Medical use of e. coronatum

Abstract

The present application relates to the field of medicine, in particular to the application of Eurotium cristatum from Fuzhuan brick tea in the preparation of drugs for degrading uric acid, the application of Eurotium cristatum in the preparation of drugs for preventing and treating hyperuricemia, and the application of Eurotium cristatum in the preparation of drugs for preventing and treating kidney damage caused by hyperuricemia.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of medicine, and relates to an application of Eurotium cristatum from Fuzhuan tea in resisting hyperuricemia and protecting the kidney. BACKGROUND

[0002] Hyperuricemia has become one of the most common metabolic diseases in the world, causing a huge burden on public health. Hyperuricemia is a disease state in which the concentration of blood uric acid exceeds the normal range, and the main physiological mechanisms include excessive uric acid production mediated by xanthine oxidase and insufficient uric acid excretion dominated by the kidney. Long-term high blood uric acid levels induce the body to present a chronic inflammatory state, especially promoting the occurrence of kidney inflammation, and further inducing renal fibrosis. Therefore, hyperuricemia is considered an independent predictor of the occurrence and progression of chronic kidney disease. In recent years, probiotic supplementation therapy has been recommended as a preventive or auxiliary non-drug treatment for hyperuricemia, and has been accepted by an increasing number of patients. Probiotics isolated from fermented foods have attracted attention due to their advantages of easy availability, economy, and high safety, which has also prompted researchers to find more probiotics from fermented foods that have the potential to alleviate hyperuricemia.

[0003] Eurotium cristatum is the dominant species of Fuzhuan tea. Currently, Eurotium cristatum has been shown to survive in the gastrointestinal tract and have probiotic-like effects on obesity and colitis by regulating body metabolism. SUMMARY

[0004] The purpose of the present application is to find a new use of Eurotium cristatum from Fuzhuan tea.

[0005] The first aspect of the present application is to provide an application of Eurotium cristatum in the preparation of a drug for degrading uric acid.

[0006] The second aspect of the present application is to provide an application of Eurotium cristatum in the preparation of a drug for preventing and treating hyperuricemia.

[0007] The third aspect of the present application is to provide an application of Eurotium cristatum in the preparation of a drug for preventing and treating kidney damage caused by hyperuricemia.

[0008] The details of each aspect of the present application will be described in the subsequent chapters. The features, purposes and advantages of the present application will be more apparent through the following description and claims. BRIEF DESCRIPTION OF DRAWINGS

[0009] Figure 1 is a chromatogram of Eurotium cristatum directly degrading uric acid in vitro and a mass spectrum of the degradation product being allantoin

[0010] (A) High performance liquid chromatography was used to detect the ability of E. turcicum to degrade uric acid

[0011] (a) Representative chromatogram of the standard (b) Representative chromatogram of the sample co-incubated with uric acid by E. turcicum

[0012] (B) Quantitative analysis of the supernatant uric acid of the sample co-incubated with uric acid by E. turcicum for different time

[0013] (C) Identification of the product degraded by E. turcicum from uric acid using liquid chromatography-mass spectrometry (a) Standard mass spectrum of uric acid (b) Mass spectrum data of the sample co-incubated with uric acid by E. turcicum Indicated, *P<0.05, **P<0.01 and ***P<0.001 indicate statistically significant differences.

[0014] Figure 2 is a graph showing the results of E. turcicum in relieving the abnormal uric acid metabolism of hyperuricemic mice (A) Plasma uric acid level (B) Urinary uric acid level (C) Content of hypoxanthine in fecal samples (D) Content of xanthine in fecal samples (E) Content of hypoxanthine in urine samples (F) Content of xanthine in urine samples

[0015] (G) Representative mass spectrum of the standard and sample analyzed using liquid chromatography-mass spectrometry

[0016] (a) Mass spectrum of xanthine standard (b) Mass spectrum of xanthine in fecal samples (c) Mass spectrum of hypoxanthine standard (d) Mass spectrum of hypoxanthine in fecal samples

[0017] Data are mean ± standard deviation Indicated, *P<0.05, **P<0.01 and ***P<0.001 indicate statistically significant differences.

