CN118496234A - Sorbicillin hybrid dimer compound sorbicillalanine A, preparation method thereof and application thereof in preparation of anti-inflammatory drugs - Google Patents

Abstract

The present invention discloses a sorbicillin mixed dimer compound, a preparation method thereof and an application thereof in the preparation of anti-inflammatory drugs. The present invention prepares a compound sorbicillalanine A with anti-inflammatory activity from the liquid fermentation product of deep-sea penicillium SCSIO06871. Sorbicillalanine A is a new compound through structural identification, and its structure is shown in formula (I). Anti-inflammatory activity evaluation found that sorbicillalanine A has a significant inhibitory effect on NO produced by mouse macrophage cell line RAW264.7 induced by lipopolysaccharide (LPS), and simultaneously inhibits the expression of pro-inflammatory factors IL-6 and MCP-1 genes, promotes the expression of anti-inflammatory factors IL-4, IL-10 and Arg-1 genes, and has no cytotoxic activity on cell line RAW264.7 at the same concentration, and can be used as a candidate lead compound for the development of anti-inflammatory drugs.

Description

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

Technical field:

The present invention belongs to the field of natural products, and in particular relates to the separation and purification of natural products from microorganisms and their application in the preparation of anti-inflammatory drugs.

Background technology:

Inflammation is a complex biological reaction regulated by various chemical mediators released by cells after the body is stimulated by various damages. Appropriate inflammatory response can help the body eliminate pathogens and protect the body from damage, but if the inflammatory response is too intense or persists, it may lead to autoimmune or autoinflammatory diseases, neurological diseases 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 the development of new anti-inflammatory drugs and other human disease drugs. Microorganisms have the unique advantages of repeatable fermentation and sustainable development. The special ecological environment of the deep sea has created the uniqueness of the secondary metabolites of deep-sea microorganisms, which has become an important source of innovative drug discovery. Therefore, screening and discovering drug lead compounds with good anti-inflammatory activity from deep-sea microorganisms is of great significance for the rational use of deep-sea microbial drug resources.

Summary of the invention:

The first object of the present invention is to provide a sorbicillin mixed dimer compound sorbicillalanine A with anti-inflammatory activity.

The sorbicillin mixed dimer compound sorbicillalanine A of the present invention has a structure as shown in formula (I):

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

The third object of the present invention is to provide the use of deep-sea source Penicillium sp.SCSIO06871 in the preparation of the above-mentioned sorbicillin mixed dimer compound sorbicillalanineA.

The fourth object of the present invention is to provide a method for preparing the above-mentioned sorbicillin mixed dimer compound sorbicillalanine A, comprising the following steps:

A. preparing a 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 the fermentation broth extract; cut the mycelium into pieces, soak it in ethyl acetate, and extract it with ultrasonic wave, and concentrate the ethyl acetate extract to obtain the mycelium extract; combine the fermentation broth extract and mycelium extract, separate them with normal phase silica gel, and use dichloromethane:methanol from 100:0, 100:1, 100:2, 100:3, 100:5, 100:10, 100:20, 0:100, v/v gradient elution to obtain 9 components Fr1-Fr9, collect the component Fr8 eluted with dichloromethane:methanol 100:5 v/v, and first pass through Sephadex The product was purified by LH-20 column chromatography with methanol as the eluent, and then by medium-pressure reverse phase column chromatography with acetonitrile: water in a gradient ratio of 10:90, 20:80, 25:75, 35:65, 60:40, 100:0, v/v. The product after elution at 25:75, v/v was purified by high performance liquid phase to obtain the sorbicillin mixed dimer compound sorbicillalanine A.

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

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

Preferably, the high performance liquid phase purification is performed by semi-preparative high performance liquid phase separation using HPLC (YMC-pack ODS-A, 10×250mm, 5μm) with detection at wavelengths of 210 and 270nm, a flow rate of 2ml/min, and isocratic elution with methanol:water=65:35, v/v, to obtain the sorbicillin mixed dimer compound sorbicillalanine A at a retention time t _R 12.0min.

