| Objective:1. Extract and enrich the iridoid glycosides from Lamiophlomis rotata (Benth.) Kudo (IGLR), and study on its antinociceptive and anti-inflammatory activities and mechanisms in mice.2. Study the anti-inflammatory activity and mechanism of iridoid glycosides in mice with several classical animal models3. Study the anti-inflammatory activity and mechanism of iridoid glycosides in vitro by determining the concentrations of plasma nitrite (NO) and prostaglandin E2 (PGE2) in the lipopolysaccharide (LPS) treated-macrophage RAW264.7.4. Study the anti-inflammatory activity and mechanism of several iridoid glycosides, 8-O-acetyl-shanzhiside methylester, phloyosideⅡand shanzhiside methylester, in vitro by determining the concentrations of plasma nitrite (NO) and prostaglandin E2 (PGE2) in the lipopolysaccharide (LPS) treated-macrophage RAW264.7,5. Study on the compositions and the contents of iridoid glycosides between Lamiophlomis rotata and Phlomis umbrosa.Methods:1. Combining with the macro-porous adsorptive resins chromatographic method and polyamide chromatographic method, the iridoid glycosides of Lamiophlomis rotata (Benth.) Kudo (IGLR) were extracted and enriched. At the same time the ultraviolet spectrophotometry (UV) and high-performance liquid chromatography (HPLC) were used to qualitation and quantification analyse the iridoid glycosides.2. After IGLR at doses of 100,200 and 400 mg/kg were administrated by intravenous injection each mouse. The acetic acid-induced writhing response test, hot plate test, formalin test, carrageenan-induced paw edema test, xylene-induced ear swelling test, peritoneal capillary permeability and leukocyte infiltration caused by acetic acid test were adopted to evaluate the antinociceptive and anti-inflammatory activities of IGLR.3. The concentrations of NO and PGE2 in LPS-stimulated macrophages RAW264.7 were determined and used to evaluate the anti-inflammatory activity of IGLR. At the same time MTT test was employed to evaluate the cytotoxicity of IGLR.4. The concentrations of NO and PGE2 in LPS-stimulated macrophages RAW264.7 were determined and used to evaluate the anti-inflammatory activities of 8-O-acetyl-shanzhiside methylester, phloyoside II and shanzhiside methylester. At the same time MTT test was employed to evaluate the cytotoxicitie of these compounds.5. Thin layer chromatography (TLC) was used to identify iridoid glycosides between L. rotata and P. umbrosa; and high-performance liquid chromatography (HPLC) was used to determine the content of 8-O-acetylshanzhiside methylester in the roots and aerial parts of these two medical materials.Results:1. The results of the qualitative and quantitative analysis of iridoid glycoside demonstrated: the ultraviolet absorption of the total iridoid glycoside was very similar to that of 8-O-acetyl shanzhiside methylester and the maximum absorption peak was at 237 nm. The content of iridoid glycoside in the total iridoid glycoside was 66.92%. RP-HPLC showed 8-dehydroxy shanzhiside, phloyosideⅡ, shanzhiside methylester,6-O-acetylshanzhiside methylester and 8-O-acetyl-shanzhiside methylester are the main components in the aqueous and iridoid glycosides extracts2. The iridoid glycosides could decrease acetic-acid-induced writhings times and formalin-induced lickings times, inhibit carrageenan-induced hind paw edema and xylene-induced ear swelling, and suppress peritoneal capillary permeability and leukocyte infiltration also induced by acetic acid in mice in vivo. 3. The iridoid glycosides at doses of 25μg/ml,10μg/ml,1μg/ml treated the LPS-stimulated macrophages RAW264.7 could inhibit the release of NO and PGE2, but no dose dependent was showed. The MTT tests showed that IGLR at the given doses would not affect the macrophages RAW264.7 viability.4.8-O-acetyl-shanzhiside methylester, phloyoside II and shanzhiside methylester exhibited significant anti-inflammatory effects in vitro. The inhibitory rate of shanzhiside methylester (10μg/ml,1μg/ml) and 8-O-acetyl-shanzhiside methylester (10μg/ml) to NO release were 30%,44% and 21%. All of these three compounds could inhibition the PGE2 release significantly at dose of 10μg/ml. MTT tests showed that they at the given dose would not affect the macrophages RAW264.7 viability.5. L. rotata and P. umbrosa have the plenty of iridoid glycosides and similar chromatograms. The content of 8-O-acetylshanzhiside methylester in the aerial parts of L. rotata and P. umbrosa were 17.42 and 0.5 mg/g, accompanying with 6.81 and 8.50 mg/g in the roots respectively.Conclusions:1. The IGLR was enriched and extracted successfully from the aqueous extract with the macro-porous adsorptive resins chromatographic method and polyamide chromatographic method.2. IGLR have the significant antinociceptive and anti-inflammatory effects in vivo.3. IGLR have the significant anti-inflammatory effect in vitro, and no cell toxicity.4. 8-O-acetyl-shanzhiside methylester, phloyoside II, shanzhiside methylester have the significant anti-inflammatory effects in vitro, and no cell toxicity.5. The roots of L. rotata and P. umbrosa have the similar iridoid glycosides.
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