Marine Biology

Oligophenalenone dimers with anti-inflammatory activities from fungus Talaromyces stipitatus

  • ZHU Ling ,
  • PAN Chao ,
  • HUA Silu ,
  • JIANG Wei
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  • School of Environmental Science and Engineering, Yangzhou University, Yangzhou 225127, China
JIANG Wei. email:

Copy editor: LIN Qiang

Received date: 2023-02-03

  Revised date: 2023-03-21

  Online published: 2023-03-24

Supported by

National Natural Science Foundation of China(81903772)

Abstract

An extract of the fungus Talaromyces stipitatus showed inhibitory activity against the nitric oxide (NO) production in the lipopolysaccharide (LPS)-activated RAW264.7 macrophage cell line, and bioassay-guided separation of the extract provided six oligophenalenone dimers, which were characterized to be bacillisporin A (1), 9a-epi-bacillisporin E (2), bacillisporin F (3), duclauxin (4), bacillisporin B (5) and bacillisporin C (6), respectively, by comparison of the nuclear magnetic resonance (NMR) and high resolution electrospray ionization mass spectroscopy (HRESIMS) data with literature data. 2-5 were noncytotoxic on macrophage cells at 30 µmol·L-1, and displayed inhibitory effects against NO production in LPS-induced RAW264.7 macrophage cells, which were all more active than the positive control indomethacin (AG). The half inhibitory concentration (IC50) values of 2-4 were 11.82±1.25, 11.44±1.58 and 23.92±2.86 µmol·L-1, respectively. 5 and 6 were isolated from T. stipitatus for the first time. Duclauxin and its homologues were firstly discovered to show significant anti-inflammatory activities in vitro, which provide a new structural model for further research of anti-inflammatory activity.

Cite this article

ZHU Ling , PAN Chao , HUA Silu , JIANG Wei . Oligophenalenone dimers with anti-inflammatory activities from fungus Talaromyces stipitatus[J]. Journal of Tropical Oceanography, 2023 , 42(6) : 150 -155 . DOI: 10.11978/2023012

近年来, 从海洋真菌中发现新的海洋天然产物数量呈明显上升趋势, 到目前为止, 研究最多的是青霉属(Penicillium)和曲霉属 (Aspergillus) (马丽丽 等, 2021)。柄蓝状菌(Talaromyces stipitatus)是一种腐生型丝状真菌, 在海洋底泥、陆地土壤、腐烂植物以及动物粪便中均有发现。目前从该种中发现了具有丁酰胆碱酯酶抑制活性的二萜 (Zhang et al, 2020), 具有细胞毒活性的麦角甾醇 (Noinart et al, 2017; Zhang et al, 2021), 具有抗菌活性或细胞毒活性的聚酮类化合物 (Zang et al, 2015; Zang et al, 2016a; Zang et al, 2016b; Noinart et al, 2017; Chaudhary et al, 2020)。其中以duclauxin (4)为代表的系列oligophenalenone芳香聚酮二聚体是T. stipitatus 代谢的一类结构特殊且具有不同生物活性的化合物 (Zang et al, 2015; Zang et al, 2016a; Zang et al, 2016b; Chaudhary et al, 2020)。Duclauxin及其同系物主要分布于TalaromycesPenicillium属 (Cao et al, 2015), 其他属鲜有报道。Gao等人发现duclauxins聚酮骨架的生物合成需要非还原聚酮合成酶 (NR-PKS) PhnA和黄素依赖型单加氧酶 PhnB介入, 以乙酰辅酶A和丙二酰辅酶A为前体, 经多步氧化还原生成duclauxins (Gao et al, 2016; Gao et al, 2018)。由于构成二聚体的单体结构差异以及单体间连接方式的不同, 形成了结构不同的duclauxin同系物 (目前共报道43个化合物), 在已报道的这类化合物中3环-3环单体聚合较为常见, 此外, 也有极少数是3环-2环、3环-4环或者3环-5环的二聚体。常见的单体聚合方式是通过C7-C8'和C8-C9'a连接形成1个环戊环 (15), 此外, 也有少部分化合物是通过C7-O-C9'和C8-C9'a连接形成呋喃环 (6)。
本课题组前期从威海附近海域的近海底泥中分离筛选出一株具有抗菌活性的真菌T. stipitatus (WH4-2), 从中发现了具有显著广谱抗菌活性的dibenzo[b, e]oxepinone类化合物 (姜薇 等, 2019)。近期, 课题组对实验室已有真菌发酵产物进行抗炎活性筛选时, 发现T. stipitatus 以土豆液 (PDB)为培养基, 其发酵产物具有显著的抗炎活性, 以体外抗炎活性分析为导向, 从该浸膏的中等偏低极性段部位分离鉴定6个oligophenalenone二聚体。本文将对化合物的提取分离、结构鉴定及抗炎活性检测等工作进行报道。

