A seven-membered ring indole terpene compound and its preparation method and use

Abstract

The invention discloses a seven-membered ring indole terpenoid compound and a preparation method and use thereof. The compound is characterized in that the structural formula is shown in I. The preparation method comprises the steps of obtaining a fermentation product of a seven-membered ring indole terpenoid compound by fermenting and culturing Cladosporium marineum with a preservation number of CCTCC NO: M2014098, then soaking the fermentation product in ethyl acetate to extract a crude extract, and then subjecting the crude extract to normal phase silica gel column chromatography, reverse medium pressure column chromatography and reverse phase semi-preparative high performance liquid chromatography separation and purification. The seven-membered ring indole terpenoid compound has an anti-candida albicans effect and can be used to develop a drug for preventing and treating fungal infections of the human upper respiratory tract caused by candida albicans.

Description

Technical Field

[0001] The invention relates to a seven-membered ring indole terpenoid compound, in particular to a seven-membered ring indole terpenoid compound extracted from marine fungi, and a preparation method and application thereof. Background Art

[0002] Indole terpenoids are a class of natural products that share a common structural skeleton consisting of a cyclic diterpene linked to an indole moiety. These compounds are usually isolated from fungi and have great structural diversity based on the size of the macrocyclic ring and the different substituents on the ring. Since their first isolation in 1975, indole terpenoids have attracted great attention due to their powerful and diverse biological activities. Indole terpenoids have been shown to have antiviral, anti-inflammatory, antibacterial, and anticancer activities.

[0003] The inventors have discovered a new natural product that resists Candida albicans in a chemical investigation of an ethyl acetate extract of a marine fungus fermented in a rice culture medium. There are no reports on the chemical structure of the compound and its anti-Candida albicans activity, and therefore no related drugs have been found on the market. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a seven-membered ring indole diterpenoid compound having an inhibitory effect on Candida albicans, and a preparation method and use thereof.

[0005] The technical solution adopted by the present invention to solve the above technical problems is:

[0006] 1. A seven-membered ring indole terpene compound, the structural formula of which is shown in (I);

[0007]

[0008] 2. A method for preparing a seven-membered ring indole terpene compound, comprising the following steps:

[0009] (1) Fermentation production

[0010] The Cladosporium sphaerospermum YS3-1-5 with a preservation number of CCTCC NO: M2014098 was streaked on a plate of a PDA medium, and after being cultured and activated in an incubator at 28° C. for 7 days, a single colony was picked and inoculated into a PDB liquid medium, and the culture was placed on a shaker at a temperature of 28° C. and a speed of 180 rpm / min to increase the bacteria, and the seed liquid was collected after culturing for 3 days, and then the seed liquid was inoculated into a rice solid medium at an inoculum amount of 10% by volume, and the culture was carried out at a temperature of 28° C. for 30 days to obtain a fermentation product;

[0011] (2) Extraction

[0012] Add an equal amount of ethyl acetate to the fermented product obtained in step (1), extract repeatedly for 3 times, and then evaporate the ethyl acetate extract under reduced pressure to obtain a crude extract;

[0013] (3) Isolation and preparation of compounds

[0014] The crude extract obtained in step (2) is first dissolved in a mixed solvent of dichloromethane and methanol in a volume ratio of 1:1, and then 200-300 mesh silica gel is added to mix the sample, and normal phase medium pressure column chromatography is performed, and petroleum ether-ethyl acetate solution in a volume ratio of (100:1) to (0:1) is used as an eluent for gradient elution, and the eluted fractions are collected and arranged from small to large according to the polarity of the fractions, and 6 components are obtained by merging; the collected second component is subjected to reverse phase medium pressure column chromatography gradient elution, and acetonitrile-water with a volume percentage of 25-100% is used as an eluent, and the eluted fractions are collected and arranged from large to small according to the polarity of the fractions, and 38 components are obtained by merging; the obtained eighth component is separated and purified by semi-preparative reverse phase high performance liquid chromatography using a solution of acetonitrile and water in a volume ratio of 34:66 as a mobile phase to obtain a compound, whose structure is shown in (I)

[0015]

[0016] Furthermore, the rice culture medium described in step (1) is prepared as follows: 90 g of rice and 25 g of sea salt are dissolved in 110 mL of sea water.

