Use of bacillus subtilis and cyclic depsipeptide metabolites thereof in the preparation of an anti-melanoma medicament

Abstract

The application discloses a bacillus subtilis and purposes of the bacillus subtilis in preparing a cyclic depsipeptide compound and an anti-melanoma drug, and has the characteristics that the strain has a preservation number of CCTCC NO: M20261136, and a preparation method of the bacillus subtilis includes the following steps: fermenting and culturing the bacillus subtilis LP strain with the preservation number of CCTCC NO: M20261136, harvesting a fermentation product, and after the fermentation product is separated through a silica gel column chromatography, the fermentation product is further purified through a reversed-phase semi-preparative HPLC to obtain a target compound Turnagainolide B; the application also provides purposes of the Turnagainolide B or a pharmaceutically acceptable salt, solvate, prodrug or derivative of the Turnagainolide B in preparing a drug for preventing and / or treating melanoma; and the Turnagainolide B has the advantages of good anti-melanoma activity and a treatment safety window, and has a direct tumor killing effect and an immune microenvironment regulating function.

Description

Technical Field

[0001] This invention belongs to the fields of marine natural product chemistry and tumor molecular pharmacology, specifically relating to the use of Bacillus subtilis and its cyclic peptide metabolites in the preparation of anti-melanoma drugs. Background Technology

[0002] Melanoma is one of the most aggressive and deadly malignant skin tumors. Although the clinical application of BRAF / MEK targeted therapy and PD-1 / PD-L1 immune checkpoint inhibitors has significantly improved the prognosis of some patients, high metastatic potential and acquired resistance remain major challenges in clinical practice. Current treatment strategies mainly rely on inducing tumor cell apoptosis, but tumor cells can avoid apoptosis by activating pro-survival pathways such as autophagy, thus limiting the efficacy of treatments. Therefore, there is an urgent need for lead compounds with novel mechanisms of action.

[0003] Turnagainolides B was initially discovered in 2011 by marine-derived Bacillus strains and was confirmed as a 17-membered cyclic peptide compound with a five-residue backbone of Hppa-Val-Ala-Ile-Val through chemical degradation and total synthesis. A 2020 study reported that Turnagainolide B possesses SHIP1 phosphatase activating activity (increasing SHIP1 activity by approximately 12% at 10 μM), similar to the activity of AQX-MN100. However, its research has been limited to in vitro enzyme activity assays, and its therapeutic potential and mechanism of action in solid tumors (especially melanoma) have never been revealed. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a Bacillus subtilis and its use in the preparation of cyclic peptide compounds and anti-melanoma drugs.

[0005] The technical solution adopted by this invention to solve the above-mentioned technical problems is: a Bacillus subtilis strain, which is classified and named Bacillus subtilis (…). Bacillus subtilis The strain, LP, with accession number CCTCC NO: M20261136, was deposited on June 1, 2026. The depositary institution is the China Center for Type Culture Collection, and the deposit address is Wuhan University, Wuhan, China.

[0006] This invention also provides a method for preparing the cyclic peptide compound Turnagainolide B, comprising the following steps: Step 1: Ferment Bacillus subtilis with accession number CCTCC NO: M20261136 and harvest the fermentation product; Step 2: After separation by silica gel column chromatography, the fermentation product was further purified by reversed-phase semi-preparative HPLC to obtain the target compound Turnagainolide B, whose structural formula is as follows: .

[0007] Further, step 1 is as follows: The seed culture of Bacillus subtilis from marine source with accession number CCTCC NO: M20261136 is inoculated into R2A liquid medium containing HP-20 macroporous adsorption resin at a volume percentage of 1-3% to 3% and fermented at a temperature of 25-30°C for 5-10 days; the fermentation product is adsorbed by HP-20 macroporous adsorption resin, eluted with methanol and concentrated to obtain the fermentation product.