1 材料与方法

1.1 实验材料

主要仪器与试剂: Bruker AV-600核磁共振仪; Anton Paar MCP 300自动旋光仪; maXis高分辨质谱仪 (HRESI-TOF-MS), 半制备高效液相色谱仪为LabTech LC600配UV600紫外检测器, 色谱柱Kromasil RP-18 (10mm×250mm); 318-MC高速双波长酶标仪; MCO-5AC型CO2培养箱; E200倒置显微镜; CCK-8和NO检测试剂盒购于上海碧芸天生物技术有限公司; 色谱用硅胶(200~300目)和ODS (50µm)分别购于阿拉丁和YMC公司; 分析级和色谱级的甲醇、乙腈、丙酮、乙酸乙酯、氯仿、石油醚均购于国药集团化学试剂有限公司; DMEM培养基、0.25%胰酶、胎牛血清和吲哚美辛均购自Sigma公司; 小鼠单核巨噬细胞RAW264.7购自中国医学科学院基础医学研究所基础医学细胞中心。
菌株来源: 真菌WH4-2分离自山东威海附近海域的近海底泥(采集时间为2015年10月), 经鉴定为Talaromyces stipitatus (姜薇 等, 2019), 标本现存放于扬州大学海洋科学与技术研究所。

1.2 菌株规模化发酵与提取

采用PDB液体发酵法, 静置培养40d, 获得发酵液40L (姜薇 等, 2019)。用含有5%丙酮的乙酸乙酯液萃取两次, 减压浓缩得粗提物18g。

1.3 抗炎活性导向分离

采用LPS诱导RAW264.7细胞生成NO作为体外抗炎活性筛选模型, 分离并筛选对NO生成具有抑制作用的组分 (样品浓度为100µg·mL-1) (Deng et al, 2015)。粗提物 (抑制率= 68.4%), 经200~300目减压硅胶柱色谱, 以二氯甲烷 (CDM): 甲醇 (MeOH)做流动相, 梯度洗脱, 得4个组份Fr.1 (CDM, 4.0g), Fr.2 (CDM:MeOH = 40:1, 4.5g), Fr.3 (CDM : MeOH = 25:1, 2.0g)和Fr.4 (MeOH, 6.0g)。取Fr.3 (抑制率= 80.1%)500mg, 经减压反相硅胶柱色谱 (ODS), 以MeOH:H2O梯度洗脱, 得3个组份Fr.3-1 (MeOH:H2O = 40:60, 60mg), Fr.3-2 (MeOH:H2O = 60:40, 30mg, 抑制率= 42.0%)和Fr.3-3 (MeOH:H2O = 90:10, 240mg, 抑制率= 93.6%)。Fr.3-2经半制备HPLC纯化, 流动相MeOH:H2O = 78:22得到化合物 6 (7.1mg)。Fr.3-3经半制备HPLC纯化, 流动相乙腈 (MeCN):H2O = 75:25得到化合物 1 (8.5mg)、2 (27.3mg)、3 (40.0mg)、4 (42.2mg)和5 (11.7mg)。

