Anti-inflammatory Properties Of Extracts From Chimonanthus Nitens Oliv.leaf

Chimonanthus nitens Oliv.(CN;Calycanthaceae)leaf is widely used to make traditional herbal tea in southern China and has a wide range of therapeutic effects treating common cold,influenza,cough,vomiting and heat stroke.In addition,CN leaf extracts also has potential properties for anti-inflammatory,weight loss and other functions.In the present study,the best extraction process for CN extract and its compounds and anti-inflammatory ability were examined.The main conclusions are as follows.(1)An analytical method for determining four typical compounds in CN leaf extracts was established.The suitable detection wavelength of the standards was determined by UV spectrophotometry.The solvent system was optimized to give a better separation for the compounds in the extracts.Standard curves of four standards were made using standards and HPLC.(2)Optimization of Extraction Process.The inhibitory ability of the extracts on 5-lipoxygenase was evaluated by visible spectrophotometry.100%ethanol extracts had the strongest inhibitory effect giving an inhibition rate at 19.07%.The effects of ethanol concentration,extraction temperature and ratio of solid to liquid on the yield of the compounds were studied by orthogonal analysis and principal component analysis.Based on the results of three experiment,the extraction process was optimized and settled,namely 100%ethanol,40? extraction temperature and 1:20 solid to liquid ratio.(3)Identification of ethanol extracts in CN leaves.The compounds of the extracts were identified by UPLC-QTOF-MS/MS.A total of 47 compounds were identified,including 4 organic acids,7 phenolic compounds,22 flavonoids,4 coumarins,4 fatty acids and 6 other compounds.The compound with the highest ion abundance in the identified compounds was kaempferol-3-O-?-D-rutinoside.(4)Validation of the method for determination of typical compounds.The precision,stability,reproducibility and recovery of the assay were examined by using extracts,standards and HPLC.The experimental results show that the precision of the experimental instrument is well,the results of the parallel tests are less affected;the stability is well,the sample does not change much within 21 h;the reproducibility is well and the recovery is well.This method yields stable and reliable results.(5)Determination of the active compounds of the CN extracts.The contents of the four typical compounds in the extracts were determined using standard curves and the settled method.The contents of scopoletin,quercetin,kaempferol and kaempferol-3-O-?-D-rutinoside in CN extracts were 0.0825,0.0365,0.0438,5.0366 mg/g DW,respectively.The total flavonoid content in the extracts was determined by spectrophotometry and show a concentration of 60.73 mg/g DW(rutin equivalent).(6)Evaluation of anti-inflammatory activity of CN extracts.The mRNA expression of pro-inflammatory cytokines in LPS-induced zebrafish was determined by real-time q-PCR.The extracts did not significantly inhibit the expression of IL-1?,but it significantly decreased the mRNA expression of TNF-a and IL-6.The effects of the extracts on the migration of inflammatory cells(macrophages and neutrophils)in the LPS-induced zebrafish were observed using transgenic zebrafish and microscopy.CN extracts did not show a significant effect on the macrophage migration,but strongly inhibited the migration of neutrophils in zebrafish.Extracts of CN leaves may serve as a source of nutraceutical compounds with anti-inflammatory properties and may have a promising prospect.The anti-inflammatory activity of extracts was due to its modulatory effect on the expression of pro-inflammatory cytokine and recruitment of neutrophils.