Application of indole diterpenoid compound in mangrove-derived fungus secondary metabolite in preparation of aquaculture pathogenic bacterium resisting medicine

Abstract

The invention provides an application of indole diterpenoid compounds in mangrove-derived fungus secondary metabolites in preparation of drugs for resisting aquaculture pathogenic bacteria. The indole diterpenoid compounds separated from fermentation metabolites of endophytic fungi Penicillium janthinellum D131 derived from mangrove kandelia candel rhizosphere have bacteriostasis effects on vibrio alginolyticus, vibrio frenesii, vibrio harveyi, streptococcus iniae and streptococcus agalactiae, so that the indole diterpenoid compounds can be used for preparing the medicines for resisting the harmful bacteria in aquaculture; the invention provides an important way for deeply excavating more valuable marine vibrio or streptococcus resistant new drugs used in the aquaculture process.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microbial technology, and particularly relates to application of an indole diterpenoid compound in a mangrove-derived fungal secondary metabolite in preparation of an anti-pathogenic bacterium drug for aquaculture. BACKGROUND

[0002] The aquaculture industry is an important part of China's agricultural economy, and China is the world's largest aquaculture producer, accounting for more than 60% of the world's total output. However, with the continuous upgrading of the scale, intensification and industrialization of the aquaculture industry, the environment of China's aquaculture is deteriorating, leading to increasing stress and declining immunity of aquaculture animals, and frequent diseases. Among them, Vibrio and Streptococcus are one of the main bacterial diseases of aquaculture. According to the 2022 China Aquatic Animal Health Report, the economic loss caused by diseases in the aquaculture industry in China in 2021 was about 53.9 billion yuan. Vibrio and Streptococcus diseases have seriously hindered the sustainable and healthy development of the fishing industry, and have had a major impact on the world's aquaculture industry.

[0003] Antibiotic drugs are the main means to prevent and treat bacterial diseases such as Streptococcus and Vibrio due to their convenient use, rapid and significant effect. However, with the widespread use of antibiotics, drug-resistant bacteria have emerged, persisted and spread, reducing the effectiveness of drug control. As one of the three sources of new drug lead molecules, natural products from microorganisms play an important role in the discovery and development of new drugs and are a research hotspot for drug lead compounds. Research data shows that among the 1220 new marine natural products reported in 2023, 754 compounds are derived from marine microorganisms, accounting for 61.8%, and some compounds have shown a wide range of biological activities, including antibacterial, antiviral, antitumor and other pharmacological effects, providing rich resources for the development of antibacterial drugs.

[0004] However, although marine-derived microorganisms are rich in bioactive substances, they have not been fully researched and developed in inhibiting Streptococcus and Vibrio. The Beibuwan is located in the tropics and subtropics and is one of the most biologically diverse sea areas in China, providing a unique condition for marine drug research. Therefore, it is of great significance to fully utilize the potential of sustainable marine microbial resources in the Beibuwan and further explore more valuable new drugs against Vibrio or Streptococcus. SUMMARY

[0005] In view of this, the present application provides application of an indole diterpenoid compound in a mangrove-derived fungal secondary metabolite in preparation of an anti-pathogenic bacterium drug for aquaculture.

[0006] The technical solution of the present application is implemented as follows: The invention relates to the use of an indole diterpenoid compound from a secondary metabolite of a mangrove-derived fungus in the preparation of a drug for resisting aquaculture pathogens.

[0007] Furthermore, the mangrove-derived fungus is a mangrove-derived fungus Penicillium janthinellum D131, deposited in China Center for Type Culture Collection, with the deposit number being CCTCC NO: M 2025732.

[0008] Furthermore, the mangrove-derived fungus Penicillium janthinellum The nucleotide sequence of D131 is shown in SEQ ID NO.1.

[0009] Furthermore, the SEQ ID NO.1 is as follows:.

[0010] Furthermore, the preservation unit of the mangrove-derived fungi is the China Type Culture Collection of Wuhan University, Wuhan, Hubei Province, the preservation address is Wuhan University, Wuhan, China, and the preservation time is April 9, 2025.

[0011] Furthermore, the aquaculture pathogenic bacteria include Vibrio or Streptococcus.

