Preparation method of compound colletotriones A-C and application of compound colletotriones A-C in preparation of anti-inflammatory drugs

Abstract

The invention discloses a preparation method of a compound colletotriones A-C and an application of the compound colletotriones A-C in preparation of an anti-inflammatory drug. The compound colletotriones A-C disclosed by the invention is prepared by being separated from a fermentation culture of an endophytic fungus Colletotrichum fructicola A885 of aquilaria sinensis, and the compound colletotriones A-C is prepared from the fermentation culture of the endophytic fungus Colletotrichum fructicola A885 of the aquilaria sinensis. Experiments prove that the IC50 value range of a mouse macrophage RAW264.7 inflammation model induced by the compound colletotriones A-C lipopolysaccharide LPS on inhibition of the generation of nitric oxide NO is 19.13 + / -0.65 to 78.72 + / -1.74 [mu] M, and the IC50 value range of the mouse macrophage RAW264.7 inflammation model induced by the compound colletotriones A-C lipopolysaccharide LPS on inhibition of the generation of the nitric oxide NO is 19.13 The positive control indometacin inhibits the generation of nitric oxide NO by a lipopolysaccharide LPS-induced mouse macrophage RAW264.7 inflammation model, the IC50 value is 29.44 + / -1.03 mu M, the obvious anti-inflammatory activity is shown, and the indometacin can be used for preparing anti-inflammatory drugs. The invention provides a candidate drug for research and development of novel anti-inflammatory drugs, and provides a scientific basis for development and utilization of aquilaria sinensis endophytic fungus resources. # imgabs0 #

Description

Technical Field

[0001] The invention belongs to the field of pharmaceutical biotechnology, and specifically relates to a compound colletotriones AC and a preparation method and application thereof. Background Art

[0002] Inflammation is one of the most important immune mechanisms to respond to health threats such as microorganisms, infections, cell or tissue damage, toxins, etc., and is essential for cell survival and function. However, uncontrolled inflammatory responses or chronic activation of immune cells will have serious adverse effects on internal organs, leading to many intractable diseases such as rheumatoid arthritis, diabetes, hypertension and cancer. Anti-inflammatory drugs are widely used in clinical practice, and their application is second only to anti-infective drugs, making them the second largest drug for treating diseases. Commercially available clinical anti-inflammatory drugs mainly include non-steroidal and steroid drugs. However, these drugs have more or less adverse side effects including gastric bleeding and kidney disease. Therefore, the development of new anti-inflammatory drugs with low toxicity and high efficiency is a scientific problem that needs to be solved urgently in the pharmaceutical industry.

[0003] Aquilaria sinensis (Lour.) Spreng, a unique aromatic plant medicine, is mainly formed in the injured stems of Aquilaria sinensis (Lour.) Spreng. It is a traditional Chinese medicine and is used in pharmacology to treat most chronic inflammatory diseases, such as degenerative neurological diseases, paralysis, rheumatism, asthma, etc. The formation of aquilaria mainly depends on natural or artificial physical damage and microbial infection. However, the long growth cycle and high production cost limit its wide application and further development. Fungi are important biological resources for mining active natural products. They can produce secondary metabolites with novel skeletons, diverse structures and significant activities. They have received increasing attention in the development of active natural drug molecules and the preparation of prodrug molecules.

[0004] Endophytic fungi are microorganisms that grow between the cells of host plant tissues but do not cause any pathology. These endophytic fungi can produce active secondary metabolites that are similar or identical to the host plant structure, as well as other active molecules with novel structures. They have a short growth cycle and low culture cost, and are the main source of lead drugs. Since 1993, when American scientists Stierle et al. discovered an endophytic fungus (Taxomyces anadreanae) that can produce paclitaxel from Pacific brevifolia, plant endophytes have attracted widespread attention from scholars at home and abroad. Endophytic fungi are regarded as a treasure trove of new microbial resources due to their unique biological value. Through the study of secondary metabolites of endophytic fungi in plants, on the one hand, it is helpful to analyze their metabolic network, explore active molecules, and discover natural products with potential for clinical application; on the other hand, the research has promoted the development of biochemistry and pharmaceutical industries and provided innovative ideas for solving the problem of sustainability of drug sources. Summary of the Invention

[0005] The object of the present invention is to provide, in view of the deficiencies of the prior art, new compounds colletotriones A - C with anti - inflammatory activity discovered from endophytic fungi of Aquilaria sinensis, as well as their preparation methods and applications.

