海洋本草软珊瑚共附生曲霉属真菌EGF7-0-1和EGF15-0-3共培养中甾体类成分研究Ⅱ

doi:10.11978/2022240

Abstract

Abstract:

In order to simulate the growth environment of microorganisms to change or increase the production of secondary metabolites in the presence of other microorganisms and protect the ecological environment of marine herbs, soft coral-associated two fungi Aspergillus sp. EGF7-0-1 and EGF15-0-3 were cocultured on rice medium. Firstly, GNPS (Global Natural Products Social Molecular Networking) method was used to analyze secondary metabolites. Then, the secondary metabolites were separated and purified by silica gel, ODS (Octadecylsilyl), Sephadex LH-20 and HPLC (Preparative High Performance Liquid Chromatography). Their structures were identified by HR-ESI-MS (High Resolution Electrospray Ionization Mass Spectroscopy), NMR (Nuclear Magnetic Resonance), ORD (Optical Rotatory Dispersion) and the relative physical constants were compared with the literature. Ten steroids were obtained and identified as Chaxine C (1), Herbarulide (2), dankasterones A (3), 25-hydroxyergosta-4,6,8(14),22-tetraen3-one (4), Ganodermasides D (5), Ergosta-4,6,8(14),22-tetraen-3-one (6), Ergosta-4,7,22-trien-3-one (7), Isocyathisterol (8), Stigmasta-4,22-dien-3-one (9) and β-sitostenone (10). Compounds 1~3 were steroids with oxidative rearrangement and 4, 5, 7 and 9 were firstly acquired from Aspergillus sp. compared with the single culture of EGF7-0-1 and EGF15-0-3. Based on the coculture strategy, the content types of steroids were more abundant.

Key words: soft coral, Aspergillus sp., coculture, steroid

SITU Meixia, LEI Zufa, YANG Qianru, DENG Shengyi, WU Kejian, CHEN Honghao, WEI Xia, FAN Hao, ZHANG Wei, ZHANG Cuixian. Research on the steroids from the coculture of soft coral-associated fungi Aspergillus sp. EGF7-0-1 and EGF15-0-3[J].Journal of Tropical Oceanography, 2023, 42(5): 161-170.

Figures/Tables 7

Fig. 1

Tab. 1

Tab. 2

Tab. 3

13C NMR Data of 1~10 in CDCl3"

