A strain of Bifidobacterium bifidum for sobering up, protecting liver and reducing uric acid, and its postbiotics, composition and application

Abstract

The present invention relates to the technical field of microbial application, and specifically to a strain of Bifidobacterium bifidum for sobering up, protecting the liver, and reducing uric acid, and its postbiotics, compositions, and applications. The Bifidobacterium bifidum is Bifidobacterium bifidum ( Bifidobacterium bifidum )WZA‑BB02, Bifidobacterium bifidum WZA‑BB02 was deposited in the China Center for Type Culture Collection on July 1, 2024, with the deposit number CCTCC NO: M 20241442. The postbiotics made from Bifidobacterium bifidum WZA‑BB02 have a high inhibition rate of xanthine oxidase activity in vitro. The rat experiment verified that Bifidobacterium bifidum WZA‑BB02 postbiotics can significantly reduce the serum uric acid level of rats with hyperuricemia, and can effectively reduce alcohol-induced liver damage and inhibit the promotion of alcohol on the increase of uric acid levels.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial application, and in particular to a strain of Bifidobacterium bifidum for sobering up, protecting the liver and reducing uric acid, and a postbiotic, composition and application thereof. Background Art

[0002] Uric acid is produced in the liver, muscles and adipose tissue. It is the end product of purine metabolism and the main antioxidant in human plasma. It can protect cells or neurons from oxidative damage. Under normal circumstances, a dynamic balance is maintained between intestinal absorption of purine, production of uric acid and excretion of uric acid through the kidneys to maintain the body's normal physiological state. Hyperuricemia refers to a disease in which purine metabolism in the human body is abnormal, uric acid production is increased or excretion is reduced, resulting in elevated blood uric acid levels.

[0003] Xanthine oxidoreductase (XOR) is a key enzyme in the production of uric acid. Xanthine oxidoreductase (XOR) exists in two forms, namely xanthine oxidase (XOD) and xanthine dehydrogenase (XDH). Both have the ability to metabolize purine into uric acid, but XOD has a higher activity in reducing oxygen molecules and plays a key role in purine metabolism. XOD is expressed in multiple tissues of the human body, mainly distributed in the liver and small intestine. The enhancement of its activity is a major cause of hyperuricemia.

[0004] Studies have shown that there is a certain correlation between blood uric acid levels and liver damage. The liver is the main site of uric acid production. This correlation may be due to liver dysfunction leading to abnormal enhancement of XOD activity, which in turn causes uric acid metabolism disorders. Hyperuricemia will induce the continuous production of pro-inflammatory and pro-oxidative mediators, further aggravating the process of liver damage, forming a vicious cycle, and further deteriorating the metabolic state. Therefore, the treatment of hyperuricemia requires not only lowering uric acid levels, but also paying attention to preventing the occurrence of liver damage.

[0005] Generally, in order to treat hyperuricemia, chemical drugs are often used to suppress the increase of uric acid levels. However, long-term use of chemical drugs will increase the metabolic burden on the liver and cause liver damage. In addition, if patients with hyperuricemia consume alcohol, the metabolism of alcohol will promote purine absorption, increase uric acid production and affect uric acid excretion; alcohol may also cause a certain degree of liver damage, further aggravating hyperuricemia. Therefore, reducing the use of chemical drugs and reducing the harm of alcohol are important ways to prevent and relieve hyperuricemia. Summary of the invention

[0006] In order to solve the technical problem that long-term use of chemical drugs for treating hyperuricemia or alcohol intake leads to liver damage, which further aggravates hyperuricemia, the present invention provides a strain of Bifidobacterium bifidum for sobering up, protecting the liver and lowering uric acid, and its postbiotics, composition and application.

[0007] In the first aspect, the present invention provides a strain of Bifidobacterium bifidum for sobering up, protecting the liver and reducing uric acid. The Bifidobacterium bifidum is Bifidobacterium bifidum ( Bifidobacterium bifidum ) WZA-BB02, Bifidobacterium bifidum WZA-BB02 was deposited in the China Center for Type Culture Collection on July 1, 2024, with the deposit number CCTCC NO: M 20241442, and the deposit address is Wuhan University, Wuhan City, Hubei Province, China.

