背景:金线莲是在热带和亚热带雨林中发现的一种小型陆生兰花。这些兰花在中国传统上被广泛使用,台湾,和越南,因为它们的药用特性和治疗益处。它们用于不同系统的治疗,比如胃部疾病,胸痛,关节炎,肿瘤,桩,沸腾,月经失调,和炎症。先前已经报道了缅甸金线莲(AB)的水提取物具有抗炎活性,然而,缺乏有关其生物活性化合物及其作用机制的证据。
目的:本研究的目的是鉴定AB的乙醇提取物中的抗炎化合物,并确定其在LPS刺激的巨噬细胞中的抗炎机制及其安全性。
方法:制备AB(ABE)的乙醇提取物,然后使用正己烷和乙酸乙酯进行极性依赖性提取,这将导致正己烷(ABH)的分离,乙酸乙酯(ABEA),和残余物或含水(ABA)级分。AB馏分进行了调查,以确定总酚和黄酮的含量,抗氧化能力,毒性,RAW264.7巨噬细胞的安全性,人PBMC,和RBC。提取后,通过一氧化氮(NO)产生测定对每种提取物进行抗炎筛选。进一步检查活性部分对促炎介质的作用。此外,使用LC-MS/MS鉴定活性组分中的kinsenoside含量。还使用果蝇的翼斑试验测试了AB的细胞毒性和遗传毒性。
结果:数据表明ABEA具有最高的酚类含量和抗氧化活性水平。ABE,ABEA,ABA,但不是ABH,显著抑制LPS刺激的巨噬细胞中NO的产生。ABEA和ABA均可降低LPS介导的TNF-α表达,IL-6,iNOS,和COX-2在mRNA和蛋白质水平。此外,只有ABEA显着减少了核易位和核因子κB(NF-κB)转录激活所需的LPS刺激的p65磷酸化。有趣的是,液相色谱-串联质谱(LC-MS/MS)分析显示ABA中含有高水平的人参皂苷,一种可能的抗炎化合物,而ABE和ABEA可能需要其他化合物与kinsenoside的组合来抑制炎症。结果表明,所有活性部分既无细胞毒性也无遗传毒性。
结论:我们的研究证明AB的亲水部分在LPS刺激的巨噬细胞中表现出抗炎活性。AB使用的机制涉及清除自由基和减少促炎介质,包括IL-1β,IL-6,TNF-α,NO,iNOS和COX-2。AB的抗炎作用涉及ABEA中存在的一些未知组分对NF-κB信号传导途径的抑制。本研究发现,人参皂苷是ABA中的主要活性化合物,可作为植物提取物质量控制的生物标志物。这项研究提供了令人信服的有关抗炎机制的重要信息,并初步证明了金线莲的安全性。因此,从这项研究中获得的知识将为开发和标准化使用该植物的提取物作为替代药物或功能食品来预防或治疗与慢性炎症相关的非传染性慢性疾病提供支持性证据。
BACKGROUND: Anoectochilus species is a small terrestrial orchid found in tropical and subtropical rain forest. These orchids are traditionally used extensively in China, Taiwan, and Vietnam due to their medicinal properties and therapeutic benefits. They are employed for treatment in different systems, such as stomach disorders, chest pain, arthritis, tumor, piles, boils, menstrual disorders, and inflammation. Aqueous extract of Anoectochilus burmannicus (AB) has been previously reported to exhibit anti-inflammatory activities, however there is a lack of evidence regarding its bioactive compounds and the mechanism of its actions.
OBJECTIVE: The objectives of this study were to identify the anti-inflammatory compound(s) in an ethanolic extract of AB and to determine its anti-inflammatory mechanisms in LPS-stimulated macrophages and also its safety.
METHODS: The ethanolic extract of AB (ABE) was prepared and subsequently subjected to polarity-dependent extraction using n-hexane and ethyl acetate, which would result in isolation of the n-hexane (ABH), ethyl acetate (ABEA), and residue or aqueous (ABA) fractions. The AB fractions were investigated to determine total phenolic and flavonoid content, antioxidant capacity, toxicity, and safety in RAW 264.7 macrophages, human PBMCs, and RBCs. After extraction anti-inflammation screening of each extract was performed by nitric oxide (NO) production assay. The active fractions were further examined for their effect on proinflammatory mediators. In addition, kinsenoside content in the active fractions was identified using LC-MS/MS. Cellular toxicity and genotoxicity of AB were also tested using the wing spot test in Drosophila melanogaster.
RESULTS: The data showed that ABEA had the highest phenolic content and level of antioxidant activities. ABE, ABEA, and ABA, but not ABH, significantly inhibited the LPS-stimulated NO production in the macrophages. Both ABEA and ABA reduced LPS-mediated expression of TNF-α, IL-6, iNOS, and COX-2 at both mRNA and protein levels. Besides, only ABEA notably diminished the LPS-stimulated p65 phosphorylation required for nuclear translocation and transcriptional activation of the nuclear factor-κB (NF-κB). Interestingly, liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis revealed ABA contained a high level of kinsenoside, a likely anti-inflammatory compound, while ABE and ABEA might require other compounds in combination with kinsenoside for the inhibition of inflammation. It was shown that all active fractions were neither cytotoxic nor genotoxic.
CONCLUSIONS: Our study demonstrated that the hydrophilic fractions of AB exhibit anti-inflammatory activity in LPS-stimulated macrophages. The mechanism used by the AB involves the scavenging of free radicals and the reduction of proinflammatory mediators, including IL-1β, IL-6, TNF-α, NO, iNOS and COX-2. The anti-inflammatory action of AB involves the suppression of the NF-κB signaling pathway by some unknown component(s) present in ABEA. This study found that kinsenoside is a major active compound in ABA which could be used as a biomarker for the quality control of the plant extraction. This study provides convincing significant information in vitro regarding the anti-inflammatory mechanism and preliminary evidence of the safety of Anoectochilus burmanicus. Therefore, the knowledge acquired from this study would provide supportive evidence for the development and standardization of the use of the extract of this plant as alternative medicine or functional food to prevent or treat non-communicable chronic diseases related to chronic inflammation.