A sorbicillin hybrid dimer compound, sorbicillalanine A, its preparation method, and its application in the preparation of anti-inflammatory drugs.

Abstract

The application discloses a sorbicillin hybrid dimer compound, a preparation method thereof and application of the compound in preparation of anti-inflammatory drugs. An anti-inflammatory compound sorbicillalanine A is prepared from liquid fermentation of a deep-sea penicillium SCSIO06871, the sorbicillalanine A is a new compound, and the structure is shown as formula (I). Anti-inflammatory activity evaluation shows that the sorbicillalanine A has a significant inhibitory effect on NO produced by a mouse macrophage cell strain RAW264.7 induced by lipopolysaccharide (LPS), can inhibit expression of pro-inflammatory factors IL-6 and MCP-1, promote expression of anti-inflammatory factors IL-4, IL-10 and Arg-1, and has no cytotoxic activity on the cell strain RAW264.7 at the same concentration, so the sorbicillalanine A can be used as a candidate lead compound for development of anti-inflammatory drugs.

Description

Technical fields:

[0001] This invention belongs to the field of natural products, specifically relating to the isolation and purification of microbial natural products and their application in the preparation of anti-inflammatory drugs. Background technology:

[0002] Inflammation is a complex biological response regulated by various chemical mediators released by cells in response to various stimuli of injury. Appropriate inflammatory responses help the body eliminate pathogens and protect it from harm. However, if the inflammatory response is too intense or persistent, it may lead to autoimmune or autoinflammatory diseases, neurological disorders, or cancer. Inflammation has become a pathological feature of most prevalent diseases, such as diabetes, atherosclerosis, cancer, epilepsy, asthma, and neurodegenerative diseases. Natural products are valuable resources for developing novel anti-inflammatory drugs and other medications for human diseases. Microorganisms possess unique advantages such as repeatable fermentation and sustainable development. The unique characteristics of the deep-sea marine ecosystem have created unique secondary metabolites from deep-sea microorganisms, making them an important source of innovative drug discovery. Therefore, screening for drug lead compounds with good anti-inflammatory activity from deep-sea microorganisms is of great significance for the rational utilization of deep-sea microbial drug resources. Summary of the Invention:

[0003] The first objective of this invention is to provide a sorbicillin hybrid dimer compound, sorbicillalanine A, which has anti-inflammatory activity.

[0004] The sorbicillin hybrid dimer compound sorbicillalanine A of the present invention has the structure shown in formula (I):

[0005]

[0006] A second object of the present invention is to provide the use of sorbicillin hybrid dimer compound sorbicillalanine A, as shown in formula (I), in the preparation of anti-inflammatory drugs.

[0007] A third objective of this invention is to provide the use of deep-sea-derived Penicillium sp. SCSIO06871 in the preparation of the aforementioned sorbicillin hybrid dimer compound sorbicillalanine A.

[0008] The fourth objective of this invention is to provide a method for preparing the above-mentioned sorbicillin hybrid dimer compound sorbicillalanine A, comprising the following steps:

[0009] A. Preparation of fermentation culture of Penicillium sp. SCSIO06871 from deep sea;

[0010] B. Separate the fermentation broth and mycelium. Extract the fermentation broth with ethyl acetate, and concentrate the ethyl acetate extract to obtain a fermentation broth extract. Cut the mycelium into small pieces, soak it in ethyl acetate, and extract it using ultrasound. Concentrate the ethyl acetate extract to obtain a mycelium extract. Combine the fermentation broth extract and the mycelium extract, and separate them using normal-phase silica gel. Elute the fractions Fr1-Fr9 in a dichloromethane:methanol gradient from 100:0, 100:1, 100:2, 100:3, 100:5, 100:10, 100:20, 0:100, v / v. Collect fraction Fr8 eluted with dichloromethane:methanol at 100:5 v / v, and first process it using Sephadex. LH-20 column chromatography was performed using methanol as the eluent, followed by medium-pressure reversed-phase column chromatography with acetonitrile:water gradients of 10:90, 20:80, 25:75, 35:65, 60:40, 100:0, v / v. The product eluted at 25:75, v / v was purified by high-performance liquid chromatography to obtain the sorbicillin hybrid dimer compound sorbicillalanineA.

