Institute of Biochemistry Molecular Biology and Biotechnology, University of Colombo, Sri Lanka.
J Ethnopharmacol. 2021 Jun 28;274:114030. doi: 10.1016/j.jep.2021.114030. Epub 2021 Mar 17.
Vernonia zeylanica (L.) Less (Family: Compositae) is a medicinal plant used as external applications for boils, bone fractures, eczema and internally for asthma in traditional medicine in Sri Lanka. Anti-nociceptive, anti-bacterial and anti-proliferative activities have been reported previously.
To investigate the anti-inflammatory activity of methanol/dichloromethane extract (MDE) of leaves of V. zeylanica by assessing in vivo inhibition of rat paw-edema, in vitro inhibition of the production of nitric oxide (NO) and superoxide and inhibitory effect on inducible nitric oxide synthase (iNOS) gene expression.
In vivo anti-inflammatory activity of MDE was tested at the dose of 1500 mg/kg using rat paw-edema model. Indomethacin and Gum acacia was used as the positive and vehicle control respectively. In vitro NO inhibitory activity of 7.8-250 μg/ml MDE was tested using lipopolysaccharide (LPS)-stimulated (1 μg/ml) mouse macrophages (RAW264.7 cells) and rat peritoneal cells (RPC) obtained following carrageenan-induction (5 mg/Kg). Griess method was used to quantify the nitrite levels in culture supernatants. In vitro inhibition of superoxide production of Phorbal 12-myristate 13-acetate (PMA)-stimulated RAW cells was determined by quantitative Nitroblue Tetrazolium (NBT) assay. N-monomethyl-L-arginine acetate (NMMA) (1 mM) and Diphenyleneiodonium chloride (DPI) (10 μM) were used as the positive controls for inhibitory activity of NO and superoxide production respectively. Reverse transcriptase polymerase chain reaction (RT-PCR) analysis was carried out to test the inhibitory effect on mRNA expression of iNOS gene.
Treatment with MDE of V. zeylanica at 1500 mg/kg showed significant inhibition of paw-edema from 1-5 hour (P < 0.01) compared with the control. The reference drug, indomethacin showed a biphasic pattern and its highest inhibition was (98.3 ± 7.1%) at 4 h (P < 0.01). MDE of V. zeylanica showed similar inhibition of paw-edema with highest inhibition recorded as 94.5 ± 5.28%, at 5 h (P < 0.01). The inhibitory concentration (IC) of MDE for in vitro NO inhibitory activity was 105 μg/ml for RAW cells and 80 μg/ml for RPCs. Both NO inhibitory activities showed significant dose-dependency (r = 0.998 and r = 0.915 respectively; p < 0.05). MDE concentration of 250 μg/ml showed 55% inhibition of ROS production in RAW cells. NMMA showed 78% and 70.1% inhibition of NO production with RAW cells and RPCs whereas DPI showed 61% superoxide inhibitory activity with RAW cells. NO inhibitory activity of MDE on RAW cells was confirmed by the significant reduction (99.1%) in iNOS gene expression.
These results demonstrated potent anti-inflammatory activity of MDE of V. zeylanica reflected by its significant in vivo inhibition of rat paw-edema, in vitro inhibition of NO and superoxide production, and the reduction of iNOS gene expression. Thus, further purification and isolation of bioactive compounds from V. zeylanica are emphasized.
泽兰 Vernonia zeylanica（菊科）在斯里兰卡传统医学中作为一种药用植物，用于治疗疖子、骨折、湿疹和哮喘等疾病，其可以外用或内服。先前已经报道过其具有止痛、抗菌和抗增殖作用。
通过评估大鼠足肿胀的体内抑制作用、测定一氧化氮（NO）和超氧化物的体外产生抑制作用以及抑制诱导型一氧化氮合酶（iNOS）基因表达，研究泽兰甲醇/二氯甲烷提取物（MDE）的抗炎活性。
采用大鼠足肿胀模型，以 1500mg/kg 的剂量测试 MDE 的体内抗炎活性。吲哚美辛和阿拉伯胶分别作为阳性对照和载体对照。采用脂多糖（LPS）（1μg/ml）刺激的（1μg/ml）小鼠巨噬细胞（RAW264.7 细胞）和角叉菜胶诱导的（5mg/kg）大鼠腹腔细胞（RPC），测试 7.8-250μg/ml MDE 的体外 NO 抑制活性。使用格里斯法测定培养上清液中亚硝酸盐水平。采用定量硝基四氮唑蓝（NBT）测定法测定 Phorbal 12-myristate 13-acetate（PMA）刺激的 RAW 细胞中超氧化物的产生抑制。N-单甲基-L-精氨酸乙酸盐（NMMA）（1mM）和二苯基碘（DPI）（10μM）分别用作 NO 和超氧化物产生抑制活性的阳性对照。进行逆转录聚合酶链反应（RT-PCR）分析，以测试 iNOS 基因表达的抑制作用。
1500mg/kg 的泽兰 MDE 处理显示，与对照组相比，1-5 小时内（P<0.01）足肿胀明显抑制。参考药物吲哚美辛呈双相模式，其最高抑制率（98.3±7.1%）在 4 小时（P<0.01）。泽兰 MDE 对足肿胀的抑制作用相似，最高抑制率为 94.5±5.28%，在 5 小时（P<0.01）。MDE 的体外 NO 抑制活性的 IC 为 105μg/ml，用于 RAW 细胞，80μg/ml 用于 RPCs。两种 NO 抑制活性均表现出显著的剂量依赖性（r=0.998 和 r=0.915；P<0.05）。250μg/ml 的 MDE 浓度可抑制 RAW 细胞中 55%的 ROS 产生。NMMA 对 RAW 细胞和 RPC 中 NO 的产生分别有 78%和 70.1%的抑制作用，而 DPI 对 RAW 细胞中超氧化物的产生有 61%的抑制活性。NO 抑制活性通过 RAW 细胞中 iNOS 基因表达的显著降低（99.1%）得到证实。
这些结果表明，泽兰 MDE 具有显著的抗炎活性，表现在其对大鼠足肿胀的显著体内抑制作用、体外抑制 NO 和超氧化物的产生以及 iNOS 基因表达的减少。因此，强调从泽兰中进一步纯化和分离生物活性化合物。
