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Journal of Tropical Oceanography >

2023 , Vol. 42 >Issue 5: 154 - 160

DOI: https://doi.org/10.11978/2022232

Exploitation of Marine Resources

Study on the secondary metabolites of fungus Aspergillus sp. GXIMD02003 derived from marine sediment in the Weizhou island

  • XING Nannan , 1, 2 ,
  • REN Runxin 1, 3 ,
  • TANG Zhenzhou 1, 3 ,
  • LUO Zhihong 1, 3 ,
  • XIA Chenxi 1, 3 ,
  • LIU Yonghong 1, 3 ,
  • PENG Liang , 4 ,
  • CHEN Xianqiang , 1, 3
Expand
  • 1. Institute of Marine Drugs, Guangxi University of Chinese Medicine, Nanning 530200, China
  • 2. Guangxi Key Laboratory of Chinese Medicine Foundation Research, Guangxi University of Chinese Medicine, Nanning 530200, China
  • 3. Guangxi Key Laboratory of Marine Drugs, Guangxi University of Chinese Medicine, Nanning 530200, China
  • 4. Engineering Center for Fine Chemicals, School of Pharmacy, Jiangxi Science & Technology Normal University, Nanchang 330013, China
CHEN Xianqiang. email: ;
PENG Liang. email:

Received date: 2022-11-01

  Revised date: 2022-12-30

  Online published: 2023-01-03

Supported by

Guangxi Science and Technology Base and Talent Special Project(AD21075016)

Natural Science Foundation of Guangxi Province(2020GXNSFAA297163)

Natural Science Foundation of Guangxi Province(2020GXNSFGA297002)

Special Fund for Bagui Scholars of Guangxi(05019055)

Open Project of Engineering Center of Jiangxi University for Fine Chemicals(JFCEC-KF-2101)

Special Scientific Research Fund for Team of Institute of Marine Drugs of Guangxi University of Chinese Medicine(2018ZD005-A02)

Project of Guangxi Key Laboratory of Chinese Medicine Foundation Research(22-065-53-01)

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Abstract

To explore the diversity of secondary metabolites of fungus Aspergillus sp. GXIMD02003 derived from marine sediment, its secondary metabolites were separated based on the various chromatographic techniques. Chemical structures were identified using mass spectroscopy, nuclear magnetic resonance spectroscopy, and optical rotation combined with comparison of literature data. Eight compounds were isolated from Aspergillus sp. GXIMD02003, and identified as (3S, 11aS)-3-[(1H-indol-3-yl)methyl]-7, 9-dihydroxy-8-methoxy-2,3,11,11a-tetrahydro-6H-pyrazino[1,2-b]isoquinoline-1,4-dione (1), cyclo(L-Pro-L-Tyr) (2), cyclo(L-4-Hyp-D-phe) (3), cyclo(L-4-Hyp-L-Phe) (4), cyclo(L-4-Hyp-L-leucine) (5), cyclo(L-4-Hyp-D-leucine) (6), desmethyldiaportinol (7), 2-(2ʹ-hydroxypropyl)-5-methyl-7-hydroxychromone (8). Compounds 1~8 showed no inhibitory activities against cancer cell proliferation, α-glucosidase and pathogenic bacteria at testing concentration. Compounds 1~8 were isolated from fungus derived from marine sediment of the Weizhou island for the first time. The project enriched the diversity of secondary metabolites from Aspergillus sp. GXIMD02003, laid a foundation for exploring the bioactivity of fungal secondary metabolites from marine sediment in the Weizhou island.