[0006] The first object of the present invention is to provide compounds colletotriones A - C represented by formula (I),

[0007]

[0008] The second object of the present invention is to provide a preparation method of compounds colletotriones A - C, which is separated and prepared from the fermentation culture of the endophytic fungus Colletotrichum fructicola A885 of Aquilaria sinensis.

[0009] Preferably, the preparation method includes the following steps:

[0010] a. Prepare the fermentation culture of the fungus Colletotrichum fructicola A885, separate the mycelium and the fermentation broth, extract the fermentation broth with ethyl acetate, and obtain an extract after concentration;

[0011] b. The extract is fractionated by gradient elution through silica gel column chromatography in a petroleum ether / ethyl acetate v / v, 1:0 → 0:1 solvent system, and divided into 5 fractions Fr.1 - Fr.5 according to TLC color development analysis. TLC thin - layer chromatography is developed with hexane:ethyl acetate = 4:1 (v / v) to obtain fraction Fr.2 with Rf = 0.4 - 0.8, and TLC thin - layer chromatography is developed with hexane:ethyl acetate = 2:1 v / v to obtain fraction Fr.3 with Rf = 0.3 - 0.8;

[0012] Fr.2 is passed through C 18Reverse-phase column chromatography was performed with elution using MeOH-H2O at a ratio of 1:5→1:0, v / v. The fraction Fr2.8 eluted with a MeOH-H2O volume ratio of 1:5 was collected. This fraction was then subjected to silica gel column chromatography with petroleum ether / ethyl acetate as the eluent, and gradient elution was carried out at volume ratios of 4:1, 2:1, and 1:1. The fractions were collected and then combined by TLC thin-layer chromatography. The fraction Fr.2.8.1 with an Rf value of 0.5 - 0.6 obtained by developing with n-hexane:ethyl acetate = 2:1 v / v in TLC thin-layer chromatography was collected; the fraction Fr.2.8.2 with an Rf value of 0.4 - 0.5 obtained by developing with n-hexane:ethyl acetate = 4:1 v / v in TLC thin-layer chromatography was collected. Colletotrione A was obtained from Fr.2.8.1 by semi-preparative high-performance liquid chromatography; Colletotrione B was obtained from Fr.2.8.2 by semi-preparative high-performance liquid chromatography;

[0013] Fr.3 was subjected to C 18 Reverse-phase column chromatography was performed with elution using MeOH-H2O at a ratio of 2:5→1:0, v / v, resulting in eight fractions Fr.3.1 - Fr.3.8. The fraction Fr.3.8 eluted with a MeOH-H2O volume ratio of 1:4 was collected. This fraction was then subjected to silica gel column chromatography with gradient elution at volume ratios of 4:1, 2:1, and 1:1. The fractions were collected and then combined by TLC thin-layer chromatography. The fraction Fr.3.8.1 with an Rf value of 0.7 - 0.8 obtained by developing with n-hexane:ethyl acetate = 1:1 v / v in TLC thin-layer chromatography was collected. Colletotrione C was obtained from Fr.3.8.1 by further semi-preparative high-performance liquid chromatography.

[0014] Preferably, the colletotrione A obtained from the fraction Fr.2.8.1 by semi-preparative high-performance liquid chromatography was obtained using -IC column, with the mobile phase being isopropanol / n-hexane at a volume ratio of 20:80 and a flow rate of 2 mL / min, to obtain enantiomers 1a and 1b.

[0015] Preferably, the colletotrione B obtained from the fraction Fr.2.8.2 by semi-preparative high-performance liquid chromatography was obtained using -IC column, with the mobile phase being isopropanol / n-hexane at a volume ratio of 13:87 and a flow rate of 2 mL / min, to obtain enantiomers 2a and 2b.

[0016] Preferably, the colletotrione C obtained from the fraction Fr.3.8.1 by semi-preparative high-performance liquid chromatography was obtained using -IC column, with the mobile phase being isopropanol / n-hexane at a volume ratio of 5:95 and a flow rate of 2 mL / min, to obtain two isomers 3a and 3b.

[0017] Preferably, the fermentation culture of the fungus Colletotrichum fructicola A885 is prepared in step a.

[0018] The third object of the present invention is to provide the use of the compounds colletotriones A-C or their pharmaceutically acceptable salts in the preparation of anti-inflammatory drugs.