[0012] Furthermore, the Vibrio includes Vibrio alginolyticus, Vibrio harveyi and Vibrio furnissi; the Streptococcus includes Streptococcus iniae and Streptococcus agalactiae.

[0013] Furthermore, the structural formula of the indole diterpenoid compound is shown in any one of 1 to 6 below: .

[0014] Furthermore, the indole diterpenoid compound is prepared from the fungus of Kandelia candel Penicillium janthinellum D131 was isolated by fermentation.

[0015] Furthermore, the mangrove-derived fungus Penicillium janthinellum The fermentation culture of D131 is to Penicillium janthinellum The seed liquid of D131 was inoculated into a rice solid culture medium, and the culture was kept at a constant temperature of 28° C. for 35 days for static fermentation. The crude secondary metabolites were extracted by soaking with an organic solvent, and the crude secondary metabolites were separated and purified to obtain indole diterpenoid compounds 1-5.

[0016] Furthermore, the preparation method of the indole diterpenoid compound comprises the following steps: S1. Activation of fungi: Extracting preserved mangrove-derived fungi Penicillium janthinellum D131 was pre-cultured in an incubator for 24-26 hours, then transferred to a sterile PDA plate for activation and cultured in an incubator for 2-4 days; S2. Seed solution preparation: Take the bean-sized mangrove-derived fungus from step S1. Penicillium janthinellum The D131 bacteria were inoculated into sterile potato liquid culture medium and cultured in a constant temperature shaker at 28-30°C for 2-3 days to obtain seed solution; S3, extract preparation: the seed liquid is inoculated into a sterile rice solid culture medium, and the fermentation culture is kept at a constant temperature of 28-30 ° C for 30-35 days to obtain a fermented product, and the fermented product is soaked in an organic solvent for 2-3 times, and concentrated under reduced pressure to obtain a crude extract; S4, chromatography: the crude extract was subjected to silica gel column chromatography, the eluent was petroleum ether-ethyl acetate solvent 100:0-0:100, v / v, gradient elution was carried out in turn, about 0.5 L was collected each time, similar fractions were combined by TLC detection, and were divided into five components, namely Fr. A-E. Components Fr. B, Fr. C and Fr. D were separated by comprehensively using normal phase silica gel column chromatography, reverse phase silica gel column chromatography, recrystallization and high performance liquid preparation separation method, and their structures were identified by comprehensively using physical and chemical analysis and NMR, MS modern spectroscopy method, and five indole diterpenoid compounds with structural formula 1-5 were separated from the fermentation product of the strain.

[0017] Further, in step S1, the mangrove-derived fungus is Penicillium janthinellum D131 was preserved in a PDB-30% glycerol medium at -80℃, and the constant culture temperature of the incubator was 28℃.

[0018] Further, the rice medium formula in steps S2 and S3 comprises rice 75-80 g and sea salt 0.2-0.3 g / mL.

[0019] Further, in step S3, the fermentation product is mycelium, and an equal volume of ethyl acetate is used as an organic solvent for soaking extraction 2-3 times, the extract is combined, and is concentrated under reduced pressure to obtain a crude extract.

[0020] Compared with the prior art, the present application has the following beneficial effects: The indole diterpenoid compounds 1-5 obtained by the present application have significant antibacterial activity on Vibrio and Streptococcus in aquaculture, and can be used for preparing antibacterial drugs for Vibrio and Streptococcus in aquaculture. Among them, the inhibitory effect of indole diterpenoid compound 1 on Vibrio and Streptococcus is the most obvious, and the MIC values are 3.125, 3.125, 1.56, 0.78 and 0.78 μg / mL, respectively.

