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2025 , Vol. 45 >Issue 8: 3033 - 3038

DOI: https://doi.org/10.6023/cjoc202412029

研究简报

海洋来源真菌Phellinus sp. HN-22倍半萜类化合物及其抗炎活性研究

  • 小红 a, b, † ,
  • 覃玉月 a, b, c, † ,
  • 陈烁 a, b ,
  • 岑福凌 a, b, d ,
  • 高转生 a, b ,
  • 黎婉珊 , a, b, * ,
  • 陈光英 , a, b, *
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  • a 海南师范大学热带药用资源化学教育部重点实验室 海口 571158
  • b 海南师范大学化学与化工学院 热带药用植物化学海南省重点实验室 海口 571158
  • c 北部湾大学海洋学院 广西北部湾海洋生物多样性养护重点实验室 广西钦州 535011
  • d 福建中医药大学药学院 福州 350122

†共同第一作者.

收稿日期: 2024-12-27

  修回日期: 2025-02-19

  网络出版日期: 2025-03-13

基金资助

国家自然科学基金(22177023)

国家自然科学基金(22477021)

海南省自然科学基金(222QN303)

及海南省研究生创新科研(Qhys2023-332)

及海南省研究生创新科研(202411658005)

及海南省研究生创新科研(202411658013)

收起

Research on Sesquiterpenoids from a Marine-Derived Fungus Phellinus sp. HN-22 and Their Anti-inflammatory Activities

  • Hong Xiao a, b ,
  • Yuyue Qin a, b, c ,
  • Shuo Chen a, b ,
  • Fuling Cen a, b, d ,
  • Zhuansheng Gao a, b ,
  • Wanshan Li , a, b, * ,
  • Guangying Chen , a, b, *
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  • a Key Laboratory of Tropical Medicinal Resources Chemistry of Ministry of Education, Hainan Normal University, Haikou 571158
  • b Key Laboratory of Tropical Medicinal Plant Chemistry of Hainan Province, College of Chemistry and Chemical Engineering, Hainan Normal University, Haikou 571158
  • c Guangxi Key Laboratory of Beibu Gulf Marine Biodiversity Conservation, Ocean College, Beibu Gulf University, Qinzhou, Guangxi 535011
  • d College of Pharmacy, Fujian University of Traditional Chinese Medicine, Fuzhou 350122
*E-mail:;

†The authors contributed equally to this work.

Received date: 2024-12-27

  Revised date: 2025-02-19

  Online published: 2025-03-13

Supported by

National Natural Science Foundation of China(22177023)

National Natural Science Foundation of China(22477021)

Hainan Provincial Natural Science Foundation(222QN303)

Hainan Provincial Postgraduate Innovation Research Project(Qhys2023-332)

Hainan Provincial Postgraduate Innovation Research Project(202411658005)

Hainan Provincial Postgraduate Innovation Research Project(202411658013)

Copyright

© 2025 Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences
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摘要

运用硅胶柱色谱、凝胶柱色谱和高效液相色谱等分离技术对一株海洋沉积物来源真菌Phellinus sp. HN-22大米发酵提取物的倍半萜类成分进行研究, 从中发现1个新的倍半萜类化合物(3S,6S,7S,10S)-tremulene-10,12-dihydroxy- 11-ylacetate (1)和7个已知的倍半萜类衍生物[6α-hydroxycinnamolide (2), 6β-hydroxycinnamolide (3), 3β-acetoxycinna- molide (4), Ceriponol D (5), 3β-hydroxycinnamolide (6), 2α,3β-dihydroxycinnamolide (7), 3β,6β-dihydroxycinnamolide (8)]. 新化合物1的结构通过高分辨电喷雾电离质谱(HR-ESI-MS)、核磁共振波谱法(NMR)和量子化学计算方法进行确定. 通过抑制炎症因子(NO)生成的活性测试显示, 化合物3能较好地抑制NO的生成, 其半抑制浓度(IC50)值为(27.78±0.04) μmol/L, 与阳性药物地塞米松(IC50值32.56±0.34 μmol/L)活性相当, 表现出较好的抗炎活性.

