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Journal of Tropical Oceanography >

2024 , Vol. 43 >Issue 4: 174 - 180

DOI: https://doi.org/10.11978/2023122

Exploitation of Marine Resources

Study on the secondary metabolites from the Weizhou Island coral Acropora austera associated fungus Arachniotus ruber GXIMD 02510 and their antibacterial activity

  • WANG Jiaxi ,
  • LU Humu ,
  • QI Xin ,
  • GAO Chenghai ,
  • LIU Yonghong ,
  • LUO Xiaowei
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  • Institute of Marine Drugs, Guangxi University of Chinese Medicine, Guangxi Key Laboratory of Marine Drugs, Nanning 530200, China
LUO Xiaowei. email:

Copy editor: SUN Cuici

Received date: 2023-08-16

  Revised date: 2023-09-03

  Online published: 2023-10-18

Supported by

Natural Science Foundation of Guangxi(2024GXNSFFA999005)

Natural Science Foundation of Guangxi(2021GXNSFDA075010)

National Natural Science Foundation of China(U20A20101)

High-Level Talent Training Project Foundation of Guangxi University of Chinese Medicine(202407)

High-Level Talent Training Project Foundation of Guangxi University of Chinese Medicine(2022C038)

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Abstract

The secondary metabolites and antibacterial activities of the Weizhou Island coral Acropora austera associated fungus Arachniotus ruber GXIMD 02510 were investigated. Various modern chromatographic techniques were used to isolate and purify the fermentation products of this strain. Their structures were determined by comprehensive spectroscopic methods, including nuclear magnetic resonance and by comparison with literature data. The antibacterial activity was determined. A total of 13 compounds were obtained and characterized, which were auxarthrols B, E, F, and H (1—4), paradictyoarthrin A (5), chrysoqueen (6), ocauxarthrol A (7), 1, 3, 6-trihydroxy-8-methylxanthone (8), N-acetyltyramine (9), methyl 2-(4-hydroxyphenyl)acetate (10), m-hydroxylphenylacitic acid (11), L-tenuazonic acid (12), and 2, 3, 22, 23-tertrahydroxy-2, 6, 10, 15, 19, 23-hexamethyl-tetracosa-6, 10, 14, 18-tetraene (13). Antibacterial activity test showed that compounds 1, 4—6, and 11 showed inhibitory activity against Pseudomonas aeruginosa, and the minimum inhibitory concentration (MIC) values were ranging from 0.078 mg·mL-1 to 0.312 mg·mL-1. This study will expand the chemical diversity of the genus Arachniotus and also provide chemical entities for the development of antibiotics.

Key words： Weizhou Island; Acropora austera; Arachniotus ruber; secondary metabolites; antibacterial activity

Cite this article

WANG Jiaxi , LU Humu , QI Xin , GAO Chenghai , LIU Yonghong , LUO Xiaowei . Study on the secondary metabolites from the Weizhou Island coral Acropora austera associated fungus Arachniotus ruber GXIMD 02510 and their antibacterial activity[J]. Journal of Tropical Oceanography, 2024 , 43(4) : 174 -180 . DOI: 10.11978/2023122