Key words： marine fungus; Aspergillus; secondary metabolites; marine sediment; Weizhou Island

Cite this article

XING Nannan , REN Runxin , TANG Zhenzhou , LUO Zhihong , XIA Chenxi , LIU Yonghong , PENG Liang , CHEN Xianqiang . Study on the secondary metabolites of fungus Aspergillus sp. GXIMD02003 derived from marine sediment in the Weizhou island[J]. Journal of Tropical Oceanography, 2023 , 42(5) : 154 -160 . DOI: 10.11978/2022232

海洋沉积物占地球表面积的三分之二以上, 其中的微生物类群主要含有细菌、古菌和真菌等, 这些微生物类群对于地球的碳和氮循环具有重要的作用(Baker et al, 2021)。由于海洋沉积物中的微生物长期共存并相互竞争生产空间, 真菌相对丰度显著低于细菌和古菌, 但是这也导致海洋沉积物来源真菌产生结构新颖、活性良好的次级代谢产物。海洋沉积物来源真菌是活性物质的重要生产源, 在2016至2020年间海洋沉积物来源真菌中报道了246个次级代谢产物, 具有抗病毒、抗菌、抗炎、细胞毒等多种生物活性, 主要包括聚酮、生物碱、萜类等结构类型, 其中41%的次级代谢产物来自曲霉Aspergillus (Yurchenko et al, 2021)。海洋来源曲霉Aspergillus的次级代谢产物具有丰富的化学多样性和重要的药用价值, 是天然衍生候选药物的宝藏。据统计, 在2015至2020年间从海洋来源曲霉Aspergillus中分离的361个次级代谢产物具有抗癌、抗炎、抗菌、抗病毒、细胞毒性、杀虫、神经保护和抗氧化等活性, 包括聚酮、甾醇、生物碱、萜类、多肽、丁烯内酯等结构类型(Orfali et al, 2021)。广西涠洲岛是火山喷发堆凝而成的火山岛, 具有极为特殊的地貌和生态环境, 引起了学者们对该地区真菌代谢产物的关注。从涠洲岛海洋沉积物来源真菌Aspergillus carbonarius WZ-4-11中分离得到两个新化合物(carbonarone A 和 carbonarone B), 对人类白血病细胞系K562有细胞毒作用(Zhang et al, 2018)。Zhang等(2018)从涠洲岛采集的海绵共附生青霉菌Penicillium brasilianum中发现了6个新的混源萜brasilianoids A-F, brasilianoid A促进聚丝蛋白和caspase-14表达, brasilianoids B和C具有抗炎作用。王长云教授课题组从涠洲岛的珊瑚共附生真菌分离得到了一系列具有抗病毒、抗肿瘤作用的甾醇和酚酸类化合物(Yu et al, 2018; Zhao et al, 2019)。虽然涠洲岛的海洋来源真菌次级代谢产物受到一定的关注, 但其沉积物真菌次级代谢产物研究较少被报道。
我们前期从涠洲岛海洋沉积物中发现一株曲霉Aspergillus sp. GXIMD02003是高产曲酸的菌株, 并优化了其发酵曲酸的条件(梁芳萍 等, 2020)。为了进一步探索该菌株次级代谢产物的多样性, 采用单菌多次级代谢产物(one-strain-many-compounds, OSMAC)策略发现该菌株在真菌4号培养基中发酵有较丰富代谢产物。因此, 本文以Aspergillus sp. GXIMD02003为研究对象, 采用各种色谱技术分离该菌株的次级代谢产物以及质谱、核磁共振波谱等技术鉴定代谢产物的结构, 拟探讨该菌株次级代谢产物的多样性, 为从涠洲岛海洋沉积物来源真菌中发现活性物质奠定基础。

1 试验部分

1.1 仪器与试剂

Avance Ⅲ HD 500M超导核磁共振波谱仪(Bruker公司); Waters Xevo G2-S 型高分辨ESI质谱(Waters公司); LC-2030C 3D Plus型高效液相色谱仪(日本岛津公司); EYELA N-1300D型旋转蒸发仪(东京理化器械株式会社); sepacore型中压制备色谱仪(瑞士Buchi公司); SHZ-CB型循环水真空泵(巩义市予华仪器有限公司); KQ-250DB型超声仪(巩义市予华仪器有限公司); MCP 100/150旋光仪(安东帕公司); Victor Nivo多功能酶标仪(珀金埃尔默); Digipol-M70全自动熔点仪(上海佳航仪器仪表有限公司); 培养箱(上海精宏实验设备有限公司); 薄层色谱硅胶、100~200目硅胶和薄层硅胶板均购于烟台江友硅胶开发有限公司; 反相硅胶(YMC公司); 氯仿、乙酸乙酯、二甲基亚砜(DMSO)等分析纯试剂均购于广东光华科技股份有限公司。

