| Objective:1. Isolate and enrich each iridoid glycosides from Lamiophlomis rotata (Benth.) Kudo with reversed-phase preparative liquid chromatography.2. Study the hematostatic activity of each iridoid glycoside monomer and screen the effective constituents by several classical animal models.3. Study the effects of the active constituent of L. rotata (Benth.) Kudo on coagulation and fibrinolytic system, and research the hematostatic mechanism of the selected ingredient.4. Establish a RP-HPLC method for the determination of shanzhiside methyl ester in preparations of L. rotata (Benth.) Kudo.Methods:1. Combining with the polyamide chromatography, macroporous adsorptive resins and reversed-phase preparative liquid chromatography method, the iridoid glycosides of L. rotata were enriched and extracted. Further, four higher purity monomer compounds were separated and purified by the silica gel column chromatography, including:8-O-acetyl-shanzhizide methyl ester, shanzhiside methyl ester, loganin and phloyosideⅡ.2. After the selected iridoid glycoside compounds were administrated by tail intravenous injection to each mouse, the capillary coagulation and eye hemorrhage were tested and to evaluate the hemostatic activities of each monomer compound.3. After the selected compounds were administrated by intravenous injection to each mouse, the blood samples were withdrawn immediately from the heart. Then the related index of endogenous blood coagulation, exogenous blood coagulation and fibrinolysis system were measured and used to evaluate the hemostatic mechanism of the active compounds. 4. The high performance liquid chromatography (HPLC) method was performed with Symmetry C18 column to establish a method for the determination of shanzhiside methylester in the preparations of L. rotata. Gradient elution was used to determination the content of shanzhiside methyl ester in preparation of L. rotata.Results:1. Combining with liquid chromatography and reverse high performance liquid chromatography,4 iridoid glycoside compounds were isolated from L. rotata in the optimal elution and detection conditions. Their structures were elucidated by the retention time in liquid chromatography, mass spectrum and NMR spectra methods. Finally, they were identified as 8-O-acetyl-shanzhizide methyl ester, shanzhiside methyl ester, loganin and phloyosideⅡ, respectively.2. The capillary coagulation tests (CT) and eye hemorrhage test induced by picking the eyeball in mice indicatied that the active compound was 8-O-acetyl-shanzhizide methyl ester which significantly shorten the clotting time in mice. At the same time, it was effective in reducing blood loss in mice with a dose dependent manner, showing a good hemostatic activity.3. Compared with control, the blood coagulation parameters in the coagulation system of mice showed that 8-O-acetyl-shanzhizide methyl ester at dose of 400, 200 and 100 mg/kg had no effect on activated partial thromboplastin time (APTT), prothrombin time (PT) and thrombin time (TT), but significantly increased fibrinogen levels with a dosage dependent manner in blood plasma of mice.4. The fibrinolytic activity tests showed that 8-O-acetyl-shanzhizide methyl ester at dose of 400,200 and 100 mg/kg could prolong euglobulin lysis time in mice with a good dose-dependent manner compared with control. The results of enzyme-labeled immunosorbent assay of plasmin (PL) and FDP showed 8-O-acetyl-shanzhizide methyl ester could significantly inhibit the activity of PL and reduce the FDP level in mice plasma at dosage of 200 mg/kg.5. The standard curve of shanzhiside methyl ester was liner in the range of 9.6-115.2μg/ml (r=0.9998) and the average recovery was 97.69%. The shanzhiside methyl ester could be detected from all preparations of L. rotata selected from market and its content varied grandly. Conclusions:1. The reverse preparative liquid chromatography could be used for isolating the 4 major iridoid glycoside compounds. The method was simple, convenient and efficient. It is a effective method for the preparation of iridoid glycoside from L. rotata.2. The pharmacodynamic screening tests showed 8-O-acetyl-shanzhizide methyl ester had good hemostatic activity, and indicatied important clinical potentiality. At the same time, the pharmacological and clinical profile based on in vitro, in vivo studies of it still need further investigation.3. 8-O-acetyl-shanzhizide methyl ester had no significant effect on the coagulation system, but inhibited the activity of fibrinolytic system in mice. Its main hemostasis mechanism may incriminate by inhibiting fibrinolysis and the specific target on fibrinolytic system still required further study.4. In this study, the HPLC method is convenient, accurate, specificity and reproducible. It is applied to determine the content of shanzhiside methyl ester in preparations of L. rotata. In addition, the antiinflammatory and hemostatic result showed that it is necessary to control the quality of preparations of L. rotata by determination content of shanzhiside methyl ester and 8-O-acetyl-shanzhizide methyl ester. In addition, the HPLC method was adopted by the Chinese Pharmacopoeia Commission, and was recorded in the 2010 edition of "Chinese Pharmacopoeia."... |
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