| ObjectivesThe systematic research of chemical constituents and anti-inflammatory activities had been carried out in order to find novel bioactive substance of Smilax glabra Roxb, Meanwhile provided a scientific foundation for the rational and efficient application of Smilax glabra Roxb.Methods1. Screening active siteThe Preliminary separation of Smilax glabra Roxb:The70%ethanol extract of Smilax glabra Roxb was concentrated individually on reduced pressure until no ethanol taste, and then sequentially partitioned by macroporous resin column with water,30%ethanol,60%ethanol,95%ethanol, roughly divided Smilax polar range and get four parts.the composition analysis of Smilax glabra Roxb different parts:The total flavones, total polyphenols, total saponins, total polysaccharides of Smilax glabra Roxb were analyzed by UV spectrophotometry respectively, with the each colour system of NaNO2-Al (N03)3-NaOH, Folin-ciocalteu, vanillin-perchloric acid, sulfuric acid-phenol.the activity evaluation of Smilax glabra Roxb different parts:Antioxidant activity:compared with ascorbic acid(VC), the vitro Antioxidant activity of Smilax glabra Roxb (Concentration:12.5μg/mL,25μg/mL,50μg/mL,100μg/mL) were evaluated by three experiments, which included DPPH and ABTS radical scavenging assay, reducing power measured.Anti-inflammatory activity:LPS-inducted RAW264.7macrophage cells were treated with Smilax glabra Roxb(Concentration:25μg/mL,50μg/mL,100μg/mL) for24h, then the culture meium was collected and nitrite was measured by the Griess reaction (nitrite reductase assay).2. Isolation and identification of Smilax glabra Roxb compounds:The7Kg Smilax was heated to extract compounds with70%ethanol, and then sequentially partitioned by macroporous resin column with water,60%ethanol,95%ethanol, roughly divided Smilax polar range and get three parts.60%ethanol extract was separated systematically, And the structures of compounds were identified on the basis of various modern spectroscopic analysis (UV, IR, ESI-MS,’H-NMR,13N-NMR, DEPT, HSQC).3. Anti-inflammatory activity of Smilax glabra Roxb compounds:RAW264.7macrophage cells were treated with Smilax glabra Roxb compounds for24h, MTT assay was adopted to screen safe concentration range of Smilax glabra Roxb.LPS-inducted RAW264.7macrophage cells were treated with Smilax glabra Roxb compounds(within safe concentration) for24h, then the culture meium was collected, nitrite was measured by the Griess reaction (nitrite reductase assay), TNF-α were measured by ELISA kit.4. Anti-inflammatory activity of astilbinRAW264.7macrophage cells were treated with astilbin (Concentration:10μg/mL,20μg/mL,40μg/mL)for24h, MTT assay was adopted to determine the effects of astilbin on cell viability.LPS-inducted RAW264.7macrophage cells were treated with astilbin(Concentration:10μg/mL,20μg/mL,40μg/mL)for24h, then the culture meium was collected, nitrite was measured by the Griess reaction (nitrite reductase assay), TNF-α were measured by ELISA kit.LPS-inducted RAW264.7macrophage cells were treated with astilbin (Concentration:10μg/mL,20μg/mL,40μg/mL)for12h, Real-time PCR was used to study the effect of astilbin on iNOS, IL-6, TNF-α mRNA expression level in LPS-induced RAW264.7.LPS-inducted RAW264.7macrophage cells were treated with astilbin (Concentration:5μg/mL,10μg/mL,20μg/mL,40μg/mL) for1h, Western blotting was used to analysis P-P65、P-P38、P-JNK and P-ERK1/2protein expression level.Results1. the results of preliminary experimentsAfter preliminary separation, the70%ethanol extract of Smilax glabra Roxb (TFL-Z) was sequentially partitioned and got4parts:water part (TFL-1),30%ethanol part (TFL-2),60%ethanol part(TFL-3),95%ethanol part (TFL-4). the compounds were redistributed after the changing in polarity of the solvent, total flavones, total polyphenols and total saponins were mainly concentrated in TFL-2and TFL-3parts (P<0.01), total polysaccharides were enriched in TFL-4 part (P<0.01).It showed that Each part of Smilax glabra Roxb were found to have different levels of antioxidant activity. TFL-2and TFL-3had better antioxidant activity than other parts (P<0.01), and their ABTS radical scavenging capacity were significantly stronger than VC(P<0.01).at the concentration of25,50,100μg/mL, each part of Smilax glabra Roxb significant-ly inhibited the release of NO from LPS-inducted RAW264.7macrophage cells (P<0.01), at the concentration of50μg/mL, the effect of them were stronger than dexamethasone(P<0.01), the IC50of TFL-Z, TFL-1, TFL-2, TFL-3, TFL-4respectively was:92.62μg/mL,81.56μg/mL,58.53μg/mL,65.71μg/mL,51.34μg/mL.2.21compounds were isolated from Smilax glabra Roxb, and the structures of9compounds were elucidated by spectroscopic methods, these compounds were determined as:Syringaresinol(1), lasiodiplodin(2), syringic acid (10),1,4-bis (3,4,5-trimethoxyphenyl)-2,3-bis (hydroxymethyl)-1,4-butanediol (12),(-)-lyoniresinol (14), caffeic acid methyl ester (15), trans-resveratrol (21). and compounds2,5,8were the first time isolated from Smilax glabra Roxb.3. The safe concentration range of Smilax glabra Roxb were:compounds5,11,16≤200μg/mL, compounds4,7,9,14,17,20≤100μg/mL, compounds2≤50μg/mL, compounds3,6,8,13,18≤25μg/mL, compounds1≤10μg/mL, compounds10,15,19,21≤1μg/mL, within safe concentration, the Smilax glabra Roxb compounds1,2,5,8,9,10,11,12,14,15,16,21significantly inhibited the release of NO from LPS-inducted RAW264.7macrophage cells (P<0.05), and compounds1,14significantly inhibited the release of TNF-α from cells.4. astilbin together with LPS significant activate NF-κB and MAPK signaling pathways, so that the phosphorylation of P65, P38, JNK, ERK1/2signaling pathways were highly expressed, the iNOS, TNF-α, IL-6mRNA relative expression levels were down-regulated, the secretion of NO was inhibited.Conclusion1. the70%ethanol extract of Smilax glabra Roxb had good antioxidant and anti-inflammatory activity,30%and60%ethanol part were the major part of the active site2.21compounds were isolated from Smilax glabra Roxb, and the structures of9compounds were elucidated by spectroscopic methods, compounds2,5,8were the first time isolated from Smilax glabra Roxb, and compounds1,2,5,8,9, 10,11,12,14,15,16,21had good anti-inflammatory activity.3. In the upstream, astilbin enhanced immune function of macrophages through activate NF-κB and MAPK signaling pathways, In the downstream, astilbin inhibited excessive inflammatory response of macrophages through downregulating inflammatory genes and inhibiting the secretion of NO. By this two-way adjustment, astilbin played a anti-inflammatory activity and maintained a stable internal environment. |
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