[0008] In a second aspect, the present invention provides a Bifidobacterium bifidum postbiotic, which is prepared by inactivating Bifidobacterium bifidum WZA-BB02 after fermentation.

[0009] Furthermore, the specific preparation method comprises the following steps:

[0010] Bifidobacterium bifidum WZA-BB02 was inoculated into bifidobacterium culture medium and fermented at 37°C for 48 h to obtain Bifidobacterium bifidum WZA-BB02 fermentation liquid, which was diluted with deionized water to obtain a viable cell count of 1×10 8 CFU / g of Bifidobacterium bifidum WZA-BB02 bacterial liquid; the Bifidobacterium bifidum WZA-BB02 bacterial liquid was heat-inactivated at 115° C. for more than 30 minutes to obtain Bifidobacterium bifidum WZA-BB02 postbiotics.

[0011] In a third aspect, the present invention provides a Bifidobacterium bifidum composition, comprising Bifidobacterium bifidum WZA-BB02 postbiotics and Flammulina velutipes extract.

[0012] Further, the preparation method of Flammulina velutipes extract is as follows:

[0013] The Flammulina velutipes was dried at 60°C and crushed through a 50-mesh sieve; the Flammulina velutipes was mixed with a 90% ethanol solution at a solid-liquid volume ratio of 1:10, and ultrasonically treated for 20 minutes at an ultrasonic frequency of 40kHz and an ultrasonic power of 300W; the ultrasonically treated mixed solution was centrifuged at 4000r / min for 15 minutes to obtain a supernatant; the supernatant was rotary evaporated at 70°C to obtain a dry matter, which is the Flammulina velutipes extract.

[0014] In a fourth aspect, the present invention provides a use of the above-mentioned Bifidobacterium bifidum postbiotic in the preparation of a drug for sobering up, protecting the liver and lowering uric acid.

[0015] In a fifth aspect, the present invention provides a use of the above-mentioned Bifidobacterium bifidum composition in the preparation of drugs for sobering up, protecting the liver and lowering uric acid.

[0016] Furthermore, the drugs for sobering up, protecting the liver and lowering uric acid have at least one of the following functions:

[0017] (1) Inhibit xanthine oxidase activity, reduce serum uric acid levels, and relieve hyperuricemia;

[0018] (2) Alleviate alcohol-induced liver damage;

[0019] (3) Inhibit the effect of alcohol on the increase of uric acid levels.

[0020] The beneficial effects of the present invention are:

[0021] 1. The Bifidobacterium bifidum WZA-BB02 postbiotic provided by the present invention has a high in vitro xanthine oxidase activity inhibition rate. According to rat experiments, the Bifidobacterium bifidum WZA-BB02 postbiotic can significantly reduce the serum uric acid level of rats with hyperuricemia, and can effectively reduce alcohol-induced liver damage and inhibit the promoting effect of alcohol on the increase of uric acid levels.

[0022] 2. The Bifidobacterium bifidum composition provided by the present invention includes Bifidobacterium bifidum WZA-BB02 postbiotics and Flammulina velutipes extract. Since the Flammulina velutipes extract has good antioxidant properties, after acute alcohol gavage, the Flammulina velutipes extract and the Bifidobacterium bifidum WZA-BB02 postbiotics act synergistically, and the effect of relieving alcohol toxicity is more obvious, which can further inhibit liver damage, reduce serum XOD, ALT and AST activities, and the inhibitory effect on the increase of serum uric acid levels is more significant. DETAILED DESCRIPTION

[0023] In order to enable those skilled in the art to better understand the technical solutions in the present invention, the technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0024] Example 1 Strain screening and identification

[0025] (1) Strain isolation and purification

[0026] In February 2024, infant feces were collected in Tai'an City, Shandong Province as samples and transported to the laboratory for standby use. 9 mL of sterilized saline was used to prepare a sample homogenate with 1 g of sample, and then diluted 10 times in increments to obtain a concentration of 10 -1 ~10 -7Sample dilution. Pipette 0.1mL of dilution for each dilution, spread evenly on the bifidobacterium agar plate, and culture anaerobically at 37℃ for 48h. Then select colonies according to the following colony characteristics: diameter 0.5~1mm, round, irregular edges, opaque, white on the front, raised in the middle, smooth and bright surface, moist and sticky texture. Pick 5 single colonies, inoculate them on the bifidobacterium agar plate, culture anaerobically at 37℃ for 72h, pick a single colony, and store it in a glycerol tube at -70℃.

