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热带海洋学报 >

2024 , Vol. 43 >Issue 4: 181 - 188

DOI: https://doi.org/10.11978/2023126

海洋资源开发

信号芋螺内生真菌Talaromyces sp. XXH006次级代谢产物研究

  • 林应婷 ,
  • 孙颖 ,
  • 胡江南 ,
  • 董帅
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  • 热带生物资源教育部重点实验室, 药学院, 海南大学, 海南 海口 570228
董帅。email:

林应婷 (1996—), 女, 海南省昌江人, 硕士研究生, 从事海洋天然产物研究。email:

Copy editor: 孙翠慈

收稿日期: 2023-08-17

  修回日期: 2023-08-31

  网络出版日期: 2023-10-23

基金资助

海南省自然科学基金资助(820RC584)

收起

Study on secondary metabolites of the endophytic fungus Talaromyces sp. XXH006 Isolated from Conus literatus

  • LIN Yingting ,
  • SUN Ying ,
  • HU Jiangnan ,
  • DONG Shuai
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  • Key Laboratory of Tropical Biological Resources of Ministry of Education, School of Pharmaceutical Sciences, Hainan University, Haikou 570228, China
DONG Shuai. email:

Copy editor: SUN Cuici

Received date: 2023-08-17

  Revised date: 2023-08-31

  Online published: 2023-10-23

Supported by

Natural Science Foundation of Hainan Province(820RC584)

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摘要

文章旨在对信号芋螺内生真菌Talaromyces sp. XXH006的次级代谢产物进行结构和活性研究。运用多种分离材料和技术, 如正反相硅胶柱层析、葡聚糖凝胶 (sephadex LH-20)、聚苯乙烯反相树脂填料 (MCI GEL CHP-20/P120)、半制备高效液相等进行分离纯化, 通过核磁共振、质谱等现代波谱技术进行结构鉴定。采用CCK8 (cell counting kit)法研究化合物1—14对脑胶质瘤细胞U87和肺癌细胞A549的抑制作用。从信号芋螺内生真菌Talaromyces sp. XXH006的次级代谢产物中分离获得14个化合物, 分别是ergosteryl stearate (1)、麦角甾醇 (2)、β-谷甾醇 (3)、2-hydroxyemodin (4)、(+)-rugulosin (5)、1, 1′, 3, 3′, 5, 5′-hexahydroxy-7, 7-dimethyl [2, 2′-bianthracene]-9, 9′, 10, 10′-tetrone (6)、rel-(6R, 7R)-5, 6, 7, 8-tetrahydro-6-hydroxy-7-methyl-8-oxo-3-propyl-1H-2-benzopyran (7)、rel-(6R, 7R)-5, 6, 7, 8-tetrahydro-7-hydroxy-7-methyl-8-oxo-3-propyl-1H-2-benzopyran (8)、scorpinone (9)、orsellinic acid (10)、6-methyl-1, 2, 4-benzenetriol (11)、2-methyl-5-isopropyl-phenol (12)、3-methoxy-5-methyl-phenol (13)、5-methyl-benzene-1, 3-diol (14)。化合物1—4和7—14为首次从Talaromyces sp. 中分离获得。化合物8和12对脑胶质瘤细胞U87的半数抑制浓度 (half maximal inhibitory concentration, IC50)分别为21.07μmol·L-1和15.74μmol·L-1, 同时对肺癌细胞A549的IC50分别为20.88μmol·L-1和22.93μmol·L-1。

关键词： 信号芋螺内生菌; 篮状菌属; 次级代谢产物; 生物活性

本文引用格式

林应婷 , 孙颖 , 胡江南 , 董帅 . 信号芋螺内生真菌Talaromyces sp. XXH006次级代谢产物研究[J]. 热带海洋学报, 2024 , 43(4) : 181 -188 . DOI: 10.11978/2023126