[0018] Figure 3 is a graph showing the results of E. turcicum in relieving kidney injury caused by high uric acid

[0019] (A) Plasma creatinine level (B) Plasma urea nitrogen level (C) Urinary creatinine level (D) Urinary urea nitrogen level (E) Analysis results of the proportion of positive area of immunohistochemical staining of Ki67 (F) Representative kidney section images of H&E staining and Ki67 staining (G) Representative kidney section images of Masson staining, TGF-β1 and α-SMA staining (H) Analysis results of the proportion of fibrosis area of Masson staining (I) Analysis results of the proportion of positive area of immunohistochemical staining of TGF-β1 and α-SMA Data are mean ± standard deviation Indicated, *P<0.05, **P<0.01 and ***P<0.001 indicate statistically significant differences. DETAILED DESCRIPTION

[0020] The Eurotium cristatum used in the present application is purchased from China General Microbiological Culture Collection Center (CICC), and the strain number is CICC 2099.

[0021] The following implementation high-performance liquid chromatography system is Waters 2695 series HPLC system, and the chromatographic column used is Agilent ZorBax Eclipse XDB-C18 (4.6mmx250mm, 5μm); the mass spectrometer is a Waters H-Class UPLC / Sciex Triple TOF 4600 combined system, and the chromatographic column used is ACQUITY UPLC Amide (2.1mmx100mm, 1.7μm); the liquid phase uses acetonitrile and methanol as chromatographic grade, water as distilled water, and formic acid as analytical grade; the mass spectrometer uses acetonitrile as mass spectrometry grade, water as distilled water treated by Mill-Q, and formic acid as mass spectrometry grade.

[0022] The experimental data are statistically analyzed by using GraphPad Prism 8.0 software, and the numerical value is expressed as mean ± standard deviation. Student's unpaired t-test is used to compare the data between two groups, and one-way ANOVA method is used to compare the data between multiple groups. The three expressions of *p<0.05, **p<0.01 and ***p<0.001 are used to show that there are different degrees of significant statistical differences between each group of data.

[0023] Unless otherwise defined, all professional and scientific terms used herein have the same meaning as familiar to those skilled in the art. In addition, any method and material similar or equivalent to those described can be applied to the method of the present application. The preferred implementation methods and materials described herein are only for demonstration.

[0024] The above-mentioned features mentioned in the present application, or the features mentioned in the embodiments, can be combined arbitrarily. All features disclosed in the present patent specification can be used with any combination, and each feature disclosed in the specification can be replaced by any alternative feature that can provide the same, equivalent or similar purpose. Therefore, unless otherwise specified, the disclosed features are only general examples of equivalent or similar features.

[0025] The present application will be further described in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present application and not to limit the scope of the present application. The experimental methods not specified in the following examples are usually carried out under conventional conditions or under the conditions recommended by the manufacturer.

[0026] Example 1 Determination of the in vitro uric acid-lowering ability of Eurotium cristatum

[0027] Euricde was inoculated into potato dextrose broth and incubated for 48 hours, then centrifuged at 4000 rpm for 10 minutes to obtain the strain. The precipitate was washed with sterile phosphate buffer solution (PBS) for three times, and finally resuspended in 900 μL PBS containing 0.2 mg / mL uric acid, and incubated at 28°C for 1, 2 and 4 hours, respectively. Then the supernatant was obtained by centrifugation at 10000 rpm for 10 minutes, and 100 μL HCIO4 (0.1 mol / L) was added to avoid further degradation of the compounds. After filtration with 0.22 μm filter membrane, 10 μL sample was injected into the HPLC system to detect the change of uric acid.

[0028] The HPLC conditions were as follows: isocratic elution was performed with 0.005% aqueous phosphoric acid and methanol (9:1, v / v) at a flow rate of 1 mL / min for 60 minutes. The column temperature was maintained at 30°C, and the detection wavelength was 283 nm.

[0029] At the same time, 5 μL sample was injected into the UPLC-MS system to identify the metabolic products of uric acid degraded by Euricde.

[0030] The UPLC conditions were as follows: the column temperature was maintained at 40°C. Isocratic elution was performed with acetonitrile (A) and water containing 0.2% formic acid, with 40% mobile phase A at a flow rate of 0.3 mL / min for 10 minutes.

[0031] The chromatogram of uric acid degradation by Euricde in vitro and the mass spectrum of the degradation products are shown in Figure 1. It can be seen that Euricde can directly degrade uric acid to allantoin.

