A new sorbicillinoids compound trichosorbicil B, its extraction and separation method and use

Abstract

The application discloses a new Sorbicillinoids compound Trichosorbicil B, an extraction and separation method thereof and application, and belongs to the technical field of natural compound extraction, separation and natural medicinal chemistry. The application takes Trichoderma reesei fermentation liquor as a raw material, extracts a new Sorbicillinoids compound from Trichoderma reesei metabolites, and names the compound as Trichosorbicil B. The compound has remarkable in-vitro anti-tumor activity, in-vitro anti-inflammatory activity, in-vitro anti-pulmonary fibrosis activity and an effect of reducing acute inflammation of skin caused by ultraviolet light, and provides a basis for further developing in-depth pharmacological and clinical research and developing a new anti-tumor drug, an anti-inflammatory drug and a drug for preventing and treating pulmonary fibrosis with strong curative effect and small side effect.

Description

Technical Field

[0001] This invention relates to the fields of natural compound extraction, separation, and natural medicinal chemistry, and in particular to a novel Sorbicillinoid compound, Trichosorbicil B, its extraction and separation methods, and its uses. Background Technology

[0002] Sorbicillinoids are a class of naturally occurring compounds produced by fungal secondary metabolism. They were first isolated from various fungal strains of marine and terrestrial origin, identified as a family of characteristic yellow hexadecosine compounds with a characteristic absorption at 370 nm. In 1948, Cram and Tishler reported the isolation and structural identification of several Sorbicillinoid compounds. Subsequently, researchers isolated more members of this family from fungal strains collected from different environments. A common structural feature of this family of compounds is the presence of sorbityl side chains at C1-C6. Given the structural similarity of these compounds and the fact that this family does not yet have a formal name, the term "Sorbicillinoids" is used to encompass the entire family of compounds.

[0003] Sorbicillinoids are not only structurally complex and diverse, but also exhibit a wide range of biological activities. Many Sorbicillinoids have been isolated and their structures identified. The discovery and identification of novel Sorbicillinoids, and their potential pharmaceutical and biotechnological applications, can serve as a potential source of new therapeutic agents and drugs, and thus have extremely high research value.

[0004] *Trichoderma reesei* is an important industrial production strain, meeting GRAS (Generally Regulated as Safe) standards, and its fermentation products are widely used in the food and feed industries. *Trichoderma reesei* is easy to scale up and has been applied in enzyme production industries worldwide. Literature reports that *Trichoderma reesei* produces 11% of the world's enzymes. Within the *Trichoderma* genus, *Trichoderma reesei* is the only species capable of producing sorbidiloids, and in high yield, with the fermentation broth exhibiting a characteristic yellow color. Previous studies have shown that crude extracts of *Trichoderma reesei* sorbidiloids possess antioxidant and anticancer activities, particularly significant in inhibiting malignant melanoma. However, whether novel, previously unreported members exist among the sorbidiloids derived from *Trichoderma reesei* remains a worthy scientific investigation. The isolation and identification of novel sorbidiloid structures will help reveal new synthetic pathways and synthases related to sorbidiloids, and hold promise as potential sources for new therapeutic agents and drugs, possessing significant theoretical value and practical application prospects. Summary of the Invention

[0005] The purpose of this invention is to provide a novel Sorbicillinoid compound, Trichosorbicil B, along with its extraction and separation method and uses, to address the aforementioned problems in the prior art. This invention uses *Trichoderma reesei* fermentation broth as raw material to extract a novel Sorbicillinoid compound, named Trichosorbicil B, from *Trichoderma reesei* metabolites. This compound exhibits significant in vitro antitumor activity, in vitro anti-inflammatory activity, and in vitro anti-pulmonary fibrosis activity, as well as the ability to alleviate acute skin inflammation induced by ultraviolet light. This provides a foundation for further in-depth pharmacological and clinical research, and for the development of novel antitumor drugs, anti-inflammatory drugs, and drugs for the prevention and treatment of pulmonary fibrosis with potent efficacy and minimal side effects.

