Cordyceps sobolifera fermentation extract for reducing uric acid as well as preparation method and application of cordyceps sobolifera fermentation extract

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a cordyceps sobolifera fermentation extract for reducing uric acid as well as a preparation method and application of the cordyceps sobolifera fermentation extract. The method comprises the following steps: concentrating cordyceps sobolifera fungus fermentation liquor, then leaching with ethanol, concentrating leaching liquor, then sequentially extracting with n-butyl alcohol, ethyl acetate and petroleum ether, and concentrating extracts to respectively obtain an n-butyl alcohol extraction part, an ethyl acetate extraction part, a petroleum ether extraction part and a residual part, the obtained petroleum ether extract is the cordyceps sobolifera fermentation extract for reducing uric acid. The cordyceps sobolifera fermentation extract disclosed by the invention has the effects of remarkably relieving kidney injury of a mouse with hyperuricemia and reducing serum uric acid of the mouse with hyperuricemia. As a novel natural uric acid-reducing active substance, the cordyceps sobolifera fermentation extract disclosed by the invention can be applied to the fields of health foods and medicines, and has good development and application values.

Description

Technical Field

[0001] The 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, which can lead to elevated uric acid levels in the blood. Hyperuricemia has become the "fourth high" after the "three highs" - high blood pressure, high blood lipids, and high blood sugar, and is second only to diabetes as the second largest metabolic disease.

[0003] At present, the drugs that reduce uric acid synthesis in clinical practice include allopurinol and febuxostat, and the drugs that promote the decomposition or excretion of uric acid include probenecid and benzbromarone. Although these drugs have been proven to have good uric acid-lowering effects, they also generally have large toxic side effects, such as irritating the gastrointestinal tract, causing liver and kidney damage, multiple arthritis and allergies, and are prone to drug resistance. Therefore, the research and development of highly efficient, low-toxic natural active ingredients with uric acid-lowering effects has become a research hotspot in the field of big health and medical care products.

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

[0005] Isaria cicadae Miq, also known as golden cicada flower, is an insect-borne fungus belonging to the genus Cordyceps of the family Ergotaceae. Paecilomyces cicadae The insect-fungus complex formed by parasitizing on cicada nymphs is the asexual stage of the traditional Chinese medicine cicada fungus. The pharmacopoeia "Zhenglei Bencao" records that cicada fungus has sedative, tranquilizing, anticonvulsant and other effects, and is a precious 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, which can improve renal function, relieve chronic renal failure, regulate lipid metabolism, relieve fever and relieve pain, calm and hypnotize, enhance the body's immunity, resist radiation and improve eyesight, and have certain preventive and auxiliary therapeutic effects on chronic metabolic diseases. In recent years, artificial Cordyceps sinensis has been developed based on microbial liquid deep fermentation technology. Studies have confirmed that the artificial culture of Paecilomyces cicadae is 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 stable raw material sources, low cost, and short fermentation cycle. It is an effective way to find, extract and prepare more bioactive substances, and it is also a development direction to further promote the deep development and utilization of Cordyceps sinensis fungi.

[0007] At present, domestic and foreign research mainly focuses on the activities of Cordyceps cicadae fruiting bodies and mycelia, and the research on its fermentation broth needs to be further deepened. Wang Yuqi et al. selected Paecilomyces cicadae as the fermentation strain and Astragalus membranaceus as the fermentation substrate, and carried out in-depth research using the two-way liquid fermentation technology. Through research, it was found that in the fermented Astragalus membranaceus, the contents of the main active substances such as saponins and flavonoids such as astragaloside increased significantly compared with those before fermentation. This change enhanced the activity of Astragalus membranaceus in intervening hyperuricemia and significantly improved the bioavailability of Astragalus membranaceus. The inventor of the present invention has previously found that the polysaccharide of Cordyceps cicadae fruiting body has a significant effect in preventing renal interstitial fibrosis and relieving acute chemical liver injury. Recent research has found that the ethanol extract of the fermentation broth of Paecilomyces cicadae has significant uric acid-lowering activity and also has a positive effect on improving kidney injury. Therefore, further determining the effective part of the ethanol extract of the fermentation broth of Paecilomyces cicadae that plays a uric acid-lowering role, so as to play a better uric acid-lowering role is the technical problem to be solved by the present invention. Summary of the Invention

[0008] To solve the above technical problems, the present invention provides a Cordyceps cicadae fermentation extract for lowering uric acid, its preparation method and application.

