| This thesis consists of five parts:Chapter one presented a simple overview on inflammation and its related contents. The second part discussed the NO production inhibitory activity of natural products and the crude samples of medicinal plants in LPS-induced RAW 264.7 cells. The anti-inflammatory effects and anti-inflammatory molecular mechanisms of neougonin A which was a prenylated flavonoid has been discussed in chapter three. And the last chapter was the conclusion of the whole thesis.Natural products were composed of monomers with diverse structure types which were isolated from a variety of medicinal plants by our group. The crude samples of medicinal plants were prepared as follows:firstly, dried herbs were crushed and extracted by 75% acetone, then extracted by petroleum ether, ethyl acetate or others, at last the extacts were divided into different sections by column chromatography. Anti-inflammatory effects of natural products have been widely reported, the paper aims to find out the more effective anti-inflammatory compounds from our natural products library included flavonoids, triterpenoid, diterpenoid, monoterpenes, lignans and others. Pharmacological activity screening with the crude samples could enhance the selective of separating work, and made the separating more effective, more valuable. In the study we had found 9 compounds having good effect on inhibiting NO production from 125 natural products, and they are compounds 57(17.1μM),60(3.3μM),61(16.6μM), 68(2.0μM),69(3.8μM),70(10.9μM),72(10.6μM),94(18.8μM),106(14.2μM), L-NMMA, an inhibitor of NOS, as the postive control with an IC50 value of 17.4μM. Besides, compounds 57,60 and 61 were prenylated flavonoids, compounds 68-70 and 72 were tanshinones, and 94 was lignan. By the structure-activity relationship analysis, we found that in flavonoids, tanshinones, triterpenoids, lignans, when the hydrophobic groups of isopentenyl, methoxy, acetyl, propionyl introduced or increased the NO inhibitory activity will enhance, but the hydrophilic groups of sugar or hydroxyl will cause the opposite result. In the NO assay to the crude samples of Vaccinium bracteatum and Euphorbia neriifolia, the petroleum ether extracts (VBP) and the small polar part of ethyl acetate extracts (VBE4) of V. bracteatum showed good activity with the IC50 value of 25.2 and 30.9μg/mL. And the ethyl acetate extracts of E. neriifolia (ENE) also had effective activity on NO inhibited (16.6μg/mL). Furthermore, ENE was chromatographed over MCIgel CHP20/P120 eluting with CH3OH/H2O gradient system (30%-90%) to afford four fractions (ENE1～4), and the franctions ENE1 and ENE2 showed the IC50 value of 0.4 and 8.6μg/mL. In addition, ENE1 separated by silica gel to give fractions ENE1-1～4, and they all inhibited the NO production significantly, with the IC50 value from 1.4 to 14.μg/mL. So, the franctions ENE1 and ENE2 were worthy for us to further separate.Prenylated flavonoid neougonin A was isolated from Helminthostachys zeylanica by our group, with the IC50 value of 3.3μM in NO assay. And in order to clarify the molecules mechanism of the anti-inflammatory activity we did this research. Firstly, to confirm the anti-inflammatory activity of neougonin A, ELISA assay was used to determine the expression of pro-inflammatory cytokines in LPS-stimulated RAW 264.7 cells, and the results showed PGE2, TNFa, IL-1β and IL-6 were significantly inhibited (P＜0.001) by neougonin A pre-treated when the concentration was greater than 1.25μM. Then the Western Blot assay informed us neougonin A downregulated the NF-κB pathway activation by degradation of phospho-IκBα, but with no obvious effect on MAPKs. Moreover, we were sure neougonin A supressed the NF-κB pathway from the nuclear translocation of p65 was markedly attenuated in a dose-dependent manner by neougonin A treatment basied on the immunofluorescent staining experiment. Taken together, these inhibitory effects of neougonin A on the expression of inflammatory proteins were found to be mediated through the inactivation of NF-κB pathway, and these findings demonstrate the pharmacological roles and molecular mechanisms of neougonin A in regulating inflammatory responses, and suggest further evaluation and development of neougonin A as a potent therapeutic agent for the treatment of inflammatory disorders.To clear up why Onosma paniculatum can be used as substitute of arnebiae radix, and to comfirm the material basis for anti-inflammatory activity, we made the tracking separation to O. paniculatum systematically. Eight compouds with NO inhibitory activity (IC50 from 0.4 to 16.5μM) had been yield from O. paniculatum, with a high effective rate of 72%. In addition, there were 7 naphthoquinone compounds in these activated compouds, it demonstrated naphthoquinone compounds were the main material basis with anti-inflammatory activity in O. paniculatum. Furthermore, study on the molecular mechanism to shikonofuran E (ICso=3.5μM) implied that NF-κB and MAPKs might be the potential molecular targets for clarifying the protective effects of shikonofuran E on LPS-induced inflammatory cytokines (PGE2、TNFα、IL-1β、IL-6) production in macrophages (when the concentrations of shikonofuran E higher than 2.5μM, the P value will less than 0.05). |
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