Unsaturated olefin aldehyde compound having Anti-inflammatory and phlegm-eliminating activity, and preparation method therefor and use thereof

Abstract

Disclosed in the present invention are an unsaturated olefin aldehyde compound having an anti-inflammatory and phlegm-eliminating activity, and a preparation method therefor and the use thereof. An unsaturated olefin aldehyde compound as represented by formula I, or a pharmaceutically acceptable salt thereof, or a solvate thereof, wherein R1, R2, R3, R4 and R5 are independently selected from methyl or hydroxyl. The intestinal endophyte strain of Hepiaua larva of the present invention can be fermented to produce a new compound which has a stable performance, a low cytotoxicity and a significant anti-inflammatory and phlegm-eliminating activity.

Description

Unsaturated olefinic aldehyde compound with anti-inflammatory and expectorant activity, preparation method and application thereof Technical field:

[0001] The present invention belongs to the technical field of natural products, and in particular relates to an endophytic fungus strain from the intestinal tract of a bat moth and a compound produced by fermentation thereof and having anti-inflammatory and expectorant activity. Background technology:

[0002] Chronic obstructive pulmonary disease (COPD) is a chronic airway inflammatory disease characterized by airflow limitation and progressive decline in lung function, severely impacting patients' ability to work and quality of life. It is a heterogeneous, systemic disease that progressively worsens and is not fully reversible in modern society. It is the third leading cause of death worldwide and has risen to fifth place in the global economic burden of disease. COPD is primarily associated with nonspecific chronic inflammatory responses in the bronchial mucosa, blood vessels, and alveoli, caused by long-term exposure to harmful gases such as chemical particles or cigarette smoke. One of the key pathological features of COPD is mucus hypersecretion. Under normal circumstances, moderate mucus secretion has a protective effect on the airways. However, under pathological conditions, abnormalities in mucus quantity or quality often occur, and excessive mucus accumulation exacerbates local respiratory inflammation and infection. Mucin 5AC (5AC) is the major protein component of airway mucus, and excessive secretion of MUC5AC is the primary cause of mucus hypersecretion. COPD also has a high mortality rate in my country, especially during acute exacerbations of COPD (AECOPD). Patients experience severe respiratory inflammation, which worsens irreversible airway obstruction and respiratory muscle fatigue, often progressing to type II respiratory failure. This increases mortality and complicates clinical treatment. Inhibiting the development and progression of inflammation and mucin is an effective means of alleviating and treating chronic diseases like COPD. Therefore, the development of safe and effective anti-inflammatory and expectorant preparations is essential.

[0003] Fungi are an important source of compounds with novel structures and unique activities. The applicant previously isolated a new macrolide compound with significant anti-inflammatory activity from the metabolites of the bat moth's intestinal endophytic fungus GIZ45-A37. Further research on the metabolites of the bat moth's intestinal endophytic fungus GIZ45-A37 (Deposit Number: GDMCC No. 64050) has led to the discovery of a new unsaturated olefinic aldehyde compound with significant anti-inflammatory and expectorant activity.

[0004] Summary of the invention:

[0005] The object of the present invention is to provide a novel compound obtained by fermenting Talaromyces rugulosus GIZ45-A37 strain and having significant anti-inflammatory and expectorant activity, namely an unsaturated aldehyde compound.

[0006] The present invention provides an unsaturated olefinic aldehyde compound represented by formula I, or a pharmaceutically acceptable salt thereof, or a solvate thereof:

[0007] Wherein, R1, R2, R3, R4 and R5 are independently selected from methyl or hydroxyl.

[0008] Furthermore, the above R1 is a methyl group.

[0009] Furthermore, the above R2 is a methyl group.

[0010] Furthermore, the above R3 is a methyl group.

[0011] Furthermore, the above R4 is a hydroxyl group.

[0012] Furthermore, the above R5 is a methyl group.

[0013] Furthermore, the structure of the above compound is as follows:

[0014] The present invention also provides a method for preparing the above compound, which is isolated from the fermentation culture of Talaromyces rugulosus GIZ45-A37.