Abstract

To investigate the chemical constituents and bioactivities of secondary metabolites extracted from Talaromyces sp., which was isolated from Conus literatus. Various of separate materials and technologies such as silica gel, reverse phase silica gel, sephadex LH-20, MCI and semi-preparative high performance liquid chromatography were employed to obtain pure compounds. These compounds were then identified by nuclear magnetic resonance, mass spectrometry etc. Subsequently, bioactivities of fourteen compounds were assessed using the CCK8 assay to explore the inhibition against tumor cell line U87 and A549. These compounds, expect compound 5 and 6 identified for the first time in Talaromyces sp., included ergosteryl stearate (1), ergosterol (2), β-sitosterol (3), 2-hydroxyemodin (4), (+)-rugulosin (5), 1, 1′, 3, 3′, 5, 5′-hexahydroxy-7, 7′-dimethyl[2, 2′-bianth-racene]-9, 9′, 10, 10′-tetrone (6), rel-(6R, 7R)-5, 6, 7, 8-tetrahydro-6-hydroxy-7-methyl-8-oxo-3-propyl-1H-2-benzo-pyran (7), rel-(6R, 7R)-5, 6, 7, 8-tetrahydro-7-hydroxy-7-methyl-8-oxo-3-propyl-1H-2-benzopyran (8), scorpinone (9), orsellinic acid (10), 6-methyl -1, 2, 4-benzene-triol (11), 2-methyl-5-isopropyl-phenol (12), 3-methoxy-5-methyl-phenol (13) and 5-methyl-benzene-1, 3-diol (14). Compound 8 and 12 exhibited inhibitory effects on glioblastoma cell (U87) with an IC50 value of 21.07μmol·L-1 and 15.74μmol·L-1 respectively. They also show inhibitory effects on lung cancer cells (A549) with an IC50 value of 20.88μmol·L-1 and 22.93μmol·L-1 respectively.

Key words： endophytic fungus of Conus literatus; Talaromycessp.; secondary metabolites; bioactivities

海洋天然产物具有结构新颖活性多样等特点, 海洋动植物内生菌是开发具有多样生物活性海洋天然产物的重要源泉。内生菌因具有丰富的次级代谢产物及多样的活性, 加之具有易获取, 易扩大培养等优点而成为天然产物的研究热点。芋螺属热带地区海洋食肉性软体动物, 在我国海洋中分布70余种(Reynaud et al, 2022), 其内生菌次级代谢产物具有巨大的药物研发价值。目前已从芋螺内生细菌中分离获得35个化合物, 包括噻唑啉类、聚酮类以及甾体类化合物等, 具有较好的神经保护活性(Lin et al, 2010, 2013a, b)。但目前对芋螺内生真菌次级代谢产物的研究未见报道。Talaromyces sp.次级代谢产物结构多样, 已报道包括萜类、生物碱类、聚酮类、蒽醌类等超过220个化合物, 具有抗肿瘤、抗菌、抗病毒、抗炎等广泛药理活性(Zhai et al, 2016)。为进一步揭示芋螺内生真菌Talaromyces sp.次级代谢产物的结构与活性, 本文对信号芋螺内生真菌Talaromyces sp. XXH006次生代谢产物进行研究, 从中分离鉴定14个化合物(图1), 化合物1—4和7—12为首次从Talaromyces sp.获得。化合物1—14的细胞毒活性评价结果表明, 8和12在50μmol·L-1浓度下对脑胶质瘤细胞U87及肺癌细胞A549均表现出抑制作用。
图1 化合物1—14的化学结构

Fig. 1 Chemical Structures of Compounds 1—14 from Talaromyces sp. XXH006

1 试验部分

1.1 仪器与材料

Bruker AVANCE NEO 400型核磁共振仪(德国布鲁克公司)和LCMS-IT-TOF型离子阱飞行时间液质联用 (日本岛津公司); ZF-7A型紫外检测器(上海勤科分析仪器有限公司)和Thermo Nicolet IS10红外光谱仪(美国Thermo Scientific公司); 半制备型岛津高效液相色谱仪(日本岛津公司); DPE-1250型旋转蒸发仪(东京理化器械株式会社); 旋光仪(ATR-W2 HHW5, 德国SCHMIDT+HAENSCH有限公司); X-bridge OBD色谱柱(250mm×10mm, 5μm, 美国Waters有限公司); 柱色谱填料Sephadex LH-20, YCM反相硅胶(慧德易有限公司); MCI gel CHP-20/P120 (日本三菱化学有限公司); 薄层色谱硅胶GF254, 柱色谱硅胶(100~200目、200~300目、300~400目, 青岛海洋化工厂); 甲醇(色谱纯、Sigma-Aldrich有限公司); 乙腈(色谱纯、Sigma-Aldrich有限公司); 所用其他分析纯试剂均为西陇科学有限公司生产。

1.2 菌株鉴定、发酵、提取分离

1.2.1 XXH006菌种鉴定

菌种鉴定采用ITS技术, 通过ITS1和ITS4引物扩增真菌18SDNA序列, 所得序列与美国国家生物技术信息中心(National Center for Biotechnology Information, NCBI)数据库进行对比, 最终确认菌种的归属。菌株XXH006(冻存于热带生物资源教育部重点实验室)经18SDNA序列测定, 确证菌株XXH006为Talaromyces sp.。系统发育树见图2。
图2 Talaromyces sp. XXH006菌系统发育树