[0017] Furthermore, the preparation method of the PDB liquid culture medium described in step (1) is as follows: 200 grams of potatoes, 20 grams of glucose, 20 grams of agar, 5 grams of peptone, 3 grams of potassium dihydrogen phosphate, and 1.5 grams of magnesium sulfate are dissolved in 1000 ml of seawater; the preparation method of the PDA solid culture medium is as follows: 200 grams of potatoes, 20 grams of glucose, 20 grams of agar, 5 grams of peptone, 3 grams of potassium dihydrogen phosphate, 1.5 grams of magnesium sulfate, and 18 grams of agar powder are dissolved in 1000 ml of seawater.

[0018] Furthermore, the elution gradient volume ratios of the petroleum ether-ethyl acetate solution in step (3) are 100:1, 50:1, 20:1, 10:1, 5:1, 2:1, 1:1 and 0:1, respectively.

[0019] Furthermore, in the reverse phase medium pressure column chromatography gradient elution in step (3), the volume percentage of acetonitrile is from 25 to 100%, and the elution time is 200 min.

[0020] Furthermore, the flow rate of the compound separation preparation by semi-preparative reverse-phase HPLC described in step (3) is 2.0 mL / min.

[0021] 3. Use of the above seven-membered ring indole terpenoid compounds in the preparation of Candida albicans and cell line inhibitors.

[0022] Compared with the prior art, the advantages of the present invention are: the present invention separates a seven-membered ring indole terpene compound, obtains the fermentation product of indole terpene by microbial fermentation and culture, then extracts the fermentation product by soaking in ethyl acetate to obtain a crude extract, and then separates and purifies the crude extract by medium-pressure forward silica gel column chromatography, medium-pressure reverse column chromatography, and reverse-phase semi-preparative high performance liquid chromatography; in addition, through the screening of antifungal activity, it is found that the compound has effective anti-Candida albicans activity and effective inhibitory effect on cell lines, and can be used for the development of drugs for inhibiting related human upper respiratory tract fungal infections caused by Candida albicans.

[0023] The above-mentioned Cladosporium sphaerospermum YS3-1-5 was deposited in China Center for Type Culture Collection on March 21, 2014, with a deposit number of CCTCC No: M2014098, and the deposit address is Wuhan University, Wuhan, China. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 is the hydrogen nuclear magnetic resonance spectrum of the compound of the present invention;

[0025] Figure 2 The carbon NMR spectrum of the compound of the present invention is

[0026] Figure 3 is the nuclear magnetic resonance DEPT-135 spectrum of the compound of the present invention;

[0027] Figure 4 is the COSY nuclear magnetic resonance spectrum of the compound of the present invention;

[0028] Figure 5 is the nuclear magnetic resonance HSQC spectrum of the compound of the present invention;

[0029] Figure 6 is the nuclear magnetic resonance HSQC spectrum of the compound of the present invention;

[0030] Figure 7 is the nuclear magnetic resonance NOESY spectrum of the compound of the present invention;

[0031] Figure 8 is the ECD absolute configuration fitting curve of the compound of the present invention Figure 1 ;

[0032] Fig. 9 is the ECD absolute configuration fitting curve of the compound of the present invention Figure 2 . DETAILED DESCRIPTION

[0033] The present invention is further described in detail below with reference to the embodiments.

[0034] Example 1

[0035] The structural formula of a seven-membered ring indole terpene compound is shown in (I):

[0036]

[0037] Example 2

[0038] The method for preparing the indole terpenoid compound shown in structural formula (I) of Example 1 specifically comprises the following steps:

[0039] (1) Fermentation production

[0040] Cladosporium sphaerospermum YS3-1-5 with a preservation number of CCTCC NO: M2014098 was streaked on a plate of PDA solid culture medium, and after being cultured and activated in an incubator at 28° C. for 7 days, a single colony was picked and inoculated into PDB liquid culture medium, and the culture was placed on a shaker at a temperature of 28° C. and a speed of 180 rpm / min to increase the bacteria. After culturing for 3 days, the seed liquid was collected, and then the seed liquid was inoculated into a rice culture medium at an inoculum amount of 10% by volume, and the culture was carried out at a temperature of 28° C. for 30 days to obtain a fermentation product, wherein the rice The preparation method of the culture medium is as follows: 90g of rice and 25g of sea salt are dissolved in 110mL of seawater; the preparation method of the PDB liquid culture medium is as follows: 200g of potato, 20g of glucose, 20g of agar, 5g of peptone, 3g of potassium dihydrogen phosphate, and 1.5g of magnesium sulfate are dissolved in 1000ml of seawater; the preparation method of the PDA solid culture medium is as follows: 200g of potato, 20g of glucose, 20g of agar, 5g of peptone, 3g of potassium dihydrogen phosphate, 1.5g of magnesium sulfate, and 18g of agar powder are dissolved in 1000ml of seawater;

