A sesterterpene compound, its preparation method and application

Abstract

The present invention discloses a sesterterpenoid compound, a preparation method thereof and an application thereof. In the present invention, two novel-structured sesterterpenoid compounds are obtained by co-culturing and fermenting two edible and medicinal fungi, Pleurotus ostreatus and Trametes robiniophila. The structural formulas are shown as Formula I or Formula II. The two sesterterpenoid compounds have significant activities of inhibiting Candida albicans and Cryptococcus neoformans, and can be applied to the research and development of antibacterial drugs; moreover, the preparation method described in the present invention opens up a new way for preparing new antibacterial preparations in the antibacterial field.

Description

Technical Field

[0001] The present invention relates to the field of fermentation culture of biological materials, and particularly relates to a sesterterpenoid compound, a preparation method thereof, and an application thereof. Background Art

[0002] In recent years, due to the increase in medical interventions such as surgical intervention and organ transplantation, as well as the prevalence of diseases such as respiratory coronavirus and HIV infections, the number of people with low immunity has been increasing continuously, resulting in a sharp rise in the fungal infection rate. The number of deaths caused by pathogenic bacteria infections each year reaches about 1.5 million. Among them, Candida albicans and Cryptococcus neoformans are both very common pathogenic fungi. At present, the drugs for treating fungal infections mainly include echinocandins (such as caspofungin), polyenes (such as amphotericin B), azoles (such as fluconazole, etc.), and 5-fluorocytosine, etc., all of which have certain toxic side effects, and the drug resistance rate of pathogenic bacteria to the above drugs is gradually increasing. It is reported that the number of deaths due to antibiotic resistance globally has exceeded 10 million per year, and it is expected that by 2050, the number of deaths will exceed the number of cancer deaths. Therefore, there is an urgent need to develop new antibacterial drugs.

[0003] Edible and medicinal fungi have a long history of medicinal and nutritional value. Since prehistoric times, mushrooms have been widely used in the field of edible and medicinal uses. In recent years, extracts of edible and medicinal fungi and their secondary metabolites have attracted much attention due to their biological activities such as antibacterial, anti-tumor, liver protection, and immunomodulation. Therefore, exploring new active compounds in the secondary metabolites of edible and medicinal fungi is of great significance for the development of antibacterial drugs. Based on this, the present invention simulates the state of coexisting with other microorganisms in the natural environment by co-culturing and fermenting Pleurotus ostreatus and Trametes robiniophila, activates their silent biosynthetic gene clusters, and chemically analyzes the obtained mixed fermentation concentrate; through research, it is found that by using the above co-culture method, two sesterterpenoid compounds with antibacterial activity can be obtained. This sesterterpenoid compound with antibacterial activity can be applied to the research and development of antibacterial drugs. Summary of the Invention

[0004] Based on the medical value of Pleurotus ostreatus and Trametes robiniophila, the present invention provides a sesterterpenoid compound, a preparation method thereof, and an application thereof. The present invention prepares a sesterterpenoid compound with activity against Candida albicans and Cryptococcus neoformans by co-culturing and fermenting Pleurotus ostreatus and Trametes robiniophila, thus opening up a new way for the preparation of antibacterial agents.

[0005] In order to solve the above technical problems, the technical solution that the present invention intends to adopt is:

[0006] The present invention provides a sesterterpenoid compound, and its structural formula is shown as Formula I or Formula II:

[0007]

[0008] The present invention also provides a method for preparing the diterpenoid compound, which comprises the following steps:

[0009] S1: Solid-culturing Pleurotus ostreatus and Trametes robiniophila Murr.;

[0010] S2: Taking the Pleurotus ostreatus and Trametes robiniophila Murr. obtained by culturing in the step S1, respectively performing liquid culture to obtain a Pleurotus ostreatus fermentation broth and a Trametes robiniophila Murr. fermentation broth;

[0011] S3: Mixing the Pleurotus ostreatus fermentation broth and the Trametes robiniophila Murr. fermentation broth in the step S2 and performing co-culture to obtain a co-culture fermentation broth;

[0012] S4: Filtering, organically extracting and concentrating under reduced pressure the co-culture fermentation broth in the step S3 to obtain a crude extract;

[0013] S5: Performing column chromatography and HPLC separation and purification on the crude extract in the step S4 to obtain the diterpenoid compound.

[0014] Further, the temperature of the solid culture in the step S1 is 26°C to 30°C, and the culture time is 6 days to 8 days.