Fig. 2 Evolutionary relationships of taxa of Talaromyces sp. XXH006 strain

1.2.2 培养基配制

马铃薯葡萄糖琼脂 (potato dextrose agar, PDA)真菌培养基: 马铃薯(从中提取浸出粉) 300g、葡萄糖20g、琼脂15g、氯霉素0.1g、1L蒸馏水。按上述配方配制1L PDA培养基, 灭菌处理高压蒸汽灭菌法(121℃, 101kPa, 20min), 冷却至60℃取出至超净台操作。将未凝固的PDA倒入培养皿, 每皿倾倒15mL。待冷却至室温, 即可用于真菌的培养。
M1种子液培养基: 葡萄糖4g、酵母提取粉4g、麦芽糖提取物5g、青霉素G 0.1g、海水1L。按上述配方配制2L M1培养基, 灭菌同上。将2L M1培养液分装至100mL锥形瓶, 并将Talaromyces sp. XXH006接种至装有M1培养液的锥形瓶中, 共接种20瓶种子液。置于摇床恒温培养5d(28℃, 150r·s-1)。
大米发酵培养基: 大米100g、100mL海水。配制200瓶大米培养基, 灭菌操作同上。将制备好的种子液接种至大米培养基上(1mL种子液/瓶)接种全过程在超净台中进行。接种完成后, 密封好置于室温(28℃)条件培养64天, 完成大发酵。

1.2.3 发酵、提取分离

菌株Talaromyces sp. XXH006大发酵64天, 共发酵200瓶, 每1L培养瓶加入100g大米、100mL海水。发酵产物用乙酸乙酯浸泡提取, 搅拌机助提, 反复提取5次。提取液旋蒸获得浸膏粗提物178.6g。甲醇:石油醚=1:1 (体积比)对粗提取物进行萃取, 萃取过程中有沉淀析出。取沉淀物100mg用硅胶纯化(石油醚:乙酸乙酯=50:1等度洗脱)得化合物1 (85mg)。取甲醇相(108.5g) 进行硅胶粗划段, 梯度洗脱[石油醚1L、石油醚:乙酸乙酯(25:1、15:1、10:1、5:1、2:1、1:1、1:2)、乙酸乙酯、甲醇各4L]点板合并获得9个粗组分(Fr1—Fr9)。
取Fr5 (石油醚:乙酸乙酯=10:1洗脱, 1.1g)经硅胶纯化(油醚/乙酸乙酯体系梯度洗脱)得Fr5.8, 经甲醇复溶析出白色沉淀物, 即化合物3 (14mg)。Fr6 (石油醚:乙酸乙酯=5:1洗脱、2g )经200~300目硅胶纯化(油醚/乙酸乙酯梯度洗脱)得Fr6.2; Fr6.2 (85.6mg)经甲醇复溶析出化合物2 (60mg)。取2g Fr6样品进行葡聚糖凝胶(Sephadex LH-20)分离纯化(甲醇洗脱), 得化合物4 (9mg)。Fr7 (石油醚:乙酸乙酯=10:1洗脱、2g)葡聚糖凝胶(Sephadex LH-20)纯化(甲醇洗脱)得化合物5 (12mg)、化合物6 (18mg)、化合物10 (15mg)。Fr7.1 (18mg)经半制备高效液相色谱(OBD prep column, 5µm, 250×10mm; 90%甲醇+含0.1%甲酸水,10min; 99.9%甲醇+0.1%甲酸, 10min; 3mL·min-1)制备得到化合物11 (5mg、tR=14.5min)。取Fr8 (石油醚:乙酸乙酯=5:1洗脱) 2g进行200~300目硅胶纯化(乙酸乙酯:石油醚梯度洗脱)得Fr8.6 (500mg), 经葡聚糖凝胶(Sephadex LH-20)纯化(甲醇洗脱), 得组分Fr8.6.1 (25mg)经半制备高效液相色谱(OBD prep column, 5µm, 250×10mm; 80%甲醇水, 10min, 3mL·min-1)制备得到化合物12 (18mg、tR =7.4min)。得组分Fr8.6.4 (103.4mg)经MCI纯化(石油醚/乙酸乙酯体系梯度洗脱)得Fr8.6.4.2 (50.8mg), 又经过MCI gel (CHP20/P120)填料纯化(甲醇/水梯度洗脱)得Fr8.6.4.2.2 (40mg), 最后经300~400目硅胶纯化(二氯甲烷:丙酮=25:1等度洗脱)得化合物7 (6mg)和化合物8 (10mg)。Fr8.2 (1g)经葡聚糖凝胶(Sephadex LH-20)纯化(甲醇洗脱)得Fr8.2.4 (10mg), 经半制备高效液相色谱(OBD prep column, 5µm, 250×10mm; 80%甲醇水, 20min, 3mL·min-1)制备得化合物13 (3mg、tR =17.5min)。Fr9 (乙酸乙酯、甲醇洗脱, 2g)进行200~300目硅胶纯化(二氯甲烷/甲醇梯度洗脱)得Fr9.1—Fr9.5; Fr9.1 (45mg)经半制备高效液相色谱(OBD prep column, 5µm, 250mm×10mm; 50%甲醇水, 10min, 3mL·min-1)制备得化合物14 (4.6mg、tR =6.5min)。Fr9.2 (125mg)经反相硅胶(YCM*GEL, ODS-A-HG, 50µm)纯化(50%~100%甲醇-水梯度洗脱)得化合物9 (22.5mg)。