海洋微生物长期栖息于复杂独特的生态环境中, 如高盐、高压、低温(局部高温)、低氧、黑暗等, 适应并进化出了独特的生理代谢机制, 易产生结构新颖、活性优良的次级代谢产物, 近年来受到了天然产物化学家的广泛关注 (Carroll et al, 2023)。近年来从海洋微生物中发现新的海洋天然产物数量呈明显的上升趋势, 结构类型涵盖了萜类、甾醇类、生物碱类、甙类、多糖、肽类等, 具有广谱显著的生物活性(马丽丽 等, 2021)。珊瑚体内外共附生大量的海洋微生物, 其产生的次级代谢产物构成了珊瑚重要的化学防御机制。珊瑚共附生真菌次级代谢产物结构丰富多样, 包括萜类、生物碱、多肽、酚酸类、内酯类和甾体等, 具有抗肿瘤、抗菌、抗污损等生物活性, 是药物先导化合物或海洋候选药物的重要来源 (Chen et al, 2022)。鹿角珊瑚属于造礁珊瑚, 是珊瑚礁生态系统中主要的建筑师, 其内附生有大量的微生物且与珊瑚共生藻协同合作维持着寡营养条件下珊瑚礁的高生产力水平, 2022年Wu Qiaoling等人从南海海域分离的鹿角珊瑚 (Acropora austera)和珊瑚沙中, 分离出4株放线菌, 并对菌株Streptomyces diacarni SCSIO 64983的次级代谢产物进行分离鉴定, 得到了4个新颖的halocarbazomycins类化合物, 进一步丰富了鹿角珊瑚来源微生物次级代谢产物 (Wu et al, 2022)。
北部湾是中国南海西北部的半封闭海湾, 拥有珊瑚礁、红树林、海草床、滨海湿地等4种典型海洋生态系统, 蕴育着极为丰富的海洋(微)生物资源。近期本团队首次全面系统地总结了2003年至2022年期间报道的477个北部湾来源海洋天然产物, 围绕其来源、化学结构和生物活性等方面进行重点介绍和阐释。这些海洋天然产物主要来源于海洋药用宏生物(48%)及其微生物(52%), 包括聚酮类(43%)、萜类(40%)、含氮类(12%)和苷类(5%), 具有细胞毒、抗菌和抗炎等生物活性 (Wang et al, 2023), Arachniotus ruber由Van Tieghem于1877年首次发现 (Kuehn et al, 1964), 在1977年Orr Ghosh和Roy将该物种归入Pseudoarachniotus属 (Orr et al, 1977), 此前有研究者从部分动物体内分离得到该菌株(Ogórek et al, 2020), 但是关于该菌株次级代谢产物研究报道稀少。
本团队长期从事海洋微生物来源活性代谢产物发掘研究, 近期从南海礁栖海藻共附生真菌 Pestalotiopsis neglecta SCSIO 41403中分离得到了6个聚酮类化合物, 其中化合物7 - hydroxy - 5 - methoxy - 4, 6 - dimethylphthalidcdui对白色念珠菌有明显抑制活性 (叶禹秀 等, 2022)。最近团队从涠洲岛鹿角珊瑚新鲜组织中分离得到一株真菌Arachniotus ruber GXIMD 02510, 从其发酵产物中获得以auxarthrol类蒽醌衍生物为主的化合物13个(图1), 即auxarthrols B, E, F和H (1—4)、paradictyoarthrin A (5)、chrysoqueen (6)、ocauxarthrol A (7)、1, 3, 6-三羟基-8-甲基占吨酮 (8)、N-乙酰酪胺 (9)、对羟基苯乙酸甲酯 (10)、间羟基苯乙酸 (11)、l-tenuazonic acid (12)和2, 3, 22, 23-tertrahydroxy-2, 6, 10, 15, 19, 23-hexamethyl-tetracosa-6, 10, 14, 18-tetraene (13)。对所有化合物进行抗MRSA (methicillin resistant Staphylococcus aureus)、铜绿假单胞菌 (Pseudomonas aeruginosa)和表皮葡萄球菌 (Staphylococcus epidermidis)等致病菌抑制活性研究, 结果显示化合物1、4—6和11具有一定的抗铜绿假单胞菌活性。
图1 化合物1—13的化学结构

Fig. 1 Chemical structures of compounds 1—13

1 材料和方法

1.1 仪器试剂及测试病原菌

超净工作台(SW-CJ-2F型, 苏州安泰空气技术有限公司); 中压制备色谱仪(瑞士Buchi公司); 旋转蒸发仪(Eyelan-1100V-W型, 日本东京理化株式会社); 高效液相色谱仪(Prominence-LC2030, 日本岛津公司); 超导核磁共振波谱仪(nuclear magnetic resonance, NMR)(Avance III HD 500M, 美国Bruker公司); 薄层色谱及柱色谱用硅胶(烟台江友硅胶开发有限公司); YMC-Pack ODS-A色谱柱(10 mm×250 mm, 5μm, 日本YMC公司); 分析纯甲醇、二氯甲烷、乙酸乙酯、石油醚等化学试剂(上海泰坦科技股份有限公司); 色谱甲醇及乙腈(上海星可高纯溶剂有限公司)。
MRSA (methicillin resistant Staphylococcus aureus), 铜绿假单胞菌 (P. aeruginosa ATCC9027)和表皮葡萄球菌 (S. epidermidis ATCC 12228)等3株测试病原菌购自北京北纳创联生物技术研究院, 菌株保藏于广西中医药大学海洋药物研究院。