1.2 菌株来源与发酵培养

菌株采集于广西涠洲岛的海洋沉积物, 鉴定为Aspergillus sp. (梁芳萍 等, 2020)。菌株保藏于广西中医药大学海洋药物研究院, 编号为GXIMD02003。
试验用培养基包括MB培养基: 麦芽提取物 15g, 海盐 30g, 蒸馏水 1000mL, 固体培养基加15~20g琼脂; 真菌4号培养基: 甘露醇 20g, 葡萄糖 20g, 蛋白胨10g, 酵母膏 5g, 玉米浆 1g, 七水合硫酸镁 0.3g, 磷酸二氢钾 0.5g、海盐 25g, 蒸馏水 1000mL, pH 7.4~7.6。
将保藏于4℃的菌株接种于MB固体培养基, 置于28℃下培养5d。取适量的孢子接种至MB培养液中, 在25℃, 180r·min-1下振摇培养4d。将种子液接种到真菌4号培养基中, 每瓶接种3mL, 共50瓶, 25℃下静置培养30d。

1.3 提取与分离

液体培养基发酵完毕后, 经纱布过滤得到发酵液和菌丝体。菌丝体用丙酮超声提取3次, 每次20min, 合并提取液, 减压回收溶剂, 得到浸膏。用水混悬浸膏, 乙酸乙酯萃取3次, 合并萃取液, 减压回收乙酸乙酯, 得到萃取物(31g)。中压硅胶柱色谱分离萃取物, 二氯甲烷-甲醇梯度洗脱(100:0 → 0:100, v/v), 根据TLC分析, 合并得到6个组分(Fr.1 ~ Fr.6)。Fr. 2 (8.60g)经ODS柱纯化, 甲醇-水梯度洗脱(50:50 → 100:0, v/v )得7个组分(Fr.2.1 ~ Fr.2.7)。Fr.2.1 (4.27g)经过ODS柱分离(v甲醇:v水=30:70 ~ 100:0), 再经过高效液相色谱制备(v乙腈:v水 = 23:77)得到化合物2 (7.6mg)、4 (223.4mg)和5 (46.9mg)。Fr.2.3经过凝胶柱纯化以及高效液相色谱制备(v乙腈:v水=25:75)得到化合物8 (9.7mg)。ODS柱分离Fr.3 (1.0g), 甲醇-水梯度洗脱(30:70 → 100:0, v/v )得到组分Fr.3.1 ~ Fr.3.5。Fr.3.3 (778.0mg)经高效液相色谱制备(v乙腈:v水=10:90)得到化合物3 (3.7mg)和6 (33.7mg)。Fr.4 (6.7g)经ODS柱纯化, 甲醇-水梯度洗脱(v甲醇:v水=20:80 → 100:0)得到6个组分(Fr.4.1 ~ Fr.4.6)。Fr.4.6 (389.5mg)经凝胶柱纯化以和硅胶色谱柱分离(v二氯甲烷:v丙酮=2:1)得到3个组分(Fr.4.6.1 ~ Fr.4.6.3)。Fr.4.6.1经过高效液相色谱制备(v乙腈:v水=25:75)得到化合物7 (27.0mg); Fr.4.6.3经过高效液相色谱制备(v乙腈:v水=24:76)得到化合物1 (21.1mg)。化合结构见图1。
图1 来源于Aspergillus sp. GXIMD02003的化合物1~8

