A strain of penicillium and the use of diketomorpholine alkaloids

Abstract

This invention discloses a Penicillium strain, named Penicillium sp. SCSIO 41434, taxonomically named Penicillium This invention discloses a method for preparing diketomorpholine alkaloids from the aforementioned Penicillium strain on May 14, 2025, with accession number GDMCC No. 66337. The invention also discloses a method for preparing diketomorpholine alkaloids from the aforementioned Penicillium strain and the applications of diketomorpholine alkaloids. This invention found that shornephine A exerts a therapeutic effect against cholestatic liver injury through multiple mechanisms, including improving bile acid homeostasis, inhibiting inflammation, and alleviating liver fibrosis. Simultaneously, SA can significantly improve the survival rate of hypoxia-reoxygenation-induced HK2 cells, reduce the expression of kidney injury molecules, the level of inflammatory factors, and the content of reactive oxygen species, thus exerting a therapeutic effect against acute kidney injury.

Description

Technical Field

[0001] This invention belongs to the field of microbial and natural product technology, and particularly relates to the application of a Penicillium strain and diketomorpholine alkaloids. Background Technology

[0002] Cholestatic liver disease is defined as a complex pathological phenotype resulting from obstructed bile excretion. Its pathogenesis involves multiple pathological stimuli, including primary intrahepatic bile duct lesions, bile outflow obstruction, and drug-induced metabolic toxicity. Pathological studies have shown that livers exposed to a high concentration of bile acids over a long period are prone to reactive bile duct hyperplasia and accelerated pathological deposition of collagen fibers, thus exposing patients to a significant risk of progression from chronic inflammation to cirrhosis. Currently, drug intervention for this disease faces severe limitations, and conventional therapies are insufficient to fundamentally reverse the natural disease course. Therefore, there is an urgent need to find novel structural compounds to treat cholestatic liver injury.

[0003] Acute kidney injury (AKI), an acute and critical illness with an extremely high clinical mortality rate, arises from the susceptibility of the renal parenchyma to pathological stimuli such as ischemic hypoxia, exposure to toxic substances, and exogenous infections. It manifests as a decline in glomerular filtration rate, accumulation of metabolic waste products, and electrolyte imbalance. AKI often leads to multiple organ dysfunction syndrome (MODS), severely threatening patients' lives. However, there are currently no first-line clinical drugs specifically targeting renal parenchymal repair and functional reversal. Therefore, the development of innovative drugs for the treatment of AKI is imperative.

[0004] Marine fungal natural products, as an important group of secondary metabolites of marine microorganisms, are mainly synthesized by marine-specific or symbiotic fungi such as Penicillium, Aspergillus, and Trichoderma. These compounds are characterized by novel structural skeletons and rich chemical diversity, making them a valuable resource for innovative drug development. Among them, diketomorpholine alkaloids are characterized by a unique diketomorpholine skeleton composed of a six-membered morpholine ring containing nitrogen and oxygen atoms and two carbonyl groups. Previous studies have shown that these compounds exhibit highly efficient and low-toxicity P-glycoprotein inhibitory activity, effectively reversing multidrug resistance in tumor cells. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a Penicillium strain and the application of the diketomorpholine alkaloid Shornephine. This invention isolated the diketomorpholine alkaloid Shornephine A from a Penicillium strain found in the Gaoqiao Mangrove Nature Reserve, and discovered that this diketomorpholine alkaloid shows promise as a leading compound for the development of treatments for cholestatic liver disease and acute kidney injury.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: On the one hand, the present invention provides a Penicillium strain, which is namedPenicillium sp. SCSIO 41434, taxonomically named Penicillium sp. was deposited at the Guangdong Provincial Center for Microbial Culture Collection on May 14, 2025, with accession number GDMCC No. 66337.

[0007] In another aspect, the present invention provides a method for preparing diketomorpholine alkaloids from the above-mentioned Penicillium strain, wherein the diketomorpholine alkaloid is Shornephine A (SA), and its chemical structure is shown in formula (I); Formula (I) The diketomorpholine alkaloid was isolated from the fermentation products of the above-mentioned Penicillium strain.