1.3 生物活性测试

选用脑胶质瘤U87和肺癌细胞A549进行细胞毒测试。用含10%胎牛血清的培养液(DMEM高糖培养基)配成单个细胞悬液, 以每孔1000个细胞数接种到96孔板, 每孔体积100μL培养24h后加入样品。分组给样, 每组3个复孔, 空白组加入新的完全培养液, 实验组分别加入含待测样品的完全培养液 (每孔100μL), 单体化合物用二甲基亚砜(dimethyl sulfoxide, DMSO)溶解。初次筛选终浓度为50μmol·L-1, 根据初筛结果进行复筛单体化合物终浓度分别为 1.25、2.5、5、10、15、20、25、37、550μmol·L-1, 培养箱中培养72h。弃去贴壁细胞孔内的培养液并加入CCK8(cell counting kit 8)液80μL。培养箱中孵育1.5h后测定光密度值(optical density, OD)。多功能酶标仪于450nm波长处读取并记录各孔OD值。半数抑制浓度 (half maximal inhibitory concentration, IC50) 计算公式如下:
IC50=Antilog[B+(50-B)/(A-B)]×C
其中A为log> 50%化合物浓度, B为log< 50%化合物浓度, C为log稀释倍数。

2 结果与分析

2.1 结构鉴定

化合物1: 白色粉末。 [ α ] D 20-50 (c = 1mg·mL-1, CHCl3), ESI-MS m/z: 663.49 [M+H]+, 分子式C46H78O2。1H-NMR (400 MHz、CDCl3) δ: 5.56 (dd, J = 5.9, 2.4 Hz, 1H, H-6), 5.38 (dt, J = 5.6, 2.7 Hz, 1H, H-7), 5.26 - 5.12 (m, 2H, H-22, 23), 4.71 (tt, J = 11.3, 4.4 Hz, 1H, H-3), 2.58 - 2.21 (m, 5H), 2.16 - 1.42 (m, 16H), 1.42 - 1.19 (m, 36H), 0.87 (ddd, J = 25.3, 12.8, 6.6 Hz, 13H), 0.63 (s, 3H, H-28), 13C-NMR (101 MHz、CDCl3) δ: 173.5 (C-1′ 141.6 (C-5), 138.8 (C-8), 135.7 (C-22), 132.1 (C-23), 120.3 (C-6), 116.5 (C-7), 72.6 (C-3), 55.9 (C-17), 54.7 (C-14), 46.2 (C-9), 430 (C-13 ), 40.6 (C-20), 39.2 (C-12), 38.1 (C-2′), 37.3 (C-10), 36.9 (C-1), 34.9 (C-4), 33.2 (C-25), 29.9 (C-3′), 29.9 (C-4′), 29.8 (C-5′), 29.8 (C-6′), 29.8 (C-7′), 29.6 (C-8′), 29.5 (C-9′), 29.4 (C-2), 28.4 (C-10′), 28.3 (C-11′), 25.2 (C-12′), 23.2 (C-13′), 22.9 (C-14′), 21.3 (C-15′), 21.2 (C-16′), 20.1 (C-17′), 19.8 (C-26), 17.8 (C-25), 16.3 (C-21), 14.3 (C-28), 12.2 (C-18′)。以上数据与文献 (Li et al, 2014)进行对比, 确定1为ergosteryl stearate。
化合物2: 白色粉末。 [ α ] D 20-120 (c = 1mg·mL-1, CHCl3), ESI-MS m/z: 397.34 [M+H]+, 分子式C28H44O。1H-NMR (400 MHz、CDCl3) δ: 5.57 (dd, J = 5.7, 2.5 Hz, 1H, H-6), 5.38 (dt, J = 5.7, 2.7 Hz, 1H, H-7), 5.27 - 5.11 (m, 2H, H-22, 23), 3.63 (tt, J = 11.2, 4.3 Hz, 1H, H-3), 2.46 (ddd, J = 14.4, 4.9, 2.3 Hz, 1H, H-14), 2.37 - 2.15 (m, 1H, H-17), 2.12 - 1.21 (m, 25H), 1.03 (d, J = 6.6 Hz, 3H, H-19), 0.94 (s, 3H, H-18), 0.91 (d, J = 6.8 Hz, 4H, H-20, 21), 0.83 (t, J = 6.4 Hz, 7H, H-25, 26, 27), 0.62 (s, 3H, H-28)。13C-NMR (101 MHz、CDCl3) δ: 141.8 (C-5), 140.3 (C-8), 136.0 (C-22), 132.5 (C-23), 120.1 (C-6), 116.8 (C-7), 70.9 (C-3), 56.2 (C-17), 55.0 (C-14), 46.7 (C-9), 43.3 (C-24), 43.3 (C-13), 41.3 (C-4), 40.9 (C-20), 39.6 (C-12), 38.8 (C-1), 37.5 (C-10), 33.6 (C-25), 32.5 (C-2), 28.8 (C-16), 23.5 (C-15), 21.6 (C-11), 21.6 (C-26), 20.4 (C-27), 20.1 (C-21), 18.1 (C-19), 16.8 (C-18), 12.5 (C-28)。以上数据与文献 (Seo et al, 2009)进行对比, 确定2为麦角甾醇。
