A sphingolipid compound and its preparation method and application

Abstract

This invention discloses a sphingolipid compound, its preparation method, and application. The invention utilizes co-cultivation and fermentation of two medicinal fungi, Coriolus versicolor and Ganoderma lucidum, to produce two sphingolipid compounds, with the structural formulas shown in Formula I or Formula II, respectively. Formula I represents a novel compound, and Formula II represents a novel natural product. These two sphingolipid compounds exhibit significant inhibitory activity against Candida albicans and Cryptococcus neoformans, and are potentially applicable to the development of antibacterial drugs. The preparation method of this invention opens up a new avenue for the preparation and development of novel antibacterial preparations. #imgabs0#

Description

Technical Field

[0001] The present invention relates to the field of biomaterial fermentation and culture, and in particular to a sphingolipid compound and a preparation method and application thereof. Background Art

[0002] Invasive fungal infections (IFIs) are infections caused by opportunistic fungi such as Candida albicans and Cryptococcus neoformans, primarily infecting deep-seated solid organs and / or the blood. Susceptible populations include immunocompromised patients, such as those with immunodeficiency, tumors, and organ transplants. The mortality rate is as high as 45%, resulting in approximately 1.7 million deaths worldwide each year. One of the main causes of high morbidity and mortality is antibiotic resistance, which is observed in almost all clinically used antibiotics. In addition to the worrying incidence of antibiotic resistance, many antibiotics, such as polyenes, echinocandins, and azoles, have been shown to be highly toxic or to cause acute adverse events. Given these challenges, the development of new antibiotic drugs is urgently needed.

[0003] Medicinal fungi are valuable sources of medicine, and their crude extracts have demonstrated diverse activities, including antibacterial, antitumor, and hypoglycemic properties. Among them, Coriolus versicolor and Ganoderma lucidum, two of the most sought-after medicinal fungi, possess enormous metabolic potential and could serve as important resources for discovering antimicrobial compounds. Summary of the Invention

[0004] Based on the medicinal value of Coriolus versicolor and Ganoderma lucidum, the present invention provides a sphingolipid compound, its preparation method, and application. This invention uses a co-culture and fermentation method of Coriolus versicolor and Ganoderma lucidum to produce a sphingolipid compound capable of inhibiting the activity of Candida albicans and Cryptococcus neoformans, thereby opening up a new approach for the preparation of antibacterial preparations.

[0005] In order to solve the above technical problems, the technical solution to be adopted in the present invention is:

[0006] The present invention provides a sphingolipid compound, the structural formula of which is shown in Formula I or Formula II:

[0007] .

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

[0009] S1: solid culture of Versicolor and Ganoderma;

[0010] S2: taking the versicolor and ganoderma lucidum cultured in step S1, and co-culturing them in a liquid to obtain a co-cultured fermentation liquid of versicolor and ganoderma lucidum;

[0011] S3: performing organic extraction and reduced-pressure concentration on the co-culture fermentation broth of step S2 to obtain a crude extract;

[0012] S4: separating and purifying the crude extract of step S3 to obtain sphingolipid compounds.

[0013] Furthermore, the solid culture temperature in step S1 is 26° C. to 30° C., and the culture time is 6 to 8 days.

[0014] Furthermore, the temperature of the liquid culture in step S2 is 26° C. to 30° C., and the culture time is 12 days to 16 days.

[0015] Furthermore, the components and content of the culture medium for liquid culture in step S2 are: glucose 8-12 g / L, peptone 1-3 g / L, MgSO4·7H2O 0.3-0.7 g / L, KH2PO4 0.5-1.5 g / L, and the balance is water.

[0016] Furthermore, the components and content of the liquid culture medium in step S2 are: glucose 10 g / L, peptone 2 g / L, MgSO4·7H2O 0.5 g / L, KH2PO4 1 g / L, and the balance is water.

[0017] Furthermore, the specific steps of step S3 are: using ethyl acetate to extract the co-culture fermentation liquid to obtain an extract, and using a rotary evaporator to concentrate under reduced pressure to obtain a crude extract.