No.	1	2	3	4	5	6	7	8	9	10
1	32.1, t	33.9, t	39.0, t	34.1, t	27.6, t	34.3, t	33.2, t	35.2, t	35.7, t	35.7, t
2	24.8, t	33.2, t	34.5, t	34.1, t	34.0, t	34.3, t	34.3, t	33.8, t	34.0, t	32.1, t
3	148.8, d	198.4, s	199.3, s	199.7, s	199.5, s	199.7, t	199.6, s	199.9, s	199.7, s	199.8, s
4	128.1, d	114.7, d	126.7, d	123.2, d	126.4, d	123.1, d	122.8, d	125.3, d	123.8, d	123.7, d
5	195.8, s	173.9, s	156.2, s	124.7, s	161.4, s	124.6, s	169.2, s	163.5, s	171.7, s	171.9, s
6	164.3, d	162.4, s	200.2, s	124.7, d	124.8, d	124.6, d	33.2, t	128.2, d	33.0, t	33.0, t
7	117.5, d	113.3, d	41.0, t	134.1, d	130.9, d	134.2, d	115.7, d	139.4, d	32.0, t	33.9, t
8	156.2, s	159.5, s	62.3, s	164.5, s	126.7, s	164.5, s	139.7, s	71.9, s	35.6, d	35.6, d
9	204.6, s	47.2, d	49.5, d	44.5, d	72.6, s	44.5, d	45.9, d	53.3, d	53.8, d	53.8, d
10	81.2, s	40.4, s	36.1, s	36.9, s	42.4, s	36.9, s	38.2, s	36.3, s	38.6, s	38.7, s
11	39.0, t	25.4, t	25.3, t	19.0, t	25.5, t	19.1, t	22.1, t	18.0, t	21.0, t	21.0, t
12	38.0, t	39.1, t	38.5, t	35.6, t	32.1, t	35.7, t	39.1, t	40.9, t	39.5, t	39.4, t
13	46.5, s	47.0, s	54.1, s	44.2, s	44.6, s	44.1, s	43.5, s	44.2, s	42.3, s	42.5, s
14	57.9, d	58.1, d	215.0, s	155.7, s	158.3, s	156.3, s	55.1, d	57.2, d	56.0, d	56.0, d
15	21.9, t	22.6, t	38.1, t	25.3, t	25.7, t	25.5, t	23.0, t	19.1, t	24.3, t	24.2, t
16	29.1, t	27.7, t	23.3, t	27.7, t	27.8, t	27.9, t	28.2, t	28.2, t	28.9, t	28.2, t
17	55.3, d	56.3, d	49.5, d	55.6, d	55.7, d	55.8, d	55.9, d	56.6, d	55.9, d	55.9, d
18	12.1, q	12.5, q	17.2, q	19.1, q	17.9, q	19.1, q	12.3, s	14.6, q	12.2, q	12.0, s
19	21.9, q	20.0, q	24.2, q	16.8, q	20.9, q	16.8, q	21.4, q	19.0, q	17.4, q	19.0, s
20	40.1, d	40.2, d	37.4, d	39.5, d	39.3, d	39.4, d	40.6, d	39.9, d	40.5, d	36.1, d
21	21.0, q	21.0, q	23.8, q	21.2, q	21.3, q	21.4, q	21.2, q	20.7, q	19.0, q	18.7, q
22	134.6, d	134.7, d	132.5, d	138.4, d	134.9, d	135.2, d	135.6, d	135.4, d	138.1, d	34.0, t
23	132.9, d	132.8, d	135.3, d	129.9, d	132.9, d	132.7, d	132.4, d	132.3, d	129.5, d	29.1, t
24	42.8, d	42.8, d	43.4, d	48.3, d	43.0, d	43.0, d	42.9, d	43.0, d	51.2, d	45.8, d
25	33.0, d	33.1, d	33.2, d	72.5, s	33.2, d	33.2, d	33.0, d	33.2, d	31.9, d	26.1, d
26	19.7, q	20.0, q	19.8, q	27.1, q	19.8, q	20.1, q	20.1, q	19.8, q	21.2, q	17.4, q
27	20.0, q	19.6, q	20.2, q	26.6, q	20.1, q	19.8, q	19.8, q	20.1, q	21.1, q	19.8, q
28	17.6, q	17.6, q	17.1, q	15.8, q	17.8, q	17.8, q	17.7, q	17.8, q	25.4, t	23.1, t
29									12.3, q	12.0, q