[0011] Preferably, the fermentation culture for preparing the deep-sea-derived Penicillium sp. SCSIO06871 is obtained by inoculating the deep-sea-derived Penicillium sp. SCSIO06871 into a fermentation medium and culturing it. The fermentation medium contains, per 1000 mL: 10 g glucose, 20 g maltose, 20 g mannitol, 1 g corn steep liquor, 10 g monosodium glutamate, 0.5 g dipotassium hydrogen phosphate, 0.3 g magnesium sulfate heptahydrate, 3 g yeast extract, and 15 g sea salt, dissolved in an appropriate amount of water and diluted to 1000 mL with water.

[0012] Preferably, the culture is carried out at 28°C and 180 rpm for 48 hours to obtain a seed culture, which is then inoculated into the fermentation medium at a volume ratio of 3% and cultured statically at 25°C for 32 days to obtain the fermentation product of Penicillium sp.SCSIO06871.

[0013] Preferably, the high-performance liquid chromatography (HPLC) purification is performed using 210 nm and 270 nm wavelengths for detection, a flow rate of 2 ml / min, and isometric elution with methanol:water = 65:35, v / v for semi-preparative HPLC separation. The HPLC (YMC-pack ODS-A, 10×250 mm, 5 μm) is used, and the result is recorded at retention time t. R The sorbicillin hybrid dimer compound sorbicillalanineA was obtained after 12.0 min.

[0014] The inventors obtained compound 1 by amplifying the liquid static fermentation of a deep-sea Penicillium sp. SCSIO06871 and purifying the fermentation extract. Structural analysis identified it as sorbicillin A, a heterodimer of sorbicillin, as shown in formula (Ⅰ). Evaluation of the anti-inflammatory activity of compound 1 revealed that it significantly inhibited NO production induced by lipopolysaccharide (LPS) in mouse macrophage cell line RAW264.7, while simultaneously inhibiting the expression of pro-inflammatory cytokines IL-6 and MCP-1 genes and promoting the expression of anti-inflammatory cytokines IL-4, IL-10, and Arg-1 genes. Furthermore, at the same concentration, it showed no cytotoxic activity against RAW264.7 cell line, making it a potential lead compound for anti-inflammatory drug development.

[0015] The deep-sea-derived Penicillium sp. SCSIO06871 of this invention was deposited on February 4, 2024, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, 510070, China, with accession number GDMCC No: 64307. Attached image description:

[0016] Figure 1 Sorbicillalanine A(1) main 1 H- 1 H COSY and HMBC information;

[0017] Figure 2 Sorbicillalanine A inhibits NO release activity; where control represents the blank control, LPS represents lipopolysaccharide treatment (100 ng / mL), L-NMMA is a NO synthase inhibitor (10 μM), which represents the positive control group, i.e., LPS plus L-NMMA treatment, and 1, 5, 10 represent LPS plus Sorbicillalanine A at concentrations of 1 μM, 5 μM, and 10 μM, respectively.

[0018] Figure 3 Sorbicillalanine A inhibits the activity of pro-inflammatory factors and promotes the activity of anti-inflammatory factors. In the chart, control represents the blank control, LPS represents lipopolysaccharide treatment, and LPS+1 represents LPS plus compound 1 treatment. Each group of bars, from left to right, represents control, LPS, and LPS+Sorbicillalanine A. Detailed implementation method:

[0019] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0020] Example 1: Preparation and structural identification of compound 1

[0021] I. Preparation of Compound 1 as shown in Formula (I)

[0022] 1. Microbial culture conditions:

[0023] Each 1000mL culture medium contains: 10g glucose, 20g maltose, 20g mannitol, 1g corn steep liquor, 10g monosodium glutamate, 0.5g dipotassium hydrogen phosphate, 0.3g magnesium sulfate heptahydrate, 3g yeast extract, and 15g sea salt. Dissolve these ingredients in an appropriate amount of water, bring the volume to 1000mL, and sterilize at 121℃ for 20 minutes.

