Use of inonotus obliquus extract in preparation of drugs for relieving and / or improving hyperuricemia and related diseases

Abstract

The application belongs to the technical field of medicine, and particularly relates to application of an extract of Inonotus obliquus in preparation of a medicine for relieving and / or improving hyperuricemia and related diseases. The application takes one or more of a water extract of Inonotus obliquus, an ethanol extract of Inonotus obliquus and an ethyl acetate extract of Inonotus obliquus as an active ingredient, can reduce levels of uric acid (UA), creatinine (CRE), urea nitrogen (BUN), xanthine oxidase (XOD) and adenosine deaminase (ADA) in a hyperuricemia model mouse, slow down a heart injury effect of a high uric acid level, and relieve and / or improve hyperuricemia and related diseases.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medicine, and particularly relates to application of an Inonotus obliquus extract in preparation of a medicine for relieving and / or improving hyperuricemia and related diseases. BACKGROUND

[0002] Hyperuricemia (HUA) is a metabolic disease caused by disorders of purine metabolism and / or abnormal excretion of uric acid in the body, which leads to high blood uric acid in the body. At present, the incidence of hyperuricemia shows a rapid growth trend. Hyperuricemia, as an independent risk factor, also induces metabolic syndrome, kidney disease, cardiovascular disease and other diseases as an inducing factor.

[0003] At present, the chemical drug treatment of hyperuricemia has been mature, and drugs with different mechanisms of action continue to be applied in clinical practice. However, with the universalization of the disease, the frequency and scope of use of chemical drugs have gradually increased, and their side effects have also been manifested to varying degrees. Existing studies have shown that in the early stage of hyperuricemia, changing the dietary structure and regulating the body function can have a certain control effect on the uric acid level in the body. However, at the present stage, people's neglect of hyperuricemia has caused attention only after the body has a serious reaction. By continuously using chemical drugs to quickly reduce the uric acid level, the use dose and cycle of chemical drugs are increased, various adverse reactions are induced, and the safety of drug use is greatly reduced. Therefore, it is necessary to develop anti-hyperuricemia drugs with high safety and significant efficacy.

[0004] Inonotus obliquus (Ach.ex Pers.) Pilat is a wood-decaying fungus growing in the cold zone, belonging to Basidiomycota, Russulales, Hymenochaetaceae and Inonotus. It mainly parasitizes birch trees and is also called birch fungus. Inonotus obliquus is a food and medicinal fungus with strong medical and health care value. The active substances isolated from it include polysaccharides, polyphenols, flavonoids, triterpenes, alkaloids, steroids, melanin and lignin compounds, which have anti-tumor, anti-viral, anti-aging, immune regulation, hypoglycemic and other pharmacological effects. There is no related research on hyperuricemia. SUMMARY

[0005] The application aims to provide application of an Inonotus obliquus extract in preparation of a medicine for relieving and / or improving hyperuricemia and related diseases, which significantly relieves and / or improves hyperuricemia and hyperuricemia-related diseases and has high safety.

[0006] The application provides application of an Inonotus obliquus extract in preparation of a medicine for relieving and / or improving hyperuricemia and related diseases.

[0007] The Inonotus obliquus extract is one or more of an Inonotus obliquus water extract, an Inonotus obliquus ethanol extract, and an Inonotus obliquus ethyl acetate extract.

[0008] Preferably, the preparation method of the Inonotus obliquus water extract comprises the following steps:

[0009] The Inonotus obliquus and water are mixed and decocted 2-3 times at a solid-liquid ratio of 1 g: 10-20 mL, each time for 3-5 h;

[0010] After the decocting liquid is combined, solid-liquid separation is performed, and the filtrate is collected; the filtrate contains the Inonotus obliquus water extract.

[0011] Preferably, after the decocting liquid is combined, the method further comprises: concentrating the decocting liquid to a extract state, drying, to obtain the Inonotus obliquus water extract.

[0012] Preferably, the preparation method of the Inonotus obliquus ethanol extract comprises the following steps:

[0013] The Inonotus obliquus and an ethanol aqueous solution with a volume concentration of 50%-90% are mixed at a solid-liquid ratio of 1 g: 10-20 mL, and heated to reflux and extract 2-3 times, each time for 3-5 h;

[0014] After the extract liquid is combined, solid-liquid separation is performed, and the filtrate is collected; the filtrate contains the Inonotus obliquus ethanol extract.

[0015] Preferably, after the filtrate is obtained, the method further comprises: concentrating the filtrate to a extract state, drying, to obtain the Inonotus obliquus ethanol extract.

[0016] Preferably, the preparation method of the Inonotus obliquus ethyl acetate extract comprises the following steps:

[0017] The Inonotus obliquus and an ethanol aqueous solution with a volume concentration of 80%-95% are mixed at a solid-liquid ratio of 1 g: 10-20 mL, heated to reflux and extract 1-3 times for 3-5 h, to obtain a first extract liquid and a filter residue;

[0018] The filter residue and an ethanol aqueous solution with a volume concentration of 50-70% are mixed, heated to reflux and extract 1-3 times for 3-5 h, and solid-liquid separation is performed, to obtain a second extract liquid; the volume concentration of the ethanol aqueous solution with a volume concentration of 50%-70% is 1:1 relative to the volume concentration of the ethanol aqueous solution with a volume concentration of 80%-95%;

[0019] The first extract liquid and the second extract liquid are combined, concentrated to a extract state, and resuspended, to obtain a suspension;

[0020] The suspension and ethyl acetate are mixed, extracted, and the ethyl acetate layer solution is collected; the ethyl acetate layer solution contains the Inonotus obliquus ethyl acetate extract.

