Polyketide compounds having anti-inflammatory activity and uses thereof

Abstract

The present invention discloses a polyketide compound with anti-inflammatory activity and its preparation and use, which belongs to the field of pharmaceutical technology. The present application isolates Alternaria sp. HJT-Y7 from the leaf part of Rhodiola tibetica, and the obtained Alternaria sp. HJT-Y7 is subjected to solid fermentation, and two polyketide compounds are obtained by a series of isolation and purification methods, both of which have good anti-inflammatory activity, providing a new approach for the development of anti-inflammatory drugs.

Description

[Technical Field]

[0001] The present invention relates to the field of pharmaceutical technology, and more particularly to a novel polyketide epimer having anti-inflammatory activity, and its preparation method and use. [Background technology]

[0002] Medicinal plant endophytic fungi are diverse, and each endophytic fungus has a wealth of secondary metabolites. Currently, a large number of biologically active substances, such as alkaloids, polypeptides, polyketides, and terpenoids, have been discovered from the secondary metabolites of endophytic fungi. These secondary metabolites not only have the same or similar biological activities as those of the host plant, such as antibacterial, anti-inflammatory, antitumor, antihyperglycemic, and antiparasitic, but also possess many new biological activities. Therefore, the secondary metabolites of medicinal plant endophytic fungi represent a vast potential resource of medicinal substances, with good prospects for development. Summary of the Invention [Problem to be solved by the invention]

[0003] The present invention has been made in view of the above, and aims to provide a polyketide compound with anti-inflammatory activity, as well as a production method and use thereof. Research into plant endophytic fungi has been conducted to fully utilize plant endophytic fungal resources, and Alternaria sp. HJT-Y7 has been isolated from the leaf parts of Tibetan Rhodiola tibetica. The fungus was deposited with the Center for Ordinary Microorganisms of the China Commission for the Preservation and Management of Microorganisms on May 6, 2022, under the accession number CGMCC No. 40166. The depository address is No. 3, Courtyard No. 1, Beichen West Road, Chaoyang District, Beijing. The obtained Alternaria sp. HJT-Y7 was subjected to solid-state fermentation and isolated using a series of separation and purification methods to obtain a pair of polyketide epimers with novel structures, providing a new lead compound for drug development. [Means for solving the problem]

[0004] The object of the present invention can be achieved by the following technical means.

[0005] According to one aspect of the present invention, there is provided a polyketide compound having the structure shown in the following formula, or a pharmaceutically acceptable salt thereof: [ka]

[0006] According to another aspect of the present invention, there is provided an endophytic fungus of the genus Alternaria (Alternaria sp.) HJT-Y7, which was deposited with the Center of Ordinary Microorganisms of the China Commission for the Preservation and Management of Microorganisms on May 6, 2022, with deposit number CGMCC No. 40166, and the address of the depository institution is No. 3, Courtyard No. 1, Beichen West Road, Chaoyang District, Beijing.

[0007] According to another aspect of the present invention, there is provided a method for producing the polyketide compound, which mainly comprises the following steps (1) and (2): Step (1): The Alternaria sp. HJT-Y7 endophytic fungus is inoculated into a fungal No. 4 medium and cultured with shaking. The resulting fermented liquid is inoculated into a solid rice medium together with the mycelium, and left to stand at 20-30°C for 10-40 days for fermentation, yielding a solid fermentation product. Step (2): The solid fermentation product obtained in step (1) was extracted with methanol and filtered. The extract was loaded onto a silica gel chromatography column and subjected to gradient elution using a chloroform / methanol mixture in a volume ratio of 100:0 to 0:100 as the mobile phase. Based on the TLC results, fractions 1 to 16 were obtained sequentially. Fraction 7 was loaded onto a silica gel chromatography column and subjected to gradient elution using a petroleum ether / ethyl acetate mixture in a volume ratio of 10:1 to 10:9 as the mobile phase. Based on the TLC results, fractions 7-1 to 7-10 were obtained sequentially. Fraction 7-6 was loaded onto a Sephadex LH-20 gel column and subjected to elution using a dichloromethane / methanol mixture in a volume ratio of 1:1 as the mobile phase. Fractions 7-6-1 to 7-6-6 were obtained sequentially. Fraction 7-6-3 was loaded onto an Agilent C18 high-performance liquid chromatography column and subjected to elution using a 55% aqueous methanol solution as the mobile phase. Compounds 1 and 2 were obtained.

[0008] Furthermore, the conditions for the shaking culture in step (1) are 20 to 30° C. and 100 to 200 r / min.