Tab. 3

Fig. 2

Fig. 3

Fig. 4

References 35

[1]	蔡金旋, 冯冉奇, 韦霞, 等, 2022. 两株软珊瑚共附生曲霉属真菌 EGF7-0-1和EGF15-0-3共培养中杂萜类成分研究[J]. 中国海洋药物, 41(4): 1-8.
	CAI JINXUAN, FENG RANQI, WEI XIA, et al, 2022. Research on the meroterpenoids from the coculture of soft coral-associated fungi Aspergillus sp. EGF7-0-1 and EGF15-0-3[J]. Chinese Journal of Marine Drugs, 41(4): 1-8 (in Chinese with English abstract).
[2]	黎咏怡, 蔡金旋, 方越, 等, 2022. 南海软珊瑚共附生真菌 Aspergillus sp. EGF15-0-3中色酮、蒽醌及其二聚体类化合物[J]. 中山大学学报, (4): 70-78.
	LI YONGYI, CAI JINXUAN, FANG YUE, et al, 2022. Xanthones, anthraquinone and their dimers from soft coral-associated symbiotic and epiphytic fungi Aspergillus sp. EGF15-0-3[J]. Journal of Sun Yat-sen University, (4): 70-78 (in Chinese with English abstract).
[3]	刘炳新, 韦霞, 肖细姬, 等, 2021. 南海软珊瑚共附生真菌 Aspergillus sp. EGF15-0-3苯甲醛类化合物研究[J]. 热带海洋学报, 40(4): 63-69. doi: 10.11978/2020085
	LIU BINGXIN, WEI XIA, XIAO XIJI, et al, 2021. Research on benzaldehydes from the soft coral-associated symbiotic fungus Aspergillus sp. EGF15-0-3[J]. Journal of Tropical Oceanography, 40(4): 63-69 (in Chinese with English abstract).
[4]	肖细姬, 邓芸, 谢路凤, 等, 2019. 柳珊瑚 Leptogorgia rigida中甾体类成分研究[J]. 热带海洋学报, 39(4): 64-69.
	XIAO XIJI, DENG YUN, XIE LUFENG, et al, 2019. Research on Sterols from Leptogorgia rigida[J]. Journal of Tropical Oceanography, 39(4): 64-69 (in Chinese with English abstract).
[5]	AMAGATA T, TANAK M, YAMADA T, et al, 2007. Variation in cytostatic constituents of a sponge-derived Gymnascella dankaliensis by manipulating the carbon source[J]. Journal of Natural Products, 70(11): 1731-1740. doi: 10.1021/np070165m
[6]	CHAN YUYI, JUANG SHINHUN, HUANG GUANJHONG, et al, 2014. The constituents of michelia compressa var. formosana and their bioactivities[J]. International Journal of Molecular Sciences, 15(6): 10926-10935. doi: 10.3390/ijms150610926
[7]	CHANG HUICHIU, CHANG FANGRONG, WANG YUCHU, et al, 2007. A bioactive withanolide Tubocapsanolide A inhibits proliferation of human lung cancer cells via repressing Skp2 expression[J]. Molecular Cancer Therapeutics, 6(5): 1572-1578. doi: 10.1158/1535-7163.MCT-06-0812
[8]	CHOI J H, AKIHIRO O, NOBUO A, et al, 2009. Chaxines B, C, D, and E from the edible mushroom Agrocybe chaxingu[J]. Tetrahedron, 65(47): 9850-9853. doi: 10.1016/j.tet.2009.09.064
[9]	DARMAS H T, MOOTOO B S, REYNOLDS W F, et al, 2000. Steroidal compounds from the Caribbean Octocoral Eunicea laciniata[J]. Journal of Natural Products, 63(12): 1669-1671. doi: 10.1021/np000315s
[10]	DING KANG, TAN YANYAN, DING YANG, et al, 2019. β-Sitosterol improves experimental colitis in mice with a target against pathogenic bacteria[J]. Journal of Cellular Biochemistry, 120(4): 5687-5694. doi: 10.1002/jcb.27853 pmid: 30548286
[11]	DUECKER F L, HEINZE R C, MUELLER M, et al, 2020. Synthesis of the alleged structures of fortisterol and herbarulide and structural revision of herbarulide[J]. Organic Letters, 22(4): 1585-1588. doi: 10.1021/acs.orglett.0c00180 pmid: 31999123
[12]	DUECKER F L, REU F, HERETSCH P, 2019. Rearranged ergostane-type natural products: chemistry, biology, and medicinal aspects[J]. Organic & Biomolecular Chemistry. 17(7): 1624-1633.
[13]	FUJIMOTO H, NAKAMURA E, OKUYAMA E, et al, 2004. Six immunosuppressive features from an ascomycete, Zopfiella longicaudata, found in a screening study monitored by immunomodulatory activity[J]. Chemical and Pharmaceutical Bulletin, 52(8): 1005-1008. doi: 10.1248/cpb.52.1005
[14]	HIRATA Y, NAKAZAKI A, KAWAGISHI H, et al, 2017. Biomimetic synthesis and structural revision of chaxine B and its analogues[J]. Organic Letters, 19(3): 560-563. doi: 10.1021/acs.orglett.6b03724 pmid: 28121455
[15]	HU ZHENGXI, WU YE, XIE SHUANGSHUANG, et al, 2017. Phomopsterones A and B, Two functionalized ergostane-type steroids from the endophytic fungus Phomopsis sp. TJ507A[J]. Organic Letters, 19(1): 258-261. doi: 10.1021/acs.orglett.6b03557
[16]	KROHN K, BIELE C, AUST H J, et al, 1999. Herbarulide, a ketodivinyllactone steroid with an unprecedented homo-6-oxaergostane skeleton from the endophytic fungus Pleospora herbarum[J]. Journal of Natural Products, 62(4): 629-630. doi: 10.1021/np980422a
