A sesquiterpene compound, and a preparation method and application thereof

Abstract

The application discloses a sesquiterpene compound and a preparation method and application thereof, and belongs to the technical field of marine fungus active ingredient analysis. A novel structure sesquiterpene compound is obtained by extraction and separation from a fermentation culture of marine fungus Epicoccum sorghinum HL-53. Through an in-vitro anti-HSV virus test, it is shown that the sesquiterpene compound provided by the application has good anti-HSV virus activity and low cytotoxicity, and has development prospects in the preparation of anti-HSV virus drugs.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of active ingredient analysis of marine fungi, and particularly relates to a sesquiterpene compound extracted from a fungus (Mycococcus xilanicus) HL-53 and application thereof. Epicoccum sorghinum ) HL-53 and application thereof. BACKGROUND

[0002] Herpes simplex virus infection (HSV) refers to repeated infections of skin, mouth, lips, eyes and genitalia caused by herpes simplex virus (divided into type 2, HSV-1 and HSV-2). Herpes simplex virus is a highly prevalent pathogen in the world, which has the characteristics of long-term latency, repeated attack and neurotropic tissue.

[0003] HSV small molecule drugs play a key role in fighting herpes simplex virus infection. The current research and development of these drugs aims to interfere with the life cycle of the virus, thereby preventing its replication and spread in the host body. For example, nucleoside inhibitors of DNA polymerase, such as acyclovir and valacyclovir, interfere with viral DNA synthesis by inhibiting polymerase activity, thereby effectively inhibiting viral replication. However, long-term and extensive use of these drugs can easily lead to some adverse reactions and induce the emergence of drug-resistant virus strains. Therefore, there is an urgent need to find new anti-herpes virus preparations with low toxicity and high efficiency.

[0004] Compared with terrestrial microorganisms, marine microorganisms can tolerate extreme conditions such as high salt, high pressure, low oxygen and low light that are unique to the ocean. The specificity of the living environment leads to the diversity of marine microorganisms in species, genetic composition and ecological function. The particularity of the marine environment, combined with the progress of marine microbial resource acquisition technology, has brought unprecedented opportunities for the research of natural drug compounds from marine microorganisms.

[0005] At present, in the research of marine active substances, many compounds from marine organisms have been found to show different activities, such as anticancer, antidiabetic, antiviral, anticoagulant, anti-inflammatory and other pharmacological activities. So far, many studies have shown that compounds with anti-HSV activity can be extracted from marine organisms such as algae, sponges, tunicates, echinoderms, mollusks, bacteria and fungi (Xu C, Li W, Wang W. Research progress of marine compounds with anti-herpes simplex virus activity [J]. China Marine Drugs, 2019, 38(04): 65-72.).

[0006] Marine fungi in marine microorganisms are a rich source of active secondary metabolites. It is of great significance to discover natural products with specific structural types from marine fungi as raw materials for the research and development of marine drugs. Epicoccum*Plasmodium* is a ubiquitous genus of filamentous fungi, widely found inside plants, in the surrounding air, soil, and decaying vegetation. It has attracted considerable attention as a biocontrol agent for plant pathogens. *Plasmodium* contains a variety of chemical components, such as polyketides, peptides, and diketopiperazines, which possess broad biological activities, including antibacterial, antioxidant, anticancer, and antiviral properties, making *Plasmodium* an important producer of compounds with potential biotechnological applications. Summary of the Invention

[0007] The purpose of this invention is to obtain *Pseudomonas sorghum* (… Epicoccum sorghinum Natural active substances with medicinal value are extracted from them.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] This invention is derived from *Pseudomonas sorghum* (… Epicoccum sorghinum A new compound was isolated from the fermentation products of HL-53. After structural identification, the specific structural formula of the new compound is shown in formula (Ⅰ), and it is named epicolide C.

[0010] (I).

[0011] The sorghum cocci ( Epicoccum sorghinum HL-53 is a large marine plant isolated from hydrothermal vent sediments on the seabed of Guishan Island, Taiwan Province, China. Its accession number is CGMCC 3.23793.

[0012] This invention also provides a method for obtaining *Pseudomonas sorghum* (… Epicoccum sorghinum The method for separating and extracting the above-mentioned new compounds from the fermentation products of HL-53 is described, but the preparation method of the above-mentioned compounds in this invention is not limited to this.