[0027] Among them, the preparation method of bifidobacterium agar plate is as follows: take 15.0g of peptone, 20.0g of glucose, 2.0g of yeast extract, 0.5g of soluble starch, 5.0g of sodium chloride, 0.5g of L-cysteine, 5.0g of tomato extract powder, 2.0g of liver powder, 801mL of Tween and 20.0g of agar, dissolve them with 1.0L of distilled water, adjust the pH value to 7.0±0.1, sterilize at 121°C for 15min, pour into a plate and let cool.

[0028] (2) Strain screening

[0029] The purified strain was inoculated into a liquid culture medium of bifidobacterium and activated for 24 hours to obtain a seed solution. The seed solution was inoculated at a 1% inoculation amount into liquid culture medium of bifidobacterium containing different concentrations (3%, 6%, 9%, 12%, 15%) of ethanol. After culturing at 37°C for 48 hours, the OD of the culture in each culture medium was detected. 600 OD of the culture in Bifidobacterium medium containing 15% ethanol 600 The pH value was 0.72. After purification, an alcohol-tolerant strain was obtained, numbered WZA-BB02, and stored in a glycerol tube at -70°C.

[0030] (3) Strain identification

[0031] The strain WZA-BB02 was sent for identification by the Henan Industrial Microbial Strain Engineering Technology Research Center. The primers used in the identification process were as follows:

[0032] 27F: 5'-AGAGTTTGATCMTGGCTCAG-3' (SEQ NO. 1);

[0033] 1492R: 5'-GGTTACCTTGTTACGACTT-3' (SEQ NO. 2);

[0034] The 16S rRNA gene sequence of strain WZA-BB02 was obtained as follows:

[0035]

[0036] The identification result is: Bifidobacterium bifidum , whose taxonomic units are: Bacteria ; Actinobacteria ; Bifidobacteriales ; Bifidobacteriaceae ; Bifidobacterium .

[0037] The identified strain was named Bifidobacterium bifidum WZA-BB02 and sent to China Center for Type Culture Collection for preservation. The preservation information is as follows:

[0038] Strain name: Bifidobacterium bifidum WZA-BB02; Classification name: Bifidobacterium bifidum Bifidobacterium bifidum WZA-BB02; Deposit date: July 1, 2024; Deposit number: CCTCC NO: M 20241442; Deposit address: Wuhan University, Wuhan, Hubei, China.

[0039] Example 2 Preparation of Bifidobacterium bifidum WZA-BB02 postbiotics

[0040] Bifidobacterium bifidum WZA-BB02 was inoculated into bifidobacterium culture medium and fermented at 37°C for 48 h to obtain Bifidobacterium bifidum WZA-BB02 fermentation liquid, which was diluted with deionized water to obtain a viable cell count of 1×10 8 CFU / g of Bifidobacterium bifidum WZA-BB02 bacterial liquid; the Bifidobacterium bifidum WZA-BB02 bacterial liquid was heat-inactivated at 115° C. for more than 30 minutes to obtain Bifidobacterium bifidum WZA-BB02 postbiotics.

[0041] Comparative Example 1 Preparation of Bifidobacterium bifidum CICC 10395 postbiotics

[0042] Bifidobacterium bifidum CICC 10395 postbiotics were prepared using Bifidobacterium bifidum CICC 10395, and the preparation process was the same as that in Example 2. Bifidobacterium bifidum CICC 10395 was purchased from China Industrial Microbiological Culture Collection Administration Center.

[0043] Example 3 Ability of Bifidobacterium bifidum to inhibit xanthine oxidase (XOD) in vitro

[0044] (1) Take 50 μL of the Bifidobacterium bifidum WZA-BB02 postbiotic prepared in Example 2 and the Bifidobacterium bifidum CICC 10395 postbiotic prepared in Comparative Example 1, add 50 μL of XOD working solution (0.05 U / mL, purchased from Merck, Germany), mix thoroughly and keep warm at 25°C for 10 min, then add 150 μL of xanthine solution (1 mmol / L, purchased from Merck, Germany), react at 25°C for 30 min, and then add 100 μL of HCI solution (1 mol / L) to terminate the reaction. The system in which only the XOD working solution and the xanthine solution were mixed and reacted was used as the control group.