[0008] Further, step 2 is as follows: The crude fermentation extract is separated by silica gel column chromatography, using 100% petroleum ether, a 1:1 volume mixture of petroleum ether and ethyl acetate, 100% ethyl acetate, a 9:1 volume mixture of ethyl acetate and methanol, and 100% methanol as eluents in a gradient elution process; the eluent obtained from the 9:1 volume mixture of ethyl acetate and methanol is collected, and the eluent is purified by semi-preparative reversed-phase high-performance liquid chromatography using a 60:40 volume mixture of acetonitrile and water as the mobile phase to obtain Turnagainolide B.

[0009] The present invention also provides the use of Turnagainolide B or a pharmaceutically acceptable salt, solvate, prodrug, or derivative thereof in the preparation of medicaments for the prevention and / or treatment of melanoma.

[0010] Furthermore, the melanoma includes: cutaneous melanoma, acral melanoma, mucosal melanoma, uveal melanoma, and metastatic melanoma.

[0011] This invention also provides the use of Turnagainolide B, or a pharmaceutically acceptable salt, solvate, prodrug, or derivative thereof, in the preparation of a medicament having at least one of the following functions: (a) Inhibits the proliferation of melanoma cancer cells; (b) Inhibit histone deacetylase (HDAC) activity; (c) Inhibits lipopolysaccharide (LPS)-induced nitric oxide (NO) production, i.e., anti-inflammatory; (d) Inhibit Ki67 expression in tumor tissue; (e) Promotes CD4 in tumor tissue + T cells and / or CD8 + T cell infiltration; (f) Inhibit melanin production.

[0012] Compared with the prior art, the advantages of the present invention are as follows: 1. In vitro activity: Turnagainolide B exhibits dose-dependent inhibitory effects on the proliferation of melanoma cells in B16-F10 mice, with an IC50 concentration of [missing value]. 50 The concentration was approximately 50 μM. Its toxicity to normal skin cells (HaCaT, MEF CF1, BEAS-2B, C3H10) was significantly lower than that to tumor cells, indicating that it has a good therapeutic safety window. At the same time, it effectively inhibited the migration of melanoma cells.

[0013] 2. In vivo antitumor activity: In the C57BL / 6J mouse B16-F10 subcutaneous tumor model, Turnagainolide B significantly inhibited tumor growth, reduced Ki67 proliferation index and tumor melanin content (H&E staining), and increased intratumoral CD8. + and CD4 + T cell infiltration suggests that it has both direct tumor-killing effects and immune microenvironment regulatory functions.

[0014] In summary, this invention provides the first systematic demonstration that Turnagainolide B has significant therapeutic activity against melanoma (B16-F10 cell line and in vivo tumor model), filling a gap in the application of this compound in the field of solid tumors. Attached Figure Description

[0015] Figure 1 The experiment was conducted to determine the inhibitory effect of Turnagainolide B on the proliferation of four types of cancer cells and its toxicity to four types of normal cells using the MTT assay. Figure 2 Comparison of cell morphology before and after treatment with Turnagainolide B, scale bar = 100 μm; Figure 3 The results show the inhibition of NO production in BV2 cells by Turnagainolide B; Figure 4 Results of the assay for HDAC inhibition activity of Turnagainolide B; Figure 5 For Nanolive holographic tomography live-cell imaging, showing the dynamic cell death process of B16-F10 cells treated with 50 μM Turnagainolide B for 24 hours, scale bar = 10 μm; Figure 6 The timeline of in vivo anti-tumor experiments, along with gross photographs of the tumor and spleen; Figure 7The growth curves for B16-F10 melanoma tumors; Figure 8 Quantitative analysis of the positive area of ​​proliferation markers in tumor tissue after melanoma treatment with Turnagainolide B; Figure 9 The images show pathological tissue sections and staining results for different treatment groups. Scale bar = 100 μm.

[0016] In the above figure, * indicates that P is less than 0.05, ** indicates that P is less than 0.01, *** indicates that P is less than 0.001, and **** indicates that P is less than 0.0001. Detailed Implementation

[0017] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0018] Example 1: Isolation and identification of strains.

