Chromone derivative derived from red sea olive fungi and application of chromone derivative

Abstract

The invention belongs to the field of marine natural medicinal chemistry, and particularly relates to a chromone derivative derived from red sea olive fungi and application of the chromone derivative. According to the application disclosed by the invention, a series of chromone derivatives with novel structures and unique activity are obtained by researching the endophytic fungi Daldinia sp.HJX1P2 of the red sea olive, a crude extract of the fungi Daldinia sp.HJX1P2 shows remarkable anti-inflammatory activity and is dose-dependent, and the IC50 value is 65.8 mu g / mL. Particularly, the compound 1 has an inhibiting effect on NO generated by RAW264.7 cells induced by LPS, the IC50 value is 62.9 [mu] M, and the compound 1 is a positive control dexamethasone (IC50 = 136.8 [mu] M).

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of marine natural medicinal chemistry, and particularly relates to a chromone derivative from a fungus of Rhizophora stylosa and application thereof. BACKGROUND

[0002] Mangrove is a unique intertidal ecosystem in tropical and subtropical regions, which has been living in the environment of acid, high salt, high radiation and frequent tides for a long time. The special growing environment makes it contain rich microbial resources, which not only plays a crucial role in maintaining this ecosystem, but also can metabolize a variety of secondary metabolites with novel structure and excellent biological activity. Rhizophora stylosa, also known as red mangrove or chicken claw olive, is a plant of Rhizophoraceae Rhizophora, which is a traditional medicinal plant in folk and is often used as an astringent, and has the effects of treating leprosy, pain, tuberculosis, antibacterial, antimalarial and antitumor. At present, glycosides, phenols, polyketides, triterpenes and other compounds have been isolated from Rhizophora stylosa, which shows that Rhizophora stylosa has important medicinal development value. A series of chromone derivatives with anti-inflammatory, antibacterial and antioxidant activities are isolated and identified from the endophytic fungus Daldinia sp. HJX1P2 in Rhizophora stylosa. SUMMARY

[0003] The present application provides an endophytic fungus Daldinia sp. HJX1P2 of Rhizophora stylosa, which is characterized by the strain preservation information: the name of the preservation unit is Guangdong Microbial Culture Collection Center; the address of the preservation unit is No. 59, Building 5, Guangdong Academy of Microbiology, 100, Xianlie Middle Road, Guangzhou; the preservation date is April 25, 2025; the preservation number is GDMCC No. 66212; and the classification name is Daldinia sp. The marine fungus is isolated from the stem of the mangrove plant Rhizophora stylosa in Dongzhaigang Mangrove Nature Reserve.

[0004] Another embodiment of the present application provides a chromone derivative or a pharmaceutically acceptable salt thereof, which is characterized in that the chromone derivative has the structure shown in compounds 1, 2, 3, 4 and 5:

[0005]

[0006] Another embodiment of the present application provides a preparation method of the above-mentioned compounds 1, 2, 3, 4 and / or 5, which is characterized by comprising the following steps:

[0007] (1) inoculating the fungus Daldinia sp. HJX1P2 into a rice solid culture medium, and culturing at room temperature for 40-45 days to obtain a fermentation product;

[0008] (2) the fermentation obtained in step (1) is extracted with equal volume of ethyl acetate for 2-4 times, the extraction liquid is combined and concentrated under reduced pressure to obtain a crude extract;

[0009] (3) the crude extract obtained in step (2) is subjected to reduced pressure silica gel column chromatography, and gradient elution is carried out by using petroleum ether-ethyl acetate as an eluent, and the elution gradient is 100:0, 90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 20:80, 10:90 and 0:100, two column volumes are collected for each gradient, the eluent obtained by using petroleum ether: ethyl acetate as 80:20 and 70:30 is combined and concentrated, then gradient elution is carried out by using MeOH-H2O as an eluent by means of reverse phase ODS column, and the elution gradient is 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, 90:10 and 100:0, two column volumes are collected for each gradient, the eluent obtained by using MeOH:H2O as 50:50 and 60:40 is combined and concentrated, and high performance liquid chromatography (HPLC) preparation is carried out, the chromatographic column is Agilent C18, 9.4*250mm, 7μm, the flow rate is 2mL / min, the mobile phase is MeOH:H2O=65:35, and compounds 1, 2 and 3 are obtained; the mobile phase is MeOH:H2O=30:70, and compounds 4 and 5 are obtained.

[0010] wherein the ratio of the eluent or the mobile phase is volume ratio; and the formula of the rice solid culture medium preferably contains 50g of rice, 50g of water and 1.6g of sea salt per 1L of Erlenmeyer flask.

[0011] Another embodiment of the present application provides a crude extract of the fungus Daldinia sp. HJX1P2, and the preparation method of the crude extract comprises the following steps:

[0012] (1) the fungus Daldinia sp. HJX1P2 is inoculated into a rice solid culture medium, and the fermentation is obtained after standing and culturing at room temperature for 40-45 days;

[0013] (2) the fermentation obtained in step (1) is extracted with equal volume of ethyl acetate for 2-4 times, the extraction liquid is combined and concentrated under reduced pressure to obtain a crude extract.