1.4 化合物对LPS诱导的RAW264.7巨噬细胞NO生成的影响

运用CCK-8法检测化合物(浓度为30µmol·L-1)对RAW264.7巨噬细胞的细胞毒活性(岗云芹, 2020), 筛选出样品作用后细胞存活率>80%的化合物, 用于下一步抗炎活性测试。随后, 加入不同浓度的样品(30、15、7.5和3.75µmol·L-1)作用于LPS诱导的巨噬细胞, 用NO试剂盒检测NO浓度, 得NO抑制率, 并求IC50值(Deng et al, 2015)。

2 结果

2.1 化合物的结构鉴定

化合物1: 淡黄色无定型粉末; [α]+18.0° (c 0.025, MeOH); HRESIMS m/z 517.1141 [M + H]+ (计算值为: C28H21O10, 517.1135); 1H NMR (600 MHz, CDCl3): δH 11.96 (1H, brs, 4’-OH), 11.19 (1H, brs, 4-OH), 6.92 (1H, s, H-5), 6.85 (1H, s, H-5’), 5.79 (1H, brs, H-9’), 5.74 (1H, d, J = 15.0 HZ, H-1ɑ), 5.66 (1H, d, J = 15.0 HZ, H-1β), 5.07 (1H, d, J = 12.3 HZ, H-1’ɑ), 5.03 (2H, overlap, H-1’β, H-8’), 3.00 (3H, s, 6-CH3), 2.62 (3H, s, 6’-CH3), 2.06 (3H, s, OCOCH3); 13C NMR (CDCl3, 150 MHz): 190.3 (C, C-7’), 170.3 (C, OCOCH3), 169.5 (C, C-3), 167.2 (C, C-3’), 165.0 (C, C-4’), 163.2 (C, C-4), 155.4 (C, C-6’), 146.9 (C, C-9), 146.2 (C, C-3’b), 145.8 (C, C-6), 136.6 (C, C-8), 132.1 (C, C-7), 128.8 (C, C-3b), 121.9 (CH, C-5’), 121.2 (CH, C-5), 119.8 (C, C-6a), 117.6 (C, C-6’a), 110.9 (C, C-9a), 102.8 (C, C-3’a), 97.5 (C, C-3a), 85.1 (C, C-9’), 69.8 (CH2, C-1), 67.4 (CH2, C-1’), 61.7 (CH, C-8’), 50.1(C, C-9’a), 25.4 (CH3, 6-CH3), 24.2 (CH3, 6’-CH3), 21.0 (CH3, OCOCH3)。通过比对文献 (Dethoup et al, 2006), 确定1为bacillisporin A, 化学结构见图1
图1 化合物16的结构