[0021] Preferably, after obtaining the ethyl acetate layer solution, the method further comprises: concentrating the ethyl acetate layer solution to a dry extract state, and drying to obtain the Inonotus obliquus ethyl acetate extract.

[0022] Preferably, the volume ratio of the suspension and ethyl acetate is 1:1-3.

[0023] The mass-volume ratio of the Inonotus obliquus and the suspension is 2g:1-5mL.

[0024] Preferably, the alleviating and / or improving of hyperuricemia and related diseases is reducing the level of one or more of serum uric acid, creatinine, urea nitrogen, xanthine oxidase and adenosine deaminase up-regulated by hyperuricemia; the related diseases include cardiomyopathy associated with hyperuricemia.

[0025] The application also provides a medicine for alleviating and / or improving hyperuricemia and related diseases, wherein the active ingredient of the medicine comprises an Inonotus obliquus extract.

[0026] The Inonotus obliquus extract is one or more of an Inonotus obliquus water extract, an Inonotus obliquus ethanol extract and an Inonotus obliquus ethyl acetate extract.

[0027] Beneficial effects:

[0028] The application provides application of an Inonotus obliquus extract in preparation of a medicine for alleviating and / or improving hyperuricemia and related diseases; the Inonotus obliquus extract is one or more of an Inonotus obliquus water extract, an Inonotus obliquus ethanol extract and an Inonotus obliquus ethyl acetate extract. The Inonotus obliquus extract as the active ingredient can reduce the level of uric acid (UA), creatinine (CRE), urea nitrogen (BUN), xanthine oxidase (XOD) and adenosine deaminase (ADA) in a hyperuricemia model mouse, slow down the damage of high uric acid level to the heart, and alleviate and / or improve hyperuricemia and related diseases. BRIEF DESCRIPTION OF DRAWINGS

[0029] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the drawings needed in the embodiments will be briefly introduced below.

[0030] Figure 1 Effects of different samples of Example 1 on xanthine oxidase activity;

[0031] Figure 2 Changes in body weight of mice in different treatment groups;

[0032] Figure 3The results of cardiac ultrasound function detection; wherein, * indicates that the administration group (BA, BB, HT-A, HT-B, HC-A, HC-B) has difference (P<0.05) compared with the model group (M), # indicates that the model group (M) has difference (P<0.05) compared with the control group (C);

[0033] Figure 4 The content of uric acid (UA) in serum of mice in different treatment groups; wherein, * indicates that the administration group (BA, BB, HT-A, HT-B, HC-A, HC-B) has difference (P<0.05) compared with the model group (M), # indicates that the model group (M) has difference (P<0.05) compared with the control group (C);

[0034] Figure 5 The content of creatinine (CRE) in serum of mice in different treatment groups; wherein, * indicates that the administration group (BA, BB, HT-A, HT-B, HC-A, HC-B) has difference (P<0.05) compared with the model group (M), # indicates that the model group (M) has difference (P<0.05) compared with the control group (C);

[0035] Figure 6 The content of urea nitrogen (BUN) in serum of mice in different treatment groups; wherein, * indicates that the administration group (BA, BB, HT-A, HT-B, HC-A, HC-B) has difference (P<0.05) compared with the model group (M), # indicates that the model group (M) has difference (P<0.05) compared with the control group (C);

[0036] Figure 7 The content of xanthine oxidase (XOD) in serum of mice in different treatment groups; wherein, * indicates that the administration group (BA, BB, HT-A, HT-B, HC-A, HC-B) has difference (P<0.05) compared with the model group (M), # indicates that the model group (M) has difference (P<0.05) compared with the control group (C);

[0037] Figure 8 The content of adenosine deaminase (ADA) in serum of mice in different treatment groups; wherein, * indicates that the administration group (BA, BB, HT-A, HT-B, HC-A, HC-B) has difference (P<0.05) compared with the model group (M), # indicates that the model group (M) has difference (P<0.05) compared with the control group (C);

[0038] Figure 9Figure 6 shows the results of histopathological observation (HE staining) of heart tissue of mice in different treatment groups; wherein, A~H are blank control group (C), high uric acid model group (M), positive control allopurinol group (BA), positive control drug benzbromarone group (BB), low-dose Inonotus obliquus ethanol extract group (HT-A), high-dose Inonotus obliquus ethanol extract group (HT-B), low-dose Inonotus obliquus ethyl acetate extract group (HC-A), and high-dose Inonotus obliquus ethyl acetate extract group (HC-B), respectively;

[0039] Figure 10 Figure 7 shows the effects of different samples on H9C2 cell toxicity; wherein, # indicates that the model group (M) has a difference (P<0.05) compared with the control group (C). DETAILED DESCRIPTION

[0040] The application provides application of Inonotus obliquus extract in preparation of a medicine for relieving and / or improving hyperuricemia and related diseases.

[0041] The Inonotus obliquus extract is one or more of Inonotus obliquus water extract, Inonotus obliquus ethanol extract and Inonotus obliquus ethyl acetate extract.

[0042] In the application, the Inonotus obliquus extract is one or more of Inonotus obliquus water extract, Inonotus obliquus ethanol extract and Inonotus obliquus ethyl acetate extract, preferably the Inonotus obliquus extract is Inonotus obliquus water extract, Inonotus obliquus ethanol extract or Inonotus obliquus ethyl acetate extract, and more preferably the Inonotus obliquus extract is Inonotus obliquus ethyl acetate extract. The Inonotus obliquus in the application is purchased from Yanbian Fengyi Special Local Products Co., Ltd.

