An Oxirapentyn diterpenoid derivative derived from shikimic acid, its preparation method and application

Abstract

The present invention belongs to the technical field of biomedicine, and particularly relates to an Oxirapentyn diterpene derivative derived from shikimic acid, and its preparation method and application. This compound has good anti-inflammatory activity, can effectively inhibit the production of NO by inflammatory cells, and is significantly superior to the positive control drug indomethacin; moreover, natural compounds derived from marine microorganisms are not prone to resistance and have high safety. The present invention also proves that it has low cytotoxicity (IC 50 >50 μM), high safety, and is very suitable for being prepared into anti-inflammatory drugs for application. On the other hand, this derivative is isolated from the fermentation product of the marine fungal strain Beauveria felina SYSU-MS7908, and can be fermented and separated on a large scale by microorganisms, with characteristics such as simple production process, short cycle, and low product cost.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine. More specifically, it relates to an Oxirapentyn diterpenoid derivative derived from shikimic acid, and its preparation method and application. Background Art

[0002] Inflammation is an important defense response that occurs when the body is stimulated by harmful factors such as microbial infection or injury. Usually, the inflammatory response is accompanied by the activation of immune cells, especially macrophages. When a persistent and excessive inflammatory response occurs, it will cause damage to body tissues and functional disorders, and often induce various diseases such as obesity, neurodegenerative diseases, and cancer, especially chronic inflammations such as type 2 diabetes, atherosclerosis, and arthritis. For example, the infection of the novel coronavirus COVID-19 has caused large-scale inflammation, resulting in respiratory failure and organ damage. It can be seen that inflammation poses a serious threat to the health of humans globally.

[0003] Currently, the commonly used anti-inflammatory drugs in clinical practice are mainly divided into two categories: steroidal and non-steroidal. For example, Chinese Patent Application CN114404423A discloses the application of pregnane-type C21-steroidal compounds in the preparation of anti-inflammatory drugs. Among them, deacetyllauroside and kedroside have good inhibitory effects on lipopolysaccharide-induced inflammatory cytokines in primary peritoneal macrophages of mice, have certain anti-inflammatory activities, and are non-toxic to cells, providing a new option for the research and development of anti-inflammatory drugs. Chinese Patent Application CN109912595A discloses an anti-inflammatory drug, which is an unsaturated pyrrolidone compound and its similar heterocyclic lactone compounds, and has a significant anti-inflammatory effect, and is expected to become a new class of non-steroidal anti-inflammatory drugs. However, whether it is steroidal or non-steroidal anti-inflammatory drugs, long-term use often brings serious side effects, such as kidney and gastrointestinal mucosal damage and sudden myocardial infarction.

[0004] Therefore, the research and development of anti-inflammatory drugs remains a hot issue in the field of pharmacy, and it is of great significance to find more new anti-inflammatory drugs with high efficiency and low toxicity. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the defects and deficiencies of various side effects existing in the long-term use of existing steroidal or non-steroidal anti-inflammatory drugs, and provide an Oxirapentyn diterpenoid derivative derived from the marine natural product shikimic acid, which has good anti-inflammatory effects and high safety.

[0006] The object of the present invention is to provide a preparation method of the Oxirapentyn diterpenoid derivative derived from shikimic acid.

[0007] Another object of the present invention is to provide the application of the Oxirapentyn diterpenoid derivative derived from shikimic acid in the preparation of anti-inflammatory drugs.

[0008] The above object of the present invention is achieved by the following technical solutions:

[0009] A shikimic acid-derived Oxirapentyn diterpene derivative, and the shikimic acid-derived Oxirapentyn diterpene derivative has any one of the following structures:

[0010]

[0011] In addition, the present invention also provides a preparation method of the shikimic acid-derived Oxirapentyn diterpene derivative, and the shikimic acid-derived Oxirapentyn diterpene derivative is isolated and purified from the mycelium of the marine ascidian-derived fungal strain Beauveria felina SYSU-MS7908 with the preservation number GDMCC 61059. Among them, the marine ascidian-derived fungal strain Beauveria felina SYSU-MS7908 was preserved in the Guangdong Provincial Culture Collection Center of Microorganisms on July 22, 2020, with the preservation number GDMCC No: 61059, and the preservation address: 5th Floor, Building 59, 100th Yard, Xianlie Middle Road, Guangzhou, and has been publicly disclosed and protected in the Chinese patent application CN112646729A, and the public can obtain it.

