Preparation of marine-derived indole alkaloid aspertoryadin K and application thereof in treatment of renal fibrosis

Abstract

The application discloses preparation of an indole alkaloid aspertoryadin K of marine origin and application thereof in treatment of renal fibrosis. The application discloses, for the first time, a preparation method of a novel structure indole alkaloid aspertoryadin K containing quinazolinone through microbial fermentation, and discloses that the indole alkaloid aspertoryadin K has medicinal activity of improving activation of renal fibroblasts and reducing overexpression and deposition of type I collagen, and can be applied to drug research and development of renal fibrosis. The research result not only provides a candidate drug lead for the treatment field of renal fibrosis which is not satisfied in clinical treatment, but also lays an important foundation for development and utilization of marine microbial drug resources.

Description

Technical Field

[0001] This invention belongs to the field of natural product application technology, specifically relating to a method for preparing a novel indole alkaloid, aspertoryadin K, from marine fungi, and its application in the treatment of renal fibrosis. Background Technology

[0002] Chronic kidney disease (CHD) is a significant global health burden, affecting approximately 10% of the general population and placing a heavy burden on healthcare systems and society. This disease is not only closely associated with cardiovascular events and metabolic syndrome, but its progression to end-stage renal disease is also a leading cause of death and disability. In this process, renal fibrosis, as the core pathological mechanism, permeates almost the entire course of CHD, manifesting as excessive extracellular matrix deposition, destruction of renal parenchymal structure, and gradual loss of function. Currently, clinical interventions such as dialysis and nonspecific drugs cannot halt the progression of fibrosis or reduce related mortality. Commonly used clinical interventions, including dialysis and kidney transplantation, while replacing some renal function, cannot reverse or halt the fibrosis process and are accompanied by high costs and a significant decline in quality of life. Regarding drug treatment, apart from basic therapies such as angiotensin-converting enzyme inhibitors, drugs directly targeting fibrosis remain very limited. Compounds such as pirfenidone, which have shown some anti-fibrotic potential in clinical trials, have limited widespread use due to unstable efficacy, large individual variability in response, and side effects such as rash and gastrointestinal discomfort. Furthermore, patients with impaired renal function often experience impaired drug metabolism and excretion, further increasing the complexity of medication use and the risk of toxicity, creating a treatment dilemma. This situation highlights the urgent and unmet clinical need for innovative antifibrotic drugs that possess greater potency and better tolerability, as well as superior safety, tissue specificity, and pharmacokinetic properties suitable for patients with impaired renal function.

[0003] Marine fungi are important producers of structurally novel and functionally unique secondary metabolites, containing a wealth of untapped bioactive substances, making them a treasure trove for the development of novel functional products and natural drugs. Among them, *Aspergillus marineensis* stands out, producing a rich variety of structurally diverse natural products that exhibit multiple pharmacological effects, including anti-inflammatory, antioxidant, and cytotoxic activities. This broad-spectrum bioactivity often stems from the multi-target potential of its mechanisms of action, making it an ideal resource for discovering novel drug leads for addressing the unmet clinical need of renal fibrosis. Summary of the Invention

[0004] The first object of this invention is to provide an indole alkaloid, aspertoryadin K, or a pharmaceutical salt thereof, for treating or delaying renal fibrosis, with the chemical structural formula shown in formula (I): Formula (I)

[0005] A second objective of this invention is to provide a method for preparing the aforementioned compound aspertoryadin K from marine fungi. Aspergillus sp. SCSIO 41420 ( Natural Product Research It was isolated and prepared from the fermentation product of ( , 2025, 39:1939-1944.). The specific steps include:

[0006] (a) Epiphytic fungi of sponges Aspergillus Preparation of fermentation product of sp. SCSIO 41420;

[0007] (b) The fermentation product obtained in step (a) was extracted with ethyl acetate, and the solvent was recovered under reduced pressure to obtain an extract. The extract was eluted by a gradient on a normal silica gel column at the following volume ratios: petroleum ether: dichloromethane 5:1; petroleum ether: dichloromethane 1:1; petroleum ether: dichloromethane 0:1; dichloromethane: methanol 50:1; dichloromethane: methanol 5:1; dichloromethane: methanol 1:1, resulting in 6 elution fractions Fr.1–Fr.6. The fractions Fr.5 and Fr.6 eluted by dichloromethane: methanol 5:1 and dichloromethane: methanol 1:1 were combined and mixed with octadecylsilane-bonded silica gel for medium-pressure column chromatography. The eluent was methanol / water at a volume ratio of 10%–100%, resulting in 7 elution fractions Fr.a–Fr.g. Fr.e eluted by methanol / water at a volume ratio of 50%. The extract was purified to obtain the compound aspertoryadin K.