[0014] Another embodiment of the present application provides the application of the fungus Daldinia sp. HJX1P2 in preparing compounds 1, 2, 3, 4 and / or 5.

[0015] Another embodiment of the present application provides the application of the fungus Daldinia sp. HJX1P2 in preparing the crude extract of the fungus Daldinia sp. HJX1P2.

[0016] Another embodiment of the present application provides the use of one or more of the above-mentioned compounds 1, 2, 3, 4, 5 or pharmaceutically acceptable salts thereof or the above-mentioned crude extract of fungus Daldinia sp. HJX1P2 in the preparation of an anti-inflammatory or antibacterial medicament.

[0017] Another embodiment of the present application provides a pharmaceutical composition characterized in that the pharmaceutical composition comprises one or more of the above-mentioned compounds 1, 2, 3, 4, 5 or pharmaceutically acceptable salts thereof or the above-mentioned crude extract of fungus Daldinia sp. HJX1P2 as an effective ingredient. The pharmaceutical composition optionally further comprises a pharmaceutically acceptable adjuvant. The pharmaceutical composition optionally further comprises other anti-inflammatory or antibacterial active ingredients.

[0018] Compared with the prior art, the present application obtains a series of novel and structurally unique chromone derivatives by studying the endophytic fungus Daldinia sp. HJX1P2 in Rhizophora stylosa, and the crude extract of fungus Daldinia sp. HJX1P2 exhibits significant anti-inflammatory activity in a dose-dependent manner, with an IC 50 value of 65.8 μg / mL. In particular, compound 1 has an inhibitory effect on the production of NO by LPS-induced RAW264.7 cells, with IC 50 values of 62.9 μM, respectively, and the positive control dexamethasone (IC 50 = 136.8 μM). BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 is a strain morphology diagram of the endophytic fungus Daldinia sp. HJX1P2 in Rhizophora stylosa of the present application;

[0020] Figure 2 is a key 1 H- 1 H COSY and HMBC correlation signal diagram of compounds 1 and 2;

[0021] Figure 3 is a key NOESY correlation signal diagram of compounds 1 and 2;

[0022] Figure 4 is a test CD and calculated ECD spectrum diagram of compounds 1 and 2;

[0023] Figure 5 is a key 1 H- 1 H COSY and HMBC correlation signal diagram of compound 3;

[0024] Figure 6 is a NOESY correlation signal diagram of compound 3;

[0025] Figure 7is the test CD and calculated ECD spectra of compound 3;

[0026] Figure 8 is the key 1 H- 1 H COSY, HMBC and NOESY correlation maps;

[0027] Figure 9 is the test CD spectra of compounds 4 and 5;

[0028] Figure 10 is the key 1 H- 1 H COSY and HMBC correlation maps;

[0029] Figure 11 is the anti-inflammatory activity results of Daldinia sp. HJX1P2 crude extract;

[0030] Figure 12 is the effect of compound 1 on RAW264.7 cell viability; n = 3, ***P < 0.001. Con: blank control;

[0031] Figure 13 is the inhibitory activity of compound 1 on NO in LPS-treated RAW264.7 cells; n = 3, ***P < 0.001. Con: blank control, Dexa: dexamethasone;

[0032] Figure 14 is the effect of compound 1 on the levels of inflammatory factors in LPS-treated RAW264.7 cells; ELISA was used to determine the levels of tumor necrosis factor alpha (TNF-a), interleukin 1 beta (IL-1b) and interleukin 6 (IL-6), n = 3, ***P < 0.001. Con: blank control, Dexa: dexamethasone;

[0033] Figure 15 is the effect of compound 1 on the levels of iNOS and COX-2 proteins in LPS-treated RAW264.7 cells. n = 3, ***P < 0.001. Con: blank control, Dexa: dexamethasone;

[0034] Figure 16 is the binding site of compound 1 with iNOS protein;

[0035] Figure 17 is the DEPT-135 spectrum of compound 1 (CD3OD);

[0036] Figure 18 is the 1 H- 1H COSY spectrum (CD3OD);

[0037] Figure 19 HSQC spectrum (CD3OD) of Compound 1;

[0038] Figure 20 HMBC spectrum (CD3OD) of Compound 1;

[0039] Figure 21 H NMR spectrum (CD3OD) of Compound 1; 1 H- 1 H NOESY spectrum (CD3OD);

[0040] Figure 22 High resolution mass spectrum (CD3OD) of Compound 1;

[0041] Figure 23 DEPT-135 spectrum (DMSO-d6) of Compound 2;

[0042] Figure 24 H NMR spectrum (DMSO-d6) of Compound 2; 1 H- 1 H COSY spectrum (DMSO-d6);