1.2 菌株来源及鉴定

菌株GXIMD 02510分离自广西北海涠洲岛海域来源鹿角珊瑚(Acropora austera)的新鲜组织, 鹿角珊瑚于2020年6月采集自广西北部湾涠洲岛海域, 由刘昕明副研究员鉴定为鹿角珊瑚(Acropora austera)。菌株保藏于广西中医药大学海洋药物研究院。利用PCR技术对菌株的18S rDNA进行扩增测序[生工生物工程(上海)股份有限公司], 通过Blast分析初步将其鉴定为Arachniotus ruber GXIMD 02510 (Genbank编号: OR077406)。

1.3 菌株的培养

将菌株接种至MB种子液(麦芽提取粉15g, 海盐15g, 蒸馏水1000mL, pH 7.4~7.8), 然后放置在28℃摇床培养箱(180r·min-1)中动态培养3d, 培养结束后, 然后将种子液接种至已经高压蒸汽灭菌(121℃, 20min)的大米固体培养基(1000mL三角瓶: 大米150g, 海盐3.6g, 蒸馏水180mL), 在28℃静置培养发酵45d, 共计接种100瓶, 合计发酵大米15kg。

1.4 发酵产物的提取分离

利用乙酸乙酯对大米发酵产物进行反复萃取。减压蒸馏得到粗浸膏598g。经中压正相硅胶柱色谱分离, 采用梯度洗脱(石油醚/CH2Cl2, 体积比100:0~0:100; CH2Cl2/CH3OH, 体积比100:0~1:1), 根据薄层色谱分析后合并得到10个流份。流份Fr.6和Fr.10进行进一步分离。Fr.6经中压反相硅胶柱色谱分离, 采用梯度洗脱(甲醇/水, 体积比10:90~100:0)得到13个流份, 其中流份Fr.6-5采用半制备高效液相色谱(CH3CN/H2O, 体积比63:37, 2mL·min−1)分离得到化合物12 (tR=37min, 25mg)。流份Fr.10经中压反相硅胶柱色谱分离, 采用梯度洗脱(甲醇/水, 体积比10:90~100:0)得到9个流份。
流份Fr.10-1经中压正相硅胶柱色谱分离, 采用梯度洗脱(CH2Cl2/CH3OH, 体积比100:0~1:1)薄层色谱分析后合并得到13个流份, 其中的流份Fr.10-1-3采用半制备高效液相色谱(CH3CN/H2O, 体积比60:40, 5mL·min-1)分离得到流份Fr.10-1-3-2 (tR=16min, 75.8mg)。流份Fr.10-1-3-2继续采用半制备高效液相色谱(CH3CN/ H2O, 体积比35:65, 2mL·min-1)纯化得到化合物8 (tR=25 min, 34.6mg)和化合物5 (tR=48min, 14.7mg)。流份Fr.10-1-6采用半制备高效液相色谱(CH3CN/H2O, 体积比32:68, 2mL·min-1)分离得到化合物10 (tR=18min, 9.7mg)和流份Fr.10-1-6-5, 流份Fr.10-1-6-5继续采用半制备高效液相色谱(CH3CN/H2O, 体积比50:50, 2mL·min-1)纯化得到化合物7 (tR=21min, 15.8mg)。
流份Fr.10-1-8采用半制备高效液相色谱(CH3CN/ H2O, 体积比32:68, 2mL·min-1)分离得到化合物1 (tR= 36 min, 13.3 mg)、化合物9 (tR =16min, 9.3mg)和流份Fr.10-1-8-4, 流份Fr.10-1-8-4继续采用半制备高效液相色谱(CH3CN/H2O, 体积比32:68, 2mL·min-1)纯化得到化合物6 (tR=20min, 7.5mg)。流份Fr.10-1-9采用半制备高效液相色谱(CH3CN/H2O, 体积比70:30, 2 mL·min-1)分离得到流份Fr.10-1-9-2, 流份Fr.10-1-9-2继续采用半制备高效液相色谱(CH3CN/H2O, 体积比18:82, 2mL·min-1)纯化得到化合物2 (tR=27min, 19.4mg)、化合物3 (tR = 35 min, 4.7 mg)和化合物4 (tR = 37min, 29.3mg)。流份Fr.10-1-3采用半制备高效液相色谱(CH3CN/H2O, 体积比58:42, 2mL·min-1)分离得到化合物11 (tR = 16min, 6.3mg)。流份Fr.10-1-5采用半制备高效液相色谱(CH3CN/H2O, 体积比90:10, 5mL·min-1)分离得到化合物13 (tR=40min, 22.3mg)。