[0013] From sorghum cocci ( Epicoccum sorghinum A method for extracting the sesquiterpene compound from the fermentation products of HL-53 includes the following steps:

[0014] (1) The fungus *Pseudomonas sorghum* with accession number CGMCC 3.23793 ( Epicoccum sorghinum After activation, HL-53 was inoculated into rice culture medium for fermentation.

[0015] (2) After the fermentation culture was completed, the fermentation broth was extracted with ethyl acetate to obtain the extract;

[0016] (3) After concentrating the extract, normal phase silica gel column chromatography was performed. Gradient elution was performed with petroleum ether / ethyl acetate mixtures with volume ratios of 10:0, 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9, and 0:10. The fractions eluted from petroleum ether / ethyl acetate mixtures with volume ratios of 7:3 to 5:5 were collected and then separated by reverse phase silica gel column chromatography and high performance liquid chromatography to obtain the sesquiterpene compound.

[0017] In step (1), *Sorghum streptococcus* ( Epicoccum sorghinum HL-53 was used for fermentation culture.

[0018] Sorghum cocci ( Epicoccum sorghinum HL-53 is a fungus that can be fermented using conventional PDA medium or rice medium. To increase yield and provide sufficient nutrients for microbial growth and metabolism, the rice medium preferably comprises the following ingredients: rice and water, with 30 g of rice added to every 50 mL of water.

[0019] The fermentation culture conditions are static culture at 25-32℃ for 15-40 days. The static culture method refers to culture without shaking flasks.

[0020] Preferably, the fermentation culture temperature is 28-30°C. More preferably, the culture is carried out at 28°C for 21 days, under which the yield of the sesquiterpene compounds is the highest.

[0021] In step (2), an extract containing the sesquiterpene compound is obtained by extracting the fermentation product with ethyl acetate.

[0022] In step (3), the separation and purification method is as follows: the extract is separated by normal-phase silica gel column chromatography, and the obtained fraction is further separated by reversed-phase silica gel column chromatography and high-performance liquid chromatography. Through multi-step separation and purification, sesquiterpene compounds with high purity can be obtained.

[0023] Preferably, the fraction is subjected to reversed-phase silica gel column chromatography, with gradient elution sequentially using methanol / water mixtures at volume ratios of 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, and 9:1, each gradient being eluted three times. The collected fractions are numbered sequentially from 1 to 27, and fractions numbered 13 to 18 are combined. Subsequently, the fractions are separated by high-performance liquid chromatography. The peak with a retention time of 51 minutes using a methanol / water mixture at a volume ratio of 50:50 is the compound epicolide C, as shown in formula (I).

[0024] This invention demonstrates that the above method can be used to isolate *Pseudomonas sorghum* (…). Epicoccum sorghinumSesquiterpenoid compounds isolated from HL-53 fermentation cultures exhibit good anti-herpes simplex virus (HSV) activity. This invention uses the compounds' anti-Namalwa lymphoma cell activity as its cytotoxicity data to verify that while exerting anti-HSV virus effects, it has low toxicity to human cells and possesses biosafety.

[0025] Therefore, the present invention provides the use of the sesquiterpene compounds in the preparation of drugs against HSV virus.

[0026] The herpes simplex virus is herpes simplex virus type 1 (HSV-1) and / or herpes simplex virus type 2 (HSV-2).

[0027] The beneficial effects of this invention are as follows:

[0028] (1) This invention is derived from marine fungi Epicoccum sorghinum A novel compound was extracted and isolated from the fermentation culture of HL-53. The method is simple to operate, has a high extraction yield and high product purity, and is suitable for large-scale production.

[0029] (2) In vitro anti-HSV virus assays showed that the compound epicolide C provided by this invention has good anti-HSV-1 and HSV-2 virus activity, IC50. 50 The values ​​were 11.4 and 10.83 μM, respectively. Further cytotoxicity tests showed that the compound epicolide C provided by this invention has low cytotoxicity and has good development prospects in the preparation of anti-HSV drugs and preventive health foods. Attached Figure Description

[0030] Epicoccum sorghinum For the compound epicolide C 1 H NMR data (in DMSO- d 6,600 MHz).

[0031] Figure 1 For the compound epicolide C 13 C NMR data (in DMSO- d 6,150 MHz).

[0032] Figure 2 For the compound epicolide C 1 H- 1 H-COSY data (in DMSO- d 6).

[0033] Figure 3 HSQC data for compound epicolide C (in DMSO-d 6).