[0043] Figure 25 HSQC spectrum (DMSO-d6) of Compound 2;

[0044] Figure 26 HMBC spectrum (DMSO-d6) of Compound 2;

[0045] Figure 27 H NMR spectrum (DMSO-d6) of Compound 2; 1 H- 1 H NOESY spectrum (DMSO-d6);

[0046] Figure 28 High resolution mass spectrum (CD3OD) of Compound 2;

[0047] Figure 29 DEPT-135 spectrum (DMSO-d6) of Compound 3;

[0048] Figure 30 H NMR spectrum (DMSO-d6) of Compound 3; 1 H- 1 H COSY spectrum (DMSO-d6);

[0049] Figure 31 HSQC spectrum (DMSO-d6) of Compound 3;

[0050] Figure 32 HMBC spectrum (DMSO-d6) of Compound 3;

[0051] Figure 33 is a high resolution mass spectrum of compound 3 (CD3OD); 1 H- 1 H COSY spectrum (CDC13);

[0052] Figure 34 is a high resolution mass spectrum of compound 3 (CD3OD);

[0053] Figure 35 is a DEPT-135 spectrum of compound 4 (CDC13);

[0054] Figure 36 is a high resolution mass spectrum of compound 4 (CDC13); 1 H- 1 H COSY spectrum (CDC13);

[0055] Figure 37 is a HSQC spectrum of compound 4 (CDC13);

[0056] Figure 38 is a HMBC spectrum of compound 4 (CDC13);

[0057] Figure 39 is a high resolution mass spectrum of compound 4 (CDC13); 1 H- 1 H NOESY spectrum (CDC13);

[0058] Figure 40 is a high resolution mass spectrum of compound 4 (CDC13);

[0059] Figure 41 is a DEPT-135 spectrum of compound 5 (CDC13);

[0060] Figure 42 is a high resolution mass spectrum of compound 5 (CDC13); 1 H- 1 H COSY spectrum (CDC13);

[0061] Figure 43 is a HSQC spectrum of compound 5 (CDC13);

[0062] Figure 44 is a HMBC spectrum of compound 5 (CDC13);

[0063] Figure 45 is a high resolution mass spectrum of compound 5 (CDC13); 1 H- 1 H NOESY spectrum (CDC13);

[0064] Figure 46 is a high resolution mass spectrum of compound 5 (CDC13). DETAILED DESCRIPTION

[0065] In order to facilitate a further understanding of the present application, the following examples are provided to make a more detailed description of the present application. However, these examples are only for better understanding of the present application and not intended to limit the scope or principles of the present application, and the embodiments of the present application are not limited to the following.

[0066] Example 1

[0067] (1) Daldinia sp. HJX1P2 was inoculated into rice solid medium (200 bottles, formula: 50 g rice, 50 g water, 1.6 g sea salt per 1 L conical flask), and incubated at room temperature for 45 days to obtain the fermentation product;

[0068] (2) The fermentation product obtained in step (1) was extracted with an equal volume of ethyl acetate for 3 times, and the combined extract was concentrated under reduced pressure to obtain the crude extract (60.0 g).

[0069] Example 2

[0070] The crude extract obtained in Example 1 was subjected to reduced pressure silica gel column chromatography, and gradient elution was performed using petroleum ether-ethyl acetate as the eluent, and the elution gradient was 100:0, 90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 20:80, 10:90, and 0:100, respectively, and two column volumes were collected for each gradient. The eluent obtained by using petroleum ether: ethyl acetate as 80:20 and 70:30 was combined and concentrated, and then gradient elution was performed using MeOH-H2O as the eluent by reversed-phase ODS column, and the elution gradient was 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, 90:10, and 100:0, respectively, and two column volumes were collected for each gradient. The eluent obtained by using MeOH:H2O as 50:50 and 60:40 was combined and concentrated, and then prepared by high performance liquid chromatography (HPLC), and the chromatographic column was Agilent C18, 9.4x250mm, 7μm, the flow rate was 2mL / min, and the mobile phase was MeOH:H2O=65:35, and compound 1 (8.0mg), 2 (12.6mg), 3 (14.0mg) were obtained; the mobile phase was MeOH:H2O=30:70, and compound 4 (2.0mg), 5 (2.6mg) were obtained.