[0021] In addition, the mangrove-derived fungus Penicillium janthinellum The indole diterpenoid compounds from the secondary metabolites of D131 are obtained by using the secondary metabolites of the mangrove-derived fungus as raw materials, through artificial fermentation culture, solvent soaking extraction, and separation means of normal phase silica gel column chromatography, reverse phase silica gel column chromatography, recrystallization and high performance liquid preparation, and five indole diterpenoid compounds 1-5 are obtained. Through high performance liquid chromatography analysis, the results show that the content of the indole diterpenoid compounds extracted from the secondary metabolites is more than 60%, and the method is simple, feasible and low in cost. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 H NMR of the indole diterpene compound 1 of the present invention 1 H NMR (600 MHz, DMSO- d 6); Figure 2 C NMR of the indole diterpene compound 1 of the present invention 13 C NMR (150 MHz, DMSO- d 6); Figure 3 H NMR of the indole diterpene compound 2 of the present invention 1 H NMR (600 MHz, DMSO- d 6); Figure 4 C NMR of the indole diterpene compound 2 of the present invention 13 C NMR (150 MHz, DMSO- d 6); Figure 5 H NMR of the indole diterpene compound 3 of the present invention 1 H NMR (600 MHz, DMSO- d 6); Figure 6 C NMR of the indole diterpene compound 3 of the present invention 13 C NMR (150 MHz, DMSO- d 6); Figure 7 H NMR of the indole diterpene compound 4 of the present invention 1 H NMR (600MHz, DMSO- d 6); Figure 8 C NMR of the indole diterpene compound 4 of the present invention 13 C NMR (150 MHz, DMSO- d 6); Figure 9 H NMR of the indole diterpene compound 5 of the present invention 1 H NMR (600MHz, DMSO- d 6); Figure 10 C NMR of the indole diterpene compound 5 of the present invention 13 C NMR (150 MHz, DMSO- d 6). DETAILED DESCRIPTION

[0023] In order to better understand the technical content of the present application, the following specific examples are provided to further illustrate the present application.

[0024] The experimental methods used in the embodiments of the present application are all conventional methods unless otherwise specified.

[0025] The materials, reagents, etc. used in the embodiments of the present application can be obtained from commercial channels unless otherwise specified.

[0026] Example 1 - Strain source Mangrove-derived fungus of the present application Penicillium janthinellum D131 is provided by the strain library of the research group, and the mangrove sample is collected in the mangrove forest protection zone of Maowei Sea in Qinzhou City, Guangxi Zhuang Autonomous Region. The strain is isolated from the rhizosphere of mangrove Kandelia candel plants in Qinzhou City, Guangxi. After isolation and purification, the strain is identified as Penicillium by morphology and molecular biology.

[0027] The strain is sent to a sequencing company for identification (test company: Shanghai SunGene Biotech Co., Ltd.). The obtained base sequence is compared for similarity in the GenBank database, and the BLAST program is used to search and compare homologous sequences. It is determined that the D131 strain is Penicillium (Penicillium sp.) and the sequence similarity is 99%. Penicillium janthinellum

[0028] According to the fermentation culture conditions of the mangrove-derived fungus Penicillium janthinellum D131 of the present application, the content of indole diterpenoid compounds in the fermentation product is greater than 60%, the preparation method is simple, and the prepared indole diterpenoid compounds have high content.

[0029] Example 2 - Preparation of indole diterpenoid compounds 1-5 1. Mangrove-derived fungus Penicillium janthinellum D131 fermentation culture: (1) Strain culture: D131 strain is cultured in PDB-glycerol medium and stored at -80°C. The PDB-glycerol medium culture is as follows: 200 g / L of potatoes, 20 g / L of glucose, and 30% of glycerol. Penicillium janthinellum (2) Strain activation: The strain D131 stored at -80°C is pre-cultured in a 28°C incubator for 24 hours, and then inoculated on a PDA plate in a sterile clean bench for activation and cultured in a 28°C incubator for 3 days. (3) Fermentation culture: The fungus D131 with the size of a broad bean is inoculated into sterile potato liquid medium, and cultured in a 28°C constant temperature shaker for 3 days to obtain seed liquid. The seed liquid is removed by a pipette gun in a sterile clean bench, and 5 mL of the seed liquid is inoculated into sterilized rice solid medium, and fermented at 28°C for 35 days to obtain fermentation product. Penicillium janthinellum Penicillium janthinellum ​​​​The rice solid culture medium is: rice 70-80 g / L, sea salt 0.3 g / mL; conical flasks with a capacity of 1000 mL, a total of 300 bottles are inoculated.