[0015] The specific steps include:

[0016] S1. inoculating Talaromyces rugulosus GIZ45-A37 into a fermentation medium and culturing to obtain a fermentation product;

[0017] S2, adding ethyl acetate to the fermentation product obtained in S1, mixing, extracting, and filtering to obtain an extract;

[0018] S3, concentrating the extract obtained in S2 under reduced pressure to obtain a crude extract;

[0019] S4. The crude extract obtained in S3 was subjected to silica gel column chromatography, and gradient elution was performed with an eluent consisting of petroleum ether and ethyl acetate in a volume ratio of petroleum ether to ethyl acetate of 50:1, 25:1, 10:1, 5:1, 2:1, and 1:1, respectively. The eluate with a volume ratio of petroleum ether to ethyl acetate of 5:1 was collected and dried to obtain a crude product;

[0020] S5. The crude product obtained in S4 is subjected to reverse-phase ODS column chromatography, and gradient elution is performed with an eluent consisting of methanol and water, with a methanol concentration of 20%, 40%, 60%, and 80% in sequence, and the eluate with 40% to 60% methanol / water is collected and dried to obtain a crude product;

[0021] S6. The crude product obtained in S5 was subjected to gel chromatography on a Sephadex LH-20 column using dichloromethane-methanol v / v 2:1 as an eluent, and the eluate was collected for high performance liquid chromatography (HPLC) using a C18 column, 60% methanol / water, and a wavelength of 254 nm as the chromatographic conditions. The eluate containing the main peak was collected and dried to obtain a crude product.

[0022] S7. Purify the crude product obtained in S6 by medium- and high-pressure preparative liquid phase purification.

[0023] Furthermore, the fermentation medium described in step S1 above is composed of the following components in parts by weight: 90-100 g of rice, 100 ml of water, and sterilized by high-pressure steam at 120° C. for 20 min.

[0024] Furthermore, the culturing in step S1 is performed by placing the culture medium at a constant temperature of 25° C. for 30 days;

[0025] Furthermore, in step S2, ethyl acetate is added to the fermentation product obtained in S1, the mass volume ratio of the fermentation product to ethyl acetate is 1:3, the extraction is ultrasonic extraction for 15 minutes, the number of extractions is 3, the power used for ultrasonic extraction is 400W, and the operating frequency is 40KHz.

[0026] Furthermore, in the high pressure preparative liquid chromatography purification in step S7, the mobile phase is a mixed solution of acetonitrile and water, wherein the volume percentage of acetonitrile is 70%.

[0027] The present invention also provides the use of the above-mentioned compound, or a pharmaceutically acceptable salt thereof, or a solvate thereof in the preparation of an anti-inflammatory and expectorant drug; preferably, the drug is a drug for preventing and treating chronic obstructive pulmonary disease (COPD).

[0028] The present invention also provides a medicine, which is a preparation prepared by using the above-mentioned compound, or a pharmaceutically acceptable salt thereof, or a solvate thereof as an active ingredient, and adding pharmaceutically acceptable excipients or auxiliary ingredients.

[0029] Beneficial effects of the present invention:

[0030] (1) The bat moth intestinal endophytic bacteria strain of the present invention can ferment to produce a new compound, which has stable performance, low cytotoxicity, and significant anti-inflammatory and expectorant activity.

[0031] (2) The compound of the present invention is produced by solid fermentation of fungi and can be obtained by extracting the fermentation product with ethyl acetate and then separating and purifying it by silica gel column chromatography, reversed-phase ODS column chromatography, gel column chromatography and high-performance liquid chromatography. The separation steps are simple, the implementation is convenient, and it is easy to industrialize and mass-produce, and it has broad application prospects.

[0032] Deposit of biological materials:

[0033] Talaromyces rugulosus GIZ45-A37 was deposited in the Guangdong Provincial Microbiological Culture Collection Center (GDMCC) on November 22, 2023. The deposit address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province, Postal Code 510070, and the deposit number is GDMCC No. 64050.

[0034] Obviously, based on the above contents of the present invention, according to common technical knowledge and customary means in this field, without departing from the above basic technical ideas of the present invention, other various forms of modifications, replacements or changes can be made.

[0035] The following further describes the above content of the present invention in detail through specific embodiments in the form of examples. However, this should not be construed as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention fall within the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] FIG1 is a high-resolution mass spectrometry result graph of the compound having the structure of Formula I of the present invention.

[0037] FIG2 is a diagram of a compound having a structure of formula I according to the present invention. 1 H NMR spectrum.