[0071] Structure analysis of compound 1

[0072]

[0073] Compound 1 is a brown oily substance with obvious dark spots under 254nm ultraviolet light. It turns purple after heating with sulfuric acid-vanillin color developer. According to high-resolution mass spectrometry HR-ESI-MS (m / z 335.1291[M–H] – , the calculated value is C 21 H 19 O4 – ,335.1289), indicating a molecular weight of 336. Based on the hydrogen and carbon spectra, the molecular formula is estimated to be C 21 H 20 O4, unsaturation is 12. 1 In the H NMR spectrum, two active hydrogen signals [δ H 9.62 (1H, s, 8'-OH) and 9.36 (1H, s, 5-OH)], 8 aromatic proton signals [δ H 6.60 (1H, d, J = 8.0 Hz, H-2), 6.80 (1H, d, J = 8.0 Hz, H-3), 6.84 (1H, d, J = 7.8 Hz, H-6), 7.45 (1H, dd, J = 8.4, 7.8 Hz, H-7), 7.70 (1H, d, J = 8.4 Hz, H-8), 6.35 (1H, d, J = 8.4 Hz, H-5'), 6.97 (1H, dd, J = 8.4, 8.0 Hz, H-6'), 6.35 (1H, t, J = 8.0 Hz, H-7')]; there is one oxygen-linked methine hydrogen signal in the oxygen-linked region [δ H 3.87 (1H, m, H-3')], 1 methine hydrogen signal [δ H 4.80 (1H, d, J = 5.6 Hz, H-1')] and one methoxy signal δ H 3.96 (3H, s, 4-OMe); a group of methylene proton signals appeared in the high field region [δ H 1.86 (1H, d, J = 13.6 Hz, H-2'b) and 1.95 (1H, ddd, J = 13.6, 11.6, 5.6 Hz, H-2'a)], one methyl signal [δ H 1.18 (3H, d, J = 6.2 Hz, 3'-Me)]. 13 CNMR and DEPT-135 spectra show that the compound has 21 carbon signals, including 16 aromatic carbon signals [δ C134.3 (C-1), 125.5 (C-2), 103.5 (C-3), 154.4 (C-4), 155.0 (C-5), 110.0 (C-6), 127.6 (C-7), 114.2 (C-8), 133.3 (C-9), 114.8 (C-10), 106.8 (C-5'), 127.7 (C-6'), 106.5 (C-7'), 155.7 (C-8'), 156.4 (C-9'), 110.6 (C-10'), 2 methine carbon signals [δ C 31.3 (C-1'), 66.8 (C-3'), 1 methine carbon signal [δ C 35.0 (C-2'), 1 methoxyl carbon signal [δ C 56.1 (4-OMe) and 1 methyl carbon signal [δ C 21.0 (3'-Me)]. Due to the overlap of solvent and sample peaks in NMR spectra, deuterated methanol and deuterated DMSO were used to assist the NMR analysis. According to the above 1 H NMR spectral and 13 C NMR spectral data, it was speculated that the compound had a chromone nucleus and was similar to the data of compound 4 (nodulisporin D), which was a chromonaphthol dimer. The difference was that the methoxy group was in different positions. In the HMBC spectrum, 4-OMe (δ H 4.04) was related to C-4 (δ C 154.4), indicating that the methoxy group was connected to C-4. Further through HSQC, 1 H- 1 H COSY and HMBC spectra, the planar structure of the compound was determined. Through the NOESY spectrum, H-1' was related to H-2'a, H-2'a was related to 3'-CH3, combined with the coupling constants (J 1',2'β = 5.8 Hz, J 2'β,3'α = 11.6 Hz), it was determined that H-1' and 3'-Me were on the same side, thus the relative configuration of compound 1 was determined. Further through CD test and ECD calculation, the absolute configuration of compound 1 was determined to be 1'S, 3'R, named as daldinaphchrom A.

[0074] Daldinaphchrom A (1): UV (MeOH) λ max (log ε) = 307, 323, 337; IR (KBr) v max 3414, 2928, 1697, 1744 cm -1 .

[0075] Compound 1 and 2 NMR data (DMSO-d6 / CD3OD-d4)

[0076]

[0077] Structure elucidation of compound 2

[0078]