[17]	LEE K, KIM M A, LEE S Y, et al, 2008. Phytochemical constituents of Schizonepeta tenuifolia Briquet[J]. Natural Product Sciences, 14(2): 100-106.
[18]	LEE T H, CHEN C C, CHEN J J, et al, 2014. New and cytotoxic components from Antrodia camphorate[J]. Molecules, 19(12): 21378-21385. doi: 10.3390/molecules191221378
[19]	LI GANG, KUSARI S, KUSARI P, et al, 2015. Endophytic Diaporthe sp. LG23 produces a potent antibacterial tetracyclic titerpenoid[J]. Journal of Natural Products, 78(8): 2128-2132. doi: 10.1021/acs.jnatprod.5b00170
[20]	LI HONGLEI, LI XIAOMING, YANG SUIQUN, et al, 2020. Induced terreins production from marine red algal-derived endophytic fungus Aspergillus terreus EN-539 co-cultured with symbiotic fungus Paecilomyces lilacinus EN-531[J]. Journal of Antibiotics, 73(2): 108-111. doi: 10.1038/s41429-019-0242-4
[21]	LIU LIN, DUAN FANGFANG, GAO YING, et al, 2021. Aspersteroids A-C, three rearranged ergostane-type steroids from Aspergillus ustus NRRL 275[J]. Organic Letters, 23(24): 9620-9624. doi: 10.1021/acs.orglett.1c03863
[22]	MCCLOSKEY S, NOPPAWAN S, MONGKOLTHANARUK W, et al, 2017. A new cerebroside and the cytotoxic constituents isolated from Xylaria allantoidea SWUF76[J]. Natural Product Research, 31(12): 1422-1430. doi: 10.1080/14786419.2016.1258559
[23]	MELTZER B S, COLQUHOUN H, RIESENBERG R, et al, 2018. Brexanolone injection in post-partum depression: two multicentre, double-blind, randomised, placebo-controlled, phase 3 trials[J]. The Lancet, 392(10152): 1058-1070. doi: 10.1016/S0140-6736(18)31551-4
[24]	MIGLIUOL A, PICCIAUI V, SICA D, 1990. Steroidal ketones from the sponge Geodza Cydonzum[J]. Journal of Natural Products, 53(5): 1262-1266. doi: 10.1021/np50071a019
[25]	OH D C, JENSEN P R, KAUFFMAN C A, et al, 2005. Libertellenones A-D: Induction of cytotoxic diterpenoid biosynthesis by marine microbial competition[J]. Bioorganic and Medicinal Chemistry, 13(17): 5267-5273. doi: 10.1016/j.bmc.2005.05.068
[26]	OKADA B K, SEYEDSAYAMDOST M R, 2017. Antibiotic dialogues: induction of silent biosynthetic gene clusters by exogenous small molecules[J]. FEMS Microbiology Reviews, 41(1): 19-33. doi: 10.1093/femsre/fuw035 pmid: 27576366
[27]	QUANG D N, BACH D D, 2008. Ergosta-4,6,8(14),22-tetraen-3-one from Vietnamese Xylaria sp. Possessing inhibitory activity of nitric oxide production[J]. Natural Product Research, 22(10): 901-906. doi: 10.1080/14786410701642706
[28]	REN YUHAO, LIU QUANFANG, CHEN LI, et al, 2019. Urceoloids A and B, two C19 steroids with a rearranged spirocyclic carbon skeleton from Urceola quintaretii[J]. Organic Letters, 21(6): 1904-1907. doi: 10.1021/acs.orglett.9b00539
[29]	SULTANA N, 2018. Microbial biotransformation of bioactive and clinically useful steroids and some salient features of steroids and biotransformation[J]. Steroids, 136: 76-92. doi: S0039-128X(18)30015-1 pmid: 29360535
[30]	VAITKEVICIUS H, HUSAIN A M, ROSENTHAL E S, et al, 2017. First-in-man allopregnanolone use in super-refractory status epilepticus[J]. Annals of Clinical and Translational Neurology, 4(6): 411-414. doi: 10.1002/acn3.408 pmid: 28589168
[31]	WANG QIUYUE, CUI GUANGXIN, WU JICHUN, et al, 2015. Steroids from Trigonostemon heterophyllus[J]. Chemistry of Natural Compounds, 51(6): 1196-1198. doi: 10.1007/s10600-015-1531-1
[32]	WEI XIA, SU JUNCHENG, HU JINSHAN, et al, 2022. Probing indole diketopiperazine-based hybrids as environmental-induced products from Aspergillus sp. EGF 15-0-3[J]. Organic Letters, 24(1): 158-163. doi: 10.1021/acs.orglett.1c03795
[33]	WENG YUFANG, LIU JUN, XIANG LAN, et al, 2011. Ganodermasides C and D, two new anti-aging ergosterols from spores of the medicinal mushroom Ganoderma lucidum[J]. Bioscience Biotechnology and Biochemistry, 75(4): 800-803. doi: 10.1271/bbb.100918
[34]	ZHANG WEN, DRAEGER S, SCHULZ B, et al, 2009. Ring B aromatic steroids from an endophytic fungus, Colletotrichum sp.[J]. Natural Product Communications, 4(11): 1449-1454.
[35]	ZHU XIOAXIA, LIU YING, HU YUNWEN, et al, 2021. Neuroprotective activities of constituents from Phyllosticta capitalensis, an endophyte fungus of Loropetalum chinense var. rubrum[J]. Chemistry & Biodiversity, 18(8): e2100314.

