Sesquiterpene compound as well as preparation method and application thereof

Abstract

The invention discloses a sesquiterpene compound as well as a preparation method and application thereof, and belongs to the technical field of active ingredient analysis of marine fungi. According to the present invention, the sesquiterpene compound having the novel structure is extracted and separated from the fermentation culture of the marine fungus Epicoccum soghhinum HL-53, and the in vitro anti-HSV virus test results show that the sesquiterpene compound provided by the present invention has good anti-HSV virus activity, has low cytotoxicity, and has development prospects in the preparation of anti-HSV virus drugs.

Description

Technical Field

[0001] The invention relates to the technical field of analysis of active components of marine fungi, and in particular to a sesquiterpene compound extracted from Epicoccum sorghinum HL-53 and application thereof. Background Art

[0002] Herpes simplex virus infection (HSV) refers to recurrent infections of the skin, mouth, lips, eyes and genitals caused by the herpes simplex virus (divided into 2 types, HSV-1 and HSV-2). Herpes simplex virus is a highly prevalent pathogen worldwide, with the characteristics of long-term latency, recurrent outbreaks and tropism for neural tissues.

[0003] HSV small molecule drugs play a key role in combating 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. For example, nucleoside inhibitors of DNA polymerase, such as acyclovir and valacyclovir, inhibit polymerase activity and interfere with viral DNA synthesis, thereby effectively inhibiting viral replication. However, the long-term and widespread use of these drugs is prone to some adverse reactions and induces 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 unique to the ocean, such as high salt, high pressure, low oxygen, and low light. The specificity of the living environment leads to the diversity of marine microorganisms in species, genetic composition, and ecological functions. The particularity of the marine environment and the advancement of marine microbial resource acquisition technology have brought unprecedented opportunities for the research of natural drug compounds derived from marine microorganisms.

[0005] At present, in the research of marine active substances, it is found that many compounds from marine organisms show different activities, such as anti-cancer, anti-diabetic, anti-viral, anti-coagulant, 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 Cuijing, Li Wenmiao, Wang Wei. Research progress of marine compounds with anti-herpes simplex virus activity [J]. Chinese Journal of Marine Drugs, 2019, 38(04): 65-72.)

[0006] Marine fungi among marine microorganisms are a rich source of bioactive secondary metabolites. Discovering natural products with specific structural types from marine fungi is of great significance for the research and development of marine drugs. Epicoccum is a widespread genus of mitosporic fungi, widely present in plants, surrounding air, soil, and decaying vegetation, and has received extensive attention as a biological control agent for plant pathogens. Epicoccum contains various chemical components such as polyketides, peptides, and diketopiperazines, which have a wide range of biological activities including antibacterial, antioxidant, anticancer, and antiviral, making Epicoccum an important producer of compounds with potential biotechnological application value. Summary of the Invention

[0007] The object of the present invention is to extract natural active substances with medicinal value from Epicoccum sorghinum.

[0008] To achieve the above object, the present invention adopts the following technical solutions:

[0009] One new compound was isolated from the fermentation product of Epicoccum sorghinum HL-53. Through structure identification, the specific structural formula of this new compound is shown in formula (Ⅰ), and it is named epicolide C.

[0010]

[0011] The Epicoccum sorghinum HL-53 was isolated from a large marine plant in the hydrothermal vent sediment of Guishan Island, Taiwan Province, and the preservation number is: CGMCC 3.23793.

[0012] The present invention also provides a method for isolating and extracting the above new compound from the fermentation product of Epicoccum sorghinum HL-53, but the preparation method of the above compound in the present invention is not limited thereto.

[0013] The method for extracting the sesquiterpene compound from the fermentation product of Epicoccum sorghinum HL-53 includes the following steps:

[0014] (1) Activate the Epicoccum sorghinum HL-53 with the preservation number of CGMCC 3.23793 and inoculate it into a rice medium for fermentation culture;

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

[0016] (3) Concentrate the extract and then perform normal-phase silica gel column chromatography separation. Gradient elution is carried out successively 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. Collect the fractions eluted by the petroleum ether / ethyl acetate mixture with a volume ratio of 7:3 to 5:5, and then perform reverse-phase silica gel column chromatography and high-performance liquid chromatography separation to obtain the sesquiterpene compound.

[0017] In step (1), Epicoccum sorghinum HL-53 is fermented and cultured.

[0018] Epicoccum sorghinum HL-53 is a fungus, and conventional PDA medium or rice medium can be used for fermentation and culture. To increase the yield and provide sufficient nutrients for the growth and metabolism of microorganisms, preferably, the rice medium includes the following raw materials: rice and water, and 30 g of rice is added to every 50 mL of water.

[0019] The conditions for fermentation and culture are static culture at 25 - 32 °C for 15 - 40 days. The static culture method means no shake-flask culture.