[0019] 1. Strains Isolation One sponge sample was collected and identified as *Plakortis halichondrioides* (specimen XSSP-8), from a mesopelagic coral reef at a depth of 18 m in the Xisha Islands, China. Two g of the sponge sample was washed with distilled water, shredded with a scalpel, and ground in liquid nitrogen. The resulting homogenate was mixed with 1 mL of 50% glycerol and serially diluted 10-fold with the same 50% glycerol solution. 50 µL of the diluted bacterial suspension was spread onto agar plates containing 30% R2A medium supplemented with one of the following antibiotic combinations: nalidixic acid (20 mg / mL) and nystatin (25 mg / mL), or polymyxin (10 mg / mL) and nystatin (25 mg / mL). The inoculum was evenly dispersed on the plate surface using 8–10 sterile glass beads (5 mm in diameter). The plates were incubated at 26°C for 7 days to isolate the target colonies. Single colonies were picked and further purified by streaking on fresh agar plates to obtain pure cultures.

[0020] 2. Strain identification The 16S rRNA gene was amplified by colony PCR to confirm that the obtained strain was a pure culture (sterile). Universal primers used were 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3'). The PCR reaction program was: 95℃ pre-denaturation for 5 min; followed by 30 cycles, each cycle consisting of 95℃ denaturation for 30 s, 55℃ annealing for 30 s, and 72℃ extension for 2 min; finally, a final extension at 72℃ for 10 min. The amplified 16S rRNA gene product was sent to Genewiz Biotechnology Co., Ltd. (Suzhou, China) for sequencing using an ABI 3730 sequencing platform. The obtained sequence was compared with the GenBank database using BLAST, confirming the strain as *Bacillus subtilis*.

[0021]

[0022] Example 2: A method for preparing cyclic peptide compounds, comprising the following steps: Step 1, Fermentation Production: Marine-derived Bacillus subtilis (CCTCC NO: M20261136) was inoculated into R2A liquid medium (400 mL / 1 L Erlenmeyer flask, 2 flasks) and cultured at 28℃ and 160 rpm for 2 days to obtain a seed culture. The seed culture was then transferred at a volume percentage of 1% to 125 1 L Erlenmeyer flasks, each containing 400 mL of R2A liquid medium supplemented with 2% (w / v) HP-20 macroporous adsorption resin. Fermentation was carried out at 28℃ and 160 rpm for 7 days. The HP-20 macroporous adsorption resin was collected by filtration, washed with distilled water, and completely eluted with methanol. The extract was concentrated under reduced pressure to obtain approximately 13 g of crude fermentation extract. The R2A liquid medium was prepared as follows: yeast extract 0.5 g, proteose peptone 0.5 g, casein amino acids (acid-hydrolyzed casein) 0.5 g, glucose 0.5 g, soluble starch 0.5 g, sodium pyruvate 0.3 g. Dissolve g of dipotassium hydrogen phosphate (K2HPO4), 0.3 g of magnesium sulfate heptahydrate (MgSO4·7H2O), and 15.0 g of agar in 1 L of distilled or deionized water.

[0023] Step 2: Separate the crude fermentation extract by silica gel column chromatography (300-400 mesh, 50×4 cm column) using gradient elution with 100% petroleum ether, petroleum ether / ethyl acetate mixed in a 1:1 volume ratio, 100% ethyl acetate, ethyl acetate / methanol mixed in a 9:1 volume ratio, and 100% methanol as eluents.

[0024] Step 3: Collect the eluent obtained by elution with ethyl acetate / methanol (9:1 volume ratio). Use a mixed solution of acetonitrile and water (60:40 volume ratio) as the mobile phase and separate and purify the fraction by semi-preparative reversed-phase high-performance liquid chromatography (Luna C18 column, 250×10 mm, 5 μm, isocratic elution for 120 min, 2 mL / min) to obtain Turnagainolide B, which has the chemical structure of a cyclic peptide compound with the skeleton shown in formula (1). .