[0009] To achieve the above object, the present invention adopts the following technical solutions: In the first aspect, the present invention provides a preparation method of a Cordyceps cicadae fermentation extract for lowering uric acid, and the preparation method includes the following steps: Filter the whole fermentation broth of Cordyceps cicadae fungus to obtain the fermentation broth of Cordyceps cicadae fungus; Concentrate the fermentation broth of Cordyceps cicadae fungus to 1 / 3 - 1 / 4 of the original volume to obtain a concentrated solution; Extract the concentrated solution with an ethanol solution with a volume fraction of 80% after freeze-drying, and concentrate the obtained ethanol extract to obtain an ethanol extract of the fermentation broth of Cordyceps cicadae fungus; Extract the ethanol extract of the fermentation broth of Cordyceps cicadae fungus with n-butanol, ethyl acetate, and petroleum ether in sequence, and the obtained petroleum ether extract is the Cordyceps cicadae fermentation extract for lowering uric acid.

[0010] In some specific embodiments, inoculate the Cordyceps cicadae fungus strain into a glucose-potato liquid medium and culture it on a shaker at 28°C for 7 days, and the obtained mixture of Cordyceps cicadae mycelia and fermentation broth is the whole fermentation broth of Cordyceps cicadae fungus.

[0011] In some specific embodiments, the glucose-potato liquid medium is prepared by the following steps: Chop 200 g of peeled potatoes, boil them in boiling water for 30 min until they are mushy, filter them through four layers of gauze, collect the filtrate, make up the volume to 1.0 L, add 20 g of glucose, 3.0 g of KH2PO4, 1.5 g of MgSO4·7H2O, 10 mg of vitamin B1, and sterilize at 121 °C for 2 h.

[0012] In some specific embodiments, the conditions for freeze-drying are -60 °C for 4 d to 5 d.

[0013] In some specific embodiments, the extraction is carried out with an ethanol solution with a volume fraction of 80% at 70 °C for 2 times, 90 min each time. Combine the ethanol extracts obtained twice and concentrate them to 1 / 3 to 1 / 4 of the original volume to obtain the ethanol extract of the Cordyceps cicadae fungal fermentation broth.

[0014] In some specific embodiments, the volume ratio of the concentrated solution after freeze-drying to the ethanol solution is 1:25.

[0015] In some specific embodiments, the volume ratios of n-butanol, ethyl acetate, petroleum ether to the ethanol extract of the Cordyceps cicadae fungal fermentation broth are all 1:1. Each organic reagent is extracted 3 times, 20 min each time.

[0016] In a second aspect, the present invention provides the Cordyceps cicadae fermentation extract prepared by the preparation method described above.

[0017] In a third aspect, the present invention provides the application of the Cordyceps cicadae fermentation extract in the treatment of hyperuricemia.

[0018] Compared with the prior art, the advantages of the present invention are as follows: The present invention first discovers that the petroleum ether extraction phase of the ethanol extract of the Cordyceps cicadae fungal fermentation broth is the effective part for exerting the uric acid-lowering effect, and the components are preliminarily identified and analyzed by gas chromatography-mass spectrometry.

