Fermented extract of Cordyceps sinensis for lowering uric acid, preparation method and application thereof

Abstract

The present invention belongs to the field of biomedicine technology, and specifically relates to a Cordyceps sinensis fermentation extract for lowering uric acid, and its preparation method and application. The present invention concentrates the Cordyceps sinensis fungal fermentation liquid and then extracts it with ethanol. After the extract is concentrated, it is extracted with n-butanol, ethyl acetate, and petroleum ether in sequence. The extract is concentrated to obtain an n-butanol extraction part, an ethyl acetate extraction part, a petroleum ether extraction part, and the remaining part. The obtained petroleum ether extract is the Cordyceps sinensis fermentation extract for lowering uric acid. The Cordyceps sinensis fermentation extract of the present invention has the effect of significantly reducing renal damage in hyperuricemia mice and reducing serum uric acid in hyperuricemia mice. As a new natural uric acid-lowering active substance, the Cordyceps sinensis fermentation extract of the present invention can be applied to the fields of health food and medicine, and has great development and application value.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and in particular relates to a fermented extract of Cordyceps sinensis for lowering uric acid, and a preparation method and application thereof. Background Art

[0002] Hyperuricemia is a chronic metabolic disease characterized by abnormal purine metabolism, leading to elevated uric acid levels in the blood. It has become the fourth most common metabolic disease after the "three highs" of hypertension, hyperlipidemia, and hyperglycemia, and the second most common metabolic disease after diabetes.

[0003] Currently, clinically used drugs that reduce uric acid synthesis include allopurinol and febuxostat, while those that promote uric acid breakdown or excretion include probenecid and benzbromarone. While these drugs have been shown to be effective in lowering uric acid, they also commonly carry significant side effects, such as gastrointestinal irritation, liver and kidney damage, polyarthritis, and allergies, and are prone to developing drug resistance. Therefore, the development of highly effective, low-toxic natural active ingredients with uric acid-lowering properties has become a research hotspot in the healthcare and medical product fields.

[0004] It has been discovered that many edible and medicinal fungi, such as Cordyceps sinensis, Armillaria igniarius, Cephalosporium sinensis, Cordyceps militaris, etc., can reduce kidney and intestinal inflammation, repair the intestinal barrier, and regulate intestinal flora disorders, thereby achieving the effects of anti-hyperuricemia and relief of gout, and are safer and more reliable. The fungal fruiting bodies and mycelium polysaccharides play a key role.

[0005] Isaria cicadae Miq, also known as golden cicada flower, is an entomogenous fungus belonging to the genus Cordyceps of the family Clavicipitaceae. Paecilomyces cicadae The insect-fungus complex, formed by parasitizing cicada nymphs, is the asexual stage of the traditional Chinese medicine cicada fungus. The pharmacopoeia "Zheng Lei Ben Cao" records that cicada fungus has sedative, tranquilizing, and anticonvulsant properties, making it a valuable medicinal and edible fungus.

[0006] Modern medical research shows that Cordyceps sinensis contains active ingredients such as cordycepic acid, cordyceps polysaccharides, adenosine, various alkaloids, and ergosterol. It has the effects of improving renal function, alleviating chronic renal failure, regulating lipid metabolism, relieving fever and pain, providing sedation and hypnosis, enhancing immunity, resisting radiation, and improving eyesight. It has certain preventive and auxiliary therapeutic effects on chronic metabolic diseases. In recent years, artificial Cordyceps sinensis has been developed based on microbial liquid submerged fermentation technology. Studies have confirmed that artificial cultures of Paecilomyces cicadae are similar to the components of Cordyceps sinensis and can be used as an excellent substitute for Cordyceps sinensis. This fermentation technology has the advantages of a stable raw material source, low cost, and a short fermentation cycle. It is an effective way to find, extract, and prepare more bioactive substances, and it is also a development direction for further promoting the in-depth development and utilization of Cordyceps sinensis fungi.

[0007] Currently, research at home and abroad focuses primarily on the activity of Cordyceps militaris fruiting bodies and mycelium, while further research on its fermentation broth is urgently needed. Wang Yuqi et al. selected Paecilomyces cicadae as the fermentation strain and Astragalus membranaceus as the fermentation substrate, conducting in-depth research using a two-way liquid fermentation technique. The study found that the content of major active substances in Astragalus membranaceus, such as saponins and flavonoids, after fermentation, showed significant changes compared to pre-fermentation levels. This change enhances Astragalus membranaceus's activity in treating hyperuricemia and significantly improves its bioavailability. The present inventors previously discovered that polysaccharides from the fruiting bodies of Cordyceps militaris are effective in preventing renal interstitial fibrosis and alleviating acute chemical liver injury. Recent studies have shown that an ethanol extract of the fermentation broth of Paecilomyces cicadae exhibits significant uric acid-lowering activity and also has a positive effect on improving renal injury. Therefore, the technical problem addressed by the present invention is to further identify the effective sites in the ethanol extract of the fermentation broth of Paecilomyces cicadae that exert uric acid-lowering effects, thereby achieving a more optimal uric acid-lowering effect. Summary of the Invention

[0008] In order to solve the above technical problems, the present invention provides a Cordyceps sinensis fermentation extract for lowering uric acid, and a preparation method and application thereof.

