| Objective:1. Optimization of the process about national invention patent in the preparation of iridoid glycosides with high purity from Lamioplomis rotata.2. Screen the laxative activity ingredients and make a study of its dose correlation from Lamiophlomis rotata.3. Discuss the laxative mechanism of iridoid glycosides from Lamiophlomis rotata..4. Establish a rapid chromatographic method to separate the iridoid glycosides and identify the target compounds with PDA and MS.Methods:1. Analyse the characteristics of the impurities of iridoid glycosides use HPLC and UV visible wavelength sanning. Optimize the process of macroporous adsorptive resins chromatotraphic column and polyamide chromatographic column on the basis of those characteristics to obtain products with high purity. Analyse the iridoid glycosides with equal concentration before and after the purification.2. Total flavonoids ingredients (P1), total iridoid glycoside ingredients (P2), and maximus polarity ingredients (P3) were isolated from the aqueous extract of Lamiophlomis rotata (AELR). Compound diphenoxylate tablet was used to establish constipation model. Marenwan was used as positive drug. Using charcoal powder as observed indicator. The time of first darkened defecation, the stool grains number, the time points of defecation in 6 h, the average grain wet weight, water content of feces, and the charcoal powder propelling ration were detected.3. Select 10 female wistar rats as the blank group. The rest rats were given compound diphenoxylate by intragastric administration for 4 weeks. Serve charcoal powder as the maker, observe and determine the time of first darkened defecation to screen the constipation model rats. The constipation rats were divided into model recovery group, maren pills positive control group, and the active ingredient P2 low, medium, high dose. Observe each index. Detect the content of 5-HT and the expression of AQP3 protein.4. Collect the chromatographic effluents according to the chromatogram. The effluents were dried with cryodesiccation to obtain the target monomer compounds. Use RP-HPLC to determine the mass fraction of corresponding compounds. Through the result of PDA, MS and compared with related document literature to make qualitative identification.Results:1. The content of iridoid glycosides was 90.60%after the process purification. The yield was 16.70%. The transfer rate was 74.72%. Compared with the early preparation technology the content was promoted 21.54%.2. Total iridoid glycosides of Lamiophlomis rotata can significantly shorten the first black time in constipation mice, increase the number of black row, and increase the rate of constipation intestinal propulsion. The water extract of Lamiophlomis rotata contained iridoid glycosides. Thus it also showed the weaker therapeutic effect. Total flavonoids and large polarity parts had no such effect.3. The content of 5-HT in the constipation rats proximal colon compared with the blank group decreased obviously (P< 0.01). In the treatment groups the content of 5-HT increased significantly compared with the model group, and the difference was statistically significant (P< 0.05). The P2-M, H and MRW had significant difference (P< 0.01).The intestinal transmission of constipation rats had different degree of recovery after given the experimental drug and MRW. The immunohistochemical result showed that MRW and P2 group can decrease the expression of mucosa AQP3.4.9 compounds were identified as iridoid glycosides, which were phlorigidoside C> schismoside, sesamoside, shanzhiside methylester,6-0-acetylshanzhiside methylester, phloyoside II, penstemoside, loganin and 8-0-acetylshanzhiside methylester.Conclusion:1. The purification process was simple and practicable with high efficiency, which made the significant promote in the content of the iridoid glycosides.2. Iridoid glycosides had laxative activity. The effect was remarkable with high dose, which can be used as a natural medicine to treat constipation.3. P2 hold significant therapeutic effects on STC by means of improving the content of 5-HT and reducing the expression of AQP3 with ideal treatment effect.4. The prep-HPLC method was simple and practicable with high efficiency. It can be used to the qualitative analysis of 9 iridoids glycosides and its preparation. |
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