[0012] Marine natural products have the characteristics of novel structures and significant activities, and among them, natural products derived from marine microorganisms can be obtained by modern microbial fermentation production, which has the advantages of not destroying the ecological balance and being easy to industrialize.

[0013] Furthermore, the preparation method specifically includes the following steps:

[0014] S1. Expand and culture the ascidian-derived fungal strain Beauveria felina SYSU-MS7908 to obtain mycelium;

[0015] S2. Extract the mycelium obtained in step S1 with an organic solvent, concentrate the extract to obtain an extract, separate it by silica gel column chromatography with gradient elution, and the eluent is ethyl acetate - petroleum ether with a volume ratio of 10% - 100% (preferably the eluent is ethyl acetate - petroleum ether with volume ratios of 10%, 20%, 30%, 45%, 60%, 100%), and collect the fraction with a volume ratio of 10% - 30% (preferably 20%);

[0016] S3. Subject the fraction obtained in step S2 to Sephadex LH-20 chromatography, and the eluent is methanol - dichloromethane with a volume ratio of 1:2 - 2:1, and concentrate the eluent to obtain a crude product;

[0017] S4. Subject the crude product obtained in step S3 to fractionation on an ODS-C18 chromatography column with a mobile phase of methanol-water, collect the fractions, and separate and purify them on an RP-HPLC chromatography column to obtain the product.

[0018] Further, in step S1, for the scale-up culture, seed culture is first carried out, followed by solid fermentation culture.

[0019] Furthermore, the seed culture medium for the seed culture comprises the following components in parts by weight: 100 parts of potato, 20 parts of glucose, 5 - 30 parts of sea salt, and 1000 parts of water. Specifically, after mixing the medium components, sterilize them at 121 °C for 30 minutes.

[0020] Preferably, the temperature for the seed culture is 24 - 28 °C, the shaker speed is 140 - 180 rpm, and the culture time is 84 - 108 hours.

[0021] Further, the fermentation medium for the solid fermentation culture comprises the following components in parts by weight: 100 parts of rice, 5 - 30 parts of sea salt, 100 parts of water, and 0.3 - 1.0 part of peptone. Specifically, after mixing the medium components, sterilize them at 121 °C for 30 minutes.

[0022] Preferably, the temperature for the fermentation culture is 22 - 28 °C, and static culture is carried out for 22 - 32 days.

[0023] Furthermore, in step S2, the organic solvent for extraction is selected from one or more of acetone, ethyl acetate, methanol, and ethanol.

[0024] Preferably, in step S2, the extract can be extracted first before silica gel column chromatography, and the extraction solvent is ethyl acetate and / or chloroform.

[0025] Preferably, in step S2, the concentration is carried out at a temperature below 50 °C.

[0026] Further, in step S4, the volume fraction of methanol in the methanol-water is 30 - 100%. Preferably, the volume fractions of methanol in the methanol-water are 30%, 60%, 90%, and 100% in sequence.

[0027] Furthermore, in step S4, the collected fraction is the fraction with a methanol volume fraction of 90%.

[0028] Further, in step S4, the RP-HPLC chromatography column is an ACE-5-C18-AR chromatography column with a specification of 4.6 × 250 mm, 5 μm.

[0029] Further, in step S4, when separating and purifying through the RP-HPLC chromatographic column, the mobile phase is methanol-water with a volume ratio of 70:30 to 80:20, and the flow rate is 2 to 4 mL / min.

[0030] In addition, the present invention also claims the application of the shikimic acid-derived Oxirapentyn diterpene derivative or any one of the following compounds in the preparation of anti-inflammatory drugs:

[0031]

[0032] Preferably, the drug further contains a pharmaceutically acceptable excipient.

[0033] Preferably, the drug is an oral preparation, an injection, an inhalant or an external preparation.

