Study On The Hemostatic Effect And Mechanism Of Lamiophlomis Rotata (Benth.) Kudo And Extraction Of The Effective Compositions

Objective:Tibet medicine Herba Lamiophlomis Rotata is one of the common used traditional Chinese drugs and has been used to treat hemorrhagic disease in clinic for a long time. In this thesis, we will study its aqueous extract's (HLRE) effects and mechanisms on hemorrhage, screen the effective composition, compare the toxicities of HLRE and Total Iridoid Glycoside (P₂), separate the HLRE to get the effective component and set up the standard of quality control (QC). All of these studies will offer the theoretical basis for the exploitation of a hemostatic new drug. Methods:(1) After intragastric administration (i.g.) of HLRE or effective compositions, bleeding time (BT) and clotting time (CT) of mice were determined. After i.g. HLRE or effective compositions with different doses and different time, blood agglutinate parameter, biochemical indicator and hematologic index of rats were determined. (2) Acute toxic test of mice once taken orally by HLRE or P₂, or once intraperitoneal injection (i.p.) respectively, was used to investigate the toxic effect of HLRE and P₂. (3) First derivative spectrophotometry was established firstly to determine P₂, which will be used as the detection method for the extraction the effective component from HLRE. (4) Cross test of three factors and three levels was used to study the extract technique of HLRE. Polyamide—macroreticular resin was used to separate the components of HLRE. The preparation technique of P₂ with macroreticular resin was studied too. Results:(1) As compared to the control, low dose(0.5g/kg), middle dose( 1.0g/kg) and high dose(2.0g/kg) of HLRE could shorten mice BT by 5.47%, 2.67%,39.33% respectively , high dose and middle dose had significantly changes (P<0.05) . As compared to the control, low dose (0. 5g/kg), middle dose (1.0g/kg) and high dose (2.0g/kg) of HLRE could shorten mice CT by 14.33%, 17.30%, and 22.77% respectively. High dose had significantly changes (P<0.05). After i.g. different doses for different time, HLRE could increase the content of FIB and shorten TT of rats, and the changes have the dose-effect relationship. It also could decrease PT of the high dose groups in 21 days.As compared to the control, high dose (2.0g/kg) of P₂ taken orally for 7 days, could shorten BT and CT of mice;as compared to the control, low dose (0.5g/kg), middle dose (1.0g/kg) and high dose (2.0g/kg) of P₂could increase the content of FIB and shorten TT of rats, and all the changes have the dose-effect relationship. But high dose (2.0g/kg) of P2 could increase APTT significantly (PO.05) .(2) After i.p. HLREto mice, the LD50of HLRE is 3. 654 g/kg;the maximum tolerated dose is 1. 44g/kg. Intraperitoneal injection P2 to mice, the LD50 is 7. 834g/kg;the maximum tolerated dose is 5. 31g/kg. i. g. P;to mice, the LD50 and the maximum tolerated dose couldn't be determined.(3) First derivative spectrophotometry with polyamide chromatographic column as precolumn and 252 nm as the maximal absorption wavelength was established firstly to determine the content of P2 in preparation. This method was convenient and reliability(4) Through the cross test of HLRE, according to aqueous extract weight, the influence order was extract frequency>extract solvent volume>extract time;according to total iridoid glycoside weight, the influence order was extract solvent volume>extract frequency>extract time. The optimum condition is 10 times solvent volume, 3 times, once 1.5 hours.Total Flavones (Pi), Total Iridoid Glycoside (P2) and Maxims Polarity Components (P3) of Herba Lamiophlomis Rotata were obtained by using polyamide column and macroreticular resin, the yields were 10.25%, 66.92% and 44.30 % respectively.In 10 kinds of macroreticular resin, XDA-1 resin of Lanxiao Company Xian has the best absorbability, its static adsorbing ratio is 16.95mg/ml.resin, and kinetic adsorbing ratio is 15.18mg/ml.resin. The purity of the total iridoid glycoside could reach 67.53 % with 50% alcohol was used as adsorption stripping and recovery rate reach 90.06% ? Conclusion:The aqueous extract of Herba Lamiophlomis rotate (HLRE) and Total Iridoid Glycoside (P2) had hemostatic and thrombosis effects, which were mediated through increasing the contents of FIB, Alb and shortening TT value. The toxicity of P2 is lower than HLRE. Using polyamide column and macroreticular resin, Total Flavones (Pi), Total Iridoid Glycoside (P2) and Maxims Polarity Components (P3) could be separated from HLRE with gradient elution. First derivative spectrophotometry with polyamide chromatographic column as precolumn could be used to determine the contents of P2 in preparation.