[0006] To achieve the above objectives, the present invention provides the following technical solution: One of the technical solutions of this invention is to provide a new Sorbicillinoid compound, Trichosorbicil B, with the molecular formula C. 22 H 24 O7, the system corresponding to its structure is named: (2S,2aS,2a) 1 S,3S,4aS,7R,7aS,8R,Z)-2a 13-dihydroxy-6-((2E,4E)-1-hydroxyhexa-2,4-dien-1-ylidene)-2,4a,7a,8-tetramethylhexahydro-3,7,2-(epiethane[1,1,2]triyl)indeno[7,1-bc]furan-4,5,9(2H,4aH)-trione, with the following structural formula: .

[0007] The second technical solution of the present invention provides a method for extracting and separating the above-mentioned novel Sorbicillinoid compound Trichosorbicil B, comprising the following steps: The fermentation broth of Trichoderma reesei, after removing mycelia, was extracted using an extraction solvent to obtain a crude extract of Sorbicillinoids. The crude extract of Sorbicillinoids was separated by gel column chromatography, then eluted with methanol, and the same fractions were collected and combined to obtain 12 fractions. The fraction containing Trichosorbicil B was obtained in the 5th fraction. After removing the solvent from the fraction containing Trichosorbicil B, the fraction was separated using preparative high-performance liquid chromatography (HPLC). The preparative HPLC conditions were as follows: Sep Basic C18 column; acetonitrile and water as the mobile phase; acetonitrile to water volume ratio: 0-15 min, 40-99% acetonitrile; 15-20 min, 99% acetonitrile; 20-25 min, 40% acetonitrile; detection wavelength: 370 nm; column eluent collected for 6.8-7.1 min, solvent removed, to obtain the novel Sorbicillinoid compound Trichosorbicil B.

[0008] Preferably, the extraction solvent is ethyl acetate.

[0009] Preferably, the Trichoderma reesei fermentation broth is obtained by inoculating Trichoderma reesei into a fungal culture medium and then fermenting it.

[0010] The third technical solution of the present invention provides the application of the above-mentioned novel Sorbicillinoid compound Trichosorbicil B in the preparation of drugs for treating tumors.

[0011] Preferably, the tumor is acute T-lymphoblastic leukemia, colorectal adenocarcinoma, neuroblastoma, breast cancer, malignant melanoma, cervical cancer, or liver cancer.

[0012] The fourth technical solution of the present invention provides a drug for treating tumors, the active ingredient of which includes the above-mentioned novel Sorbicillinoid compound Trichosorbicil B.

[0013] Preferably, the medicament for treating tumors further includes pharmaceutically acceptable excipients.

[0014] Fifth technical solution of the present invention: to provide the application of the above-mentioned novel Sorbicillinoid compound Trichosorbicil B in the preparation of anti-inflammatory drugs.

[0015] The sixth technical solution of the present invention provides an anti-inflammatory drug, the active ingredient of which includes the above-mentioned novel Sorbicillinoid compound Trichosorbicil B.

[0016] Preferably, the anti-inflammatory drug further includes pharmaceutically acceptable excipients.

[0017] The novel Sorbicillinoid compound Trichosorbicil B of this invention has a significant inhibitory effect on the release of NO from LPS-induced mouse macrophages RAW264.7, and can be used in anti-inflammatory drugs or veterinary drugs.

[0018] Preferably, the application is in the preparation of UV-induced epidermal inflammation protective products.

[0019] The novel Sorbicillinoid compound Trichosorbicil B of this invention can inhibit the upregulation of pro-inflammatory gene transcription in human epidermal cells caused by ultraviolet radiation, and reduce acute inflammatory damage to the skin caused by ultraviolet radiation; the application includes two administration methods: pretreatment protection and post-irradiation repair.

[0020] The seventh technical solution of the present invention is to provide the application of the above-mentioned novel Sorbicillinoid compound Trichosorbicil B in the preparation of drugs for the prevention and treatment of pulmonary fibrosis.