关键词： 海洋来源真菌; Tremulane型倍半萜; Drimane型倍半萜; 分离与鉴定; 抗炎活性

本文引用格式

小红 , 覃玉月 , 陈烁 , 岑福凌 , 高转生 , 黎婉珊 , 陈光英 . 海洋来源真菌Phellinus sp. HN-22倍半萜类化合物及其抗炎活性研究[J]. 有机化学, 2025 , 45(8) : 3033 -3038 . DOI: 10.6023/cjoc202412029

Abstract

The study on sesquiterpenoids from a marine-derived fungus Phellinus sp. HN-22 led to the isolation and purifi- cation of a new sesquiterpene compound, (3S,6S,7S,10S)-tremulene-10,12-dihydroxy-11-ylacetate (1), and seven known sesquiterpenes derivatives, 6α-hydroxycinnamolide (2), 6β-hydroxycinnamolide (3), 3β-acetoxycinnamolide (4), Ceriponol D (5), 3β-hydroxycinnamolide (6), 2α,3β-dihydroxycinnamolide (7), and 3β,6β-dihydroxycinnamolide (8). The structure absolute configuration of the new compound was determined by high resolution electrospray ionization mass spectroscopy (HRESIMS), nuclear magnetic resonance (NMR) and electronic circular dichroism calculations (ECD) methods. Based on inhibiting effects of the production of inflammatory factor NO, compound 3 exhibited similar anti-inflammatory activity with a half maximal inhibitory concentration (IC50) value of (27.78±0.04) μmol/L as that of the positive control, dexamethasone (IC50=(32.56±0.34 μmol/L)).

Key words： marine-derived fungi; tremulane-type sesquiterpenes; drimane-type sesquiterpenes; separation and identification; anti-inflammatory activities

针层孔菌属(Phellinus)真菌隶属于担子菌亚门(Basidiomycotina)层菌纲(Hymenomycetes)非褶菌目(Aphyllophorales)锈革孔菌科(Hymenochaetaceae)[1], 广泛分布于全球寒带、温带和热带地区. 据统计, 目前全世界已知的针层孔菌属真菌有250多种, 我国已发现约有70种[2]. 该属真菌多为重要的林木病原菌, 一些种类具有重要的药用价值, 如针层孔菌(P. igniarius)、裂蹄针层孔菌(P. linteus)和鲍氏针层孔菌(P. baumii)等被历史记载为重要的药用真菌资源[3]. 文献调研发现, 该属真菌含多种类型的代谢产物, 多糖类、黄酮类、萜类和甾体类化合物是该属真菌的主要代谢成分[4-9]. 这些成分具有多种生物活性, 包括抗肿瘤、抗糖尿病、抗流感病毒、免疫调节、抗炎症和抗氧化等[10-16]. 因此, 该属药用真菌资源的开发利用越来越受到药物化学家的关注.
海洋, 作为一个重要的天然资源宝库, 其高压、高盐等特殊的环境压力促使海洋生物在生命活动中产生大量具有特殊结构和良好生物活性的次级代谢产物[17]. 本课题组长期致力于海洋生物活性物质的研究, 从多种海洋来源真菌中发现了大量的活性天然产物, 尤其是混源萜和聚酮类化合物[18-20]. Phellinus sp. HN-22为一株较为稀有的来源于南海海底沉积物的针层孔菌属真菌. 前期经课题组对该菌的初步分析发现该菌富含倍半萜类化合物, 具有特征的紫外吸收, 其最大特征吸收波长(λmax)约为240 nm. 倍半萜类化合物为该属真菌中重要的生物活性成分, 结构新颖丰富, 包括drimane、tremulane和illudan型等多种结构骨架, 多具有血管松弛[21-22]、细胞毒[23]、抗炎[24-25]和抗流感病毒[26]等活性. 因此, 基于最大特征紫外吸收波长, 我们对该菌的倍半萜类成分进行研究, 从大米发酵提取物中发现1个新的tremulane倍半萜化合物(3S,6S,7S,10S)-tremulene-10,12- dihydroxy-11-ylacetate (1), 1个已知的tremulane倍半萜化合物(5), 6个已知的drimane倍半萜类衍生物(2~4, 6~8), 分别被鉴定为6α-hydroxy-cinnamolide (2)[27], 6β-hydroxycinnamolide (3)[27], 3β-acetoxycinnamolide (4)[28], Ceriponol D (5)[29], 3β-hydroxycinnamolide (6)[27], 2α,3β-dihydroxycinnamolide (7)[30], 3β,6β-dihydroxycin- namolide (8)(图1)[31]. 测试了所有化合物对炎症因子NO生成的影响, 结果显示化合物3具有较好的抑制作用, 其半抑制浓度(IC50)值为(27.78±0.04) μmol/L, 与阳性药物地塞米松的IC50值[32.56±0.34) μmol/L]活性相当, 表现出较好的抗炎活性. 本文报道新化合物1的分离纯化和结构鉴定, 以及化合物1~8的抗炎活性.
图1 化合物1~8的结构图