[0019] The present invention discovers through experiments that the compounds colletotriones A-C inhibit the production of nitric oxide (NO) in a lipopolysaccharide (LPS)-induced inflammatory model of mouse macrophages RAW264.7, and the IC 50 value ranges from 19.13 ± 0.65 to 78.72 ± 1.74 μM. The positive control indomethacin inhibits the production of nitric oxide (NO) in a lipopolysaccharide (LPS)-induced inflammatory model of mouse macrophages RAW264.7, and the IC 50 value is 29.44 ± 1.03 μM. This result indicates that the compounds colletotriones A-C of the present invention have relatively significant anti-inflammatory activities.

[0020]

[0021] The fourth object of the present invention is to provide an anti-inflammatory drug, which comprises at least one of the compounds colletotriones A-C or their pharmaceutically acceptable salts as an active ingredient.

[0022] The fifth object of the present invention is to provide the use of the marine fungus Colletotrichum fructicola A885 in the preparation of the compounds colletotriones A-C.

[0023] Compared with the prior art, the advantages of the present invention are as follows:

[0024] The compounds colletotriones A-C are prepared and isolated from the endophytic fungus Colletotrichum fructicola A885 of Aquilaria sinensis. They have anti-inflammatory activities and can be used in the preparation of anti-inflammatory drugs, providing candidate compounds for the research and development of new anti-inflammatory drugs and scientific basis for the development and utilization of natural active substances of endophytic fungi in medicinal plants.

[0025] The endophytic fungus Colletotrichum fructicola A885 of the present invention was also publicly disclosed in a non-patent literature (webpage) on August 2, 2022, and its website is: https: / / www.ncbi.nlm.nih.gov / nuccore / OL517768.1 / . The applicant himself also holds this strain and guarantees to provide it to the public within 20 years from the filing date. Description of the Drawings

[0026] Figure 1 is the 1 1H-NMR spectrum of compound 1 (colletotrione A);

[0027] Figure 2 is the 13 13C-NMR spectrum of compound 1 (colletotrione A);

[0028] Figure 3 is the COSY spectrum of compound 1 (colletotrione A);

[0029] Figure 4 is the HSQC spectrum of compound 1 (colletotrione A);

[0030] Figure 5 is the HMBC spectrum of compound 1 (colletotrione A);

[0031] Figure 6 is the NOESY spectrum of compound 1 (colletotrione A);

[0032] Figure 7 is the circular dichroism spectrum of compound 1a;

[0033] Figure 8 is the ultraviolet spectrum of compound 1a;

[0034] Figure 9 is the circular dichroism spectrum of compound 1b;

[0035] Figure 10 is the ultraviolet spectrum of compound 1b;

[0036] Figure 11 is the infrared spectrum of compound 1 (colletotrione A);

[0037] Figure 12 is the HR-ESIMS spectrum of compound 1 (colletotrione A);

[0038] Figure 13is that of compound 2 (colletotrione B) 1 1H-NMR spectrum;

[0039] Figure 14 is that of compound 2 (colletotrione B) 13 13C-NMR spectrum;

[0040] Figure 15 is the COSY spectrum of compound 2 (colletotrione B);

[0041] Figure 16 is the HSQC spectrum of compound 2 (colletotrione B);

[0042] Figure 17 is the HMBC spectrum of compound 2 (colletotrione B);

[0043] Figure 18 is the NOESY spectrum of compound 2 (colletotrione B);

[0044] Figure 19 is the circular dichroism spectrum of compound 2a;

[0045] Figure 20 is the ultraviolet spectrum of compound 2a;

[0046] Figure 21 is the circular dichroism spectrum of compound 2b;

[0047] Figure 22 is the ultraviolet spectrum of compound 2b;

[0048] Figure 23 is the infrared spectrum of compound 2 (colletotrione A);

[0049] Figure 24 is the HR-ESIMS spectrum of compound 2 (colletotrione B).

[0050] Figure 25 is that of compound 3 (colletotrione C) 1 1H-NMR spectrum;

[0051] Figure 26 is that of compound 3 (colletotrione C) 13 13C-NMR spectrum;

[0052] Figure 27 is the COSY spectrum of compound 2 (colletotrione B);

[0053] Figure 28 It is the HSQC spectrum of compound 3 (colletotrione C);

[0054] Figure 29 It is the HMBC spectrum of compound 3 (colletotrione C);

[0055] Figure 30 It is the NOESY spectrum of compound 3 (colletotrione C);

[0056] Figure 31 It is the circular dichroism spectrum of compound 3a;

[0057] Figure 32 It is the ultraviolet spectrum of compound 3a;

[0058] Figure 33 It is the circular dichroism spectrum of compound 3b;

[0059] Figure 34 It is the ultraviolet spectrum of compound 3b;

[0060] Figure 35 It is the infrared spectrum of compound 3 (colletotrione C);

[0061] Figure 36 It is the HR-ESIMS spectrum of compound 3 (colletotrione C). Detailed implementation manners

[0062] The following examples are further illustrations of the present invention rather than limitations thereof.