[0045] (2) After the reaction, 3.5 mL of ultrapure water was added to each reaction solution and mixed thoroughly. The mixture was filtered through a 0.2 μm microporous nylon filter membrane and then determined using a high performance liquid chromatograph (Essentia LC-16, purchased from Shimadzu Corporation, Japan). The chromatographic conditions were as follows: the mobile phase was a mixture of pH 7.4 phosphate buffer solution (0.02 mol / L) and 1% methanol, the volume ratio of 0.02 mol / L phosphate buffer to 1% methanol was 95:5, isocratic elution was 10 min, the flow rate was set to 1.0 mL / min, the detection wavelength was set to 290 nm, the column temperature was room temperature, and the injection volume was 20 μL.

[0046] (3) The XOD inhibition rate (%) of Bifidobacterium bifidum WZA-BB02 postbiotics and Bifidobacterium bifidum CICC 10395 postbiotics was calculated according to the following formula.

[0047]

[0048] The results showed that the inhibition rate of Bifidobacterium bifidum WZA-BB02 postbiotics on the activity of milk-derived XOD was 57.6%, while the inhibition rate of Bifidobacterium bifidum CICC 10395 postbiotics on the activity of milk-derived XOD was 20.5%, indicating that the ability of Bifidobacterium bifidum WZA-BB02 postbiotics to inhibit XOD activity in vitro is significantly better than that of Bifidobacterium bifidum CICC 10395 postbiotics.

[0049] Example 4 Preparation of a Bifidobacterium bifidum WZA-BB02 composition

[0050] (1) Preparation of Flammulina velutipes extract

[0051] The Flammulina velutipes was dried at 60°C and crushed through a 50-mesh sieve. The Flammulina velutipes was mixed with a 90% ethanol solution at a solid-liquid volume ratio of 1:10, and ultrasonically treated for 20 minutes at an ultrasonic frequency of 40kHz and an ultrasonic power of 300W; the ultrasonically treated mixed solution was centrifuged at 4000r / min for 15 minutes to obtain a supernatant; the supernatant was rotary evaporated at 70°C to obtain a dry matter, which was the Flammulina velutipes extract.

[0052] (2) Preparation of Bifidobacterium bifidum WZA-BB02 composition

[0053] The Flammulina velutipes extract was added to the Bifidobacterium bifidum WZA-BB02 postbiotic prepared in Example 2 at a mass percentage of 2%, and the mixture was evenly mixed to obtain a Bifidobacterium bifidum WZA-BB02 composition.

[0054] Example 5 Effects of Bifidobacterium bifidum WZA-BB02 postbiotics and their composition on hyperuricemia rat model

[0055] 1. Build the model

[0056] A total of 80 SPF SD rats were selected, 0.4 mL of blood was collected from the fasting tail vein in a 1.5 mL EP tube, and the blood was placed in a 4°C refrigerator for 1 hour. The blood was centrifuged at 4°C and 3000 r / min for 15 minutes, and serum was collected. The uric acid level was determined by the phosphotungstic acid method to exclude animals with unstable uric acid levels. 75 SD rats with normal uric acid levels were selected and adaptively fed for one week.

[0057] After one week of adaptive feeding, SPF SD male rats were randomly divided into a normal control group (12 rats) and a modeling group (63 rats). The rats in the modeling group were gavaged with potassium oxonate (300 mg / kg) and hypoxanthine (300 mg / kg) every day, and the rats in the normal control group were gavaged with an equal volume of normal saline. After 7 consecutive days, blood was collected from the tip of the tail, and the serum uric acid content was detected using a uric acid determination kit (Nanjing Jiancheng Bioengineering Institute). Rats with serum uric acid levels higher than 120 μmol / L were determined to have successfully established a model.