[0025] The compound is a 17-membered cyclic depsipeptide composed of five residues: Hppa-1, Val-2, Ala-3, Ile-4, and Val-5. It contains an S-configured 3-hydroxy-4-pentenoic acid (Hppa) residue, with an S stereoconfiguration at the C-3 position. All amino acid residues are L-configured, except for Ala, which is D-configured. It contains an E-configured olefin double bond with a coupling constant J = 16.0 Hz.

[0026] Structural identification of the cyclic peptide compound Turnagainolide B prepared in Example 3 and Example 2.

[0027] The compound is a white powder. HRESIMS analysis: [M+H] + m / z = 557.3328 (calculated value C) 30 H 45 O6N4 is 557.3339), determining the molecular formula C. 30 H 44 O6N4, high-resolution mass spectrometry [M+H] + The m / z range is 557.3324–557.3328, and the degree of unsaturation is 11. This compound... 1 H and 13 The nuclear magnetic resonance data of C are shown in Table 1.

[0028] The ¹H and ¹³C NMR spectra of Turnagainolide B contain characteristic resonance signals such as amide / ester carbonyl groups, α-methylene groups of amino acids, and side chains, indicating the presence of peptide components in the molecule. Detailed analysis of COSY, HSQC, and HMBC data confirms the presence of alanine (Ala), isoleucine (Ile), and two valine (Val) residues in the molecule. The characteristic signal of the alanine residues includes an α-methylene group at δC 48.84 (δ¹³C). H 4.32, p, J = 6.8 Hz), side chain methyl δC 16.37 (δ H 1.18, d, J = 7.0 Hz) and amide proton δ H 8.56 (d, J = 5.8 Hz); characteristic signals of isoleucine residues include α-methine δ C 57.22 (δH 4.26) and sidechain signal δ C 35.57, 11.95, 23.55, and 15.51; the α-methyl signals of the two valine residues were δ C 57.53 (δ) H 4.14) and δ C 58.28 (δ) H 4.24), each connected to two geminal dimethyl groups.

[0029] The non-peptide portion of the molecule was analyzed as follows: COSY spectroscopy showed that the hydroxymethyl group signal δ H 5.50 (Hppa-H-3) and the δ-alpha signal of olefinic methylene H 6.28 (Hppa-H-4) is associated with and is related to a pair of geminal methylene proton signals δ H The correlations of 2.88 and 2.42 (Hppa-H-2 / H-2′) indicate that the oxymethine group is flanked by an alkene bond and an aliphatic methylene group, respectively. The proton signal of the oxymethine group is located at a lower field (δ). H 5.50) and no COSY correlation was observed with the hydroxyl proton, suggesting that the methine is attached to an acylated alcohol hydroxyl group. Enyl hydrogen signal δ H 6.28 (Hppa-H-4) and another olefinic hydrogen signal δ H The δC signal at 6.68 (Hppa-H-5) correlates with the aromatic ring carbon signal at δC 135.76 (Hppa-C-6) in the HMBC spectrum, thus linking the disubstituted alkene bond to the benzene ring. The methylene proton (Hppa-H-2 / H-2′) correlates with the carbonyl carbon signal at δC 135.76 (Hppa-C-6). C The HMBC correlation of 168.75 (Hppa-C-1) indicates that the methylene group is attached to the carbonyl group. A coupling constant of approximately 16.0 Hz between the two olefinic hydrogen signals confirms that the olefinic bond is in the E configuration. Based on this, the non-peptide residues in the molecule are identified as derivatives of (E)-3-hydroxy-5-phenylpentan-4-enoic acid (Hppa).

[0030] Based on the linkage relationships of the above residues and HMBC-related information, it was determined that the compound Turnagainolide B is composed of Hppa, two Val, Ile and Ala residues, and is a cyclic condensed peptide (cyclic ester peptide) compound.