[0063] Example 1:

[0064] I. Isolation, purification and identification of the endophytic fungus A885 from Aquilaria sinensis

[0065] The endophytic fungus A885 of the present invention was isolated from the stem of Aquilaria sinensis collected from Huidong County, Guangdong Province in August 2022. It was identified by ITS sequence analysis, and the GenBank gene accession number is: OL517768.1. After blast alignment and homologous analysis, this strain was identified as Colletotrichum fructicola and named Colletotrichum fructicola strain NYG, hereinafter referred to as strain A885.

[0066] II. Liquid fermentation of strain A885

[0067] The culture medium is potato dextrose liquid medium, and each liter of the culture medium is prepared by the following method: 200 g of potatoes are boiled with 500 mL of water for 20 min, filtered to obtain potato juice, then 20 g of glucose, 3 g of KH2PO4, 1.5 g of MgSO4, 10 mg of vitamin B1 are added, and the volume is made up to 1000 mL with water, sterilized at 121 °C under high pressure for 20 min, and cooled for use.

[0068] An appropriate amount of mycelium of strain A885 is picked and inoculated into potato dextrose liquid medium, and cultured at 28 °C and 120 r / min for 5 days to obtain a seed solution. Then the seed solution is inoculated into a solid medium (3000 mL conical flask) containing 250 g of rice and 350 mL of water at an inoculation amount of 10% by volume, and a total of 40 bottles are fermented, and cultured at 28 °C for 30 days to obtain a solid fermentation culture of strain A885.

[0069] III. Preparation of Compounds Colletotriones A - C

[0070] The solid fermentation culture is extracted 4 - 5 times with twice the volume of ethyl acetate, and the extract is concentrated by vacuum distillation to finally obtain 150.1 g of a brownish-black crude extract. The crude extract is fractionated by gradient elution on a silica gel column chromatography (200 - 300 mesh) in a petroleum ether / ethyl acetate (v / v, 1:0 → 0:1) solvent system, and divided into 5 fractions (Fr.1 - Fr.5) according to TLC color development analysis.

[0071] Fraction Fr.2 (5.0 g, developed by TLC thin layer chromatography with hexane:ethyl acetate = 4:1 (v / v) to obtain Rf = 0.4 - 0.8) is subjected to 18 reverse phase column chromatography, eluted with MeOH - H2O (1:5 → 1:0, v / v) to obtain nine fractions (Fr.2.1 - Fr.2.9). The fraction Fr2.8 eluted with a methanol - water volume ratio of 1:5 is collected, subjected to silica gel column chromatography, eluted with petroleum ether / ethyl acetate as the eluent, and gradient eluted at a volume ratio of 4:1, 2:1, 1:1. The fractions are collected, then combined by TLC thin layer chromatography, and the fraction Fr2.8.1 with Rf = 0.5 - 0.6 developed by TLC thin layer chromatography with hexane:ethyl acetate = 2:1 (v / v) is collected; the fraction Fr2.8.2 with Rf = 0.4 - 0.5 developed by TLC thin layer chromatography with hexane:ethyl acetate = 4:1 (v / v) is collected. Colletotrione A is obtained from Fr.2.8.1 by semi-preparative high performance liquid chromatography; Colletotrione B is also obtained from Fr.2.8.2 by semi-preparative chiral HPLC.

[0072] The component Fr.3 (4.3 g, developed by TLC with n-hexane:ethyl acetate = 2:1 (v / v) to give Rf = 0.3 - 0.8) was subjected to C 18 reverse-phase column chromatography and eluted with MeOH-H2O (2:5 → 1:0, v / v) to obtain eight components (Fr.3.1 - Fr.3.8). The fraction Fr.3.8 eluted with a MeOH-H2O volume ratio of 1:4 was collected. Component Fr.3.8 was then subjected to silica gel column chromatography and gradient eluted with volume ratios of 4:1, 2:1, and 1:1. The fractions were collected and then combined by TLC. The component Fr.3.8.1 with Rf = 0.7 - 0.8 developed by TLC with n-hexane:ethyl acetate = 1:1 (v / v) was collected. Fr.3.8.1 was further purified by semi-preparative HPLC to obtain colletotrione C.