[0020] Preferably, the temperature for fermentation and culture is 28 - 30 °C. More preferably, it is cultured at 28 °C for 21 days, and the yield of the sesquiterpene compound is the highest under this condition.

[0021] In step (2), ethyl acetate is used to extract from the fermentation product to obtain an extract containing the sesquiterpene compound component.

[0022] In step (3), the separation and purification method is: perform normal-phase silica gel column chromatography separation on the extract, and then perform reverse-phase silica gel column chromatography and high-performance liquid chromatography separation on the obtained fractions. Through multi-step separation and purification, a sesquiterpene compound with a higher purity can be obtained.

[0023] Preferably, for the reverse-phase silica gel column chromatography of the fractions, gradient elution is carried out successively with methanol / water mixtures with volume ratios of 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, 9:1. Each gradient is eluted 3 times, and the collected fractions are numbered 1 - 27 in sequence. The fractions numbered 13 - 18 are combined; then, high-performance liquid chromatography separation is carried out. Under the eluent of a methanol / water mixture with a volume ratio of 50:50, the peak with a retention time of 51 minutes is the compound epicolide C with the structural formula as shown in formula (Ⅰ).

[0024] The research of the present invention shows that the sesquiterpene compound isolated from the fermentation culture of Epicoccum sorghinum HL-53 by the above method has good anti-herpes simplex virus (HSV) activity. The present invention uses the anti-Namalwa lymphoma cell activity of the compound as its cytotoxicity data to verify that it has low toxicity to human cells while exerting the anti-HSV virus effect and has biological safety.

[0025] Therefore, the present invention provides the application of the described sesquiterpene compound in the preparation of drugs for anti-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 the present invention are as follows:

[0028] (1) The present invention extracts and isolates a compound with a novel structure from the fermentation culture of the marine fungus Epicoccum sorghinum HL-53. This method is simple to operate, has a high extraction yield, and high product purity, and is suitable for large-scale production.

[0029] (2) Through in vitro anti-HSV virus tests, it shows that the compound epicolide C provided by the present invention has good anti-HSV-1 and HSV-2 virus activities, and the IC 50 values are 11.4 and 10.83 μM respectively. Further cytotoxicity tests show that the compound epicolide C provided by the present invention has low cytotoxicity and has good development prospects in the preparation of drugs for anti-HSV virus and preventive health foods. Description of the Drawings

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

[0031] Figure 2 For the 13 13C NMR data of compound epicolide C (in DMSO-d 6 , 150 MHz).

[0032] Figure 3 For the 1 1H- 1 1H-COSY data of compound epicolide C (in DMSO-d 6 ).

[0033] Figure 4 HSQC data of compound epicolide C (in DMSO-d 6 )

[0034] Figure 5 HMBC data of compound epicolide C (in DMSO-d 6 )

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

[0036] Figure 7 Electronic circular dichroism data of compound epicolide C (in CH 3 OH)

[0037] Figure 8 Structural formula of compound epicolide C

[0038] Figure 9 Analysis of the anti-HSV-1 virus activity of compound epicolide C

[0039] Figure 10 Analysis of the anti-HSV-2 virus activity of compound epicolide C Detailed implementation manners

[0040] The present invention will be further described below in conjunction with specific embodiments. The following embodiments are only used to illustrate the present invention and are not used to limit the scope of application of the present invention. Without departing from the spirit and essence of the present invention, any modification or replacement of the methods, steps or conditions of the present invention shall fall within the scope of the present invention

[0041] Unless otherwise specified, the test methods used in the following examples are all conventional methods; the materials, reagents, etc. used, unless otherwise specified, are reagents and materials that can be obtained from commercial channels

[0042] Example 1: Isolation and identification of fungi of the genus Epichloë

[0043] Large marine plants were collected from the hydrothermal vent submarine sediments of Guishan Island, Taiwan Province. After the samples were brought back to the laboratory, they were first rinsed 3 times with sterile seawater to remove non-attached microorganisms. The plants were placed in centrifuge tubes, then a small amount of seawater was added, and they were shaken for 10 min with a vortex oscillator. Then the suspension after removing the algal bodies was centrifuged for 20 min (5000 r / min), and the supernatant was poured off. The precipitate was suspended with a small amount of sterile seawater, and 0.1 mL was taken and spread on a Martin's medium (containing 8 U / L of gentamicin) plate. After culturing at room temperature of 20 °C for 10 d, single colonies were picked, purified by streaking, and transferred to a slant and stored at 4 °C for later use.

[0044] The isolated fungi were cultured on PDA, and the ITS sequences of the strains were determined. The ITS sequence of this strain is shown as SEQ ID No. 1.