Fig. 1 Compounds 1~8

1.4 细胞增殖抑制试验

试验参照文献方法(陈显强 等, 2020), 样品用DMSO溶解配置成初始浓度为20mmol·L-1母液, 测试时稀释至所需浓度的工作液。胰腺癌细胞SW1990和PNAC1、结直肠癌细胞DLD-1、肝癌细胞Bel-7402在对数期分别接种至96孔板中, 37℃孵育过夜, 加入各测试化合物, 0.2% DMSO为阴性对照组, 设3个复孔。培养48h后, 每孔加入10μL CCK-8 (Cell Counting Kit-8)试剂, 置于37℃培养箱中放置 2h。用微孔板酶标仪读数, 测定波长为450nm, 计算抑制率。

1.5 抑菌试验

采用滤纸片琼脂扩散法测试化合物的抑菌活性。化合物用DMSO溶解配成50mg·mL-1母液。阳性对照为氨苄青霉素钠和环丙沙星溶于DMSO配成0.1mg·mL-1母液。DMSO作为阴性对照。吸取3µL待测样品到直径为6mm的无菌滤纸片上, 载样滤纸片贴于有指示菌的LB平板上, 37℃培养16h。采用十字交叉法记录抑菌圈的直径。

1.6 α-葡萄糖酶抑制试验

试验参照文献方法(Chen et al, 2018), 化合物用10% DMSO溶解配成900μg·mL-1母液。阳性药物阿卡波糖用PBS缓冲液溶解配置成1mg·mL-1母液。试验在96孔板中执行, 每孔加入α-葡萄糖苷酶(0.2U·mL-1)、4-硝基苯-α-D-吡喃葡萄糖苷(2.5mmol·L-1)、被测化合物各20μL, 对照孔中用PBS缓冲液溶代替α-葡萄糖苷酶, 空白孔中用PBS缓冲液溶代替被测化合物, 设3个复孔。37℃, 孵育15min, 每孔加入80μL Na2CO3 (0.2mol·L-1)终止反应, 测定405nm处OD值。