[0043] In the application, the preparation method of the Inonotus obliquus water extract preferably comprises the following steps: mixing Inonotus obliquus and water according to a solid-liquid ratio of 1g:10-20mL, decocting for 2-3 times, each time for 3-5h; after the decocting liquid is combined, performing solid-liquid separation, and collecting the filtrate; the filtrate contains the Inonotus obliquus water extract. In the application, the solid-liquid ratio is preferably 1g:15mL, and the decocting time is preferably 4h. After the filtrate is obtained, the application preferably concentrates the filtrate to a extract state, dries, and obtains the Inonotus obliquus water extract. The drying mode in the application is preferably vacuum reduced-pressure drying.

[0044] In the present application, the preparation method of the ethanol extract of Inonotus obliquus preferably comprises the following steps: mixing Inonotus obliquus and an aqueous ethanol solution with a volume concentration of 50% to 90% according to a solid-liquid ratio of 1 g: 10-20 mL, heating and refluxing for 2-3 times, each time for 3-5 h; after the extraction solutions are combined, solid-liquid separation is performed, and the filtrate is collected; the filtrate contains the ethanol extract of Inonotus obliquus. After the filtrate is obtained, the present application preferably concentrates the filtrate to a state of extract, and then dries the extract to obtain the ethanol extract of Inonotus obliquus. In the present application, Inonotus obliquus and an aqueous ethanol solution with a volume concentration of 60% to 80% are mixed according to a solid-liquid ratio of 1 g: 10-20 mL, and further preferably, Inonotus obliquus and an aqueous ethanol solution with a volume concentration of 70% are mixed according to a solid-liquid ratio of 1 g: 10-20 mL. In the present application, the solid-liquid ratio is preferably 1 g: 15 mL; and the time for each decoction is preferably 4 h.

[0045] In the present application, the preparation method of the ethanol extract of Inonotus obliquus preferably comprises the following steps: mixing Inonotus obliquus and an aqueous ethanol solution with a volume concentration of 50% to 90% according to a solid-liquid ratio of 1 g: 10-20 mL, heating and refluxing for 2-3 times, each time for 3-5 h; after the extraction solutions are combined, solid-liquid separation is performed, and the filtrate is collected; the filtrate contains the ethanol extract of Inonotus obliquus. After the filtrate is obtained, the present application preferably concentrates the filtrate to a state of extract, and then dries the extract to obtain the ethanol extract of Inonotus obliquus. In the present application, Inonotus obliquus and an aqueous ethanol solution with a volume concentration of 60% to 80% are mixed according to a solid-liquid ratio of 1 g: 10-20 mL, and further preferably, Inonotus obliquus and an aqueous ethanol solution with a volume concentration of 70% are mixed according to a solid-liquid ratio of 1 g: 10-20 mL. In the present application, the solid-liquid ratio is preferably 1 g: 15 mL; and the time for each decoction is preferably 4 h.

[0046] In the present application, the preparation method of the ethanol extract of Inonotus obliquus preferably comprises the following steps: mixing Inonotus obliquus and an aqueous ethanol solution with a volume concentration of 50% to 90% according to a solid-liquid ratio of 1 g: 10-20 mL, heating and refluxing for 2-3 times, each time for 3-5 h; after the extraction solutions are combined, solid-liquid separation is performed, and the filtrate is collected; the filtrate contains the ethanol extract of Inonotus obliquus. After the filtrate is obtained, the present application preferably concentrates the filtrate to a state of extract, and then dries the extract to obtain the ethanol extract of Inonotus obliquus. In the present application, Inonotus obliquus and an aqueous ethanol solution with a volume concentration of 60% to 80% are mixed according to a solid-liquid ratio of 1 g: 10-20 mL, and further preferably, Inonotus obliquus and an aqueous ethanol solution with a volume concentration of 70% are mixed according to a solid-liquid ratio of 1 g: 10-20 mL. In the present application, the solid-liquid ratio is preferably 1 g: 15 mL; and the time for each decoction is preferably 4 h.

[0047] In the present application, the alleviation and / or improvement of hyperuricemia and related diseases preferably refers to reducing the level of one or more of serum uric acid, creatinine, urea nitrogen, xanthine oxidase and adenosine deaminase that are up-regulated by hyperuricemia; and the related diseases preferably include heart damage associated with hyperuricemia, and further preferably, cardiomyopathy associated with hyperuricemia.

[0048] The present application takes Inonotus obliquus extract as an active ingredient, can reduce the levels of uric acid (UA), creatinine (CRE), urea nitrogen (BUN), xanthine oxidase (XOD) and adenosine deaminase (ADA) in the body of a hyperuricemia model mouse, slow down the damage of high uric acid level to the heart, and relieve and / or improve hyperuricemia and related diseases.

[0049] The present application also provides a medicine for relieving and / or improving hyperuricemia and related diseases, wherein the active ingredient of the medicine comprises Inonotus obliquus extract.

[0050] The Inonotus obliquus extract is one or more of Inonotus obliquus water extract, Inonotus obliquus ethanol extract and Inonotus obliquus ethyl acetate extract.

[0051] In order to further illustrate the present application, the application of the Inonotus obliquus extract provided by the present application in the preparation of the medicine for relieving and / or improving hyperuricemia and related diseases is described in detail below in combination with the drawings and examples, but they should not be understood as limiting the protection scope of the present application.

