不同产源艾纳香油化学成分及其抗炎活性研究

Analysis of Chemical Constituents and Anti-inflammatory Activity of Blumea balsamifera Oil from Different Sources

目的探讨不同产源艾纳香油的化学成分及其抗炎活性。方法利用GC-MS法对不同产源艾纳香油(B1~B5)进行分析,比较确定其主要成分,并采用峰面积归一化法计算相对百分含量。通过小鼠耳廓肿胀法从不同产源中筛选抗炎效果最佳的艾纳香油(0.6 g·kg-1)。采用脂多糖(LPS)诱导小鼠腹腔巨噬细胞建立炎症细胞模型;MTT法检测不同浓度艾纳香油(4.24、12.72、38.16μg·mL-1)对小鼠腹腔巨噬细胞活性的影响;ELISA法检测肿瘤坏死因子(TNF-α)、白细胞介素-1β(IL-1β)、白细胞介素-6(IL-6)及前列腺素E2(PGE2)含量;紫外-可见分光光度法检测一氧化氮(NO)含量及一氧化氮合酶(iNOS)活力;qRT-PCR法检测iNOS、环氧合酶-2(COX-2)mRNA表达。结果不同产源的艾纳香油中左旋龙脑、反式石竹烯、樟脑、石竹烯氧化物、香树烯、花椒素、芳樟醇含量相对较高,不同产源艾纳香油主要化学成分相对含量存在差异。与吐温-80溶剂组比较,艾纳香油B1、B2、B3、B5组均能明显降低二甲苯致小鼠的耳廓肿胀度(P<0.05,P<0.01);后续选择抗炎活性最佳的B2组(抑制率为37.45%)艾纳香油进行实验。与空白组比较,LPS模型组及艾纳香油低、中、高浓度组的细胞活力无明显变化(P>0.05);LPS模型组小鼠巨噬细胞的TNF-α、IL-1β、IL-6及PGE2的分泌量均显著升高(P<0.01),细胞上清液中NO、iNOS含量显著上升(P<0.01),iNOS、COX-2 mRNA表达量明显升高(P<0.01)。与LPS模型组比较,艾纳香油各浓度组的TNF-α、IL-1β分泌量明显降低(P<0.05,P<0.01);艾纳香油低、高浓度组的IL-6分泌量明显降低(P<0.01);艾纳香油低、中浓度组的PGE2分泌量明显降低(P<0.01);艾纳香油各浓度组细胞上清液中NO、iNOS含量以及iNOS、COX-2 mRNA表达量显著降低(P<0.01)。结论不同产源艾纳香油各主要成分相对含量不同,且具有不同程度的抗炎作用,其抗炎机制可能是通过抑制TNF-α、IL-1β、IL-6、NO及PGE2的生成,降低iNOS活力,抑制iNOS及COX-2 mRNA的表达。

Objective To preliminarily determine the chemical composition and anti-inflammatory activity of Blumea balsamifera oil.Methods GC-MS was used to analyze oils of Blumea balsamifera from different sources(B1-B5),to determine and compare the main components.The relative contents were calculated by area normalization method.The strongest anti-inflammatory activity of Blumea balsamifera oils(0.6 g·kg-1)from different sources were screened using mouse ear swelling method.Inflammatory cell model in mouse peritoneal macrophages was established by lipopolysaccharide(LPS)induction;the toxicity of Blumea balsamifera oils(4.24,12.72,38.16μg·mL-1)on mouse peritoneal macrophages were detected by MTT assay.The secretion of tumour necrosis factor-α(TNF-α),interleukin(IL)-1 beta(IL-1β),interleukin-6(IL-6)and Prostaglandin E2(PGE2)in the culture medium of mouse peritoneal macrophages were detected by ELISA.Content of nitric oxide(NO)and the activity of inducible nitric oxide synthase(iNOS)were detected by UV-VIS spectrophotometry.Relative mRNA expression of iNOS and cyclooxygenase-2(COX-2)were determined by quantitative real-time polymerase chain reaction(qRT-PCR).Results The contents of(-)-Borneol,trans-Caryophyllene,Camphor,Caryophyllene-oxide,Alloaromadendrene,Xanthoxylin,Linalool are relatively high in Blumea balsamifera oils of different sources.Compared with Tween-80 solvent group,Blumea balsamifera oil B1,B2,B3,B5 groups can significantly reduce the degree of xyleneinduced ear swelling in mice(P<0.05,P<0.01).The B2 group with the best anti-inflammatory activity(inhibition rate of 37.45%)was chosen for further investigation.Compared with the control group,cell viabilities of the low-,medium-,and high-concentration groups of Blumea balsamifera oil and LPS model group had no change(P>0.05).In the LPS model group,the secretion of TNF-α,IL-1β,IL-6 and PGE2 in mouse macrophages were all significantly increased(P<0.01);the contents of NO and iNOS in cell supernatant were also significantly increased(P<0.01),and the expression levels of iNOS and COX-2 mRNA were vastly increased(P<0.01).Compared with LPS model group,the secretion of TNF-αand IL-1βin all concentration groups of Blumea balsamifera oil were significantly decreased(P<0.05,P<0.01);the secretion of IL-6 in the low and high concentration groups of Blumea balsamifera oil were all reduced(P<0.01);secretion of PGE2 in the low and middle concentration groups of Blumea balsamifera oil were decreased obviously(P<0.01);and the contents of NO,iNOS in the supernatant and the experssion of iNOS,COX-2 mRNA were significantly lower in each concentration group of Blumea balsamifera oil(P<0.01).Conclusion Blumea balsamifera oils from different sources showed different contents,and they had anti-inflammatory effects to different extents.The anti-inflammatory mechanism may be reducing the secretion of TNF-α,IL-1β,IL-6,PGE2,NO production and iNOS activity,and decreasing the expression of iNOS and COX-2 mRNA.