2 试验结果

2.1 结构鉴定

化合物1: 红褐色粉末, 分子式为C22H21N3O5。HRESIMS m/z 408.1562 [M+H]+, $\left[ \alpha \right]_{\mathrm{D}}^{20}$-124.7° (c 0.1, MeOH), 熔点189.1~191.5℃。1H NMR (500MHz, CD3OD): δH 7.46(1H, d, J=8.0Hz, H-16), 7.20(1H, d, J=8.0Hz, H-19), 6.90(1H, td, J=9.0, 1.5Hz, H-17), 6.81(1H, td, J=9.0, 1.0Hz, H-18), 6.92(1H, s, H-14), 5.68(1H, s, H-10), 5.16(1H, d, J=17.0Hz, H-6a), 4.36(1H, t, J=17.0Hz, H-3), 3.78(1H, dd, J=12.0, 3.5Hz, H-11a), 3.69(1H, d, J=17.0Hz, H-6b), 3.72(3H, s, H-20), 3.47(1H, dd, J=15.0, 3.5Hz, H-12a), 3.11(1H, dd, J=15.0, 4.0Hz, H-12b), 2.24(1H, dd, J=16.0, 4.0Hz, H-11), 0.58(1H, t, J=13.0Hz, H-11); 13C NMR (125MHz, CD3OD): δC 168.7(C-1), 166.5(C-4), 149.8(C-9), 147.8(C-7), 137.6(C-15a), 135.0(C-8), 129.1(C-10a), 129.0(C-19a), 125.5(C-14), 122.5(C-18), 120.1(C-16), 119.7(C-17), 112.0(C-19), 110.9(C-6a), 109.1(C-13), 107.4(C-10), 60.9(8-OCH3), 57.7(C-3), 56.5(C-11a), 41.1(C-6), 33.1(C-11), 31.4(C-12); 13C NMR (125MHz, DMSO-d6): δC 165.5(C-1), 164.2(C-4), 148.6(C-9), 146.5(C-7), 135.7(C-15a), 133.8(C-8), 127.8(C-10a), 127.8(C-19a), 124.2(C-14), 120.9(C-18), 118.80(C-16), 118.35(C-17), 111.00(C-19), 110.05(C-6a), 108.19(C-13), 106.36(C-10), 59.94(8-OCH3), 55.7(C-3), 54.57(C-11a), 39.8(C-6), 32.16(C-11), 29.9(C-12)。上述数据与文献(Lin et al, 2008)报道基本一致, 化合物1鉴定为(3S,11aS)-3-[(1H-indol-3-yl)
methyl]-7,9-dihydroxy-8-methoxy-2,3,11,11a-tetrahydro-
6H-pyrazino[1,2-b]isoquinoline-1,4-dione。
化合物2: 白色粉末, 分子式为C14H16N2O3。HRESIMS m/z 261.1241 [M+H]+, $\left[ \alpha \right]_{\mathrm{D}}^{20}$-36.3° (c 0.1, MeOH), 熔点157.1~158.4℃。1H NMR (500MHz, CDCl3): δH 7.05(2H, d, J=8.4Hz, H-11, 15), 6.78(2H, d, J=8.5Hz, H-12, 14), 5.83(1H, s, H-8), 4.21(1H, dd, J=3.0, 10.0Hz, H-7), 4.08(1H, t, J=7.5Hz, H-2), 3.59(2H, m, H-5), 3.47(2H, dd, J=14.5, 4.0Hz, H-9a), 2.76(1H, dd, J=14.5, 4.0Hz, H-9b), 2.33(1H, m, H-3a), 2.00(1H, m, H-3b), 1.92(2H, m, H-4); 13C NMR (125MHz, CDCl3): δC 169.7(C-6), 165.2(C-1), 155.6(C-13), 130.3(C-11, 15), 127.0(C-10), 116.2(C-12, 14), 59.2(C-2), 56.3(C-7), 45.5(C-3), 36.0(C-9), 28.3(C-5), 22.5(C-4)。上述数据与文献(Jayatilake et al, 1996)报道基本一致, 化合物2鉴定为环-(L-脯氨酸-L-酪氨酸)[cyclo(L-Pro-L-Tyr)]。