Figure 1 Structures of compounds 1~8

1 结果与讨论

化合物1为无色油状化合物, 易溶于甲醇, 根据高分辨质谱(HR-ESI-MS)给出的分子离子峰m/z 295.1914 [M－H]－, 可推测化合物1的分子式为C17H28O4, 不饱和度为4. 600 MHz核磁共振1H NMR数据(表1)和异核单量子关系(HSQC)相关信号显示, 化合物1存在4个甲基质子[δH 2.07 (s, 3H, H-17), 1.03 (s, 3H, H-15), 0.79 (s, 3H, H-14), 0.86 (d, J=7.0 Hz, 3H, H-13)]; 5个亚甲基质子信号, 包括3个连碳亚甲基质子[δH 1.66~1.71 (m, 1H, H-4α), 1.76~1.81 (overlapped, 1H, H-4β), 1.58~1.63 (m, 1H, H-5α), 1.92~2.01 (m, 1H, H-5β), 1.38 (dd, J=11.6, 8.2 Hz, 1H, H-8β), 1.72 (dd, J=11.6, 11.6 Hz, 1H, H-8α)], 以及2个连氧亚甲基质子[δH 4.58 (dd, J=11.6, 1.0 Hz, 1H, H-11α), 4.80 (dd, J=11.6, 1.0 Hz, 1H, H-11β), 3.58 (dd, J=10.8, 6.0 Hz, 1H, H-12α), 3.79 (dd, J=10.8, 6.0 Hz, 1H, H-12β)]; 3个次甲基质子[δH 2.49~2.55 (m, 1H, H-3), 1.76~1.81 (overlappped, 1H, H-6), 3.02 (dd, J=11.6, 8.2 Hz, 1H, H-7)], 以及1个连氧次甲基质子[δH 4.1 (br s, 1H, H-10)]. 13C NMR(表1)和DEPT135谱图数据显示, 该化合物中含有17个碳信号, 包括4个甲基碳信号[δC 13.0 (C-13), 26.2 (C-14), 22.6 (C-15), 21.0 (C-17)]; 5个亚甲基碳信号[δC 21.0 (C-4), 32.6 (C-5), 42.6 (C-8), 60.6 (C-11), 69.1 (C-12)]; 4个次甲基碳信号[δC 45.1 (C-3), 32.3 (C-6), 47.1 (C-7), 81.1 (C-10)]; 2个烯烃碳信号[δC 149.8 (C-11), 134.9 (C-2)]; 以及1个酯基碳信号[δC 173.6 (C-16)].
表1 化合物1的1H NMR (600 MHz)和13C NMR (150 MHz)核磁数据(CD3OD, J in Hz)

Table 1 1H NMR (600 MHz) and 13C NMR (150 MHz) data of compound 1 (CD3OD, J in Hz)

