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Journal of Tropical Oceanography >

2021 , Vol. 40 >Issue 4: 63 - 69

DOI: https://doi.org/10.11978/2020085

Marine Biology

Research on benzaldehydes from the soft coral-associated symbiotic fungus Aspergillus sp. EGF15-0-3

  • LIU Bingxin , 1, 2 ,
  • WEI Xia 1 ,
  • XIAO Xiji 1 ,
  • ZHANG Qi 1 ,
  • ZHANG Cuixian , 1
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  • 1. School of Pharmaceutical Sciences, Guangzhou University of Chinese Medicine, Guangzhou 510006, China
  • 2. Kangmei Pharmaceutical Co., Ltd, Guangzhou 510627, China
ZHANG Cuixian. email:

Copy editor: YAO Yantao

Received date: 2020-08-01

  Revised date: 2020-10-16

  Online published: 2020-12-24

Supported by

Special Project for Marine Economic Development (Six Major Marine Industries) of Guangdong Province Department of Natural Resources of Guangdong Province([2020]039)

Science and Technology Planning Project of Guangdong Province(2017A020217008)

Science and Technology Planning Project of Guangdong Province(2015A020216017)

Fire Plan Project of the Guangzhou University of Chinese Medicine(XH20170110)

National Natural Science Foundation of China(81741160)

Natural Science Foundation of Guangdong Province(2014A030313411)

Copyright

Copyright reserved © 2021. Office of Acta Agronomica Sinica All articles published represent the opinions of the authors, and do not reflect the official policy of the Chinese Medical Association or the Editorial Board, unless this is clearly specified.
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Abstract

To research the benzaldehydes of the soft coral-associated symbiotic fungus Aspergillus sp. EGF 15-0-3 with the activity of overcoming tumor resistance, the secondary metabolites were isolated by silica gel column chromatography, ODS, Sephadex LH-20, thin layer chromatography, and high performance liquid chromatography (HPLC). The structures of compounds were identified by nuclear magnetic resonance (NMR), mass spectrometry(MS), and literature analysis. Nine benzaldehydes were obtained and elucidated as follows: flavoglaucin (1), tetrahydroauroglaucin (2), isoaspergin (3), isotetrahydroauroglaucin (4), dihydroauroglaucin (5), isodihydroauroglaucin (6), 2-(1',5'-heptadienyl)-3,6-dihydroxy-5- (3''-methyl-2''-butenyl) benzaldehyde (7), auro- glaucin (8), and 2-(2',3-epoxy-1'-heptenyl)-6- hydroxy-5-(3''methyl-2''-butenyl) benzal-dehyde (9). Using the MTT [3-(4, 5)-dimethylthiahiazo (-z-y1)-3, 5-di-phenytetrazoliumromide] method, compound 9 showed strong activity to drug-resistant tumor cell lines H1975/GR. This may be due to the structure of a pyran ring between the 2-position side chain and 3-hydroxyl group.

Key words： Soft coral-associated symbiotic epiphytic fungus; Aspergillus sp. EGF15-0-3; benzaldehydes; overcoming tumor resistance activity

Cite this article

LIU Bingxin , WEI Xia , XIAO Xiji , ZHANG Qi , ZHANG Cuixian . Research on benzaldehydes from the soft coral-associated symbiotic fungus Aspergillus sp. EGF15-0-3[J]. Journal of Tropical Oceanography, 2021 , 40(4) : 63 -69 . DOI: 10.11978/2020085