[0009] Furthermore, the solid rice medium in step (1) is obtained by preparing rice and pure water in a mass / volume ratio of 80:80 to 120 g / ml.

[0010] Furthermore, the above-mentioned fungal No. 4 medium in step (1) is composed of 2% mannitol, 2% glucose, 0.5% yeast extract, 1% peptone, 0.05% KH2PO4, 0.03% MgSO4·7H2O, 0.1% corn steep liquor, and deionized water.

[0011] Furthermore, during the gradient elution in step (2), the volume ratios of chloroform to methanol are 100:0, 100:1, 100:2, 100:3, 100:5, 100:10, and 0:100.

[0012] Furthermore, the volume ratios of petroleum ether to ethyl acetate during the gradient elution in step (2) are 10:1, 10:3, 10:5, 10:6, 10:7, and 10:9.

[0013] The present invention further provides a pharmaceutical composition comprising the above polyketide compound or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable additive.

[0014] The present invention further provides use of the above-mentioned polyketide compound or a pharmaceutically acceptable salt thereof, or the above-mentioned pharmaceutical composition for the production of an anti-inflammatory drug. [Effects of the Invention]

[0015] Compared with the prior art, the present invention has the following advantages: The present invention utilizes endophytic fungal resources and conducts research on plant endophytic fungi to discover new polyketide-based active compounds. As a result of anti-inflammatory activity experiments, compared with the model group, Compound 1 and Compound 2 inhibited NO release to different degrees depending on the concentration, and at a concentration of 5 μM, they showed better inhibitory ability than the positive control group, demonstrating that Compound 1 and Compound 2 have good anti-inflammatory effects. [Brief explanation of the drawings]

[0016] In order to more clearly explain the embodiments of the present invention, the drawings relating to the embodiments will be briefly described below.

[0017] [Figure 1] 1H-NMR diagram of Compound 1 isolated in Example 1. [Figure 2] 1 is a 13C-NMR diagram of Compound 1 isolated in Example 1. [Figure 3] FIG. 1 is an HSQC diagram of Compound 1 isolated in Example 1. [Figure 4] 1 is an HMBC diagram of Compound 1 isolated in Example 1. [Figure 5] FIG. 1 is a NOESY diagram of Compound 1 isolated in Example 1. [Figure 6] 1H-NMR diagram of compound 2 isolated in Example 1. [Figure 7] FIG. 13C-NMR diagram of Compound 2 isolated in Example 1. [Figure 8] FIG. 1 is an HSQC diagram of Compound 2 isolated in Example 1. [Figure 9] FIG. 1 is an HMBC diagram of Compound 2 isolated in Example 1. [Figure 10] FIG. 1 is a NOESY diagram of Compound 2 isolated in Example 1. [Figure 11] FIG. 1 is a CD diagram of Compound 1-2 isolated in Example 1. [Figure 12] FIG. 1 is a DP4+ diagram of compound 1-2 isolated in Example 1. [Figure 13] FIG. 1 is a graph showing the anti-inflammatory effect of Compound 1-2 isolated in Example 1. DETAILED DESCRIPTION OF THE INVENTION

[0018] The present invention will be described in detail below with reference to examples, but the embodiments of the present invention are not limited thereto. The examples described below are only a portion of the examples of the present invention, and it is clear that other similar examples obtained by those skilled in the art without any creative work are within the scope of protection of the present invention. Unless otherwise specified, all experimental methods used in the present invention are conventional methods, and all experimental equipment, materials, reagents, etc. used are commercially available.

[0019] Example 1 The method for producing a polyketide compound mainly includes steps (1) and (2).

[0020] (1) Fungal fermentation: Alternaria sp. HJT-Y7, an endophytic fungus of the genus Alternaria collected from the leaves of Tibetan Rhodiola rosea, was cultured in Fungal Medium No. 4 (prepared with 2% mannitol, 2% glucose, 0.5% yeast extract, 1% peptone, 0.05% KH2PO4, 0.03% MgSO4·7H2O, 0.1% corn steep liquor, and deionized water) with shaking at 28°C and 180 rpm. The fermented liquid obtained by shaking was inoculated into an Erlenmeyer flask containing rice solid medium (prepared with rice and purified water in a mass / volume ratio of 80 g:110 mL) and left to ferment at 28°C for 40 days to obtain a solid fermented product.