[0031] Table 1. NMR data of compound turnagainolides B (600 / 150 MHz, DMSO- d 6)

[0032] Example 4: Assay of cell proliferation inhibition activity (MTT method).

[0033] 1. Turnagainolide B cytotoxicity assay B16-F10 melanoma cells (CRL-6475, ATCC), pancreatic cancer Panc02 cells (STCC20054P, ServiceBio, China), bladder cancer MB49 cells (SCC148, Sigma-Aldrich, St. Louis, MO, USA), and lung cancer LLC cells (CRL-1642, ATCC, USA) were used at a rate of 5 × 10⁻⁶ cells / year. 3 Cells were seeded in 96-well plates and cultured at 37°C with 5% CO2 for 24 hours. Different concentrations of Turnagainolide B (0, 12.5, 25, 50, 100 μM, DMSO final concentration ≤0.1%) were added for 24 hours. 10 μL of MTT (5 mg / mL) was added to each well, and the plates were incubated at 37°C for 4 hours. The culture medium was discarded, and formazan crystals were dissolved in 100 μL of DMSO. The absorbance was measured at 550 nm. Cell viability was calculated with the untreated group as 100%, and the IC50 was fitted. 50 The toxicity of normal cell lines such as HaCaT, MEF (CF1), BEAS-2B, and MSC (C3H10) was evaluated.

[0034] The results are as follows Figure 1 As shown, Turnagainolide B exhibited significant, dose-dependent cytotoxic activity against various cancer cell lines, with particularly prominent inhibitory activity against B16-F10 mouse melanoma cells, inducing apoptosis in B16-F10 cells at a concentration of 50 μM. Within the investigated concentration range (0, 6.25, 12.5, 25, 50, 100 μM), the survival rate of Turnagainolide B against cancer cell lines such as Panc02 (pancreatic cancer), MB49 (bladder cancer), LLC (lung cancer), and B16-F10 (melanoma) gradually decreased with increasing concentration, showing a clear concentration-dependent relationship.

[0035] Notably, Turnagainolide B exhibited significantly lower cytotoxicity to normal skin-related cells compared to cancer cells. The normal cell types examined included epidermal keratinocytes (HaCaT), mouse embryonic fibroblasts (MEF, CF1), bronchial epithelial cells (BEAS-2B), and mesenchymal stem cells (MSCs, C3H10). Within the same concentration range, the survival rate of these normal cells remained high at all tested concentrations, indicating that Turnagainolide B possesses good selectivity and safety for normal cells.

[0036] In summary, Turnagainolide B exhibits potent and dose-dependent cytotoxic activity against cancer cells (especially B16-F10 melanoma cells) and can induce apoptosis in cancer cells, while showing low cytotoxicity to a variety of normal cells and good safety profile, suggesting its potential application value in anti-tumor therapy (especially local treatment of melanoma).

[0037] The aforementioned differential effects were further verified by phase contrast microscopy. The results are as follows: Figure 2 As shown, after 24 hours of treatment with 50 μM compound Turnagainolide B, B16-F10 melanoma cells exhibited significant morphological changes, including cytoplasmic vacuolization, cell adhesion and detachment, and cell death. Under the same treatment conditions, normal epidermal keratinocytes (HaCaT) remained largely unaffected, maintaining their normal morphology. These results further confirm that the compound Turnagainolide B has a significantly different effect on cancer cells (B16-F10) and normal cells (HaCaT), exhibiting potent cytotoxicity against cancer cells while showing low cytotoxicity against normal cells, indicating good cell selectivity.

[0038] 2. Analysis of the HDAC inhibitory activity and anti-inflammatory effect of turnagainolide B Further investigation was conducted into other activities of turnagainolide B related to its anticancer potential. Given that histone deacetylase (HDAC) inhibition is a classic mechanism of action for many clinically approved anticancer drugs, the HDAC inhibitory activity and anti-inflammatory effects of turnagainolide B were also evaluated.

