Division of Immunopathology of Nervous System, Department of Neuropathology, Institute of Pathology, University of Tübingen, Germany.
J Ethnopharmacol. 2013 Aug 26;149(1):162-8. doi: 10.1016/j.jep.2013.06.016. Epub 2013 Jun 21.
Entada africana is a plant used in African traditional medicine for the treatment of stomachache, fever, liver related diseases, wound healing, cataract and dysentery.
This study aimed at evaluating the anti-inflammatory activity of fractions of the stem bark extract of the plant using lipopolysaccharide (LPS)-induced inflammation in RAW 264.7 macrophages model.
The crude extract was prepared using the mixture CH2Cl2/MeOH (1:1, v/v) and fractionated by flash chromatography using solvents of increasing polarity to obtain five different fractions. The effects of the fractions on the cells viability were studied by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and their inhibitory activity against LPS-induced nitric oxide (NO) production screened by Griess test. The most active fraction was further investigated for its effects on reactive oxygen species (ROS) production using flux cytometry, the expression of inducible nitric oxide synthase (iNOS), pro-and anti-inflammatory cytokines (IL1β, TNFα, IL6, IL10 and IL13) by RT-PCR, and the activity of the enzyme p38 MAPK kinase by enzyme-linked immunosorbent assay (ELISA).
The fractions presented no significant effect on the viability of macrophages at 100 μg/ml after 24h incubation. The CH2Cl2/MeOH 5% (Ea5) fraction was found to be the most potent in inhibiting NO production with a half inhibition concentration (IC50)=18.36 μg/ml, and showed the highest inhibition percentage (89.068%) in comparison with Baicalin (63.34%), an external standard at 50 μg/ml. Ea5, as well as Baicalin significantly (P<0.05) inhibited the expression of TNFα, IL6 and IL1β mRNA, attenuated mRNA expression of inducible NO synthase in a concentration-dependent manner, stimulated the expression of anti-inflammatory cytokines (IL10 and IL13), and showed a 30% inhibition of the activity of p38 MAPK kinase.
The results of the present study indicate that the fraction Ea5 of Entada africana possesses most potent in vitro anti-inflammatory activity and may contain compounds useful as a therapeutic agent in the treatment of inflammatory related diseases cause by over-activation of macrophages.
非洲刺桐是一种在非洲传统医学中用于治疗胃痛、发热、肝脏相关疾病、伤口愈合、白内障和痢疾的植物。
本研究旨在使用脂多糖 (LPS) 诱导 RAW 264.7 巨噬细胞模型评估该植物茎皮提取物的馏分的抗炎活性。
使用 CH2Cl2/MeOH（1:1，v/v）混合物制备粗提取物，并通过使用极性逐渐增加的溶剂进行闪式色谱分离来获得五种不同馏分。通过 3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四唑溴化物 (MTT) 测定法研究馏分对细胞活力的影响，并通过格里斯试验筛选其对 LPS 诱导的一氧化氮 (NO) 产生的抑制活性。对最活跃的馏分进行进一步研究，以评估其对活性氧 (ROS) 产生的影响，方法是使用通量细胞术，通过 RT-PCR 检测诱导型一氧化氮合酶 (iNOS)、促炎和抗炎细胞因子 (IL1β、TNFα、IL6、IL10 和 IL13) 的表达，并通过酶联免疫吸附测定 (ELISA) 测定丝裂原活化蛋白激酶激酶 (p38 MAPK) 的活性。
在孵育 24 小时后，浓度为 100μg/ml 时，各馏分对巨噬细胞的活力没有显著影响。发现 CH2Cl2/MeOH 5%（Ea5）馏分在抑制 NO 产生方面最为有效，半抑制浓度 (IC50)=18.36μg/ml，与 50μg/ml 时的外部标准黄芩苷（63.34%）相比，抑制率最高（89.068%）。Ea5 以及黄芩苷显著（P<0.05）抑制 TNFα、IL6 和 IL1βmRNA 的表达，以浓度依赖的方式减弱诱导型一氧化氮合酶 mRNA 的表达，刺激抗炎细胞因子（IL10 和 IL13）的表达，并抑制 p38 MAPK 激酶活性的 30%。
本研究结果表明，非洲刺桐的馏分 Ea5 具有最强的体外抗炎活性，可能含有可作为治疗因巨噬细胞过度激活引起的炎症相关疾病的治疗剂的有用化合物。