[0041] (2) Extraction

[0042] Add an equal amount of ethyl acetate to the fermented product obtained in step (1), and soak the mixture for three times, then evaporate the ethyl acetate extract under reduced pressure to obtain a crude extract;

[0043] (3) Isolation and preparation of compounds

[0044] First, it was dissolved in a mixed solvent of dichloromethane and methanol in a volume ratio of 1:1, and then 200-300 mesh silica gel was added to mix the sample. Normal phase medium pressure column chromatography was performed, and gradient elution was performed using a petroleum ether-ethyl acetate solution in a volume ratio of (100:1) to (0:1) (the elution gradient volume ratio of the petroleum ether-ethyl acetate solution was 100:1, 50:1, 20:1, 10:1, 5:1, 2:1, 1:1 and 0:1, respectively) as the eluent. The eluted fractions were collected and sorted according to the polarity of the fractions. Arrange from small to large, and merge to obtain 6 components; the collected second component is subjected to reverse phase medium pressure column chromatography gradient elution, using acetonitrile-water with a volume percentage of 25-100% as an eluent, collecting the eluted fractions, and arranging them from large to small according to the polarity of the fractions, and merging to obtain 38 components; the obtained eighth component is separated and purified by semi-preparative reverse phase high performance liquid chromatography using a solution of acetonitrile and water mixed in a volume ratio of 34:66 as a mobile phase to obtain a compound, whose structure is shown in (I):

[0045]

[0046] Example 3

[0047] Structural identification of compounds and attribution of NMR signals

[0048] Compound I of the present invention is a white powder. High-resolution mass spectrometry (HR-ESI-MS) in positive ion mode gives its quasi-molecular ion peak m / z 536.3376 [M+H] + . Combined 13 C NMR spectrum confirmed that its molecular formula is C 33 H 45 NO5, by treating compound I 1 H- 1 For analysis of H coupling constants and NOESY data, see Figures 1 to 7 The relative configurations of 3, 4, 9, 12 and 18, 20 were determined, and the absolute configurations were simulated and calculated by NMR. Finally, the ECD simulation data was verified to be consistent with the actual ECD data, see Figure 8 and 9 Therefore, the absolute configuration of compound I is 3S, 4R, 9S, 12S, 18R, 20S.

[0049] The compound 1 H and 13 C NMR spectrum data are shown in Table 1:

[0050] Table 1. 1D NMR data of compound I (DMSO-d6, δ in ppm, J in Hz)

[0051]

[0052]

[0053] Note 1: s—singlet, d—doublet, t—triplet, q—quartet, m—multiplet;

[0054] Note 2: 1 H was obtained at 600 MHz NMR; 13 C was obtained at 150 MHz NMR.

[0055] Example 4

[0056] Antifungal activity and application of indole terpenoid compounds described in Example 1

[0057] (1) Experimental samples

[0058] Preparation of the sample solution: The test sample is the pure compound I separated and purified in Example 1 above. Accurately weigh an appropriate amount of the sample and use DMSO to prepare a solution of the required concentration for testing antifungal activity. The indicator bacteria used in this experiment is Candida albicans (ATCC10231).

[0059] (2) Experimental methods

[0060] Oxford cup antifungal test method: 100 μL of spore suspension was evenly applied on the PDA plate, and an Oxford cup was placed in the center of the plate. 100 μL of compound solutions of different concentrations were added to the Oxford cups, with concentrations of 128 μg / mL, 64 μg / mL, 32 μg / mL, 16 μg / mL, 8 μg / mL, 4 μg / mL, and 2 μg / mL. DMSO was used as a negative control, and the same concentration of prochloraz was used as a positive control. After culturing at 28°C for 5 days, the fungal growth was observed. Four culture plates were set up for each treatment, and the entire experiment was repeated 3 times.