化合物3: 白色粉末。 [ α ] D 20-25 (c = 0.1mg·mL-1, CHCl3), ESI-MS m/z: 415.39 [M+H]+, 分子式C29H50O。1H-NMR (400 MHz、CDCl3) δ: 5.32 (s, 1H, H-6), 3.81 - 3.20 (m, 2H, H-3), 2.62 - 0.30 (m, 44H)。 13C-NMR (101 MHz、CDCl3) δ: 140.9 (C-5), 121.8 (C-6), 71.9 (C-3), 56.9 (C-14), 56.2 (C-17), 50.2 (C-9), 45.9(C-22), 42.4 (C-13), 42.4 (C-4), 39.9 (C-12), 37.4 (C-1), 36.6 (C-10), 36.3 (C-18), 34.1 (C-20), 32.1 (C-2), 32.0, 31.7 (C-8), 29.3 (C-27), 28.4 (C-16), 26.2 (C-21), 24.4 (C-15), 23.2 (C-23), 21.2 (C-11), 19.9 (C-24), 19.5 (C-29), 19.2 (C-19), 18.9 (C-25), 12.1 (C-28), 12.0 (C-26)。以上数据与文献 (Wei et al, 2013)进行对比, 确定3为β-谷甾醇。
化合物4: 红色固体粉末。ESI-MS m/z: 287.05 [M+H]+, 分子式C15H10O6。1H-NMR (400 MHz、Acetone-d6) δ: 7.29 (s, 1H, H-5), 7.05 (s, 1H, H-4), 6.71 (s, 1H, H-7), 2.35 (s, 3H, H-11)。13C-NMR (101 MHz、Acetone-d6) δ : 191.4 (C-9), 183.9 (C-10), 166.1 (C-1), 162.6 (C-6, 8), 149.1 (C-2), 133.0 (C-3), 124.0 (C-5), 121.2 (C-4), 114.4 (C-8a, 9a), 110.4 (C-4a, 10a), 108.2 (C-7), 21.9 (C-11)。以上数据与文献 (Lu et al, 2016)进行对比, 确定4为2-hydroxyemodin。
化合物5: 黄色固体粉末。 [ α ] D 20+205.7 (c = 0.35mg·mL-1, MeOH), ESI-MS m/z: 543.12 [M+H]+。分子式C30H22O10。1H-NMR (400 MHz、Acetone-d6) δ: 7.47 (s, 1H, H-4), 7.12 (s, 1H, H-2, 4.59 (d, J=5.4 Hz, 1H, H-7), 3.52 (d, J = 1.8 Hz, 1H, H-6), 2.91 (d, J = 5.5 Hz, 1H, H-8), 2.46 (s, 3H, 3-CH3)。13C-NMR (101 MHz、Acetone-d6) δ: 195.2 (C-10), 184.9(C-5), 180.5 (C-1), 160.5 (C-9), 149.3 (C-3), 133.4 (C-4a), 124.5 (C-4, 121.1 (C-2), 115.3 (C-10a), 107.1 (C-9a), 69.9(C-7), 59.9 (C-5a), 57.6 (C-6), 49.2 (C-8), 21.9 (3-CH3)。以上数据与文献 (Yamazaki et al, 2010)进行对比, 确定5为(+)-rugulosin。
化合物6: 橘红色固体粉末。ESI-MS m/z: 539.09 [M+H]+, 分子式C30H18O10。1H-NMR (400 MHz、DMSO-d6) δ: 12.78 (s, 1H), 12.03 (s, 1H), 7.25 (d, J = 1.7 Hz, 1H), 7.09 (d, J = 1.7 Hz, 1H), 6.68 (s, 1H), 2.30 (s, 3H)。 13C-NMR (101 MHz、DMSO-d6) δ: 189.4 (C-10), 182.0 (C-9), 164.9 (C-3), 164.4 (C-1), 161.0 (C-5), 148.0 (C-7), 133.2 (C-8a), 131.2 (C-4a), 123.9 (C-2), 123.5 (C-6), 120.4 (C-8), 113.1 (C-10a), 108.7 (C-9a), 107.1 (C-4), 21.5 (C-11)。以上数据与文献(Chen et al, 2011)进行对比, 确定6为1, 1′, 3, 3′, 5, 5′-hexahydroxy-7, 7′-dimethyl[2, 2′-bianth-racene]-9, 9′, 10, 10′-tetrone。