化合物3: 白色粉末, 分子式为C14H16N2O3。HRESIMS m/z 261.1240 [M+H]+, $\left[ \alpha \right]_{\mathrm{D}}^{20}$ +32.4° (c 0.1, MeOH), 熔点158.6~159.8℃。1H NMR (500MHz, CD3OD): δH 7.29(3H, m, H-11, 13, 15), 7.17(2H, dd, J=3.0, 7.5Hz, H-12, 14), 4.21(2H, m, H-7, 4), 3.59(1H, dd, J=12.5, 4.0Hz, H-5a), 3.27(3H, dd, J=12.5, 6.0Hz, H-5b), 3.18(1H, dd, J=14.0, 5.0Hz, H-9a), 2.99(1H, dd, J=14.0, 5.0Hz, H-9b), 2.76(1H, t, J=8.5Hz, H-2), 2.22(1H, m, H-3a), 1.90(1H, m, H-3b); 13C NMR (125MHz, CD3OD): δC 171.0(C-1), 167.7(C-6), 136.8(C-10), 131.2(C-12, 14), 129.7(C-11, 15), 128.5(C-13), 68.4(C-4), 59.6(C-2), 57.2(C-5), 54.0(C-7), 40.9(C-9), 38.0(C-3)。上述数据与文献(Chen et al, 2015)报道基本一致, 化合物3鉴定为环-(L-4-羟基脯氨酸-D-苯丙氨酸)[cyclo(L-4-Hyp-D-phe)]。
化合物4: 白色粉末, 分子式为C14H16N2O3。HRESIMS m/z 261.1239 [M+H]+, $\left[ \alpha \right]_{\mathrm{D}}^{20}$-49.6° (c 0.1, MeOH), 熔点156.4~158.2℃。1H NMR (500MHz, CD3OD): δH 7.26(5H, m, Ar-H), 4.50(1H, dt, J=5.5, 2.0Hz, H-7), 4.38(1H, dq, J=6.0, 2.0Hz, H-2), 4.29(1H, t, J=5.0Hz, H-4), 3.72(1H, dd, J=13.0, 5.0Hz, H-5a), 3.30(1H, d, J=13.0Hz, H-5b), 3.18(2H, m, H-9), 2.08(1H, dd, J=14.0, 6.0Hz, H-3a), 1.38(1H, ddd, J=13.0, 4.5, 1.5Hz, H-3b); 13C NMR (125MHz, CD3OD): δC 171.3(C-1), 167.1(C-6), 137.4(C-10), 131.0(C-11, 15), 129.4(C-12, 14), 128.1(C-13), 68.5(C-4), 58.3(C-2), 57.6(C-7), 55.2(C-5), 38.9(C-3), 38.0(C-9)。上述数据与文献(Xiang et al, 2020)报道基本一致, 化合物4鉴定为环-(L-4-羟基脯氨酸-L-苯丙氨酸)[cyclo(L-4-Hyp-L-Phe)]。
化合物5: 白色粉末, 分子式为C11H18N2O3。HRESIMS m/z 227.1400 [M+H]+, $\left[ \alpha \right]_{\mathrm{D}}^{20}$ -90.1° (c 0.1, MeOH), 熔点180~181℃。1H NMR (500MHz, CD3OD): δH 4.51(1H, dd, J=11.0, 6.5Hz, H-7), 4.46(1H, t, J=4.5Hz, H-4), 4.16(1H, m, H-2), 3.65(1H, dd, J=12.5, 4.5Hz, H-5a), 3.42(1H, d, J=12.5Hz, H-5b), 2.27(1H, dd, J=6.5, 12.5Hz, H-3a), 2.08(1H, m, H-3a), 1.89(2H, m, H-9a, 10), 1.51(1H, m, H-9b), 0.94(3H, d, J=6.5Hz, H-11), 0.95(3H, d, J=6.5Hz, H-12); 13C NMR (125MHz, CD3OD): δC 173.1(C-6), 169.0(C-1), 69.1(C-4), 58.7(C-7), 55.2(C-2), 54.6(C-5), 39.4(C-9), 38.2(C-3), 25.8(C-10), 23.3(C-11), 22.2(C-12)。上述数据与文献(Cronan et al, 1998)报道的基本一致, 化合物5鉴定为环-(L-4-羟基脯氨酸-L-亮氨酸)[cyclo(L-4-Hyp-L-leucine)]。