[0009] To achieve the above object, the present invention adopts the following technical solutions:

[0010] In a first aspect, the present invention provides a method for preparing a fermented extract of Cordyceps sinensis for lowering uric acid, the preparation method comprising the following steps:

[0011] Taking the whole fermentation liquid of Cordyceps sinensis fungus and filtering it to obtain the fermentation liquid of Cordyceps sinensis fungus;

[0012] Concentrating the Cordyceps sinensis fungus fermentation liquid to 1 / 3 to 1 / 4 of the original volume to obtain a concentrated liquid;

[0013] The concentrated solution is freeze-dried and extracted with an 80% ethanol solution by volume, and the obtained ethanol extract is concentrated to obtain an ethanol extract of the Cordyceps sinensis fungus fermentation liquid;

[0014] The ethanol extract of the Cordyceps sinensis fermentation liquid is extracted with n-butanol, ethyl acetate and petroleum ether in sequence, and the obtained petroleum ether extract is the Cordyceps sinensis fermentation extract for lowering uric acid.

[0015] In some specific embodiments, the Cordyceps militaris strain is inoculated into a glucose-potato liquid culture medium and cultured in a shaking incubator at 28° C. for 7 days. The obtained mixture of Cordyceps militaris mycelium and fermentation liquid is the Cordyceps militaris fungal fermentation liquid.

[0016] In some specific embodiments, the glucose-potato liquid culture medium is prepared by the following steps:

[0017] Chop 200 g of peeled potatoes, boil in boiling water for 30 min until cooked, filter through four layers of gauze, collect the filtrate, adjust the volume to 1.0 L, add 20 g of glucose, 3.0 g of KH2PO4, 1.5 g of MgSO4·7H2O, and 10 mg of vitamin B1, and sterilize at 121°C for 2 h.

[0018] In some specific embodiments, the freeze-drying condition is -60°C for 4 to 5 days.

[0019] In some specific embodiments, the extraction is performed using an 80% by volume ethanol solution at 70° C. for two times, each time for 90 minutes. The ethanol extracts obtained from the two times are combined and concentrated to 1 / 3 to 1 / 4 of the original volume to obtain an ethanol extract of the Cordyceps sinensis fermentation broth.

[0020] In some specific embodiments, the volume ratio of the freeze-dried concentrate to the ethanol solution is 1:25.

[0021] In some specific embodiments, the volume ratio of n-butanol, ethyl acetate, petroleum ether and the ethanol extract of the Cordyceps sinensis fermentation broth is 1:1, and each organic reagent is extracted three times, each time for 20 minutes.

[0022] In a second aspect, the present invention provides a Cordyceps sinensis fermentation extract prepared by the preparation method.

[0023] In a third aspect, the present invention provides the use of the Cordyceps sinensis fermented extract in treating hyperuricemia.

[0024] Compared with the prior art, the present invention is beneficial in that:

[0025] The present invention firstly discovered that the petroleum ether extract of the ethanol extract of the Cordyceps sinensis fungus fermentation liquid is the effective part that exerts the effect of lowering uric acid, and preliminarily identified and analyzed its components by gas chromatography-mass spectrometry technology.

[0026] The present invention uses potassium oxonate combined with adenine to induce an acute hyperuricemia mouse model, and proves the uric acid-lowering effect of the fermented extract of Cordyceps sinensis through animal experiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The weight changes of mice in the model group and the control group within 10 days after oral administration of potassium oxonate combined with adenine.

[0028] Figure 2 The uric acid levels of mice in the model group and the control group within 10 days after oral administration of potassium oxonate combined with adenine.

[0029] Figure 3This is the effect of the fermented extract of Cordyceps sinensis on the serum uric acid (SUA) level in hyperuricemia model mice after 10 days of administration.

[0030] Figure 4 Figure 2 is the body weight changes of mice in each group after treatment.

[0031] Figure 5 is the serum xanthine oxidase (XOD) level of mice in each group.