[0030] 2. Extraction of crude extract: The fermented product obtained after the fermentation culture was extracted three times with ethyl acetate, concentrated under reduced pressure, and the ethyl acetate extracts were combined to obtain a crude extract of 56.3 g.

[0031] 3. Separation and purification of compounds: The obtained crude ethyl acetate extract (56.3 g) was subjected to gradient elution by silica gel column chromatography (200-300 mesh silica) ( v / v , petroleum ether / ethyl acetate, 100:0-0:100), analyzed by TLC, and five components Fr.AE were obtained; component Fr.C was purified by reverse phase C 18 Column chromatography gradient elution ( v / v , methanol / water, 30:80-100:0), and 18 subfractions Fr.C.1-C.18 were obtained by HPLC and TLC analysis. Subfraction Fr.C.4 was subjected to semi-preparative high performance liquid chromatography ( v / v , acetonitrile / water, 65:35) to further separate and purify, respectively, to obtain indole diterpenoid compound 1 (13.2 mg); subfraction Fr.C.6 was subjected to semi-preparative high performance liquid chromatography ( v / v , acetonitrile / water, 68:32) were further separated and purified to obtain indole diterpenoid compounds 2 (29.5 mg) and 4 (24.1 mg); component Fr.D was purified by reverse phase C 18 Column chromatography gradient elution ( v / v , methanol / water, 30:70-100:0), and 23 subfractions Fr.D.1-D.23 were obtained by HPLC and TLC analysis; among them, subfraction Fr.D.12 was purified by semi-preparative high performance liquid chromatography ( v / v , acetonitrile / water, 70:30) to further separate and purify the indole diterpene compound 5 (18.9 mg); component Fr.E was purified by reverse phase C 18 Column chromatography gradient elution ( v / v , methanol / water, 20:80-100:0), and then analyzed by HPLC and TLC, and 21 subfractions Fr.E.1-E.21 were obtained; subfraction Fr.E.9 was purified by semi-preparative high performance liquid chromatography ( v / v, acetonitrile / water, 67:33) to give compound 3 (24.1 mg). The structures of these five indole diterpene compounds are shown below: .

[0032] Structure identification of indole diterpene compounds 1-5 in Example 3 The chemical structures of the indole diterpene compounds 1-5 obtained in Example 2 were determined by using modern structure identification techniques such as spectroscopy and MS.

[0033] The structure identification data are as follows: Indole diterpene compound 1, white solid powder, dissolved in methanol. The nuclear magnetic resonance data of indole diterpene compound 1: 1 HNMR (600 MHz, DMSO- d 6) δ H: 10.76 (s, 1H, NH-1), 7.27 (t, J = 7.0 Hz, 2H, H-20 / H-23), 6.94 (t, J = 7.6 Hz, 1H, H-22), 6.89 (d, J = 7.0 Hz, 1H, H-21), 5.76 (s, 1H,H-11), 5.03 (s, 1H, OH-7), 4.32 (s, 1H, OH-27), 4.16 (s, 1H, H-9), 2.77 (m,1H, H-16), 2.62 (m, 1H, H-17a), 2.51 (m, 1H, H-5a), 2.33 (m, 1H, H-17b), 2.18(m, 1H, H-6a), 2.13 (m, 1H, H-6b), 1.94 (m, 1H, H-15a), 1.85 (m, 1H, H-14a),1.83 (m, 1H, H-5b), 1.70 (m, 1H, H-14b), 1.64 (m, 1H, H-15b), 1.26 (s, 3H, H-25), 1.19 (s, 3H, H-28), 1.18 (s, 3H, H-29), 0.93 (s, 3H, H-26). 13 C NMR (150MHz, DMSO- d 6) δ C:198.26 (C-10), 161.43 (C-12), 152.68 (C-2), 139.79 (C-24), 124.49 (C-19), 120.47 (C-11), 119.31 (C-22), 118.47 (C-21), 117.70 (C-20), 114.97 (C-18), 111.81 (C-23), 93.76 (C-7), 78.06 (C-9), 76.09 (C-13), 71.14(C-27), 50.35 (C-3), 49.40 (C-16), 42.56 (C-4), 35.07 (C-6), 32.57 (C-14), 26.90 (C-17), 26.24 (C-29), 26.09 (C-28), 25.28 (C-5), 20.96 (C-15), 18.99(C-26), 16.20 (C-25)。