Position δH δC Position δH δC
1 149.8 10 4.10 (1H, br s) 81.1
2 134.9 11α 4.58 (dd, J=11.6, 1.0 Hz, 1H) 60.6
11β 4.80 (dd, J=11.6, 1.0 Hz, 1H)
3 2.49~2.55 (m, 1H) 45.1 12α 3.58 (dd, J=10.8, 6.0 Hz, 1H) 69.1
12β 3.79 (dd, J=10.8, 6.0 Hz, 1H)
4α 1.66~1.71 (m, 1H) 21.0 13 0.86 (d, J=7.0 Hz, 3H) 13.0
4β 1.76~1.81 (overlapped, 1H)
5α 1.58~1.63 (m, 1H) 32.6 14 0.79 (s, 3H) 26.2
5β 1.92~2.01 (m, 1H)
6 1.76~1.81 (overlapped, 1H) 32.3 15 1.03 (s, 3H) 22.6
7 3.02 (dd, J=11.6, 8.2 Hz, 1H) 47.1 16 173.6
8α 1.72 (dd, J=11.6, 11.6 Hz, 1H) 42.6 17 2.07 (s, 3H) 21.0
8β 1.38 (dd, J=11.6, 8.2 Hz, 1H)
9 41.9
化合物1的平面结构通过HMBC (Heteronuclear multiple-bond correlation)和1H-1H COSY (图2)相关信号进行确认. 1H-1H COSY谱图显示化合物1具有一个质子自旋耦合体系, 如图2所示, OCH2CHCH2CH2CH- (CH3)CHCH2 (C-3到C-8, C-12和C-13)片段. 结合H2-12与C-2、H2-11与C-1/C-2/C-3、H-7与C-1/C-2、H2-8与C-1/C-9、H-10与C-2/C-7/C-9、H3-14与C-8/C-9/C-10/ C-15以及H3-15与C-8/C-9/C-10/C-14的HMBC相关信号, 推测出化合物1具有与已知物(＋)-(3S,6S,7S,10S)- tremulene-10,11,12-triol[21]相似的结构, 主要区别在于化合物1比(＋)-(3S,6S,7S,10S)-tremulene-10,11,12-triol的C-11位多了一个乙酰氧基取代. C-11的乙酰氧基取代可通过乙酰基信号[δH 2.07 (s, 3H, H-17); δC 21.0 (C-17), 173.6 (C-16)]以及H2-11与C-16、H3-17与C-16的HMBC相关信号得以确定.
图2 化合物1的主要1H-1H COSY, HMBC和NOESY相关信号

Figure 2 Key 1H-1H COSY, HMBC and NOESY correlations of compound 1

化合物1的相对构型通过NOESY(图2)相关信号进行确定. H3-14/H-10, H3-14/H-7、H3-15/H-8α的NOE相关信号以及H-8α与H-7的偶合常数11.6Hz确定H3-15为α构型, H3-14、H-10和H-7为β构型. 此外, H-7/ H2-12、H-7/H-5β、H-5β/H2-12、H3-13/H-5α以及H3-13/ H-4α的NOE相关信号确定H-7、H2-12和H-6处于同一面为β构型, H3-13和H-3为α构型.
化合物1的绝对构型通过量子计算化学进行进一步确认. 由于实测的电子圆二色谱(ECD)曲线与3S,6S,7S, 10S构型的计算ECD曲线相吻合(图3), 因此, 确定化合物1的绝对构型为3S,6S,7S,10S, 并命名为(3S,6S,7S, 10S)-tremulene-10,12-dihydroxy-11-ylacetate.
图3 化合物1的实验和计算ECD谱图

Figure 3 Experimental and calculated ECD spectra of compound 1

经1H NMR和13C NMR数据分析及文献检索比对, 已知化合物2~8分别鉴定为6α-hydroxycinnamolide (2), 6β-hydroxycinnamolide (3), 3β-acetoxycinnamolide (4), Ceriponol D (5), 3β-hydroxycinnamolide (6), 2α,3β-dihy- droxycinnamolide (7), 3β,6β-dihydroxycinnamolide (8).
抗炎活性测试结果显示化合物3在30 μmol/L浓度下对Lipopolysaccharide (LPS)刺激的RAW264.7细胞产生的炎症因子(NO)具有明显抑制作用, IC50值为(27.78±0.04) μmol/L, 与阳性药物地塞米松[IC50为(32.56±0.34) μmol/L]活性相当, 表现出较好的抗炎活性.

2 结论

运用多种色谱分离方法和波谱鉴定技术对海洋来源真菌Phellinus sp. HN-22的倍半萜类化合物进行了研究, 共分离鉴定了8个倍半萜类化合物, 其中化合物1为新化合物. 对所有化合物进行了抗炎评价, 化合物3显示具有较好的抗炎活性, IC50值为(27.78±0.04) μmol/L, 与阳性药地塞米松活性相当. 本研究为海洋来源的Phellinus属真菌的药用研究提供了理论依据, 具有重要参考意义和价值.

3 实验部分

3.1 仪器与试剂

Nicolet 6700型红外光谱仪; JASCO DIP-370型数字旋光仪; Bruker AV-400型核磁共振波谱仪; 日本电子株式会社JNM-ECZ600型核磁共振仪, 使用DMSO-d6或CD3OD等溶剂; Agilent G3250AA LC/MSDTOF质谱仪; 岛津LC-20A半制备高效液相色谱仪, Agilent Eclipse XDB-C18色柱(250 mm×9.4 mm, 5 μm); ELISA 酶标仪(BioTek ELX800); 反相硅胶(YMC公司; 12~50 μm), 硅胶色谱和薄层色谱硅胶板(青岛海洋化工厂), 色谱试剂均为分析纯.