1.5 抑菌活性实验

参考本团队前期报道的实验方法 (张艳婷 等, 2022)。简言之, 采用滤纸片琼脂扩散法对所有获得的单体化合物(20mg·mL-1甲醇溶液, 每片取5μL)进行抑菌活性初步筛选, 包括MRSA, 铜绿假单胞菌和表皮葡萄球菌。针对有抑菌圈的活性化合物(1、4—6、11), 继续采用改良肉汤稀释法测定其最小抑菌浓度, 链霉素(streptomycin)作为阳性对照。

2 结果

2.1 结构鉴定

化合物1: 淡红色油状, 分子式为 C16H18O9; ECD (electronic circular dichroism) (0.25μg·mL-1, methanol) λmax(∆ε) 216 (-0.32), 237 (+8.59), 264 (-9.31), 275 (-6.72), 290 (-10.84), 340 (+8.86) nm。1H NMR (500 MHz, methanol-d4) δH: 7.05 (1H, d, J = 2.4 Hz, H-8), 6.74 (1H, d, J = 2.5 Hz, H-6), 4.72 (1H, d, J = 3.3 Hz, H-4), 3.92 (3H, s, H-12), 3.76 (1H, dd, J = 3.2, 1.3 Hz, H-3), 2.38 (1H, d, J = 15.2 Hz, H-1), 1.83 (1H, dd, J = 15.2, 1.4 Hz, H-1), 1.38 (3H, s, H-11); 13C NMR (125 MHz, methanol-d4) δC: 27.2 (CH3, C-11), 32.6 (CH2, C-1), 56.6 (CH3, C-12), 65.4 (CH, C-3), 74.3 (qC, C-2), 78.3 (CH, C-4), 81.1 (qC, C-1a), 81.2 (qC, C-4a), 106.8 (CH, C-6), 107.3 (CH, C-8), 111.6 (qC, C-10a), 137.0 (qC, C-9a), 165.7 (qC, C-5), 167.7 (qC, C-7), 194.4 (qC, C-9), 202.1 (qC, C-10)。以上数据与文献报道 (Alvi et al, 2004)的基本一致, 鉴定为auxarthrol B。
化合物2: 白色粉末, 分子式为C16H20O9; ECD (0.25μg·mL-1, methanol) λmax(∆ε) 218 (-37.88), 242 (+10.32), 257 (-5.17), 298 (+9.44) nm; HR-ESIMS m/z 355.1208 [M-H]-(calcd. for C16H19O9, 355.1209)。1H NMR (500 MHz, methanol-d4) δH: 6.77 (1H, dd, J = 2.5, 1.3 Hz, H-8), 6.35 (1H, d, J = 2.4 Hz, H-6), 4.62 (1H, d, J = 3.2 Hz, H-4), 3.85 (3H, s, H-12), 3.74 (1H, dd, J = 3.2, 1.4 Hz, H-3), 2.13 (1H, d, J = 14.8 Hz, H-1), 2.01 (1H, dd, J = 14.7, 1.5 Hz, H-1), 1.35 (3H, s, H-11); 13C NMR (125 MHz, methanol-d4) δC: 27.3 (CH3, C-11), 35.0 (CH2, C-1), 56.1 (CH3, C-12), 66.7 (CH, C-4), 71.3 (CH, C-9), 74.5 (qC, C-2), 78.4 (CH, C-3), 78.9 (CH, C-4a), 80.1 (qC, C-1a), 100.6 (CH, C-6), 107.1 (CH, C-8), 109.4 (qC, C-10a), 148.4 (qC, C-9a), 166.8 (qC, C-5), 168.4 (qC, C-7), 203.0 (qC, C-10)。以上数据与文献报道 (Ge et al, 2019)的基本一致, 鉴定为auxarthrol E。
化合物3: 浅黄色油状, 分子式为C16H20O8; ECD (0.25μg·mL-1, methanol) λmax(∆ε) 213 (-21.99), 242 (+4.57), 254 (-0.41), 284 (+13.67), 315 (-8.11), 340 (+3.41)nm。1H NMR (500 MHz, methanol-d4) δH: 6.69 (1H, dd, J = 2.5, 1.3 Hz, H-8), 6.24 (1H, d, J = 2.4 Hz, H-6), 4.54~4.44 (1H, m, H-9), 3.75 (3H, s, H-12), 3.55 (1H, dd, J = 3.0, 1.2 Hz, H-3), 2.82 (1H, d, J = 9.9 Hz, H-4a), 1.91 (1H, dd, J = 14.3, 1.3 Hz, H-1), 1.81 (1H, d, J = 14.4 Hz, H-1), 1.22 (3H, s, H-11); 13C NMR (125 MHz, methanol-d4) δC: 27.4 (CH3, C-11), 39.5 (CH2, C-1), 53.5 (CH, C-4a), 56.1 (CH3, C-12), 68.0 (CH, C-4), 74.2 (qC, C-2), 75.1 (CH, C-9), 76.6 (CH, C-3), 79.1 (qC, C-1a), 100.5 (CH, C-6), 106.8 (CH, C-8), 111.5 (qC, C-10a), 148.8 (qC, C-9a), 165.8 (qC, C-5), 168.3 (qC, C-7), 206.3 (qC, C-10)。以上数据与文献报道 (Ge et al, 2019)的基本一致, 鉴定为auxarthrol F。
