Department of Pharmacy, Faculty of Science, National University of Singapore, 18 Science Drive 4, Singapore, 117543, Singapore.
Department of Pharmacology, Yong Loo Lin School of Medicine, 16 Medical Drive, Block MD3, #04-01S, Singapore, 117600, Singapore.
BMC Complement Med Ther. 2020 Mar 18;20(1):91. doi: 10.1186/s12906-020-02884-w.
Dysregulation of pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) form the basis of immune-mediated inflammatory diseases. Vitex trifolia L. is a medicinal plant growing in countries such as China, India, Australia and Singapore. Its dried ripe fruits are documented in Traditional Chinese Medicine to treat ailments like rhinitis and dizziness. Its leaves are used traditionally to treat inflammation-related conditions like rheumatic pain.
This study aimed to investigate the effects of V. trifolia leaf extracts prepared by different extraction methods (Soxhlet, ultrasonication, and maceration) in various solvents on cytokine production in human U937 macrophages, and identify phytoconstituents from the most active leaf extract.
Fresh leaves of V. trifolia were extracted using Soxhlet, ultrasonication, and maceration in hexane, dichloromethane, methanol, ethanol or water. Each extract was evaluated for its effects on TNF-α and IL-1β cytokine production by enzyme-linked immunosorbent assay in lipopolysaccharide-stimulated human U937 macrophages. The most active extract was analyzed and further purified by different chemical and spectroscopic techniques.
Amongst 14 different leaf extracts investigated, extracts prepared by ultrasonication in dichloromethane and maceration in ethanol were most active in inhibiting TNF-α and IL-1β production in human U937 macrophages. Further purification led to the isolation of artemetin, casticin, vitexilactone and maslinic acid, and their effects on TNF-α and IL-1β production were evaluated. We report for the first time that artemetin suppressed TNF-α and IL-1β production. Gas chromatography-mass spectrometry analyses revealed the presence of eight other compounds. To the best of our knowledge, this is the first report of butylated hydroxytoluene, 2,4-di-tert-butylphenol, campesterol and maslinic acid in V. trifolia leaf extracts.
In conclusion, leaf extracts of V. trifolia obtained using different solvents and extraction methods were successfully investigated for their effects on cytokine production in human U937 macrophages. The findings provide scientific evidence for the traditional use of V. trifolia leaves (a sustainable resource) and highlight the importance of conservation of medicinal plants as resources for drug discovery. Our results together with others suggest further investigation on V. trifolia and constituents to develop novel treatment strategies in immune-mediated inflammatory conditions is warranted.
肿瘤坏死因子-α（TNF-α）和白细胞介素-1β（IL-1β）等促炎细胞因子的失调是免疫介导的炎症性疾病的基础。三叶鬼针草是一种药用植物，生长在中国、印度、澳大利亚和新加坡等国家。传统中医记载其干成熟果实可治疗鼻炎、头晕等疾病。其叶子传统上用于治疗风湿痛等炎症相关疾病。
本研究旨在探讨不同提取方法（索氏提取、超声提取和浸渍提取）用不同溶剂制备的三叶鬼针草叶提取物对人 U937 巨噬细胞细胞因子产生的影响，并从最活跃的叶提取物中鉴定植物成分。
用索氏提取、超声提取和浸渍提取法分别用己烷、二氯甲烷、甲醇、乙醇和水提取三叶鬼针草新鲜叶片。采用酶联免疫吸附试验（ELISA）测定各提取物对脂多糖刺激的人 U937 巨噬细胞产生 TNF-α和 IL-1β细胞因子的影响。对最活跃的提取物进行分析，并通过不同的化学和光谱技术进一步纯化。
在所研究的 14 种不同叶提取物中，超声提取二氯甲烷提取物和浸渍提取乙醇提取物在抑制人 U937 巨噬细胞 TNF-α和 IL-1β产生方面最为活跃。进一步的纯化导致了 artemetin、casticin、vitexilactone 和 maslinic acid 的分离，并评估了它们对 TNF-α和 IL-1β产生的影响。我们首次报道 artemetin 抑制 TNF-α和 IL-1β的产生。气相色谱-质谱分析显示存在其他八种化合物。据我们所知，这是首次在三叶鬼针草叶提取物中发现丁基化羟基甲苯、2,4-二叔丁基苯酚、菜油甾醇和齐墩果酸。
综上所述，采用不同溶剂和提取方法获得的三叶鬼针草叶提取物成功地研究了其对人 U937 巨噬细胞细胞因子产生的影响。研究结果为三叶鬼针草叶（一种可持续资源）的传统用途提供了科学依据，并强调了药用植物作为药物发现资源的保护的重要性。我们的研究结果与其他研究结果一起表明，进一步研究三叶鬼针草及其成分有望开发出治疗免疫介导的炎症性疾病的新治疗策略。