[0034] Figure 4 HMBC data for compound epicolide C (in DMSO- d 6).

[0035] Figure 5 ROESY data for compound epicolide C (in DMSO- d 6).

[0036] Figure 6 Electronic circular dichroism data for compound epicolide C (in CH3OH).

[0037] Figure 7 Structural formula for compound epicolide C.

[0038] Figure 8 Analysis of anti-HSV-1 viral activity of compound epicolide C.

[0039] Figure 9 Analysis of anti-HSV-2 viral activity of compound epicolide C. DETAILED DESCRIPTION

[0040] The application will be further described below in connection with specific embodiments. The following examples are intended to be illustrative only and not limiting of the scope of the application. Any modification or replacement of the method, steps or conditions of the application, without departing from the spirit and essence of the application, shall fall within the scope of the application.

[0041] The test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents, etc. used are commercially available reagents and materials unless otherwise specified.

[0042] Example 1: Isolation and identification of epiphytic fungi

[0043] The large marine plants were collected from the hydrothermal vent sediments of Guishan Island, Taiwan Province, China. After being brought back to the laboratory, the samples were first washed with sterile seawater for 3 times to remove the non-attached microorganisms. The plants were then placed in a centrifuge tube, and a small amount of seawater was added and shaken for 10 min using a vortex shaker. The suspension after removing the algal bodies was then centrifuged for 20 min (5000 r / min), and the supernatant was discarded. The precipitate was suspended with a small amount of sterile seawater, and 0.1 mL was spread on a Martin's medium (containing gentamicin 8 U / L) plate. After incubation at room temperature (20℃) for 10 d, single colonies were picked and purified by streaking, and then stored on a slant at 4℃ for preservation.

[0044] The isolated fungus was cultured on a PDA, and the ITS sequence of the strain was determined. The ITS sequence of the strain is shown in SEQ ID No. 1.

[0045] Based on the morphological characteristics and ITS sequence analysis results, the strain was identified as belonging to the genus *Plasmodium*. Figure 10 The fungus was named *Saccharomyces sorghum* (sp.) and named *Saccharomyces Epicoccum HL-53. This strain is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at the Institute of Microbiology, Chinese Academy of Sciences, Beijing, China, with accession number CGMCC 3.23793, and deposited on August 21, 2024.

[0046] Example 2: Fermentation culture of *Plasmodium* fungi

[0047] The *Pseudomonas sorghum* isolated and identified in Example 1 ( Epicoccum sorghinum After activation, HL-53 was inoculated into rice culture medium and statically fermented at 28°C for 21 days.

[0048] The rice culture medium formula is: 30 g rice and 50 mL water. It is then autoclaved at 121℃ for 20 min.

[0049] Example 3: Preparation of sesquiterpenoid compounds

[0050] Sorghum cocci ( Epicoccum sorghinum After fermentation, 9 kg of rice fermentation medium was taken from HL-53, extracted and concentrated with ethyl acetate, and separated by normal phase silica gel column chromatography (200-300 mesh, 1 kg; silica gel column size L 50 mm, Ø12 mm). Gradient elution was performed with petroleum ether / ethyl acetate mixtures with volume ratios of 10:0, 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9, and 0:10. The fractions eluted from the petroleum ether / ethyl acetate mixtures with volume ratios of 7:3 to 5:5 were collected.

[0051] The fraction was separated by reversed-phase silica gel column chromatography, eluent was methanol / water (1:9-9:1), gradient elution was performed with methanol / water mixture in the volume ratio of 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, 9:1, respectively, 1.5 L for each gradient elution, automatic collector was used to collect the eluate in the unit of 0.5 L, totally 27 fractions were collected, numbered as 1-27 in turn. According to the similarity of the composition of the fractions, 15 sub-fractions were formed, named as 5-1-5-15, specifically, 5-1: 1-3; 5-2: 4; 5-3: 5; 5-4: 6-9; 5-5: 10-11; 5-6: 12; 5-7: 13-18; 5-8: 19; 5-9: 20-24; 5-10: 25-26; 5-11: 27.

[0052] Then, the sub-fraction 5-7 was separated by high performance liquid chromatography, the peak with a retention time of 51 min in the eluent of methanol / water mixture in the volume ratio of 50:50 was recorded as compound 1.