[0079] Compound 2 was a brown oil, with obvious dark spots under the irradiation of 254 nm ultraviolet light, and purple after heating with sulfuric acid-vanillin color reagent. According to the high resolution mass spectrometry HR-ESI-MS (m / z 359.1258 [M+Na] + ), combined with hydrogen spectrum and carbon spectrum data, the molecular formula was determined as C 21 H 20 O4, with 12 degrees of unsaturation. In the 1 H NMR spectrum, two active hydrogen signals [δ H 9.34 (1H, s, 4-OH) and 8.90 (1H, s, 8'-OH)] appeared in the low field region, 8 aromatic proton signals [δ H 7.77 (1H, m, H-8), 7.37 (1H, m, H-7), 6.94 (1H, m, H-6), 6.93 (1H, m, H-6'), 6.90 (1H, m, H-2), 6.62 (1H, d, J = 7.8 Hz, H-3), 6.35 (1H, d, J = 7.4 Hz, H-5'), 6.25 (1H, d, J = 7.4 Hz, H-7')]; two methine proton signals [δ H 4.79 (1H, s, H-1') and 4.10 (1H, m, H-3')] and one methoxy signal [δ H 4.02 (3H, s, 5-OMe)] appeared in the high field region; one set of methylene signals [δ H 2.48 (1H, m, H-2'a) and 1.67 (1H, m, H-2'b)] and one methyl signal [δ H 1.24 (3H, d, J = 6.2 Hz, 3'-Me)] appeared in the high field region. Combined with 13 C NMR and DEPT-135 spectrum, it was found that the compound had 21 carbon signals, including 16 aromatic carbon signals [δ C127.1(C-1),127.1(C-2),110.1(C-3),152.0(C-4),156.6(C-5),104.1(C-6),125.7(C-7),117.0(C-8),133.7(C-9),114.7(C-10),107.6(C-5'),127.1(C-6'),107.5(C-7'),156.6(C-8'),158.0(C-9'),113.6(C-10')], 2 methine carbon signals [δ C 35.3 (C-1'), 71.8 (C-3')], 1 methylene carbon signal [δ C 41.7 (C-2')], one methoxy carbon signal [δ C 56.3 (5-OMe)] and one methyl carbon signal [δ C 21.1(3'-Me)]. 1 H NMR spectra and 13 The C NMR spectrum data showed that the compound was very similar to the known compound 4 (nodulisporin D), and it was speculated to be a chromone naphthol dimer. 1 H- 1 H COSY and HMBC spectra confirmed that compounds 2 and 4 share the same planar structure, differing in stereochemistry. The relative configuration of compound 2 was determined by NOESY spectroscopy, where H-1' and H-3' correlated, indicating that H-1' and 3'-Me are on the same side. ECD calculations confirmed the absolute configuration of the new compound to be 1'S, 3'S, and it was named daldinaphchrom B.

[0080] Daldinaphchrom B(2): UV(MeOH)λ max (logε)307,315,337; IR(KBr)ν max 3534,3442,1697,1264cm -1 .

[0081] Structural elucidation of compound 3

[0082]

[0083] Compound 3 is a yellow oily substance with obvious dark spots under 254nm ultraviolet light. It turns purple after heating with sulfuric acid-vanillin color developer. According to high-resolution mass spectrometry HR-ESI-MS (m / z 359.1259 [M+Na] + , the calculated value is C 21 H 20O4Na + ,359.1254), the molecular formula was determined as C 21 H 20 O4, with 12 degrees of unsaturation. 1 H NMR data showed two active hydrogens in the low field region [δ H 9.82 (1H, s, 4-OH), 9.18 (1H, s, 8'-OH)], eight aromatic proton signals in the aromatic region [δ H 7.15 (1H, d, J = 8.4 Hz, H-1), 6.70 (1H, d, J = 8.4 Hz, H-2), 6.94 (1H, d, J = 8.0 Hz, H-6), 7.30 (1H, dd, J = 8.0, 8.0 Hz, H-7), 7.34 (1H, d, J = 8.0 Hz, H-8), 6.34 (1H, d, J = 8.0 Hz, H-5'), 6.97 (1H, dd, J = 8.0, 8.0 Hz, H-6'), 6.33 (1H, d, J = 8.0 Hz, H-7')], and one methylene signal in the high field region [δ H 2.07 (1H, d, J = 13.6 Hz, H2'a), 1.83 (1H, dt, J = 13.8, 5.8 Hz, H2'b)]. H 4.62 (1H, d, J = 4.8 Hz, H-1'), 3.87 (1H, dq, J = 12.4, 6.0 Hz, H-3')], one methoxy signal [δ H 4.06 (3H, s, 5-OMe)], and one methyl signal [δ H 1.21 (3H, d, J = 6.2 Hz, 3'-Me)]. 13 C NMR and DEPT-135 spectral data showed that there were 16 aromatic carbon signals [δ C 117.2 (C-1), 127.6 (C-2), 126.4 (C-3), 149.9 (C-4), 155.7 (C-5), 104.4 (C-6), 125.5 (C-7), 120.9 (C-8), 134.7 (C-9), 114.3 (C-10), 106.9 (C-5'), 127.8 (C-6'), 106.4 (C-7'), 155.7 (C-8'), 156.5 (C-9'), 110.5 (C-10')], two methine carbon signals [δ C 29.5 (C-1'), 67.3 (C-3')], one methoxy carbon signal [δ C 56.3 (5-OMe)], and one methylene carbon signal [δ C34.8(C-2'), 1 methyl carbon signal [δ C 21.3 (3'-Me)]. In combination with the above 1 HNMR spectrum and 13 C NMR spectrum information found that the compound was highly similar to the known compound nodulisporin E data, speculated as similar chromophore naphthol dimer. By 1 H- 1 H COSY and HMBC related signals, determined that the compound has the same plane structure with the known compound nodulisporin E. From the NOESY spectrum, H-1' is related to H2'b, H2'b is related to 3'-Me, indicating that H-1' and 3'-Me are on the same side, and through ECD calculation to determine the absolute configuration of the new compound is 1'S, 3'R, named daldinaphchrom C.