The inventors obtained compound 1 by liquid static fermentation of a deep-sea Penicillium sp.SCSIO06871 and extraction and purification of the fermentation extract. After structural analysis, it was determined to be a sorbicillin mixed dimer compound sorbicillalanine A, and the specific structure is shown in formula (I). Through the evaluation of the anti-inflammatory activity of compound 1, it was found that compound 1 had a significant inhibitory effect on the NO produced by mouse macrophage cell line RAW264.7 induced by lipopolysaccharide (LPS), and at the same time inhibited the expression of pro-inflammatory factors IL-6 and MCP-1 genes, promoted the expression of anti-inflammatory factors IL-4, IL-10 and Arg-1 genes, and had no cytotoxic activity on cell line RAW264.7 at the same concentration, and can be used as a candidate lead compound for the development of anti-inflammatory drugs.

The deep-sea Penicillium sp.SCSIO06871 of the present invention was deposited in the Guangdong Microbiological Culture Collection Center (GDMCC) on February 4, 2024, address: Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Postal Code: 510070, Deposit Number: GDMCC No: 64307.

Description of the drawings:

Figure 1: Main ¹ H- ¹ H COSY and HMBC information of Sorbicillalanine A (1);

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

Figure 3. Sorbicillalanine A inhibits the activity of pro-inflammatory factors and promotes the activity of anti-inflammatory factors. Control represents blank control, LPS represents lipopolysaccharide treatment, LPS+1 represents LPS plus compound 1 treatment, and each group of bar graphs from left to right are control, LPS, and LPS+Sorbicillalanine A.

Specific implementation method:

The following examples are provided to further illustrate the present invention, rather than to limit the present invention.

Example 1: Preparation and structural identification of compound 1

1. Preparation of compound 1 represented by formula (I)

1. Microbial culture conditions:

Each 1000mL culture medium contains: 10g glucose, 20g maltose, 20g mannitol, 1g corn steep liquor, 10g MSG, 0.5g dipotassium hydrogen phosphate, 0.3g magnesium sulfate heptahydrate, 3g yeast extract powder, 15g sea salt, which are then dissolved in appropriate amount of water, made up to 1000mL with water, sterilized at 121℃ for 20min, and set aside.

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

2. Extraction and separation:

The fermentation product of the deep-sea Penicillium sp.SCSIO06871 was centrifuged to obtain supernatant and mycelium. The supernatant fermentation liquid was extracted three times with equal volumes of ethyl acetate. The ethyl acetate extract was concentrated under reduced pressure at below 40°C to obtain a fermentation liquid extract. The mycelium was cut into pieces with scissors, soaked in ethyl acetate solution, and extracted by ultrasonic. The extract was concentrated after reduced pressure distillation to obtain a mycelium extract. The fermentation liquid extract and mycelium extract were combined to obtain a total of about 72.5g of extract. The extract was separated by normal phase silica gel. After mixing and dry column loading, the extract was eluted with dichloromethane: methanol (100:0, 100:1, 100:2, 100:3, 100:5, 100:10, 100:20, 0:100, v/v) in a gradient elution sequence to obtain 9 components Fr1-Fr9. Component Fr8 (component eluted with dichloromethane: methanol 100:5 v/v) was first purified by Sephadex LH-20 column chromatography with methanol as the eluent, and then medium-pressure reverse phase column chromatography with acetonitrile:water from 10:90, 20:80, 25:75, 35:65, 60:40, 100:0, v/v gradient elution, wherein the product after elution at 25:75, v/v was detected at 210 and 270 nm wavelengths, with a flow rate of 2 mL/min, and isocratic elution with methanol:water (65:35, v/v) for semi-preparative high performance liquid separation, HPLC (YMC-pack ODS-A, 10×250 mm, 5 μm), to obtain new compound 1 (7.0 mg, retention time t _R 12.0 min).

2. Structural identification of compound 1

Compound 1 was subjected to high-resolution mass spectrometry (HRESIMS), one-dimensional and two-dimensional nuclear magnetic resonance (NMR) data tests to determine the chemical structure of compound sorbicillalanineA.