*本论文中克服肿瘤耐药活性的药理部分由暨南大学张冬梅教授团队完成, 在此表示衷心的感谢！
苯甲醛类化合物(图1)广泛分布于自然界(Li et al, 2008; 黄玉玲 等, 2012), 结构的1位为醛基取代, 2位常为直链庚烷取代, 5位常为异戊烯基取代, 3位和6位存在羟基。当2位侧链为直链时, 常与苯环形成1~3个共轭与非共轭双键(图1a), 或者与3位羟基形成呋喃(图1b)或吡喃环(图1c、1d)。此类物质主要来源于曲霉属, 且具有较高的生物活性(Umeda et al, 1974; Nazar et al, 1984; Miyake et al, 2010; Smetanina et al, 2007; Gao et al, 2012; Kim et al, 2014), 如抗氧化、细胞毒活性和抑菌活性, 是国内外学者们的重要研究课题。本文作者所在课题组一直在寻找海洋生物中结构新颖的次生代谢产物, 在OSMAC(One strain many compounds)和GNPS(Global Natural Product Social Molecular Networking)的共同指导下, 继续对南海软珊瑚共附生真菌Aspergillus sp. EGF15-0-3 PDA (Potato Dextrose Agar (Medium))培养基次生代谢产物具有克服肿瘤活性的苯甲醛部位(浓度为200ug·ml-1时克服肿瘤耐药活性为56.89%)进行研究(李秀婷 等, 2016; Feng et al, 2020; Wei et al, 2020), 并分离得到9个苯甲醛类化合物(图2)。通过核磁共振和质谱等现代波谱研究, 确定了这9个苯甲醛类化合物的结构为: flavoglaucin (1)、tetrahydroauroglaucin(2)、 isoaspergin (3)、 isotetrahydroauroglaucin (4)、dihydroauroglaucin (5)、isodihydroauroglaucin (6)、2-(1',5'-heptadienyl)-3, 6-dihydroxy-5-(3''-methyl-2''-butenyl) benzaldehyde (7)、auro-glaucin (8)和2-(2',3-epoxy-1'-heptenyl)- 6-hydroxy-5-(3''methyl-2''-butenyl) benzal-dehyde (9)。体外克服耐药肿瘤H1975/GR细胞株的活性研究表明, 在浓度为50μg·mL-1时, 化合物3具有较弱的细胞毒活性, 抑制率62.66%; 而化合物9具有较强细胞毒活性, 抑制率为93.71%。
图1 海洋微生物中苯甲醛类衍生物常见结构类型

图c中的R为H或OH

Fig. 1 Common structural types of benzadehyde derivatives in marine microorganisms

图2 来源于软珊瑚共附生真菌Aspergillus sp.EGF15-0-3的苯甲醛类化合物

化合物2~8后的Δ代表双键

Fig. 2 The benzadehyde compounds derived from soft coral-associated symbiotic fungus Aspergillus sp. EGF15-0-3

1 实验部分

1.1 仪器与试剂

仪器: GZX-9070MBE恒温培养箱(上海博迅实业有限公司); HYG-A全温摇瓶柜(常州市三盛仪器制造有限公司); 超净工作台(单人)(苏州净化设备厂); DZ-900振荡器(太仓市强乐实验设备厂); LX- B75L型立式自动电热压力蒸汽灭菌器(合肥华泰医疗设备有限公司); YLD-2000烘箱(上海索谱仪器有限公司); 巩义市予华仪器有限责任公司生产的SHZ-DIII循环水真空泵、XHDLSB-5/25低温冷却循环泵、XHRE-2000A旋转蒸发仪和KQ-500DB超声波清洗机; QuikSep高效液相色谱仪(北京慧德易科技有限责任公司); AVANCE AV400超导核磁共振仪(瑞士Bruker公司); AB SCIEX三重四级杆飞行时间串联质谱(美国ABSciex公司); POLARTRONIC HH W5旋光仪(德国SCHMIDT+ HAENSCH公司)。
试剂: 土豆(市场购买); 葡萄糖(AR, 天津市大茂化学试剂厂); 速溶海水晶(江西盐通科技有限公司); 甲醇、乙酸乙酯、石油醚、氯仿、正丁醇、乙酸、浓硫酸、二甲基亚砜(AR, 广州东巨精细化工有限公司); 色谱甲醇、乙腈和乙醇(上海迈瑞尔化学技术有限公司); 开放性ODS柱层析(12nm, S-50μm, 日本分光株式会社); Sephadex LH-20葡聚糖凝胶(General Electrics医疗集团); 柱层析硅胶(200~300目, 青岛海洋化工厂); GF254薄层层析(青岛海洋化工厂)。
PDA培养基: 葡萄糖2.0%, 土豆200g·L-1(土豆汁用陈海水作溶剂配制), 盐度35‰, pH自然。

1.2 微生物材料

菌种: 菌株EGF15-0-3由采集于中国南海三亚海域的软珊瑚分离而来, 根据其形态特征和分子协议, 对rRNA基因ITS区域的DNA序列进行扩增和测序, 种属鉴定为曲霉属真菌Aspergillus sp., 菌种样本保存在广州中医药大学中药学院海洋微生物实验室(编号EGF15-0-3)。
细胞株: 吉非替尼耐药株(H1975/GR, 由暨南大学药学院张冬梅教授提供)。