化合物4: 浅黄色油状, 分子式为C16H20O8; ECD (0.25μg·mL-1, methanol) λmax(∆ε) 211 (-29.20), 243 (+5.51), 284 (+23.55), 315 (-17.16), 340 (+6.47) nm; HR-ESIMS m/z 339.1021 [M-H]- (calcd. for C16H19O8, 339.1023)。1H NMR (500 MHz, methanol-d4) δH: 6.75 (1H, dd, J = 2.5, 1.3 Hz, H-8), 6.39 (1H, d, J = 2.4 Hz, H-6), 4.77 (1H, d, J = 4.1 Hz, H-4), 3.86 (3H, s, H-12), 3.67 (1H, d, J = 4.1 Hz, H-3), 2.51 (1H, d, J = 15.4 Hz, H-1), 2.46 (1H, d, J = 15.5 Hz, H-1), 1.37 (3H, s, H-11); 13C NMR (125 MHz, methanol-d4) δC: 25.3 (CH3, C-11), 42.0 (CH2, C-1), 56.2 (CH3, C-12), 64.1 (qC, C-4a), 66.1 (CH, C-1a), 66.8 (CH, C-4), 70.4 (CH, C-9), 71.7 (qC, C-2), 74.0 (CH, C-3), 100.9 (CH, C-6), 107.3 (CH, C-8), 108.1 (qC, C-10a), 146.0 (qC, C-9a), 166.5 (qC, C-5), 168.5 (qC, C-7), 197.6 (qC, C-10)。以上数据与文献报道 (Ge et al, 2019)的基本一致, 鉴定为auxarthrol H。
化合物5: 无色油状, 分子式为C16H19ClO8; ECD (0.25μg·mL-1, methanol) λmax(∆ε) 221 (-5.52), 229 (-3.66), 235 (-4.65), 248 (+0.18), 255 (-1.16), 307 (+6.75), 337 (-1.71) nm; HR-ESIMS m/z 397.0692 [M + Na]+(calcd. for C16H19ClNaO8, 397.0666)。1H NMR (500 MHz, methanol-d4) δH: 6.81 (1H, dd, J = 2.5, 1.3 Hz, H-8), 6.41 (1H, d, J = 2.4 Hz, H-6), 5.02 - 4.96 (1H, m, H-4), 4.81 (1H, d, J = 3.6 Hz, H-9), 3.89 (3H, s, H-12), 3.80 (1H, dd, J = 3.6, 1.4 Hz, H-3), 2.24 (1H, d, J = 14.7 Hz, H-1), 2.10 (1H, dd, J = 14.7, 1.5 Hz, H-1), 1.39 (3H, s, H-11); 13C NMR (125 MHz, methanol-d4) δC: 27.4 (CH3, C-11), 33.7 (CH2, C-1), 56.2 (CH3, C-12), 66.8 (CH, C-4), 71.7 (CH, C-9), 71.8 (qC, C-4a), 74.4 (qC, C-2), 77.6 (CH, C-3), 80.1 (qC, C-1a), 100.9 (CH, C-6), 107.8 (CH, C-8), 108.6 (qC, C-10a), 147.1 (qC, C-9a), 166.7 (qC, C-5), 168.4 (qC, C-7), 198.4 (qC, C-10)。以上数据与文献报道 (Isaka et al, 2015)的基本一致, 鉴定为paradictyoarthrin A。