[0073] The above-mentioned component Fr.2.8.1 was further purified by semi-preparative HPLC to obtain the compounds colletotrione A (1a and 1b) using -IC column, with the mobile phase being isopropanol / n-hexane at a volume ratio of 20:80 and a flow rate of 2 mL / min, to obtain the enantiomers 1a (10 mg, t R = 12.5 min) and 1b (17.5 mg, t R = 17 min). The compounds colletotrione B (2a and 2b) were obtained using -IC column, with the mobile phase being isopropanol / n-hexane at a volume ratio of 13:87 and a flow rate of 2 mL / min, to obtain the enantiomers 2a (2.0 mg, t R = 7.5 min) and 2b (2.5 mg, t R = 12 min). The compounds colletotrione C (3a and 3b) were obtained using -IC column, with the mobile phase being isopropanol / n-hexane at a volume ratio of 5:95 and a flow rate of 2 mL / min, to obtain the two isomers 3a (5.0 mg, t R = 10 min) and 3b (7.0 mg, t R = 13 min).

[0074] IV. Structure Identification of Compounds Colletotriones A - C

[0075] 1 1H NMR, 13The 13C NMR and HMBC nuclear magnetic resonance spectra were measured using a Bruker Advance-500 nuclear magnetic resonance spectrometer with tetramethylsilane (TMS) as the internal standard; the ESI-MS data were measured using a VG Autospec-3000 mass spectrometer; the ultraviolet spectrum was measured using a UV6000 ultraviolet-visible spectrophotometer from Shanghai Yuanxi Instruments Co., Ltd.

[0076] As Figures 1-34 shown, Figure 1 is the 1 1H-NMR spectrum of compound 1 (colletotrione A); Figure 2 is the 13 13C-NMR spectrum of compound 1 (colletotrione A); Figure 3 is the COSY spectrum of compound 1 (colletotrione A); Figure 4 is the HSQC spectrum of compound 1 (colletotrione A); Figure 5 is the HMBC spectrum of compound 1 (colletotrione A); Figure 6 is the NOESY spectrum of compound 1 (colletotrione A); Figure 7 is the CD spectrum of compound 1a; Figure 8 is the ultraviolet spectrum of compound 1a; Figure 9 is the circular dichroism spectrum of compound 1b; Figure 10 is the ultraviolet spectrum of compound 1b; Figure 11 is the infrared spectrum of compound 1 (colletotrione A); Figure 12 is the HR-ESIMS spectrum of compound 1 (colletotrione A); Figure 13 is the 1 1H-NMR spectrum of compound 2 (colletotrione B); Figure 14 is the 13 13C-NMR spectrum of compound 2 (colletotrione B); Figure 15 is the COSY spectrum of compound 2 (colletotrione B); Figure 16 is the HSQC spectrum of compound 2 (colletotrione B); Figure 17 is the HMBC spectrum of compound 2 (colletotrione B); Figure 18 is the NOESY spectrum of compound 2 (colletotrione B); Figure 19 is the CD spectrum of compound 2a; Figure 20 is the ultraviolet spectrum of compound 2a; Figure 21 is the circular dichroism spectrum of compound 2b;Figure 22 is the UV spectrum of compound 2b; Figure 23 is the IR spectrum of compound 1 (colletotrione A); Figure 24 is the HR-ESIMS spectrum of compound 2 (colletotrione B). Figure 25 is of compound 3 (colletotrione C) 1 1H-NMR spectrum; Figure 26 is of compound 3 (colletotrione C) 13 13C-NMR spectrum; Figure 27 is the COSY spectrum of compound 3 (colletotrione B); Figure 28 is the HSQC spectrum of compound 3 (colletotrione C); Figure 29 is the HMBC spectrum of compound 3 (colletotrione C); Figure 30 is the NOESY spectrum of compound 3 (colletotrione C); Figure 31 is the CD spectrum of compound 3a; Figure 32 is the UV spectrum of compound 3a; Figure 33 is the circular dichroism spectrum of compound 3b; Figure 34 is the UV spectrum of compound 3b; Figure 35 is the IR spectrum of compound 3 (colletotrione C); Figure 36 is the HR-ESIMS spectrum of compound 3 (colletotrione C).

[0077] The new compound 1, named (±)-colletotriones A (1a / 1b), is a yellow transparent oil, soluble in MeOH, Based on the HRESIMS m / z 455.2080 ([M-H] - , calculated value 455.5350) of the high-resolution mass spectrum of the compound, its molecular formula was determined to be C 26 H 32 O7, with an unsaturation degree of 11. In the infrared spectrum, hydroxyl signals at 3649 cm -1 , carbonyl signals at 1607 cm -1 and ether bond signals at 1220 cm -1 were shown.