Fig.1 The structures of compounds 1-6

化合物2: 无色无定型粉末; [α]+10.0° (c 0.020, MeOH); HRESIMS m/z 533.1092 [M + H]+ (计算值为: C28H21O11, 533.1084); 1H NMR (600 MHz, CDCl3): δH 11.85 (1H, brs, 4’-OH), 11.23 (1H, brs, 4-OH), 6.91 (1H, s, H-5), 6.81 (1H, s H-5’), 5.68 (1H, s, H-9’), 4.87 (1H, d, J = 13.1 HZ, H-1ɑ), 4.84 (1H, d, J = 12.4 HZ, H-1’β), 4.75 (1H, d, J = 12.4 HZ, H-1’ɑ), 4.72 (1H, s, H-8’), 4.31 (1H, d, J = 13.1 HZ, H-1β), 2.87 (3H, s, 6-CH3), 2.61 (3H, s, 6’-CH3), 2.13 (3H, s, OCOCH3); 13C NMR (CDCl3, 150 MHz): 191.1 (C, C-9), 187.9 (C, C-7’), 169.8 (C, OCOCH3), 167.3 (C, C-3), 167.2 (C, C-3’), 164.8 (C, C-4’), 163.0 (C, C-4), 155.5 (C, C-7), 153.8 (C, C-6’), 148.0 (C, C-6), 145.4 (C, C-8), 143.0 (C, C-3b), 135.7 (C, C-3’b), 122.5 (CH, C-5), 121.2 (CH, C-5’), 116.8 (C, C-6’a), 116.6 (C, C-6a), 108.0 (C, C-3a), 104.0 (C, C-3’a), 83.8 (CH, C-9’), 71.0 (CH2, C-1), 65.8 (C, C-9a), 68.0 (CH2, C-1’), 63.6 (CH, C-8’), 47.8 (C, C-9’a), 24.7 (CH3, 6-CH3), 23.8 (CH3, 6’-CH3), 20.8 (CH3, OCOCH3)。通过比对文献(Zang et al, 2016a), 确定2为9a-epi-bacillisporin E, 化学结构见图1
化合物3: 淡黄色无定型粉末; [α]+28.5° (c 0.020, MeOH); HRESIMS m/z 547.1244 [M + H]+ (计算值为: C29H23O11, 547.1240); 通过比对文献 (Zang et al, 2016a), 样品在CD3OD中的核磁数据与文献吻合, 确定3为bacillisporin F, 化学结构见图1。这里报道一下以CDCl3为溶剂测得的核磁数据1H NMR (600 MHz, CDCl3): δH 11.86 (1H, brs, 4’-OH), 11.60 (1H, brs, 4-OH), 6.94 (1H, s, H-5), 6.82 (1H, s, H-5’), 6.50 (1H, s, H-1), 5.81 (1H, s, H-9’), 5.12 (1H, overlap, H-1’ɑ, H-8’), 4.97 (1H, d, J = 12.6 HZ H-1’β), 3.80 (3H, s, 1-OCH3), 3.01 (3H, s, 6-CH3), 2.59 (3H, s, 6’-CH3), 2.05 (3H, s, OCOCH3); 13C NMR (CDCl3, 150 MHz): 190.3 (C, C-7’), 170.3 (C, OCOCH3), 168.0 (C, C-3), 167.6 (C, C-3’), 164.9 (C, C-4’), 164.1 (C, C-4), 154.7 (C, C-6’), 150.0 (C, C-9), 146.6 (C, C-6), 146.5 (C, C-3’b), 139.5 (C, C-8), 131.6 (C, C-7), 130.7 (C, C-3b), 121.6 (CH, C-5’), 121.0 (CH, C-5), 119.6 (C, C-6a), 117.1 (C, C-6’a), 109.1 (C, C-9a), 103.2 (C, C-3’a), 100.4 (CH, C-1), 97.2 (C, C-3a), 85.7 (CH, C-9’), 69.3 (CH2, C-1’), 62.0 (CH, C-8’), 56.8 (CH3, 1-OCH3), 49.3 (C, C-9’a), 25.5 (CH3, 6-CH3), 24.1 (CH3, 6’-CH3), 21.0 (CH3, OCOCH3)。