化合物6: 无色油状, 分子式为C17H18O10; ECD (0.25μg·mL-1, methanol) λmax(∆ε) 217 (-14.44), 242 (+5.41), 253 (-0.27), 283 (+5.69)nm。1H NMR (500 MHz, methanol-d4) δH: 6.78 (1H, dd, J = 2.5, 1.2 Hz, H-8), 6.39 (1H, d, J = 2.4 Hz, H-6), 5.42 (1H, d, J = 6.2 Hz, H-4), 3.87 (3H, s, H-12), 2.26 (1H, dd, J = 14.9, 1.7 Hz, H-1), 2.16 (1H, d, J = 14.8 Hz, H-1), 1.44 (3H, s, H-11); 13C NMR(125 MHz, methanol-d4) δC: 27.1 (CH3, C-11), 34.6 (CH2, C-1), 56.2 (CH3, C-12), 70.5 (qC, C-2), 71.1 (CH, C-9), 75.9 (CH, C-4), 76.5 (qC, C-1a), 79.5 (qC, C-4a), 83.4 (CH, C-3), 100.7 (CH, C-6), 107.1 (CH, C-8), 109.3 (qC, C-10a), 147.5 (qC, C-9a), 157.0 (qC, C-13), 166.6 (qC, C-5), 168.3 (qC, C-7), 199.6 (qC, C-10)。以上数据与文献报道 (Xu et al, 2021)的基本一致, 鉴定为chrysoqueen。
化合物7: 白色粉末, 分子式为C17H16O10; ECD (0.25μg·mL-1, methanol) λmax(∆ε) 214 (+3.53), 235 (+21.80), 264 (-21.33), 276 (-13.30), 291 (-20.48), 332 (+14.51) nm; HR-ESIMS m/z 381.0820 [M + H]+(calcd for C17H17O10, 381.0822)。1H NMR (500 MHz, methanol-d4) δH: 7.07 (1H, d, J = 2.4 Hz, H-8), 6.77 (1H, d, J = 2.5 Hz, H-6), 5.58 (1H, d, J = 6.1 Hz, H-4), 4.71 (1H, dd, J = 6.3, 1.6 Hz, H-3), 3.91 (3H, s, H-12), 2.28 (1H, d, J = 15.4 Hz, H-1), 2.16 (1H, dd, J = 15.3, 1.6 Hz, H-1), 1.46 (3H, s, H-11) ; 13C NMR (125 MHz, methanol-d4) δC: 27.1 (CH3, C-11), 32.4 (CH2, C-1), 56.7 (CH3, C-12), 70.3 (qC, C-2), 74.6 (CH, C-4), 78.1 (qC, C-1a), 80.1 (qC, C-4a), 83.1 (CH, C-3), 107.0 (CH, C-8), 107.6 (CH, C-6), 111.0 (qC, C-10a), 136.3 (qC, C-9a), 156.6 (qC, C-13), 165.8 (qC, C-7), 167.9 (qC, C-5), 193.1 (qC, C-9), 199.3 (qC, C-10)。以上数据与文献报道 (Xu et al, 2021)的基本一致, 鉴定为ocauxarthrol A。
化合物8: 淡红色油状, 分子式为C15H12O4。1H NMR (500 MHz, methanol-d4) δH: 6.67~6.56 (2H, m, H-5, H-7), 6.24 (1H, d, J = 2.2 Hz, H-4), 6.13 (1H, d, J = 2.2 Hz, H-2), 2.79 (3H, s, H-10); 13C NMR (125 MHz, methanol-d4) δC: 23.6 (CH3, C-10), 94.2 (CH, C-4), 98.8 (CH, C-2), 101.6 (CH, C-5), 104.0 (qC, C-1a), 112.9 (qC, C-8a), 117.0 (CH, C-7), 144.8 (qC, C-8), 158.6 (qC, C-1), 160.8 (qC, C-5a), 164.2 (qC, C-6), 164.9 (qC, C-4a), 166.1 (qC, C-3), 183.5 (qC, C-9)。以上数据与文献报道 (Mutanyatta et al, 2003)的基本一致, 鉴定为1, 3, 6-三羟基-8-甲基占吨酮。
化合物9: 淡红色油状, 分子式为C10H13NO2。1H NMR (500 MHz, methanol-d4) δH: 7.04 (2H, d, J = 8.0 Hz, H-2, H-6), 6.72 (2H, d, J = 8.1 Hz, H-3, H-5), 3.40~3.29 (2H, m, H-8), 2.69 (2H, t, J = 7.4 Hz, H-7), 1.92 (3H, s, H-10); 13C NMR (125 MHz, methanol-d4) δC: 22.5 (CH3, C-10), 35.6 (CH2, C-7), 42.4 (CH2, C-8), 116.2 (CH, C-3, C-5), 130.7 (CH, C-2, C-6), 131.2 (qC, C-1), 156.9 (CH, C-4), 173.3 (qC, C-9)。以上数据与文献报道 (杜康平 等, 2001)的基本一致, 鉴定为N-乙酰酪胺。