[0078] From the 1 1H NMR data of compound 1 (Table 1), signals of a pentasubstituted benzene ring were shown: [δ H 6.88 (1H, s, H-2)], a tetrasubstituted benzene ring: [δH 6.53 (1H, t, J = 8.2 Hz, H-5), δ H 7.31 (1H, d, J = 8.2 Hz, H-6)], one olefinic hydrogen signal: [δ H 5.32 (1H, J = 7.3 Hz, H-18)], one methoxy signal: [δ H 3.37 (3H, s, H-16)] and five methyl functional group signals: [δ H 2.33 (3H, s), δ H 1.35 (3H, s), δ H 1.34 (3H, s), δ H 1.72 (3H, s), δ H 1.76 (3H, s)]. Further combined with 13 C NMR (Table 3-2) and HMQC data showed a total of 26 carbon signals, including 6 methyl, 3 methylene, 5 methine and 2 ketone carbonyl carbon signals. The above aromatic rings and functional groups together occupied 10 degrees of unsaturation of the molecule, and the remaining 1 degree of unsaturation was speculated to possibly contain a tricyclic system of benzoxazole.

[0079] Through the 2D NMR spectral data, the planar structure of 1 was further determined. To further determine the configuration of compound 1, we measured the CD curve spectrum and compared it with the theoretically calculated ECD curve, and deduced its absolute configuration. Since the specific rotation of 1 was very close to zero, it indicated that compound 1 might exist as a mixture of a pair of racemic isomers. Subsequently, the racemate 1 was successfully separated into a pair of optically pure enantiomers 1a and 1b by semi-preparative HPLC using a Chiralpak IC chiral chromatographic column, and the data of compounds 1a and 1b were verified by experimental circular dichroism (ECD) to confirm that they were enantiomeric relationships. By comparing the experimental ECD curve with the theoretically calculated ECD curve, the absolute configuration of compound 1a was finally determined to be 4c-R and the absolute configuration of compound 1b was 4c-S.

[0080] Table 1 NMR data of compounds 1-3

[0081]

[0082] a Chloroform-d; b Methanol-d

[0083] The new compound 2, named (±)-colletotriones B (2a / 2b), was a yellow transparent oil and was soluble in MeOH, Based on the high-resolution mass spectrum of the compound, HRESIMS m / z 441.1912 ([M+H] + , with a calculated value of 441.5360), its molecular formula was determined to be C 26 H 32 O6, and the degree of unsaturation was 11. In the infrared spectrum, signals of hydroxyl group at 3420 cm -1 , carbonyl group at 1607 cm -1 , and ether bond at 1220 cm -1 were shown.

[0084] The 1 H and 13 C NMR (Table 1) data of compound 2 were highly similar to those of compound 1. The only difference was that the hydroxyl group at C-13 in compound 1 was replaced by H in 2 (δ of C-13 in 1 C 70.1; δ in 2 C 28.2), which was further confirmed by the speculation in the 2D NMR spectrum ( Figures 3-7 ). Subsequently, compound 2 was subjected to chiral separation to obtain two isomers (2a and 2b). To further determine the absolute configuration of compound 2, the experimental CD curve data of compounds 2a and 2b were measured by circular dichroism (ECD) respectively to verify their enantiomeric relationship. Since the planar structures of compounds 1 and 2 were highly similar, we further determined the absolute configurations of compounds 2a and 2b to be 4c-R and 4c-S respectively by comparing the ECD spectra of compounds 1a and 1b.

[0085] The new compound 3, named (±)-colletotriones C (3a / 3b), was a yellow transparent oil, soluble in MeOH, By analyzing the HRESIMS m / z 439.2122 ([M+H] + , with a calculated value of 439.5200) of the high-resolution mass spectrum of the compound, its molecular formula was determined to be C 26 H 30 O6, and the degree of unsaturation was 12. In the infrared spectrum, signals of hydroxyl group at 3354 cm -1 , carbonyl group at 1624 cm -1 , and ether bond at 1022 cm -1 were shown.

[0086] By analyzing 1 H and 13 C NMR spectral data (Table 4-4), compound 3 had similar NMR data to 1, suggesting that they might contain the same tricyclic skeleton. By further analyzing the NMR data of compounds 1 and 3, it was found that C-13 of compound 3 (δ C= 139.0) and C-12 (δ C 119.2, δ H The chemical shift of 5.56, H-12) increased. Considering the additional degree of unsaturation in compound 3 compared to 1, it was speculated that the 13-OH in compound 1 was dehydrated to form a 12-enyl group in compound 3. The 2D NMR spectral data further confirmed this conclusion. Further chiral separation of compound 3 yielded two optically purified isomers (3a and 3b). Finally, by comparing the ECD spectra of compound 1a and 1b, the absolute configurations of compound 3a and 3b were determined to be 4c-R and 4c-S, respectively.