[0052] Example 1

[0053] Preparation of Inonotus obliquus extract

[0054] 1. Inonotus obliquus water extract

[0055] Take 100 g of Inonotus obliquus, crush it into blocks, and add distilled water according to the solid-liquid ratio of 1 g:10 mL to decoct twice, each for 3 h. Combine the extract and filter it. Take the filtrate, heat and concentrate it to the state of extract, and then reduce it to dry powder by vacuum decompression. Grind it, weigh it, and obtain the Inonotus obliquus water extract, which is recorded as sample S1 and stored for use.

[0056] 2. Inonotus obliquus ethanol extract

[0057] (1) Take 100 g of Inonotus obliquus, crush it into blocks, and add ethanol solution with a volume concentration of 50% according to the solid-liquid ratio of 1 g:10 mL to extract by heating reflux twice, each for 3 h. Combine the two extract and filter it. Recover the solvent of the filtrate by a rotary evaporator and concentrate it to the state of wet extract. Then reduce it to dry powder by vacuum decompression. Grind it, weigh it, and obtain the Inonotus obliquus 50% ethanol extract, which is recorded as sample S2 and stored for use.

[0058] (2) Refer to the mode of step (1), replace the ethanol solution with a volume concentration of 50% with an ethanol solution with a volume concentration of 60% to extract, and finally obtain the Inonotus obliquus 60% ethanol extract, which is recorded as sample S3 and stored for use.

[0059] (3) Refer to the manner of step (1), replace the 50% ethanol solution with a 70% ethanol solution to extract, and finally obtain the 70% ethanol extract of Inonotus obliquus, recorded as sample S4, and store for later use;

[0060] (4) Refer to the manner of step (1), replace the 50% ethanol solution with an 80% ethanol solution to extract, and finally obtain the 80% ethanol extract of Inonotus obliquus, recorded as sample S5, and store for later use;

[0061] (5) Refer to the manner of step (1), replace the 50% ethanol solution with a 90% ethanol solution to extract, and finally obtain the 90% ethanol extract of Inonotus obliquus, recorded as sample S6, and store for later use;

[0062] 3. Organic solvent extract of Inonotus obliquus

[0063] Take 400 g of Inonotus obliquus, crush it into blocks, first add 4000 mL of 95% ethanol solution, heat and reflux to extract once for 3 h, filter the filtrate after extraction; add 4000 mL of 70% ethanol solution to the residue, heat and reflux to extract once for 3 h, combine the two extraction liquids and filter, and then concentrate the filtrate to an alcohol-free extract state (concentrate the extract with an appropriate amount of water to make it into a suspension state) using a rotary evaporator, and then suspend the extract in water to 200 mL to obtain a suspension liquid;

[0064] (1) Take the suspension liquid, add petroleum ether (60-90°C) to the suspension liquid at a solid-liquid ratio of 1:1 to extract until it is colorless, collect the petroleum ether extract layer, concentrate it to an extract state using a rotary evaporator, and then vacuum reduce pressure to a dry powder state, grind it, weigh it, and obtain the petroleum ether extract of Inonotus obliquus, recorded as sample S7, and store for later use;

[0065] (2) Take the suspension liquid, add chloroform to the suspension liquid at a solid-liquid ratio of 1:1 to extract until it is colorless, collect the chloroform extract layer, concentrate it to an extract state using a rotary evaporator, and then vacuum reduce pressure to a dry powder state, grind it, weigh it, and obtain the chloroform extract of Inonotus obliquus, recorded as sample S8, and store for later use;

[0066] (3) Take the suspension liquid, add ethyl acetate to the suspension liquid at a solid-liquid ratio of 1:1 to extract until it is colorless, collect the ethyl acetate extract layer, concentrate it to an extract state using a rotary evaporator, and then vacuum reduce pressure to a dry powder state, grind it, weigh it, and obtain the ethyl acetate extract of Inonotus obliquus, recorded as sample S9, and store for later use;

[0067] (4) Take the suspension, add n-butanol to the suspension at a material-liquid ratio of 1:1, extract until colorless, collect the n-butanol extract layer, concentrate to the extract state by a rotary evaporator, vacuum reduce to a dry powder, grind finely, weigh, obtain the Inonotus obliquus n-butanol extract, mark as sample S10, and reserve for use; heat and concentrate the remaining water phase part to dryness, mark as sample S11, and reserve for use.

[0068] Example 2

[0069] Concentration-effect determination of different samples on xanthine oxidase inhibition

[0070] 1. Establishment of in vitro incubation system

[0071] The in vitro incubation condition is maintained under a total amount of 5 mL, with a xanthine solution as the substrate, 800 μL of the substrate and xanthine oxidase are taken and placed in a sample bottle, shaken to start the reaction, the sample bottle is placed in a constant temperature water bath to fix the reaction time, after the reaction is completed, it is taken out and placed in 95℃ hot water for 5 min to terminate the reaction, and then 3.4 mL of PBS is used to make up to the mark and shake to the scale. The reaction solution is filtered through a fine filter head, and HPLC analysis is performed using an Agilent 6000 liquid chromatograph to detect the content of the reaction product uric acid.

[0072] In the enzymatic reaction process, the final concentration of xanthine oxidase is 0.06 U / mL, the final concentration of the substrate is 400 μmol·L -1 , the reaction time is 30 min, and the reaction temperature is 25℃;

[0073] The chromatographic conditions are as follows: mobile phase A (acetonitrile): mobile phase B water (containing 0.1% formic acid solution) = 3:97; elution time: 10 min; injection volume: 10 μL; flow rate: 0.8 mL / min; column oven: 30℃; uric acid detection wavelength: 293 nm.