[0021] The eighth technical solution of the present invention: provides a drug for preventing and treating pulmonary fibrosis, the active ingredient of which includes the above-mentioned novel Sorbicillinoid compound Trichosorbicil B.

[0022] Preferably, the drug for preventing and treating pulmonary fibrosis also includes pharmaceutically acceptable excipients.

[0023] The novel Sorbicillinoid compound Trichosorbicil B of this invention prevents and treats pulmonary fibrosis by inhibiting the proliferation of activated pulmonary fibrotic cells, promoting apoptosis of pulmonary fibrotic cells, and reducing fibrin and collagen.

[0024] The beneficial technical effects of the present invention are as follows: This invention uses Trichosorbicil fermentation broth as raw material to extract a novel sorbicillinoid compound from Trichosorbicil metabolites, named Trichosorbicil B. This compound exhibits significant in vitro antitumor activity, in vitro anti-inflammatory activity, in vitro reduction of UV-induced acute skin inflammation, and in vitro anti-pulmonary fibrosis activity. This provides a foundation for further in-depth pharmacological and clinical research, and for the development of novel antitumor drugs, anti-inflammatory drugs, and drugs for the prevention and treatment of pulmonary fibrosis with potent efficacy and minimal side effects.

[0025] The novel compound Trichosorbicil B of this invention is the first to be obtained from Trichoderma reesei fermentation products, and this discovery provides a basis for further research on the biosynthetic pathway of Sorbicillinoids in Trichoderma reesei. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0027] Figure 1 The LC-MS chromatogram of fraction Fr.5 is shown below. Figure 2 High-performance liquid chromatography (HPLC) chromatogram of the column eluent for fraction Fr.5 at 6.8–7.1 min; Figure 3 The HR-ESI-MS spectrum of the positive ion of compound Trichosorbicil B; Figure 4 For the compound Trichosorbicil B 1 H-NMR spectrum; Figure 5 For the compound Trichosorbicil B 13 C-NMR spectrum; Figure 6 For the compound Trichosorbicil B 1 H- 1 H COSY spectrum; Figure 7 The HSQC spectrum of compound Trichosorbicil B; Figure 8 The HMBC spectrum of compound Trichosorbicil B; Figure 9 The NOESY spectrum of compound Trichosorbicil B; Figure 10 The ROESY spectrum of compound Trichosorbicil B; Figure 11 For the compound Trichosorbicil B 1 H- 1 Schematic diagrams related to H COSY and HMBC; Figure 12 A schematic diagram showing the NOESY and ROESY of compound Trichosorbicil B; Figure 13 The single-crystal X-ray diffraction ORTEP ellipsoid structure of the compound Trichosorbicil B is shown. Figure 14 The results show the effects of compound Trichosorbicil B on the cytotoxicity (a) and NO release level (b) of RAW264.7. Figure 15 Figure showing the effect of compound Trichosorbicil B on epithelial-mesenchymal related proteins in lung fibroblasts; Figure 16 The results show the effects of compound Trichosorbicil B on alleviating acute skin inflammation caused by ultraviolet light; (a) shows the effect of Trichosorbicil B on the proliferation of immortalized human keratinocytes HaCaT; (b) shows the effect of Trichosorbicil B on the proliferation of immortalized human keratinocytes HaCaT after ultraviolet light irradiation. Detailed Implementation

[0028] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention. It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the present invention.

[0029] Furthermore, regarding the numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, are also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0030] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. It should be noted that any aspects of this invention not described in detail are conventional practices in the art and are not the focus of this invention.

[0031] The terms “comprising,” “including,” “having,” “containing,” etc., used in this invention are all open-ended terms, meaning that they include but are not limited to.

[0032] Experimental methods not specifically described in the following examples are generally performed under conventional conditions, such as those described in Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989).

[0033] The cell culture media and reagents used in this invention are shown in Table 1.

[0034] Table 1 Cell culture media and reagents Unless otherwise specified, "room temperature" in this invention refers to 20±10℃.

[0035] Unless otherwise specified, "overnight" in this invention refers to 12 hours.