[0087] Thus, it was determined that the chemical structures of compounds colletotriones A-C are as shown in formula (I).

[0088]

[0089] Example 2:

[0090] The anti-inflammatory activities of compounds colletotriones A-C were tested using the Griess method.

[0091] 1. Test reagents: The compounds colletotriones A-C prepared in this invention were dissolved in dimethyl sulfoxide (DMSO) to obtain a stock solution with a concentration of 10 mM, and then diluted to the required concentration with DMEM medium. The positive control was an aqueous solution of indomethacin.

[0092] The cell line used in this experiment was mouse macrophage RAW264.7.

[0093] 2. Experimental method: The Griess method was used to determine the effect of the compounds on the release of nitric oxide (NO) in the RAW264.7 cell inflammation model induced by bacterial lipopolysaccharide (LPS), so as to evaluate the anti-inflammatory activities of the compounds. RAW264.7 cells in the logarithmic growth phase were digested with trypsin, stained with trypan blue for counting. After the cell viability was detected by the trypan blue exclusion experiment to be greater than 95%, the cell concentration was adjusted to 5×10 5cells / mL, the cells were seeded in a 96-well plate, 180 μL of cell suspension was added to each well, and 3 blank wells were set for zero adjustment. The cells were cultured in an incubator at 37 °C and 5% CO2. After 24 h, the original culture medium was discarded, and 200 μL of medium containing a certain concentration of the above compound and 1 μg / mL LPS was added. The negative control was added with 200 μL of 1 μg / mL LPS medium, and the positive control was added with 200 μL of medium containing a certain concentration of indomethacin and 1 μg / mL LPS. After incubation in an incubator at 37 °C and 5% CO2 for 24 h, 50 μL of the supernatant from each well was aspirated and transferred to a new 96-well plate. The Griess method was used to determine the effect of the compound on the release of nitric oxide (NO). 50 μL of Griess A solution and 50 μL of Griess B solution were added to each well, mixed evenly, and the OD value of each well was measured with an enzyme-linked immunosorbent assay (ELISA) reader at a wavelength of 540 nm. Each experiment was repeated 3 times, and its IC 50 value was calculated. The inhibition rate of the drug on NO production was calculated using the following formula: Inhibition rate of NO production (%) = (1 - A 样品组 / A 对照组 ) × 100%.

[0094] 3. Experimental results: The compounds colletotriones A-C prepared in the present invention inhibited the production of nitric oxide (NO) in the lipopolysaccharide (LPS)-induced RAW264.7 macrophage inflammation model in mice, and the IC 50 value range was 19.13 ± 0.65 - 78.72 ± 1.74 μM. The positive control indomethacin inhibited the production of nitric oxide (NO) in the lipopolysaccharide (LPS)-induced RAW264.7 macrophage inflammation model in mice, and the IC 50 value was 29.44 ± 1.03 μM. This result indicates that the compounds colletotriones A-C of the present invention have relatively significant anti-inflammatory activity. Therefore, the present invention provides candidate compounds for the research and development of new anti-tumor drugs and provides a scientific basis for the development and utilization of natural active substances derived from plant endophytic fungi.

[0095] Table 2 Inhibitory effects of compounds colletotriones A-C on tumor cells

[0096]

[0097]

Claims

1. Compounds colletotriones A - C represented by formula (I): Among them, 1 is compound colletotrione A, 2 is compound colletotrione B, and 3 is compound colletotrione C.

2. A method for preparing the compound colletotrione A-C according to claim 1, characterized in that, They are isolated and prepared from the fermentation culture of the fungus Colletotrichum fructicola A885.

3. The preparation method according to claim 2, wherein It includes the following steps: a. Prepare the fermentation culture of the fungus Colletotrichum fructicola A885, separate the mycelium and the fermentation broth. The fermentation broth is extracted with ethyl acetate, and the extract is concentrated to obtain an extract paste; b. The extract paste is fractionated by gradient elution of the extract through silica gel column chromatography in a petroleum ether / ethyl acetate v / v, 1:0 → 0:1 solvent system. According to TLC color development analysis, it is divided into 5 fractions Fr.1 - Fr.