[0034] The present invention has the following beneficial effects:

[0035] The present invention provides a shikimic acid-derived Oxirapentyn diterpene derivative, which has good anti-inflammatory activity and can effectively inhibit the production of NO by inflammatory cells. The IC 50 is 10 to 20 μM, which is significantly better than the positive control drug indomethacin (IC 50 = 35.8 μM); moreover, natural compounds derived from marine microorganisms are not prone to resistance and have high safety. The present invention also proves that the shikimic acid-derived Oxirapentyn diterpene derivative has low cytotoxicity (IC 50 > 50 μM) and high safety, and is very suitable for being prepared into anti-inflammatory drugs for application. On the other hand, this shikimic acid-derived Oxirapentyn diterpene derivative is isolated from the fermentation product of the marine fungal strain Beauveria felina SYSU-MS7908, and can be fermented and separated on a large scale by microorganisms, with the characteristics of simple production process, short cycle and low product cost. Specific Embodiments

[0036] The following specific examples are used to further illustrate the present invention, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field.

[0037] Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0038] Example 1 A Preparation Method of a Shikimic Acid-Derived Oxirapentyn Diterpene Derivative

[0039] The sea squirt symbiotic fungus strain Beauveria felina SYSU-MS7908 (deposited at the Guangdong Microbial Culture Collection Center on July 22, 2020, accession number GDMCC No: 61059, deposition address: 5th Floor, Building 59, 100th Yard, Xianlie Middle Road, Guangzhou, has been publicly disclosed and protected in Chinese patent application CN112646729A and is publicly available) was used for fermentation, and the fermentation product was separated and extracted to obtain Compound I and Compound II.

[0040] The specific fermentation, separation, and extraction processes are as follows:

[0041] S1. Seed culture:

[0042] S1-1. Preparation of seed medium: 100 g of potatoes, 20 g of glucose, 30 g of sea salt, and 1 L of tap water were evenly divided and filled into 4 500-mL conical flasks, and sterilized at 121 °C for 15 minutes;

[0043] S1-2. Seed culture: The strain of sea squirt symbiotic fungus was inoculated into the seed medium, and cultured on a shaker at a temperature of 26 °C and a rotation speed of 180 rpm for 96 hours to obtain a seed culture solution.

[0044] S2. Fermentation culture:

[0045] S2-1. Preparation of fermentation medium: Each 1-L Erlenmeyer flask contained 100 g of rice, 3 g of sea salt, 100 ml of tap water, and 0.5 g of peptone.

[0046] S2-2. Fermentation culture: 5 mL of the seed solution was aseptically transferred into a conical flask containing the fermentation medium, and statically cultured at 26 °C for 28 days to obtain fermented bacteria.

[0047] S3. Separation and purification of compounds:

[0048] S3-1. The fermented bacteria were soaked in methanol, and the soaking solution was concentrated under reduced pressure at a temperature below 50 °C to obtain 105 g of an extract; the extract was separated by silica gel column chromatography and eluted with ethyl acetate-petroleum ether solutions with ethyl acetate volumes of 10%, 20%, 30%, 45%, 60%, and 100% in gradient, and divided into 6 groups (Fr.A - Fr.F).

[0049] S3-2. Collect the 20% ethyl acetate - petroleum ether gradient elution fraction Fr.B, subject it to Sephadex LH-20 chromatography, with the eluent being methanol - dichloromethane (V / V, 1:1), concentrate the collected Fr.B-2 fraction, and then fractionate it through an ODS-C18 column. The mobile phase is methanol / water (30%, 60%, 90%, and 100%), collect the fraction with 90% methanol, and perform separation and purification through RP-HPLC chromatography (ACE-5-C18-AR column, 4.6×250 mm, 5 μm, MeOH / H2O, 73 / 27, 3 mL / min) to obtain the target products, Oxirapenty-class meroterpenoids I and compound II.