[0034] Indole diterpene compound 2, white solid powder, dissolved in methanol. ESI-MS m / z 420.15 [M - H] - . NMR data of indole diterpene compound 2: 1 H NMR (600 MHz, DMSO- d 6) δ H: 10.58 (s, 1H, NH-1), 7.26 (d, J = 3.6 Hz, 1H, H-23), 7.25 (d, J = 3.2 Hz, 1H, H-20), 6.93 (t, J = 7.3 Hz, 1H, H-22), 6.89 (t, J = 7.3 Hz, 1H, H-21), 4.10 (s, 1H, OH-28), 3.17 (m, 1H, H-9),3.09 (dd, J = 11.6, 2.6 Hz, 1H, H-7), 2.97 (dd, J = 11.6, 3.5 Hz, 1H, H-16), 2.65(m, 1H, H-17a), 2.56 (m, 1H, H-11a), 2.24 (m, 1H, H-17b), 1.93 (d, J= 12.6 Hz,1H, H-5a), 1.79 (m, 1H, H-6a), 1.73 (m, 1H, H-5b), 1.70 (m, 1H, H-11b), 1.63(m, 1H, H-14a), 1.57 (m, 1H, H-15a), 1.55 (m, 1H, H-10a), 1.53 (m, 1H, H-6b),1.51 (m, 1H, H-10b), 1.47 (m, 1H, H-13), 1.45 (m, 1H, H-14b), 1.32 (m, 1H, H-15b), 1.10 (s, 3H, H-27), 1.04 (s, 6H, H-29 / H-30), 0.96 (s, 3H, H-25), 0.82(s, 3H, H-26). 13 C NMR (150 MHz, DMSO- d 6) δ C : 151.17 (C-2), 140.25 (C-24),124.44 (C-19), 119.31 (C-22), 118.39 (C-21), 117.58 (C-20), 115.82 (C-18),111.88 (C-23), 85.24 (C-7), 84.60 (C-9), 70.40 (C-28), 52.47 (C-3), 48.57 (C-16), 45.89 (C-13), 37.24 (C-11), 36.08 (C-12), 32.46 (C-5), 27.10 (C-17),26.95 (C-30), 25.09 (C-6), 24.63 (C-29), 24.54 (C-15), 21.53 (C-14), 21.08(C-10), 19.44 (C-26), 14.57 (C-25), 12.64 (C-27)。