[0008] In a preferred embodiment, the method includes the following steps: (1) The above-mentioned Penicillium strains were subjected to fermentation treatment; (2) After inactivation, extract with ethyl acetate; evaporate and concentrate the extract to obtain crude extract; (3) The crude extract was separated by normal phase silica gel column chromatography and purified by high performance liquid chromatography (HPLC) to obtain diketomorpholine alkaloids.

[0009] In a preferred embodiment, in step (3), the normal phase silica gel column chromatography separation is performed using a gradient elution of petroleum ether (PE) / ethyl acetate (EA) / methanol (CH3OH); Preferably, in the high-performance liquid chromatography (HPLC) purification, the mobile phase is 70% CH3OH / H2O; the aqueous phase of the mobile phase also contains 0.4% ( v / v The flow rate was 3 mL / min, and the retention time was 8.5 min.

[0010] In a preferred embodiment, step (1) includes: inoculating the above-mentioned Penicillium strain into PDB liquid culture medium to prepare a seed culture solution; inoculating the seed culture solution into rice culture medium and fermenting at room temperature.

[0011] In some specific embodiments, the culture conditions for the seed culture medium are: 28°C, shaker at 180 rpm for 3 to 7 days.

[0012] In some specific embodiments, the rice culture medium contains, by weight percentage: 45%~50% rice, 1% sea salt and 50%~54% water.

[0013] In the technical solution of the present invention, the sea salt is sun-dried salt.

[0014] In some specific embodiments, the rice culture medium is prepared by placing rice, sea salt, and water in a container according to a certain ratio, and then sterilizing it with high-pressure steam at 121°C and 103 kPa for 20 minutes.

[0015] Preferably, the fermentation time at room temperature is 28 to 42 days.

[0016] In another aspect, the present invention provides the use of the above-mentioned Penicillium strain and diketomorpholine alkaloid compound in the preparation of drugs for cholestasis or drugs for acute kidney injury, wherein the diketomorpholine alkaloid is Shornephine A (SA), and its chemical structure is shown in formula (I). Formula (I).

[0017] Compared with the prior art, the advantages of the present invention are as follows: This invention relates to a Penicillium strain in the Gaoqiao Mangrove Nature Reserve. Penicillium The diketomorpholine alkaloid Shornephine A (SA) was isolated from the fermentation product of sp. SCSIO 41434.

[0018] This invention constructed an in vivo cholestasis model induced by α-naphthyl isothiocyanate (ANIT). By measuring serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), total bile acid (TBA) levels, liver tissue inflammatory factors, and fibrosis markers, it was demonstrated that compound SA can reduce inflammation and fibrosis levels, improve metabolic disorders in cholestatic diseases, and alleviate cholestatic liver disease.

[0019] This invention constructed an in vitro hypoxia-reoxygenation injury model of human renal proximal tubular epithelial cells (HK2) to simulate the pathophysiological changes of renal ischemia-hypoxia and reperfusion during acute kidney injury. Cell viability assays confirmed that compound SA could improve cell viability in the hypoxia-reoxygenation cell model. Furthermore, the expression of KIM1, TNFα, and IL6, as well as ROS levels, were detected, confirming that compound SA could ameliorate ischemia-reperfusion-induced acute kidney injury and alleviate acute kidney injury.

[0020] This invention is the first to demonstrate the pharmacological activity of compound SA, providing a novel candidate compound for the preparation of drugs to prevent and treat cholestasis and acute kidney injury, and has broad prospects for development and application. Attached Figure Description

[0021] Figure 1 These are overall morphological images of mouse livers from different groups in Embodiment 2 of the present invention.

[0022] Figure 2These are the ALT, AST, ALP, and TBA levels in the plasma of different groups of mice in Example 2 of this invention.

[0023] Figure 3 This refers to the relative expression levels of bile acid-related downstream genes, fibrosis markers, and inflammatory factors in the livers of different groups of mice in Example 2 of this invention.

[0024] Figure 4 This refers to the cell viability in different groups of HK2 cells in the hypoxia-reoxygenation model of Example 3 of this invention.

[0025] Figure 5 This refers to the mRNA levels of KIM1, TNFα, and IL6 in different groups of HK2 cells in the hypoxia-reoxygenation model of this invention, as described in Example 3 of this invention.

