该成果在河南省精准营养与保健食品等项目支持下,与新西兰奥克兰大学、埃及国家农业中心合作,以“Nigella sativa: A dietary supplement as an immune-modulator on the basis of bioactive components”为题发表于中科院JCR二区《Frontier in Nutrition》杂志(IF:6.576)。
Nigella sativa种子为毛茛科黑种草属植物N. sativa干燥的种子,原产于西南亚,广泛分布在地中海、中欧、西亚等地。种子入药,广泛用于食品添加剂、防腐剂和香料,也作为食用油在埃及等中东和北非国家被广泛使用;医疗上常用作膳食营养补充剂、祛风剂、利尿剂和驱虫剂等。N. sativa种子具有多种药理作用,包括抗菌、抗氧化、抗癌、抗炎、降糖、保肝、利尿等。
文献研究表明,营养补充剂和药物的联合使用可能有良好的结果。据报道,N. sativa种子作为营养补充剂对辅助治疗COVID-19有效。此外,以N. sativa种子油作膳食补充剂对T2DM患者表现出良好的心血管保护作用。与其他营养补充剂一样,N. sativa种子也可以通过影响免疫系统来增强身体对疾病的抵抗能力。
研究表明,N. sativa种子在满足营养需求的同时有改善机体免疫系统的作用。黑种草属植物种子化学成分的研究始于20世纪中期,N. sativa种子含有多种生物活性物质,如挥发油、生物碱、甾体、黄酮、皂苷和酚类等。因此,我们选择对N. sativa种子的化学成分进行系统研究,研究其免疫抗炎等功效成分和作用机制,为寻找新的免疫活性成分提供基础。
结果与讨论
化合物结构鉴定
对N. sativa的70%乙醇提取物进行了系统的分离,并鉴定1个新的单萜苷3-methoxythymol-6-O-β-D-apiofuranosyl-(1→6)-β-D-
glucopyranoside(1)和11个已知化合物(2-12)。
图1:N. sativa种子化合物结构图
图2:化合物1的1H-1H COSY和HMBC相关
单糖衍生物(化合物5和8)的免疫调节作用
巨噬细胞是机体天然抵抗力的一种重要效应细胞,在先天性和适应性免疫反应中可发挥吞噬、呈递抗原和分泌细胞因子等作用。我们发现,在一定浓度范围内,化合物5和8可以激活巨噬细胞,促进RAW264.7巨噬细胞的吞噬作用,这可能是免疫调节所必需的。
细胞因子是免疫和非免疫细胞释放的细胞间信号蛋白,通过诱导细胞分化和增殖,从而在控制整个生物体内稳态中发挥重要作用。免疫调节剂诱导巨噬细胞合成细胞因子的能力是评估其能否增强先天免疫活性的方法之一。结果表明,化合物5和8能促进RAW264.7细胞中肿瘤坏死因子-α(TNF-α)和白细胞介素-6(IL-6)的释放,最佳剂量为200 μmol/L,在此基础上,用qRT-PCR方法测定了化合物5和8 (200 μmol/L)对正常培养RAW264.7巨噬细胞中细胞因子mRNA水平的影响。如图3(C)所示,阳性对照组在脂多糖(LPS)作用后,TNF-α和IL-6的基因转录显著升高。此外,与对照组相比,化合物5和8显著增加了TNF-α和IL-6的水平。这表明单糖衍生物可以上调巨噬细胞中TNF-α和IL-6的基因水平,促进IL-6和TNF-α的产生。
为了确定化合物5和8增强RAW264.7巨噬细胞免疫功能的作用机制,我们检测了其对NF-κB信号通路的影响。NF-κB在细胞和机体的免疫、炎症反应、细胞凋亡等过程中起至关重要作用,其通过调控相关炎性因子、趋化因子、生长因子以及环氧合酶-2(COX-2)和一氧化氮合成酶(NOS)诱导酶等表达而参与细胞凋亡、细胞免疫调节、炎症反应、肿瘤发生等多种生物过程。结果显示,与空白组相比,化合物5和8可以促进正常培养的RAW264.7细胞的NF-κB信号通路中关键蛋白p-IκBα和p-p65的表达,从而适度激活NF-κB信号通路并增强免疫力。在正常培养条件下,我们测量了化合物5和8对COX-2和诱导型一氧化氮合酶(iNOS)表达的影响。如图3(D)所示,LPS可以显著增加iNOS和COX-2 mRNA的表达,同时,化合物5和8(200 μmol/L)能显著上调RAW264.7巨噬细胞中iNOS和COX-2 mRNA的转录水平。此外,与空白组相比,化合物5和8 (50、100和200 μmol/L)显著促进了COX-2和iNOS蛋白的产生。总之,化合物5和8可以在基因和蛋白质水平上显著促进COX-2和iNOS的产生。
图3:化合物5和8对正常培养RAW264.7细胞中NF-κB信号通路的影响
(A)NO的释放量,(B)炎性细胞因子TNF-α、IL-6 mRNA转录水平,(C)TNF-α、IL-6的分泌水平,(D)COX-2和iNOS的mRNA和蛋白表达水平,(E)NF-κB信号通路中关键蛋白的表达水平。GAPDH served as control. Data shown are means ± SEM. ***p < 0.001, **p < 0.01, *p < 0.05.