[0035] Indole diterpene compound 3, white solid powder, dissolved in methanol. NMR data of indole diterpene compound 3: 1 HNMR (600 MHz, DMSO- d 6) δ H: 10.75 (s, 1H, NH-1), 7.29 (m, 1H, H-20), 7.27 (m, 1H,H-23), 6.96 (t, J= 7.5 Hz, 1H, H-22), 6.91 (t, J = 7.4 Hz, 1H, H-21), 6.62 (s,1H, OH-27), 5.63 (s, 1H, H-11), 4.28 (s, 1H, OH-7), 4.10 (s, 1H, H-9), 2.71(m, 1H, H-16), 2.67 (m, 1H, H-17a), 2.62 (m, 1H, H-5a), 2.32 (m, 1H, H-17b),2.11 (m, 1H, H-6a), 2.04 (m, 1H, H-6b), 2.01 (m, 1H, H-15a), 1.97 (m, 1H, H-14a), 1.72 (m, 1H, H-15b), 1.64 (m, 1H, H-5b), 1.55 (m, 1H, H-14b), 1.45 (m,1H, H-13), 1.20 (s, 3H, H-25), 1.18 (s, 3H, H-28), 1.01 (s, 3H, H-29), 0.86(s, 3H, H-26). 13 C NMR (150 MHz, DMSO- d 6) δ C: 197.54 (C-10), 166.08 (C-12), 150.01(C-2), 140.13 (C-24), 124.36 (C-19), 121.19 (C-11), 119.53 (C-22), 118.52 (C-21), 117.75 (C-20), 115.95 (C-18), 111.89 (C-23), 93.21 (C-7), 77.07 (C-9),71.18 (C-27), 49.90 (C-3), 48.67 (C-16), 42.10 (C-4), 41.33 (C-13), 35.47 (C-6), 30.18 (C-14), 26.91 (C-17), 26.37 (C-29), 25.70 (C-28), 24.99 (C-5),23.89 (C-15), 15.17 (C-26), 14.66 (C-25)。 Indole diterpene compound 4, white solid powder, dissolved in methanol. NMR data of indole diterpene compound 4: 1 HNMR (600 MHz, DMSO-d 6) δ H： 10.60 (s, 1H, NH-1), 7.28 (m, 1H, H-20), 7.26 (m, 1H,H-23), 6.93 (t, J = 7.5 Hz, 1H, H-22), 6.90 (t, J = 7.2 Hz, 1H, H-21), 5.73 (s,1H, H-11), 5.08 (s, 1H, OH-13), 4.41 (s, 1H, H-9), 2.73 (m, 1H, H-16), 2.69(m, 1H, H-17a), 2.60 (m, 1H, H-5a), 2.45 (m, 1H, H-6a), 2.32 (m, 1H, H-17b),2.01 (m, 1H, H-6b), 1.95 (m, 1H, H-15a), 1.91 (m, 1H, H-14a), 1.86 (m, 1H, H-5b), 1.80 (m, 1H, H-14b), 1.63 (m, 1H, H-15b), 1.36 (s, 3H, H-25), 1.31 (s,3H, H-28), 1.11 (s, 3H, H-29), 1.08 (s, 3H, H-26). 13 C NMR (150 MHz, DMSO- d 6) δ C: 196.95 (C-10), 169.71 (C-12), 152.82 (C-2), 140.10 (C-24), 124.64 (C-19),119.27 (C-11), 118.46 (C-22), 117.69 (C-21), 116.80 (C-20), 114.87 (C-18),111.90 (C-23), 104.21 (C-7), 86.89 (C-9), 77.92 (C-27), 75.95 (C-13), 50.96(C-3), 48.47 (C-16), 39.15 (C-4), 31.83 (C-14), 28.54 (C-28), 27.99 (C-6),27.20 (C-17), 25.94 (C-5), 22.92 (C-29), 22.52 (C-26), 20.96 (C-15), 16.38(C-25)。

[0036] Indole diterpene compound 5, white solid powder, dissolved in methanol. NMR data of indole diterpene compound 5: 1 HNMR (600 MHz, DMSO- d 6) δ H: 10.60 (s, 1H, NH-1), 7.26 (m, 1H, H-23), 7.25 (m, 1H,H-20), 6.92 (t, J = 7.6 Hz, 1H, H-22), 6.89 (t, J = 7.6 Hz, 1H, H-21), 4.06 (s,1H, OH-28), 3.12 (m, 1H, H-9), 3.10 (m, 1H, H-7), 2.62 (m, 1H, H-16), 2.57(m, 1H, H-17a), 2.54 (m, 1H, H-6a), 2.36 (m, 1H, H-11a), 2.24 (m, 1H, H-17b),1.98 (m, 1H, H-5a), 1.84 (m, 1H, H-5b), 1.80 (m, 1H, H-14a), 1.70 (m, 1H, H-15a), 1.67 (m, 1H, H-13), 1.64 (m, 1H, H-10a), 1.62 (d, J = 7.2 Hz, 1H, H-14b),1.51(m, 1H, H-15b), 1.27 (m, 1H, H-6b), 1.23 (m, 1H, H-11b), 1.19 (m, 1H, H-10b), 1.09 (s, 1H, H-30), 1.00 (s, 1H, H-29), 0.97 (s, 1H, H-25), 0.92 (s,1H, H-26). 13 C NMR (150 MHz, DMSO- d 6) δ C:175.29 (C-27), 150.96 (C-2), 140.24 (C-24), 124.40 (C-19), 119.36 (C-22), 118.42 (C-21), 117.62 (C-20), 115.88 (C-18), 111.90 (C-23), 84.24 (C-9), 83.61 (C-7), 70.35 (C-28), 51.69 (C-3), 48.63 (C-16), 47.26 (C-12), 46.03 (C-13), 40.06 (C-4), 35.28 (C-11), 32.41(C-5), 27.03 (C-17), 25.18 (C-6), 24.77 (C-15), 24.44 (C-14), 23.41 (C-29), 22.81 (C-10), 16.17 (C-26), 14.51 (C-25).