[0053] Example 4: Structure identification of sesquiterpene compound

[0054] The prepared compound was identified for purity by HPLC, the sample with a purity greater than 98% was identified for structure by mass spectrometry and nuclear magnetic resonance technology, the nuclear magnetic resonance was determined by JEOL 600 MHz spectrometer, TMS was used as an internal standard; the high resolution mass spectrometry was determined by AB Sciex 5500 Q-TRAP.

[0055] According to the one-dimensional NMR (see Table 1, Epicoccum sorghinum ) and mass spectrometry analysis results of compound 1, it can be seen that the molecular formula of compound 1 is C 15 H 22 O4.

[0056] Table 1. NMR data of compound 1

[0057]

[0058] The structure of the compound was confirmed by two-dimensional NMR and electronic circular dichroism analysis results (see Figures 1-2 ), by consulting databases and literature, compound 1 was identified as a new compound epicolide C, the structure is shown as Figures 3-7 .

[0059] Example 5: Analysis of anti-HSV virus activity of epicolide C compound

[0060] (1) Preparation of solution

[0061] MEM complete medium: 89% MEM basal medium + 10% FBS + 1% double antibiotics (10000 units / mL Penicillin, 10000 μg / mL Streptomycin);

[0062] MEM maintenance medium: 98.8% MEM basal medium + 1% double antibiotics;

[0063] PBS (1x) 1 L: NaCl 8 g + KCl 0.2 g + Na2HPO4•12H2O 3.58 g + KH2PO4 0.24 g, filter through 0.22 μm membrane, tighten the bottle cap and sterilize, naturally cool and tighten;

[0064] Cell freezing solution: 80% MEM basal medium + 10% FBS + 10% DMSO.

[0065] (2) Cytopathic effect (CPE) inhibition assay

[0066] Vero cells were infected with herpes simplex virus HSV-1 (F strain) and HSV-2 (333 strain) (both viruses from Wuhan Institute of Virology, Chinese Academy of Sciences) to establish a cell model, and the cell model was used to determine the in vitro anti-herpes virus activity by combining with cytopathic effect (CPE) inhibition test.

[0067] First, Vero cells in logarithmic growth phase were taken, 5x10 4 cells per well were inoculated in a 96-well plate, and the plate was incubated in a 37℃, 5% CO2 incubator for 24 h. Vero cells in the 96-well plate were infected with HSV virus (MOI=0.1), and different concentrations of epicolide C compound were added after removing the virus inoculum. The experiment set blank group, virus group, positive control group (acyclovir) and experimental group, and each group set 3 replicate wells. After incubation at 37℃, 5% CO2 for 48 h, the culture medium was aspirated and the cells were fixed with 4% paraformaldehyde at room temperature for 20 min. After removing the paraformaldehyde, the cells were stained with 0.1% crystal violet staining solution at room temperature for 30 min. The plate was washed and dried, and the OD value was detected at 570 nm.

[0068] The CPE inhibition rate of compound epicolide C on HSV-1 virus at a concentration of 20 μM was calculated, and the inhibition rate (%) = (sample group OD average value - model group OD average value) / (blank group OD average value - model group OD average value) x 100%.

[0069] The above compound was further rescreened, and the IC 50 was calculated.

[0070] (3) Experimental results

[0071] Table 2. HSV virus inhibition rate and IC of compounds 50 values

[0072]

[0073] The primary screening data of the compounds (Table 2) showed that the compound epicolide C had a CPE inhibition rate of HSV-1 virus of more than 80% (86.6% ± 5.0%) at a concentration of 20 μM; the above-mentioned compounds were further rescreened, and the results are shown in Table 2 and Figure 8 Figures 9-10 , which showed that the onset drug concentration of epicolide C was lower than that of the antiviral drug acyclovir, and the IC 50 values (11.40 ± 1.9 μM and 10.83 ± 1.3 μM) of HSV-1 and HSV-2 were less than those of acyclovir (IC 50 = 16.7 ± 1.6 μM and 10.50 ± 1.2 μM). This indicated that epicolide C had a good in vitro inhibitory effect on the proliferation of HSV virus.

[0074] Example 6: Cytotoxicity analysis of sesquiterpene compounds

[0075] (1) Preparation of reagents

[0076] 0.4% SRB solution: weigh 0.8 g of SRB and dissolve in 200 mL of 1% acetic acid, and store at room temperature.

[0077] 50% TCA solution: weigh 50 g of TCA, add water to make up to 100 mL, and store at 4°C.