[0038] FIG3 is a diagram of a compound having a structure of formula I according to the present invention. 13 C NMR spectrum.

[0039] FIG4 is a diagram of a compound having a structure of formula I according to the present invention. 1 H- 1 H COSY spectrum.

[0040] FIG5 is a HSQC spectrum of the compound having the structure of Formula I of the present invention.

[0041] FIG6 is the HMBC spectrum of the compound having the structure of Formula I of the present invention.

[0042] FIG7 is a NOESY spectrum of the compound having the structure of Formula I of the present invention.

[0043] FIG8 shows the cell growth rate at different concentrations of the compound having the structure of Formula I of the present invention.

[0044] FIG9 shows the results of the assay for inhibiting cellular IL-6 secretion by the compound having the structure of Formula I of the present invention.

[0045] FIG10 shows the results of the assay for inhibiting cell MUC5AC secretion by the compound having structure I of the present invention. Specific implementation method:

[0046] The raw materials and equipment used in the present invention are all known products and are obtained by purchasing commercially available products.

[0047] Example 1. Preparation of the compound of the present invention

[0048] 1. Fermentation of bacteria

[0049] 1. Inoculate the strain Talaromyces rugulosus GIZ45-A37 onto a PDA culture medium plate and culture in the dark at 28°C for 7 days to obtain plate seeds.

[0050] 2. Transfer the seeds obtained in step 1 to liquid seed culture medium and culture in the dark at 28°C and 150 rpm for 4 days to obtain seed culture solution.

[0051] Seed culture medium: The solvent is potato juice (potato juice preparation method: 200g potatoes, add about 1L of water, boil for 30min, filter, and make up the filtrate to 1L). The solute and its content are as follows: glucose 20g / L, agar 20g, sterilize and set aside.

[0052] 3. Inoculate approximately 10 mL of seed culture solution into a flask containing fermentation medium and culture at a constant temperature of 25°C for 30 days.

[0053] 4. Preparation of fermentation medium: Take 100g of rice, add 100ml of water, put it into a 500mL Erlenmeyer flask, and sterilize it under high pressure (120℃, 20min).

[0054] 2. Extraction, separation and purification of the compounds of the present invention

[0055] 1. Add 300 ml of ethyl acetate to the fermented flask, perform ultrasonic extraction at 30°C for 15 min, extract three times, with an ultrasonic power of 400 W and an operating frequency of 40 kHz, filter the extract, combine all the extracts and concentrate under reduced pressure at 40°C to obtain a total extract.

[0056] 2. The total extract was dissolved in ethyl acetate and then stirred and mixed with column chromatography silica gel in a mass ratio of 1:1 and dried. The sample was dry loaded and subjected to silica gel column chromatography (filler: 200-300 mesh silica gel powder). The mixture was eluted with a petroleum ether / ethyl acetate mixed solution (v / v: 50:1, 25:1, 10:1, 5:1, 2:1, 1:1, 0:100). The eluate with a volume ratio of petroleum ether to ethyl acetate of 5:1 was collected and rotary evaporated to obtain crude product A.

[0057] 3. The crude product A was further chromatographed on a reverse column (filler: ODS C-18 column chromatography silica gel, gradient elution, eluent: methanol / water mixed solvent, eluted with volume fractions of 20%, 40%, 60%, and 80% methanol / water, respectively, for 2 column volumes, and the 40% to 60% methanol / water eluate was collected and concentrated to dryness under reduced pressure to obtain crude product A-1.

[0058] 4. The crude product A-1 was further separated by gel chromatography on a Sephadex LH-20 column using dichloromethane-methanol (v / v 2:1) as the eluent. The eluate was collected and analyzed by high-performance liquid chromatography using a C18 column (4.6×100 mm, 2.7 μM) with 60% methanol / water by volume, a flow rate of 0.5 ml / min, and a wavelength of 254 nm. The eluate containing the main peak (retention time 7.15 min) was collected and dried to obtain the crude product A-2. The crude product A-2 was purified by medium- and high-pressure preparative liquid chromatography (C18 column: 10×250 mm, 5 μM; flow rate: 1 ml / min, mobile phase: 70% acetonitrile / water, v / v; wavelength: 254 nm; Rt = 21.5 min) to obtain a white powder:

[0059] 5. Figure 1 is a high-resolution mass spectrometry result of a compound having a structure of Formula I according to the present invention. Figure 2 is a high-resolution mass spectrometry result of a compound having a structure of Formula I according to the present invention. 1 H NMR spectrum. Figure 3 is a graph of the compound of formula I of the present invention. 13 C NMR spectrum. Figure 4 is a graph of the compound having the structure of formula I of the present invention. 1 H- 1 H COSY spectrum. Figure 5 is the HSQC spectrum of the compound having the structure of Formula I of the present invention. Figure 6 is the HMBC spectrum of the compound having the structure of Formula I of the present invention. Figure 7 is the NOESY spectrum of the compound having the structure of Formula I of the present invention.

[0060] The compound of the present invention is a white solid powder, easily soluble in chloroform, methanol and DMSO, HR-ESI-MS: m / z 235.13399 [MH] - (Figure 1), 1 H NMR (600MHz, CDCl3) spectrum H :9.62 (1H, d, J = 7.8 Hz), possibly the hydrogen signal of the aldehyde group, δ H:7.41 (1H, dd, J1 = 15.0 Hz, J2 = 15.0 Hz), 6.20 (1H, dd, J1 = 15.0 Hz, J2 = 7.8 Hz), 6.40 (1H, d, J = 11.4 Hz). Based on the displacement and coupling constants, it can be inferred that the three hydrogen atoms are trans double bond hydrogen atoms and are in adjacent positions. 5.73 (1H, s) may also be a double bond hydrogen atom. The other hydrogen atom signals are distributed in δ H :4.60(1H,s),3.50(2H,m),1.87(3H,d,J=1.2Hz),1.51(3H,s),1.24(3H,m),1.17(3H,s). 13 The C NMR (150MHz, CDCl3) spectrum showed 14 carbon atom signals, of which δ C : The six carbon atom signals at 147.8, 146.9, 135.2, 132.4, 131.3 and 128.0 are double bond carbon atom signals, δ C :194.0 is the signal of the aldehyde carbon atom. DEPT spectrum indicates that the compound has 3 quaternary carbons (C), 7 tertiary carbons (CH), and 4 primary carbons (CH3). 1 H- 1 H COSY spectrum shows δ H :9.62 and 6.20, δ H :7.41 and 6.20,6.40,δ H :3.50 and 1.24 respectively, indicating that the hydrogen atoms are in adjacent positions. H :6.40 and δ C :128.0,δ H :5.73 and δ C :131.3,δ H :4.60 and δ C :84.7,δ H :3.50 and δ C :77.2,δ H :1.87 and δ C :14.1,δ H :1.51 and δ C :18.4,δ H :1.24 and δ C :12.9,δ H :1.19 and δ C :23.6 show correlated signals, which can be assigned to carbon atoms and corresponding hydrogen atoms. The HMBC spectrum shows that hydrogen atoms are remotely coupled to carbon atoms, such as δ H :9.62 and δ C:132.4 has long-range coupling related signals. Based on the above spectrum information, the structure of the compound was identified.

[0061] 6. NMR characterization data are as follows:

[0062] 1 H NMR (600 MHz, CDCl3) δ H :1.17(3H,s),1.23(3H,d,J=6.0Hz),1.51(3H,s),1.87(3H,d,J=1.2Hz),3.50(1H,q,J=6.0Hz),4.46(1H,s),5.73(1H,s),6.19(1H,dd,J1=7.8Hz,J2=15.0Hz),6.39(1H,d,J=11.4Hz),7.41(1H,dd,J1=11.4Hz,J2=15.0Hz),9.62(1H,d,J=7.8Hz), see Figure 2.

[0063] 13 C NMR (150 MHz, CDCl3) δ C :12.7(q),14.1(q),18.4(q),23.6(q),67.1(s),77.3(d),84.7(d),128.0(d),131.3(d),132.4(d),135.2(s),146.9(d),147.8(s),194.0(d),see Figure 3.

[0064] The structure of the compound was identified and its molecular formula was C 14 H 20 O3, the structure is as follows:

[0065] The compound was named TRX.

[0066] The following experimental examples demonstrate the beneficial effects of the compounds of the present invention.