[0039] The inhibitory effect of the compound Turnagainolide B on lipopolysaccharide (LPS)-induced NO release was evaluated using BV2 microglia. BV2 cells were seeded in culture plates and cultured to an appropriate density. They were then pretreated with Turnagainolide B at a final concentration of 50 μM for 1 h, followed by incubation with LPS at a final concentration of 1 μg / mL for another 24 h. After culture, the cell supernatant was collected, and assays were performed using a Nitric Oxide Assay Kit (E1030, Applygen Technologies Inc., Beijing, China) according to the manufacturer's instructions. Absorbance was measured at 550 nm, and NO concentration was calculated based on the nitrite standard curve. The LPS-stimulated group served as a control group. The inhibition rate of the compound on LPS-induced NO release was calculated to evaluate its anti-inflammatory activity. Results are as follows: Figure 3As shown, in the lipopolysaccharide (LPS)-stimulated BV2 microglia model, higher concentrations of the compound turnagainolide B effectively inhibited LPS-induced nitric oxide (NO) production, indicating that it has anti-inflammatory effects.

[0040] Using Amplite TM The Fluorimetric HDAC Activity Assay Kit (13601, AAT Bioquest, USA) was used to determine the inhibitory effect of compounds on HDAC activity. MB49 cells were collected and lysed, and protein concentrations were measured and then adjusted to the same level. Cell lysates were incubated with either the test compound (50–400 μM) or the positive control HDAC inhibitor (Trichostatin A), followed by the addition of HDAC Green. TM The fluorescent substrate was reacted at 37°C for 30–60 min. Fluorescence signals were detected using a fluorescence microplate reader with excitation wavelength of 490 nm and emission wavelength of 525 nm. Using the carrier control group as a reference, the relative HDAC activity and inhibition rate were calculated to evaluate the inhibitory effect of the compound on HDAC enzyme activity. Figure 4 As shown, in MB49 cells, 50 μM Turnagainolide B significantly inhibited HDAC activity, with the negative control referring to the experimental group without the compound.

[0041] The above results indicate that Turnagainolide B possesses both anti-inflammatory and HDAC inhibitory activities, further supporting its potential as a therapeutic agent.

[0042] 3. Morphological changes and dynamic response characteristics of melanoma cells during Turnagainolide B treatment The Nanolive 3D Cell Explorer holographic tomographic microscopy system (Nanolive, Switzerland) was used for real-time, label-free monitoring of dynamic changes in cell morphology. B16-F10 melanoma cells were seeded in μ-Dish culture dishes (35 mm glass-bottomed dishes, ibidi GmbH, Germany) for confocal microscopy and cultured overnight to allow for full cell adhesion. Before the experiment, the medium was replaced with 50 μM Turnagainolide B, with a 0.1% DMSO treatment group serving as a control. The culture dishes were then placed on a microscope stage equipped with a constant temperature and humidity system and continuously observed at 37°C, 5% CO2, and suitable humidity. For each group, a field of view containing 3–10 cells was selected, and real-time three-dimensional holographic tomographic imaging was performed continuously for 24 h, with three-dimensional refractive index images of the cells acquired at time intervals of 30 s to 15 min. The obtained images were processed and analyzed using Nanolive STEVE and ImageJ software to evaluate the morphological changes and dynamic response characteristics of cells during Turnagainolide B treatment.

[0043] The morphological changes of B16-F10 melanoma cells after treatment with Turnagainolide B were continuously monitored. Results are as follows: Figure 5 The study showed that after treatment with 50 μM Turnagainolide B, cells gradually lost their migration ability and pseudopodia activity, eventually leading to cell death. Two typical morphological processes of cell death were observed: first, some cells remained relatively quiescent for approximately 18 hours after treatment, followed by significant membrane blebbing, eventually resulting in cell membrane rupture and the retention of cell outline-like structures; second, another group of cells gradually shrank in size within approximately 12–14 hours after treatment, accompanied by vigorous cytoplasmic movement and vesicle-like structure formation, ultimately losing their complete cell morphology and dying. In contrast, control group cells maintained normal adherent growth throughout the observation period, exhibiting active cell migration, pseudopodia extension, and cell division.