[0019] The present invention uses a potassium oxonate combined with adenine-induced acute hyperuricemia mouse model to prove the uric acid-lowering effect of the Cordyceps cicadae fermentation extract through animal experiments. Description of the Drawings

[0020] Figure 1 It is the body weight change of the model group and the control group mice within 10 d after intragastric administration of potassium oxonate combined with adenine.

[0021] Figure 2 It is the uric acid level of the model group and the control group mice within 10 d after intragastric administration of potassium oxonate combined with adenine.

[0022] Figure 3 It is the effect of the Cordyceps cicadae fermentation extract on the serum uric acid (SUA) level of hyperuricemia model mice after 10 d of administration.

[0023] Figure 4 It is the change in the body weight of mice in each group after drug administration treatment.

[0024] Figure 5 It is the level of xanthine oxidase (XOD) in the serum of mice in each group.

[0025] Figure 6 It is the levels of blood urea nitrogen (BUN) and creatinine (CRE) in the serum of mice in each group. a is the level of blood urea nitrogen (BUN), and b is the level of creatinine (CRE).

[0026] Figure 7 It is the H&E staining diagram of the kidneys of mice in each group. The glomeruli are indicated by blue arrows, the renal tubules are indicated by black arrows, and the scale is 1:100. Detailed implementation manners

[0027] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments, but it should not be construed as a limitation of the present invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well-known to those skilled in the art. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0028] The cicada flower cordyceps used in the following embodiments is Isaria cicadae, with the Latin name Isaria cicadae Miq. provided by the manufacturer "Anhui Cordyceps Source Biotechnology Co., Ltd.".

[0029] Example 1: Preparation of fermented extract of cicada flower cordyceps.

[0030] 1. Inoculate 8 wt% of Isaria cicadae strains into 150 ml of glucose-potato liquid medium.

[0031] The preparation of the glucose-potato liquid medium is as follows: Cut 200 g of peeled potatoes into pieces, boil them in boiling water for 30 min until they are cooked, filter them with four layers of gauze, collect the filtrate, and make up 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. Sterilize at 121 °C for 2 h to obtain the glucose-potato medium, and culture it on a shaker at 28 °C and 120 rmp for 7 d to obtain a mixture of Isaria cicadae mycelium and fermentation broth, which is the whole liquid of Isaria cicadae fungal fermentation.

[0032] 2. Filter the whole liquid of Isaria cicadae fungal fermentation obtained in step 1 through a 60-mesh sieve to obtain the Isaria cicadae fungal fermentation broth.

[0033] 3. Concentrate the fermentation broth obtained in step 2 by rotary evaporation to 1 / 3 of the original volume.

[0034] 4. Vacuum freeze-dry the concentrated solution obtained in step 3 at -60°C for 4 days; extract it twice with an ethanol solution with a volume fraction of 80% at 70°C for 90 minutes each time. The volume ratio of the freeze-dried concentrated solution to the ethanol solution is 1:25. Combine the two ethanol extracts and vacuum concentrate them to 1 / 3 of the original volume at 55°C to obtain the ethanol extract of the Cordyceps cicadae fungal fermentation broth.

[0035] 5. Extract the ethanol extract of the Cordyceps cicadae fungal fermentation broth obtained in step 4 successively with n-butanol, ethyl acetate, and petroleum ether to obtain the n-butanol extraction part, ethyl acetate extraction part, petroleum ether extraction part, and the remaining part. Among them, the volume ratio of petroleum ether, ethyl acetate, and n-butanol to the ethanol extract is 1:1. Each organic reagent is extracted 3 times for 20 minutes each time. The petroleum ether extraction part is the Cordyceps cicadae fermentation extract.

[0036] Example 2: Determination of the uric acid-lowering activity of the Cordyceps cicadae fermentation extract.

[0037] Using the Cordyceps cicadae fermentation extract prepared in Example 1 as the raw material, the uric acid-lowering effect of the Cordyceps cicadae fermentation extract was experimentally evaluated using a hyperuricemia mouse model.

[0038] 1. Preparation of a hyperuricemia mouse model.