[0058] 2. Experimental Grouping and Treatment

[0059] 60 successfully modeled hyperuricemia rats were randomly selected and divided into model control group, positive control group, WZA-BB02 postbiotic group, CICC 10395 postbiotic group and WZA-BB02 combination group, with 12 rats in each group. The experimental treatments of each group were as follows:

[0060] Normal control group: gavage with 10 mL of normal saline once a day;

[0061] Model control group: gavage with 10 mL of normal saline once a day;

[0062] Positive control group: gavage once a day with 10 mL of allopurinol solution, the dosage is 5 mg allopurinol / kg;

[0063] WZA-BB02 postbiotic group: 10 mL of WZA-BB02 postbiotic solution was gavaged once a day, and the dosage of WZA-BB02 postbiotic was 1 g / kg;

[0064] CICC 10395 postbiotic group: 10 mL of CICC 10395 postbiotic solution was gavaged once a day, and the dosage of CICC 10395 postbiotic was 1 g / kg;

[0065] WZA-BB02 composition group: 10 mL of WZA-BB02 composition solution was gavaged once a day, and the dosage of WZA-BB02 composition was 1 g / kg.

[0066] In the above treatments, each solution was prepared by diluting physiological saline.

[0067] The above-mentioned gavage method was used for intervention for 14 days. Every 7 days of intervention, blood was collected from the tail of each group of rats (30 minutes after the last administration), and the uric acid content in the serum was measured using a uric acid determination kit (Nanjing Jiancheng Bioengineering Institute). The results are shown in Table 1.

[0068] Table 1 Results of serum uric acid content in rats (mean ± SD)

[0069]

[0070] Note: * indicates significant difference compared with the normal control group, P < 0.5; # indicates significant difference compared with the model control group, P < 0.5.

[0071] As can be seen from Table 1, the uric acid content of the model control group was significantly higher than that of the normal control group, indicating that the model was successfully established. Compared with the model control group, the uric acid content of rats in the positive control group, WZA-BB02 postbiotic group, and WZA-BB02 combination group decreased significantly after two weeks of intervention, and there was no significant difference with the normal control group, indicating that WZA-BB02 postbiotics and WZA-BB02 combination have good uric acid-lowering effects, and the uric acid-lowering effect is close to that of allopurinol. However, the uric acid-lowering effect of Bifidobacterium bifidum CICC 10395 postbiotics is not ideal.

[0072] Example 6 Changes in Physiological Indexes of Hyperuricemic Rats after Acute Alcohol-Purine Oral Administration

[0073] 1. Test treatment

[0074] For the rats whose tails were bled in Example 5, the rats in the normal control group were randomly divided into an alcohol control group and a saline control group, with 6 rats in each group. The rats in the alcohol control group, model control group, positive control group, WZA-BB02 postbiotic group, and WZA-BB02 composition group were treated with acute alcohol-purine gavage (10 mL / kg of 56-degree liquor and 300 mg / kg of hypoxanthine), and the rats in the saline control group were treated with saline-purine gavage (10 mL / kg of saline and 300 mg / kg of hypoxanthine).

[0075] 2. Index detection and result analysis

[0076] Four hours after acute alcohol-purine gavage or saline gavage, blood was collected from the tail tip of each group of rats. After blood collection, the blood was centrifuged at 3500r / min for 4min, and the upper serum was taken out and stored in a -80℃ refrigerator for detection.

[0077] The uric acid level in rat blood was determined using a uric acid assay kit (Nanjing Jiancheng Bioengineering Institute), the XOD activity in serum was determined using a XOD activity assay kit (Nanjing Jiancheng Biotechnology Co., Ltd.), the ALT activity in serum was determined using an ALT activity assay kit (Beijing Zhongsheng Beikong Biotechnology Co., Ltd.), and the AST activity in serum was determined using an AST activity assay kit (Beijing Zhongsheng Beikong Biotechnology Co., Ltd.). The results are shown in Table 2.

[0078] Table 2 Results of serum index tests in rats after acute alcohol gavage

[0079]

[0080] From the results in the table above, we can see that the uric acid level, serum XOD activity, ALT activity and AST activity of the rats in the alcohol control group treated with acute alcohol-purine gavage were higher than those in the saline control group, indicating that alcohol can promote purine absorption, uric acid production, and affect the normal excretion of uric acid, leading to increased uric acid levels in the body and causing certain damage to the liver. Compared with the rats in the alcohol control group, the uric acid level, serum XOD activity, ALT activity and AST activity of the rats in the model control group were significantly increased, and were also significantly higher than the test results before alcohol intervention, indicating that alcohol will further aggravate the symptoms of hyperuricemia and cause obvious damage to the liver.