[0067] Example 2: Identification of the endophytic fungus strain Talaromyces rugulosus of the present invention

[0068] The Talaromyces rugulosus GIZ45-A37 of the present invention is isolated from the intestine of a bat moth, identified by the Guangdong Institute of Microbiology, and stored in a refrigerator at 4°C using a PDA slant culture medium.

[0069] Based on the DNA sequence of the internal transcribed spacer (ITS), the fungal strain was identified as Talaromyces rugulosus by NCBI BLAST analysis. The following sequences were used in the BLAST search.

[0070] SEQ ID NO.1:

[0071] The strain, named Talaromyces rugulosus GIZ45-A37, was deposited with the Guangdong Provincial Microbiological Culture Collection (GDMCC) on November 22, 2023, at Building 59, 5th Floor, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong Province, 510070, China, with the accession number GDMCC No. 64050. The strain is disclosed in CN202311729129.1 and is subject to a Chinese patent application titled "A Macrolide Compound with Anti-inflammatory Activity, Its Preparation Method, and Application."

[0072] Example 3: Anti-inflammatory activity test of the compounds of the present invention

[0073] Interleukin -6 (IL-6) is an important inflammatory factor, and IL-6 expression increases and promotes the development of inflammation. Therefore, IL-6 can be used as an indicator substance for the development of inflammation, and may also be a potential target for the treatment of COPD (Winslow S, Odqvist L, Diver S, et al. Multi-omics links IL-6 trans-signalling with neutrophil extracellular trap formation and Haemophilus infection in COPD[J]. Eur Respir J. 2021, 58 (4): 1-14.). The present invention acts on human bronchial epithelial cells Beas2b induced by lipopolysaccharide (LPS) by acting on the compound (hereinafter referred to as TRX) prepared by Example 1, and the anti-inflammatory activity of TRX is evaluated by detecting the IL-6 content produced in the cell culture supernatant.

[0074] 1. Reagents and Instruments

[0075] Human bronchial epithelial cells Beas-2b; lipopolysaccharide (LPS); DMEM high-glucose medium; fetal bovine serum (FBS); phosphate-buffered saline (PBS); dimethyl sulfoxide (DMSO); 1% P / S double-antibody; enzyme-linked immunosorbent assay kit (ELISA kit); precision electronic balance; biological safety cabinet; carbon dioxide incubator; inverted microscope; low-speed refrigerated centrifuge; microplate reader; digital constant-temperature water bath.

[0076] 2. Culture of Beas-2b cells

[0077] The basal culture medium is DMEM culture medium + 10% FBS + 1% P / S double antibody; the culture conditions are 37°C, 5% CO2 and saturated humidity; after culturing for 2 to 3 days, the cell density reaches 90% and can be used for experiments.

[0078] 3. Cell Viability Assay

[0079] Beas-2b cells in the logarithmic growth phase were taken and prepared into 2×10 5 A single-cell suspension of 100 μl / well was seeded into a 96-well plate (three replicates per group). After the cells attached to the wall, they were cultured until the confluence was approximately 80%. The original culture medium was discarded. The control group was added with normal culture medium (1 μl of compound solvent and 99 μl of DEME medium). The TRX group was added with culture medium containing different concentrations of compounds (1 μl of TRX compound solution and 99 μl of DEME medium, with final TRX concentrations of 100 μM, 50 μM, 25 μM, 12.5 μM, and 6.25 μM). The cells were cultured for 24 h and the cell growth status and culture medium changes were observed under an inverted microscope. After the culture was completed, 10 μl of CCK-8 working solution was added to each well, incubated at 37°C for 2 h, and detected at 450 nm.

[0080] Cell inhibition rate = 1-(drug-added well-background value) / (control well-background value) × 100%

[0081] The experimental results are shown in FIG8 : TRX had no significant inhibitory effect on Beas2b cells within the concentration range of 6.25 μM to 100 μM, indicating that TRX is safe within this range and has very low cytotoxicity.