[0045] Based on the morphological characteristics of the strain and the results of ITS sequence analysis, this strain was identified as a fungus of the genus Epicoccum sp., and it was named Epicoccum sorghinum HL-53. This strain was deposited in the China General Microbiological Culture Collection Center (CGMCC). The deposit address: Institute of Microbiology, Chinese Academy of Sciences, Beijing, China. The deposit number is: CGMCC 3.23793, and the deposit date is August 21, 2024.

[0046] Example 2: Fermentation culture of fungi of the genus Epicoccum

[0047] The Epicoccum sorghinum HL-53 isolated and identified in Example 1 was inoculated into a rice medium after activation and statically fermented at 28 °C for 21 days.

[0048] Among them, the formula of the rice medium is: 30 g of rice, 50 mL of water. Autoclaved at 121 °C for 20 min.

[0049] Example 3: Preparation of sesquiterpene compounds

[0050] After the Epicoccum sorghinum HL-53 was fermented and cultured, 9 kg of the rice fermentation medium was taken, 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, 12 mm), and gradient elution was carried out successively with petroleum ether / ethyl acetate mixed solutions with volume ratios of 10:0, 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9, 0:10. The fractions eluted by the petroleum ether / ethyl acetate mixed solutions with volume ratios of 7:3 to 5:5 were collected.

[0051] The fraction was subjected to reverse-phase silica gel column chromatography with eluent of methanol / water (1:9 - 9:1). Gradient elution was carried out successively with 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 was eluted with 1.5 L, and collected in 0.5 L units using an automatic collector, resulting in a total of 27 fractions numbered 1 - 27 in sequence. The fractions were combined based on their compositional similarity to form 15 subcomponents, named 5-1 to 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] Subsequently, separation by high performance liquid chromatography was performed. For subcomponent 5-7, the peak with a retention time of 51 minutes under the eluent of methanol / water mixture with a volume ratio of 50:50 was designated as Compound 1.

[0053] Example 4: Structure Identification of Sesquiterpene Compounds

[0054] The purity of the prepared compounds was identified by HPLC. Samples with a purity greater than 98% were subjected to structure identification using mass spectrometry and nuclear magnetic resonance techniques. Nuclear magnetic resonance was measured using a JEOL 600 MHz spectrometer with TMS as the internal standard; high-resolution mass spectrometry was measured using an AB Sciex 5500 Q-TRAP.

[0055] Based on the one-dimensional NMR of Compound 1 (see Table 1, Figure 1-2 ) and the mass spectrometry analysis results, it was found that the molecular formula of Compound 1 is C 15 H 22 O 4 .

[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 Figure 3-7 ). By consulting the database and literature, Compound 1 was identified as a new compound, epicolide C, with the structure as shown in Figure 8 .

[0059] Example 5: Anti-HSV Virus Activity Analysis of Epicolide C Compound

[0060] (1) Preparation of Solutions

[0061] MEM Complete Medium: 89% MEM Basal Medium + 10% FBS + 1% Antibiotic-Antimycotic (10000 units / mL Penicillin, 10000 μg / mL Streptomycin);

[0062] MEM Maintenance Medium: 98.8% MEM Basal Medium + 1% Antibiotic-Antimycotic;

[0063] PBS (1×) 1L: 8 g NaCl + 0.2 g KCl + Na 2 HPO 4 ·12H 2 O 3.58 g + KH 2 PO 4 0.24 g, filtered through a 0.22 μm membrane by suction filtration, tighten the bottle cap for sterilization, and tighten again after natural cooling;

[0064] Cell Freezing Medium: 80% MEM Basal Medium + 10% FBS + 10% DMSO.

[0065] (2) Cytopathic Effect (CPE) Inhibition Assay

[0066] African green monkey kidney cells (Vero) were infected with herpes simplex virus HSV-1 (F strain) and HSV-2 (333 strain) (both strains of virus were from Wuhan Institute of Virology, Chinese Academy of Sciences) to establish a cell model, and the antiviral activity against herpes virus in vitro was determined by using this cell model combined with the cytopathic effect (CPE) inhibition test.

[0067] First, Vero cells in the logarithmic growth phase were seeded at 5×10 4 per well in a 96-well plate and cultured in an incubator at 37°C and 5% CO 2 for 24 h. The Vero cells in the 96-well plate were infected with HSV virus (MOI = 0.1), and after removing the virus inoculum, different concentrations of epicolide C compound were added. The experiment was set up with a blank group, a virus group, a positive control group (acyclovir), and an experimental group, with 3 replicates in each group. After culturing in an incubator at 37°C and 5% CO 2 for 48 h, the 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 stain at room temperature for 30 min. The plate was washed and dried, and the OD value was measured at 570 nm.

[0068] Calculate the CPE inhibition rate of the compound epicolide C against HSV-1 virus at a concentration of 20 μM. Inhibition rate (%) = (Average OD value of the sample group - Average OD value of the model group) / (Average OD value of the blank group - Average OD value of the model group) × 100%.

