetin and rhamnose was converted into huaicarbon A and huaicarbon B by heating at 250℃ or 200 ℃. The heating should be carefully controlled at 250℃ for 10-15 min or at 200℃for 25-30 min to ensure efficient conversion. The yields of huaicarbon A and huaicarbon B, which increased with rising initial molar ratio of quercetin to rhamnose, reached maxima at the ratio of 1:3. A highest huacarbon A yield of 11% was obtained at 220℃in 20 min, and the huaicarbon A yield decreased to 4% in a reaction time of 3 min when the reaction temperature was increased to 300 ℃. In order to obtain a high huaicaibon A/B yield, the temperature should controlled from 200℃to 250 ℃. Negative results were achieved when the mixtures of quercetin and glucose, isorhamnetin and rhamnose and isorhamnetin and glucose were heated. This heating reaction can be used to prepare huaicarbon A/B.Chapter 5. Preliminary study on platelet aggregation mechanism of compound huaicarbon A/B, cecarbonTo preliminary elucidare the collagen induced aggregation mechanism of huaicarbon A/B and cecarbon, this paper using in vitro platelet aggregation assay to observe the effect of huaicarbon A/B and cecarbon on human platelets aggregation and on intracellular Ca2+ level before and after addition of GPVI receptor specific inhibitor. The results showed that huaicarbon A/B and cecarbon could increase the accumulation of intracellular Ca+ and promote collagen induced platelet aggregation, which could be blocked by GPVI receptor specific inhibitor. Huaicarbon A/B and cecarbon promoted collagen induced platelet aggregation by activating signaling pathway mediated through platelet GPVI receptor.In this paper, flavonoids in Flos Sophorae, Platycladi Cacumen and Pollen Typhae were obtained and choosed as the research object. The component direct heating method was used to simulate processing, and investigated heating effect on flavonoids transformation. The results showed that three new components generated after heating, and these components were material basis of three kinds of Chinese herbal medicines which the hemostatic effect were enhanced after charring. The flavonoids in Flos Sophorae, Platycladus Cacumen and Pollen Typhae in the heating process existe the common rules, hemostatic ingredients generated in the process of heating are due to the three drugs itself contains quercetin and rhamnose is generated in the heating process. This paper reveals the correlations between therapeutic effects of Flos Sophorae Carbonisatus, Platycladi Cacumen Carbonisatus and Pollen Typhae Carbonisatus and transformation of flavonoids during heating. And from the point of view of common components and common effect, revealing the mechanism of "Chao Tang Zhi Xue" and "Chao Tang Cun Xing" of Flos Sophorae, Platycladi Cacumen and Pollen Typhae, providing a reasonable research ideas and methods for the theoretical study on carbon medicine hemostasis.