鹿衔草内生真菌Penicillium solitum次生代谢产物的分离鉴定与神经保护活性筛选

Isolation, identification, and neuroprotective activity screening of secondary metabolites of endophytic Penicillium solitum from Pyrola incarnata Fisch

摘要:

【背景】鹿衔草(Pyrola incarnata Fisch)化学成分丰富，而且具有多种药理活性，在食品、医药领域得到广泛应用，但尚未见其内生真菌次生代谢产物的相关报道。【目的】对鹿衔草内生真菌Penicillium solitum (P.solitum)的次生代谢产物进行分离鉴定，筛选出具有抗神经炎症的活性成分。【方法】采用柱层析和制备型高效液相色谱对鹿衔草内生真菌P.solitum次生代谢产物进行分离和纯化，通过核磁共振和高分辨质谱进行结构鉴定，利用反向分子对接技术筛选出抗神经炎症活性的化合物，采用脂多糖(lipopolysaccharide，LPS)诱导的BV-2细胞炎症模型进行验证。【结果】从鹿衔草内生真菌P.solitum次生代谢产物中分离鉴定出12个化合物，分别为cyclopenin (1)、dehydrocyclopeptin (2)、viridicatin (3)、对羟基苯甲酸(4)、吲哚乙酸(5)、methyl compactin (6)、cyclopeptine (7)、cyclopenol (8)、原儿茶酸(9)、viridicatol (10)、N-[2-(3-indolyl) ethyl]malonamic acid (11)和solitumidine A (12)。分子对接结果显示化合物11与炎症相关的丝裂原活化蛋白激酶(mitogen-activated protein kinase，MAPK)信号通路中p38丝裂原活化蛋白激酶(p38 mitogen-activated protein kinase，简称p38)和细胞外信号调节激酶1(extracellular regulated protein kinase 1，ERK1)有较高的结合活性。BV-2细胞实验表明，化合物11 (10、20μmol/L)能显著提高BV-2细胞存活率，并有效抑制炎症因子(肿瘤坏死因子-α、白介素-1β、白介素-6)和一氧化氮的释放。【结论】化合物11是新天然产物，具有抗神经炎症活性，其作用机制可能与抑制MAPK信号通路中p38和ERK蛋白的表达有关。

Abstract:

[Background] Pyrola incarnata Fisch (P. incarnata) has been widely used in the fields of food and medicine owing to the abundant chemical components with a variety of pharmacological activities. However, no report on the secondary metabolites of its endophytic fungi is available. [Objective] To isolate and identify the secondary metabolites of endophytic Penicillium solitum from P. incarnata and screen out the ones with anti-neuroinflammatory activity. [Methods] They were isolated and purified by column chromatography and preparative high performance liquid chromatography (HPLC). The structures were identified by nuclear magnetic resonance (NMR) and high-resolution mass spectrometry (HRMS). Moreover, the anti-neuroinflammatory activity was verified in LPS-induced inflammatory BV-2 cells by reverse docking. [Results] A total of 12 compounds were isolated and identified:cyclopenin (1), dehydrocyclopeptin (2), viridicatin (3), p-hydroxybenzoic acid (4), indoleacetic acid (5), methyl compactin (6), cyclopeptine (7), cyclopenol (8), protocatechuic acid (9), viridicatol (10), N-[2-(3-indolyl)ethyl]malonamicacid (11), solitumidine A (12). Molecular docking showed that compound 11 had remarkable binding activity to p38 mitogen-activated protein kinase (p38) and extracellular regulated protein kinase 1 (ERK1) in mitogen-activated protein kinase (MAPK) signaling pathway which was associated with inflammation. In the experiment on BV-2 cells, compound 11 (10, 20 μmol/L) significantly improved the survival rate of BV-2 cells. Moreover, it inhibited the release of inflammatory factors (tumor necrosis factor-α, interleukin-1β, and interleukin-6) and nitric oxide (NO) in BV-2 cells induced by LPS. [Conclusion] Compound 11 is a new natural product with potential anti-neuroinflammatory activities, and the mechanism is the likelihood that it inhibits the expression of p38 and ERK proteins in MAPK signal pathway, which needs further investigation.