[0032] Figure 6 The serum urea nitrogen (BUN) and creatinine (CRE) levels of mice in each group (a) are serum urea nitrogen (BUN) levels, and (b) are creatinine (CRE) levels.

[0033] Figure 7 These are H&E staining images of the kidneys of mice in each group. The blue arrows point to the glomeruli, the black arrows point to the renal tubules, and the scale bar is 1:100. DETAILED DESCRIPTION

[0034] The present invention will be described in detail below with reference to the accompanying drawings and specific examples, but they should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0035] The Cordyceps sinensis used in the following examples is the Cordyceps sinensis, also known as Isaria cicadae Miq. Provided by the manufacturer "Anhui Cordyceps Source Biotechnology Co., Ltd."

[0036] Example 1: Preparation of Cordyceps sinensis fermentation extract.

[0037] 1. Take 8 wt% of Cordyceps sinensis fungus and inoculate it into 150 ml of glucose-potato liquid culture medium.

[0038] The glucose-potato liquid culture medium is prepared by chopping 200 g of peeled potatoes, boiling them in boiling water for 30 minutes until they are soft, filtering the filtrate through four layers of gauze, collecting the filtrate, and adjusting the volume to 1.0 L. 20 g of glucose, 3.0 g of KH2PO4, 1.5 g of MgSO4·7H2O, and 10 mg of vitamin B1 are added. The glucose-potato culture medium is sterilized at 121°C for 2 hours, and then cultured in a shaking incubator at 28°C and 120 rpm for 7 days to obtain a mixture of mycelium and fermentation liquid, namely, the fermentation liquid of the Cordyceps sinensis fungus.

[0039] 2. Filter the whole fermentation liquid of Cordyceps sinensis fungi obtained in step 1 through a 60-mesh sieve to obtain Cordyceps sinensis fungi fermentation liquid.

[0040] 3. The fermentation liquid obtained in step 2 was concentrated by rotary evaporation to 1 / 3 of the original volume.

[0041] 4. The concentrated solution obtained in step 3 was freeze-dried at -60°C for 4 days; extracted twice with 80% ethanol solution at 70°C for 90 minutes each time, with the volume ratio of the freeze-dried concentrate to the ethanol solution being 1:25. The two ethanol extracts were combined and concentrated under vacuum at 55°C to 1 / 3 of the original volume to obtain the ethanol extract of the Cordyceps sinensis fungus fermentation liquid.

[0042] 5. Extract the ethanol extract of the Cordyceps militaris fermentation broth obtained in step 4 with n-butanol, ethyl acetate, and petroleum ether, respectively, to obtain an n-butanol-extracted fraction, an ethyl acetate-extracted fraction, a petroleum ether-extracted fraction, and a remaining fraction. The volume ratio of petroleum ether, ethyl acetate, and n-butanol to the ethanol extract is 1:1, and extraction is performed three times with each organic reagent, each extraction lasting 20 minutes. The petroleum ether-extracted fraction is the Cordyceps militaris fermentation extract.

[0043] Example 2: Determination of uric acid-lowering activity of the fermented extract of Cordyceps sinensis.

[0044] The fermented extract of Cordyceps sinensis prepared in Example 1 was used as raw material, and the uric acid-lowering efficacy of the fermented extract of Cordyceps sinensis was experimentally evaluated using a hyperuricemia mouse model.

[0045] 1. Preparation of hyperuricemia mouse model.

[0046] Forty-eight male Kunming mice, aged 5 to 6 weeks and weighing 20 ± 2 g, were randomly divided into eight groups, with six mice in each group.

[0047] Blank control group; referred to as group A.

[0048] Model group: abbreviated as group B.

[0049] Positive drug allopurine group: abbreviated as group C.

[0050] The ethanol extract of Cordyceps sinensis fermentation liquid group: abbreviated as Group D.

[0051] n-Butanol extraction part group: abbreviated as E group.

[0052] Ethyl acetate extraction part group: abbreviated as F group.

[0053] The petroleum ether extraction part group, i.e. the Cordyceps sinensis fermentation extract group: abbreviated as Group G.

[0054] The remaining parts group: abbreviated as H group.

[0055] The blank control group received an equal volume of normal saline by gavage, while the remaining groups received 500 mg / kg of potassium oxonate and 200 mg / kg of adenine suspension for 10 consecutive days to establish an acute hyperuricemia mouse model. Following model establishment, the model group continued to receive the same dose of potassium oxonate and adenine suspension daily. The allopurine group received 10 mg / kg of allopurine, while the remaining groups received 200 mg / kg of the ethanol extract, n-butanol extract, ethyl acetate extract, petroleum ether extract, and the remaining extracts of the Cordyceps sinensis fungus fermentation broth, respectively, for 10 days. Daily body weight was measured in each group, and serum samples were analyzed for uric acid levels according to the uric acid test kit instructions during the modeling phase to determine the optimal dosing and blood collection times to ensure successful model establishment.