[0024] The deep-sea-derived Penicillium sp.SCSIO06871 was inoculated into the above-mentioned culture medium and cultured on a shaker at 28°C and 180 rpm for 2 days to obtain a seed culture. The seed culture was then inoculated into the above-mentioned culture medium at a volume ratio of 3% and cultured statically at 25°C for 32 days to obtain the fermentation product of the deep-sea-derived Penicillium sp.SCSIO06871.

[0025] 2. Extraction and separation:

[0026] The fermentation product of the deep-sea-derived Penicillium sp. SCSIO06871 was centrifuged to obtain the supernatant and mycelium. The supernatant fermentation broth was extracted three times with equal volumes of ethyl acetate. The ethyl acetate extract was concentrated under reduced pressure below 40°C to obtain a fermentation broth extract. The mycelium was shredded with scissors, soaked in ethyl acetate solution, and ultrasonically extracted. The extract was concentrated under reduced pressure by distillation to obtain a mycelial extract. The fermentation broth extract and mycelial extract were combined to obtain approximately 72.5 g of extract. This extract was separated using normal-phase silica gel. After mixing and dry packing, a gradient elution of dichloromethane:methanol (100:0, 100:1, 100:2, 100:3, 100:5, 100:10, 100:20, 0:100, v / v) was used to obtain nine fractions Fr1-Fr9. Fraction Fr8 (the fraction eluted by dichloromethane:methanol 100:5 v / v) was first subjected to Sephadex... LH-20 column chromatography, using methanol as eluent, followed by medium-pressure reversed-phase column chromatography with acetonitrile:water gradient elution at 10:90, 20:80, 25:75, 35:65, 60:40, 100:0, v / v. The product eluted at 25:75, v / v was detected at 210 and 270 nm wavelengths. Semi-preparative high-performance liquid chromatography (HPLC) was performed at a flow rate of 2 mL / min with isogradient elution of methanol:water (65:35, v / v). The new compound 1 (7.0 mg, retention time t) was obtained. R 12.0 min).

[0027] II. Structural Identification of Compound 1

[0028] The chemical structure of compound sorbicillalanine A was determined by high-resolution mass spectrometry (HRESIMS), one-dimensional and two-dimensional nuclear magnetic resonance (NMR) data analysis.

[0029] Compound 1: Colorless oil; High-resolution mass spectrometry (HRESIMS) m / z 420.1648 [M+H] + (calcd forC 21 H 26 NO8), the suggested molecular formula is C 21 H 25 NO8 has an unsaturation degree of 10. 1 H and 13 The C10 NMR data are shown in Table 1; the NMR data show that the molecule contains 21 carbon atoms, including 4 methyl groups, 3 methylene groups, 4 methine groups, and 10 quaternary carbons. Observation of one-dimensional and two-dimensional proton NMR spectra revealed 5 ester carbonyl groups, 2 double bond signals, and 3 unsaturation levels, indicating that there are 3 cyclic structural segments in the molecule. 1 H-1 The 1H COSY spectrum shows a two-spin coupled system containing H2-2' / H2-3' / H-4' / H-5' / H3-6' and H-2” / H2-3” segments. Combined with HMBC C-H long-range correlation, compound 1 was found to be very similar to the N-containing sorbicillinoid structure sorbicillactone B. Careful comparison revealed that compound 1 forms a new chemical bond at the C-2” and C-1 positions of sorbicillactone B, adding a six-membered ring compared to sorbicillactone B. Furthermore, HMBC correlation signals from H-2” and H2-3” to C-1; H-2” to C-1, C-2, and C-6; and H-6 to C-2” confirm this inference. A SciFinder search identified compound 1 as a novel compound, named sorbicillalanine A. The main components of compound 1 are... 1 H- 1 For H COSY and HMBC information, please see [link / details]. Figure 1 .