3.2 菌株材料

真菌Phellinus sp. HN-22是于2020年7月在南海北部(114.1896°E, 20.085°N) 670 m海底沉积物中分离得到, 菌株保存于海南师范大学化学与化工学院热带药用资源化学教育部重点实验室. 该真菌是根据分子生物学特征来鉴定, 通过测定真菌的rRNA/ITS片段序列, 与美国国家生物信息中心(NCBI)数据库比对, 确认菌株为Phellinus sp. (NCBI登录号PQ772088), 编号为Phellinus sp. HN-20.

3.3 真菌发酵、提取、分离

真菌Phellinus sp. HN-22接种至马铃薯葡萄糖水(1 L培养瓶加入400 mL)培养基中, 25 ℃、140 r/min条件下振摇3 d得到种子液, 以每瓶5 mL种子液接种至0.3%盐的大米培养基(1 L培养瓶中加入50 g大米, 50 mL水, 0.15 g海盐, 共280瓶)中培养28 d. 培养28 d后发酵物及菌体颜色变黄, 加入乙酸乙酯反复提取, 浓缩乙酸乙酯提取液得到油状粗浸膏300 g.
粗提物进行硅胶(100~200目)柱色谱分离, 用石油醚/乙酸乙酯(梯度V∶V=100∶0~0∶100)和乙酸乙酯/甲醇(梯度V∶V=100∶0~0∶100)梯度洗脱, 得到13个流份(Fr.1~Fr.13). 将Fr.5 (5.1 g)通过反相C18硅胶柱色谱分离, 使用水/甲醇(梯度V∶V=95∶5~0∶100)梯度洗脱, 获得10个亚流份(Fr.5A~Fr.5J). Fr.5C经半制备型高效液相色谱制备, 以乙腈/水(V∶V=35∶65)为流动相, 得到化合物1 (3.3 mg), 化合物2 (4.5 mg), 化合物3 (10.4 mg), 化合物4 (3.6 mg). Fr.7 (26 g)经反相硅胶柱色谱分离, 水/甲醇(V∶V=95∶5~0∶100)梯度洗脱, 得到8个亚流份(Fr.7A~Fr.7H), Fr.7D经半制备型高效液相色谱制备, 以乙腈/水(V∶V=38∶62)为流动相, 得到化合物5 (3.8 mg), 6 (4.1 mg). Fr.7F经半制备型高效液相色谱制备, 以乙腈/水(V∶V=45∶55)为流动相, 得到化合物7 (3.5 mg), 8 (4.4 mg).
(3S,6S,7S,10S)-Tremulene-10,12-dihydroxy-11-ylace-tate (1): 无色油状物, $[\alpha ]_{\text{D}}^{\text{25}}$＋8.8 (c 0.25, CH3OH); UV λmax [log ε/(L•mol－1•cm－1)]: 201 (2.78), 210 (2.73) nm; ECD (3.4 mmol/L, CH3OH) λmax (∆ε/(L•mol－1•cm－1)]: 201 (－15.0), 206 (－21.9), 254 (＋1.7) nm; 1H NMR and 13C NMR (600/150 MHz, CD3OD) see Table 1; IR (MeOH) νmax 3446.76, 1653.13, 1576.51, 504.29 cm−1. HR-ESI-MS calcd for C17H27O4 [M－H]－ 295.1915, found 295.1914.
6α-Hydroxycinnamolide (2): 黄色无定形固体; [ α ] D 25＋73.13 (c 0.08, CH3OH); 1H NMR (600 MHz, CDCl3) δ: 6.72 (t, J=3.4 Hz, 1H, H-7), 4.49 (overlapped, 1H, H-6), 4.39 (t, J=9.0 Hz, 1H, H-11α), 4.06 (t, J=9.0 Hz, 1H, H-11β), 2.89~2.94 (m, 1H, H-9), 1.58 (overlapped, 1H, H-2α), 1.43~1.50 (m, 3H), 1.37 (d, J=9.4 Hz, 1H, H-5), 1.23~1.33 (m, 3H), 1.16 (s, 3H, H-14), 1.06 (s, 3H, H-13), 0.86 (s, 3H, H-15); 13C NMR (150 MHz, CDCl3) δ: 170.2 (C-12), 136.3 (C-7), 129.0 (C-8), 69.0 (C-6), 67.5 (C-11), 58.0 (C-5), 50.3 (C-9), 43.4 (C-3), 40.2 (C-4), 39.7 (C-1), 36.2 (C-14), 33.4 (C-10), 22.3 (C-13), 18.6 (C-2), 14.7 (C-15). HR-ESI-MS calcd for C15H21O3 [M－H]－ 249.1496, found 250.1495. ECD (4.0 mmol/L, CH3OH) λmax [∆ε/(L• mol－1•cm－1)]: 203 (－28.5), 212 (＋14.3), 223 (＋8.5), 259 (＋143.6) nm.