[0081] Compared with the alcohol control group, the uric acid level in the positive control group increased instead of decreased, and the serum XOD activity, ALT activity and AST activity were higher than those in the alcohol control group. This indicates that in the presence of alcohol, the uric acid-lowering effect of allopurinol will be inhibited, and both will aggravate the degree of liver damage.

[0082] Compared with the alcohol control group, all indicators of rats in the WZA-BB02 postbiotic group and the WZA-BB02 combination group were lower, and their serum uric acid content and XOD activity were even lower than those in the normal saline control group, indicating that both WZA-BB02 postbiotics and the WZA-BB02 combination can reduce the damage of alcohol to the liver and effectively inhibit uric acid production and the increase of uric acid caused by alcohol.

[0083] Although the present invention has been described in detail by way of preferred embodiments, the present invention is not limited thereto. Without departing from the spirit and essence of the present invention, a person of ordinary skill in the art may make various equivalent modifications or substitutions to the embodiments of the present invention, and such modifications or substitutions shall be within the scope of the present invention. Any person of ordinary skill in the art may easily conceive of changes or substitutions within the technical scope disclosed by the present invention, and such changes or substitutions shall be within the scope of protection of the present invention.

Claims

1. A strain of Bifidobacterium bifidum for sobering up, protecting the liver and reducing uric acid, characterized in that: Bifidobacterium bifidum is Bifidobacterium bifidum ( Bifidobacterium bifidum ) WZA-BB02, Bifidobacterium bifidum WZA-BB02 was deposited in the China Center for Type Culture Collection on July 1, 2024, with the deposit number CCTCC NO: M 20241442, and the deposit address is Wuhan University, Wuhan City, Hubei Province, China.

2. A Bifidobacterium bifidum postbiotic, characterized in that The method is prepared by fermenting and inactivating the Bifidobacterium bifidum WZA-BB02 as claimed in claim 1.

3. The Bifidobacterium bifidum postbiotic according to claim 2, characterized in that The specific preparation method comprises the following steps: Bifidobacterium bifidum WZA-BB02 was inoculated into bifidobacterium culture medium and fermented at 37°C for 48 h to obtain Bifidobacterium bifidum WZA-BB02 fermentation liquid, which was diluted with deionized water to obtain a viable cell count of 1×10 8 CFU / g of Bifidobacterium bifidum WZA-BB02 bacterial liquid; the Bifidobacterium bifidum WZA-BB02 bacterial liquid was heat-inactivated at 115° C. for more than 30 minutes to obtain Bifidobacterium bifidum WZA-BB02 postbiotics.

4. Use of the Bifidobacterium bifidum postbiotic as claimed in claim 2 in the preparation of drugs for sobering up, protecting the liver and lowering uric acid.

5. A Bifidobacterium bifidum composition, characterized in that The invention comprises the Bifidobacterium bifidum WZA-BB02 postbiotic and Flammulina velutipes extract as claimed in claim 2.

6. The Bifidobacterium bifidum composition according to claim 5, characterized in that The preparation method of Flammulina velutipes extract is as follows: The Flammulina velutipes was dried at 60°C and crushed through a 50-mesh sieve; the Flammulina velutipes was mixed with a 90% ethanol solution at a solid-liquid volume ratio of 1:10, and ultrasonically treated for 20 minutes at an ultrasonic frequency of 40kHz and an ultrasonic power of 300W; the ultrasonically treated mixed solution was centrifuged at 4000r / min for 15 minutes to obtain a supernatant; the supernatant was rotary evaporated at 70°C to obtain a dry matter, which is the Flammulina velutipes extract.

7. Use of the Bifidobacterium bifidum composition as claimed in claim 5 in the preparation of drugs for sobering up, protecting the liver and lowering uric acid.

8. The use according to claim 4 or 7, characterized in that Drugs for alcohol detoxification, liver protection and uric acid reduction have at least one of the following functions: (1) Inhibit xanthine oxidase activity, reduce serum uric acid levels, and relieve hyperuricemia; (2) Alleviate alcohol-induced liver damage; (3) Inhibit the effect of alcohol on the increase of uric acid levels.

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