图4:化合物5和8对LPS诱导的RAW264.7细胞中NF-κB信号通路的影响
(A)细胞存活率,(B)RAW264.7细胞的吞噬活性,(C)NO释放量,(D)COX-2和iNOS的mRNA和蛋白表达水平,(E)NF-κB通路中关键蛋白的表达水平,(F)活性氧的产生水平。β-Actin served as a control. Data shown are means ± SEM. ***p < 0.001, **p < 0.01, *p < 0.05. ###p < 0.001, ##p < 0.01, #p < 0.05.
图5:化合物5和8对RAW264.7巨噬细胞中NF-κB信号通路影响的潜在机制图
结论
N. sativa种子中首次分离的单糖衍生物5和8通过调控NF-κB信号通路对免疫和抗炎产生双向调节作用。免疫机制可能与增加正常RAW264.7细胞中细胞因子的释放和mRNA转录水平有关,抗炎可能与抑制LPS刺激下促炎细胞因子的过度释放和mRNA水平的过度转录有关。为进一步研究N. sativa种子作为免疫调节的膳食补充剂提供理论基础。
Nigella sativa: A dietary supplement as an immune-modulator on the basis of bioactive components
Yun Niu1,2‡, Baoguang Wang1,2‡, Li Zhou1,2,3, Changyang Ma1,3, Geoffrey I.N. Waterhouse1,5, Zhenhua Liu1,3*, Adel F. Ahmed3,4*, Dongxiao Sun-Waterhouse1,5*, Wenyi Kang1,2,3*
1National R & D Center for Edible Fungus Processing Technology, Henan University, Kaifeng 475004, China
2Functional Food Engineering Technology Research Center, Henan, Kaifeng, 475004, China
3Joint International Research Laboratory of Food & Medicine Resource Function, Henan Province, Kaifeng 475004, China
4Medicinal and Aromatic Plants Researches Department, Horticulture Research Institute, Agricultural Research Center, Egypt
5 School of Chemical Sciences, University of Auckland, Auckland 1142, New Zealand
‡These authors contributed equally to this work.
*Corresponding authors to: Zhenhua Liu, Email: 10200113@vip.henu.edu.cn; Adel F. Ahmed: Email: adelfahmi_2008@yahoo.com; Dongxiao Sun-Waterhouse, Email: dxsun72@hotmail.com Wenyi Kang, E-mail: kangweny@hotmail.com
Abstract: Nutrients that can be considered as functional foods and exert physiological benefits on immune system. The seeds of Nigella sativa with many active constituents are mainly used for medicine, food spice and nutritional supplements in Egypt. Much attention has been paid to N. sativa seeds for their anticancer, antibacterial, anti-inflammatory and immune properties. However, it is unclear that active constituents and mechanisms underlying functions from N. sativa seeds. Thus, the bioactive constituents with immune regulation from N. sativa seeds were systematically studied. A new compound (3-methoxythymol-6-O-β-D-apiofuranosyl-(1→6)-β-D-glucopyranoside 1) and eleven known compounds (2-12) were separated from the N. sativa seeds by chromatographic methods. Their structures were elucidated by spectroscopic analysis of MS, UV, IR, 1H- and 13C-NMR. Furthermore, immunomodulatory effects of those compounds in RAW 264.7 cells were evaluated by phagocytosis, NO and cytokine release, related mRNA transcription and key proteins expression in vitro. Monosaccharide derivatives, Ethyl-α-D-furaarabinose (5) and Ethyl-β-D-fructofuranoside (8), played a bidirectional regulatory role in immunity and anti-inflammation through regulating NF-κB signaling pathways. The results showed that active compounds and the mechanism of immune regulation in N. sativa, it indicated that N. sativa seeds could be used as dietary supplements in immunomodulation.
Reference:
Niu Y, Wang B G, Zhou L, et al. Nigella sativa: A dietary supplement as an immune-modulator on the basis of bioactive components[J]. frontier in Nutrition, 2021. DOI: 10.3389/fnut.2021.722813.