6β-Hydroxycinnamolide (3): 无色晶体, [ α ] D 25－83.30 (c 0.1, CH3OH); ECD (4.0 mmol/L, CH3OH) λmax [∆ε/(L•mol－1•cm－1)]: 203 (－26.0), 212 (＋11.0), 223 (＋6.2), 259 (＋142.7); 1H NMR (400 MHz, CDCl3) δ: 6.81 (t, 1H, J=3.6 Hz, H-7), 4.72 (m, 1H, H-6), 4.44 (t, J=9.1 Hz, 1H, H-11α), 4.10 (t, J=9.1 Hz, 1H, H-11β), 2.66~2.74 (m, 1H, H-9), 1.79~1.57 (m, 2H), 1.55~1.42 (m, 2H), 1.34 (s, 3H, H-13), 1.33~1.19 (m, 3H), 1.12 (s, 3H, H-14), 1.06 (s, 3H, H-15); 13C NMR (100 MHz, CDCl3) δ: 170.3 (C-12), 135.8 (C-7), 128.2 (C-8), 67.7 (C-11), 65.9 (C-6), 54.9 (C-5), 52.1 (C-9), 44.9 (C-3), 41.8 (C-1), 34.4 (C-4), 34.2 (C-10), 32.8 (C-14), 25.0 (C-13), 18.5 (C-2), 15.8 (C-15). HR-ESI- MS calcd for C15H21O3 [M－H]－ 249.1496, found 249.1495.
3β-Acetoxycinnamolide (4): 黄色无定形固体; [ α ] D 25＋7.50 (c 0.1, CH3OH); ECD (3.4 mmol/L, CH3OH) λmax (∆ε/(L•mol－1•cm－1)]: 202 (－37.1), 208 (－0.7), 213 (－22.1), 220 (－8.5), 238 (＋17.4), 261 (＋539.9) nm; 1H NMR (400 MHz, CDCl3) δ: 6.86 (dd, J=8.0, 3.6 Hz, 1H, H-7), 4.53 (dd, J=11.4, 4.5 Hz, 1H, H-3), 4.38 (t, J=9.1 Hz, 1H, H-11α), 4.02 (t, J=9.1 Hz, 1H, H-11β), 2.88~2.98 (m, 1H, H-9), 2.42 (dq, J=20.2, 4.3 Hz, 1H, H-6α), 2.13~2.25 (m, 1H, H-6β), 2.05 (s, 3H), 1.82~1.57 (m, 3H), 1.34~1.49 (m, 2H), 0.97 (s, 3H, H-14), 0.91 (s, 3H, H-13), 0.81 (s, 3H, H-15); 13C NMR (100 MHz, CDCl3) δ: 170.9 (C-16), 167.0 (C-12), 136.0 (C-7), 127.2 (C-8), 80.2 (C-3), 67.1 (C-11), 50.6 (C-9), 49.3 (C-5), 37.7 (C-10), 36.9 (C-1), 34.1 (C-4), 27.9 (C-13), 24.7 (C-6), 23.6 (C-2), 21.3 (C-17), 16.1 (C-14), 13.6 (C-15). HR-ESI-MS calcd for C17H23O4 [M－H]－ 291.1601, found 291.1607.
Ceriponol D (5): 白色无定形固体; [ α ] D 25＋115.303 (c 0.1, CH3OH); ECD (4.0 mmol/L, CH3OH) λmax (∆ε/(L•mol－1• cm－1)]: 207 (－32.8), 218 (＋33.5), 230 (－16.9), 201 (＋201.6) nm; 1H NMR (400 MHz, CD3OD) δ: 4.42 (dd, J=8.4, 8.4 Hz, 1H, H-12α), 3.67 (dd, J=10.3, 8.4 Hz, 1H, H-12β), 3.39~3.47 (1m, H, H-7), 3.00~3.12 (m, 1H, H-3), 2.84 (dt, J=19.2, 2.6 Hz, 1H, H-10α), 2.44 (dt, J=19.2, 2.6 Hz, 1H, H-10β), 1.96 (overlapped, 1H, H-5α), 1.92 (overlapped, 1H, H-5β), 1.80~1.88 (m, 1H, H-4α), 1.70~1.75 (m, 1H, H-4β), 1.75~1.79 (m, 1H, H-8α), 1.61 (t, J=12.4 Hz, 1H, H-8β), 1.18 (s, 3H, H-13), 1.17 (s, 3H, H-15), 1.00 (s, 3H, H-14); 13C NMR (100 MHz, CD3OD) δ: 173.27 (C-11), 161.20 (C-1), 123.09 (C-2), 75.32 (C-6), 72.29 (C-12), 52.78 (C-7), 49.70 (C-10), 43.86 (C-5), 43.07 (C-8), 40.82 (C-3), 37.20 (C-9), 29.52 (C-15), 27.61 (C-14), 25.85 (C-4), 25.37 (C-13). HR-ESI-MS calcd for C15H22O3Na [M＋Na]＋ 273.1462, found 273.1454.