[0056] At six time points, 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, and 8 hours after modeling, six mice were randomly selected from each of the model and normal groups for blood collection from the fundus venous plexus. Blood was placed in clean EP tubes, incubated at 37°C for 30 minutes, and then centrifuged at 5000 rpm for 10 minutes at 4°C to collect serum. Serum uric acid levels in each group were measured using the kit, and the data were analyzed for significance using GraphPad software. The time point with the highest uric acid level was selected for subsequent studies on the uric acid-lowering effects of different components of Cordyceps sinensis fermented extracts, providing the optimal time point for drug administration and blood collection.

[0057] 2. Experimental results.

[0058] (1) Oral administration of potassium oxonate combined with adenine can significantly increase the SUA level in mice and cause damage to the body. Compared with the control group, 10 days of oral administration of potassium oxonate combined with adenine can significantly reduce the weight of mice, P < 0.01. The results show that high uric acid can cause emaciation in mice. In addition, high uric acid can significantly increase the SUA level in mice, P < 0.01, indicating that the established hyperuricemia mouse model is successful. The results are as follows Figure 1 and Figure 2 shown.

[0059] (2) The petroleum ether extract of the ethanol extract of the Cordyceps sinensis fermentation broth can reduce the SUA level in mice with hyperuricemia. The uric acid level in serum can be used as one of the most intuitive evaluation indicators of hyperuricemia. After 10 days of intervention treatment with the extracts of the Cordyceps sinensis fermentation broth, the SUA level of mice with hyperuricemia was significantly reduced compared with the model group, P < 0.01, indicating that the petroleum ether extract of the ethanol extract of the Cordyceps sinensis fermentation broth can effectively alleviate hyperuricemia. Figure 3 shown.

[0060] (3) Fermented extracts of Cordyceps sinensis can reduce the damage to the body caused by hyperuricemia (HUA). After 10 days of administration, the body weight of HUA mice still showed a significant downward trend compared with the control group (P < 0.01). Compared with the model group, the body weight levels of mice in different groups showed varying degrees of upward trends (P < 0.01). Figure 4 shown.

[0061] (4) The petroleum ether extract of the ethanol extract of the Cordyceps sinensis fungus fermentation liquid inhibited the activity of xanthine oxidase XOD in HUA mice. XOD is a key enzyme in uric acid synthesis and plays a key role in the purine nucleoside metabolic pathway. Compared with the normal group of mice, the serum XOD in the model group was significantly more active, indicating that potassium oxonate combined with adenine modeling can significantly increase the XOD activity in mice, thereby promoting uric acid synthesis, P < 0.01. Compared with the model group, the serum XOD activity of mice in the positive drug allopurine group and the petroleum ether extract group was reduced, P < 0.05. This shows that the components in the petroleum ether extract can inhibit the activity of XOD enzyme, reduce the synthesis pathway of uric acid, and thus exert the effect of lowering uric acid. The results are as follows Figure 5 shown.

[0062] (5) The petroleum ether extract of the ethanol extract of the Cordyceps sinensis fungus fermentation liquid alleviates the renal damage caused by HUA. Urea nitrogen BUN and creatinine Cre are commonly used indicators for testing renal function. Compared with the normal group, the serum urea nitrogen and creatinine levels of the mice in the model group were significantly increased, P < 0.01. This shows that the high level of uric acid in hyperuricemia mice will lead to impaired renal function, thereby hindering the normal excretion of uric acid. Compared with the model group, the serum urea nitrogen and creatinine levels of each drug group decreased after oral administration, P < 0.01, especially the petroleum ether extract group had the most obvious decrease, indicating that it can alleviate the renal damage caused by hyperuricemia. The results are as follows Figure 6 and Figure 7 shown.

[0063] (6) The petroleum ether extract of the ethanol extract of the Cordyceps sinensis fungus fermentation liquid improved the pathological damage caused by HUA. Compared with the blank control group, the kidney tissue of the model group showed obvious edema, glomerular atrophy and tubular dilatation, inflammatory cell infiltration, and abnormalities of the distal and proximal convoluted tubules. However, these pathological changes were improved after drug treatment.

[0064] Example 3: Preliminary identification of the uric acid-lowering active ingredients of the fermented extract of Cordyceps sinensis.