[0018] Furthermore, the step S4 is to dissolve the crude extract and then separate and purify it by HPLC, with the mobile phase being methanol and water.

[0019] Furthermore, the specific steps of step S4 are: setting the mobile phase gradient elution method to: 0-2 min, 5% methanol, 95% water; 2-30 min, gradually adjusting from 5% methanol, 95% water to 100% methanol; 30-40 min, 100% methanol; the prepared components are then purified using the following gradient elution method: 0-2 min, 50% methanol, 50% water; 2-30 min, gradually adjusting from 50% methanol, 50% water to 100% methanol; 30-40 min, 100% methanol; thereby obtaining sphingolipid compounds.

[0020] The present invention also provides the use of the sphingolipid compound in the preparation of antibacterial preparations or antibacterial drugs.

[0021] Furthermore, the pathogenic bacteria inhibited by the antibacterial preparation or antibacterial drug include Candida albicans and Cryptococcus neoformans.

[0022] Compared with the existing technology, the advantages and beneficial effects of the present invention are as follows: the present invention simulates the state of competition or antagonism between Yunzhi and Ganoderma lucidum in the natural environment by co-culturing and fermenting them, aiming to activate or up-regulate their antibiotic biosynthesis pathways, and conducts activity detection and chemical analysis on the obtained mixed fermentation concentrate, thereby obtaining novel highly active antibacterial substances; research has found that the above co-cultivation method can obtain two sphingolipid compounds with antibacterial activity.

[0023] The present invention obtains two sphingolipid compounds by co-culturing Yunzhi and Ganoderma lucidum, including a new compound and a new natural product. Experimental data confirm that these two sphingolipid compounds can significantly inhibit the growth of Candida albicans and Cryptococcus neoformans. They can be used as antibacterial preparations and can also be used in the preparation of new antibacterial drugs, and have broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0025] Figure 1 The structural compound shown in Formula I in the embodiment of the present invention is 1 H NMR spectrum;

[0026] Figure 2 The structural compound shown in Formula I in the embodiment of the present invention is 13 C NMR spectrum;

[0027] Figure 3 HSQC spectrum of the compound of formula I in the embodiment of the present invention;

[0028] Figure 4 The HMBC spectrum of the compound of formula I in the embodiment of the present invention is shown;

[0029] Figure 5 This is the HRESIMS spectrum of the compound of formula I in the embodiment of the present invention;

[0030] Figure 6 The structural compound shown in formula II in the embodiment of the present invention is 1 H NMR spectrum;

[0031] Figure 7 The structural compound shown in formula II in the embodiment of the present invention is 13 C NMR spectrum;

[0032] Figure 8 This is the ESIMS spectrum of the compound represented by formula II in the embodiment of the present invention; Implementation Method

[0033] The present invention will be further described in detail below with reference to the examples. The following examples are intended to explain the present invention but the present invention is not limited to the following examples.

[0034] Example 1: Method for preparing sphingolipid compounds by fermenting and co-culturing versicolor and ganoderma lucidum

[0035] The specific preparation steps of the sphingolipid compound are as follows:

[0036] Step 1: Take appropriate amounts of mycelia of Coriolus versicolor and Ganoderma lucidum (from commercially available Coriolus versicolor and Ganoderma lucidum), inoculate them onto PDA solid plate culture medium, and culture them in an incubator at 28°C for 6-8 days, preferably 7 days.

[0037] Step 2: Take 3 pieces of Yunzhi and Ganoderma lucidum obtained in step 1, each with a diameter of about 0.5 mm, and inoculate them into a 500 mL conical flask containing 200 mL of culture medium. Ferment and culture at 28°C and 180 rpm for 12 to 16 days, preferably 14 days; wherein the components and content of the culture medium used are as follows: glucose 8 to 12 g / L, peptone 1 to 3 g / L, MgSO4·7H2O 0.3 to 0.7 g / L, KH2PO4 0.5 to 1.5 g / L, and the balance is water. In this embodiment, the preferred components and their contents are: glucose 10 g / L, peptone 2 g / L, MgSO4·7H2O 0.5 g / L, KH2PO4 1 g / L, and the balance is water.