3β-Hydroxycinnamolide (6): 无色晶体, [ α ] D 25－8.70 (c 0.1, CH3OH); ECD (4.0 mmol/L, CH3OH) λmax (∆ε/(L•mol－1•cm－1)]: 202 (－31.8), 210 (＋20.7), 223 (＋20.4), 252 (＋220.0) nm; 1H NMR (400 MHz, CDCl3) δ: 6.88 (1H, q, J=3.6 Hz, H-7), 4.37 (t, J=9.1 Hz, 1H, H-11α), 4.03 (t, J=9.1 Hz, 1H, H-11β), 3.29 (dd, J=10.8, 4.6 Hz, 1H, H-3 ), 2.71~2.83 (m, 1H, H-9), 2.49~2.36 (m, 1H, H-6eq), 2.20 (dddd, J=20.1, 11.6, 4.8, 3.3 Hz, 1H, H-6ax), 1.76~1.56 (m, 3H), 1.43~1.29 (m, 2H), 1.03 (s, 3H, H-14), 0.90 (s, 3H, H-13), 0.79 (s, 3H, H-15); 13C NMR (100 MHz, CDCl3) δ: 170.1 (C-12), 136.4 (C-7), 127.2 (C-8), 78.7 (C-3), 67.2 (C-11), 50.8 (C-9), 49.2 (C-5), 38.8 (C-4), 37.4 (C-1), 34.2 (C-10), 27.9 (C-14), 27.0 (C-2), 24.9 (C-6), 15.0 (C-13), 13.6 (C-15). HR-ESI-MS calcd for C15H21O3 [M－H]－ 249.1496, found 250.1496.
2α,3β-Dihydroxycinnamolide (7): 无色晶体; [ α ] D 25－28.80 (c 0.1, CH3OH); ECD (3.8 mmol/L, CH3OH) λmax [∆ε/(L•mol－1•cm－1)]: 212 (－58.1), 230 (＋21.7), 250 (＋177.9) nm; 1H NMR (600 MHz, CD3OD) δ: 6.87 (dd, J=6.8, 3.5 Hz, 1H, H-7), 4.46 (dd, J=9.2, 9.2 Hz, 1H, H-11α), 4.12 (t, J=9.2, 9.2 Hz, 1H, H-11β), 3.63~3.69 (m, 1H, H-2), 3.00 (d, J=9.6 Hz, 1H, H-3), 2.92~2.98 (m, 1H, H-9), 2.43~2.51 (m, 1H, H-6α), 2.23~2.32 (m, 1H, H-6β), 1.89 (dd, J=12.7, 4.4 Hz, 1H, H-1β), 1.52 (dd, J=11.6, 5.3 Hz, 1H, H-5), 1.30 (t, J=12.1 Hz, 1H, H-1α), 1.06 (s, 3H, H-13), 0.94 (s, 3H, H-14), 0.89 (s, 3H, H-15); 13C NMR (150 MHz, CD3OD) δC: 172.3 (C-12), 137.8 (C-7), 128.3 (C-8), 83.9 (C-3), 68.7 (C-11), 68.4 (C-2), 51.9 (C-9), 50.2 (C-5), 46.4 (C-1), 40.3 (C-4), 36.0 (C-10), 28.8 (C-14), 26.0 (C-6), 16.5 (C-13), 14.5 (C-15); HR-ESI-MS calcd for C15H21O4 [M－H]－ 265.1445, found 265.1449.
3β,6β-Dihydroxycinnamolide (8): 无色晶体, [ α ] D 25－126.00 (c 0.1, CH3OH); ECD (3.8 mmol/L, CH3OH) λmax [∆ε/(L•mol－1•cm－1)]: 203 (－38.1), 211 (＋42.8), 221 (＋30.8), 245 (－91.3) nm; 1H NMR (400 MHz, DMSO- d6) δ: 6.59 (t, J=3.5 Hz, 1H, H-7), 4.54 (dd, J=5.5, 1.8 Hz, 1H, H-6), 4.49~4.33 (overlapped, 1H, H-11α), 4.06 (td, J=9.0, 1.3 Hz, 1H, H-11β), 3.09~2.95 (m, 1H, H-3), 2.62~2.71 (m, 1H, H-9), 1.61~1.38 (m, 3H, H-2/1α), 1.20~1.31 (m, 1H, H-1β), 1.15 (s, 3H, H-13), 1.08 (s, 3H, H-14), 1.04 (d, J=4.9 Hz, 1H, H-5), 0.90 (s, 3H, H-15); 13C NMR (100 MHz, DMSO-d6) δ: 169.8 (C-12), 137.0 (C-7), 126.3 (C-8), 77.4 (C-3), 67.3 (C-11), 64.3 (C-6), 53.6 (C-5), 50.70 (C-9), 39.3 (C-4), 38.1 (C-1), 33.4 (C-10), 26.9 (C-2/14), 17.4 (C-13), 14.8 (C-15). HR-ESI- MS calcd for C15H21O4 [M－H]－ 265.1445, found 265.1447).