[0036] All raw materials used in the following embodiments and comparative examples of the present invention are commercially available products.

[0037] The technical solution of the present invention will be further illustrated by the following embodiments.

[0038] Example 1 1. Extraction of new compounds The fungal culture medium consisted of: 100 g / L glucose, 6 g / L tryptone, 0.15 g / L KH₂PO₄, 0.15 g / L K₂HPO₄, 0.10 g / L CaCl₂·2H₂O, 0.10 g / L MgSO₄·7H₂O, 0.05 g / L NaCl, 1 mL / L trace element solution, and 20 g / L calcium carbonate. The trace element solution consisted of: 1.6 g / L MnSO₄·4H₂O, 5 g / L FeSO₄·7H₂O, 2 g / L CoCl₂·6H₂O, and 1.4 g / L ZnSO₄·7H₂O.

[0039] The fungal culture medium was dispensed into 20 250 mL shake flasks, with 50 mL of fungal culture medium in each flask. After sterilization, conidia of Trichoderma reesei strain QM6a (ATCC 13631) were inoculated into the fungal culture medium at an inoculum size of 10 mL per shake flask. 7 One spore. After inoculation, the shake flask was placed in a shaker and cultured at 200 rpm and 28℃ for 12 days to obtain the fermentation broth.

[0040] The fermentation broth was centrifuged to remove mycelia, yielding a fermentation supernatant. The supernatant was mixed with ethyl acetate at a volume ratio of 3:1, shaken thoroughly, and extracted. The upper ethyl acetate extract was collected. The extraction was repeated three times, and the upper ethyl acetate extracts from the three extractions were combined. The upper ethyl acetate extract was then concentrated under vacuum at 40°C using a rotary evaporator to obtain a crude Sorbicillinoids extract.

[0041] All crude Sorbicillinoids extracts were redissolved in 20 mL of methanol and preliminarily separated by gel column chromatography: elution with methanol was performed, and identical fractions were collected and combined to obtain 12 fractions, designated Fr.1 to Fr.12. Chromatography of Fr.5 showed the presence of the target compound (…). Figure 1 ).

[0042] Figure 1 The image shows the LC-MS chromatogram of fraction Fr.5.

[0043] The above-mentioned fraction Fr.5 was concentrated under vacuum at 40℃ using a rotary evaporator, then redissolved in 5 mL of methanol. The redissolved fraction Fr.5 was then separated using a preparative high-performance liquid chromatography (PHPLC) system (Cyprister preparative grade HPLC system). The preparative HPLC conditions were as follows: Sep Basic C18 column; acetonitrile and water as the mobile phase; acetonitrile to water volume ratio: 0-15 min, 40-99% acetonitrile; 15-20 min, 99% acetonitrile; 20-25 min, 40% acetonitrile; detection wavelength 370 nm. The column effluent from 6.8 to 7.1 min was collected. Figure 2The compound was concentrated under vacuum at 40°C using a rotary evaporator to obtain a yellow powder, which was the extracted new compound and named Trichosorbicil B.

[0044] Figure 2 High-performance liquid chromatography (HPLC) chromatogram of the column eluent for fraction Fr.5 at 6.8–7.1 min.

[0045] 2. Structural identification One-dimensional (1D) nuclear magnetic resonance using high-resolution mass spectrometry (HR-ESI-MS) and nuclear magnetic resonance 1 H-NMR, 13 C-NMR), two-dimensional technology ( 1 H- 1 The structure of the new compound Trichosorbicil B was identified using H COSY, HSQC, HMBC, NOESY and X-ray single-crystal diffraction techniques.

[0046] The new compound, Trichosorbicil B, is a yellow amorphous powder. In HR-EISI-MS, the m / z value is 401.1580, and the calculated molecular formula is C0. 22 H 24 O7 (M = 400.1500), such as Figure 3 As shown.

[0047] Figure 3 The image shows the HR-ESI-MS spectrum of the positive ion of compound Trichosorbicil B.