[0038] Step 3: The co-culture fermentation broth (18 L) obtained in step 2 was extracted three times with ethyl acetate. The ethyl acetate extracts were combined and concentrated under reduced pressure using a rotary evaporator to obtain a crude extract (2.9 g).

[0039] Step 4: The crude extract obtained in step 3 was dissolved in methanol and separated and purified by HPLC using methanol and water as the mobile phase.

[0040] First, the mobile phase gradient elution method was set as follows: 0-2 min, 5% methanol, 95% water; 2-30 min, gradually adjusted from 5% methanol, 95% water to 100% methanol; 30-40 min, 100% methanol; 4 components were prepared. Component 3 was further purified using the following gradient elution method: 0-2 min, 50% methanol, 50% water; 2-30 min, gradually adjusted from 50% methanol, 50% water to 100% methanol; 30-40 min, 100% methanol; finally, compound I (3.2 mg, t R =16.7 min) and compound II (27.0 mg, tR =18.0 min)

[0041] The structural formulas of the compound I and compound II are shown in Formula I and Formula II, respectively:

[0042] .

[0043] The 500 MHz nuclear magnetic resonance spectrometer was used to determine the 1 H NMR, 13 C NMR, HSQC and HMBC spectra, such as Figure 1-4 The compound represented by formula I was subjected to high resolution mass spectrometry (HRESIMS) analysis using a high resolution electrospray ionization mass spectrometer. The results are shown in FIG. Figure 5 As shown by Figure 5 The [M+H] of the compound shown in formula I is obtained. + The peak is 300.2893, and the image was collected in positive ion mode. It is inferred that the molecular formula of the compound is C 18 H 37 Therefore, the structure of the compound shown in Formula I was confirmed by NMR, mass spectrometry and other data.

[0044] The 500 MHz nuclear magnetic resonance spectrometer was used to determine the 1 H NMR and 13 C NMR spectrum, such as Figure 6 and 7 The compound represented by Formula II was analyzed by low-resolution mass spectrometry (ESIMS) using a triple quadrupole mass spectrometer. The results are shown in Figure 8 As shown by Figure 8 The [M+H] of the compound shown in formula I is obtained. + The peak is 302.3, and the image was collected in positive ion mode, from which the molecular weight of the compound was inferred to be 301. Therefore, the structure of the compound represented by Formula II was determined by analyzing NMR, mass spectrometry and other data and comparing with literature data.

[0045] Example 2: Antibacterial activity test of the sphingolipid compound

[0046] 1. Experimental Samples: Test samples were Compounds I and II isolated and purified in Example 1. Accurately weigh an appropriate amount of sample and prepare a 5 mg / mL solution in methanol for activity measurement. Dissolve appropriate amounts of nystatin and fluconazole in methanol to a 5 mg / mL concentration as positive controls.

[0047] 2. Antibacterial activity experimental method:

[0048] Candida albicans ( Candida albicans ) and Cryptococcus neoformans ( Cryptococcus neoformans ) were inoculated onto YPD solid medium and incubated at 37°C in an incubator for activation. After 2-3 days of incubation, a single colony was picked and inoculated into YPD liquid medium and incubated at 30°C, 200 rpm until OD 600 When the value is about 0.6, dilute 1000 times and use it for activity detection. μ Place L bacterial solution in the first row of a 96-well plate and add 2 μ L of the test compound or positive control solution, dilute it twice to the last row. In addition, set up a blank control group (bacterial solution) and a negative control group (add 2 μ L methanol). Place the 96-well plate in a 37 °C constant temperature incubator and incubate for 12-24 h. Then observe the minimum inhibitory concentration (MIC) of the sample, which is the minimum concentration that completely inhibits the growth of the strain.