[0030] Table 1. Nuclear magnetic resonance spectral data of Sorbicillalanine A (500MHz, CD3OD, ppm)

[0031]

[0032]

[0033] The structural formula of compound 1 is shown in formula (I), and it is named Sorbicillalanine A:

[0034]

[0035] Example 2: Experimental data on the anti-inflammatory activity of Sorbicillalanine A

[0036] The effects of sorbicillalanine A on NO levels in RAW264.7 cells were detected using the Griess method (V. Dirschl et al., Planta Medica, 1998, 64, 423-426). The effects of sorbicillalanine A on pro-inflammatory cytokine levels in RAW264.7 cells were detected using an ELISA kit. The gene expression levels of macrophage polarization-related factors were detected by RT-PCR (M. Conget et al., Phytochemistry, 2023, 208, 113593). The experimental results showed that sorbicillalanine A (1) could effectively inhibit the trend of LPS-induced pro-inflammatory cytokine (IL-6, TNF-α, and MCP-1) levels. Figure 3 A), and can also effectively inhibit the mRNA expression of IL-6 and MCP-1 pro-inflammatory factors (A). Figure 3 B), and promotes the expression of anti-inflammatory factors IL-4, IL-10 and Arg-1. Figure 3 C). This is consistent with the inhibitory effect of sorbicillalanine A(1) on NO release. Figure 2 At the same concentration, it showed no cytotoxic activity against RAW264.7 cells, and can serve as a lead compound for the development of anti-inflammatory drugs, which is of great significance for the rational utilization of deep-sea microbial drug resources.

Claims

1. The structure of sorbicillalanine A, a heterodimer of sorbicillin, is shown in formula (I): Formula (I).

2. The use of the sorbicillin hybrid dimer compound sorbicillalanine A as described in claim 1 in the preparation of anti-inflammatory drugs.

3. An anti-inflammatory drug, characterized in that, The active ingredient is sorbicillalanine A, a heterodimer compound of sorbicillin as described in claim 1.

4. A method for preparing the sorbicillin hybrid dimer compound sorbicillalanine A according to claim 1, characterized in that, It was isolated from the fermentation culture of Penicillium sp. SCSIO06871, which has the accession number GDMCC No. 64307.

5. The preparation method according to claim 4, characterized in that, Includes the following steps: A. Preparation of fermentation culture of Penicillium sp. SCSIO06871 from deep sea; B. Separate the fermentation broth and mycelium. Extract the fermentation broth with ethyl acetate, and concentrate the ethyl acetate extract to obtain a fermentation broth extract. Shred the mycelium and extract it with ethyl acetate, and concentrate the ethyl acetate extract to obtain a mycelial extract. Combine the fermentation broth extract and mycelial extract, and separate them using normal-phase silica gel. Elute in a gradient of dichloromethane:methanol from 100:0, 100:1, 100:2, 100:3, 100:5, 100:10, 100:20, 0:100, v / v to obtain nine fractions Fr1–Fr9. Collect fraction Fr8 eluted with dichloromethane:methanol 100:5 v / v, first by Sephadex LH-20 column chromatography with methanol as the eluent, then by medium-pressure reversed-phase column chromatography with acetonitrile:water from 10:90, 20:80, 25:75, 35:65, 60:40, ... Gradient elution of 100:0, v / v, with the product eluted at 25:75, v / v, was purified by high performance liquid chromatography to obtain sorbicillin hybrid dimer compound sorbicillalanine A.

6. The preparation method according to claim 5, characterized in that, The preparation of the fermentation culture of deep-sea Penicillium sp. SCSIO06871 involves inoculating the deep-sea Penicillium sp. SCSIO06871 into a fermentation medium. Each 1000 mL of the fermentation medium contains: 10 g glucose, 20 g maltose, 20 g mannitol, 1 g corn steep liquor, 10 g monosodium glutamate, 0.5 g dipotassium hydrogen phosphate, 0.3 g magnesium sulfate heptahydrate, 3 g yeast extract, 15 g sea salt, and the remainder is water.

7. The preparation method according to claim 5, characterized in that, The high-performance liquid chromatography (HPLC) purification method used 205 nm and 265 nm wavelengths for detection, a flow rate of 2 ml / min, and isogradient elution with methanol:water = 65:35, v / v for semi-preparative HPLC separation. The HPLC was performed using a YMC-pack ODS-A filter, 10 × 250 mm, 5 µm, with values ​​recorded at retention time t. R The sorbicillin hybrid dimer compound sorbicillalanine A was obtained after 12.0 min.

8. The use of Penicillium sp. SCSIO06871 in the preparation of the sorbicillin hybrid dimer compound sorbicillalanine A as described in claim 1, wherein the Penicillium sp. SCSIO06871 has the accession number GDMCC No. 64307.

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