[0032] Example 2 Verification of the anti-hyperuricemia activity of Euricde in vivo

[0033] Male C57BL / 6J mice were randomly divided into three groups of 8: control group, model group and Euricde group. To induce hyperuricemia, the model group and the Euricde group were given intragastrically 60 mg / kg of adenine and 240 mg / kg of potassium oxonate. In addition, the mice in the Euricde group were given 200 μL of Euricde at a concentration of 10 6 CFU / mL daily, and the control group was given the same volume of sterile water. The anti-hyperuricemia activity of Euricde in vivo was evaluated by detecting the content of uric acid in the plasma and urine of the mice using a kit, and detecting the content of xanthine and hypoxanthine, the precursors of uric acid, in the feces and urine of the mice using UPLC-MS.

[0034] For kit detection, a uric acid detection kit from Nanjing Jiancheng was used according to the instructions. The specific steps were as follows:

[0035] 1) Use a clean 96-well plate, mark the samples and groups;

[0036] 2) Take 5 μL distilled water / sample / standard and add to the corresponding well, then add 250 μL reagent one;

[0037] 3) After mixing the sample to be tested and reagent one, place the plate in an incubator at 37°C for 10 minutes;

[0038] 4) Adjust the detection wavelength of the microplate reader to 510 nm, and read the absorbance value A of each well.

[0039] The formula for calculating the concentration of uric acid is as follows: Uric acid concentration (μmol / L) = ((A measured - A blank) / (A standard - A blank)) * C 标准品 , where A measured represents the absorbance value of the sample; A blank represents the absorbance value of distilled water; A standard represents the absorbance value of the standard; C 标准品 is the concentration of the standard, which is 500 μmol / L. As shown in FIGS. Figure 2A and B, Eurotium cristatum can reduce the uric acid level in hyperuricemic mice.

[0040] For UPLC-MS detection, the sample processing method is as follows: the urine sample is mixed with methanol (1:3, v / v) uniformly, and then vortexed for 30 seconds. 95 mg of fecal sample is mixed with 800 μL of 80% methanol, and then the sample is broken up using a full-automatic sample rapid grinder at a frequency of 60 Hz for 2 minutes. The extracted sample is ultrasonically treated in an ice water bath for 30 minutes, and then placed in a -20°C refrigerator for 30 minutes. All the supernatants of the samples are centrifuged at 15000 rpm for 20 minutes at 4°C, and then the samples are dried in a centrifugal concentrator. Before analysis, the samples are reconstituted in water containing 30% acetonitrile and 30% methanol. The resulting solution is filtered before being injected. The liquid phase conditions are slightly modified from those described in Example 2, i.e., 90% A phase isocratic elution, and the injection volume is 1 μL. The instrument is set to multiple reaction monitoring (MRM) mode. The monitored compound ion pairs are as follows: hypoxanthine (m / z) 135.35 [M-H]-→92.10, xanthine (m / z) 151.20 [M-H]-→108.05. The results show that Eurotium cristatum can affect the metabolic pathway of uric acid in vivo Figure 2C -F). FIG. 2G shows representative mass spectra of the control and sample.

[0041] Example 3 Improvement of renal damage caused by high uric acid by Eurotium cristatum

[0042] (1) The levels of urea nitrogen and creatinine can be used to evaluate the condition of kidney function, so commercial kits purchased from Nanjing Jiancheng are used to detect the levels of urea nitrogen and creatinine in plasma and urine according to the instructions.

[0043] For urea nitrogen detection, first prepare the required reagents for detection, as follows:

[0044] Buffer enzyme solution preparation: when used, prepare the buffer enzyme solution according to enzyme stock solution: enzyme diluent = 3:1000, and prepare it immediately before use.

[0045] 100mmol / L urea nitrogen standard stock solution preparation: before use, take one of the powders in reagent four and add 1mL double distilled water to prepare a 100mmol / L standard stock solution, which is stored at 4°C.

[0046] 10mmol / L urea nitrogen standard application solution preparation: dilute the 100mmol / L standard stock solution with double distilled water at a ratio of 1:9 to prepare a 10mmol / L urea nitrogen standard application solution.