[0082] 4. Determination of the anti-inflammatory activity of compound TRX

[0083] Beas-2b cells in the logarithmic growth phase were taken and the cell density was adjusted to 2×10 6Cells were seeded into a 48-well plate at a volume of 500 μl / well. After the cells adhered to the wall, they were cultured until the confluence reached about 80%. The culture medium was discarded. A control group, a model group, and a TRX group were set up. LPS and TRX were not added to the control group. LPS solution (final LPS concentration was 1 μg / ml) was added to the culture medium of the model group. Fresh DMEM culture medium containing LPS (final LPS concentration was 1 μg / ml) and TRX solution (final TRX concentration was 10 μM and 20 μM) was added to the TRX group. The culture was continued for 24 h. After the culture was completed, the supernatant of each well was collected into a 1.5 ml centrifuge tube and centrifuged at 6000 rpm for 5 min. The supernatant was transferred to another 1.5 ml centrifuge tube and stored at -20°C for ELISA detection. The IL-6 content was detected according to the instructions of the ELISA kit.

[0084] The experimental data were expressed as mean ± standard deviation (x ± s), and the inter-group comparison was performed using SPSS 22.0 statistical software for single-factor analysis of variance to analyze the significant differences. # p<0.05, ## p<0.01 indicates a significant difference, which is statistically significant; comparison between the sample group and the model group * p<0.05, ** p<0.01 indicates a significant difference and is statistically significant.

[0085] As shown in Figure 9, the IL-6 content in the model group was significantly different from that in the control group, indicating that the model was successfully established. The IL-6 content in the TRX group was significantly lower than that in the model group, indicating that TRX has the ability to inhibit IL-6 expression activity, thereby exerting an anti-inflammatory effect.

[0086] Example 4: Expectorant activity test of the compounds of the present invention

[0087] One of the key pathological features of chronic inflammatory airway diseases is mucus hypersecretion. While moderate mucus secretion protects the airways under normal circumstances, abnormalities in mucus quantity or quality often occur under pathological conditions, and excessive mucus accumulation can aggravate local respiratory inflammation and infection. Mucin 5AC (5AC) is the primary protein component of airway mucus, and excessive secretion of MUC5AC is the primary cause of mucus hypersecretion (Zhao et al. Effects of PM2.5 on mucus secretion and tissue remodeling in a rabbit model of chronic rhinosinusitis. Int Forum Allergy Rhinol, 2018, 8(11):1349-13552).

[0088] 1. Reagents and Instruments

[0089] Human bronchial epithelial cells Beas-2b; exogenous epidermal growth factor (EGF); DMEM high-glucose medium; fetal bovine serum (FBS); phosphate-buffered saline (PBS); dimethyl sulfoxide (DMSO); 1% P / S double-stranded antibody; mucin 5AC (MUC5AC) enzyme-linked immunosorbent assay kit (ELISA kit); precision electronic balance; biological safety cabinet; carbon dioxide incubator; inverted microscope; low-speed refrigerated centrifuge; microplate reader; and digital constant-temperature water bath.

[0090] 2. Culture of Beas-2b cells

[0091] The basal culture medium is DMEM culture medium + 10% FBS + 1% P / S double antibody; the culture conditions are 37°C, 5% CO2 and saturated humidity; after culturing for 2 to 3 days, the cell density reaches 90% and can be used for experiments.

[0092] 3. Determination of Expectorant Activity of Compounds

[0093] Human Beas-2b cells were cultured in DMEM medium supplemented with 10% FBS. Control, model, and sample groups were established. Cells were seeded into 48-well plates and incubated at 37°C in a 5% CO2 incubator. When cell confluence reached 70%, serum-free medium was replaced in all three groups. The model group was stimulated with exogenous epidermal growth factor (EGF) (25 ng / ml), while the TRX group was stimulated with fresh DMEM medium containing LPS and TRX solution (final concentrations of 10 μM and 20 μM, respectively). The cells were stimulated for 24 hours, and the culture supernatants were collected. The cell supernatants were centrifuged at 6000 rpm for 5 minutes, transferred to a 1.5 ml centrifuge tube, and stored at -20°C for ELISA analysis. MUC5AC content was determined according to the ELISA kit instructions.

[0094] The experimental data are expressed as mean ± standard deviation The comparison between the groups was performed using SPSS22.0 statistical software for one-way analysis of variance for significant differences. * indicates a significant difference p < 0.05, and ** indicates a significant difference p < 0.01.

[0095] As shown in Figure 10, the MUC5AC content in the model group was significantly different from that in the control group, indicating that the model was successfully established. The MUC5AC content in the TRX group was significantly lower than that in the model group, indicating that TRX has the ability to inhibit the expression activity of cellular MUC5AC, thereby exerting an expectorant effect.