[0021] (2) Extraction and separation of metabolites: The solid fermentation product obtained in step (1) was subjected to ultrasonic extraction with an equal volume of methanol, filtered through eight layers of gauze, and the extract was separated from the mycelium and rice. The extract was concentrated and loaded onto a silica gel chromatography column for separation. 300-400 mesh silica gel was used as the stationary phase, and chloroform / methanol was used as the mobile phase for gradient elution. The gradient was chloroform:methanol = 100:0, 100:1, 100:2, 100:3, 100:5, 100:10, 0:100 (v / v), and the flow rate of the eluent (mobile phase) was 30 mL / min. 10,000 mL was eluted per gradient. The eluate (one bottle for every 500 mL) was collected and concentrated. Based on the TLC results, the bottles were combined to sequentially obtain fractions 1 to 16. Fraction 7 was loaded onto a silica gel chromatography column for separation. 200-300 mesh silica gel was used as the stationary phase, and petroleum ether / ethyl acetate was used as the mobile phase for gradient elution. The gradient was petroleum ether:ethyl acetate = 10:1, 10:3, 10:5, 10:6, 10:7, 10:9 (v / v) in order. The eluate flow rate was 30 mL / min. The eluate (one bottle for every 500 mL) was collected and concentrated. Based on the TLC results, the bottles were combined to sequentially obtain fractions 7-1 to 7-10. Fraction 7-6 was loaded onto a Sephadex column. The mixture was loaded onto an LH-20 gel column for separation. The mobile phase was a 1:1 (v / v) mixture of dichloromethane and methanol at a flow rate of 0.3 mL / min. The eluate (collected every 10 mL) was collected and four eluates were combined into one fraction, yielding fractions 7-6-1 to 7-6-6, respectively. Fraction 7-6-3 was loaded onto an Agilent C18 high-performance liquid chromatography column for separation. The mobile phase was a 55% aqueous methanol solution at a flow rate of 3 mL / min. The detector was a 210 nm UV detector. Monomer compound 1 was obtained at a retention time of 59 minutes, and monomer compound 2 was obtained at a retention time of 62 minutes.

[0022] (3) Structural identification: Using deuterium-substituted dimethyl sulfoxide as the solvent, a Bruker Avance II 500M nuclear magnetic resonance spectrometer was used to measure the nuclear magnetic resonance spectra of the separated compounds 1 and 2, and the mass spectra were used to estimate the molecular formulas. A J-810-150S circular dichroism analyzer was used to measure the circular dichroism spectra of compounds 1 and 2, and DP4+ software was used to calculate the three-dimensional structures of compounds 1 and 2. The structure of the product was finally identified based on the nuclear magnetic resonance spectra, CD results, and calculation results.

[0023] The spectral data of Compound 1 is as follows: 1 H-NMR (500 Hz, DMSO-d6) δ : 3.14 (1H, d, J = 13.5 Hz, H-1a), 4.17 (1H, d, J = 13.5 Hz, H-1b), 9.45 (1H, s, H-3OH), 6.63 (1H, d, J = 8.0 Hz, H-4), 7.00 (1H, t, J = 8.0 Hz, H-5), 6.72 (1H, d, J = 8.0 Hz, H-6), 6.56 (1H, d, J = 12.5 Hz, H-8), 6.01 (1H, d, J = 12.5Hz, H-9), 3.72 (1H, q, J = 6.0 Hz, H-11), 0.87 (3H, d, J = 6.0 Hz, H-12), 5.04 (1H, d, J = 12.5 Hz, H-1'a), 5.23 (1H, d, J = 12.5 Hz, H-1'b), 9.67 (1H, s, H-3'OH), 6.83 (1H, d, J = 8.0 Hz, H-4'), 7.05 (1H, t, J = 8.0 Hz, H-5'), 6.76 (1H, d, J = 8.0 Hz, H-6'), 4.04 (1H, d, J = 10.0Hz, H-8'), 4.54 (1H, d, J = 10.0 Hz, H-9'), 3.66 (1H, s, H-10'), 2.17 (3H, s, H-12'). 13C-NMR (125 Hz, DMSO-d6) δ : 59.5 (C-1), 126.8 (C-2), 153.3(C-3), 114.4 (C-4), 128.3 (C-5), 121.2 (C-6), 137.4 (C-7), 131.1 (C-8), 137.1 (C-9), 93.7 (C-10), 81.7 (C-11), 12.9 (C-12), 61.0 (C-1'), 122.8 (C-2'), 156.1 (C-3'), 115.3 (C-4'), 128.6 (C-5'), 123.3 (C-6'), 139.3 (C-7'), 59.8 (C-8'), 79.3 (C-9'), 79.7 (C-10'), 209.5 (C-11'), 28.0 (C-12').

