| Pouzolzia zeylanica (L.) Benn. Var. microphylla (Wedd.)W.T.Wang(PZB) hasbeen known as Shi Zhu and Shi Zhu seed, recorded in “Sheng Cao Yao Xing Bei Yao”with the main effect of the detoxification swelling, bone and using for the carbuncle,syphilis and tuberculosis. The previous studies showed PZB has good effects on theskin ulcers abscess and inflammation pain, and the active parts were screenedpreliminary. The research about chemical ingredients declared that this plant hasflavones glycosides, triterpenes and lignans et al. It’s necessarily to study the effectiveparts and ingredients with the anti-inflammatory and analgesic effect of PZB, andexplore the anti-inflammatory mechanism. This paper investigated theanti-inflammatory and analgesic action in vivo, and the anti-inflammatory activity, theantibacterial activities in vitro of different polar parts from PZB, and carried on thepreliminary discussion about anti-inflammation mechanism. On the basis of chemicalresearch, the anti-inflammatory effects and mechanism of several representativecompounds were examined. And the purification process of macroporous adsorptionresin for PZB active extracts also was assayed. The main researchs are as follows:1. To divide the aqueous extract(AE) of PZB into different polar parts withmacroporous adsorption resin, including water elution part(WP),30%ethanol elutionpart(30%EP),60%ethanol elution part(60%EP) and85%ethanol elutionpart(85%EP).And flavones was acquired according to the patent method.(1) We established acetic acid induced body torsion in mice and xylene inducedmouse ear swelling model to study the anti-inflammatory and analgesic action. The results showed that ear swelling rate and body torsion times in mice of AE、WP and30%EP groups were significant difference compared with the control group,60%EPand85%EP have no obvious inhibitory effect for mouse auricular swelling and bodytorsion in mice. So we know that AE、WP and30%EP show the anti-inflammatoryand analgesic effect,60%EP and85%EP have no effect.(2) The mouse macrophage inflammation model was established by LPSstimulated the mouse peritoneal macrophages. The contents of NO、TNF-α and PGE2in the medium supernatantrespectively were measured by using Griess Regent andELISA. The expression of mRNA of TNF-α、iNOS and COX-2was detected byreal-time RT-PCR, in order to studing the anti-inflammatory activity and mechanismof AE、WP、30%EP and flavones. The80μg/ml concentration group of SZ, the40and80μg/ml concentration groups of WP,30%EP all concentration groups and the40and80μg/ml concentration groups of flavones significantly reduced the contents ofNO; The PGE2and TNF-α level were decreased by the80μg/ml concentration groupof AE, all30%EP concentration groups and the40and80μg/ml concentrationgroups of flavones; And all WP concentration groups reduced the PGE2as well as the40and80μg/ml concentration groups of WP lowered TNF-α level. We speculate thatAE、WP、30%EP and flavones develop anti-inflammatory activity by inhibiting therelease of inflammatory factor in mouse peritoneal macrophage, and flavones may beone of the effective components.(3)To study the anti-staphylococcus aureus effects of different polar partsthrough the determination the bacteriostatic ring size with punching method. Theresult illustrates that AE has antibacterial activities because of the9mm inhibitoryzone diameter, and other parts have no antibacterial activity on account of no obviousbacteriostatic ring.2. We made research about the anti-inflammatory effects and mechanism ofrepresentative compounds. The compounds are as follows:1. Pouzolignan D,2.Pouzolignan C,3. Pouzolignan E,4. Pouzolignan F,5. Pouzolignan G,6. friedelin,7.vitexin,8. quercetin-3,7-O-2-α-L--pyran buckthorn glycoside,9. quercetin-3-[O-β -rutinoside]-7-O-α-L-rhamnopyranoside. Compounds1-5are lignas, compound6istriterpenoids, compounds7-9are flavones glycosides.The mouse macrophage inflammation model was established by LPS stimulatedthe peritoneal macrophages in vitro. The contents of NO,TNF-α and PGE2weredetermined by Griess Regent and ELISA. The gene expression of TNF-α, iNOS andCOX-2were detected by real-time RT-PCR. The results showed that the release ofNO was inhibited by compounds1-7, compounds8and9had no obvious inhibition.The release of PGE2and TNF-α were reduced, and compound7lowered PGE2level.Compound1and2reduced the gene expression of COX-2and TNF-α; Compound2and3decreased the expression of mRNA of iNOS. According to the results, itindicates compounds1-7have anti-inflammatory effect, compounds8and9have noeffect. Compound1displays anti-inflammatory effect with restraining the NO andreducing the release of PGE2and TNF-α by inhibiting the expression of mRNA ofTNF-α and COX-2. Compound2has anti-inflammatory activity because the lowergene expression of iNOS, TNF-α and COX-2and the level of NO、PGE2and TNF-αbe down. Compound3has anti-inflammatory effect because it could decrease theexpression of mRNA of iNOS so that lower level, as well as reduce the contents ofNO、PGE2and TNF-α.Compound7shows anti-inflammatory effect through theinhibition of PGE2and NO.3. To survey purification process of PZB effective parts with macroporousadsorption resin. UV spectrophotometry was adopted to construct the contentdetermination method for flavones, saponins and phenolic acid. With the threecomponent contents as indexes, the macroporous adsorption resin HP20was selectedfrom nine different polarity resins with the better adsorption and desorptionperformance, furthermore each parameter was investigated. The results showed theoptimum process is to choose the3.3BV sample liquid with0.2g/ml concentration andpH4, then use2BV distilled water washing after the sample standing for3h, finallyelute with4BV60%ethanol,2.00ml/min elution velocity. The adsorption anddesorption rate of flavones in sample liquid come to90%, the adsorption and desorption rate of phenolic acids is over85%, and the adsorption and desorption ratesof saponins improve double compared with condition investigation before. It has laida foundation for further developing medicinal parts of PZB. |
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