[0084] Daldinaphchrom C (3): UV (MeOH) λ max (logε)310,319,335; IR (KBr) v max 3534, 3438, 1690 cm -1 .

[0085] NMR spectral data of compound 3 (DMSO-d6)

[0086]

[0087] Structure analysis of compound 4:

[0088]

[0089] Compound 4 is a yellow oil, under the irradiation of 254 nm ultraviolet light, there is a dark spot, after heating with sulfuric acid-vanillin color reagent into light red. According to the high resolution mass spectrometry HR-ESI-MS (m / z 285.1098 [M+Na] + , calculated value of C 15 H 18 O4Na + , 285.1097), the molecular weight is 262, combined with the hydrogen spectrum and carbon spectrum data, the molecular formula is C 15 H 18 O4, the unsaturation degree is 7. In 1 H NMR spectrum, there are three aromatic hydrogen signals δ H6.47 (1H, d, J = 8.4 Hz, H-2), 7.34 (1H, dd, J = 8.4, 8.4 Hz, H-3), 6.36 (1H, d, J = 8.4 Hz, H-4), 2 methine proton signals in the aliphatic region δ H 4.13 (2H, m, H-6), 2 methine proton signals in the aliphatic region δ H 1.86 (1H, m, H-8), 1.85 (1H, br s, H-8a), 2 methine proton signals [δ H 2.41 (1H, m, H-5a), 1.39 (1H, m, H-5b) and 2.06 (1H, d, J = 12.0 Hz, H-7a), 1.09 (1H, m, H-7b)] and 2 methyl hydrogen signals [δ H 1.35 (3H, s, H-11), 0.98 (3H, d, J = 6.2 Hz, H-12)]. The 13 C NMR and DEPT-135 spectral data, the compound has 15 carbon signals, including 6 aromatic carbon signals [δ C 162.2 (C-1), 108.9 (C-2), 138.2 (C-3), 107.7 (C-4), 158.9 (C-5), 106.0 (C-6)], 3 methine carbon signals [δ C 66.0 (C-6), 30.6 (C-8), 58.5 (C-8a)], 2 methine carbon signals [δ C 45.1 (C-5), 42.6 (C-7)], 2 methyl carbon signals [δ C 24.4 (C-11), 19.5 (C-12)]. From the above 1 HNMR spectral data and 13 C NMR spectral information, the compound is presumed to have a chromone skeleton structure. 1 H- 1 The H-5 / H-6 / H-7 / H-8 / H-12 fragment correlation signals are given in the H COSY spectrum; in the HMBC spectrum, 12-Me is correlated with C-8 / C-8a, H-7 is correlated with C-8a, and 11-Me is correlated with C-5 / C-8a / C-10. From the above 1 H- 1H COSY and HMBC spectral data, the planar structure of the new compound was determined. From the NOESY spectrum, 11-Me was associated with H-8, H-8 was associated with H-6, indicating that 11-Me and H-6 and H-8 were on the same side, 12-Me and 6-OH were on the same side, thus determining the relative configuration of compound 7. The absolute configuration of compound 7 was determined by CD spectral data, compared with the literature value of known compound (monodictysin C), which showed a positive Cotton effect at 218 nm (+8.93), monodictysin C showed a positive Cotton effect at 210 nm, and the absolute configuration was determined to be 6S, 8S, 8aS, 10aS, and named daldichrome C.

[0090] Daldichrom C (4): UV (MeOH) λ max (logε) 310, 322, 336; IR (KBr) v max 3605, 3362, 1688, 1343 cm -1 .

[0091] NMR spectral data of compounds 4 and 5 (CDC13)

[0092]

[0093] Structure analysis of compound 5:

[0094]

[0095] Compound 5 is a yellow oil, which has obvious dark spots under the irradiation of 254 nm ultraviolet light, and turns purple after heating with sulfuric acid-vanillin color reagent. According to the high resolution mass spectrum HR-ESI-MS (m / z 233.0780 [M+Na] + , the calculated value is C 11 H 14 O4Na + , 233.0784), the molecular formula is C 11 H 14 O4, and the unsaturation degree is 5. According to the nuclear magnetic spectral information, it is speculated that the compound has a similar xanthone mother nucleus structure. 1 H NMR spectrum low field region exists 3 aromatic hydrogen signals [δ H 7.17 (1H, dd, J = 8.4, 8.4 Hz, H-7), 6.51 (1H, d, 8.4 Hz, H-8), 6.47 (1H, d, J = 8.4 Hz, H-6)], 3 methine hydrogen signals [δ H4.97 (1H, d, J = 4.2 Hz, H-4), 4.06 (1H, dq, J = 9.6, 6.0 Hz, H-2), 3.61 (1H, br s, H-3)], one methoxy hydrogen signal [δ H 3.88 (3H, s, 5-OMe)] and one methyl hydrogen signal [δ H 1.49 (3H, d, J = 6.0 Hz, 2-Me)]; in combination with 13 CNMR and DEPT-135 spectra showed 11 carbon signals, including 3 quaternary carbon signals [δ C 158.9 (C-5), 155.2 (C-8a), 112.2 (C-4a)], 3 aromatic carbon signals [δ C 130.3 (C-7), 109.9 (C-8), 102.4 (C-6)]; 3 carbon signals of the methine carbons [δ C 71.1 (C-2), 71.2 (C-3), 61.5 (C-4)], one methoxy carbon signal [δ C 55.8 (5-OMe)] and one methyl carbon signal [δ C 17.6 (2-Me)]. The chromone moiety was confirmed by HMBC correlation signals, but the carbonyl group was reduced to hydroxyl group. The planar structure of the compound was determined by HSQC and 1 H- 1 COSY signals. The relative configuration of the compound was determined by J 2,3 = 9.6 Hz, it was deduced that H-2 and H-3 were in the syn relationship, J 3,4 = 4.2 Hz, it was deduced that H-3 and H-4 were in the syn relationship, thus the relative configuration of compound 5 was determined, and named as daldichrome D.