[0039] Select 48 male Kunming white rats, 5 - 6 weeks old, with a body weight of 20 ± 2 g. Randomly divide them into 8 groups, with 6 rats in each group. The groups are as follows: Blank control group; briefly recorded as group A.

[0040] Model group: briefly recorded as group B.

[0041] Positive drug allopurinol group: briefly recorded as group C.

[0042] Ethanol extract group of Cordyceps cicadae fungal fermentation broth: briefly recorded as group D.

[0043] n-butanol extraction part group: briefly recorded as group E.

[0044] Ethyl acetate extraction part group: briefly recorded as group F.

[0045] Petroleum ether extraction part group, that is, the Cordyceps cicadae fermentation extract group: briefly recorded as group G.

[0046] Remaining part group: briefly recorded as group H.

[0047] The blank control group was intragastrically administered with an equal volume of normal saline, and the remaining groups were given a dose of 500 mg / kg potassium oxonate and 200 mg / kg adenine suspension. They were intragastrically administered continuously for 10 days to establish an acute hyperuricemia mouse model. After the model was established, the model group was still given the same dose of potassium oxonate and adenine suspension daily, the positive drug allopurinol group was given 10 mg / kg allopurinol, and the remaining groups were respectively given 200 mg / kg ethanol extract of Cordyceps cicadae fungal fermentation broth, n-butanol extraction part, ethyl acetate extraction part, petroleum ether extraction part and the remaining part for a 10-day intervention treatment. The body weight of each group of mice was measured daily, and during the modeling stage, the uric acid level in the serum samples was analyzed according to the instructions of the uric acid detection kit to determine the optimal drug administration and blood sampling time points to ensure the successful establishment of the model.

[0048] At six time points of 1 h, 2 h, 3 h, 4 h, 6 h, and 8 h after modeling, 6 mice were randomly selected from the model group and the normal group at each time point for blood collection from the fundus venous plexus, placed in clean EP tubes, incubated at 37 °C for 30 min, and then centrifuged at 5000 rpm for 10 min at 4 °C to obtain serum. The serum uric acid levels of each group were detected according to the kit, and the obtained data were analyzed for significance using GraphPad software. The time point with the highest uric acid was selected for the study on the effect of reducing uric acid of different components of Cordyceps cicadae fermentation extract, providing the optimal drug administration and blood sampling time points.

[0049] 2. Experimental results.

[0050] (1) Intragastric administration of potassium oxonate combined with adenine can cause a significant increase in the SUA level of mice and cause damage to the body. Compared with the control group, intragastric administration of potassium oxonate combined with adenine for 10 days can significantly reduce the body weight of mice, P<0.01, indicating that hyperuricemia can cause weight loss symptoms in mice. In addition, hyperuricemia can cause a significant increase in the SUA level of mice, P<0.01, indicating that the established hyperuricemia mouse model is successful, and the results are as Figure 1 and Figure 2 shown.

[0051] (2) The petroleum ether extraction part of the ethanol extract of Cordyceps cicadae fungal fermentation broth can reduce the SUA level of hyperuricemia mice. The uric acid level in the serum can be used as one of the most intuitive evaluation indicators for hyperuricemia. After 10 days of intervention treatment with each extract of Cordyceps cicadae fungal fermentation broth, compared with the model group, the SUA level of hyperuricemia mice was significantly reduced, P<0.01, indicating that the petroleum ether extraction part of the ethanol extract of Cordyceps cicadae fungal fermentation broth can effectively relieve hyperuricemia, and the results are Figure 3 shown.

[0052] (3) The fermented extract of Cordyceps cicadae can reduce the body damage caused by hyperuricemia (HUA). After 10 days of administration, compared with the control group, the body weight of HUA mice still showed an obvious downward trend, P<0.01. Compared with the model group, the body weight levels of mice in different groups showed an upward trend to varying degrees, P<0.01, and the results were as Figure 4 shown.