3溶解在甲醇溶液中, 长时间放置在日光下会形成1对3:1的混合物, 经核磁分析混合物是由3份bacillisporin F (1S)和1份1-epi-bacillisporin F (1R)组成。1-epi-bacillisporin F的核磁数据如下1H NMR (600 MHz, CD3OD): δH 6.93 (1H, s, H-5), 6.80 (1H, s, H-5’), 6.56 (1H, s, H-1), 5.83 (1H, s, H-9’), 5.18 (1H, d, J =12.6 HZ H-1’ɑ), 5.14 (1H, s, H-8’), 5.06 (1H, d, J = 12.6 HZ H-1’β), 3.65 (3H, s, 1-OCH3), 3.00 (3H, s, 6-CH3), 2.54 (3H, s, 6’-CH3), 2.04 (3H, s, OCOCH3) (Zang et al, 2016a)。
化合物4: 淡黄色无定型粉末; [α]+75.0° (c 0.025, MeOH); HRESIMS m/z 547.1242 [M + H]+ (计算值为: C29H23O11, 547.1240); 1H NMR (600 MHz, CDCl3): δH 11.69 (1H, brs, 4’-OH), 10.65 (1H, brs, 4-OH), 7.70 (1H, s, H-1), 6.89 (1H, s, H-5), 6.63 (1H, s, H-5’), 5.20 (1H, brs, H-9’), 5.08 (1H, d, J = 12.0 HZ, H-1’ɑ), 4.77 (1H, d, J = 12.0 HZ, H-1β’), 4.13 (1H, s, H-8’), 2.96 (3H, s, 7-OCH3), 2.73 (3H, s, 6-CH3), 2.21 (3H, s, OCOCH3), 2.10 (3H, s, H-CH3-6’)。13C NMR (CDCl3, 150 MHz): 193.7 (C, C-9), 190.9 (C, C-7’), 169.5 (C, OCOCH3), 167.4 (C, C-3’), 164.8 (C, C-4’), 163.9 (C, C-3), 161.8 (C, C-4), 152.1 (C, C-6), 152.0 (C, C-6’), 148.8 (CH, C-1), 142.9 (C, C-3’b), 133.0 (C, C-3b), 121.4 (CH, C-5’), 121.0 (C, C-5), 120.9 (CH, C-6’a), 118.3 (C, C-6a), 113.3 (C, C-9a), 104.9 (C, C-3’a), 101.5 (C, C-3a), 88.8 (C, C-7), 78.9 (CH, C-9’a), 71.4 (CH2, C-1’), 67.4 (CH, C-8’), 64.1 (CH, C-8), 51.9 (CH3, 7-OCH3), 51.2 (C, C-9’a), 22.7 (CH3, 6’-CH3), 22.2 (CH3, 6-CH3), 21.0 (CH3, OCOCH3)。将核磁数据与文献 (Dethoup et al, 2006; Chaudhary et al, 2020)比对, 发现两篇文献均以氘代二甲基亚砜 (DMSO-d6)为溶剂, 但核磁数据出入较大, 本论文里使用CDCl3测得的核磁数据与文献 (Dethoup et al, 2006)数据吻合, 推测其可能标注错了溶剂。最终通过2D NMR数据的归属, 确定4为duclauxin, 化学结构见图1