[0026] Figure 6 It refers to the level of reactive oxygen species (ROS) in different groups of HK2 cells in the hypoxia-reoxygenation model of the present invention in Example 3.

[0027] Preservation Instructions Penicillium in this invention Penicillium sp. SCSIO 41434, its classification name is Penicillium sp. was deposited on May 14, 2025 at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No. 66337, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. Detailed Implementation

[0028] The following embodiments are merely some, not all, of the embodiments of the present invention. Therefore, the detailed descriptions of the embodiments provided below are not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0029] In this invention, unless otherwise specified, all equipment and raw materials are commercially available or commonly used in the industry. The methods described in the following embodiments are conventional methods in the art, unless otherwise specified.

[0030] Example 1: Purification and fermentation of the strain Isolation and Preservation of the Strains: Strains were isolated from sediment samples collected from the Gaoqiao Mangrove Nature Reserve (21.573°N, 109.767°E). The ITS sequence of the fungus was amplified by PCR, and the sequenced data was compared with similar sequences retrieved from the GenBank database. The fungus was identified as *Penicillium*. Penicillium sp.), and named strain Penicillium sp. SCSIO 41434. The corresponding ITS sequence has been uploaded to GenBank, accession number PV611825. This strain is currently deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), accession number GDMCCNo. 66337.

[0031] Preparation and separation of SA: Will Penicillium The seed culture solution was prepared by inoculating sp. SCSIO 41434 into potato glucose broth (PDB) liquid medium and culturing it at 28°C and 180 rpm on a shaker for 4 days.

[0032] Place 150 g of rice, 3.6 g of sea salt and 180 mL of water in a 1 L Erlenmeyer flask and autoclave (121 °C, 103 kPa, 20 min).

[0033] The seed culture was inoculated into 45 sterilized 1 L Erlenmeyer flasks containing rice culture medium and incubated statically at room temperature for 37 days. The fermented mycelium was inactivated with ethyl acetate and sonicated for 15 minutes. The mixture was extracted three times thoroughly with ethyl acetate until the extract was colorless. The ethyl acetate extracts were combined and concentrated under reduced pressure using a rotary evaporator to obtain a dark brown crude extract (100.0 g). The crude extract was separated by normal-phase silica gel column chromatography: a gradient elution was performed using petroleum ether (PE) / ethyl acetate (EA) / methanol (CH3OH) with the following volume ratios: 100 / 0 / 0, 80 / 20 / 0, 60 / 40 / 0, ​​40 / 60 / 0, 20 / 80 / 0, 0 / 100 / 0, 0 / 99 / 1, 0 / 98 / 2, 0 / 95 / 5, 0 / 90 / 10, 0 / 70 / 30, 0 / 50 / 50, 0 / 25 / 75, 0 / 0 / 100; after purification by high-performance liquid chromatography (HPLC) (70% CH3OH / H2O, with 0.4% (v / v) trifluoroacetic acid added to the aqueous phase, flow rate 3 mL / min), compound SA (529.5 mg, retention time t) was obtained. R = 8.5 min).

[0034] Example 2: This embodiment investigates the effect of SA on liver function in ANIT-induced cholestasis model mice. Experimental animals: Male C57 BL / 6 mice of SPF grade, weighing 22 - 25 g, were provided by the Experimental Animal Center of Southern Medical University (License number: SCXK (Yue) 2016 - 0041). All mice were housed in an environment with a temperature of 25 ± 1 °C and a humidity of 55 ± 5%. The light and dark cycles alternated (12 h light, 12 h dark), and they had free access to water and food. They were housed in the SPF animal room for one week before the experiment to adapt to the environment.

[0035] 1. Establishment of ANIT-induced cholestasis model and grouped administration The ANIT-induced cholestasis mouse model was intragastrically administered ANIT at 75 mg / kg for 48 hours to establish a bile duct injury model.

[0036] Twenty-four male C57 BL / 6 mice were randomly divided into 4 groups, with 6 mice in each group, namely: Intragastric solvent control olive oil (Oil) group; Intragastric ANIT group; ANIT + SA-L (0.5 mg / kg) group; ANIT + SA-H (1 mg / kg) group.