[0074] 2. The concentration-effect determination of the samples (S1-S11) in Example 1 was performed, and the test solutions were diluted with PBS buffer to 1, 2, 4, 6, 8, and 10 mg / mL, respectively, to obtain 6 concentration gradients. 400 μL of each of the different concentrations of test solution was added to the reaction system of step 1 for injection analysis, and the absorption peak area (E) of different test solutions was determined. The average value was recorded in triplicate, the inhibition rate was calculated according to the following formula, and the results are shown in Table 1 and Figure 1 .

[0075] Inhibition rate (%) = (E 样品 -E 空白 ) / E 空白 ×100%

[0076] E 空白Absorbance peak area value of uric acid generated by the reaction system without adding the sample to be tested; E 样品 Absorbance peak area value of uric acid generated by the reaction system after adding the sample to be tested.

[0077] Table 1 Inhibitory effect of samples to be tested on xanthine oxidase

[0078]

[0079]

[0080] According to Table 1 and Figure 1 It can be seen that S1 (water extract), S2 (50% ethanol extract), S3 (60% ethanol extract), sample S4 (70% ethanol extract), sample S5 (80% ethanol extract), sample S6 (90% ethanol extract) and S9 (ethyl acetate extract) all have inhibitory activity on xanthine oxidase, and S9 shows obvious inhibitory activity. Since no absorbance peak area was detected for S7 (petroleum ether extract), S8 (trichloromethane extract), S10 (n-butanol extract) and S11 in the experiment, i.e. no xanthine oxidase inhibitory activity, they are not embodied in Table 1. Figure 1

[0081] Example 3

[0082] Animal experiment

[0083] 1. Animal model establishment

[0084] Potassium oxonate as a uric acid oxidase inhibitor inhibits the self-decomposition of uric acid in mice, and hypoxanthine as a supplementary synthetic uric acid precursor increases the synthesis of uric acid. After continuous drug intervention, the uric acid level of mice is maintained in a state of continuous increase. The present application uses the modeling mechanism of combined medication to select potassium oxonate and hypoxanthine to establish a hyperuricemia (HUA) mouse model, and the specific steps are as follows:

[0085] 48 6-8-week-old C57BL / 6J male mice (SPF level) were randomly divided into 8 groups according to body weight, 6 mice in each group, and adaptively fed for one week. The mice in each group were allowed to freely eat, and the light and dark alternation was maintained at 12h / 12h environment. All animal experiment operations met the requirements of the animal ethics committee of Ruian People's Hospital;

[0086] ① Blank control group (C): 0.5% sodium carboxymethyl cellulose (CMC-Na) solution was used for gavage at a dose of 0.3 mL / day;

[0087] ​② High uric acid model group (M): 300 mg / kg / day of potassium oxonate and 500 mg / kg / day of hypoxanthine were administered, wherein the potassium oxonate was administered by intraperitoneal injection and the hypoxanthine was administered by gavage;

[0088] ③ Positive control allopurinol group (BA): 5 mg / kg / day of allopurinol was administered by gavage;

[0089] ④ Positive control drug benzbromarone group (BB), 7.8 mg / kg / day of benzbromarone was administered by gavage;

[0090] ⑤ Low-dose Inonotus obliquus ethanol extract group (HT-A): 50 mg / kg / day of the 80% ethanol extract of Inonotus obliquus prepared in Example 1 was administered by gavage;

[0091] ⑥ High-dose Inonotus obliquus ethanol extract group (HT-B): 100 mg / kg / day of the 80% ethanol extract of Inonotus obliquus prepared in Example 1 was administered by gavage;

[0092] ⑦ Low-dose Inonotus obliquus ethyl acetate extract group (HC-A): 50 mg / kg / day of the ethyl acetate extract of Inonotus obliquus prepared in Example 1 was administered by gavage;

[0093] ⑧ High-dose Inonotus obliquus ethyl acetate extract group (HC-B): 100 mg / kg / day of the ethyl acetate extract of Inonotus obliquus prepared in Example 1 was administered by gavage.

[0094] Except for the blank control group (C), the remaining 7 groups of mice were administered with the high uric acid model group (M) at the same dose and administration method for 14 days, and on the 14th day, one mouse from each group was randomly taken out to determine the blood uric acid concentration by a kit to determine whether the model was established. After the modeling was successful, the corresponding drugs were administered according to the set group for intervention treatment, and all drugs were prepared using 0.5% CMC-Na as a solvent.

[0095] 2. General state observation of mice

[0096] The mental state, activity, diet state, and color change of the mice in each group were observed, and the body weight of the mice was monitored and recorded every week.

[0097] The results show that during the experimental period of 6 weeks, the body weight of the blank control group mice showed a normal upward trend, the mental state was good without any abnormalities, and the diet and excretion were normal without any abnormalities and the reaction was agile. Under the action of continuous drug administration, the body weight of the model group mice increased slowly, and after 4 weeks, it showed a downward trend. At the same time, the mental state was slightly abnormal compared with normal mice, the activity level was also reduced, and the fur color changed to brown-gray, the food intake decreased, and the water intake increased. Compared with the model group, the other drug administration groups showed different degrees of improvement. There was no significant difference in body weight between the positive drug group and the treatment drug group, and the trend was basically the same, with a small decrease in individual cases. Figure 2

[0098] 3 Heart ultrasound function detection

[0099] Before gavage and on the 28th day of gavage (a total of 42 days), the heart function of each group of mice was detected by ultrasound, and the specific operation was as follows:

[0100] The mouse chest and abdomen were depilated, and the isoflurane gas concentration in the anesthetic machine was adjusted to 2%. The mouse was anesthetized. After the induction of anesthesia was completed, the maintenance anesthesia concentration was set to 1-1.5%, and then the switch was converted to flow into the anesthesia mask. The mouse's head / nose was placed in the mask, and the state was confirmed to be good before the subsequent detection. After the coupling agent was applied, the probe was parallel to the mouse's midline and placed on its left chest, with the probe head pointing towards the mouse's head. At this time, the probe head was rotated 45° to the right, so that the probe was parallel to the mouse's left ventricular long axis. The picture obtained was the long axis section of the left ventricle. On the basis of the long axis, the probe head was rotated 90° to the left. The probe was moved regularly to display the papillary muscle level, and the image obtained was the short axis papillary muscle section of the left ventricle.

[0101] Left heart function test: 8 points were selected, namely diastolic interventricular septum upper wall, diastolic interventricular septum lower wall, diastolic free wall upper edge, diastolic free wall lower edge, systolic interventricular septum upper wall, systolic interventricular septum lower wall, systolic free wall upper edge, and systolic free wall lower edge. The ejection fraction (EF), short axis shortening rate (FS), diastolic left ventricular internal diameter (LVIDd), systolic left ventricular internal diameter (LVIDs), diastolic left ventricular wall thickness (LVPWd), systolic left ventricular wall thickness (LVPWs), diastolic left ventricular volume (LVEDV), and systolic left ventricular volume (LVESV) were measured.

[0102] ​The results showed that compared with the normal group, the heart function indexes of the model group mice, LVEF and E / A, decreased, indicating that the myocardium was damaged. After administration, the LVEF and E / A of the administration group were higher than those of the control group, among which the heart function indexes of the BB group, the HT-B group and the HC-B group increased significantly (P<0.05). It was proved that the high-dose groups of the ethanol extract and the ethyl acetate extract of Inonotus obliquus could improve the myocardial injury induced by high uric acid Figure 3 ).

[0103] 4. Effect of Inonotus obliquus on serum biochemical indexes of hyperuricemia mice

[0104] (1) Preparation of serum and tissue samples

[0105] After 28 days of intragastric administration according to step 1 (a total of 42 days), the mice in each group were anesthetized with isoflurane, blood was taken, centrifuged at 4℃ at 3000r / min for 20min, the supernatant was taken and stored at -80℃. After anesthesia, the mouse heart was taken, a part was fixed in 4% paraformaldehyde, the other was placed in liquid nitrogen and stored at -80℃ for later use.

[0106] (2) Determination of biochemical indexes in serum

[0107] ① Determination of uric acid (UA) content in mouse serum

[0108] According to the kit instructions (Nanjing Jiancheng Biological Engineering Institute), accurately add reagents and each determination sample, incubate at 37℃ for 10min, detect the absorbance value (510nm wavelength) by enzyme label instrument, and calculate the concentration value of uric acid in serum according to the formula in the instructions. The results showed that compared with the blank group mice, the UA value of the model group mice increased significantly (p<0.05), indicating that the model was successfully established; compared with the model group, after continuous administration of Inonotus obliquus ethanol extract and Inonotus obliquus ethyl acetate extract, the administration dose groups all played different degrees of pharmacological effect, significantly reduced the UA value of the mice (p<0.05), the high-dose group of Inonotus obliquus ethanol extract and the low-dose group of Inonotus obliquus ethyl acetate extract to a certain extent close to the UA value of the normal mice in the blank group, and the UA values of the mice in the two positive control drug groups also decreased to different degrees and tended to be lower than or lower than the UA value of the mice in the blank group (p<0.01) Figure 4 ).

[0109] ② Determination of creatinine (CRE) content in mouse serum

[0110] According to the kit instructions (Nanjing Jiancheng Bioengineering Institute) operation, accurate addition of reagents and each determination sample, incubated at 37℃ for 5 min, enzyme marker instrument detects absorbance value A1 (546 nm wavelength), then add reagent liquid two mix, incubated at 37℃ for 5 min, enzyme marker instrument detects absorbance value A2 (546 nm wavelength), according to the formula in the instructions to calculate the concentration of serum creatinine value. The results show that, compared with the blank group mice, the CRE value of model group mice appears extremely significant degree of increase (p<0.5), compared with the model group, after the continuous administration of inonotus obliquus ethanol extract and inonotus obliquus ethyl acetate extract treatment intervention, inonotus obliquus ethanol extract and inonotus obliquus ethyl acetate extract two dose groups all play different degree of pharmacological effect, significantly reduced the CRE value of mice (p<0.05), and to a certain extent close to the CRE value of normal mice in the blank group, while the two positive control drug group of mice, the CRE value also appears different degree of reduction and close to the CRE value of mice in the blank group (p<0.05), but the CRE of inonotus obliquus ethyl acetate extract low dose group has no significant difference Figure 5

[0111] ③Determination of urea nitrogen (BUN) content in mouse serum

[0112] According to the kit instructions (Nanjing Jiancheng Bioengineering Institute) operation, accurate addition of reagents and each determination sample, incubated at 37℃ for 10 min, enzyme marker instrument detects absorbance value (640 nm wavelength) according to the formula in the instructions to calculate the concentration of serum urea nitrogen value. The results show that, compared with the blank group mice, the BUN value of model group mice appears extremely significant degree of increase (p<0.5), compared with the model group, after the continuous administration of inonotus obliquus ethanol extract and inonotus obliquus ethyl acetate extract treatment intervention, inonotus obliquus ethanol extract and inonotus obliquus ethyl acetate extract two dose groups all play different degree of pharmacological effect, significantly reduced the BUN value of mice (p<0.05), and to a certain extent close to the BUN value of normal mice in the blank group, while the two positive control drug group of mice, BUN value also appears different degree of reduction and close to the BUN value of mice in the blank group (p<0.05) Figure 6