[0037] Example 4 Antibacterial Activity Test of Indole Diterpenoid Compounds 1-5 1. Test strains Streptococcus iniae ( Streptococcus iniae ), Streptococcus agalactiae ( S. agalactiae )、Vibrio furnissis( Vibrio furnissii ), provided by Guangxi Beibu Gulf Marine Biodiversity Conservation Key Laboratory, College of Oceanography, Beibu Gulf University. V. alginolyticus ) and Vibrio harveyi ( V. harveyi ) was provided by the Key Laboratory of Tropical Medicinal Plant Chemistry, Ministry of Education, College of Chemistry and Chemical Engineering, Hainan Normal University.

[0038] 2. Activation of Streptococci and Vibrio species On a sterile, clean bench, use a sterile inoculating loop to select a small piece of Vibrio mycelium and place it in 2216E medium for activation. Similarly, use a sterile inoculating loop to select a small piece of Streptococcus mycelium and place it in LB medium for activation. Then, incubate it in a 37°C incubator for 2-3 days before use. During the activation process, regularly observe the growth of colonies on the culture medium to ensure aseptic operation and prevent contamination. After activation, the strain displays typical colony characteristics, such as a smooth surface, neat edges, and uniform color, indicating successful activation and suitable for subsequent antimicrobial activity testing.

[0039] 3. Preparation of Bacterial Suspension In a sterile operating table after sterilization, the above-mentioned activated pathogenic bacteria are picked up with a loop to inoculate 80 mL of broth liquid medium and 2216E or LB liquid medium. Then the medium is placed in a 37 °C shaker for 24 hours of shaker culture. After the culture is completed, 1 mL of bacterial suspension is diluted in 100 mL of sterile water, and after shaking well, it can be used as a standby.

[0040] To ensure that the concentration of the bacterial suspension is moderate, the OD value (optical density value) at 600 nm wavelength is also measured by spectrophotometer, and the OD value is adjusted to the range of 0.5±0.05 by adjusting the dilution multiple. Such moderate concentration of bacterial suspension is conducive to the subsequent antibacterial activity test, and ensures the accuracy and reliability of the test results. The prepared bacterial suspension should be used as soon as possible to avoid long-term placement leading to reduced bacterial activity or contamination.

[0041] 4. Antibacterial activity determination The antibacterial activity of monomeric indole diterpene compounds 1-5 against Vibrio and Streptococcus was evaluated by double dilution method.

[0042] The experimental procedure is as follows: first, the monomeric compound is dissolved in dimethyl sulfoxide (DMSO) to prepare a 2 mg / mL drug solution for use. Then, using a pipette, 10 μ L of sterilized 2216E liquid medium is added to the first hole of the first row. 10 μ L of 2.0 mg / mL sample solution is added to the first hole of the first row. After mixing well, 10 μ L is transferred to the second hole, and so on until the seventh concentration gradient is formed, and the last hole removes 10 μ L of drug solution and discards. Then, 90 μL of bacterial suspension is added to each hole and mixed well, thereby preparing 100 μ g / mL, 50 μ g / mL, 25 μ g / mL, 12.5 μ g / mL, 6.25 μ g / mL, 3.125 μ g / mL and 1.56 μ g / mL of drug solution at 7 different concentrations. The last column is used as a negative control, only adding bacterial suspension without drug solution. Ampicillin sodium is used as a positive control, and each group of drugs is tested in triplicate. The treated 96-well plate is placed in a 37 °C incubator for 18 to 24 hours, and the minimum inhibitory concentration (MIC) value is recorded.