化合物5: 淡黄色无定型粉末; [α]+20.6° (c 0.025, MeOH); HRESIMS m/z 475.1022 [M + H]+ (计算值为: C26H19O9, 475.1029); 1H NMR (600 MHz, DMSO-d6): δH 11.87 (1H, brs, 4-OH), 6.96 (1H, s, H-5), 6.83 (1H, s, H-5’), 6.24 (1H, s, 9’-OH), 5.73 (1H, d, J = 15.0 HZ, H-1ɑ), 5.65 (1H, d, J = 15.0 HZ, H-1β), 5.14 (1H, d, J = 12.3 HZ, H-1’ɑ), 4.99 (1H, d, J = 12.3 HZ, H-1’β), 4.83 (1H, brs, H-8’), 4.77 (1H, d, J = 4.5 HZ, H-9’), 2.98 (3H, s, 6-CH3), 2.49 (3H, s, 6’-CH3); 13C NMR (DMSO-d6, 150 MHz): 192.8 (C, C-7’), 169.4 (C, C-3), 167.9 (C, C-3’), 161.5 (C, C-4), 163.2 (C, C-4’), 152.4 (C, C-6’), 148.9 (C, C-9), 147.8 (C, C-3’b), 146.0 (C, C-6), 137.3 (C, C-7), 134.8 (C, C-8), 131.3 (C, C-3b), 119.7 (CH, C-5’), 119.2 (2C, C-6, C-6a), 116.8 (C, C-6’a), 109.7 (C, C-9a), 97.5 (C, C-3a), 85.3 (CH, C-9’), 70.1 (CH2, C-1’), 66.8 (CH2, C-1), 64.6 (CH, C-8’), 49.6 (C, C-9’a), 24.5 (CH3, 6-CH3), 23.3 (CH3, 6’-CH3)。比对文献 (Dethoup et al, 2006), 确定5为bacillisporin B, 化学结构见图1
化合物6: 淡黄色无定型粉末; [α]+17.3° (c 0.025, MeOH); HRESIMS m/z 491.0983 [M + H]+ (计算值为: C26H19O10, 491.0978); 1H NMR (600 MHz, DMSO-d6): δH 11.87 (1H, brs, 4’-OH), 8.62 (1H, brs, 9’-OH), 6.88 (1H, s, H-5), 6.74 (1H, s, H-5’), 5.58 (1H, d, J = 14.2 HZ, H-1ɑ), 5.43 (1H, d, J = 14.2 HZ, H-1β), 4.88 (1H, d, J = 11.2 HZ, H-1’ɑ), 4.59 (1H, d, J = 11.2 HZ, H-1’β), 3.18 (1H, d, J = 15.4 HZ, H-8’ɑ), 3.07 (1H, d, J = 15.4 HZ, H-8’β), 2.73 (3H, s, 6-CH3), 2.50 (3H, s, 6’-CH3); 13C NMR (DMSO-d6, 150 MHz): 193.2 (C, C-7’), 169.5 (C, C-3), 168.9 (C, C-3’), 162.8 (C, C-4), 162.5 (C, C-4’), 155.0 (C, C-7), 150.0 (C, C-9), 148.6 (C, C-6), 145.9 (C, C-6’), 144.1 (C, C-3’b), 131.9 (C, C-3b), 121.0 (C, C-6a), 119.9 (CH, C-5), 116.9 (CH, C-5’), 113.8 (C, C-8), 111.4 (C, C-9’), 109.5 (C, C-3a), 108.5 (C, C-6’a), 102.1 (C, C-9a), 96.8 (C, C-3’a), 73.2 (CH2, C-1’), 66.7 (CH2, C-1), 48.5 (2CH, C-8’, C-9’a ), 23.0 (CH3, 6’-CH3), 22.9 (CH3, 6-CH3)。比对文献(Dethoup et al, 2006), 确定6为bacillisporin C, 化学结构见图1