[0044] The above results indicate that Turnagainolide B can continuously induce significant morphological changes in B16-F10 melanoma cells and ultimately lead to cell death. The cell death process has a clear time dependence and characteristic morphological changes, providing experimental evidence for the application of this compound as an anti-melanoma active molecule.

[0045] Example 5: In vivo anti-tumor experiment.

[0046] 1. The inhibitory effect of Turnagainolide B on melanoma growth and its histological effects A B16-F10 melanoma allograft model was established using female C57BL / 6J mice (6-8 weeks old). B16-F10 cells (2×10⁻⁶) were used to establish the model. 5 A tumor sample (100 μL, 2 mM) was suspended in a solution containing Turnagainolide B and subcutaneously injected into the right axillary region of mice. After tumor formation, the experimental animals were randomly divided into a control group and a Turnagainolide B treatment group, with 6 animals in each group. The control group was given physiological saline containing 0.1% DMSO, while the experimental group was treated with Turnagainolide B. During the experiment, the long and short diameters of the tumor and the weight of the mice were measured every 3 days, and the results were calculated according to the formula V = 0.5 × L × W. 2 Tumor volume was calculated, where L represents the long diameter of the tumor and W represents the short diameter. Mice were sacrificed on day 19 of the experiment, and the tumor tissue was completely dissected and photographed. A portion of the tumor tissue was fixed in 4% paraformaldehyde for histopathological analysis, including hematoxylin-eosin (H&E) staining and immunohistochemical detection, to evaluate the inhibitory effect of Turnagainolide B on melanoma growth and its histological impact.

[0047] Experimental results are as follows Figure 6 The results showed that, compared with the vector control group, tumor growth was significantly inhibited in the Turnagainolide B treatment group, with both tumor volume and weight significantly reduced at the experimental endpoint. The excised tumor tissue (left tissue sample) showed that the tumor volume in the Turnagainolide B treatment group was significantly smaller than that in the control group, while no obvious abnormalities were observed in the mouse spleen (right tissue sample), suggesting that this compound exhibits good in vivo tolerability while exerting its anti-tumor effect.

[0048] like Figure 7 As shown, compared with the control group, the growth of B16-F10 melanoma was significantly inhibited after treatment with Turnagainolide B. During the first 9 days of the experiment, tumor volume growth was slow in both groups; from day 12 onwards, the tumors in the control group entered a rapid growth phase, with the tumor volume increasing rapidly, reaching approximately 1600 mm² on day 18. 3 The above results indicate that tumor growth in the Turnagainolide B treatment group was significantly inhibited, with tumor volume remaining at a low level throughout the experimental period, and the final tumor volume being significantly lower than that in the control group (P < 0.01). These results demonstrate that Turnagainolide B can effectively inhibit the growth of B16-F10 melanoma in vivo, exhibiting significant anti-melanoma activity, and can be used to prepare drugs for the prevention and / or treatment of melanoma.

[0049] To further evaluate the effects of Turnagainolide B on melanoma proliferation and the tumor immune microenvironment, immunohistochemical and quantitative histological analyses were performed on tumor tissues. Figure 8 The results showed that, compared with the control group, the area positive for the proliferation marker Ki67 in tumor tissue was significantly reduced after Turnagainolide B treatment (P < 0.0001), indicating that the proliferation capacity of tumor cells was significantly inhibited. Simultaneously, CD4+ was observed in the tumor tissue. + T cells and CD8 + The significantly increased T cell infiltration levels (P < 0.01 and P < 0.0001, respectively) indicate that Turnagainolide B can promote the recruitment of anti-tumor immune cells and enhance the body's immune response. In addition, the melanin content in tumor tissue was significantly decreased (P < 0.0001), indicating that this compound can inhibit melanin production or reduce the differentiation phenotype of melanoma cells.