化合物7: 白色固体粉末。 [ α ] D 20+73 (c = 0.2mg·mL-1, MeOH), ESI-MS m/z: 403.17 [M+H]+。分子式C22H26O7。1H-NMR (400 MHz、CD3OD) δ: 6.31 (d, J = 2.1 Hz, 1H, H-5′), 6.26 (d, J = 2.1 Hz, 1H, H-3′), 5.38 (s, 1H, H-4), 4.93 (dt, J = 12.5, 1.5 Hz, 1H, H-1), 4.82 (dd, J = 12.4, 1.5 Hz, 1H, H-1), 4.05 (dd, J = 8.2, 5.0 Hz, 1H, H-6), 3.77 (s, 3H, 2′-OCH3), 2.83 (ddt, J = 18.0, 8.2, 1.7 Hz, 1H, H-5), 2.61 (dd, J = 17.9, 5.0 Hz, 1H, H-5), 2.25 (t, J = 7.5 Hz, 2H, H-10), 2.21 (s, 3H, H-7′), 1.72 (s, 3H, H-9), 1.63 (h, J = 7.4 Hz, 2H, H-11), 1.01 (t, J = 7.4 Hz, 3H, H-12)。13C-NMR (101 MHz、CD3OD) δ: 193.1 (C-8), 169.5 (C-3), 168.9 (C-8′), 160.9 (C-4′), 159.8 (C-2′), 149.6 (C-4a), 139.1 (C-6′), 116.2 (C-1′), 114.9 (C-8a), 109.9 (C-5′), 102.4 (C-4), 97.6 (C-3′), 86.0 (C-7), 73.9 (C-6), 65.4 (C-1), 56.3 (2′-OCH3), 36.8 (C-10), 34.7 (C-5), 21.31 (C-11), 19.5 (C-7′), 16.8 (C-9), 13.9 (C-12)。以上数据与文献 (Gao et al, 2011)进行对比, 确定7为rel-(6R, 7R)-5, 6, 7, 8-tetrahydro-6-hydroxy-7-methyl-8-oxo-3-propyl-1H-2-benzopyran。
化合物8: 白色固体粉末。 [ α ] D 20+33 (c = 0.3mg·mL-1, MeOH), ESI-MS m/z: 403.17 [M+H]+, 分子式C22H26O7。1H-NMR (400 MHz、CD3OD) δ: 6.31 (d, J = 2.1 Hz, 1H, H-5′), 6.27 (d, J = 2.2 Hz, 1H, H-3′), 5.44 - 5.37 (m, 2H, H-1), 3.76 (s, 3H, H-1, 2′-OCH3), 3.12 - 2.96 (m, 1H, H-4), 2.82 (dd, J = 19.1, 3.6 Hz, 1H, H-10), 2.26 (d, J = 7.4 Hz, 2H, H-5), 2.23 (s, 3H, H-7), 1.62 (h, J = 7.4 Hz, 2H, H-11), 1.44 (s, 3H, H-9), 1.00 (t, J = 7.4 Hz, 3H, H-12)。13C-NMR(101 MHz、CD3OD) δ: 196.8 (C-8), 170.0 (C-3), 169.5 (C-8′), 161.2 (C-4′), 160.2 (C-2′), 149.6 (C-4a), 140.1 (C-6′), 115.4 (C-1′), 113.7 (C-8a), 110.3 (C-5′), 102.5 (C-4), 97.80 (C-3′), 78.0 (C-6), 75.3 (C-7), 65.0 (C-1), 56.3 (2′-OCH3), 36.8 (C-10), 32.7 (C-5), 23.0 (C-11), 21.3 (C-7′), 20.1 (C-9), 13.9 (C-12)。以上数据与文献(Gao et al, 2011; Shao et al, 2020)进行对比, 确定8为rel-(6R, 7R)-5, 6, 7, 8-tetrahydro-7-hydroxy-7-methyl -8-oxo-3-propyl-1H-2-benzopyran。