[0043] 5. Antibacterial activity test results Table 1 Anti-bacterial activity test of indole diterpene compounds 1-5

[0044] The indole diterpene compounds obtained by the present application were subjected to anti-bacterial activity research on Vibrio and Streptococcus in aquaculture, and the anti-bacterial activity test results showed that the indole diterpene compounds all had bacteriostatic effect on Vibrio alginolyticus, Vibrio furnissii, Vibrio harveyi, Streptococcus iniae and Streptococcus agalactiae, indicating that the above indole diterpene compounds can be used for preparing anti-bacterial drugs for harmful bacteria in aquaculture, and provide an important way for further mining more valuable marine anti-Vibrio or Streptococcus drugs in the process of aquaculture. Among them, indole diterpene compounds 1 and 5 showed significant bacteriostatic effect on Vibrio furnissii ( V. furnissii ), Vibrio harveyi ( V. harveyi ) and Vibrio alginolyticus ( V. alginolyticus ), and the MIC value was in the range of 0.78-1.56 μ g / mL. In addition, indole diterpene compound 5 had strong bacteriostatic activity on Streptococcus agalactiae ( Streptococcus agalactiae ), and the MIC value was 1.56 μ g / mL. Indole diterpene compound 1 also had good bacteriostatic activity on Streptococcus iniae ( S. iniae ) and Streptococcus agalactiae ( S. agalactiae ), and the MIC value was 3.125 μ g / mL.

[0045] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. The use of an indole diterpenoid compound from a secondary metabolite of a mangrove-derived fungus in the preparation of a drug against aquaculture pathogens.

2. The use according to claim 1, characterized in that The mangrove-derived fungus is a mangrove-derived fungus Penicillium janthinellum D131, deposited in China Center for Type Culture Collection, with the deposit number CCTCCNO: M 2025732.

3. The use according to claim 1, characterized in that The mangrove-derived fungus Penicillium janthinellum The nucleotide sequence of D131 is shown in SEQ ID NO.

1.

4. The use according to claim 1, characterized in that The aquaculture pathogenic bacteria include Vibrio or Streptococcus.

5. The use according to claim 4, characterized in that The Vibrio includes Vibrio alginolyticus, Vibrio harveyi and Vibrio furnissi; the Streptococcus includes Streptococcus iniae and Streptococcus agalactiae.

6. The use according to claim 1, characterized in that The structural formula of the indole diterpenoid compound is shown in any one of 1 to 6 below: 。 7. The use according to claim 1, characterized in that The indole diterpenoid compounds are obtained from the fungus Kandelia officinalis Penicillium janthinellum D131 was isolated by fermentation.

8. The use according to claim 1, characterized in that The preparation method of the indole diterpenoid compound comprises the following steps: S1. Activation of fungi: Extracting preserved mangrove-derived fungi Penicillium janthinellum D131 was pre-cultured in an incubator for 24-26 hours, then transferred to a sterile PDA plate for activation and cultured in an incubator for 2-4 days; S2. Seed solution preparation: Take the mangrove-derived fungus from step S1 Penicillium janthinellum The D131 bacteria were inoculated into sterile potato liquid culture medium and cultured in a constant temperature shaker at 28-30°C for 2-3 days to obtain seed solution; S3, extract preparation: the seed liquid is inoculated into a sterile rice solid culture medium, and the fermentation culture is kept at a constant temperature of 28-30 ° C for 30-35 days to obtain a fermented product, and the fermented product is soaked in an organic solvent for 2-3 times, and concentrated under reduced pressure to obtain a crude extract; S4. Chromatographic separation: The crude extract was subjected to silica gel column chromatography, and the eluent was petroleum ether-ethyl acetate solvent 100:0--0:100, v / v, and gradient elution was performed in sequence to separate the indole diterpenoid compounds.

9. The use according to claim 8, characterized in that In step S1, the mangrove-derived fungus Penicillium janthinellum D131 was stored at -80℃ in PDB-30% glycerol medium; the constant temperature of the incubator was 28℃.

10. The use according to claim 8, characterized in that The rice culture medium formula in step S2 and step S3 includes 75-80 g of rice and 0.2-0.3 g / mL of sea salt. In step S3, the fermented product is mycelium, which is soaked and extracted 2-3 times with an equal volume of ethyl acetate as an organic solvent, and the extracts are combined and concentrated under reduced pressure to obtain a crude extract.

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