Use of a. fumigatus coronatum for the preparation of a medicament for the treatment and / or prevention of hyperuricemia and methods

Abstract

The present application relates to the field of microorganism, disclose the application and method of the preparation for treating and / or preventing high uric acid of the medicine of the crown protuberance aspergillus, the preservation number of the crown protuberance aspergillus ( Aspergillus cristatus ) is CGMCC No.41727.The crown protuberance aspergillus provided by the present application can realize the purpose of reducing uric acid by degrading uric acid and its precursor, or inactivating the enzyme substance necessary for the conversion of uric acid precursor into uric acid, so the crown protuberance aspergillus provided has the application prospect of the preparation for treating and / or preventing high uric acid of the medicine.

Description

Technical Field

[0001] This invention relates to the field of microbiology, and more specifically to the use and method of Aspergillus cristatus in the preparation of drugs for the treatment and / or prevention of hyperuricemia. Background Technology

[0002] In the treatment of diseases related to uric acid metabolism disorders, hyperuricemia and gout are common conditions, and their clinical intervention faces significant limitations and insufficient safety issues with existing technologies. Current uric acid-lowering drugs may cause various adverse reactions. Moreover, the safety and tolerability of long-term treatment for patients with chronic hyperuricemia still need improvement, failing to meet the comprehensive management needs of clinical patients with uric acid metabolism disorders.

[0003] Microorganisms, due to their high biocompatibility and mild effects, have shown potential advantages in the treatment of metabolic diseases; however, the development of microbial resources targeting uric acid metabolism is currently insufficient. *Aspergillus cristatus*, a fungus already used in traditional fermented foods, possesses metabolites with rich biological activity. Studies have shown that it can influence uric acid metabolism pathways through various routes. Furthermore, *Aspergillus cristatus*, which can colonize the gut, can further synergistically improve uric acid metabolism by regulating gut microbiota structure.

[0004] Therefore, there is an urgent need to develop Coronavirus cristatum that can prevent or treat uric acid. Summary of the Invention

[0005] The purpose of this invention is to overcome the above-mentioned problems existing in the prior art and to provide the application and method of Aspergillus cristatus in the preparation of drugs for the treatment and / or prevention of hyperuricemia.

[0006] The first aspect of the present invention provides the use of Aspergillus cristatus in the preparation of a medicament for the treatment and / or prevention of hyperuricemia, wherein the Aspergillus cristatus has the accession number CGMCC No. 41727.

[0007] A second aspect of the present invention provides a method for reducing uric acid, the method comprising: contacting Aspergillus cristatus with accession number CGMCC No. 41727 with uric acid and / or a uric acid precursor, or contacting Aspergillus cristatus with accession number CGMCC No. 41727 with an oxidase of a uric acid precursor.

[0008] Through the above technical solution, the *Aspergillus cristatus* provided by this invention can reduce uric acid by degrading uric acid, degrading uric acid precursors, or inactivating the enzymes necessary for the conversion of uric acid precursors into uric acid. Therefore, the provided *Aspergillus cristatus* has the potential to be used in the preparation of drugs for the treatment and / or prevention of hyperuricemia.

[0009] Biological Preservation The strain provided by this invention is classified and named *Aspergillus cristatus*.Aspergillus cristatus It was deposited on December 23, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 41727 and address at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Detailed Implementation

[0010] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0011] The inventors of this invention unexpectedly obtained a strain of Aspergillus cristatus that can colonize the intestine and can be used to prepare fermented dairy products. Aspergillus cristatus CCNH008, due to the above characteristics, is more suitable than conventional Aspergillus cristatus for the preparation of drugs for the prevention and / or treatment of hyperuricemia.

[0012] The first aspect of the present invention provides Aspergillus cristatus ( Aspergillus cristatus The use of *Aspergillus cristatus* in the preparation of medicaments for the treatment and / or prevention of hyperuricemia, wherein the preservation number of *Aspergillus cristatus* is CGMCC No. 41727.

[0013] A second aspect of the present invention provides a method for reducing uric acid, the method comprising: contacting Aspergillus cristatus with accession number CGMCC No. 41727 with uric acid and / or a uric acid precursor, or contacting Aspergillus cristatus with accession number CGMCC No. 41727 with an oxidase of a uric acid precursor.

[0014] In this invention, the uric acid precursor may include at least one of nucleoside compounds, nucleotide compounds, and purine compounds (such as hypoxanthine, xanthine, guanine, adenine, etc.).

[0015] According to the present invention, the uric acid precursor may also be a substance capable of generating the uric acid precursor as described above.

[0016] Preferably, the nucleoside includes at least one of inosine nucleoside, xanthine nucleoside, uracil nucleoside, adenine nucleoside, cytosine nucleoside, guanine nucleoside, and thymine nucleoside.

[0017] Preferably, the purine includes at least one of adenine, guanine, hypoxanthine, and xanthine.

[0018] In this invention, the oxidase of the uric acid precursor can be an enzyme capable of oxidizing the uric acid precursor as described above, such as xanthine oxidase and / or adenosine deaminase.

[0019] Preferably, the oxidase of the uric acid precursor is xanthine oxidase.

[0020] It is understood that the method for reducing uric acid provided by this invention is not for therapeutic purposes, that is, the use of Aspergillus cristatus provided in this application can achieve the purpose of reducing uric acid in vitro.