化合物10: 淡黄色油状, 分子式为C9H10O3。1H NMR (500 MHz, methanol-d4) δH: 7.08~7.04 (2H, m, H-3, H-5), 6.75~6.69 (2H, m, H-2, H-6), 3.66 (3H, s, H-9), 3.52 (2H, s, H-7); 13C NMR (125 MHz, methanol-d4) δC: 40.9 (CH2, C-7), 52.4 (CH3, C-9), 116.3 (CH, C-2, C-6), 126.3 (qC, C-1), 131.3 (CH2, C-3, C-5), 157.6 (qC, C-4), 174.6 (qC, C-8)。以上数据与文献报道 (Shen et al, 2013)的基本一致, 鉴定为对羟基苯乙酸甲酯。
化合物11: 白色晶体, 分子式为C8H8O3。1H NMR (500 MHz, methanol-d4) δH: 7.16~7.01 (2H, m, H-2, H-5), 6.79 (2H, m, H-4, H-6), 3.60 (2H, s, H-7); 13C NMR (125 MHz, methanol-d4) δC: 36.5 (CH2, C-7), 115.9 (CH, C-4), 120.4 (CH, C-2), 122.8 (qC, C-1), 129.3 (CH, C-6), 132.1 (CH, C-5), 156.7 (qC, C-3), 176.2 (qC, C-8)。以上数据与文献报道 (吕海宁 等, 2015)的基本一致, 鉴定为间羟基苯乙酸。
化合物12: 黄色油状, 分子式为C10H15NO3; [ α ] D 25-12 (0.39 mg·mL-1, methanol)。1H NMR (500 MHz, methanol-d4) δH: 3.81~3.74 (1H, m, H-1), 2.34 (3H, s, H-6), 1.83 (1H, m, H-7), 1.30 (1H, m, H-8), 1.16 (1H, m, H-8 ), 0.91 (3H, d, J = 7.0 Hz, H-10), 0.82 (3H, t, J = 7.4 Hz, H-9); 13C NMR (125 MHz, methanol-d4) δC: 12.2 (CH3, C-9), 15.9 (CH3, C-6), 20.3 (CH3, C-10), 24.8 (CH, C-8), 38.4 (CH, C-7), 67.6 (CH, C-1), 103.8 (qC, C-3), 176.0 (qC, C-4), 187.5 (qC, C-5), 198.9 (qC, C-2)。以上数据与文献报道 (Gallardo et al, 2004)的基本一致, 鉴定为l-tenuazonic acid。
化合物13: 无色油状, 分子式为C30H54O4; [ α ] D 25 +37 (0.14 mg·mL-1, methanol); ECD (0.25 μg·mL-1, methanol) λmax (∆ε) 201 (+0.60), 210 (-3.62) nm; HR-ESIMS m/z 501.3930 [M + Na]+(calcd. for C30H54O4Na, 501.3920)。1H NMR (500 MHz, methanol-d4) δH: 5.28~5.08 (4H, m, H-7, H-11, H-14, H-18), 3.26 (2H, dd, J = 10.5, 1.8 Hz, H-3, H-22), 2.27 (2H, ddt, J = 15.5, 10.4, 5.1 Hz, H-5, H-20), 2.13 (6H, q, J = 7.1 Hz, H-5, H-20, H-8, H-17), 2.08~1.97 (8H, m, H-9, H-12, H-13, H-16), 1.77~1.69 (2H, m, H-4, H-21), 1.64 (12H, dd, J = 5.8, 1.3 Hz, H-26, H-27, H-28, H-29), 1.42~1.33 (2H, m, H-4, H-21), 1.18, 1.15 (each 6H, s, H-1, H-24, H-25, H-30); 13C NMR (125 MHz, methanol-d4) δC: 16.2 (CH3, C-27, C-28), 16.2 (CH3, C-26, C-29), 25.0, 25.6 (CH3, C-1, C-24, C-25, C-30), 27.8 (CH2, C-8, C-17), 29.3 (CH2, C-12, C-13), 30.9 (CH2, C-4, C-21), 37.9 (CH2, C-5, C-20), 40.9 (CH2, C-9, C-16), 73.8 (qC, C-2, C-23), 79.1 (CH, C-3, C-22), 125.5 (CH, C-11, C-14), 125.6 (CH, C-7, C-18), 136.1, 136.1 (qC C-6, C-10, C-15 and C-19)。以上数据与文献报道 (Nishiyama et al, 1996)的基本一致, 鉴定为2, 3, 22, 23-tetrahydroxy-2, 6, 10, 15, 19, 23-hexamethy1-6, 10, 14, 18-tetracosatetraene。