化合物9: 淡绿色固体粉末。ESI-MS m/z: 284.09 [M+H]+, 分子式C22H26O7。1H-NMR (400 MHz、CDCl3) δ: 9.41 (s, 1H, H-8), 7.80 (s, 1H, H-2), 7.43 (d, J = 2.4 Hz, 1H, H-4), 6.84 (d, J = 2.4 Hz, 1H, H-5), 4.02 (s, 3H, H-11), 3.99 (s, 3H, H-12), 2.74 (s, 3H, H-13)。13C-NMR (101 MHz、CDCl3) δ: 183.7 (C-10), 180.6 (C-9), 165.2 (C-6), 164.3 (C-3), 162.9 (C-8), 149.9 (C-1), 137.7 (C-11), 137.1 (C-14), 125.6 (C-13), 117.7 (C-4), 115.8 (C-12), 105.6 (C-7), 103.7 (C-5), 56.8 (C-17), 56.2 (C-C-16), 25.2 (C-24.7)。以上数据与文献(Mao et al, 2021)进行对比, 确定9为scorpinone。
化合物10: 红色固体粉末。ESI-MS m/z: 169.05 [M+H]+, 分子式C8H8O4。1H-NMR (400 MHz、Acetone-d6) δ: 6.60 (s, 1H, H-3 ), 6.54 (s, 1H, H-5), 2.41 (s, 3H, H-7)。13C-NMR (101 MHz、Acetone-d6) δ: 174.3 (C-7), 166.9 (C-4), 163.0 (C-2), 144.9 (C-6), 111.9 (C-5), 105.4 (C-1), 101.4 (C-3), 24.3 (C-8)。以上数据与文献 (Van Eijk, 1969)进行对比, 确定10为orsellinic acid。
化合物11: 灰色固体粉末。ESI-MS m/z: 141.05 [M+H]+, 分子式C7H8O3。1H-NMR (400 MHz、CD3OD) δ: 6.18 (d, J = 2.4 Hz, 1H, H-3), 6.16 - 6.12 (m, 1H, H-5), 2.49 (s, 3H, H-7)。13C-NMR (101 MHz、CD3OD ) δ: 166.9 (C-1), 163.5 (C-2), 159.3 (C-4), 145.3 (C-3), 112.2 (C-5), 101.6 (C-6), 24.3 (C-7)。以上数据与文献 (Sekiguchi et al, 1987)进行对比, 确定11为6-methyl-1, 2, 4-benzenetriol。
化合物12: 淡黄色油状物。ESI-MS m/z: 151.11 [M+H]+, 分子式C10H14O。1H-NMR (400 MHz、CD3OD) δ: 6.93 (d, J = 7.6 Hz, 1H, H-2), 6.64 (t, J = 2.5 Hz, 1H, H-4), 6.58 (dd, J = 7.7, 1.8 Hz, 1H, H-5), 2.75 (h, J = 6.9 Hz, 1H, H-8), 2.14 (d, J = 2.8 Hz, 3H, H-7), 1.19 (d, J = 7.0 Hz, 6H, H-9, 10)。13C-NMR (101 MHz、CD3OD) δ: 156.2 (C-1), 148.9 (C-2), 131.5 (C-4), 122.6 (C-5), 118.5 (C-3), 114.0 (C-6), 34.9 (C-8), 24.5 (C-7), 15.8 (C-9, 10)。以上数据与文献 (Tang et al, 2011)进行对比, 确定12为2-methyl-5- isopropyl-phenol。
化合物13: 橘黄色油状物。ESI-MS m/z: 139.07 [M+H]+, 分子式C8H10O2。1H-NMR (400 MHz、CD3OD) δ: 6.32 (s, 1H, H-4), 6.25 (dd, J = 12.0, 2.3 Hz, 2H, H-2, 6), 3.76 (s, 3H, 3-OCH3), 2.27 (s, 3H, H-7)。13C-NMR (101 MHz、CD3OD) δ: 160.9 (C-1), 156.8 (C-2), 140.7 (C-6), 108.8 (C-3), 107.4 (C-4), 98.8 (C-5), 55.4 (3-OCH3), 21.7 (C-7)。以上数据与文献 (Lu et al, 2022)进行对比, 确定13为5-methyl-benzene-1, 3-diol。
化合物14: 橘黄色油状物。ESI-MS m/z: 125.06 [M+H]+, 分子式C7H8O2。1H-NMR (400 MHz、CD3OD) δ: 6.15 (d, J = 2.2 Hz, 2H, H-4, 6), 6.10 (d, J = 2.3 Hz, 1H, H-2), 2.20 (s, 3H, H-7)。13C-NMR (101 MHz、CD3OD) δ: 159.3(C-1, 3), 141.1 (C-2), 108.6 (C-4, 6), 100.7 (C-5), 21.9 (C-7)。以上数据与文献 (Kwon et al, 2011)进行对比, 确定14为5-methyl-1, 3-dihydroxy-benzene。

