| Cirsium japonicum DC. is widely distributed all around china, its aerial parts or roots are wildely used as a clinical traditional Chinese hemostatic medcine. Studies show after been processed into Carbonized Cirsium japonicum DC., its hemostatic activity can be enhanced and its major hemostatic content pectolinarin decreases, which is apparently illogical. In order to explain this seemingly contradictory question, we made some researches in this thesis as follows.Objective:(1)Using hemostatic efficacy as indicator to optimize the selection of Cirsium japonicum DC. in different Povinces and compare the chemical constituents in Cirsium japonicum DC. from different places.(2)Study the chemical constituents in Carbonized Cirsium japonicum DC.; To clarify the chemical constituents been changed in Cirsium japonicum DC. after it is been processed; Reveal the mechanism of those changes during carbonizing process.(3)To establish experimental animal models of bleeding and study the, hemostatic mechanisms of Carbonized Cirsium japonicum DC.; To study the hemostatic fractions of Carbonized Cirsium japonicum DC.; To compare the hemostatic differences and hemostatic mechanisms of pectolinarin and pectolinaringenin; To study the absorption mechanism and absorption differences of pectolinarin and pectolinaringenin; To study metabolites of pectolinarin and pectolinaringenin and reveal its biotransformation mechanisms in rats(4)Study the pectolinarigenin content of Carbonized Cirsium japonicum DC. from different places; To optimize the processing method of carbonizing; To optimize the purification process of pectolinarin by macroporous absorption resin method; To study contents of trace elements in Carbonized Cirsium japonicum DC.Method:(1)The tail tip transection and glass test methods were applied to compare the hemostatic activity differences of Cirsium japonicum DC. from different places; HPLC method was applied to compare chemical constituents differences of Cirsium japonicum DC. from different places.(2)To investigate the chemical constituents of Carbonized Cirsium japonicum DC. by repeated silicagel column chromatography, macroporous resin adsorption chromatography, Sephadex LH-20methods, etal. The structural identification was performed by nuclear resonance spectrometry and mass spectrometry.To clarify the chemical constituents been changed in Cirsium japonicum DC. after it is been processed by HPLC method; Reveal the mechanism of those changes during carbonizing process based on structures of those chemical constituents been changed.(3)To establish animal models of hemorrhage in mice by giving an oral administration of tradional chinese herbs (Monkshood, rhizoma zingiberis, etal)、 warfarin or giving an iv of yeast or heparin sodium in mice tail; The tail tip transection and capillary tube methods were applied to study the hemostatic activity of different extracted parts from Carbonized Cirsium japonicum DC.; To compare the hemostatic differences and hemostatic mechanisms of pectolinarin and pectolinaringenin by tail tip transection methods capillary tube method, blood coagulation funtion tests and platelet activity tests; To study the absorption mechanism of pectolinarin and pectolin-aringenin by everted rat intestinal Sac Method, single-pass perfusion and HPLC mthods; To study metabolites of pectolinaringenin by LC-MS and enzymatic hydrolysis methods in vitro.(4)Study the pectolinarigenin content of Carbonized Cirsium japonicum DC. from different places by HPLC method; To optimize the processing method of carbonizing by Response Surface Methodology (RSM); To optimize the purification process of pectolinarin by macroporous absorption resin method; To study contents of trace elements in Carbonized Cirsium japonicum DC. by microwave digestion and ICP-OES methods.Results:(1)Carbonized Cirsium japonicum DC from Zhangshu and Hunan show stronger hemostatic effect than Carbonized Cirsium japonicum DC from Hebei. The Cirsium japonicum DC. from Anhui show no hemostatic effect and even promote the bleeding. There were differences of hemostatic effect and compounds between Carbonized Cirsium japonicum DC. from different places.(2)Fourteen compounds were isolated from Carbonized Cirsium japonicum DC. All their structures were identified as pectolinarin, pectolinaringenin, linarin, acacetin, dibutyl phthalate,3-hydroxy-4-methyl-benzoic acid,3,5-dihydroxy-7-methoxy-2(4-methoxy-phenyl)-chromen-4-one,3-hydroxy-benzoic acid, apigenin-7-O-β-D-glucuronide,4-ethyl-3-hydroxy-benzoic acid、hispidulin-7-O-glucuronide,5-hydroxy-2-(4-methoxy-phenyl)-7-(3,4,5-trihydroxy-6-hydroxymethyl-tetrahydro-pyran-2-yloxy)-chromen-4-one,5-hydr oxy-6-methoxy-2-(4-methoxy-phenyl)-7-(3,4,5-trihydroxy-6-hydroxymethyl-tetrahydro-pyran-2-yloxy)-chromen-4-one, apigenin by NMR and mass spectrometry method. Among those compounds,3,5-dihydroxy-7-methoxy-2(4-methoxy-phenyl)-chromen-4-one、apigenin-7-O-β-D-glucuronide、hispidulin-7-O-glucuronide、5-hydroxy-6-methoxy-2-(4-methoxy-phenyl)-7-(3,4,5-trihydroxy-6-hydroxymethyl-tetrahydro-pyran-2-yloxy)-chromen-4-one were firstly isolated from Cirsium japonicum DC.After Cirsium japonicum DC. been carboinzed., the contents of linarin and pectolinarin decreased sharply and contents of pectolina-ringenin and acacetin increased, we surmised that linarin and pectolinarin were hydrolyzed into acacetin and pectolinaringenin during the Carbonized processing based on their chemical structures. (3)The tradional chinese herbs (Monkshood、rhizoma zingiberis, etal)、warfarin and heparin sodium can all increase bleeding and coagulating time of mice, differences were significant (p<0.05), while the yeast can also increase bleeding and coagulating time of mice, but differences weren’t significant (p>0.05). Carbonized Cirsium japonicum DC. can decrease bleeding and coagulating time of mice in all bleeding animal models.Both chloroform, ethyl acetate and n-butanol extracted parts from Carbonized Cirsium japonicum DC. show hemostatic activity.Both pectolinarin and pectolinaringenin can decrease the bleeding and coagulating time of mice of bleeding model induced by heparin sodium, differences were significant (p<0.05). The bleeding and coagulating time of mice in high and median dose group of pectolinaringenin were significantly shorter than pectolinarin group (p<0.05). the hemostatic activity of pectolinaringenin is dose related.Both pectolinarin and pectolinaringenin can significantly increase Fib in blood of rats (p<0.05). High dose group of pectolinaringenin can decrease APTT in blood of rats, but there were no significant differences (p>0.05). the results show the realtionshiop between hemostatic effection of pectolinaringenin, pectolinarin and theirs’FIB promotion effection, it also show that pectolinarin may can enhance the activity of coagulation factor VIII、IX、 XI or XII.Both pectolinarin and pectolinaringenin can significantly increase positive rate of CD-62P protein expression in rat platelet (p<0.05). The positive rate of CD-62P protein expression in high dose group of pectolinaringenin is dose related higher than Yunnan Baiyao group, low dose group of pectolinarin and pectolinaringenin, the differences were significant (p>0.05)The result reveals that the absorption of pectolinarin and pectolinaringenin both increased over time. The absorption of both ingredients among jejunum and ileum parts were higher than other Small intestinal parts. The absorption rate of pectolinarin among entire small intestinal was much higher than the absorption rate of pectolinaringenin. The absorption approach of each sample is quiet different, Obviously some bioconversion take place in the process of pectolinarigenin adsorbtion, while the pectorinarin is aborbed in prototype, so the real absorbtion rate maynot been revealed in this experiment.The absorption rate constant, the effective permeability coefficients (Peff), the absorption (Ma) of pectolinarin were significantly decreased than pectolinaringenin (P<0.01); We found a few unknown components in intestinal effluent and blood of pectolinaringenin group, while the pectorinarin is aborbed in prototypeBy LC-MS and enzymatic hydrolysis methods in vitro, we inferred that metabolites of pectolinaringenin in intestinal effluent may be pectolinarin-genin-glucoside or its methylation products(4)Pectolinarigenin in carbonized cirsium japonicum DC. from different places varied greatly. The content of pectolinarigenin in carbonized cirsium japonicum DC. from Zhangshu. Zhejiang is0.22%, which is the highest among all different places. The pectolinarigenin content in carbonized cirsium japonicum DC. from Anhui、Hebei is undetectable.The best processing method of carbonizing Cirsium japonicum DC. is quantity165g processing temperature310℃、processing time13min.The best macroporous adsorption resin method to purifying pectolinarin is loading quantity of sample28BV、adsorption velocity3BV/h、cleaning solvent water、cleaning solvent volume19BV、desorption solvent50%ethanol、desorption solvent volume12BV. desorption velocity2BV/h, after purification the purity of the pectolinarin can reach68%.The content of trace elements in Carbonized Cirsium japonicum DC. varied greatly, content of Ca、K reach30mg/g, content of Fe、Mg、Mn is lower than Ca、K. content of Zn、Na、Cu reach only0. lmg/g. after Cirsium japonicum DC. been carbonized, the content of trace elements in carbonized Cirsium japonicum all increased at different extent.New ideas of the reseacrh:(1)Firstly, we made research on differences of hemostatic activity and chemical constituents in Cirsium japonicum DC. from different places in order to ensure the smooth implementation of our research.(2)14components from Cirsium japonicum DC. were identified. Among them3,5-dihydroxy-7-methoxy-2(4-methoxy-phenyl)-chromen-4-one、apigenin-7-O-β-D-glucuronide、hispidulin-7-O-glucuronide、5-hydroxy-6-methoxy-2-(4-methoxy-phenyl)-7-(3,4,5-trihydroxy-6-hydroxymethyl-tetrahydro-pyran-2-yloxy)-chromen-4-one were firstly isolated from Cirsium japonicum DC. The chemical constituents been changed、 the mechanism of those changes during carbonizing process were firstly studied.(3)The hemostatic activity difference and hemostatic mechanism between pectolinarin and pectolinaringenin was firstly studied, these results construct steady base to reveal the carbonizing processing mechanism of Cirsium japonicum DC.(4)We formulate a scientific hypothesis which the hemostatic activity difference between pectolinarin and pectolinaringenin may connect with their different absorption rate in rat. we found in the following study that their absorption rate varied greatly and metabolic route is quiet different. The metabolites of pectolinaringenin were firstly studied by LC-MS and enzymatic hydrolysis methods in vitro. According to the results we try to infer the chemical structure of metabolites of pectolinaringenin.Conclusion:The pectolinarigenin has stronger hemostatic activity than pectolinarin, it can explain why Carbonized Cirsium japonicum DC. show stronger hemostatic activity while its hemostatic content pectolinarin decreased after been carbonized.. It also reveals that the stronger hemostatic effection of pectolinarigenin may connect with its different absorbtion approach from pectolinarin. Usually, the active ingredients in chinese medcine may not be one, so, other elements such as carbonaceous、tannins、Ca2+all should be considered, this is still worth studying. |
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