1.3 菌株EGF 15-0-3规模发酵

种子液活化: 将菌种管从冰箱取出解冻后用接种环蘸取适量的菌液, 接种到含有PDA固体培养基的平板上(共5块平板), 置恒温培养箱中28℃培养2~3d。待平板长出单菌落后, 用接种环挑取单菌落转移至装有200mL PDA培养基的1L培养瓶中, 置于CO2恒温培养箱中28℃、165r·min-1培养2~3d, 获得EGF15-0-3的种子培养液。
发酵: 吸取该种子液3.0mL, 分别加入装有400mL PDA培养基的1L培养瓶中, 在28℃下静置培养60d, 与空白培养基对比并观察是否有明显的菌株生长, 生长完毕停止发酵。共培养菌株EGF15-0-3 100L。

1.4 菌株EGF 15-0-3次生代谢产物的提取分离

提取: 培养基的菌株成长完毕后, 用少量甲醇灭活后停止发酵, 经纱布过滤, 得到菌丝体和发酵液两部分。发酵液在85℃水浴条件下浓缩至10L, 依次用等体积的EtOAc和n-BuOH进行分配萃取, 各萃取3次, 分别合并EtOAc和n-BuOH后减压浓缩, 得到发酵液的EtOAc相浸膏(17.5g)及n-BuOH相浸膏(67.5g)。菌丝体用甲醇浸泡提取3次(每次3d), 所得提取液经合并及减压浓缩后再用水涅溶, 依次用等量的EtOAc和n-BuOH进行分配萃取, 分别萃取3次, 萃取液减压浓缩后得到菌丝体的EtOAc相浸膏(36.5g)和n-BuOH相浸膏(50.8g)。
分离: 根据TLC和LC-MS情况, 将PDA菌丝体的EtOAc相浸膏(36.5g)经硅胶柱层析, 以石油醚-乙酸乙酯体系0~100%洗脱后再用氯仿-甲醇体系0~100%梯度洗脱, TLC薄层跟踪并合并流分得到12个不同极性的流分(标记为Fr.1~Fr.12)。通过NMR和LC-MS共同分析后, 选择目标组流分进行下一步分离, 其中Fr.2(14.5g)经硅胶柱层析和石油醚-乙酸乙酯梯度洗脱, 分离后重结晶得到化合物8(250mg)。Fr.2-2(1.5g)甲醇溶解, 经半制备 HPLC纯化(Kromasil色谱柱, 5μm, 250mm×10mm)和纯甲醇洗脱(6mL·min-1), 分离出8个峰, 通过与化合物8的UV对照后确定3个目标峰, 分别为peak 4(201.3mg, τR=26.82min)、peak 5(534.6mg, τR= 28.38min)、peak 8(153.9mg, τR=39.08min)。peak 4进行二次半制备HPLC纯化(ACE色谱柱, 250mm×10mm C18-PFP ), 经甲醇水体系(V甲醇:V水= 8:2, 3mL·min-1)洗脱后获得化合物3(32.2mg, τR =49.43min); peak 5进行二次半制备HPLC纯化(YMC色谱柱, 250mm×10mm ), 经甲醇水体系(V甲醇: V水=8:2, 3mL·min-1)洗脱后获得化合物4(68.2mg, τR=47.26min)和5(45.1mg, τR=50.19min); peak 8进行二次半制备HPLC纯化(YMC色谱柱, 250mm×10mm ), 经甲醇水体系(V甲醇:V水=8.8:1.2, 3mL·min-1)洗脱后获得化合物9(3.8mg, τR= 52.95min)。Fr.2-3(500mg)甲醇溶解, 经半制备 HPLC纯化(Kromasil色谱柱, 5μm, 250mm×10 mm)和甲醇水体系(V甲醇:V水=85:15, 8mL·min-1)洗脱后, 依次获得化合物1(10mg, τR=13.28min)、2(20mg, τR= 20.03min)、6(10mg, τR=21.39min)和7(7mg, τR= 17.15min)。