[0053] (4) The petroleum ether extraction part of the ethanol extract of the fermented liquid of Cordyceps cicadae fungi inhibits 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 of the model group was significantly more active, indicating that after modeling with potassium oxonate combined with adenine, the activity of XOD in mice can be significantly increased, thereby promoting uric acid synthesis, P<0.01. Compared with the model group, the serum XOD activity of the mice in the positive drug allopurinol group and the petroleum ether extraction part group decreased, P<0.05. It shows that the components in the petroleum ether extraction part can inhibit the activity of XOD enzyme, reduce the uric acid synthesis pathway, and thus play the effect of reducing uric acid. The results were as Figure 5 shown.

[0054] (5) The petroleum ether extraction part of the ethanol extract of the fermented liquid of Cordyceps cicadae fungi reduces the kidney damage caused by HUA. Blood urea nitrogen (BUN) and creatinine (Cre) are commonly used indicators for testing kidney function. Compared with the normal group, the serum BUN and creatinine levels of the model group mice increased significantly, P<0.01. It shows that the high uric acid level in hyperuricemia mice can lead to kidney function damage, thus hindering the normal excretion of uric acid. Compared with the model group, after intragastric administration treatment in each administration group, the serum BUN and creatinine levels decreased, P<0.01, especially the petroleum ether extraction part group decreased the most significantly, indicating that it can reduce the renal function damage caused by hyperuricemia. The results were as Figure 6 and Figure 7 shown.

[0055] (6) The petroleum ether extraction part of the ethanol extract of the fermented liquid of Cordyceps cicadae fungi improves the pathological damage caused by HUA. Compared with the blank control group, obvious edema, glomerular atrophy and renal tubular dilation appeared in the kidney tissue of the model group, infiltration of inflammatory cells was observed, and abnormalities in the distal and proximal tubules were found. However, these pathological changes were all improved after administration treatment.

[0056] Example 3: Preliminary identification of the uric acid-lowering active components of the fermented extract of Cordyceps cicadae.

[0057] 1. In this example, the petroleum ether extraction part of the fermented liquid of Cordyceps cicadae fungi was analyzed by GC-MS. The specific inspection conditions and procedures are as follows: (1) Ionization system: Electron ionization system, ionization energy 70 eV.

[0058] (2) Carrier gas: Helium with a purity of 99.99%, constant flow rate of 1 mL / min.

[0059] (3) Injection volume: 1 μL. The sample is the extract dispersed in petroleum ether. An appropriate amount of the extract is dissolved in 2 ml of methanol, chromatographic grade ≥99.9%. After ultrasonic treatment for 15 min, it is filtered through a 0.22 μm nylon filter membrane.

[0060] (4) Split ratio: 10:1.

[0061] (5) Column oven program: Initial temperature 50°C, hold for 3 min, then increase to 280°C at a rate of 20°C / min, and then increase to 350°C at a rate of 10°C / min, hold at 280°C for 12 min.

[0062] (6) Analysis time: Total GC running time 15 min.

[0063] (7) Result calculation: Using the NIST Mass Spectra database software, based on the peaks of different mass-to-charge ratios m / z generated by its fragmentation, possible target substances are initially retrieved. At the same time, their chemical structures are predicted based on the fragmented mass spectra. By comparing the average peak area of each component with the total area, the relative percentage of each component is calculated.

[0064] 2. Experimental results.