化合物6: 白色粉末, 分子式为C11H18N2O3。HRESIMS m/z 227.1390 [M+H]+, $\left[ \alpha \right]_{\mathrm{D}}^{20}$ +56.7° (c 0.1, MeOH), 熔点183.1~184.4℃。1H NMR (500MHz, CD3OD): δH 4.42(1H, m, H-7), 4.35(1H, t, J=8.0Hz, H-4), 3.87(1H, dd, J=9.5, 6.0Hz, H-2), 3.66(1H, dd, J=12.5, 4.2Hz, H-5a), 3.44(1H, dd, J=12.0, 4.5Hz, H-5b), 2.48(1H, m, H-3a), 2.22(1H, m, H-3b), 1.74(1H, m, H-10), 1.67(1H, m, H-9a), 1.58(1H, m, H-9b), 0.98(3H, d, J=6.5Hz, H-11), 0.97(3H, d, J=6.5Hz, H-12); 13C NMR (125MHz, CD3OD): δC 171.2(C-6), 169.4(C-1), 68.8(C-4), 57.5(C-7), 56.9(C-2), 54.2(C-5), 43.3(C-9), 37.3(C-3), 25.6(C-10), 23.3(C-11), 21.9(C-12)。上述数据与文献(Cronan et al, 1998)报道的基本一致, 化合物6鉴定为环-(L-4-羟基脯氨酸-D-亮氨酸)[cyclo(L-4-Hyp-D-leucine)]。
化合物7: 黄色油状物, 分子式为C12H12O6。HRESIMS m/z 253.0711 [M+H]+, $\left[ \alpha \right]_{\mathrm{D}}^{20}$ +63.2° (c 0.1, MeOH), 熔点178.6.3~179.5℃。1H NMR (500MHz, DMSO-d6): δH 6.44(1H, s, H-5), 6.32(1H, s, H-7), 6.27(1H, s, H-4), 3.78(1H, m, H-10), 3.29(2H, m, H-11), 2.68(1H, dd, J=12.0, 3.0Hz, H-9a), 2.36(1H, dd, J=12.0, 7.5Hz, H-9b); 13C NMR (125MHz, DMSO-d6): δC 166.3(C-6), 165.7(C-1), 162.7(C-8), 155.4(C-3), 139.7(C-4a), 105.5(C-4), 102.7(C-5), 101.5(C-7), 97.9(C-8a), 69.0(C-10), 65.4(C-11), 37.7(C-9)。上述数据与文献报道(Aly et al, 2008)基本一致, 化合物7鉴定为desmethyldiaportinol。
化合物8: 白色粉末, 分子式为C13H15O4。HRESIMS m/z 235.0900 [M+H]+, $\left[ \alpha \right]_{\mathrm{D}}^{20}$ -14.3° (c 0.1, MeOH), 熔点205.3~206.8℃。1H NMR (500MHz, CD3OD): δH 6.64(1H, d, J=3.0Hz, H-8), 6.61(1H, d, J=2.4Hz, H-6), 6.04(1H, s, H-3), 4.20(1H, m, H-2ʹ), 2.69(3H, dd, J=6.0, 15.6Hz, H-CH3), 2.62(2H, dd, J=9.6, 16.8Hz, H-1ʹ), 1.26(3H, d, J=7.2Hz, H-CH3); 13C NMR (125MHz, CD3OD): δC 182.0(C-4), 167.1(C-2), 163.1(C-7), 161.5(C-4a), 143.6(C-5), 118.0(C-3), 115.8(C-8a), 112.5(C-6), 101.7(C-8), 66.3(C-10), 44.2(C-9), 23.5(C-11), 23.2(C-12)。上述数据与文献(Kashiwada et al, 1984)报道基本一致, 化合物8鉴定为2-(2ʹ-羟基丙基)-5-甲基-7-羟基色酮[2-(2ʹ-hydroxypropyl)-5-methyl-7-hydroxychromone]。

