State Key Laboratory of Biotherapy, West China Hospital, West China Medical School, Sichuan University, Chengdu 610041, China , Guangdong Zhongsheng Pharmaceutical Co. Ltd., Dongguan 440100, Guangdong, China.
Am J Chin Med. 2014;42(4):949-65. doi: 10.1142/S0192415X14500608.
The present study was designed to investigate the in vitro and in vivo anti-inflammatory activity of flavonoids isolated from Millettia pachycarpa Benth. The seeds of M. pachycarpa Benth were extracted with ethanol and subjected to chromatographic separation for the isolation of bioactive compounds. Their structures were elucidated by spectroscopic methods. The anti-inflammatory activity of the compounds was investigated by evaluating the inhibition ability of NO production, iNOS activity and iNOS protein expression induced by LPS-stimulated RAW264.7 macrophages in vitro and the carrageenan-induced hind paw edema model in vivo. Molecular docking simulation was also employed to obtain the binding parameters in the binding pocket of iNOS. Thirteen compounds (1-13) were isolated from Chinese herbal medicine M. pachycarpa Benth. Among them, 4-hydroxylonchocarpin (6) and deguelin (7) exhibited remarkable inhibitory rates of 66.5% and 57.7%, respectively, compared with that of 52.5% of indomethacin in LPS-induced macrophages cells. 4-hydroxylonchocarpin (6) with low toxicity (IC50 > 100 μm) exhibited better inhibitory effects to positive control of 1400W on iNOS activity at the concentration of 10 μm. Western blot assay revealed that 4-hydroxylonchocarpin (6) inhibited iNOS protein expression in RAW264.7 cells and molecular docking simulation showed that 4-hydroxylonchocarpin (6) fit well into the binding pocket of iNOS. In the carrageenan-induced paw edema model, our data revealed that the anti-inflammatory potential of 4-hydroxylonchocarpin (6) at 10 mg/kg showed comparable inhibitory ability to indomethacin at 5 h while a higher concentration of 4-hydroxylonchocarpin (6) at 50 mg/kg showed higher inhibitory activity than indomethacin, which was further confirmed by plasma levels of nitrite. The overall results suggest that 4-hydroxylonchocarpin (6) might be used as a potential therapeutic agent for inflammation-associated disorders.
本研究旨在探讨密花豆（Millettia pachycarpa Benth.）中分离得到的黄酮类化合物的体外和体内抗炎活性。密花豆种子用乙醇提取，经色谱分离得到生物活性化合物。通过光谱方法阐明其结构。通过评估 LPS 刺激 RAW264.7 巨噬细胞产生的 NO 产生、iNOS 活性和 iNOS 蛋白表达的抑制能力，以及体内角叉菜胶诱导的后爪水肿模型，研究了化合物的抗炎活性。还进行了分子对接模拟，以获得 iNOS 结合口袋中的结合参数。从中药密花豆中分离得到 13 种化合物（1-13）。其中，4-羟基野百合碱（6）和去甲氧基野百合碱（7）的抑制率分别为 66.5%和 57.7%，明显优于 LPS 诱导的巨噬细胞中 52.5%的吲哚美辛。毒性低（IC50 > 100 μm）的 4-羟基野百合碱（6）在 10 μm 浓度下对 1400W 阳性对照物的 iNOS 活性具有更好的抑制作用。Western blot 分析表明，4-羟基野百合碱（6）抑制 RAW264.7 细胞中 iNOS 蛋白的表达，分子对接模拟表明，4-羟基野百合碱（6）与 iNOS 的结合口袋结合良好。在角叉菜胶诱导的爪水肿模型中，我们的数据显示，10 mg/kg 的 4-羟基野百合碱（6）的抗炎潜力在 5 小时时与吲哚美辛相当，而较高浓度的 4-羟基野百合碱（6）（50 mg/kg）显示出比吲哚美辛更高的抑制活性，这进一步得到了亚硝酸盐血浆水平的证实。总体结果表明，4-羟基野百合碱（6）可能可作为治疗与炎症相关疾病的潜在治疗剂。