[0048] The structure of the compound was determined using nuclear magnetic resonance (NMR) and X-ray single-crystal diffraction (XCD) techniques. The NMR data are shown in Table 2, and the spectra are as follows: Figures 4-12 As shown, the single-crystal X-ray diffraction ORTEP ellipsoid structure is as follows: Figure 13 As shown, the structural formula is as follows: .

[0049] Table 2. NMR data of compound Trichosorbicil B (600 / 150 MHz, DMSO- d 6 ) Figure 4 For the compound Trichosorbicil B 1 H-NMR spectrum.

[0050] Figure 5 For the compound Trichosorbicil B 13 C-NMR spectrum.

[0051] Figure 6 For the compound Trichosorbicil B 1 H- 1 H COSY spectrum.

[0052] Figure 7 The image shows the HSQC spectrum of the compound Trichosorbicil B.

[0053] Figure 8 The image shows the HMBC spectrum of the compound Trichosorbicil B.

[0054] Figure 9 The NOESY spectrum of compound Trichosorbicil B is shown.

[0055] Figure 10 The image shows the ROESY spectrum of the compound Trichosorbicil B.

[0056] Figure 11 For the compound Trichosorbicil B 1 H- 1 Schematic diagram related to H COSY and HMBC.

[0057] Figure 12 This is a schematic diagram showing the NOESY and ROESY of compound Trichosorbicil B.

[0058] Figure 13 The single-crystal X-ray diffraction ORTEP ellipsoid structure of the compound Trichosorbicil B (50% thermal ellipsoid).

[0059] Example 2: In vitro antitumor activity test The novel compound Trichosorbicil B prepared in Example 1 was tested for in vitro antitumor activity. The experimental cells were human acute T-lymphoblastic leukemia cell line (Jurkat), human colorectal adenocarcinoma cells (HT-29), human neuroblastoma cells (SH-SY5Y), human breast cancer cells (MCF-7), human malignant melanoma cells (A375), human cervical cancer cells (HeLa), and human liver cancer cells (Hep G2).

[0060] The MTT assay was used to conduct in vitro cytotoxicity experiments. The specific implementation method is as follows: The novel compound Trichosorbicil B from Example 1 was dissolved in dimethyl sulfoxide to prepare a stock solution with a concentration of 200 mg / mL. Test solutions with concentrations of 0, 5.0, 10.0, 20.0, 30.0, 40.0, 50.0, 80.0, 100.0, 150.0, and 200.0 μg / mL were then prepared using DMEM high-glucose fetal bovine antibody medium (45 mL DMEM high-glucose medium, 5 mL fetal bovine serum, 0.5 mL antibiotics).

[0061] Cancer cells in the logarithmic phase were seeded at a density of 3000 cells / well in 96-well plates using DMEM high-glucose fetal bovine antibiotic medium. The control group was not seeded with cells, but only DMEM high-glucose fetal bovine antibiotic medium was added. The 96-well plates were placed in an incubator at 37°C and 5% CO2 for 24 hours.

[0062] Then, 100 μL of the above-mentioned test solution at different concentrations was added to each well of the 96-well plate. The blank group and the control group were given test solution with a concentration of 0. After culturing for 48 hours, 20 μL of tetrazolium blue-phosphate buffer was added to each well and the plate was placed in a 37°C, 5% carbon dioxide incubator for 4 hours. The absorbance OD value of each well was measured at 492 nm using a microplate reader. The experiment was repeated 3 times and the average value was taken.

[0063] The IC50 of the compound for each cell type was calculated using the Logit method. 50 The results are shown in Table 3. The results indicate that the compound Trichosorbicil B has inhibitory activity against various tumor cell lines, with the highest inhibitory activity against human acute T-lymphoblastic leukemia cell lines, IC50 value [value missing]. 50 The value reached 4.13 μg / mL.