[0037] The ANIT + SA-L (0.5 mg / kg) group and the ANIT + SA-H (1 mg / kg) group were intraperitoneally injected with the corresponding SA solution five days in advance, once a day. On the fifth day, ANIT was intragastrically administered and continuous administration was carried out for a total of 7 days. During this period, the solvent control olive oil (Oil) group and the intragastric ANIT group were intraperitoneally injected with an equal dose of blank control solvent (1% DMSO / PBS). 48 hours after model establishment, blood was collected from the orbital venous plexus into a heparin sodium-containing centrifuge tube and centrifuged at 8000 rpm for 5 min (4 °C). The upper plasma was aspirated into a new centrifuge tube for the determination of plasma biochemical indexes. Subsequently, the mice were sacrificed and the liver tissues were taken for RNA extraction.

[0038] 2. Detection of plasma alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) and total bile acid (TBA) levels According to the kits for ALT, AST, ALP and TBA produced by Nanjing Jiancheng Bioengineering Research Institute, the specific determination methods were completely carried out according to the instructions.

[0039] 3. Effects of compound SA on bile acid homeostasis-related genes, fibrosis markers, and inflammatory factors Total RNA was extracted from mouse liver tissue using an animal tissue total RNA extraction kit (Chengdu Fuji Biotechnology Co., Ltd.), and the extraction method was performed strictly according to the manufacturer's instructions. After extraction, the total RNA was reverse transcribed into cDNA, and changes in its transcriptional expression level were detected using qPCR.

[0040] 4. Experimental Results like Figure 1 As shown, SA significantly alleviated ANIT-induced liver necrosis and cirrhosis in mice and reduced gallbladder enlargement and greening. Plasma biochemical parameters were detected using serum ALT, AST, ALP, and TBA kits. Figure 2 As shown, serum ALT, AST, ALP, and TBA levels were significantly increased in the ANIT model group compared to the Oil group, while SA dose-dependently decreased these plasma parameters. Furthermore, the mRNA levels of fibrosis and inflammatory factors in the liver of mice in the ANIT model group were significantly increased, while SA dose-dependently decreased their mRNA levels, inhibiting the progression of inflammation and fibrosis. These results indicate that SA can alleviate cholestasis induced by ANIT.

[0041] Example 3: This embodiment investigates the effect of compound SA on HK2 cells induced by a hypoxia-reoxygenation model. Experimental cells: HK2 cells were used in this invention and cultured in DMEM / F12 medium containing 10% fetal bovine serum and placed in a CO2 incubator (37°C, 5% CO2).

[0042] 1. Effects of compound SA on the viability of HK2 cells induced by hypoxia-reoxygenation This invention employs a hypoxia-reperfusion (H / R) intervention protocol of 12 h hypoxia followed by 2 h reoxygenation to treat HK-2 cells. By creating an in vitro ischemia-reperfusion microenvironment, it simulates the pathophysiological changes of renal ischemia-hypoxia and blood flow reperfusion during acute kidney injury (AKI). This model can effectively induce oxidative stress, inflammatory activation, and apoptosis in renal tubular epithelial cells, and can well replicate the cellular damage characteristics of clinical AKI. It is a classic cell model for in vitro research on the pathogenesis and pharmacological protective effects of AKI.

[0043] 1.1 Cellular Intervention HK2 cells were loaded at 2×10 4 The cells were seeded at a density of 1 cell per well in 96-well plates and cultured for 12 h until they adhered. Then, the cells were divided into 8 groups and the compound was added for further culture.

[0044] Control group: Co-culture with an equal amount of DMSO.

[0045] SA (5 μM) group: 5 μM of SA was added; SA (10 μM) group: 10 μM of SA was added; SA (15 μM) group: 15 μM of SA was added; H / R group: cells were hypoxic for 12 h and reoxygenated for 2 h.

[0046] H / R + SA (5 μM) group: After adding 5 μM SA, the cells were cultured under hypoxia for 12 h and reoxygenated for 2 h.

[0047] H / R + SA (10 μM) group: After adding 10 μM SA, the cells were cultured under hypoxia for 12 h and reoxygenated for 2 h.

[0048] H / R + SA (15 μM) group: After adding 15 μM SA, the cells were cultured under hypoxia for 12 h and reoxygenated for 2 h.