仪器/试剂	型号/规格	品牌
高分辨液质联用仪	Triple TOFTM 5600⁺	美国 AB SCIEX公司
核磁共振波谱仪	400 MHz AVANCE Ⅲ型	德国Bruker公司
高效液相色谱	QuikSep半制备型	北京慧德易科技有限公司
自动旋光仪	MCP 500	安东帕公司
紫外透射反射仪	WFH-201B	上海精科实业有限公司
超净工作台	SW-CJ-1FD	苏州净化设备厂
立式压力蒸汽灭菌器	LS-100H	江阴滨江医疗设备有限公司
电子天平	PWN224ZH	奥豪斯仪器(常州)有限制造公司
旋转蒸发器	XHRE-2000A	上海宵汉实业发展有限公司
低温冷却液循环泵	XHDLSB-5/25	上海宵汉实业发展有限公司
色谱柱a: Luna 分析柱	4.6mm×100mm, 5μm	美国Phenomenex公司
色谱柱b: YMC-Pack ODS-A半制备柱	10mm×250mm, 5μm	日本YMC Co.,Ltd.公司
色谱柱c: H& EC18半制备柱	10mm×250mm, 5μm	北京慧德易科技有限公司
常规柱层析硅胶	200~300目、300~400目	青岛海洋化工厂
Sephadex LH-20	YILIMART 500G	瑞典 cytiva公司
C-18反相色谱填料	YMC*GEL ODS-A-HG	日本 YMC公司
乙酸乙酯、石油醚、甲醇、乙腈、二氯甲烷、三氯甲烷和正丁醇	分析纯	广东光华科技股份有限公司
氘代氯仿	25G	美国剑桥CIT公司
葡萄糖	分析纯	天津致远化学试剂有限公司
技术琼脂粉	分析纯	广东环凯微生物科技有限公司
土豆、大米、海盐		购于市场