[0113] ④Determination of xanthine oxidase (XOD) content in mouse serum

[0114] ​​According to the kit instructions (Nanjing Jiancheng Bioengineering Institute), the reagents and samples were accurately added, incubated at 37℃ for 20 min, then mixed with reagent liquid, and the absorbance value A (530 nm wavelength) was detected by a microplate reader. The microplate reader was set at 530 nm wavelength to detect the absorbance value A, and the concentration of xanthine oxidase in serum was calculated according to the formula in the instructions. The results showed that compared with the blank group, the XOD activity value of the model group increased significantly (p<0.05). Compared with the model group, the two dose groups of ethanol extract and ethyl acetate extract of Inonotus obliquus showed different degrees of pharmacological effect, significantly reduced the XOD activity value of mice (p<0.05), and to a certain extent, close to the XOD activity value of the blank group. The XOD activity value of the two positive control drug groups also decreased to a certain extent and tended to be close to the XOD activity value of the blank group (p<0.05). Figure 7

[0115] ⑤Determination of the content of adenosine deaminase (ADA) in mouse serum

[0116] According to the kit instructions (Nanjing Jiancheng Bioengineering Institute), the reagents and samples were accurately added, incubated at 37℃ for 20 min, then mixed with reagent liquid, and the absorbance value A (530 nm wavelength) was detected by a microplate reader. The microplate reader was set at 530 nm wavelength to detect the absorbance value A, and the concentration of xanthine oxidase in serum was calculated according to the formula in the instructions. The results showed that compared with the blank group, the XOD activity value of the model group increased significantly (p<0.05). Compared with the model group, the two dose groups of ethanol extract and ethyl acetate extract of Inonotus obliquus showed different degrees of pharmacological effect, significantly reduced the XOD activity value of mice (p<0.05), and to a certain extent, close to the XOD activity value of the blank group. The XOD activity value of the two positive control drug groups also decreased to a certain extent and tended to be close to the XOD activity value of the blank group (p<0.05). Figure 8

[0117] 5. Heart histopathological observation

[0118] According to step 4, it can be seen that the administration of the model group caused obvious pathological changes in the biochemical indicators of mice, indicating that high uric acid status had a great impact on the visceral tissue. The biochemical indicators of mice in other treatment groups were significantly improved, indicating that Inonotus obliquus may have a certain protective effect on visceral tissue. Therefore, the heart pathological sections of hyperuricemic mice were observed, and the specific steps were as follows: ​​

[0119] 5.1 Paraffin-embedded section

[0120] (1) After the mouse heart tissue was fixed with 4% paraformaldehyde for 24-48 h, the tissue was loaded into an embedding box and rinsed with running water for more than 30 min;

[0121] (2) After dehydration, transparency and embedding in wax: the tissue was placed in 40% and 70% ethanol for 20 min each, 80% ethanol I, ethanol II, anhydrous ethanol I and anhydrous ethanol II for 10 min each, dimethylbenzene I and dimethylbenzene II for 5 min each, 56-58°C soft wax for 30 min, and hard wax embedding for 40 min;

[0122] (3) Embedding: the tissue was placed in the center of the embedding box to avoid air bubbles;

[0123] (4) Sectioning: after the tissue was embedded, it was cut into 3 μm thickness;

[0124] (5) Fishing, spreading and baking: spread at 50°C water bath, and bake on a slice baking machine for 1 h to prevent slice falling off.

[0125] 5.2 HE staining

[0126] (1) Section deparaffinization to water: the section was placed in dimethylbenzene I for 10 min, dimethylbenzene II, anhydrous ethanol I, anhydrous ethanol II, 95% ethanol, 90% ethanol, 85% ethanol for 5 min each, and rinsed with running water for 5 min;

[0127] (2) HE staining: after staining with hematoxylin solution for 2 min, rinse with a small water flow for 5 min, return to blue with 1xPBS for 2 min, rinse with running water for 5 min, and stain with eosin for 6 min, and rinse with running water for 5 min;

[0128] (3) Dehydration, transparency and fixation: the section was sequentially placed in 85%, 90%, 95% I, 95% II, 100% I, 100% II ethanol concentration for 10 min each, dimethylbenzene I and dimethylbenzene II for 3 min each, and sealed with neutral balsam, and observed under a microscope, and the results are shown in Figure 9 .

[0129] According to Figure 9 It can be seen that compared with the blank group, the model group has obvious inflammatory cell infiltration, disordered arrangement of myocardial cells, and dark nucleus. Compared with the model group, the allopurinol group and the benzbromarone group improved the inflammatory infiltration of the mouse heart; the Inonotus obliquus ethanol extract and the Inonotus obliquus ethyl acetate extract group also improved the inflammatory infiltration of the mouse heart, and the improvement degree did not reach that of the positive drug group.