[0096] Daldichrom D (5): UV (MeOH) λ max (log ε) 190, 215, 260; IR (KBr) v max 2365, 1743, 1288 cm -1 .

[0097] Activity test of Example 3

[0098] 1. Anti-inflammatory activity screening of the crude extract of Example 1 and compound 1:

[0099] The inhibitory effect of the extract of the crude extract on nitric oxide (NO) in RAW264.7 cells induced by lipopolysaccharide (LPS) was detected by the Griess method at a concentration of 1 mg / mL. RAW264.7 cells (1 x 10 5 / well) were inoculated into 96-well plates for culture. After 12 hours, the culture medium was removed and 100 μL of culture medium containing the sample (1% FBS) was added. After 1 hour, 100 μL of culture medium containing LPS (2 μg / mL) was added and treated for 24 hours. According to the method steps on the NO kit, 50 μL of supernatant, Griess reagent I and II were taken respectively, and the OD value was measured at a wavelength of 540 nm. The NO content accumulated in the cell supernatant was calculated. Dexamethasone was used as a positive control, and three replicates were set up. The results showed that the crude extract exhibited significant anti-inflammatory activity in a dose-dependent manner ( Figure 11 ), IC 50 The value was 65.8 μg / mL. Compound 1 had an inhibitory effect on LPS-induced NO production in RAW264.7 cells, with an IC 50 The values ​​were 62.9 μM, and the positive control dexamethasone (IC 50 =136.8 μM), and the anti-inflammatory mechanism of compound 1 was studied.

[0100] Compound 1 had no significant effect on the viability of RAW264.7 cells at concentrations of 25, 50, and 60 μM ( Figure 12 ).

[0101] The Greiss method was used to detect the effect of compound 1 on the NO level in LPS-induced RAW264.7 cells. Compound 1, at a non-toxic dose (50 μM) to RAW264.7 cells, was able to effectively inhibit the release of NO in LPS-induced cells and had significant anti-inflammatory activity, such as ( Figure 13 )

[0102] ELISA was used to detect the effect of compound 1 on the expression of inflammatory factors TNF-α, IL-1β, and IL-6 in LPS-induced RAW264.7 cells. The results of the ELISA experiment showed that the LPS group significantly increased the release of IL-1β, IL-6, and TNF-α compared with the blank group, and the positive control (dexamethasone, 50μM) showed a good inhibitory effect on the release of IL-1β and TNF-α. When compound 1 acted on RAW264.7 cells at a concentration of 50μM, the release of proinflammatory factors IL-1β, IL-6, and TNF-α showed a downward trend and was dose-dependent. At a concentration of 50μM, compound 1 significantly inhibited the expression of the proinflammatory factor IL-6 and was stronger than the positive control dexamethasone ( Figure 14 ).

[0103] LPS stimulation can cause macrophages to be abnormally activated, promote the release of pro-inflammatory factors such as TNF-α and IL-6 through the TLR4 / NF-κB / MAPK signal axis, and up-regulate the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) to promote the generation of inflammatory mediators such as NO. The MAPK family is an important signal transduction substance in the LPS-induced inflammatory response, including JNK, ERK and p38. The phosphorylation levels of JNK, ERK and p38 were detected by Western blotting method, and relative to the protein of the control group of cells, compound 1 had no obvious inhibitory effect on the expression of p-JNK, p-ERK and p-JNK proteins in the LPS-induced MAPK pathway. To further explore the effect of compound 1 on the expression of iNOS and COX-2 proteins, Western blotting method was used for detection, and it was found that compound 1 could significantly inhibit the expression of iNOS protein in RAW264.7 cells, such as Figure 15