[0024] The HMBC spectrum of Compound 1 is shown below. [ka]

[0025] The NOESY spectrum of compound 1 is shown below. [ka]

[0026] The spectral data of Compound 2 is as follows: 1H-NMR (500 Hz, DMSO-d6) δ : 4.33 (1H, d, J = 13.5 Hz, H-1a), 5.11 (1H, d, J = 13.5 Hz, H-1b), 9.62 (1H, s, H-3OH), 6.83 (1H, d, J = 8.0 Hz, H-4), 7.16 (1H, t, J = 8.0Hz, H-5), 6.85 (1H, d, J = 8.0 Hz), 6.63 (1H, d, J = 12.5 Hz, H-8), 5.85 (1H, d, J = 12.5 Hz, H-9), 4.31 (1H, q, J = 6.5 Hz, H-11), 1.31 (3H, d, J = 6.5 Hz, H-12), 4.33 (1H, d, J = 13.5 Hz, H-1’a), 5.27 (1H, d, J = 13.5 Hz, H-1’b), 9.40 (1H, s, H-3’OH), 6.58 (1H, d, J = 7.8 Hz, H-4’), 6.53 (1H, t, J = 7.8 Hz, H-5’), 6.82 (1H, d, J = 7.8 Hz, H-6’), 3.84 (1H, d, J = 10.0 Hz, H-8’), 3.75 (1H, d, J = 10.0 Hz, H-9’), 4.01 (1H, s, H-10’), 2.14 (3H, s, H-12’)。 13 C-NMR (125 Hz, DMSO-d6) δ : 59.1 (C-1), 126.0 (C-2), 154.0 (C-3), 115.2 (C-4), 128.6 (C-5), 122.1 (C-6), 137.5 (C-7), 131.6 (C-8), 132.9 (C-9), 92.5 (C-10), 84.6 (C-11), 20.7 (C-12), 64.0 (C-1’), 126.4 (C-2’), 154.4 (C-3’), 113.8 (C-4’), 127.9 (C-5’), 119.8 (C-6’), 136.7 (C-7’), 52.5 (C-8’), 78.7 (C-9’), 91.5 (C-10’), 205.3 (C-11’), 27.7 (C-12’).

[0027] The HMBC spectrum of compound 2 is shown below. [ka]

[0028] The NOESY spectrum of compound 2 is shown below. [ka]

[0029] Example 2 Study on the anti-inflammatory activity of Compound 1-2 prepared in Example 1 Culture of mouse RAW 264.7 macrophages: RAW 264.7 macrophages were seeded in high-sugar DMEM medium containing 10% fetal bovine serum (<0.5 EU / mL) and 1% penicillin and streptomycin, and cultured in a 37°C, 5% CO2 incubator. When cell coverage reached approximately 90%, the supernatant was discarded and the cells were subcultured by spraying with 37°C medium. Cells in the logarithmic growth phase were harvested and used for subsequent experiments.

[0030] Establishment of LPS-induced RAW 264.7 macrophage inflammation model: RAW 264.7 cells in the logarithmic growth phase were harvested at 1 x 10 per well. 4The cells were seeded into a 96-well cell culture plate, and after 24 hours of cell adhesion, a normal control group, an LPS model group, a positive control group, and a compound group were placed, each group having 6 wells. For the normal control group and LPS model group, cells were first cultured in serum-free DMEM medium; for the compound group, cells were cultured in serum-free DMEM containing compound 1 or compound 2 at final concentrations of 40.00, 20.00, 10.00, and 5 μM, respectively; for the positive control group, cells were cultured in serum-free DMEM containing L-NAME (a nitric oxide synthase inhibitor) at a final concentration of 200 μM. After culturing the cells in this manner for 4 hours, the normal control group continued to be cultured in serum-free DMEM; in the other groups, LPS was added to a final concentration of 1 μg / mL to stimulate the cells. After 24 hours, 50 μL of medium per well was collected and NO secretion was detected using a nitric oxide screening kit. 10 μL of 5 g / L MTT was added to each well. After further culturing for 4 hours, the supernatant was discarded, and 150 μL of DMSO was added to each well. The cells were shaken for 10 minutes, and the absorbance of each well was measured at 570 nm.