[0065] 1. In this example, the petroleum ether extraction portion of the Cordyceps sinensis fermentation broth was subjected to GC-MS analysis. The specific inspection conditions and process were as follows:

[0066] (1) Ionization system: electron ionization system, ionization energy 70eV.

[0067] (2) Carrier gas: Helium with a purity of 99.99%, a constant flow rate of 1 mL / min.

[0068] (3) Injection volume: 1 μL. The sample is an extract dispersed in petroleum ether. Dissolve an appropriate amount of the extract in 2 ml of methanol (chromatographic grade ≥ 99.9%), and filter with a 0.22 μm nylon filter membrane after ultrasonic treatment for 15 minutes.

[0069] (4) Diversion ratio: 10:1.

[0070] (5) Column oven program: initial temperature 50°C, constant temperature for 3 min, then increase to 280°C at 20°C / min, then increase to 350°C at 10°C / min, and maintain at 280°C for 12 min.

[0071] (6) Analysis time: Total GC run time 15 min.

[0072] (7) Calculation of results: Using the NIST Mass Spectra database software, we initially searched for possible target substances based on the peaks of different mass-to-charge ratios (m / z) generated by fragmentation. At the same time, we predicted their chemical structures based on their fragmented mass spectra, and calculated the relative percentages of each component by comparing the average peak area of ​​each component with the total area.

[0073] 2. Experimental results.

[0074] Fifteen compounds were detected in the petroleum ether extract of the Cordyceps sinensis fermentation broth. Based on the retention times and peak areas of the bioactive compounds, the main compounds included esters, ketones, glycosides, terpenes, acids, furans, phenols, aldehydes, furfurals, alcohols, and aromatic hydrocarbons. The components with relative contents above 2% were preliminarily screened out by searching the NIST mass spectrometry database, including 35.78% of 5-hydroxymethylfurfural, 11.06% of α-D-pyranoglucopyranoside, O-α-D-pyranoglucopyranosyl-(1→3)-α-D-fructofuranosyl, 10.64% of 2-methylhexadecan-1-ol, 4.60% of n-hexadecanoic acid, 4.35% of 4,5-diethyl-2,3-dimethyl-2,3-dihydrofuran, 3.20% of ethyl isoallocholate, 3.12% of 2,4-di-tert-butylphenol, and 2.73% of 3,5-dihydroxy-6-methyl-2,3-dihydro-4H-pyran-4-one.

[0075] Although the preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

Claims

1. Use of a fermented extract of Cordyceps sinensis for lowering uric acid in the preparation of a medicament for treating hyperuricemia, characterized in that: The preparation method of the Cordyceps sinensis fermentation extract comprises the following steps: Taking the whole fermentation liquid of Cordyceps sinensis fungus and filtering it to obtain the fermentation liquid of Cordyceps sinensis fungus; Concentrating the Cordyceps sinensis fungus fermentation liquid to obtain a concentrated liquid; The concentrated solution was freeze-dried and then extracted with an ethanol solution. The extraction was performed twice with an 80% by volume ethanol solution at 70°C for 90 minutes each time. The ethanol extracts obtained from the two extractions were combined and concentrated to 1 / 3 to 1 / 4 of the original volume to obtain an ethanol extract of the Cordyceps sinensis fermentation liquid. The ethanol extract of the Cordyceps sinensis fermentation broth is extracted with n-butanol, ethyl acetate, and petroleum ether in sequence, and the obtained petroleum ether extract is the Cordyceps sinensis fermentation extract for lowering uric acid; the volume ratio of n-butanol, ethyl acetate, petroleum ether to the ethanol extract of the Cordyceps sinensis fermentation broth is 1:1, and each organic reagent is extracted 3 times, each time for 20 minutes.

2. The use according to claim 1, characterized in that The Cordyceps sinensis strains are inoculated into a glucose-potato liquid culture medium and cultured in a shaking incubator at 28° C. for 7 days. The obtained mixture of Cordyceps sinensis mycelium and fermentation liquid is the Cordyceps sinensis fungal fermentation liquid.

3. The use according to claim 2, characterized in that The glucose-potato liquid culture medium is prepared by the following steps: Chop 200 g of peeled potatoes, boil in boiling water for 30 min until cooked, filter through four layers of gauze, collect the filtrate, adjust the volume to 1.0 L, add 20 g of glucose, 3.0 g of KH2PO4, 1.5 g of MgSO4·7H2O, and 10 mg of vitamin B1, and sterilize at 121°C for 2 h.

4. The use according to claim 1, characterized in that The freeze-drying conditions are -60℃ for 4d~5d.

5. The use according to claim 1, characterized in that The volume ratio of the freeze-dried concentrate to the ethanol solution is 1:25.

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