[0065] Fifteen compounds were detected in the petroleum ether extract of the fermentation broth of Cordyceps cicadae fungus. According to the retention time and peak area of each bioactive compound, the main compounds include esters, ketones, glycosides, terpenes, acids, furans, phenols, aldehydes, furfural, alcohols, and aromatic hydrocarbons. Components with a relative content of more than 2% were initially screened through retrieval in the NIST mass spectrometry database, including 5-hydroxymethylfurfural at 35.78%, α-D-glucopyranoside, O-α-D-glucopyranosyl-(1→3)-α-D-fructofuranoside at 11.06%, 2-methylhexadecane-1-ol at 10.64%, n-hexadecanoic acid at 4.60%, 4,5-diethyl-2,3-dimethyl-2,3-dihydrofuran at 4.35%, ethyl isocholanate at 3.20%, 2,4-di-tert-butylphenol at 3.12%, and 3,5-dihydroxy-6-methyl-2,3-dihydro-4H-pyran-4-one at 2.73%.

[0066] Although the preferred embodiments of the present invention have been described, those skilled in the art can make additional changes and modifications once they learn the basic creative concept. Therefore, the appended claims are intended to be construed to include the preferred embodiments and all changes and modifications falling within the scope of the present invention.

Claims

1. Preparation method of cicada flower cordyceps fermentation extract for reducing uric acid, characterized in that, The preparation method comprises the following steps: Filter the whole liquid of Cordyceps cicadae fungus fermentation to obtain the Cordyceps cicadae fungus fermentation broth; Concentrate the Cordyceps cicadae fungus fermentation broth to obtain a concentrated solution; Freeze-dry the concentrated solution and then extract it with an ethanol solution. Concentrate the obtained ethanol extract to obtain the ethanol extract of Cordyceps cicadae fungus fermentation broth; Extract the ethanol extract of Cordyceps cicadae fungus fermentation broth successively with n-butanol, ethyl acetate, and petroleum ether. The obtained petroleum ether extract is the Cordyceps cicadae fermentation extract for reducing uric acid; 2. The preparation method of a cicada flower cordyceps fermented extract for reducing uric acid according to claim 1, wherein, Inoculate the Cordyceps cicadae strain into a glucose-potato liquid medium and culture it on a shaker at 28°C for 7 days. The obtained mixture of Cordyceps cicadae mycelium and fermentation broth is the whole liquid of Cordyceps cicadae fungus fermentation; 3. The preparation method of the cicada flower cordyceps fermentation extract for reducing uric acid according to claim 2, characterized in that, The glucose-potato liquid medium is prepared by the following steps: Chop 200 g of peeled potatoes, boil them in boiling water for 30 min until they are mashed, filter them through four layers of gauze, collect the filtrate, make up the volume to 1.0 L, add 20 g of glucose, 3.0 g of KH2PO4, 1.5 g of MgSO4·7H2O, 10 mg of vitamin B1, and sterilize at 121°C for 2 h; 4. The preparation method of a cicada flower cordyceps fermentation extract for reducing uric acid according to claim 1, wherein, The conditions for freeze-drying are -60°C for 4 to 5 days; 5. The preparation method of a cicada flower cordyceps fermentation extract for reducing uric acid according to claim 1, wherein, The extraction is carried out with an ethanol solution with a volume fraction of 80% at 70°C for 2 times, 90 min each time. Combine the ethanol extracts obtained twice and concentrate them to 1 / 3 to 1 / 4 of the original volume to obtain the ethanol extract of Cordyceps cicadae fungus fermentation broth; 6. The preparation method of a cicada flower cordyceps fermentation extract for reducing uric acid according to claim 1, characterized in that, The volume ratio of the freeze-dried concentrated solution to the ethanol solution is 1:25; 7. The preparation method of a cicada flower cordyceps fermentation extract for reducing uric acid according to claim 1, characterized in that, The volume ratios of n-butanol, ethyl acetate, and petroleum ether to the ethanol extract of Cordyceps cicadae fungus fermentation broth are all 1:

1. Each organic reagent is extracted 3 times, 20 min each time; 8. The Cordyceps cicadae fermentation extract prepared by the preparation method according to any one of claims 1 to 7; 9. Use of the Cordyceps cicadae fermentation extract according to claim 8 in the preparation of a drug for treating hyperuricemia.

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