[0047] For urea nitrogen level detection, the specific steps are as follows:

[0048] 1) Use a clean 5mL EP tube and label the sample and group;

[0049] 2) Take 20μL distilled water / sample / standard and add it to the corresponding EP tube, then add 250μL buffer enzyme solution;

[0050] 3) After mixing the sample to be tested and the buffer enzyme solution, place the EP tube in a 37°C water bath for 10 minutes;

[0051] 4) Add 1mL of reagent two and 1mL of reagent three to each tube and mix well, then place it again in a 37°C water bath for 10 minutes;

[0052] 5) Adjust the detection wavelength of the ultraviolet spectrophotometer to 640nm, and after zeroing with distilled water, read the absorbance value of each tube.

[0053] The urea nitrogen calculation formula is as follows: urea nitrogen concentration (mmol / L) = ((A measured - A blank) / (A standard - A blank))*C 标准品 *N, where A measured represents the sample absorbance value; A blank represents the distilled water absorbance value; A standard represents the standard absorbance value; C 标准品 is the standard concentration, which is 10mmol / L; N is the sample test dilution factor.

[0054] For creatinine detection, the specific steps are as follows:

[0055] 1) Use a clean 96-well plate and label the sample and group;

[0056] 2) Take 6μL distilled water / sample / standard and add it to the corresponding well, then add 180μL reagent one;

[0057] 3) After mixing the sample to be tested and reagent one, place the well plate in a 37°C incubator for 5 minutes;

[0058] 4) Adjust the wavelength of the microplate reader to 546 nm, and read the absorbance value of each well A1;

[0059] 5) Add reagent two, mix well, and incubate the well plate in a 37°C incubator for 5 minutes;

[0060] 6) Adjust the wavelength of the microplate reader to 546 nm, and read the absorbance value of each well A2;

[0061] 7) Calculate ΔA = A2 - K * A1.

[0062] The formula for calculating the concentration of creatinine is as follows: Creatinine concentration (μmol / L) = ((ΔA measured - ΔA blank) / (ΔA standard - ΔA blank)) * C 标准品 , where ΔA measured represents the ΔA value of the sample; ΔA blank represents the ΔA value of distilled water; ΔA standard represents the ΔA value of the standard; C 标准品 represents the standard concentration, which is 442 μmol / L; K is the dilution factor, and the value is 186 / 246.

[0063] As Figure 3A -D shows, Eurotium cristatum can effectively reduce the plasma levels of creatinine and urea nitrogen, and increase the urine levels of creatinine and urea nitrogen, indicating that Eurotium cristatum can improve renal function.

[0064] (2) Further evaluate the level of kidney injury by histopathological section staining. First, take the rehydrated kidney tissue section, and use H&E staining kit and Masson staining kit to perform staining according to the instructions. Because Ki67 is related to inflammation of kidney tissue, and TGF-β1 and α-SMA are markers of fibrosis, another tissue section is taken to perform immunohistochemical staining.

[0065] First, make a tissue section, and the specific steps are as follows:

[0066] 1) Put the fresh kidney into 4% paraformaldehyde prepared in phosphate buffer solution for fixation for 2 days;

[0067] 2) Take out the fixed kidney tissue, and rinse with running water for 3 times, each time for 5 minutes;

[0068] 3) Dehydrate the washed kidney tissue in a gradient ethanol solution from low to high concentration, and the concentration and time are as follows: 75% ethanol, 1 hour→ 85% ethanol, 1 hour→ 95% ethanol, 1 hour→ anhydrous ethanol I, 1 hour→ anhydrous ethanol II, 1 hour

[0069] → xylene I, 30 minutes→ xylene II, 30 minutes;

[0070] 4) The dehydrated and transparented kidney is sequentially soaked in paraffin I and II with a melting point of 54-56°C for 45 minutes each;

[0071] 5) The group kidney is placed in a mold containing wax liquid, embedded with the kidney broad bean-like section parallel to the bottom, and then the wax block is cooled and stored;

[0072] 6) After embedding, the paraffin block is cut into about 5 μm sections, and the sections are laid flat on water at 40°C to expand;

[0073] 7) The sections are picked up with a glass slide, then placed in a section box, and then placed in a 60°C oven for 3 hours, and then stored at room temperature for standby.