[0049] 3. The experimental results are as follows

[0050] The antibacterial activity of the compounds I and II was studied, with the antibacterial drugs nystatin and fluconazole as positive control drugs. The results showed that the compounds I and II had significant inhibitory effects on Candida albicans and Cryptococcus neoformans, with MICs of 3.13. μ g / mL (MIC of positive control drugs nystatin and fluconazole against Candida albicans and Cryptococcus neoformans were both 6.25 μ g / mL), as shown in Table 1. It is further shown that compounds I and II prepared in the present invention have obvious inhibitory effects on Candida albicans and Cryptococcus neoformans.

[0051] Table 1. Inhibitory activity of compounds Ⅰ and Ⅱ against Candida albicans and Cryptococcus neoformans

[0052]

[0053] 4. Conclusion

[0054] The compounds represented by Formula I and Formula II provided by the present invention have significant activity in inhibiting Candida albicans and Cryptococcus neoformans, can be used as antibacterial preparations, and can also be applied to the preparation of new antibacterial drugs.

[0055] Furthermore, it should be noted that the specific embodiments described in this specification may vary in the shapes and names of their components. Any equivalent or simple variations based on the structure, features, and principles described in the patented concept of this invention are included within the scope of protection of this patent. Persons skilled in the art may make various modifications, additions, or substitutions to the described specific embodiments, and these modifications, as long as they do not deviate from the structure of the invention or exceed the scope defined by the claims, shall fall within the scope of protection of this invention.

Claims

1. A sphingolipid compound, characterized in that The structural formula of the sphingolipid compound is shown in Formula I: ， Formula I.

2. The method for preparing a sphingolipid compound according to claim 1, wherein The preparation method comprises the following steps: S1: solid culture of Versicolor and Ganoderma; S2: taking the versicolor and ganoderma obtained by culture in step S1, and co-culturing them in a liquid to obtain a co-cultured fermentation liquid of versicolor and ganoderma; S3: performing organic extraction and reduced-pressure concentration on the co-culture fermentation broth of step S2 to obtain a crude extract; S4: separating and purifying the crude extract of step S3 to obtain the sphingolipid compound.

3. The method for preparing a sphingolipid compound according to claim 2, wherein: The solid culture temperature in step S1 is 26° C. to 30° C., and the culture time is 6 to 8 days.

4. The method for preparing a sphingolipid compound according to claim 2, wherein: The temperature of the liquid co-culture in step S2 is 26° C. to 30° C., and the time is 12 to 16 days.

5. The method for preparing a sphingolipid compound according to claim 2 or 4, characterized in that: The components and content of the liquid co-culture medium are as follows: glucose 8-12 g / L, peptone 1-3 g / L, MgSO4·7H2O 0.3-0.7 g / L, KH2PO4 0.5-1.5 g / L, and the balance is water.

6. The method for preparing a sphingolipid compound according to claim 2, wherein: The specific steps of step S3 are: extracting the co-culture fermentation broth with ethyl acetate to obtain an extract, and concentrating under reduced pressure to obtain a crude extract.

7. The method for preparing a sphingolipid compound according to claim 2, wherein: The separation and purification in step S4 is to dissolve the crude extract and then separate and purify it by HPLC, with the mobile phase being methanol and water.

8. The method for preparing a sphingolipid compound according to claim 7, wherein: The specific steps of the separation and purification are: setting the mobile phase gradient elution method as follows: 0-2 minutes, 5% methanol, 95% water; 2-30 minutes, gradually adjusting from 5% methanol, 95% water to 100% methanol; 30-40 minutes, 100% methanol; then purifying the obtained components using the following gradient elution method: 0-2 minutes, 50% methanol, 50% water; 2-30 minutes, gradually adjusting from 50% methanol, 50% water to 100% methanol; 30-40 minutes, 100% methanol; thereby obtaining sphingolipid compounds.

9. Use of a sphingolipid compound in the preparation of an antibacterial preparation or an antibacterial drug, characterized in that: The pathogenic bacteria inhibited by the antibacterial preparation or antibacterial drug are Candida albicans and Cryptococcus neoformans; the structural formula of the sphingolipid compound is as shown in Formula I or Formula II: ， Formula I ， Formula II.

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