[0015] Further, the temperature of the liquid culture in the step S2 is 26°C to 30°C, and the culture time is 6 days to 8 days.

[0016] Further, the components and component contents of the culture medium for the liquid culture in the step S2 are: 44 - 48 g / L of glucose, 12 - 16 g / L of sucrose, 4 - 8 g / L of peptone, 0.3 - 0.6 g / L of MgSO4·7H2O, 0.8 - 1.2 g / L of KH2PO4, and the balance is water.

[0017] Further, in the step S3, the mixing volume ratio of the Pleurotus ostreatus fermentation broth and the Trametes robiniophila Murr. fermentation broth is 1:1; the co-culture time is 6 days to 8 days.

[0018] Further, the specific steps of the step S4 are: filtering the co-culture fermentation broth to obtain mycelia and supernatant; extracting the mycelia with an organic solvent methanol, concentrating the obtained extract and then extracting with ethyl acetate to obtain an extract I; extracting the supernatant with an organic solvent ethyl acetate to obtain an extract II; combining the extract I and the extract II and concentrating under reduced pressure to obtain a crude extract.

[0019] Further, the specific steps of separation and purification in step S5 are as follows: after dissolving the crude extract, first perform gradient elution on a normal-phase silica gel column using petroleum ether-dichloromethane-methanol as the solvent, and then perform gradient elution on a C-18 reversed-phase silica gel column using methanol-water as the solvent for the obtained fractions. The obtained sub-fractions are further separated and purified by HPLC to obtain the sesterterpenoid compounds.

[0020] Further, when separating and purifying by HPLC, the mobile phase used is methanol and 2 / 1000 aqueous trifluoroacetic acid solution; the gradient elution method of the mobile phase is: 0 - 2 min, 70% methanol, 30% aqueous trifluoroacetic acid solution; 2 - 30 min, gradually adjust from 70% methanol, 30% aqueous trifluoroacetic acid solution to 100% methanol, 0% aqueous trifluoroacetic acid solution; 30 - 40 min, 100% methanol.

[0021] The present invention also provides the application of the sesterterpenoid compounds in the preparation of antibacterial preparations or antibacterial drugs.

[0022] Further, the pathogenic bacteria inhibited by the antibacterial preparation or antibacterial drug include Candida albicans and Cryptococcus neoformans.

[0023] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows:

[0024] The present invention discloses a preparation method of sesterterpenoid compounds. Two sesterterpenoid compounds with novel structural features are obtained by co-culturing Pleurotus ostreatus and Trametes robiniophila. Experimental data confirm that these two sesterterpenoid compounds can effectively inhibit Candida albicans and Cryptococcus neoformans, can be used as antibacterial preparations, and can also be applied to the preparation of novel antibacterial drugs, having broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The present invention will be further described in detail below with reference to the drawings and specific embodiments.

[0026] Figure 1 For the compound with the structure shown in Formula I in the embodiment of the present invention 1 1H NMR spectrum;

[0027] Figure 2 For the compound with the structure shown in Formula I in the embodiment of the present invention 13 13C NMR spectrum;

[0028] Figure 3 For the compound with the structure shown in Formula I in the embodiment of the present invention

[0029] Figure 4 For the compound with the structure shown in Formula I in the embodiment of the present invention

[0030] Figure 5The NOESY spectrum of the compound with the structure shown in Formula I in the embodiments of the present invention;

[0031] Figure 6 The HRESIMS spectrum of the compound with the structure shown in Formula I in the embodiments of the present invention;

[0032] Figure 7 For the compound with the structure shown in Formula II in the embodiments of the present invention 1 1H NMR spectrum;

[0033] Figure 8 For the compound with the structure shown in Formula II in the embodiments of the present invention 13 13C NMR spectrum;

[0034] Figure 9 The HSQC spectrum of the compound with the structure shown in Formula II in the embodiments of the present invention;

[0035] Figure 10 The HMBC spectrum of the compound with the structure shown in Formula II in the embodiments of the present invention;

[0036] Figure 11 The NOESY spectrum of the compound with the structure shown in Formula II in the embodiments of the present invention;

[0037] Figure 12 The HRESIMS spectrum of the compound with the structure shown in Formula II in the embodiments of the present invention. Detailed implementation manners

[0038] The following further describes the present invention in detail with reference to embodiments. The following embodiments are explanations of the present invention, and the present invention is not limited to the following embodiments.