[0104] Compound 1 showed significant anti-inflammatory activity at the cellular level, but the anti-inflammatory mechanism was not clear, which limited its application and further structure-based optimization. Computational chemistry such as molecular docking, molecular simulation and other technologies have been proven to have the ability to screen target points, and virtual reverse target fishing can narrow the range of potential binding target points. It was proved in the early stage of the experiment that compound 1 could significantly inhibit the expression of iNOS protein in RAW264.7 cells, suggesting that it could inhibit the M1-type polarization response of macrophages by reducing the release of pro-inflammatory factors such as IL-6 and TNF-α, and promote the occurrence of anti-inflammatory M2-type polarization response, thereby playing an anti-inflammatory role. Related protein targets in the pathway were collected, and molecular docking analysis was performed to calculate the binding ability of the related targets, and compared with the classical inhibitors. Compound 1 was subjected to molecular docking with iNOS-related protein targets to analyze the binding ability of compound 1 and the corresponding protein targets.

[0105] AutoDock Vina 1.2.5 was used for docking, and the search center was the molecular center of the crystal itself. The search space x, y and z axes were set to 25, and the rest of the parameters were set to default. UCSF ChimeraX and PLIP were used for result analysis.

[0106] Compound 1 showed the best fitting in the binding pocket of iNOS protein, which included 5 amino acid residues VAL537, VAL686, ARG687, ARG536 and PHE684, which helped the ligand to be correctly positioned in the iNOS protein, increasing the binding affinity and stability Figure 16

[0107] 2. Screening of antibacterial activity of compounds 1-5:

[0108] ​​The antibacterial activity of compounds 1-5 was screened by microdilution method, and the test strains were S. aureus ATCC 29213 and S. aureus ATCC 700699. Ciprofloxacin was used as a positive control, and three parallels were set. The results are shown in the following table.

[0109] Antibacterial activity of compounds 1-5 against S. aureus (MIC, μg / mL)

[0110]

[0111] “-” means not tested.

Claims

1. A red mangrove endophytic fungus Daldinia sp.HJX1P2, characterized in that Its deposit number is GDMCC No.66212.

2. A chromone derivative or a pharmaceutically acceptable salt thereof, characterized in that The chromone derivatives have the structures shown in compounds 1, 2, 3, 4, and 5:

3. The crude extract of the fungus Daldinia sp. HJX1P2 according to claim 1, characterized in that The preparation method of the crude extract comprises the following steps: (1) The fungus Daldinia sp. HJX1P2 was inoculated into a rice solid culture medium and cultured at room temperature for 40-45 days to obtain a fermentation product; (2) The fermented product obtained in step (1) was extracted 2 to 4 times with an equal volume of ethyl acetate, and the extracts were combined and concentrated under reduced pressure to obtain a crude extract.

4. A method for preparing compound 1, 2, 3, 4 and / or 5 according to claim 2, characterized in that The steps include: The crude extract of claim 3 was subjected to vacuum silica gel column chromatography, and gradient elution was performed using petroleum ether-ethyl acetate as eluent. The elution gradients were 100:0, 90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 20:80, 10:90, and 0:100, respectively. Two column volumes were collected for each gradient, and the eluates obtained with gradients of petroleum ether:ethyl acetate of 80:20 and 70:30 were combined. The product was concentrated and then gradient eluted through a reversed-phase ODS column using MeOH-H2O as the eluent. The elution gradients were 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, 90:10, and 100:0, respectively. Two column volumes were collected for each gradient. The eluates obtained with the gradients of MeOH:H2O of 50:50 and 60:40 were combined and concentrated. The product was prepared by high-performance liquid chromatography (HPLC) on an Agilent C18 column, 9.4×250 mm, 7 μm, at a flow rate of 2 mL / min and a mobile phase of MeOH:H2O=65:35 to obtain compounds 1, 2, and 3; the mobile phase was MeOH:H2O=30:70 to obtain compounds 4 and 5.

5. Use of the fungus Daldinia sp. HJX1P2 according to claim 1 in the preparation of the chromone derivative according to claim 2.

6. Use of the fungus Daldinia sp. HJX1P2 according to claim 1 in preparing the crude extract according to claim 3.

7. Use of one or more of the compounds 1, 2, 3, 4, 5 or pharmaceutically acceptable salts thereof according to claim 2 or the crude extract of the fungus Daldinia sp. HJX1P2 according to claim 3 in the preparation of anti-inflammatory or antibacterial drugs.

8. A pharmaceutical composition, characterized in that The pharmaceutical composition uses one or more of the compounds 1, 2, 3, 4, 5 or pharmaceutically acceptable salts thereof according to claim 2 or the crude extract of the fungus Daldinia sp. HJX1P2 according to claim 3 as active ingredients.

9. The pharmaceutical composition according to claim 8, characterized in that The pharmaceutical composition may optionally further comprise a pharmaceutically acceptable excipient.

10. The pharmaceutical composition according to claim 9, characterized in that The pharmaceutical composition optionally contains other anti-inflammatory or antibacterial active ingredients.

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