[0069] Further rescreen the above compounds and calculate the IC 50 .

[0070] (3) Experimental results

[0071] Table 2. HSV virus inhibition rate and IC 50 value

[0072]

[0073] The primary screening data of the compounds (Table 2) showed that the inhibition rate of the compound epicolide C on HSV-1 virus CPE was above 80% (86.6% ± 5.0%) at a concentration of 20 μM; further rescreen the above compounds, and the results are shown in Table 2 and Figure 9-10 , and the data indicated that compared with the antiviral drug acyclovir, the effective drug concentration of epicolide C was lower, and the half-maximal inhibitory concentration IC 50 values (11.40 ± 1.9 μM and 10.83 ± 1.3 μM) for 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 effect on inhibiting HSV virus proliferation in vitro.

[0074] Example 6: Cytotoxicity analysis of sesquiterpene compounds

[0075] (1) Reagent preparation

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

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

[0079] (2) Experimental instruments

[0080] CO 2 incubator (Thermo), micro oscillator (Mini shaker, Kylin-Bell Lab instruments), microplate reader (MD company, model M5).

[0081] (3) Method

[0082] Namalwa cells in the logarithmic growth phase were selected. After trypsin digestion, the cell concentration was adjusted to 2×10 4 cells / mL with RPMI 1640 medium containing 10% fetal bovine serum. The cell suspension was inoculated into a 96-well culture plate at 190 μL per well and cultured at 37°C in 5% CO 2 for 24 h. 10 μL of the sample solution (final concentration: 20 μM for the compound) was added to the drug treatment wells, and 5-FU / As with a final concentration of 20 μM was added to the positive drug wells 2 O 3 for treatment; the control wells were added with the medium containing the same volume of solvent and cultured at 37°C in 5% CO 2 for 3 d. The medium was discarded, and 100 μL of 50% TCA pre-cooled at 4°C was gently added to fix the cells. Let it stand for 5 min first, and then transfer it to 4°C for 1 h. Pour out the fixing solution, wash 5 times with distilled water to remove TCA, and air dry for 1 h. 80 μL of 0.4% SRB solution was added to each well and stained at room temperature for 30 min. Discard the staining solution, wash 5 times with 1% acetic acid to fully remove the unbound SRB, and air dry. Add 150 μL of 10 mM Tris-base (pH = 10.5) to dissolve, and oscillate on a micro oscillator for 5 min. Measure the OD 510nm value with an M5 microplate reader.

[0083] (4) Result calculation

[0084] Inhibitory rate of tumor cell growth (%) = (ODcontrol - ODdrug) / (ODcontrol - ODblank) × 100%.

[0085] (5) Experimental results

[0086] Table 3. Survival rate of Namalwa cells of the compound

[0087]

[0088] The cytotoxicity of compound 1 at a concentration of 20 μM was further determined using the Namalwa cell line. The data showed that the survival rate of the Namalwa cell line for the positive drug 5-Fu was 11.2%, and the survival rate of the Namalwa cell line for the positive drug As 2 O 3 was 5.6%. Compound 1 showed low cytotoxicity to the Namalwa cell line.

Claims

1. A sesquiterpene compound, characterized in that: The structural formula of the sesquiterpene compound is as shown in formula (I), 2. The method for preparing a sesquiterpene compound according to claim 1, characterized in that: The following steps are involved: (1) Activating the sorghum Epicoccum sorghinum HL-53 with a deposit number of CGMCC 3.23793 and inoculating it into a rice culture medium for fermentation; (2) After the fermentation culture is completed, the fermentation liquid is extracted with ethyl acetate to obtain an extract; (3) After the extract is concentrated, it is separated by normal phase silica gel column chromatography, and gradient elution is performed with a petroleum ether / ethyl acetate mixture 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, and the fractions eluted with the petroleum ether / ethyl acetate mixture with a volume ratio of 7:3 to 5:5 are collected, and then the fractions are separated by reverse phase silica gel column chromatography and high performance liquid chromatography to obtain the sesquiterpene compound.

3. The preparation method according to claim 2, characterized in that: In step (1), the rice culture medium comprises the following raw materials: rice and water, with 30 g of rice added to every 50 mL of water.

4. The preparation method according to claim 2 or 3, characterized in that: In step (1), the fermentation culture conditions are static culture at 25-32° C. for 15-40 days.

5. The preparation method according to claim 4, characterized in that: The fermentation culture conditions were static culture at 28°C for 21 days.

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

7. Use of the sesquiterpene compound as claimed in claim 1 in the preparation of drugs against herpes simplex virus.

8. The use according to claim 7, characterized in that The herpes simplex virus is herpes simplex virus type 1 and / or herpes simplex virus type 2.

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