[0039] Example 1: A method for preparing a sesterterpenoid compound by co-culturing Pleurotus ostreatus and Trametes robiniophila Murr. The specific preparation steps of the sesterterpenoid compound are as follows:

[0040] Step (1): Take appropriate amounts of Pleurotus ostreatus and Trametes robiniophila Murr., and inoculate them onto PDA solid plate media respectively. Incubate in an incubator at 28 °C for 6 - 8 days, preferably 7 days.

[0041] Step (2): Take 3 pieces each of Pleurotus ostreatus and Trametes robiniophila Murr. cultured in step (1), with each piece having a diameter of about 0.5 mm, and inoculate them separately into 500 mL conical flasks containing 100 mL of medium, and ferment and culture at 28 °C and 180 rpm for 6 - 8 days, preferably 7 days; among them, the components and component contents of the used medium are as follows: glucose 44 - 48 g / L, sucrose 12 - 16 g / L, peptone 4 - 8 g / L, MgSO4·7H2O 0.3 - 0.6 g / L, KH2PO4 0.8 - 1.2 g / L, and the balance is water. The preferred components and their contents are: glucose 45.5 g / L, sucrose 14.5 g / L, peptone 6.0 g / L, MgSO4·7H2O 0.5 g / L, KH2PO4 1.0 g / L, and the balance is water.

[0042] Step (3): Mix the Trametes robiniophila Murr. fermentation broth and Pleurotus ostreatus fermentation broth in step (2) at a volume ratio of 1:1, and continue to culture for 6 - 8 days, preferably 7 days.

[0043] Step (4): Filter the co-culture fermentation broth (60 L) obtained in step (3) with coarse gauze to separate the supernatant and mycelium; the mycelium is extracted three times with methanol, and the obtained aqueous solution is concentrated under reduced pressure, and then extracted with ethyl acetate; the supernatant is directly extracted three times with ethyl acetate, and the ethyl acetate extraction solutions are combined and concentrated under reduced pressure to obtain a crude extract (20 g).

[0044] Step (5): After dissolving the crude extract obtained in step (4) with methanol, use petroleum ether - dichloromethane - methanol as the solvent for gradient elution on a normal-phase silica gel column, and the mobile phase ratios are respectively 1.0 L of petroleum ether elution; 2.0 L of dichloromethane elution; dichloromethane:methanol = 98:2, 2.5 L; dichloromethane:methanol = 96:4, 2.5 L; dichloromethane:methanol = 94:6, 2.5 L; dichloromethane:methanol = 9:1, 3.0 L; dichloromethane:methanol = 1:1, 3.0 L; After analysis and combination, 7 components (1 - 7) are obtained.

[0045] Step (6): Further use methanol - water as the solvent for gradient elution of component 5 obtained in step (5) on a C-18 reversed-phase silica gel column, and the mobile phase ratios are respectively methanol:water = 5:95, 2 L; methanol:water 20:80 = 2.0 L; methanol:water = 40:60, 2.5 L; methanol:water = 60:40, 2.5 L; methanol:water = 80:20, 2.5 L; methanol, 2.0 L; methanol:dichloromethane = 1:1, 1 L; After analysis and combination, six sub-components (5-1 - 5-6) are obtained.

[0046] Step (7): The component 5-5 obtained in step (6) was separated and purified by HPLC. The mobile phase used in HPLC was methanol and 2 / 1000 aqueous trifluoroacetic acid solution. When separating and purifying by HPLC, the mobile phase gradient elution method was as follows: 0 - 2 min, 70% methanol, 30% aqueous trifluoroacetic acid solution; 2 - 30 min, gradually adjusted from 70% methanol, 30% aqueous trifluoroacetic acid solution to 100% methanol, 0% aqueous trifluoroacetic acid solution; 30 - 40 min, 100% methanol; finally, Compound I (15 mg, t R = 22.4 min) with the structure shown in Formula I and Compound II (3 mg, t R = 17.3 min) with the structure shown in Formula II were obtained.