Compound 1: colorless oil; high resolution mass spectrometry HRESIMS m/z 420.1648[M+H] ⁺ (calcd forC ₂₁ H ₂₆ NO ₈ ), the suggested molecular formula is C ₂₁ H ₂₅ NO ₈ , the unsaturation is 10; ¹ H and ¹³ C NMR data are shown in Table 1; 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 the one-dimensional and two-dimensional hydrogen spectra revealed that there were 5 ester carbonyl groups, 2 double bond signals, and 3 remaining unsaturations in the molecule, indicating that there were 3 cyclic structural fragments in the structure. The ¹ H- ¹ H COSY spectrum showed that it contained H ₂ -2'/H ₂ -3'/H-4'/H-5'/H ₃ -6';H-2"/H ₂ -3" two-segment spin coupling system. Combined with HMBC carbon-hydrogen long-range correlation, it was found that compound 1 was very similar to the N-containing sorbicillinoid structure sorbicillactone B. A careful comparison of the differences revealed that compound 1 formed a new chemical bond between the C-2" and C-1 positions of sorbicillactone B, and had an additional six-membered ring compared to sorbicillactone B. At the same time, the HMBC correlation signals of H-2" and H ₂ -3" to C-1; H-2" to C-1, C-2 and C-6; H-6 to C-2" can prove the above inference. According to SciFinder search, compound 1 is a new compound with a new structure and was named sorbicillalanine A. The main ¹ H- ¹ H COSY and HMBC information of compound 1 is shown in Figure 1.

Table 1. NMR spectrum data of Sorbicillalanine A (500MHz, CD ₃ OD, ppm)

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

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

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

Claims (9)

1. Sorbicillin mixed dimer compound sorbicillalanine A, whose structure is shown in formula (I): 2. Use of the sorbicillin mixed dimer compound sorbicillalanine A as shown in formula (I) in the preparation of anti-inflammatory drugs. 3. An anti-inflammatory drug, characterized in that it contains the sorbicillin mixed dimer compound sorbicillalanine A described in claim 1 as an active ingredient. 4. Penicillium sp.SCSIO06871, deposit number: GDMCC No: 64307. 5. A method for preparing the sorbicillin mixed dimer compound sorbicillalanine A as claimed in claim 1, characterized in that it is isolated from the fermentation culture of Penicillium sp.SCSIO06871 as claimed in claim 4. 6. The preparation method according to claim 5, characterized in that it comprises the following steps: A. preparing a 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 the fermentation broth extract; cut the mycelium into pieces and extract with ethyl acetate, and concentrate the ethyl acetate extract to obtain the mycelium extract; combine the fermentation broth extract and the mycelium extract, separate them with normal phase silica gel, and use dichloromethane: methanol from 100:0, 100:1, 100:2, 100:3, 100:5, 100:10, 100:20, 0:100, v/v gradient elution to obtain 9 components Fr1-Fr9, collect the component Fr8 eluted with dichloromethane: methanol 100:5 v/v, and first pass through Sephadex The product was purified by LH-20 column chromatography with methanol as the eluent, and then by medium-pressure reverse phase column chromatography with acetonitrile: water in a gradient ratio of 10:90, 20:80, 25:75, 35:65, 60:40, 100:0, v/v. The product after elution at 25:75, v/v was purified by high performance liquid phase to obtain the sorbicillin mixed dimer compound sorbicillalanine A. 7. The preparation method according to claim 6, characterized in that the preparation of deep-sea Penicillium sp. The fermentation culture of SCSIO06871 is prepared by inoculating deep-sea Penicillium sp. SCSIO06871 into a fermentation medium for cultivation. The fermentation medium contains, per 1000 mL, 10 g of glucose, 20 g of maltose, 20 g of mannitol, 1 g of corn steep liquor, 10 g of monosodium glutamate, 0.5 g of dipotassium hydrogen phosphate, 0.3 g of magnesium sulfate heptahydrate, 3 g of yeast extract powder, 15 g of sea salt, and the balance is water. 8. The preparation method according to claim 6, characterized in that the high performance liquid phase purification is carried out by semi-preparative high performance liquid phase separation with detection at wavelengths of 205 and 265 nm, a flow rate of 2 ml/min, methanol: water = 65:35, v/v, isocratic elution, HPLC (YMC-pack ODS-A, 10×250 mm, 5 μm), and sorbicillin mixed dimer compound sorbicillalanine A is obtained at a retention time t _R 12.0 min. 9. Use of the Penicillium sp. SCSIO06871 described in claim 4 in the preparation of the sorbicillin mixed dimer compound sorbicillalanine A described in claim 1.