[0050] The above results indicate that Turnagainolide B can not only inhibit the proliferation of melanoma cells, but also enhance the infiltration of immune cells in tumor tissue and reduce melanin deposition, thereby exerting an anti-melanoma effect through a dual mechanism of direct tumor suppression and immunomodulation. It can be used to prepare drugs for the prevention and / or treatment of melanoma.

[0051] 2. Effects of Turnagainolide B on the histological characteristics and tumor immune microenvironment of melanoma To further evaluate the effects of Turnagainolide B on the histological characteristics and tumor immune microenvironment of melanoma, HE staining and immunohistochemical analysis were performed on tumor tissues from tumor-bearing mice. Figure 9 HE staining results showed that, compared with the control group, the tumor tissue structure in the Turnagainolide B treatment group was significantly altered, with looser tumor cell arrangement and decreased cell density and tissue damage observed in some areas. Ki67 immunohistochemical staining results showed a significant reduction in the number of Ki67-positive cells in the Turnagainolide B treatment group, indicating a significant inhibition of tumor cell proliferation. Furthermore, CD4 and CD8 immunohistochemical staining results showed that CD4+ was significantly reduced in the tumor tissue of the Turnagainolide B treatment group. + T cells and CD8 + T cell infiltration was significantly increased, including CD8 cells. +The increase in T cells was particularly significant, suggesting that this compound can promote the recruitment of effector T lymphocytes to tumor tissue and enhance the anti-tumor immune response. These results indicate that Turnagainolide B can not only directly inhibit melanoma cell proliferation but also improve the tumor immune microenvironment, synergistically exerting an anti-melanoma effect by enhancing the body's anti-tumor immune response.

[0052] The foregoing description is not intended to limit the invention, nor is the invention limited to the examples given. Any changes, modifications, additions, or substitutions made by those skilled in the art within the scope of the invention should also be considered within the protection scope of the invention.

Claims

1. Use of Turnagainolide B or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the treatment of melanoma.

2. The use according to claim 1, characterized in that, The drug mentioned is a drug that inhibits the proliferation of melanoma cancer cells.

3. The use according to claim 1 or 2, characterized in that, The melanomas include cutaneous melanoma, acral melanoma, mucosal melanoma, uveal melanoma, and metastatic melanoma.

4. The use according to claim 1 or 2, characterized in that, The preparation method of the Turnagainolide B includes the following steps: Step 1: The Bacillus subtilis strain with accession number CCTCC NO: M20261136 (…) Bacillus subtilis Fermentation culture is carried out to harvest the fermentation products; Step 2: After separation by silica gel column chromatography, the fermentation product was further purified by reversed-phase semi-preparative HPLC to obtain the target compound Turnagainolide B, whose structural formula is as follows: .

5. The use according to claim 4, characterized in that, Step 1 is as follows: The seed culture of Bacillus subtilis from marine source with preservation number CCTCC NO: M20261136 is inoculated into R2A liquid medium containing HP-20 macroporous adsorption resin at a volume percentage of 1-3% to 3% and fermented at a temperature of 25-30℃ for 5-10 days. The fermentation product is adsorbed by HP-20 macroporous adsorption resin, eluted with methanol and concentrated to obtain the fermentation product.

6. The use according to claim 4, characterized in that, Step 2 is as follows: The crude fermentation extract was separated by silica gel column chromatography, using a gradient elution with 100% petroleum ether, a 1:1 volume mixture of petroleum ether and ethyl acetate, 100% ethyl acetate, a 9:1 volume mixture of ethyl acetate and methanol, and 100% methanol as eluents. The eluent obtained from the 9:1 volume mixture of ethyl acetate and methanol was collected. The eluent was then purified by semi-preparative reversed-phase high-performance liquid chromatography using a 60:40 volume mixture of acetonitrile and water as the mobile phase to obtain Turnagainolide B.

Images