2.2 抑菌活性

抑菌活性结果发现化合物1、4—6和11对铜绿假单胞菌具抑制活性。进一步测定其MIC值发现, 化合物1和11的MIC值均为0.312mg·mL-1, 化合物4和5的MIC值均为0.156mg·mL-1, 化合物6的MIC值为0.078 mg·mL-1, 其中阳性药streptomycin的MIC值为0.156mg·mL-1。初步构效关系分析发现化合物6中的9位羟基有利于提高抗铜绿假单胞菌活性。

3 结论与讨论

通过对涠洲岛鹿角珊瑚来源的真菌Arachniotus ruber GXIMD 02510的次级代谢产物进行研究, 共分离得到了13个单体化合物, 含8个蒽醌类化合物(1—8)、3个苯酚化合物(9—11)、1个生物碱(12)和1个三萜(13), 抑菌活性筛选发现化合物1、4—6和11对铜绿假单胞菌具一定抑制活性, MIC值为0.078 ~0.312mg·mL-1, 化合物1、4、5和11的抗铜绿假单胞菌活性与阳性药相当, 初步构效关系分析发现化合物6的9位羟基有利于提高抗铜绿假单胞菌活性。化合物1—13均为首次从该属中分离得到。该研究丰富了该菌种的次级代谢产物研究, 为新型抗生素研发提供化学实体。
本文分离得到的蒽醌类化合物主要为高度氧化的四氢蒽醌类化合物, 降低了分子结构刚性, 具有多羟基取代, 使得化合物与蛋白的靶向结合能力增强。四氢蒽醌类化合物是一类比较少见的天然结构, 以微生物次生代谢产物居多, 少量来源于植物, 具有细胞毒活性、抗菌活性、抗疟原虫等生物活性 (冯世秀 等, 2019)。
四氢蒽醌类化合物是一类非常具有潜力的抗肿瘤药物, 其中1968年由Stoessl发现的化合物altersolanol A (Stoessl, 1969)具有显著的细胞毒活性, 对34种人类肿瘤细胞的半抑制浓度(half maximal inhibitory concentration, IC50)在0.001~0.412μg·mL-1之间, 平均IC50值为0.005μg·mL-1 (Mishra et al, 2015)。化合物5被报道对人口腔表皮癌细胞(KB)、人乳腺癌细胞(MCF-7)、小细胞肺癌(NCI-H187)和Vero细胞均具有细胞毒性, 其IC50值分别为: 26、24、23和31μg·mL−1 (Isaka et al, 2015), 化合物10对烟草花叶病毒具有较强的抑制活性(Shen et al, 2013)。

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