[0064] Table 3. IC50 of compound Trichosorbicil B in various cell types. 50 value In summary, the novel sorbicillinoid compound Trichosorbicil B prepared by the present invention through isolation and purification exhibits good in vitro antitumor activity and can be applied to the preparation of drugs for the prevention and / or treatment of tumors. The method for isolating and purifying Trichosorbicil B provided by the present invention offers a research direction for the identification of new compounds. This invention provides a new method and process for the large-scale fermentation production of Trichosorbicil B and also offers new research ideas for the development of anticancer drugs.

[0065] Example 3: In vitro anti-inflammatory activity test The novel compound Trichosorbicil B prepared in Example 1 was tested for its in vitro anti-inflammatory activity using a lipopolysaccharide (LPS)-induced inflammation model in mouse macrophage RAW264.7 cells. First, the toxicity of Trichosorbicil B to RAW264.7 cells was tested using the method described in Example 2 to determine its safe toxicity range. Then, the NO release from RAW264.7 cells was detected using the Griess method, as detailed below: The anti-inflammatory activity of each monomeric compound was evaluated using an LPS-induced RAW264.7 mouse macrophage inflammation model. Logarithmically growing cells were collected, washed twice with PBS, and then mixed thoroughly with an appropriate amount of culture medium. After cell counting on cell plates, the cell concentration was diluted to 1×10⁻⁶ cells with complete culture medium. 5 LPS (1 μg / ml) was seeded into 96-well plates. After 24 h of culture, cells adhered. Diluted test sample was added to each well. The plates were divided into three groups: LPS (negative control), blank control, and LPS + drug control. Each concentration was used in triplicate. Cells were cultured for another 24 h. Fresh 96-well plates were then filled with 50 μl of culture supernatant. Following the instructions of the inflammatory factor kit, NO release was measured. Absorbance was measured at 540 nm using a microplate reader. The IC50 (inhibition rate) of the drug on NO release from mouse macrophage RAW264.7 cells was calculated using GraphPad Prism 7 biostatistics software. 50 value.

[0066] Figure 14 The figure shows the effects of compound Trichosorbicil B on the cytotoxicity (a) and NO release level (b) of RAW264.7.

[0067] Depend on Figure 14 As can be seen from (a), compared with the blank control, the Sorbicillinoids compound of the present invention showed a cell proliferation activity of more than 90% at 15 μg / ml, indicating that when the monomer concentration is ≤15 μg / ml, it has almost no effect on the proliferation of mouse macrophages RAW264.7, which can be considered a safe concentration range.

[0068] Depend on Figure 14 As shown in (b), NO release after LPS stimulation was significantly higher than that in the control group (P<0.001), indicating successful model establishment. In the test, compared with the LPS group, the Sorbicillinoids compound of this invention reduced NO release in a dose-dependent manner. From the above studies, it can be seen that Sorbicillinoids exhibit good anti-inflammatory activity.

[0069] Example 4: In vitro test of anti-pulmonary fibrosis activity Pulmonary fibrosis cells are human embryonic lung cells (MRC-5 cells). MRC-5 cells proliferate excessively under TGF-β1 inducer stimulation, accelerating epithelial-mesenchymal transition (EMT) and causing collagen deposition. Therefore, inhibiting MRC-5 cell proliferation and promoting MRC-5 cell apoptosis can inhibit EMT, reduce collagen deposition, and decrease the formation of pulmonary fibrosis. The effects of sorbidillinoids on the growth of pulmonary fibrosis cells were tested using the following method: MRC-5 cells were seeded in 6.00 mm culture dishes at a density of 90% and cultured for 12 hours to allow for full cell adhesion. Subsequently, TGF-β1 inducers were added to stimulate lung fibrosis cells for 24 hours to establish a lung fibrosis cell model. After successful model establishment, the drug treatment groups were incubated with low, medium, and high concentrations (2.5 μg / ml, 5.0 μg / ml, and 10.0 μg / ml) of Trichosorbicil B for 24 hours. Simultaneously, the positive control group (PFD) received an equal volume of pirfenidone (PFD), and the normal control group received an equal volume of culture medium. After treatment, RIPA lysis buffer was added, and the cells were repeatedly pipetted three times on ice for 10 minutes each time. The lysates were then collected in 1.5 ml centrifuge tubes using a cell scraper and centrifuged at 4 °C and 12,000 rpm for 15 minutes. The supernatant was retained, and the precipitate was discarded. The protein concentration in the supernatant was determined using the BCA kit and quantified to a final concentration of 1 ng / µl. 100 µl of protein solution was taken, 25 µl of 4× loading buffer was added, and the remainder was filled with PBS. The mixture was then frozen at -20 °C for later use. Electrophoresis conditions were as follows: stacking gel electrophoresis at 80 V for 30 minutes; separating gel electrophoresis at 120 V for 60 minutes. Transfer conditions were 100 V for 90 minutes. After transfer, the membrane was blocked with 5% milk for 1 hour, washed three times with 1×TBST buffer for 5 minutes each time, and incubated overnight at 4 °C with primary antibody. The next day, the primary antibody incubation chamber was removed and allowed to equilibrate at room temperature for 1 hour. After washing with 1×TBST, the membrane was incubated with primary antibody at room temperature for 1 hour, washed again with 1×TBST, and finally developed.