[0078] 10 mM Tris-base solution: weigh 0.6057 g of Tris-base, add water to make up to 500 mL, pH 10.5, and store at 4°C.

[0079] (2) Experimental instruments

[0080] CO2 incubator (Thermo), mini shaker (Kylin-Bell Lab instruments), microplate reader (M5 type, MD company).

[0081] (3) Method

[0082] Logarithmic growth phase Namalwa cells were selected, and after trypsin digestion, the cell concentration was adjusted to 2×10 4Cells were seeded in 96-well plates at a density of 5000 cells / mL in 190 μL cell suspension per well and incubated at 37 °C in 5% CO2 for 24 h. 10 μL of sample solution (final concentration: 20 µM) was added to the drug-treated wells, and 20 µM of 5-FU / As2O3 was added to the positive drug-treated wells. The control wells were treated with the same volume of solvent. The plates were incubated at 37 °C in 5% CO2 for 3 days. The medium was removed, and the cells were fixed with 100 μL of 50% TCA at 4 °C. The plates were incubated at 4 °C for 1 h. The TCA was removed, and the cells were washed with distilled water for 5 times to remove the TCA. The plates were air-dried for 1 h. The cells were stained with 80 μL of 0.4% SRB solution at room temperature for 30 min. The staining solution was removed, and the cells were washed with 1% acetic acid for 5 times to remove the unbound SRB. The plates were air-dried. The cells were dissolved with 150 μL of 10 mM Tris-base (pH = 10.5) and shaken for 5 min on a micro-vibrator. The OD values were measured using a M5 microplate reader. 510nm

[0083] (4) Result calculation

[0084] Tumor cell growth inhibition rate (%) = (ODcontrol- ODdrug) / (ODcontrol- ODblank) x 100%.

[0085] (5) Experimental results

[0086] Table 3. Namalwa cell survival rate of the compounds

[0087]

[0088] The cytotoxicity of compound 1 was further determined at a concentration of 20 µM using the Namalwa cell line. The data showed that the positive drug 5-Fu had a survival rate of 11.2% on the Namalwa cell line, the positive drug As2O3 had a survival rate of 5.6% on the Namalwa cell line, and compound 1 showed lower cytotoxicity on the Namalwa cell line.​

Claims

1. A sesquiterpene compound, characterized by, The structural formula of the sesquiterpene compound is shown in formula (I), 2. The method for preparing the sesquiterpene compound according to claim 1, characterized in that, The method comprises the following steps: (1) inoculating the fungus Epicoccum sorghinum HL-53 with a preservation number of CGMCC 3.23793 after activation into a rice medium to perform fermentation culture; (2) after the fermentation culture is completed, extracting the fermentation liquor by using ethyl acetate to obtain an extraction liquor; (3) concentrating the extraction liquor and then performing normal-phase silica gel column chromatography separation, gradient elution is performed in sequence by using petroleum ether / ethyl acetate mixed solution with a volume ratio of 10:0, 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9 and 0:10, the fraction eluted by the petroleum ether / ethyl acetate mixed solution with a volume ratio of 7:3 to 5:5 is collected, and then reverse-phase silica gel column chromatography and high-performance liquid chromatography separation are performed to obtain the sesquiterpene compound.

3. The production method according to claim 2, wherein In step (1), the rice medium comprises the following raw materials: rice and water, and 30 g of rice is added to 50 mL of water.

4. The production method according to claim 2 or 3, characterized by, In step (1), the fermentation culture condition is static culture at 25-32 ℃ for 15-40 days.

5. The production method according to claim 4, wherein The fermentation culture condition is static culture at 28 ℃ for 21 days.

6. The production method according to claim 2, wherein In step (3), the fraction is subjected to reverse-phase silica gel column chromatography, gradient elution is performed in sequence by using methanol / water mixed solution with a volume ratio of 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2 and 9:1, each gradient elution is performed for three times, the fractions numbered as 1-27 are collected, and the fractions numbered as 13-18 are combined; then, high-performance liquid chromatography separation is performed, the peak with a retention time of 51 minutes is obtained under the eluent of methanol / water mixed solution with a volume ratio of 50:50, and the peak is the compound epicolide C with the structural formula shown in formula (I).

7. Use of the sesquiterpene compound of claim 1 in the preparation of a medicine for resisting herpes simplex virus.

8. Use according to claim 7, wherein the compound is ###0002### The herpes simplex virus is herpes simplex virus type 1 and / or herpes simplex virus type 2.

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