[0047] The structures of Compound I and Compound II are shown in Formula I and Formula II as follows:

[0048]

[0049] The 1H NMR, 1 13C NMR, HSQC, HMBC and NOESY spectra of the compound shown in Formula I were measured using a 500 MHz nuclear magnetic resonance spectrometer, as 13 shown. The high-resolution electrospray ionization mass spectrometer was used to perform high-resolution mass spectrometry (HRESIMS) analysis on the compound shown in Formula I, and the results were as Figures 1-5 shown. From Figure 6 it was obtained that the [M+Na] Figure 6 peak of the compound shown in Formula I was 433.32874, and this figure was collected in the positive ion mode. From this, it was inferred that the molecular formula of this compound was C + 21H 25 H 46 16O4. Therefore, the structure of the compound shown in Formula I was determined through NMR, mass spectrometry and other data.

[0050] The 1H NMR, 1 13C NMR, HSQC, HMBC and NOESY spectra of the compound shown in Formula II were measured using a 500 MHz nuclear magnetic resonance spectrometer, as 13 shown. The high-resolution electrospray ionization mass spectrometer was used to perform high-resolution mass spectrometry analysis on the compound shown in Formula II, and the results were as Figures 7-11 shown. From Figure 12 it was obtained that the [M+Na] Figure 12 peak of the compound shown in Formula II was 433.32877, and this figure was collected in the positive ion mode. From this, it was inferred that the molecular formula of this compound was C + 21H 25 H 46 16O4. Therefore, the structure of the compound shown in Formula II was determined through NMR, mass spectrometry and other data.

[0051] Example 2: Antibacterial Activity Test of Sesterterpenoids

[0052] 1. Experimental Samples: The test samples were Compounds I and II separated and purified in Example 1 above. An appropriate amount of the sample was accurately weighed and dissolved in methanol to prepare a solution with a concentration of 10 mg / mL for activity testing. An appropriate amount of amphotericin B was dissolved in DMSO and prepared into 5 mg / mL.

[0053] 2. Antibacterial Activity Experimental Method:

[0054] Candida albicans (ATCC 10231) and Cryptococcus neoformans (ATCC 90012) were activated on YPD solid medium and statically cultured in an incubator at 37°C. After single colonies grew, single colonies were picked and inoculated into YPD liquid medium, and cultured overnight at 35°C and 200 rpm. When the OD 600 value was 0.8 - 1.0, 30 μL was aspirated, diluted a thousand times, and used for activity detection. In the sample group, 198 μL of the diluted bacterial solution was placed in the first column of a 96-well plate, and 2 μL of the test compound solution was added, and serially diluted two-fold to the last column. In the sample background control group, 198 μL of YPD medium was added to the first column, and 2 μL of the test compound solution was added, and serially diluted two-fold to the last column. In the bacterial solution group, 198 μL of the bacterial solution was added to the first column, and 2 μL of methanol or DMSO solvent was added, and serially diluted two-fold to the last column. Finally, a column of 200 μL of YPD medium control was set. The 96-well plate was placed in an incubator at 37°C for 12 - 24 hours, taken out and the absorbance (OD) value at 600 nm was measured on an enzyme-linked immunosorbent assay (ELISA) reader. The inhibition rate was calculated according to the following formula, and the drug concentration that inhibited 80% of the growth was taken as the minimum inhibitory concentration (MIC) 80 .

[0055] The inhibition rate was calculated according to the following formula:

[0056] Inhibition rate (%) = [1 - (OD value of the sample group - OD value of the sample background control group) / (OD value of the bacterial solution group - OD value of the medium control group)] * 100%

[0057] 3. The experimental results are as follows:

[0058] The antibacterial activities of Compounds I and II were studied, with the antibacterial drug amphotericin B as the positive control drug. The results showed that Compounds I and II had obvious inhibitory effects on both Candida albicans and Cryptococcus neoformans. The MIC of Compound I against Candida albicans and Cryptococcus neoformans 80were 0.9 μg / mL and 1.1 μg / mL respectively, and the MIC of Compound II against Candida albicans 80 was 3.1 μg / mL and 40.0 μg / mL (the MIC of the positive control drug amphotericin B against Candida albicans and Cryptococcus neoformans 80 were 0.4 μg / mL and 0.8 μg / mL respectively), as shown in Table 1. Furthermore, it shows that the prepared Compound I and II of the present invention have good inhibitory effects on Candida albicans and Cryptococcus neoformans.

[0059] Table 1. Inhibitory activities of Compound I and II against Candida albicans and Cryptococcus neoformans

[0060]

[0061]

[0062] 4. Conclusion

[0063] The compounds shown in Formula I and Formula II have good activities against Candida albicans and Cryptococcus neoformans, can be used as antibacterial preparations, and can also be applied to the preparation of new antibacterial drugs.