[0096] Chronic obstructive pulmonary disease (COPD) is a chronic disease closely associated with inflammation and mucus secretion. Inhibiting inflammation and mucus production is an important method and approach for treating COPD. The compound TRX of the present invention exhibits significant anti-inflammatory and expectorant activity at concentrations as low as 10 μM, and cell viability experiments show very low cytotoxicity. Therefore, the compound TRX of the present invention has the potential to be developed into an anti-inflammatory and expectorant drug for the treatment of COPD and other conditions.

Claims

1. An unsaturated enal compound represented by Formula I, or a pharmaceutically acceptable salt thereof, or a solvate thereof: Among them, R1, R2, R3, R4, and R5 are each independently selected from methyl or hydroxyl.

2. The unsaturated enal compound according to claim 1, wherein R1 is methyl; or R2 is methyl; or R3 is methyl; or R4 is hydroxyl; or R5 is methyl.

3. The unsaturated enal compound according to claim 2, wherein The structure of the compound is as follows:

4. A method for preparing the unsaturated enal compound according to claim 3, characterized in that, It is isolated from the fermentation culture of Talaromyces rugulosus GIZ45-A37, and the Talaromyces rugulosus GIZ45-A37 has a preservation number of GDMCC No. 64050.

5. The preparation method according to claim 4, wherein, It includes the following steps: S1. Inoculate Talaromyces rugulosus GIZ45-A37 into a fermentation medium and culture to obtain a fermentation product; S2. Add ethyl acetate to the fermentation product obtained in S1, mix and extract, and filter to obtain an extract; S3. Concentrate the extract obtained in S2 under reduced pressure to obtain a crude extract; S4. Subject the crude extract obtained in S3 to silica gel column chromatography, using an eluent composed of petroleum ether and ethyl acetate, and perform gradient elution in sequence according to the volume ratio of petroleum ether to ethyl acetate of 50:1, 25:1, 10:1, 5:1, 2:1, 1:

1. Collect the eluent with a volume ratio of petroleum ether to ethyl acetate of 5:1, and dry to obtain a crude product; S5. Subject the crude product obtained in S4 to reverse-phase ODS column chromatography, using an eluent composed of methanol and water, and perform gradient elution in sequence according to the methanol concentration of 20%, 40%, 60%, 80%. Collect the eluent of 40% - 60% methanol / water, and dry to obtain a crude product; S6. Subject the crude product obtained in S5 to gel Sephadex LH-20 column chromatography, elute with dichloromethane-methanol v / v 2:1 as the eluent, collect the eluent for high-performance liquid chromatography detection, and perform detection under the chromatographic conditions of a C18 chromatographic column, 60% methanol / water, and a wavelength of 254 nm. Collect the eluent containing the main peak, and dry to obtain a crude product; S7. The crude product obtained in S6 is purified by medium-high pressure preparative liquid chromatography to obtain the product.

6. The preparation method according to claim 5, wherein The fermentation medium described in step S1 is composed of the following components in parts by weight: 90 - 100 g of rice, 100 ml of water, and autoclaved at 120 °C for 20 min; the culture in step S1 is to place it in a constant temperature of 25 °C and statically culture for 30 days.

7. The preparation method according to claim 5, characterized in that, In step S2, when adding ethyl acetate to the fermentation product obtained in S1, the mass-volume ratio of the fermentation product to ethyl acetate is 1:3, the extraction is ultrasonic extraction for 15 min, the extraction times are 3 times, the power used for ultrasonic extraction is 400 W, and the working frequency is 40 KHz.

8. The preparation method according to claim 5, characterized in that, In the medium-high pressure preparative liquid chromatography purification described in step S7, the mobile phase is a mixed solution of acetonitrile and water, and among them, the volume percentage of acetonitrile is 70%.

9. Use of the unsaturated enal compound according to claim 1, or a pharmaceutically acceptable salt thereof, or a solvate thereof in the preparation of anti-inflammatory and expectorant drugs; preferably, the drug is a drug for preventing and treating chronic obstructive pulmonary disease.

10. A drug, characterized in that, It is a preparation prepared by using the compound described in claim 1, or a pharmaceutically acceptable salt thereof, or a solvate thereof as an active ingredient, plus a pharmaceutically acceptable excipient or adjuvant ingredient.

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