[0074] For H&E staining, the specific steps are as follows:

[0075] 1) De-waxing: the sections are placed in xylene I for 5 min and xylene II for 5 min;

[0076] 2) Hydration: the kidney tissue is sequentially soaked in 100%, 95%, 70%, 50%, 30% ethanol and pure water for 1 minute each;

[0077] 3) Staining: the kidney tissue sections are stained with hematoxylin for 5 minutes, washed with pure water for 1 minute, differentiated with 70% ethanol-1% hydrochloric acid for 1 second, washed with pure water for 1 minute, stained with eosin for 1 minute, and washed with pure water for 1 minute;

[0078] 4) Dehydration and transparency: the kidney tissue is sequentially soaked in 70% and 95% ethanol for 1 minute, and then sequentially dehydrated in 100% ethanol I, 100% ethanol II, xylene I and xylene II for 2 minutes;

[0079] 5) Mounting: neutral gum mounting, after the sections are dried, the effect is observed under a microscope.

[0080] For Masson staining, the specific steps are as follows:

[0081] 1) De-waxing: xylene I de-waxing for 3 minutes, xylene II de-waxing for 3 minutes;

[0082] 2) Hydration: the kidney tissue is sequentially soaked in 100%, 95%, 70%, 50%, 30% ethanol and pure water for 1 minute each for hydration;

[0083] 3) Staining with prepared Weigert iron hematoxylin staining solution for 8 minutes, then quickly washed with distilled water to remove excess staining solution;

[0084] 4) Differentiate with acidified ethanol differentiation solution for 5-10 seconds, then quickly wash with distilled water;

[0085] 5) return blue with Masson blue solution for 5-10 minutes, then wash with water;

[0086] 6) wash with distilled water for 1 minute;

[0087] 7) stain with Ponceau red staining solution for 8 minutes;

[0088] 8) prepare weak acid working solution with a ratio of distilled water: weak acid solution = 2:1, and wash with the weak acid working solution for 1 minute;

[0089] 9) wash with phosphomolybdic acid aqueous solution for 2 minutes;

[0090] 10) wash with the prepared weak acid working solution for 1 minute;

[0091] 11) directly place in aniline blue staining solution for 1 minute;

[0092] 12) wash with the prepared weak acid working solution for 1 minute;

[0093] 13) dehydrate the kidney tissue by sequentially placing in 95% ethanol, 100% ethanol I, 100% ethanol II for 10 seconds, and then placing in xylene I and xylene II for 1 minute;

[0094] 14) gum sealing: vertically place for 2 minutes to dry the slice, add a small amount of neutral gum sealing agent, cover with a cover glass to seal the slice;

[0095] 15) randomly select 3 non-overlapping fields under a high power lens for each slice, take photos and record under a microscope;

[0096] 16) analyze the percentage of blue-stained area in each field to the field using Image J software, take the average value to obtain the fibrosis ratio of the sample.

[0097] For immunohistochemical staining, the specific steps are as follows:

[0098] 1) use antigen repair solution containing 0.4 g / L citric acid and 3 g / L sodium citrate to repair antigens;

[0099] 2) wash with 0.25% triton-TBS twice, each for 5 minutes;

[0100] 3) activate with hydrogen peroxide for 15 minutes, then repeat the washing operation;

[0101] 4) after air drying, block with 10% sheep blood at room temperature for 2 hours, then repeat the washing operation;

[0102] 5) add Ki67, TGF-β1 and α-SMA antibodies for incubation, and place the slice at 4°C for overnight incubation.

[0103] 6) the next day, repeat the washing operation;

[0104] 7) Incubate secondary antibody for 2 hours at room temperature, then repeat washes;

[0105] 8) Develop with DAB kit;

[0106] 9) Counterstain with hematoxylin for 5 seconds, rinse with running water for 5 minutes;

[0107] 10) Dehydrate with graded ethanol and xylene, then mount with resin.

[0108] The results of the slice staining are shown in Figure 3E -I, it can be seen that Eurotium cristatum can improve the inflammation and fibrosis of the kidney caused by high uric acid.

[0109] Aspects of the present application have been described above. However, it should be understood that equivalent changes and modifications can be suggested by one skilled in the art and can be made without departing from the spirit of the present application, and it is thus intended that the application cover all such changes and modifications as fall within the scope of the appended claims.

Claims

1. Eurotium cristatum Eurotium cristatum Use in the manufacture of a medicament for the degradation of uric acid as the sole active ingredient.

2. Eurotium cristatum Eurotium cristatum Use as the sole active ingredient in the manufacture of a medicament for the prevention and treatment of hyperuricemia.

3. Eurotium cristatum Eurotium cristatum The use of the compound of formula (I) as the sole active ingredient in the preparation of a medicament for the prevention and treatment of kidney damage caused by high uric acid.

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