[0064] In addition, it should be noted that for the specific embodiments described in this specification, the shapes, names of the components, etc. can be different. Any equivalent or simple changes made according to the structure, features and principles described in the inventive concept of this patent are included in the protection scope of this patent. Those skilled in the technical field to which the present invention belongs can make various modifications or supplements to the described specific embodiments or use similar ways to replace them, as long as they do not deviate from the structure of the present invention or exceed the scope defined by this claim book, they should all belong to the protection scope of the present invention.

Claims

1. A sesterterpenoid compound, characterized in that, The structural formula of the diterpene sesquiterpenoid compound is shown in Formula I or Formula II: ， Formula I, ， Formula II; The [M+Na] peak of the compound represented by Formula I + is 433.32874; The [M+Na] of the compound represented by Formula II + has a peak at 433.32877.

2. The preparation method of the sesterterpenoid compound according to claim 1, characterized in that, The preparation method includes the following steps: S1: Solid-cultivate Pleurotus ostreatus and Trametes robiniophila; S2: Take the Pleurotus ostreatus and Trametes robiniophila obtained by cultivation in the step S1, and perform liquid cultivation respectively to obtain a Pleurotus ostreatus fermentation broth and a Trametes robiniophila fermentation broth; S3: Mix the Pleurotus ostreatus fermentation broth and the Trametes robiniophila fermentation broth in the step S2 and perform co-cultivation to obtain a co-cultivation fermentation broth; S4: Filter, perform organic extraction, and perform vacuum concentration on the co-cultivation fermentation broth in the step S3 to obtain a crude extract; S5: Perform column chromatography, separation and purification on the crude extract in the step S4 to obtain a diterpene sesquiterpenoid compound; The components and component contents of the medium for liquid cultivation in the step S2 are: 44 g / L - 48 g / L of glucose, 12 g / L - 16 g / L of sucrose, 4 g / L - 8 g / L of peptone, 0.3 g / L - 0.6 g / L of MgSO4·7H2O, 0.8 g / L - 1.2 g / L of KH2PO4, and the balance is water; The mixing volume ratio of the Pleurotus ostreatus fermentation broth and the Trametes robiniophila fermentation broth in the step S3 is 1:1; the co-cultivation time is 6 days - 8 days.

3. The preparation method of the sesterterpene compound according to claim 2, characterized in that, The temperature for solid cultivation in the step S1 is 26°C - 30°C, and the cultivation time is 6 days - 8 days; the temperature for liquid cultivation in the step S2 is 26°C - 30°C, and the cultivation time is 6 days - 8 days.

4. The preparation method of the sesterterpenoid compound according to claim 2, wherein, The specific steps of the step S4 are: Filter the co-cultivation fermentation broth to obtain mycelium and supernatant; The mycelium is extracted with the organic solvent methanol, and the obtained extract is concentrated and then extracted with ethyl acetate to obtain Extract I; The supernatant is extracted with the organic solvent ethyl acetate to obtain Extract II; Combine Extract I and Extract II and perform vacuum concentration to obtain a crude extract.

5. The preparation method of the sesterterpenoid compound according to claim 2, characterized in that, The specific steps of separation and purification in the step S5 are: After dissolving the crude extract, first perform gradient elution on a normal-phase silica gel column using petroleum ether - dichloromethane - methanol as the solvent, and the obtained fractions are subjected to gradient elution on a C-18 reversed-phase silica gel column using methanol - water as the solvent. The obtained sub-fractions are further separated and purified by HPLC to obtain a diterpene sesquiterpenoid compound.

6. The preparation method of the sesterterpenoid compound according to claim 5, wherein, When performing separation and purification by HPLC, the mobile phase used is methanol and a 2 / 1000 trifluoroacetic acid aqueous solution; The mobile phase gradient elution method is: 0 - 2 min, 70% methanol, 30% trifluoroacetic acid aqueous solution; 2 - 30 min, gradually adjust from 70% methanol, 30% trifluoroacetic acid aqueous solution to 100% methanol, 0% trifluoroacetic acid aqueous solution; 30 - 40 min, 100% methanol.

7. Use of the diterpene sesquiterpenoid compound according to claim 1 in the preparation of an antibacterial preparation or an antibacterial drug.

8. The application according to claim 7, wherein The pathogenic bacteria inhibited by the antibacterial preparation or the antibacterial drug include Candida albicans and Cryptococcus neoformans.

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