[0130] Example 4

[0131] Effect of different samples on H9C2 cell toxicity

[0132] H9C2 cells in logarithmic growth phase were harvested, digested, counted, and then adjusted to a concentration of 3 × 10⁻⁶ cells using complete culture medium. 4 Inoculated at 100 μL per well in 96-well plates, the samples were randomly divided into four treatment groups: allopurinol (BA) positive control, benzbromarone (BB) positive control, ethanol extract of Inonotus obliquus (HT), and ethyl acetate extract of Inonotus obliquus (HC). Each treatment group included a blank control group (C), a high uric acid control group (M), and a drug-treated group. After 24 h of incubation, the original culture medium was discarded. The blank control group (C) was given normal culture medium, while the other treatment groups were given high uric acid stimulation with culture medium containing 2.5 μmol / L adenosine. After 24-48 h, to avoid the influence of serum growth factors and other conditions, the culture conditions were replaced with serum-free DMEM medium for serum starvation. After 12 h, the culture medium in each well was replaced with the drug-treated medium. The control group (C) was given DMEM (Free) medium; the positive control group (allopurinol) (BA), the positive control drug group (benzbromarone) (BB), the ethanol extract of Inonotus obliquus (HT), and the ethyl acetate extract of Inonotus obliquus (HC) were serially diluted with medium to concentrations of 0.14, 1.4, 14, 28, and 70 μM, with 100 μL added to each well, and 6 replicates for each concentration. After culturing in an incubator for 24 h, the original medium was aspirated, and 100 μL of free medium containing 10% CCK-8 reagent was slowly added to each well, avoiding the generation of air bubbles during the operation. The wells were then incubated in the dark for 2 h. After incubation, the OD value of each well was detected at a wavelength of 450 nm using a microplate reader. Cell viability was calculated according to the following formula, and the data were analyzed using Excel and GraphPad Prism. The results showed that there was no significant difference in cytotoxicity between the ethanol extract (HT) and the ethyl acetate extract (HC) of Inonotus obliquus within the concentration range of 0.14–70 μM, both of which were within the safe concentration range, indicating that they have low cytotoxicity and high safety. Figure 10 ).

[0133] Cell viability (%) = OD value measured in the experimental group / OD value measured in the blank group × 100%.

[0134] As can be seen from the above, Inonotus obliquus extract has high safety and can reduce the in vivo levels of UA, CRE, BUN, XOD, and ADA in hyperuricemia model mice. It can also mitigate the heart damage caused by high uric acid levels. Its mechanism of action may be to reduce uric acid synthesis by inhibiting XOD activity and reducing XOD content.

[0135] Although the above embodiments have been described in detail, it should be understood that these are only some embodiments of the present application, but not all embodiments. Other embodiments can be obtained based on the above embodiments without creativity, and these embodiments all belong to the protection scope of the present application.

Claims

1. Use of an extract of Inonotus obliquus in the preparation of a drug for relieving and / or improving hyperuricemia and related diseases; the extract of Inonotus obliquus is an ethyl acetate extract of Inonotus obliquus; a preparation method of the ethyl acetate extract of Inonotus obliquus comprises the following steps: Inonotus obliquus and an 80%-95% volume concentration ethanol aqueous solution are mixed according to a solid-liquid ratio of 1g:10-20mL, heated to reflux and extracted for 1-3 times, with a time of 3-5h, to obtain a first extract and a residue; the residue and a 50%-70% volume concentration ethanol aqueous solution are mixed, heated to reflux and extracted for 1-3 times, with a time of 3-5h, and then solid-liquid separation is performed to obtain a second extract; a volume ratio of the 50%-70% volume concentration ethanol aqueous solution to the 80%-95% volume concentration ethanol aqueous solution is 1:1; the first extract and the second extract are combined, concentrated to a extract state, resuspended to obtain a suspension; the suspension and ethyl acetate are mixed, extracted, and an ethyl acetate layer solution is collected; the ethyl acetate layer solution is concentrated to a extract state, dried to obtain the ethyl acetate extract of Inonotus obliquus; the related disease is myocardial disease caused by hyperuricemia.

2. Use according to claim 1, characterized in that, a volume ratio of the suspension to ethyl acetate is 1:1-3; a mass-volume ratio of Inonotus obliquus to the suspension is 2g:1-5mL.

3. Use according to claim 1 or 2, characterized in that, the extract of Inonotus obliquus relieves and / or improves hyperuricemia and related diseases by reducing a level of one or more of serum uric acid, creatinine, urea nitrogen, xanthine oxidase and adenosine deaminase.

4. A medicament for alleviating and / or ameliorating hyperuricemia and related diseases, characterized by comprising the compound of claim 1. an active ingredient of the drug comprises the extract of Inonotus obliquus; the extract of Inonotus obliquus is an ethyl acetate extract of Inonotus obliquus; a preparation method of the ethyl acetate extract of Inonotus obliquus comprises the following steps: Inonotus obliquus and an 80%-95% volume concentration ethanol aqueous solution are mixed according to a solid-liquid ratio of 1g:10-20mL, heated to reflux and extracted for 1-3 times, with a time of 3-5h, to obtain a first extract and a residue; the residue and a 50%-70% volume concentration ethanol aqueous solution are mixed, heated to reflux and extracted for 1-3 times, with a time of 3-5h, and then solid-liquid separation is performed to obtain a second extract; a volume ratio of the 50%-70% volume concentration ethanol aqueous solution to the 80%-95% volume concentration ethanol aqueous solution is 1:1; the first extract and the second extract are combined, concentrated to a extract state, resuspended to obtain a suspension; the suspension and ethyl acetate are mixed, extracted, and an ethyl acetate layer solution is collected; the ethyl acetate layer solution is concentrated to a extract state, dried to obtain the ethyl acetate extract of Inonotus obliquus; the related disease is myocardial disease caused by hyperuricemia.

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