1.5 物理常数和波谱数据

化合物1: 黄色针晶(甲醇), 分子式为C19H28O3, UV 254nm下有暗斑, 香草醛-浓H2SO4显棕蓝色, HRESIMS m/z: 303.1280[M-H]-; 1H NMR(CDCl3, 500MHz) δH ppm: 11.9 (1H, s, 6-OH), 10.25 (1H, s, H-14), 6.89 (1H, s, H-4), 5.28 (1H, t, H-16), 4.4 (1H, s, 3-OH), 3.28 (2H, d, H-15), 2.88 (2H, brt, H-7), 1.76(3H, s, H-19), 1.7 (3H, s, H-18), 1.58 (2H, m, H-8), 1.39 (2H, m, H-9), 1.29 (2H, m, H-10), 1.29 (2H, m, H-11), 1.29 (2H, m, H-12), 0.88(3H, m, H-13) 和13C NMR (CDCl3, 125MHz) δC ppm: 14.04 (q, C-13), 17.76 (q, C-18), 22.61 (t, C-12), 23.94 (t, C-7), 25.77 (q, C-19), 27.01(t, C-15), 29.11(t, C-10), 29.59 (t, C-9), 31.77 (t, C-11), 31.99 (t, C-8), 133.79 (s, C-17), 117.34 (s, C-1), 121.21 (d, C-16), 125.69 (d, C-4), 128.59 (s, C-2), 128.59 (s, C-5), 144.59 (s, C-3), 155.82 (s, C-6), 195.52 (d, C-14).
化合物2: 黄色块晶(甲醇), 分子式为 C19H26O3, UV 254nm下有暗斑, 香草醛-浓H2SO4显棕蓝色, HR-ESI-MS m/z: 301.1808[M-H]-; 1H NMR (Pyr-d5, 400MHz) δH ppm: 11.18 (1H, s, 6-OH), 10.41(1H, s, H-14), 7.36 (1H, s, H-4), 7.01(1H, td, H-7), 6.0 (1H, td, H-8), 5.43 (1H, t, H-16), 4.91 (1H, s, 3-OH), 3.5 (2H, d, H-15), 2.24(2H, m, H-9), 1.65(3H, s, H-19), 1.62 (3H, s, H-18), 1.41 (2H, m, H-10), 1.26 (2H, m, H-11), 1.26 (2H, m, H-12), 0.84(3H, m, H-13) 和13C NMR(Pyr-d5, 100MHz) δC ppm: 14.56 (q, C-13), 18.04 (q, C-18), 23.11 (t, C-12), 26.09 (q, C-19), 28.04(t, C-15), 29.55(t, C-10), 32.08 (t, C-11), 34.4(t, C-9), 118.97(s, C-1), 122.6 (d, C-7), 122.71 (d, C-16), 126.1(s, C-2), 127.53 (d, C-4), 129.31(s, C-5), 133.78 (s, C-17), 141.52(d, C-8), 148.84(s, C-3), 154.92 (s, C-6), 198.09 (d, C-14).
化合物3: 黄色针晶(甲醇), 分子式为C19H26O3, HR-ESI-MS m/z: 301.1808 [M-H]- (Cal. 301.1804); 1H NMR (CDCl3, 400 MHz) δH ppm: 11.92 (1H, s, 6-OH), 10.21 (1H, s, H-14), 6.91 (1H, s, H-4), 5.45 (H, m, H-9), 5.42 (H, m, H-10), 5.28 (1H, brt, H-16), 4.5 (1H, s, 3-OH), 3.3 (2H, d, H-15), 2.96 (2H, t, H-7), 2.28 (2H, m, H-8), 1.95 (2H, m, H-11), 1.76(3H, s, H-18), 1.69(3H, s, H-19), 1.34 (2H, m, H-12), 0.86 (3H, d, H-13) 和 13C NMR (CDCl3, 100MHz) δC ppm: 13.8 (q, C-13), 17.9 (q, C-18), 22.6 (t, C-12), 24.4 (t, C-7), 26 (q, C-19), 27.2(t, C-15), 34.3 (t, C-11), 34.7 (t, C-8), 117.4 (s, C-1), 121.3 (d, C-16), 126 (d, C-4), 127.7 (s, C-5),128.4 (d, C-9), 129 (s, C-2), 132.8 (d, C-10), 134 (s, C-17),145.2 (s, C-3), 156.0 (s, C-6), 195.7 (d, C-14).