2.2 活性测试结果

化合物1~8均做了细胞毒、抗菌和α-葡萄糖酶抑制活性测试。所有化合物在40μmol·L-1浓度下对胰腺癌细胞SW1990和PNAC1、结直肠癌细胞DLD-1、肝癌细胞Bel-7402无增殖抑制作用; 在150μg·片-1给药量下对耐甲氧西林金黄葡萄球菌Methicillin-resistant Staphylococcus aureus、铜绿假单孢菌Pseudomona aeruginosa、大肠杆菌Escherichia coli、表皮葡萄球菌Staphylococcus epidermidis、粘性放线菌Actinomyces viscosus、藤黄微球菌Micrococcus luteus、枯草芽孢杆菌Bacillus subtilis、金黄葡萄球菌Staphylococcus aureus、肺炎克雷伯菌Klebsiella pneumoniae和鲍曼不动杆菌Acinetobacter baumannii的生长无抑制作用; 在300μg·mL-1浓度下对α-葡萄糖酶未显示抑制作用。

3 讨论与结论

本文从海洋沉积物来源真菌Aspergillus sp. GXIMD02003中分离并鉴定了到8个化合物, 包括6个2,5-二酮哌嗪类化合物(1~6), 1个异香豆素类化合物(7)和一个色酮类化合物(8)。化合物1~8均首次从涠洲岛海洋沉积物中报道。
化合物1~6是含有2,5-二酮哌嗪六元环重要药效基团的二酮哌嗪类化合物, 该类化合物具有抗肿瘤、抗病毒、抗炎、抗菌、抗氧化等广泛的生物活性(Borthwick, 2012; de Carvalho et al, 2012; Song et al, 2021), 尤其含有吲哚或脯氨酸基团的2,5-二酮哌嗪类化合物具有作为药物或药物先导化合物的潜力(Ma et al, 2016; Bojarska et al, 2021)。根据文献报道, 化合物1对早幼粒白血病细胞HL-60表现出较弱的细胞毒活性, IC50值为36.5μg·mL-1 (Lin et al, 2008), 但在40μg·片-1给药量下对多个致病细菌无抑制作用(Shaaban et al, 2014)。化合物2从细菌Pseudomonas fluorescens GcM5-1A (Guo et al, 2007)、Pseudomonas aeruginosa (Jayatilake et al, 1996)、放线菌Streptomyces sp. ML 1532 (Munekata et al, 1981)、溶杆菌Lysobacter capsici AZ78 (Puopolo et al, 2014)等微生物中被分离报道, 对日本黑松的悬浮细胞和幼苗有毒性, 选择性抑制SV40转化细胞系的增殖, 具有抗植物病源真菌生物活性, 但对人致病细菌无抑制作用。化合物3和4是一对手型异构体, 从海绵共附生真菌Eupenicillium brefeldianum HMP-F96中被分离得到, 化合物3由于含有D-苯丙氨酸基团能够显著地诱导烟草悬浮细胞产生H2O2, 而化合物4含有L-苯丙氨酸基团无显著活性(Chen et al, 2015)。化合物5和6从海绵共附生细菌中被分离, 能够促进植物生长和抗旱(Cronan et al, 1998)。化合物4和5从野桐属植物Mallotus nudiflorus内生细菌中被分离, 在100μg·片-1给药量下未显示抑制细菌生长作用(Xiang et al, 2020)。虽然二酮哌嗪类化合物具有丰富多样的显著生物活性, 但是我们未检测到化合物1~6的生物活性, 与前人研究的部分结果一致。这些化合物无被测生物活性的主要原因可能是由于氨基酸组成或氨基酸的立体构型差异导致。
2,5-二酮哌嗪类化合物广泛存在于细菌、真菌、海绵和高等生物中, 其生物合成与非核糖体肽合成酶或环二肽合成酶密切相关(Harken et al, 2021), 但其起源也被质疑为源于微生物培养基成分经非酶催化而形成。为了考察本研究的二酮哌嗪类化合物是否是由真菌Aspergillus sp. GXIMD02003产生, 我们运用HPLC分析了未接菌种的培养基提取物, 未检测到这些二酮哌嗪类化合物, 因此, 我们认为化合物1~6是来源于真菌Aspergillus sp. GXIMD02003的代谢产物。
OSMAC策略通常采用改变培养基、混合培养、添加酶抑制剂和基因调控等方法改变微生物的代谢途径, 在挖掘海洋微生物的代谢产物多样性方面是一种有效且操作简便的方法(Pan et al, 2019)。本文基于该策略发现真菌Aspergillus sp. GXIMD02003在真菌4号培养基中发酵可以产生丰富代谢产物。本研究表明不同培养基激活了真菌Aspergillus sp. GXIMD02003不同的代谢途径并产生不同的结构类型化合物, 增加了真菌Aspergillus sp. GXIMD02003次级代谢产物的多样性, 丰富了涠洲岛海洋沉积物来源真菌次级代谢产物的研究。

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