[0050] The structures of the target products, Oxirapenty-class meroterpenoids I and compound II, are shown as follows:

[0051]

[0052] Determination of the physicochemical properties of the compounds in Example 2

[0053] The physicochemical property data of the target products, Oxirapenty-class meroterpenoids I and compound II, are as follows:

[0054] Compound I: White oil; (c 0.20, MeOH); UV (MeOH) λ max (logε) 225 (4.02) nm; ECD (MeOH) λ max (Δε) 225 (-2.16) nm, 280 (+1.03) nm; 1 1H NMR (CDCl3, 400 MHz) and 13 13C NMR (CDCl3, 100 MHz), and the detailed data are shown in Table 1; HR-ESIMS m / z 391.1750 [M+H] + (calcd. for C 21 H 27 O7, 391.1751).

[0055] Compound II: White oil; (c 0.20, MeOH); CD (MeOH) λ max (Δε) 223 (-1.47) nm; UV (MeOH) λ max (logε) 224 (3.21) nm; 1 1H NMR (CDCl3, 400 MHz) and 1313C NMR (CDCl3, 100 MHz), detailed data are shown in Table 1; HR-ESIMS m / z 319.1528 [M+H] + (calcd. for C 18 H 22 O5, 319.1540).

[0056] Table 1 NMR data of Compounds I and II (100 MHz / 400 MHz, CDCl3, ppm)

[0057]

[0058] It can be seen that Compounds I and II are typical Oxirapenty meroterpenoids of p - diisopentenyl cyclohexanol, and contain a pyranocyclohexanol skeleton with 1,3 - enyne, in which cyclohexanol is derived from the shikimic acid pathway.

[0059] Example 3 Anti - inflammatory activity test of shikimic acid - derived Oxirapentyn meroterpenoid derivatives

[0060] Taking the two Oxirapenty meroterpenoid derivatives Compounds I and II prepared in Example 1 and the known meroterpenoid analogs III (Oxirapenty F), IV (Oxirapenty G), V (Oxirapenty B), VI (Oxirapenty E), VII (Oxirapenty E diacetate) and VIII (Oxirapenty H) as the research objects, their anti - inflammatory activities were evaluated by testing the effect on the NO release amount in LPS - induced mouse macrophages RAW264.7.

[0061]

[0062] Among them, the preparation and related data of Compound VI (Oxirapenty E) and Compound VII (Oxirapenty E diacetate) refer to the literature: Yurchenko A.N., Smetanina, et al, New oxirapentyn E from marine isolate of the fungus Isaria felina[J], Chemistry of Natural Compounds 2013, 49(5):857 - 860. doi:10.1007 / s10600 - 013 - 0764 - 0.. Those skilled in the art can obtain Compounds VI and VII with corresponding structures according to the above literature.

[0063] The specific process is as follows:

[0064] 1. Experimental materials

[0065] Lipopolysaccharide (LPS), indomethacin (positive control), mouse macrophages (RAW264.7), DMSO, tetrazolium (MTT, 5 mg / mL), Griess method NO kit (Shanghai Beyotime Biotechnology Co., Ltd.).

[0066] 2. Experimental methods

[0067] The compound was dissolved in DMSO to prepare a 10 mM stock solution, and diluted to the required concentration with DMEM medium when used (DMSO content was less than 0.5%).

[0068] The experimental groups were: blank group (100 μL DMEM medium), model group (1 μL LPS + 99 μL DMEM medium), positive control group (1 μL LPS + 25 μL indomethacin + 74 μL DMEM cell medium), sample group (1 μL LPS + 99 μL medium containing different concentrations of the sample); among them, the concentrations of LPS and indomethacin were 100 μg / mL and 200 μg / mL, respectively.

[0069] RAW264.7 cells (1×10 5 cells / mL) were cultured at 100 μL per well in a 96-well plate and incubated in an incubator at 37 °C and 5% CO2 for 12 h; according to the grouping, different concentrations of the sample containing lipopolysaccharide LPS (final concentration 1 μg / mL) were added to each well and cultured for 24 h. Then, 50 μL of the supernatant was carefully aspirated into another 96-well plate, and NO I and NO II reagents in the Griess method NO kit were added respectively. After mixing evenly, it was left standing at room temperature for 10 min. The absorbance at 540 nm was measured with a microplate reader Multiskan GO (Thermo Scientific), and the NO release level of each group of cells was calculated according to the standard curve.