[0070] Figure 15 The figure shows the effect of compound Trichosorbicil B on epithelial-mesenchymal related proteins in lung fibroblasts.

[0071] Experimental results showed that, compared with the normal group, the expression levels of Fibronectin and Collagen I proteins were significantly increased in the TGF-β1-induced model group; compared with the model group, all Trichosorbicil B treatment groups and the positive control drug PFD group significantly reduced the expression levels of Fibronectin and Collagen I proteins. These results indicate that Trichosorbicil B can effectively reduce fibrin expression levels, thereby playing a role in preventing and treating pulmonary fibrosis.

[0072] Example 5: In vitro experiment to reduce acute skin inflammation induced by ultraviolet light The novel compound Trichosorbicil B prepared in Example 1 was used in an in vitro experiment to alleviate UV-induced acute skin inflammation. The experimental model was a UVB-induced immortalized human keratinocyte (HaCaT) inflammation model. First, the toxicity of Trichosorbicil B to HaCaT cells was tested using the method described in Example 2 to determine its safe toxicity range. Then, Trichosorbicil B was applied to HaCaT cells before and after UVB irradiation, and the recovery of cell viability was detected. The specific implementation method is as follows: HaCaT cells were seeded in 96-well plates (5 × 10⁻⁶ cells per well). 4 (cells / well). After the cells had fully adhered to the plate, for the prevention experimental group, add complete culture medium containing different concentrations (10.0 μg / ml, 15.0 μg / ml, 20.0 μg / ml) of Trichosorbicil B to each well, and add an equal amount of complete culture medium to the remaining wells. After incubation for two hours, remove the culture medium, wash 2-3 times with PBS, and cover the bottom with a small amount of PBS. Place the 96-well plate under a UVB lamp (15 W, main peak wavelength at 280-380 nm, peak wavelength at 313 nm, plate 10 cm away from the UV lamp), with an irradiation dose of 60 mJ / cm². 2 After the treatment, the PBS was removed, and both the prevention and repair groups were given complete culture medium containing different concentrations of Trichosorbicil B. The control group was not irradiated with UV light and was given the same volume of fresh culture medium; the model group was irradiated with UV light of the same intensity but without the extract and was given the same volume of fresh culture medium. Cell viability was measured using the MTT assay after 24 h.

[0073] Figure 16The results show the effects of compound Trichosorbicil B on alleviating acute skin inflammation caused by ultraviolet light; (a) shows the effect of Trichosorbicil B on the proliferation of immortalized human keratinocytes HaCaT; (b) shows the effect of Trichosorbicil B on the proliferation of immortalized human keratinocytes HaCaT after ultraviolet light irradiation.

[0074] Figure 16 In the table, UVB represents the model group after ultraviolet light irradiation, NC represents the blank control group without UVB irradiation and without drug administration, "10 µg / mL-pre, 15 µg / mL-pre, 20 µg / mL-pre" represent the prevention group (treated with Trichosorbicil B first, then irradiated with ultraviolet light), and "10 µg / mL, 15 µg / mL, 20 µg / mL" represent the repair group (irradiated with ultraviolet light first, then treated with Trichosorbicil B).