[0049] 1.2 Cell viability assay Prepare the working solution by mixing CCK-8 solution with complete culture medium at a ratio of 1:10. Add 110 μL of the working solution to each well to replace the original culture medium and incubate for 2 h. Measure the absorbance at 450 nm using a microplate reader and calculate the cell viability according to the manufacturer's instructions. Careful attention must be paid to precise adjustment of cell density and strict control of culture conditions to ensure the accuracy of experimental results.

[0050] 2. Measurement of cellular KIM1, TNFα, and IL6 levels Total RNA was extracted from cells using a total RNA extraction kit (Chengdu Fuji Biotechnology Co., Ltd.), and the extraction method was performed strictly according to the manufacturer's instructions. After extraction, the total RNA was reverse transcribed into cDNA, and changes in its transcriptional expression level were detected using qPCR.

[0051] 3. Cellular reactive oxygen species measurement After H / R-induced HK-2 cell modeling was completed, the cells were washed with PBS, and then incubated with a 5 μM reactive oxygen species fluorescent probe working solution at 37°C in the dark for 30 min. After incubation, the cells were washed again with PBS to remove unbound dye. Fluorescence and bright-field images were acquired using a laser confocal microscope.

[0052] 4. Experimental Results like Figure 4As shown, SA dose-dependently increased the viability of H / R-induced HK2 cells. The expression levels of KIM1, TNFα, and IL6 in cells of the H / R group were significantly increased compared to the Control group, while SA dose-dependently reduced these damage and inflammatory markers. Furthermore, SA administration significantly reduced the significantly increased cellular ROS levels induced by H / R, confirming its definite anti-acute kidney injury efficacy. In summary, the diketomorpholine alkaloid SA provided by this invention can be used as an active ingredient in the preparation of anti-acute kidney injury drugs, providing a new lead compound for the development of drugs for the treatment of acute kidney injury.

[0053] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A strain of Penicillium, characterized in that, The Penicillium strain was named Penicillium sp. SCSIO41434, taxonomically named Penicillium sp. was deposited at the Guangdong Provincial Center for Microbial Culture Collection on May 14, 2025, with accession number GDMCC No. 66337.

2. The method for preparing diketomorpholine alkaloids using the Penicillium strain according to claim 1, characterized in that, The diketomorpholine alkaloid is Shornephine A, and its chemical structure is shown in formula (I). Equation (I); The diketomorpholine alkaloid was isolated from the fermentation products of the Penicillium strain.

3. The method according to claim 2, characterized in that, The preparation method includes the following steps: (1) The above-mentioned Penicillium strains were subjected to fermentation treatment; (2) After inactivation, extract with ethyl acetate; evaporate and concentrate the extract to obtain crude extract; (3) The crude extract was separated by normal phase silica gel column chromatography and purified by high performance liquid chromatography to obtain diketomorpholine alkaloids.

4. The method according to claim 3, characterized in that, In step (3), the normal phase silica gel column chromatography separation is performed by gradient elution with petroleum ether / ethyl acetate / methanol.

5. The method according to claim 3, characterized in that, In step (3), during the high-performance liquid chromatography purification, the mobile phase is 70% CH3OH / H2O; the aqueous phase of the mobile phase also contains 0.4% ( v / v The flow rate was 3 mL / min, and the retention time was 8.5 min.

6. The method according to claim 3, characterized in that, In step (1), the fermentation process includes: inoculating the above-mentioned Penicillium strain into PDB liquid culture medium to prepare a seed culture solution; inoculating the seed culture solution into rice culture medium and fermenting at room temperature.

7. The method according to claim 6, characterized in that, The seed culture medium was cultured at 28°C for 3-7 days on a shaker at 180 rpm.

8. The method according to claim 6, characterized in that, The rice culture medium contains, by weight percentage: 45%–50% rice, 1% sea salt and 50%–54% water.

9. The method according to claim 6, characterized in that, The fermentation time at room temperature is 28 to 42 days.

10. The use of the Penicillium strain of claim 1 or the diketomorpholine alkaloid compound or a pharmaceutically acceptable salt thereof in the preparation of an antichostatic medicament or an anti-acute kidney injury medicament, characterized in that, The diketomorpholine alkaloid is Shornephine A, and its chemical structure is shown in formula (I). Equation (I).

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