化合物4: 黄色针晶(甲醇), 分子式为C19H26O3, HRESIMS m/z: 301.1808 [M-H]- (Cal. 301.1804); 1H NMR (CDCl3, 400MHz) δH ppm: 11.91 (1H, s, 6-OH), 10.23 (1H, s, H-14), 6.89 (1H, s, H-4), 5.4 (1H, m, H-12), 5.38 (H, m, H-11), 5.27 (1H, t, H-16), 3.49 (1H, s, 3-OH), 3.27 (2H, d, H-15), 2.88 (2H, t, H-7), 2.02 (2H, q, H-10), 1.75 (3H, s, H-18), 1.69 (3H, s, H-19), 1.63 (3H, m, H-13), 1.57 (2H, m, H-9), 1.45 (2H, m, H-8) 和13C NMR (CDCl3,100MHz) δC ppm: 17.9 (q, C-13), 18.1 (q, C-18), 23.8 (t, C-7), 25.9 (q, C-19), 27.2(t, C-15), 29.4 (t, C-9), 31.4 (t, C-8), 32.4(t, C-10), 117.4 (s, C-1), 121.3 (d, C-16), 125.4(d, C-4), 125.8 (s, C-5), 128.6 (d, C-11), 128.7(s, C-2), 131.1 (d, C-12), 134 (s, C-17), 145.2 (s, C-3), 155.8 (s, C-6), 195.5 (d, C-14).
化合物5: 黄色针晶(甲醇), 分子式为C19H24O3, HR-ESI-MS m/z: 299.1650 [M-H]- (Cal. 299.1647); 1H NMR (CDCl3, 400MHz) δH ppm: 11.77 (1H, s, 6-OH), 10.08 (1H, s, H-14), 6.99 (1H, s, H-4), 6.54 (1H, d, H-7), 6.43 (1H, m, H-9), 6.27 (1H, m, H-8), 5.88 (1H, m, H-10), 5.29 (1H, brt, H-16), 3.49 (1H, s, 3-OH), 3.32 (2H, d, H-15), 2.13 (2H, q, H-11), 1.75(3H, s, H-18), 1.69(3H, s, H-19), 1.45 (2H, m, H-12), 0.94 (3H, t, H-13) 和13C NMR (CDCl3, 100MHz) δC ppm: 13.9 (q, C-13), 17.9 (q, C-18), 22.4 (t, C-11), 25.9 (q, C-19), 27.4 (t, C-15), 35 (t, C-12), 117.2 (s, C-1), 119.6 (d, C-16), 121.1 (d, C-7), 124.3(s, C-5), 125.3(d, C-4), 129.7 (d, C-9), 130.5 (s, C-2), 134.1 (s, C-17), 139 (d, C-8), 140 (d, C-10), 145.32 (s, C-3), 155.4 (s, C-6), 195.4 (d, C-14.
化合物6: 黄色针晶(甲醇), 分子式为C19H24O3, UV 254nm下有暗斑, 香草醛-浓H2SO4显棕蓝色, HRESIMS m/z: 299.1650[M-H]-; 1H NMR(CDCl3, 500MHz) δH ppm: 11.92 (1H, s, 6-OH), 10.23 (1H, s, H-14), 6.9 (1H, s, H-4), 6.08 (1H, m, H-11), 5.98(1H, m, H-9), 5.63 (1H, m, H-12), 5.55 (1H, m, H-10), 5.28 (1H, m, H-16), 4.52 (1H, s, 3-OH), 3.3 (2H, d, H-15), 2.98(2H, t, H-7), 2.35 (2H, dd, H-8), 1.76(3H, s, H-19), 1.73 (3H, t, H-13), 1.7 (3H, s, H-18) 和13C NMR (CDCl3, 125MHz) δC ppm: 15.6 (q, C-19), 17.8 (q, C-18), 18 (q, C-13), 24.2 (t, C-7), 27.2(t, C-15), 34.1 (t, C-8), 117.1 (s, C-1), 121 (d, C-16), 125.4 (d, C-4), 127.1 (s, C-2), 128.3 (d, C-9), 128.7 (s, C-5), 129.2 (d, C-10), 131 (d, C-12), 131.8 (d, C-11), 134 (s, C-17), 145.5 (s, C-3), 155.4 (s, C-6), 195.7 (d, C-14).