[0021] The present invention will be described in detail below through embodiments.

[0022] PDA culture medium: purchased from Qingdao Haibo Biotechnology, catalog number HB0233-5.

[0023] Potato glucose aqueous medium (PDB): purchased from Qingdao Haibo Biotechnology, catalog number HB0233-8; Activation process: Take the seed cryopreservation tube and place it at room temperature. After thawing, use a sterile inoculation loop to pick up the bacterial solution in a clean bench and spread it on PDA medium. After culturing at 30℃ for 7 days and growing single colonies, pick the colonies and transfer them to fresh sterile PDB medium. Culturing at 30℃ and 180r / min for 24 hours will allow them to be used for later use. Absorbance was measured using a visible light spectrophotometer.

[0024] Example 1 Take the activated fresh Aspergillus cristatus CCNH008 fermentation broth (viable count 10). 8 The *Aspergillus cristatus* (CFU / mL) was inoculated at a 3 vol% inoculum into potato dextrose agar (PDB) containing 0.5 g / L inosine, 0.5 g / L guanosine, 8 mg / L hypoxanthine, 8 mg / L xanthine, and 75 mg / L uric acid, respectively. The cultures were incubated at 30°C and 180 rpm. Samples were taken at 0 min, 15 min, 30 min, and 1 h. The concentrations of these substances in the culture medium were determined by HPLC. The degradation rate of these substances by *Aspergillus cristatus* was calculated using Formula 1 below. The results are shown in Table 1 (in %).

[0025] Degradation rate (%) = [(C0-C)] t ) / C0]×100%; In the formula C tThe values ​​represent the content of each substance at different time points (15 min, 30 min, and 1 h), with C0 representing the initial content of each substance at 0 min. The HPLC conditions were as follows: mobile phase: methanol:water = 1:9, retention time 15 min, C18 column (4.6 × 250 mm, 4.6 μm), column temperature 30℃, injection volume 20 μL, flow rate 0.7 mL / min, detection wavelength 254 nm, and UV detector.

[0026] Table 1

[0027] Furthermore, in normal biological metabolic pathways, inosine and guanosine are degraded into hypoxanthine and guanine, respectively. Hypoxanthine is then oxidized to xanthine. These metabolites still generate uric acid. Therefore, the inventors further studied the content of the metabolites corresponding to each substance in different experimental groups. The results showed that the strain of this application can utilize inosine, guanosine, hypoxanthine, and xanthine for growth and reproduction, thereby further reducing the amount of uric acid produced. In addition, the strain provided by this invention can convert highly water-soluble substances such as inosine and guanosine into less water-soluble substances. These less water-soluble products are difficult to absorb into the bloodstream through the intestinal mucosa, avoiding secondary metabolic transformation in the body. On the other hand, they can be directly excreted from the body through the intestine with feces, further reducing the total amount of uric acid precursors in body fluids such as blood and urine, thereby achieving a synergistic reduction of uric acid.

[0028] Example 2 Take the activated Aspergillus cristatus CCNH008 seed culture and inoculate it into fresh sterile PDB medium at an inoculation rate of 3 vol%. Incubate at 30℃ for 24 h, and collect the fermentation supernatant by centrifugation at 4℃ and 8000 r / min. Use PDB medium as a control.

[0029] The inhibition rate of XOD activity by Aspergillus cristatus CCNH008 fermentation supernatant was determined using a xanthine oxidase (XOD) activity assay kit (purchased from Solaribio, catalog number BC1090) and the kit's definition of XOD activity inhibition rate. Allopurinol (0.1 mmol / L) (purchased from Sigma-Aldrich, catalog number A8003) was used as a positive control. The XOD activity inhibition rates of PDB medium, Aspergillus cristatus CCNH008 fermentation supernatant, and allopurinol were 0%, 77.32±2.45%, and 89.74±2.33%, respectively. It can be seen that CCNH008 has a good inhibitory effect on XOD activity.

[0030] Example 3 Aspergillus cristatus was activated to the second generation on PDA medium, then transferred to malt extract sporulation medium and cultured for 10-15 days. The plates were washed with physiological saline and the mycelium was filtered off to prepare 10... 8 CFU / ml spore suspension. The spore suspension was inoculated into PDB medium and cultured for 4 days. The supernatant was collected to obtain crude enzyme solution. The precipitated mycelium was dried and weighed to calculate protease activity: at 30℃, one enzyme activity unit is defined as 1 nmol of tyrosine produced per gram of mycelium per minute through casein hydrolysis. The activities measured under acidic (pH=3.0), neutral (pH=7.0), and alkaline (pH=10.5) conditions are the acidic, neutral, and alkaline protease activities, respectively. The protease hydrolyzes casein to produce tyrosine, and tyrosine can reduce phosphomolybdic acid to produce tungsten blue; tungsten blue has a characteristic absorption peak at 680 nm. A standard curve of absorbance versus tyrosine concentration was established to determine the protease activity of the strain of this invention. The enzyme activity unit is nmol / (min mycelial dry weight). The results are shown in Table 2.

[0031] Table 2

[0032] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. Aspergillus cristatus ( Aspergillus cristatus The use of this drug in the preparation of a medicine for the treatment and / or prevention of hyperuricemia, characterized in that, The preservation number of the *Aspergillus cristatus* is CGMCC No. 41727.

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