[0008] Preferably, step a involves collecting sponge-associated epiphytic fungi. Aspergillus Sp. SCSIO 41420 was used for seed fermentation culture. The seed fermentation medium contained 15 g of malt extract, 24 g of crude sea salt, 15 g of agar, and pH 7.2–7.4 per liter. The culture was carried out by shaking at 180 rpm at 25 ℃ for 3 days. After 3 days of culture, 10 mL of the seed fermentation broth was taken and fermented in rice culture medium at 25 ℃ to obtain fermented product. The rice culture medium contained 200 g of rice, 250 mL of water, and 24 g / L of sea salt.

[0009] Preferably, the purification is performed by semi-preparative HPLC purification of Fr.e, using a YMC-Pack ODS-A column (5 μm, 10 × 250 mm), isocratic elution with 80% CH3CN / H2O, a flow rate of 2.5 mL / min, and a retention time of 8.0 min to obtain the pure compound aspertoryadin K.

[0010] The chemical structure of Aspertoryadin K was identified as follows: Figure 1As shown, this is a new compound that has not been previously reported.

[0011] A third objective of this invention is to provide the above-mentioned compound aspertoryadin K or its pharmaceutical salt for the preparation of a drug for treating or delaying renal fibrosis by improving myofibroblast activation and reducing type I collagen overexpression and deposition.

[0012] A fourth object of the present invention is to provide a medicament for treating renal fibrosis, comprising an effective amount of the compound aspertoryadin K or a pharmaceutically acceptable salt thereof as an active ingredient, and a pharmaceutically acceptable carrier.

[0013] The compound aspertoryadin K of this invention can be used to treat diseases directly or indirectly caused by abnormal activation of myofibroblasts and excessive deposition of the extracellular matrix, especially type I collagen. The described treatment or delay in renal fibrosis can be used to prepare drugs for the prevention or treatment of end-stage renal disease, improvement of renal function, or in combination with existing antifibrotic therapies. Therefore, this invention provides candidate compounds for the development of new drugs for the treatment or delay in renal fibrosis, and is of great significance for the development of marine drug resources in China. Attached Figure Description

[0014] Figure 1 The chemical structure of the indole alkaloid aspertoryadin K;

[0015] Figure 2 High-resolution mass spectrum of the indole alkaloid aspertoryadin K;

[0016] Figure 3 Important two-dimensional NMR related data of COSY and HMBC for the indole alkaloid aspertoryadin K;

[0017] Figure 4 NOESY important two-dimensional NMR related information diagram for the indole alkaloid aspertoryadin K;

[0018] Figure 5 Infographics related to the treatment of renal fibrosis with the indole alkaloid aspertoryadin K. (ab) Representative protein blots of fibronectin (FN) and α-smooth muscle actin (α-SMA) in HK-2 cells stimulated by TGF-β1 after treatment with different concentrations of aspertoryadin K; (cf) Representative fluorescence images of type I collagen α1 chain (Collal), α-SMA, and FN in HK-2 cells stimulated by TGF-β1 after treatment with aspertoryadin K. Detailed Implementation

[0019] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the embodiments described in this specification are merely illustrative of the invention and are not intended to limit the invention.

[0020] Example 1: Fermentation of sponge symbiotic fungus Aspergillus sp. SCSIO 41420 and isolation of aspertoryadin K

[0021] Collecting symbiotic fungi from sponges Aspergillus sp. SCSIO 41420 (published in the literature) Natural products Research The applicant also holds, and guarantees, the right to make available to the public for 20 years from the date of application, a medium for seed fermentation culture (15 g malt extract, 24 g crude sea salt, 15 g agar, 950 mL distilled water, pH 7.2–7.4, adjusted to 1000 mL). 200 mL of medium is placed in a 500 mL Erlenmeyer flask and cultured at 25 °C with shaking at 180 rpm. After 3 days of culture, 10 mL of the seed fermentation broth is transferred to a sterilized Erlenmeyer flask containing rice culture medium (200 g rice, 250 mL water, 6 g sea salt / flask, 100 flasks in total) and allowed to ferment statically at 25 °C to obtain the fermented product.