[0061] (3) Experimental results

[0062] In the Oxford cup antifungal test, the MIC of compound Ⅰ against Candida albicans (ATCC10231) was determined to be 64 μg / mL.

[0063] The above description is not intended to limit the present invention, and the present invention is not limited to the above examples. Any changes, modifications, additions or substitutions made by a person skilled in the art within the spirit and scope of the present invention shall also fall within the protection scope of the present invention.

Claims

1. A seven-membered ring indole terpene compound, characterized in that The structural formula of the indole terpenoid compound is shown as (I); 2. A method for preparing the seven-membered ring indole terpene compound according to claim 1, characterized in that The steps include: (1) Fermentation production Cladosporium sphaerospermum YS3-1-5) streaking on a plate of PDA solid medium, culturing and activating in an incubator at 28°C for 7 days, picking a single colony and inoculating it in PDB liquid medium, culturing and growing the bacteria on a shaker at 28°C and 180 rpm / min, collecting the seed liquid after culturing for 3 days, and then inoculating the seed liquid into a rice solid medium at an inoculum volume ratio of 10%, culturing at 28°C for 30 days, and obtaining a fermentation product; (2) Extraction Add an equal amount of ethyl acetate to the fermented product obtained in step (1), and soak the mixture for three times, then evaporate the ethyl acetate extract under reduced pressure to obtain a crude extract; (3) Isolation and preparation of compounds The crude extract obtained in step (2) is first dissolved in a mixed solvent of dichloromethane and methanol in a volume ratio of 1:1, and then 200-300 mesh silica gel is added to mix the sample, and normal phase medium pressure column chromatography is performed, and petroleum ether-ethyl acetate solution in a volume ratio of (100:1) to (0:1) is used as an eluent for gradient elution, and the eluted fractions are collected and arranged in descending order according to the polarity of the fractions, and 6 components are obtained by merging; the collected second component is subjected to reverse phase medium pressure column chromatography gradient elution, and acetonitrile-water in a volume ratio of 25-100% is used as an eluent, and the eluted fractions are collected and arranged in descending order according to the polarity of the fractions, and 38 components are obtained by merging; the obtained eighth component is separated and purified by semi-preparative reverse phase high performance liquid chromatography using a solution of acetonitrile and water in a volume ratio of 34:66 as a mobile phase to obtain a compound, whose structure is shown in (I):

3. The method for preparing a seven-membered ring indole terpene compound according to claim 2, characterized in that The rice culture medium described in step (1) is prepared as follows: 90 g of rice and 25 g of sea salt are dissolved in 110 mL of sea water.

4. The method for preparing a seven-membered ring indole terpene compound according to claim 2, characterized in that The preparation method of the PDB liquid culture medium described in step (1) is as follows: 200 grams of potatoes, 20 grams of glucose, 20 grams of agar, 5 grams of peptone, 3 grams of potassium dihydrogen phosphate, and 1.5 grams of magnesium sulfate are dissolved in 1000 ml of seawater to prepare the preparation; the preparation method of the PDA solid culture medium is as follows: 200 grams of potatoes, 20 grams of glucose, 20 grams of agar, 5 grams of peptone, 3 grams of potassium dihydrogen phosphate, 1.5 grams of magnesium sulfate, and 18 grams of agar powder are dissolved in 1000 ml of seawater to prepare the preparation.

5. The method for preparing a seven-membered ring indole terpene compound according to claim 2, characterized in that The elution gradient volume ratios of the petroleum ether-ethyl acetate solution in step (3) are 100:1, 50:1, 20:1, 10:1, 5:1, 2:1, 1:1 and 0:1, respectively.

6. The method for preparing a seven-membered ring indole terpene compound according to claim 2, characterized in that: In the reverse phase medium pressure column chromatography gradient elution described in step (3), the volume percentage of acetonitrile ranges from 25 to 100%, and the elution time is 200 min.

7. The method for preparing a seven-membered ring indole terpene compound according to claim 2, characterized in that: The flow rate of the semi-preparative reversed-phase HPLC compound separation preparation described in step (3) is 2.0 mL / min.

8. Use of the seven-membered ring indole terpenoid compound according to claim 1 in the preparation of a Candida albicans inhibitor.

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