No.	1	2	3	4	5
4	6.03, d, 10.1	5.74, s	6.36, s	5.74, s	5.88, s
6				6.04, d, 9.5	6.12, d, 9.6
7	5.64, d, 2.4	5.72, s	2.65, dd, 16.9, 1.5	6.60, d, 9.5	6.49, d, 9.6
9			2.81, t, 9.1
18	0.87, s	0.63, s	0.98, s	0.96, s	0.96, s
19	1.47, s	1.23, s	1.26, s	0.99, s	1.11, s
21	1.02, d, 6.6	1.02, d, 6.6	1.09, d, 7.1	1.08, d, 6.7	1.08, d, 6.7
22	5.14, dd, 15.3, 8.4	5.14, dd, 15.3, 8.4	5.24, dd, 15.2, 6.0	5.34, dd, 15.2, 7.6	5.20, dd, 15.3, 7.9
23	5.26, dd, 15.3, 7.7	5.25, dd, 15.2, 7.6	5.28, d, 15.2, 6.1	5.39, dd, 15.2, 7.8	5.27, dd, 15.3, 7.2
26	0.84, d, 6.8	0.82, d, 6.8	0.81, d, 6.8	1.14, s	0.83, d, 6.8
27	0.82, d, 6.8	0.82, d, 6.8	0.83, d, 6.8	1.17, s	0.85, d, 6.8
28	0.91, d, 6.8	0.91, d, 6.8	0.91, d, 6.8	1.01, d, 6.7	0.93, d, 6.9
No.	6	7	8	9	10
4	5.73, s	5.79, d, 2.0	5.72, s	5.72, s	5.71, s
6	6.03, d, 9.5		6.09, d, 9.6
7	6.61, d, 9.5		6.16, d, 9.6
18	0.96, s	0.60, s	1.02, s	0.73, s	0.70, s
19	0.99, s	1.17, s	1.32, s	1.18, s	1.17, s
21	1.06, d, 6.7	1.03, d, 6.6	0.99, d, 6.6	1.02, d, 6.6	.91, d, 6.5
22	5.26, dd, 15.2,7.1	5.16, dd, 15.2, 7.7	5.12, dd, 15.3, 8.2	5.15, dd, 15.2, 8.5
23	5.20, dd, 15.2,7.8	5.23, dd, 15.2, 6.9	5.22, dd, 15.3, 7.6	5.02, dd, 15.2, 8.5
26	0.83, d, 6.8	0.82, d, 6.5	0.82, d, 6.8	0.84, d, 6.3	0.83, d, 6.8
27	0.85, d, 6.8	0.84, d, 6.5	0.83, d, 6.8	0.79, d, 6.3	0.82, d, 6.8
28	0.93, d, 6.9	0.91, d, 6.5	0.91, d, 6.8
29				0.80, t, 7.3	0.84, t, 7.3

Research on the steroids from the coculture of soft coral-associated fungi Aspergillus sp. EGF7-0-1 and EGF15-0-3

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[7]	Yubin JI,Zhe ZHANG,Weihao ZHANG,Hu WANG,Ying PAN,Wei JIANG. QSI activity-oriented isolation of metabolites from Fusarium solanum [J]. Journal of Tropical Oceanography, 2019, 38(3): 98-103.
[8]	Yanhong TAN, Jixing LI, Xiuping LIN, Bin YANG, Yonghong LIU, Yunqiu LI. Study on the secondary metabolites from the South China Sea soft coral-derived fungus Eupenicillium sp. DX-SER3 (KC871024) [J]. Journal of Tropical Oceanography, 2019, 38(2): 43-47.
[9]	CHAI Xing-yun,HU Jing,HUANG Hui,LEI Hui,CHEN Xian-qiang,LI Yun-qiu,SUN Jia. Pregnane steroids from a gorgonian Menella sp. in South China Sea [J]. Journal of Tropical Oceanography, 2011, 30(1): 127-130.
[10]	QI Xin,HUANG Hai,ZHOU Wen-yi,YIN Shao-wu,ZHANG Yong,LIU Xiao-chun,. Variations in sex steroid hormone level and changes in ultrastructure of related cells during the artificial maturation of spermary of Anguilla marmorata [J]. Journal of Tropical Oceanography, 2010, 29(6): 130-136.