[0075] like Figure 16 As shown in (a), compared with the blank control, the Sorbicillinoids compound of the present invention showed a cell proliferation activity of more than 90% at 20 μg / mL, indicating that when the monomer concentration is ≤20 μg / mL, it has almost no effect on the proliferation of HaCaT cells, which can be considered a safe concentration range.

[0076] like Figure 16 As shown in (b), compared with the control group, the cell viability of the model group (after UV irradiation) was significantly decreased, while the cell viability of the prevention group (pre: treated with Trichosorbicil B first, then exposed to UV irradiation) and the repair group (treated with UV irradiation first, then treated with Trichosorbicil B) treated with Trichosorbicil B was significantly increased in a dose-dependent manner. It can be seen that in the UVB-damaged HaCaT cell photoaging model, the Trichosorbicil B of this invention exhibits good anti-UVB-induced photoaging activity, can alleviate acute inflammatory damage to the skin caused by ultraviolet radiation, and has the potential to be developed into a skin drug or cosmetic.

[0077] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A novel Sorbicillinoid compound, Trichosorbicil B, characterized in that, The molecular formula is C 22 H 24 O7, the structural formula is as follows: 。 2. A method for extracting and separating the novel Sorbicillinoid compound Trichosorbicil B as described in claim 1, characterized in that, Includes the following steps: The fermentation broth of Trichoderma reesei, after removing mycelia, was extracted using an extraction solvent to obtain a crude extract of Sorbicillinoids. The crude extract of Sorbicillinoids was separated by gel column chromatography, then eluted with methanol, and the same fractions were collected and combined to obtain 12 fractions. The fraction containing Trichosorbicil B was obtained in the 5th fraction. After removing the solvent from the fraction containing Trichosorbicil B, the fraction was separated using preparative high-performance liquid chromatography (HPLC). The preparative HPLC conditions were as follows: the column was Sep Basic C18, and the mobile phase was acetonitrile and water. The volume ratio of acetonitrile to water was: 0-15 min, 40-99% acetonitrile; 15-20 min, 99% acetonitrile; 20-25 min, 40% acetonitrile. The detection wavelength was 370 nm; the column eluent was collected for 6.8–7.1 min, the solvent was removed, and the novel Sorbicillinoid compound Trichosorbicil B was obtained.

3. The extraction and separation method according to claim 2, characterized in that, The extraction solvent is ethyl acetate.

4. The extraction and separation method according to claim 2, characterized in that, The Trichoderma reesei fermentation broth is obtained by inoculating Trichoderma reesei into a fungal culture medium and then fermenting it.

5. The extraction and separation method according to claim 4, characterized in that, The fungal culture medium comprises the following components: 100 g / L glucose, 6 g / L tryptone, 0.15 g / L KH2PO4, 0.15 g / L K2HPO4, 0.10 g / L CaCl2·2H2O, 0.10 g / L MgSO4·7H2O, 0.05 g / L NaCl, 1 mL / L trace element solution, and 20 g / L calcium carbonate; wherein the trace element solution comprises the following components: 1.6 g / L MnSO4·4H2O, 5 g / L FeSO4·7H2O, 2 g / L CoCl2·6H2O, and 1.4 g / L ZnSO4·7H2O.

6. The extraction and separation method according to claim 4, characterized in that, The fermentation culture was carried out at a temperature of 28°C, a rotation speed of 200 rpm, and a duration of 12 days.

7. The use of the novel Sorbicillinoid compound Trichosorbicil B as described in claim 1 in the preparation of a medicament for treating tumors.

8. The use of the novel Sorbicillinoid compound Trichosorbicil B as described in claim 1 in the preparation of an anti-inflammatory drug.

9. The use of the novel Sorbicillinoid compound Trichosorbicil B as described in claim 1 in the preparation of a drug for the prevention and treatment of pulmonary fibrosis.

Images