3.4 ECD计算

Spartan 14程序(Wavefunction Inc., Irvine, CA, USA)用于计算Merck分子力场(MMFF). Gaussian 16程序包用于密度泛函理论(DFT)和时间相关的密度泛函理论(TDDFT)计算. 通过MMFF模型进行构象搜索, 然后将相对能量较低(<41.84 kJ/mol)的构象在B3LYP/6-31G*水平上进行DFT方法的几何优化. 在同一水平上进行振动频率计算, 以评估它们在298.15 K下的相对热和自由能. 为了获得这些低能量构象的能量, 进一步在B3LYP/6-311G*水平上对几何优化的构象进行计算, 并考虑了基于密度的溶剂模型(SMD)的影响[32-33].

3.5 抗炎活性

体外抗炎活性筛选采用小鼠单核巨噬细胞RAW 264.7模型[34], 具体操作如下: RAW264.7细胞用含体积分数为10%的胎牛血清(FBS) DMEM (dulbecco's modified eagle medium)培养液于37 ℃、体积分数为5%的CO2培养箱中常规培养. 细胞按1×105 个/mL、100 μL/孔接种于96孔板中, 置于37 ℃、体积分数为5%的CO2细胞培养箱中, 贴壁12 h后弃去上清. 药物溶于体积分数为1%的胎牛血清(FBS) DMEM中, 将溶解的药物以每孔100 μL加入96孔板中, 1 h后加入浓度为1 μg/mL的LPS溶液100 μL, 继续培养24 h, 离心, 取上清液按照Griess法测定上清液中NO的含量. 结果发现, 化合物3表现出较好的抗炎活性, 其IC50为27.78±0.04 μmol/L, 与阳性对照地塞米松(IC50值32.56±0.34 μmol/L)活性相当. 其余化合物在50 μmol/L浓度下均未表现出明显的抗炎活性.
辅助材料(Supporting Information) 化合物1的1H NMR, 13C NMR, HSQC, HMBC和HRESIMS谱等. 这些材料可以免费从本刊网站(http://sioc-journal.cn/)上下载.
(Lu, Y.)

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