[0022] The fermentation product was extracted three times with ethyl acetate, and the solvent was recovered under reduced pressure to obtain the extract. The extract (120.0 g) was dissolved in a small amount of ethyl acetate and eluted using a silica gel column gradient with petroleum ether / dichloromethane / methanol in six stages (volume ratios of petroleum ether:dichloromethane 5:1; petroleum ether:dichloromethane 1:1; petroleum ether:dichloromethane 0:1; dichloromethane:methanol 50:1; dichloromethane:methanol 5:1; dichloromethane:methanol 1:1), yielding six elution fractions Fr.1–Fr.6. Fr.5 (dichloromethane:methanol 5:1) and Fr.6 (dichloromethane:methanol 1:1) were combined, mixed with octadecylsilane-bonded silica gel (ODS), and subjected to ODS medium-pressure column chromatography with methanol / water eluting in a volume ratio gradient from 10% to 100%, yielding seven elution fractions Fr.a–Fr.g. Fr.e (50% methanol / water volume ratio) was purified by semi-preparative HPLC using a YMC-Pack ODS-A column (5 μm, 10 × 250 mm), with isocratic elution of 80% CH3CN / H2O (2.5 mL / min) and a retention time of 8.0 min to obtain the pure compound aspertoryadin K.

[0023] Example 2: Structural Identification of aspertoryadin K

[0024] The chemical structure of the open-ring indole diterpenoid compound aspertoryadin K isolated in Example 1 is as follows: Figure 1 As shown. Structural analysis and testing were performed, and the following physicochemical property data were obtained ( Figure 2 -4):

[0025] Aspertoryadin K: Yellow solid; UV (MeOH)λ max (log ε): 204 (39.08), 227(37.87) nm; ECD (0.15 mg / mL, MeOH) λ max (Δ ε): 211 (+49.44), 230 (−39.79); IR(film) ν max 3593, 2962, 2841, 1772, 1668, 1608, 1541, 1506, 1386, 1257, 1112,1064, 1016, 952, 758, 700, 667, 603, 586, 557, 545, 538, 526cm -1 ; -102.0(c 0.1, MeOH); 1 H and 13 C data are shown in Table 1; HRESIMS m / z 475.1968 [M+H] + (calcd forC 26 H 27 N4O5 + , 475.1976).

[0026] Table 1. Aspertoryadin K 1 H NMR and 13 C NMR data (500 and 125 MHz, deuterated DMSO)

[0027] Example 3: Activity assay of aspertoryadin K in treating renal fibrosis

[0028] This study used TGF- β 1. By stimulating renal fibroblasts HK-2, a renal fibrosis cell model was constructed to evaluate the therapeutic activity of aspertoryadin K in renal fibrosis.

[0029] 3.1 Construction of a renal fibrosis cell model: HK-2 cells were cultured in a 37°C, 5% CO2 incubator in DMEM / F12 (Gibco) (ExCell, FSP500) supplemented with 10% fetal bovine serum (FBS). Cells were seeded in 6-well plates (2 × 10⁶ cells per well). 5 (1 cell), cultured for 12 hours. To establish TGF- β A HK-2 cell model was established by stimulating cells in serum-free medium for 24 hours, followed by induction with 10 ng / mL recombinant TGF-β. β 1 (240-B-002, R) & Treat with D Systems, or co-treat with aspertoryadin K (2.5 µM and 10 µM) for 48 hours.

[0030] 3.2 Western Blot (WB) Analysis: HK-2 cells were lysed in RIPA buffer containing protease inhibitors (Ncmblo, WB3100) and then centrifuged at 13,000g for 15 min at 4°C. Total protein concentration was determined using a BCA kit (Epizyme, ZJ102). Quantitative proteins were mixed with loading buffer (Epizyme, LT101S) and boiled at 100°C for 10 min, then separated by SDS-PAGE and transferred to a PVDF membrane (Millipore, ISEQ00010). After blocking the membrane at room temperature for 1 hour, it was incubated overnight at 4°C with a specific primary antibody. The membrane was then incubated with a secondary antibody (anti-rabbit or anti-mouse IgG) at room temperature for 2 hours. Western blot bands were detected by ECL substrate (Fdbio, FD8020) and analyzed using Image-J 1.52a software.