2.2 生物活性

对上述化合物进行抗肿瘤活性测试, 结果显示, 在浓度50μmol·L-1, 化合物8和化合物12对脑胶质瘤细胞U87表现抑制作用, 测得其IC50值分别为21.07μmol·L-1和15.74μmol·L-1。在浓度50μmol·L-1, 化合物8和12对肺癌细胞A549也表现出抑制作用, 并测得其IC50值分别为20.88μmol·L-1和22.93μmol·L-1, 结果见表1和图3。
表1 化合物1—14抗肿瘤细胞半数抑制浓度(IC50)

Tab. 1 IC50 Value (μmol·L-1) of Compounds 1—14 against cell line U87, A549

Compounds IC50/(μmol·L-1)
UU87 AA549
1 >50 >50
2 >50 >50
3 >50 >50
4 >50 >50
5 >50 >50
6 >50 >50
7 >50 >50
8 21.07 20.88
9 >50 >50
10 >50 >50
11 >50 >50
12
13
14		 15.74
>50
>50		 22.93
>50
>50
图3 化合物8和12分别对肿瘤细胞U87、A549的IC50拟合曲线

Fig. 3 Curve fitting of IC50 of compound 8 and 12 against tumor cell line U87, A549

3 讨论

本研究从芋螺内生真菌Talaromyces sp. XXH006中首次分离得到14个化合物, 其中化合物1—4和7—14均为首次从Talaromyces sp.中获取, 进一步丰富Talaromyces sp.的化学成分多样性。目前已报道的Talaromyces sp.次级代谢产物结构类型包括生物碱、多肽、内酯、聚酮、蒽醌、甾体等(Zhai et al, 2016), 本研究报道的苯酚类化合物10—14为该菌属中首次发现的结构类型。氮杂蒽醌骨架类型的化合物9在Talaromyces sp.次级代谢产物中鲜少报道。Talaromyces sp.次级代谢产物的结构多样性使其同样具有多样的药理活性。Ren等人从Talaromyces aculeatus分离获得具有α-葡萄糖苷酶抑制效果的新苯扎酮化合物talaraculones A和B (Ren et al, 2017)。Zhang等人从Talaromyces adpressus中分离得到的麦角甾烷talasterone A通过抑制NF-κB通路发挥抗炎活性(Zhang et al, 2022)。据文献报道, 化合物8仅涉及NO抑制作用(Shao et al, 2020)。本研究补充化合物8对肿瘤细胞U87和A549的抑制活性, 测得化合物8对肿瘤细胞系U87和A549的IC50值分别为21.07μmol·L-1和20.88μmol·L-1。而化合物12的活性报道涉及杀虫(Xu et al, 2023)、微生物抑制(Sousa et al, 2022)及抗肿瘤活性(Shinde et al, 2020), 其中抗肿瘤活性报道未涉及对肿瘤细胞U87和A549的研究。在本研究中, 化合物12对肿瘤细胞U87和A549的IC50值分别为15.74μmol·L-1和 22.93μmol·L-1。后续, 我们将继续挖掘更多芋螺内生真菌Talaromyces sp.次级代谢产物的结构和活性, 为开发活性显著的药物先导化合物奠定基石。

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