5. The TLC thin layer chromatography is developed with n - hexane:ethyl acetate = 4:1 (v / v) to obtain the fraction Fr.2 with Rf = 0.4 - 0.8, and the TLC thin layer chromatography is developed with n - hexane:ethyl acetate = 2:1 v / v to obtain the fraction Fr.3 with Rf = 0.3 - 0.8; Fr.2 via C 18 Reverse-phase column chromatography, eluted with MeOH-H2O 1:5→1:0, v / v, and the fraction eluted with a MeOH-H2O volume ratio of 1:5 was collected. Fr2.8 was subjected to silica gel column chromatography, eluted with petroleum ether / ethyl acetate as the eluent, and gradient elution was carried out at volume ratios of 4:1, 2:1, and 1:

1. The fractions were collected and then combined by TLC thin-layer chromatography. The fraction with an Rf value of 0.5-0.6 developed by TLC thin-layer chromatography with hexane:ethyl acetate = 2:1 v / v was collected as Fr.2.8.1; the fraction with an Rf value of 0.4-0.5 developed by TLC thin-layer chromatography with hexane:ethyl acetate = 4:1 v / v was collected as Fr.2.8.

2. Colletotrione A was obtained from Fr.2.8.1 by semi-preparative high-performance liquid chromatography; Colletotrione B was obtained from Fr.2.8.2 by semi-preparative high-performance liquid chromatography; Fr.3 was subjected to C 18 Reverse-phase column chromatography was performed using MeOH-H2O with a gradient of 2:5 → 1:0 (v / v) for elution, yielding eight fractions Fr.3.1 - Fr.3.

8. The fraction eluted with MeOH-H2O at a volume ratio of 1:4 was collected as Fr.3.

8. Fraction Fr.3.8 was then subjected to silica gel column chromatography with gradient elution using volume ratios of 4:1, 2:1, and 1:

1. The eluates were collected and then combined by TLC. The fraction with an Rf value of 0.7 - 0.8 developed with n-hexane:ethyl acetate = 1:1 (v / v) on TLC was collected as Fr.3.8.

1. Fr.3.8.1 was further purified by semi-preparative high performance liquid chromatography to obtain colletotrione C.

4. The preparation method according to claim 3, characterized in that, The component Fr.2.8.1 was subjected to semi-preparative high performance liquid chromatography to obtain colletotrione A using column, with the mobile phase being isopropanol / n-hexane at a volume ratio of 20:80 and a flow rate of 2 mL / min to obtain enantiomers 1a and 1b.

5. The preparation method according to claim 3, characterized in that, The colletotrione B obtained by semi-preparative high performance liquid chromatography for Fr.2.8.2 is obtained using a column with a mobile phase of isopropanol / n-hexane in a volume ratio of 13:87 and a flow rate of 2 mL / min to obtain enantiomers 2a and 2b.

6. The preparation method according to claim 3, characterized in that, The colletotrione C obtained from Fr. 3.8.1 by semi-preparative high performance liquid chromatography was obtained using a column with a mobile phase of isopropanol / n-hexane in a volume ratio of 5:95 and a flow rate of 2 mL / min, yielding two isomers 3a and 3b.

7. The preparation method according to claim 3, characterized in that, The preparation of the fermentation culture of the fungus Colletotrichum fructicola A885 in step a includes the following steps: Pick the mycelium of the fungus Colletotrichum fructicola A885 and inoculate it into a potato dextrose liquid medium, culture it at 28°C and 120 r / min for 5 days to obtain a seed liquid; then inoculate the seed liquid at an inoculation amount of 0.1 mL / g into a rice medium and culture it at 28°C for 30 days to obtain a solid fermentation culture. The potato dextrose liquid medium per liter is prepared by the following method: Boil 200 g of potatoes with 500 mL of pure water for 20 min, filter to obtain potato juice, then add 20 g of glucose, 3 g of KH2PO4, 1.5 g of MgSO4, 10 mg of vitamin B1, and make up to 1000 mL with water, and sterilize it; the rice medium is prepared by the following method: Mix 250 g of rice with 350 mL of a mass - volume ratio aqueous solution and sterilize it.

8. Use of the compounds colletotriones A, B or C according to claim 1, or their pharmaceutically acceptable salts in the preparation of anti - inflammatory drugs.

9. An anti-inflammatory drug, characterized in that, It contains the compounds colletotriones A, B or C according to claim 1, or their pharmaceutically acceptable salts as the active ingredient.

10. Use of the fungus Colletotrichum fructicola A885 in the preparation of the compounds colletotriones A - C according to claim 1.

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