[0031] The MTT results showed that the IC of Compound 1 and Compound 2 prepared in Example 1 50 is greater than 100 μM (IC 50 The results of the anti-inflammatory experiment are shown in Figure 13. Compared with the model group, Compound 1 and Compound 2 inhibited NO release to different degrees depending on the concentration, and at a concentration of 5 μM, both showed better inhibitory activity than the positive control group. This clearly shows that Compound 1 and Compound 2 have good anti-inflammatory activity.

[0032] Finally, it should be noted that the above embodiments are merely for illustrating the technical means of the present invention, and are not intended to limit the same. Although the present invention has been described in detail with reference to the above embodiments, it is understood by those skilled in the art that the technical means described in the above embodiments can be modified or equivalently replaced with part or all of the technical features, and that such modifications or replacements do not depart from the essence of the corresponding technical means within the scope of the technical means of the embodiments of the present invention.

[0033] [Note] [Appendix 1] A polyketide compound having the structure shown in the following formula or a pharmaceutically acceptable salt thereof. [ka]

[0034] [Appendix 2] The endophytic fungus of the genus Alternaria (Alternaria sp.) HJT-Y7 was deposited at the Center of Ordinary Microorganisms, China Committee for the Preservation and Management of Microbial Species of China on May 6, 2022, with the deposit number CGMCC No. 40166 and the address of the depository institution is No. 3, Courtyard No. 1, Beichen West Road, Chaoyang District, Beijing.

[0035] [Appendix 3] Step (1) of inoculating the Alternaria sp. HJT-Y7 described in Appendix 2 into a fungal No. 4 medium, culturing it with shaking, inoculating the resulting fermented liquid together with the mycelium into a rice solid medium, and allowing it to stand at 20 to 30°C for 10 to 40 days for fermentation to obtain a solid fermentation product; and (2) extracting the solid fermentation product obtained in step (1) with methanol, filtering the extract, loading the extract onto a silica gel chromatography column, and performing gradient elution using a chloroform / methanol mobile phase at a volume ratio of 100:0 to 0:100 to obtain fractions 1 to 16 in sequence based on the TLC results. Loading fraction 7 onto a silica gel chromatography column, and performing gradient elution using a petroleum ether / ethyl acetate mobile phase at a volume ratio of 10:1 to 10:9 to obtain fractions 7-1 to 7-10 in sequence based on the TLC results. Loading fraction 7-6 onto a Sephadex LH-20 gel column, and performing elution using a dichloromethane / methanol mobile phase at a volume ratio of 1:1 to obtain fractions 7-6-1 to 7-6-6 in sequence. Loading fraction 7-6-3 onto an Agilent C18 high-performance liquid chromatography column, and performing elution using a 55% aqueous methanol solution in sequence to obtain compound 1 and compound 2.

[0036] [Appendix 4] The production method according to Appendix 3, wherein the shaking culture conditions in step (1) are 20 to 30°C and 100 to 200 r / min.

[0037] [Appendix 5] The method for producing rice according to Appendix 3, wherein the solid rice medium in step (1) is obtained by preparing rice and pure water in a mass / volume ratio of 80:80 to 120 g / mL.

[0038] [Appendix 6] The production method described in Appendix 3, wherein the fungal No. 4 medium in step (1) is composed of 2% mannitol, 2% glucose, 0.5% yeast extract, 1% peptone, 0.05% KH2PO4, 0.03% MgSO4·7H2O, 0.1% corn steep liquor, and deionized water.

[0039] [Appendix 7] The production method described in Appendix 3, wherein during the gradient elution in step (2), the volume ratio of chloroform / methanol is 100:0, 100:1, 100:2, 100:3, 100:5, 100:10, or 0:100.

[0040] [Appendix 8] The method for producing the present invention described in Appendix 3, wherein in the gradient elution step (2), the volume ratio of petroleum ether / ethyl acetate is 10:1, 10:3, 10:5, 10:6, 10:7, or 10:9.

[0041] [Appendix 9] A pharmaceutical composition comprising the polyketide compound described in Appendix 1 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient.

[0042] [Appendix 10] Use of the polyketide compound or pharmaceutically acceptable salt thereof described in Appendix 1 or the pharmaceutical composition described in Appendix 9 for the manufacture of an anti-inflammatory drug.

Claims

1. A polyketide compound having the structure shown in the following formula or a pharmaceutically acceptable salt thereof. 【Chemistry 1】

2. A pharmaceutical composition comprising the polyketide compound of claim 1 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient.

3. Use of the polyketide compound according to claim 1 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition according to claim 2, for the manufacture of an anti-inflammatory drug.

Images