[0070] Carefully aspirate the remaining 50 μL of the culture medium, add 100 μL of MTT solution diluted with DMEM, and place it in the incubator for 4 h; aspirate the supernatant, add 110 μL of DMSO solution, shake for 10 min, and measure the absorbance at 490 nm with a microplate reader to evaluate the cell viability.

[0071] Calculation method:

[0072] NO release inhibition rate % = (OD 模型组 - OD 样品组 ) / (OD 模型组 - OD 空白组 ) × 100%.

[0073] Cell viability % = [(Average OD value measured in the sample group) / (Average OD value measured in the control group)] × 100%.

[0074] 3. Experimental results

[0075] Table 2 Anti-inflammatory activities of Oxirapenty sesquiterpene derivatives

[0076]

[0077] As can be seen from Table 2, Compounds I, II, VI, and VII can all effectively inhibit the production of NO by LPS-induced inflammatory cells RAW264.7, significantly stronger than other analogs, and even stronger than the positive control indomethacin (IC 50 is 35.8 ± 2.5 μM); and in the MTT test, all compounds have no cytotoxicity to RAW264.7 cells (IC 50 > 50 μM), with high safety. This indicates that all compounds have good anti-inflammatory effects.

[0078] Therefore, Oxirapenty sesquiterpene derivatives Compounds I, II, VI, and VII all have good potential as anti-inflammatory drugs.

[0079] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. An Oxirapentyn diterpenoid derivative derived from shikimic acid, characterized in that, The shikimic acid-derived Oxirapentyn diterpene derivative has any of the following structures:

2. The preparation method of the shikimic acid-derived Oxirapentyn diterpene derivative according to claim 1, characterized in that, The shikimic acid-derived Oxirapentyn diterpene derivative is isolated and purified from the mycelium of the marine ascidian-derived fungal strain Beauveria felina SYSU-MS7908 with the preservation number GDMCC 61059.

3. The preparation method according to claim 2, wherein The preparation method specifically includes the following steps: S1. Expand the culture of the ascidian-derived fungal strain Beauveria felina SYSU-MS7908 to obtain mycelium. S2. Extract the mycelium obtained in step S1 with an organic solvent, concentrate the extract to obtain an extract, separate by gradient elution on a silica gel column, the eluent is ethyl acetate-petroleum ether with a volume ratio of 10% to 100%, and collect the fraction with a volume ratio of 10% to 30%. S3. Subject the fraction obtained in step S2 to Sephadex LH-20 chromatography, the eluent is methanol-dichloromethane with a volume ratio of 1:2 to 2:1, and concentrate the eluent to obtain a crude product. S4. Segment the crude product obtained in step S3 on an ODS-C18 chromatography column, the mobile phase is methanol-water, collect the fraction and separate and purify it on an RP-HPLC chromatographic column to obtain the product.

4. The preparation method according to claim 3, wherein In step S1, the expansion culture first performs seed culture and then solid fermentation culture.

5. The preparation method according to claim 4, wherein The seed culture medium for the seed culture includes the following components in parts by weight: 100 parts of potato, 20 parts of glucose, 5 to 30 parts of sea salt, and 1000 parts of water.

6. The preparation method according to claim 4, characterized in that, The fermentation medium for the solid fermentation culture includes the following components in parts by weight: 100 parts of rice, 5 to 30 parts of sea salt, 100 parts of water, and 0.3 to 1.0 part of peptone.

7. According to the preparation method described in claim 3, characterized in that, In step S2, the organic solvent for extraction is selected from one or more of acetone, ethyl acetate, methanol, and ethanol.

8. The preparation method according to claim 3, characterized in that, In step S4, the volume fraction of methanol in the methanol-water is 30% to 100%.

9. The preparation method according to claim 8, wherein In step S4, the collected fraction is the fraction with a methanol volume fraction of 90%.

10. Use of the shikimic acid-derived Oxirapentyn diterpene derivative according to claim 1 or any of the following compounds in the preparation of an anti-inflammatory drug:

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