化合物7: 黄色针晶(甲醇), 分子式为C19H24O3, UV 254nm下有暗斑, 香草醛-浓H2SO4显棕蓝色, HR-ESI-MS m/z: 299.1650[M-H]-; 1H NMR (CDCl3, 500MHz) δH ppm: 11.77 (1H,s, 6-OH), 10.06 (1H, s, H-14), 7.0 (1H, s, H-4), 6.5 (1H, m, H-7), 6.5 (1H, m, H-8), 5.95 (1H, m, H-11), 5.95 (1H, m, H-12), 5.28 (H, m, H-16), 4.65 (1H, s, 3-OH), 3.47 (2H, m, H-15), 3.4 (2H, m, H-10), 3.21 (2H, m, H-9), 1.76 (3H,s, H-19), 1.7 (3H, s, H-18), 1.63 (3H, m, H-13).
化合物8: 橙黄色针晶, 分子式为C19H22O3, HRESIMS m/z: 297.1499[M-H]-(cal. 299.1491); 1H NMR (CDCl3, 400 MHz) δH ppm: 11.79 (1H, s, 6-OH), 10.08 (1H, s, H-14), 6.99 (1H, s, H-4), 6.65 (1H, d, H-7), 6.48 (1H, m, H-9), 6.33 (1H, m, H-8), 6.31 (1H, m, H-11), 6.15 (1H, m, H-10), 5.84 (1H, m, H-12), 5.29 (1H, brt, H-16), 4.93(1H, s, 3-OH), 3.33 (2H, d, H-15), 1.82 (3H, d, H-13), 1.76 (3H, s, H-18), 1.7(3H, s, H-19) 和13C NMR (CDCl3,100MHz) δC ppm: 17.9 (q, C-18), 18.6 (q, C-13), 26 (q, C-19), 27.4(t, C-15), 117.1 (s, C-1), 120.8 (d, C-16), 121 (d, C-7), 124.2(s, C-5), 134.2 (s, C-17), 125.3 (d, C-4), 129.1 (d, C-8), 130.7(s, C-2), 131.4(d, C-9), 132.9(d, C-11), 136.4(d, C-12), 139.7(d, C-10), 145.2(s, C-3), 155.5 (s, C-6), 196.2 (d, C-14).
化合物9: 黄色油状, 分子式为C19H24O3, HRESIMS m/z: 299.1651[M-H]- (cal. 299.1641); 1H NMR (CDCl3, 400MHz) δH ppm: 11.67 (1H, s, 6-OH), 10.24 (1H, s, H-14), 7.45 (1H, s, H-4), 6.65 (1H, s, H-7), 5.34 (1H, brt, H-16), 3.4 (2H, d, H-15), 2.78 (2H, t, H-9), 1.77 (3H, s, H-18), 1.74 (2H, m, H-10), 1.72 (3H, s, H-19), 1.38 (2H, m, H-11), 1.36 (2H, m, H-12), 0.91 (3H, t, H-13)和13C NMR (CDCl3, 100MHz) δC ppm: 14.1 (q, C-13), 17.9(q, C-18), 22.5 (t, C-12), 26 (q, C-19), 27.5(t, C-10), 27.6(t, C-15), 28.8 (t, C-9), 31.5 (t, C-11), 98.7(d, C-7), 110.9 (s, C-1), 119.6 (d, C-4), 121.6 (d, C-16), 125.5 (s, C-5), 128.7(s, C-2), 134(s, C-17), 148.6 (s, C-3), 157.6 (s, C-8), 162.8 (s, C-6), 193.3 (d, C-14).