2.2 化合物体外抑制NO生成的结果

采用CCK-8法检测化合物16对RAW264.7巨噬细胞的细胞毒活性, 实验结果表明: 样品浓度30µmol·L-1时, 1作用后的细胞存活率为77%, 26作用后的细胞存活率均大于90%(存活率<80%的化合物被视作有一定的细胞毒活性, 26将被采纳进行下一步体外抗炎活性测试)。在浓度30µmol·L-1时, 25具有NO生成抑制作用 (表1), 抑制率均高于阳性对照药AG。24的IC50值分别为11.82±1.25、11.44±1.58和23.92±2.86µmol·L-1
表1 化合物26对LPS诱导的RAW264.7细胞NO生成的抑制活性 ($\bar{x} \pm s$, n = 3)

Tab. 1 Inhibitory activities of compounds 2-6 against NO production in RAW264.7 cells induced by LPS ($\bar{x} \pm s$, n = 3)

化合物 抑制率/%a IC50/(µmol·L-1)
2 92.40±6.48 11.82±1.25
3 90.85±6.15 11.44±1.58
4 74.08±5.04 23.92±2.86
5 44.70±3.42 >30
6 9.67±1.06 >30
阳性对照(AG) 36.71±5.23 >30

注: a测试样品浓度为30.00µmol·L-1

本论文以生物活性分析为导向, 对一株具有体外抗炎活性的真菌T. stipitatus 发酵产物开展次生代谢产物研究, 从中分离鉴定6个oligophenalenone二聚体, 分别是bacillisporin A (1)、9a-epi-bacillisporin E (2)、bacillisporin F (3)、duclauxin (4)、bacillisporin B (5)和bacillisporin C (6)。化合物25 (浓度30µmol·L-1), 不影响巨噬细胞存活, 显示出不同程度的NO生成抑制作用, 抑制率均高于阳性对照药AG; 24的IC50分别为11.82±1.25, 11.44±1.58和23.92±2.86µmol·L-156是从该种中首次分离得到, 并首次发现duclauxin及其同系物具有显著的体外抗炎活性。

3 讨论

化合物16为oligophenalenone二聚体, 是一类以duclauxin (4)为代表的同系物。Shahid等(2021)对duclauxins类化合物的结构、生物活性和生物合成做了详细的综述, 截止2021年从TalaromycesPenicillium属总计报道了36个天然duclauxins。2022年李德海等人又从1株分离自南极海绵共生真菌Talaromyces sp.中分离鉴定1个罕见的具有6/6/6/5/5/5/6七环稠环体系的duclauxin类化合物Talaverrucin A (Sun et al, 2022)。Duclauxin同系物多以苷元的形式存在, 近期, 从真菌Talaromyces sp.中首次发现了duclauxins苷类化合物 (5个新的氮苷glyclauxins A-E) (Samarasekera et al, 2023)。从目前已有的结构分析, duclauxins类化合物结构中的下半部分单体的A’、B’和C’环结构相对较为固定, 主要变化在上半部分单体, 有2、3和5环, 其中绝大多数是3环, 少数为2或5环; A环的2位多数为O原子, 也有少量结构是N原子 (Chaudhary et al, 2020; Samarasekera et al, 2023); 此外, 环上的取代基也存在变化。因此, duclauxins类化合物的结构多样性尚有进一步挖掘的空间。据我们所知, 56为首次从T. stipitatus中发现。
Shahid等(2021)在综述中提到36个天然的duclauxins类化合物中目前只检索到22个相关化合物的生物活性报道, 其中41%具有抗肿瘤活性, 32%具有抗菌活性, 21%具有酶抑制活性, 6%具有抗疟活性。2022年发现的talaverrucin A具有靶向Wnt通路抑制活性(Sun et al, 2022)。目前, 我们尚未检索到关于duclauxins类化合物抗炎活性方面的研究。本课题组前期运用体外抗炎活性模型筛选菌株时, 发现真菌T. stipitatus的发酵产物具有抑制脂多糖诱导的RAW264.7细胞系NO生成的作用, 以体外抗炎活性分析为导向, 从具有活性的中低极性段部位获得6个oligophenalenone二聚体。化合物浓度为30µmol·L-1时, 除了1具有一定的巨噬细胞毒活性外, 26在该工作浓度下均无巨噬细胞毒活性, 并显示出不同程度的抗炎活性。其中6抗炎活性最弱, NO抑制率仅为(9.67±1.06)%, 远低于25。对比625的结构差别, 发现两个oligophenalenone单体连接成环 (D环)的方式不同, 25的D环为环戊烷, 6的D环为二氢呋喃, 推测D环为环戊烷是保持其高活性的重要影响因素。在浓度为30µmol·L-1时, 25的 NO抑制率均高于阳性对照药, 更值得一提的是, 24的IC50值分别为11.82±1.25、11.44±1.58和23.92±2.86µmol·L-1, 均比5的活性更强。比对分析245的结构, 发现24的C-9’位均被乙酰基所取代, 5的C-9’位为羟基取代, 推测C-9’位的乙酰基取代对其抗炎活性存在一定的贡献。
本研究丰富了T. stipitatus次生代谢产物的结构多样性, 拓展了抗炎活性研究的结构类型, duclauxins类化合物高效低毒的体外抗炎活性为抗炎药理活性的后续研究提供了可行性。
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