[0031] 3.3 Immunofluorescence assay: HK-2 cells (1 × 10⁶ cells per well) were collected in a 96-well plate. 4 (cells) using TGF- β 1. Induction of fibrosis treatment. Cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.2% Triton X-100 (Solarbio, T8200) for 15 minutes, and then blocked with 5% bovine serum albumin (BOSTER, AR0009) at room temperature for 1 hour. The fixed cells were then inoculated with the specified primary antibody (… αCells were incubated overnight at 4°C with FITC-labeled secondary antibody (ABclonal, AS053, 1:500) at room temperature, followed by incubation for 1 hour at room temperature. The nuclei were stained with Hoechst (Beyotime, C1025) for 15 minutes. Finally, images were acquired using an ImageXpress Micro confocal microscope (Molecular Devices Shanghai Corporation).

[0032] 3.4 Statistical Analysis: All experiments were performed at least three times. All data are expressed as mean ± standard deviation (SD). Statistical analysis was performed using GraphPad Prism 7 software (GraphPad Software, San Diego, CA, USA), including independent samples t-tests, one-way ANOVA, and two-way ANOVA. P < 0.05 is considered a significant difference (*) P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001).

[0033] The results showed that aspertoryadin K at 2.5 μM significantly inhibited the activity of the key fibrosis protein FN. α The expression of -SMA showed good anti-fibrotic activity. Even at a low concentration of 2.5 μM, the compound still exhibited a strong inhibitory effect. Figure 5 a−b). Aspertoryadin K can effectively downregulate TGF-β. β 1-induced Col1a1 in HK-2 cells, α - Expression of SMA and FN ( Figure 5 c−f), thus verifying its anti-fibrotic effect at the morphological level.

Claims

1. Indole alkaloid aspertoryadin K or its medicinal salt, with the chemical structural formula shown in formula (I): Equation (I).

2. A method for preparing the compound aspertoryadin K according to claim 1, characterized in that, From sponge symbiotic fungi Aspergillus It was isolated and prepared from the fermentation product of sp. SCSIO 41420.

3. The preparation method according to claim 2, characterized in that, Specifically, the following steps are included: (a) Epiphytic fungi of sponges Aspergillus Preparation of fermentation product of sp. SCSIO 41420; (b) The fermentation product obtained in step (a) was extracted with ethyl acetate, and the solvent was recovered under reduced pressure to obtain an extract. The extract was then eluted by a normal silica gel column gradient at the following volume ratios: petroleum ether: dichloromethane 5:1; petroleum ether: dichloromethane 1:1; petroleum ether: dichloromethane 0:

1. Gradient elution of dichloromethane:methanol 50:1; dichloromethane:methanol 5:1; dichloromethane:methanol 1:1 yielded 6 elution fractions Fr.1–Fr.6 sequentially, dichloromethane:methanol 5:1; Fractions Fr.5 and Fr.6 eluted with dichloromethane:methanol 1:1 were combined and mixed with octadecylsilane-bonded silica gel for medium-pressure column chromatography. The eluent was methanol / water at a gradient of 10%–100% by volume, yielding seven eluting fractions Fr.a–Fr.g. Fr.e was eluted with methanol / water at a volume ratio of 50%. After purification, the compound aspertoryadin K was obtained.

4. The preparation method according to claim 3, characterized in that, Step a is to collect sponge-associated epiphytic fungi. Aspergillus Sp. SCSIO 41420 was used for seed fermentation culture. The seed fermentation medium contained 15 g of malt extract, 24 g of crude sea salt, 15 g of agar, and pH 7.2–7.4 per liter. The culture was carried out by shaking at 180 rpm at 25 ℃ for 3 days. After 3 days of culture, 10 mL of the seed fermentation broth was taken and fermented in rice culture medium at 25 ℃ to obtain fermented product. The rice culture medium contained 200 g of rice, 250 mL of water, and 24 g / L of sea salt.

5. The preparation method according to claim 3, characterized in that, The purification described above was performed by semi-preparative HPLC purification of Fr.e. using a YMC-Pack ODS-A column (5 μm, 10 × 250 mm), eluted isocratically with 80% CH3CN / H2O at a flow rate of 2.5 mL / min, and the pure compound aspertoryadin K was obtained at a retention time of 8.0 min.

6. The use of the compound aspertoryadin K or its pharmaceutical salt as described in claim 1 in the preparation of a medicament for treating renal fibrosis.

7. The application according to claim 6, characterized in that, The application includes the preparation of a drug for treating or delaying renal fibrosis by improving renal fibroblast activation and reducing type I collagen overexpression and deposition.

8. A drug for treating renal fibrosis, characterized in that, It comprises an effective amount of the compound aspertoryadin K or a pharmaceutically acceptable salt thereof as the active ingredient, as described in claim 1, and a pharmaceutically acceptable carrier.

Images