1.6 药理活性试验

MTT法: 其原理为活细胞线粒体中的琥珀酸脱氢酶能使外源性MTT(噻唑兰, 溴化噻唑蓝四氮唑)还原为水不溶性的蓝紫色结晶甲瓒(Formazan)并沉积在细胞中, 而死细胞无此功能。二甲基亚砜(DMSO)能溶解细胞中的甲瓒, 用酶联免疫检测仪在490nm波长处测定其光吸收值, 可间接反映活细胞数量。采用96孔板法, 粗提物和单体化合物的筛药浓度分别为200μg·mL-1和50μmol·L-1 (LaKshmi et al, 2009)。耐药肿瘤细胞株为吉非替尼耐药肺癌肿瘤细胞株(H1975/GR)。

2 结果与讨论

化合物1为黄色针晶(甲醇), 紫外灯254nm下有暗斑, 香草醛-浓H2SO4显棕蓝色。由ESI-MS给出的准分子离子峰m/z为303[M-H]-, 提示其相对分子质量为304, 结合1H和13C NMR确定其分子式为 C19H28O3, 表明不饱和度为6。结合其NMR图谱信息, 结构中含有6个C、3个CH、7个CH2和3个CH3。1H NMR谱中δH 10.25(1H, s, H-14)和13C NMR谱中δC 195.52(d, C-14)的信号提示结构中含有醛基; 1H NMR中只显示了一个芳香质子信号δH 6.89(1H, s), δH 11.9(1H, s)和4.4(1H, s)为酚羟基质子信号, 结合13C NMR信息说明结构中存在五取代苯环, 为苯甲醛类化合物。1H NMR谱存在一组典型的异戊烯基片段质子信号: δH 3.28(2H, d, 7.2Hz, H-15)、5.28(1H, t, 7.2Hz, H-16)、1.7(3H, s, H-18)和1.76(3H, s, H-19); 一系列质子信号: δH 2.88(2H, br t, 7.2 Hz, H-7)、1.58(2H, m, H-8)、1.39(2H, m, H-9)、1.29(2H, m, H-10)、1.29(2H, m, H-11)、1.29(2H, m, H-12)和0.88(3H, m, H-13)。结合13C NMR信号δC 23.94(t, C-7)、31.99(t, C-8)、29.59(t, C-9)、 29.11(t, C-10)、31.77(t, C-11)、22.61(t, C-12) 及14.04(q, C-13), 可知结构中应存在一饱和的直链7碳烷烃片段。进一步将数据与黄玉玲等(2012)的flavoglaucin对比, 可确定该化合物1为灰绿曲霉黄色素(flavoglaucin)。
化合物2、3和4均为黄色晶体(甲醇), 紫外灯254nm下有暗斑, 香草醛-浓H2SO4显棕蓝色, 提示为苯甲醛类化合物。三者的HR-ESI-MS分析给出的准分子离子峰m/z均为301.1808[M-H]-(cal. 301.1804), 分子式均为C19H26O3, 不饱和度为7, 互为同分异构体。同时发现2、3、4的分子量比1少2, 不饱和度与1比较增加1, 可推断2、3、4只是比1多了1个双键。对比化合物1~4的NMR信息, 四者高度类似, 其中2的1H NMR谱中含有异戊烯基的特征吸收信号δH 5.43(1H, m, H-16)、3.50(2H, d, J=7.2Hz, H-15)、1.65(3H, s, H-19)和1.62(3H, s, H-18); 13C NMR谱中给出相应的信号为: 2 个甲基信号δC 18.04(C-18)、26.09 (C-19), 1个亚甲基δC 28.04(C-15), 1个sp2杂化的次甲基δC 122.71(C-16)和1个sp2杂化的季碳δC 133.78(C-17)。分子中庚二烯基结构片段信号δH 7.01(1H, td, J=16.0Hz, H-7)和6.0(1H, td, J=2.4,16.0Hz, H-8)表明该化合物的7、8位之间为反式双键; 信号δH 2.24(2H, m, H-9)、1.41(2H, m, H-10)和1.26(4H, m, H-11, 12)表明该化合物的9、10位和11、12位之间都为单键; 信号δH 0.84(3H, m, H-13)、1.26(4H, m, H-11, 12)表明化合物的12、13位为单键。综合以上信息, 将化合物2与文献曲霉素(aspergin)(Inoue et al, 1997)对比, 确定2为曲霉素(aspergin)或四氢金(色)灰绿曲霉素(tetrahydroauroglaucin)。化合物3、4与2对比, 唯一不同的是在直链的7碳烯烃片段中双键取代的位置存在差异。将化合物3的 NMR数据与isoaspergin (黄玉玲 等, 2012)对比, 两者基本一致, 故确定3为isoaspergin。将化合物4 的NMR数据与isotetrahydroauroglaucin对比(Hamasaki et al, 1980), 两者基本一致, 故确定4为isotetrahydroauroglaucin。
化合物5、6和7均为黄色针晶(甲醇), 三者紫外灯254nm下均有暗斑, 香草醛-浓H2SO4显棕蓝色, 提示为苯甲醛类化合物。三者的HR-ESI-MS分析给出的准分子离子峰m/z均为299.1650[M-H]-(cal. 299.1647), 分子式均为C19H24O3, 不饱和度为8, 提示化合物5、6、7为同分异构体。其次, 3个化合物的NMR图谱信息与化合物2、3、4高度相似, 唯一不同的是在直链的7碳烯烃片段中双键取代的数量存在差异。化合物5的1H NMR中, δH 6.54(1H, d, 16.0 Hz, H-7)、6.27(1H, m, H-8)、6.43(1H, m, H-9)、5.88(1H, m, H-10)、2.13(2H, q, 8.0 Hz, H-11)、1.45(2H, m, H-12)和0.94(3H, t, J=8.0 Hz, H-13), 以及13C NMR信号中的δC 121.1(d, C-7)、139.0(d, C-8)、129.7(d, C-9)、140.0(d, C-10)、22.4(t, C-11)、35.0(t, C-12)和13.9(q, C-13), 表明化合物5的7、8位和9、10位分别为双键取代, 将该化合物的NMR数据与dihydroauroglaucin(Hamasaki et al, 1981)对比, 两者基本一致, 故确定化合物5为dihydroauroglaucin。化合物6、7与5对比, 唯一不同的是在直链的7碳烯烃片段中双键取代的位置存在差异。将化合物6的数据与异二氢金(色)灰绿曲霉素(黄玉玲 等, 2012)进行比较, 两者接近一致, 故确定化合物6为异二氢金(色)灰绿曲霉素(isodihydroauroglaucin)。将化合物7的数据与2-(1',5'-heptadienyl)-3,6-dihydroxy- 5-(3''-methyl-2''-butenyl) benzaldehyde (Li et al, 2008)对比, 两者接近一致, 故将化合物7的结构定为2-(1',5'-hepta-dienyl)-3, 6-dihydroxy-5-(3''-methyl- 2''-butenyl) benzaldehyde。
化合物8为橙黄色针晶(甲醇), HR-ESI-MS分析给出的准分子离子峰m/z为297.1499[M-H]-(cal. 299.1491), 分子式为C19H22O3, 不饱和度为9。其NMR图谱信号与化合物5高度相似, 唯一不同的是在直链的7碳烯烃片段中双键取代的位置存在差异。化合物8的1H NMR中, 系列质子信号为δH 6.65(1H, d, J=16.0 Hz, H-7)、6.33(1H, m, H-8)、6.48(1H, m, H-9)、6.15(1H, m, H-10)、6.31(1H, m, H-11)、5.84(1H, m, H-12)和1.82(3H, d, 8.0 Hz, H-13), 结合13C NMR信号中的δC 121.0(d, C-7)、129.1(d, C-8)、131.4(d, C-9)、139.7(d, C-10)、132.9(d, C-11)、136.4(d, C-12)以及18.6(q, C-13), 可判断直链的7碳烯烃片段为共轭三双键取代。将化合物8的NMR数据与auroglaucin(Miyake et al, 2009)对比, 两者基本一致, 故确定化合物8为auroglaucin。
化合物9为黄色油状, HR-ESI-MS分析给出的准分子离子峰m/z为299.1651[M-H]-(cal. 299.1641), 分子式为C19H24O3, 不饱和度为8。对其NMR图谱进行分析, 发现该化合物也属于苯甲醛类化合物, 结构中存在一个五取代苯体系和一个异戊二烯基片段。1H NMR图谱仅显示烯烃质子信号δH 6.65(1H, s, H-7), 除此之外无任何不饱和度信息, 说明其结构中还存在一个环。13C NMR图谱中的烯烃信号δH 98.7(d, C-7)和157.6(s, C-8), 说明其一端为连氧烯碳。根据13C NMR图谱中给出的19个碳信号(7个C, 4个CH, 5个CH2和3个CH3), 结合1H NMR的一系列质子信号[δH 2.78(2H, t, 8.0 Hz, H-9)、1.74(2H, m, H-10)、1.38(2H, m, H-11)、1.36(2H, m, H-12)和0.91(3H, t, 4.0 Hz, H-13)], 且只存在一个酚羟基质子信号[δH 11.67(1H, s, 6-OH)], 可推测结构中直链片段的8位与苯环的3位形成了吡喃环。将化合物9的NMR数据与2-(2',3-epoxy-1'-heptenyl)-6- hydroxy-5-(3°°-methyl-2°°-butenyl) benzaldehyde(Li et al, 2008)对比, 两者基本一致, 故确定化合物9为2-(2°,3-epoxy-1°-heptenyl)-6-hydroxy-5-(3°°-methyl- 2°°-butenyl) benzaldehyde。
本文体外克服肿瘤耐药活性研究的结果表明, 苯甲醛部位在浓度为200ug·mL-1时, 其对吉非替尼耐药肺癌细胞株HT1975/GR的抑制率为58.19%。而单体化合物在浓度为50μg·mL-1时, 仅化合物3和9表现出对吉非替尼耐药肺癌细胞株HT1975/GR有抑制作用, 抑制率分别为62.66%和93.71%。这可能是由于化合物9的结构2位侧链与3羟基形成的吡喃环大大地提高了其克服吉非替尼耐药肺癌细胞株HT1975/GR的活性, 故具有一定新颖性。文献调研表明(Inoue et al, 1997), 化合物2对产气杆菌Enterobacter aerogenes、大肠杆菌Escherichia coli、枯草芽孢杆菌Bacillus subtilis具有弱的抗菌活性, 且具有弱的抗肿瘤活性。此外, 化合物1、2、3、5、6、8清除DPPH自由基的IC50值分别为17.8、10.5、18.0、9.6、19.6、7.6μmol·L-1, 阳性对照VC(维生素C)的IC50值为21.0μmol·L-1(黄玉玲 等, 2012), 提示它们具有抗氧化活性, 并且如果侧链与苯环形成共轭体系, 可能会明显提高化合物清除自由基的活性。

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