CN109394753B - Application of diosmetin in preparation of medicine for preventing and/or treating hyperuricemia nephropathy - Google Patents

Abstract

The invention belongs to the field of biological medicines, and particularly relates to application of diosmetin in preparation of a medicine for preventing and/or treating hyperuricemia nephropathy. The geraniol is used singly or the geraniol and the allopurinol are combined, so that the serum uric acid of a hyperuricemia nephropathy mouse is reduced, the serum urea nitrogen and the creatinine level of the mouse can be obviously reduced, the rise of the kidney index of the mouse caused by the hyperuricemia can be reduced, and the spleen index is also obviously increased, which indicates that the kidney function damage of the mouse caused by the rise of the serum uric acid can be reversed while the uric acid of the hyperuricemia nephropathy mouse is reduced by combining the geraniol and the allopurinol, so that the kidney function of the hyperuricemia mouse is protected, and the immune system of the hyperuricemia mouse is protected.

Description

Application of diosmetin in preparation of medicine for preventing and/or treating hyperuricemia nephropathy

Technical Field

The invention belongs to the field of biological medicine, and particularly relates to application of diosmetin in preparation of a medicine for treating hyperuricemia nephropathy.

Background

In recent years, the incidence of asymptomatic hyperuricemia and hyperuricemic nephropathy has increased significantly with the increase in the living standard and the change in dietary structure of people. Approximately 2/3 uric acid produced by the human body is excreted with urine through the kidney, and therefore, hyperuricemia may cause a decrease in renal function. Uric acid nephropathy means that a large amount of urate crystals deposit in a kidney collecting pipe, a renal pelvis and a ureter in a short time and block the renal tubules, so that the proximal end of the renal tubules expands, renal function is acutely and severely damaged, and acute renal failure is caused. At present, the medicines for treating hyperuricemia nephropathy are mainly western medicines, mainly include xanthine oxidase inhibitors, such as: allopurinol, febuxostat, and the like; or drugs that promote uric acid excretion, such as: probenecid, benzbromarone, and the like. However, the above drugs have serious side effects such as: renal toxicity, anaphylaxis, cardiovascular toxicity, hepatorenal toxicity, etc., which have caused serious limitations on the clinical use of these drugs. In recent years, the traditional Chinese medicine has low toxic and side effects, definite curative effect and strong superiority, so that the traditional Chinese medicine is widely regarded for treating the uric acid nephropathy.

Disclosure of Invention

Aiming at the defects that the existing medicine for treating hyperuricemia nephropathy has large toxic and side effects, is not easy to be tolerated by patients and is limited in clinical use, the invention aims to develop the high-efficiency and low-toxicity medicine for resisting the hyperuricemia nephropathy so as to solve the toxicity problem of the existing medicine used clinically and improve the tolerance and the compliance of the patients. The invention firstly provides that the flavone compound diosmetin can treat hyperuricemia nephropathy, and diosmetin and allopurinol have the synergistic effect of reducing uric acid and the effect of restoring renal function, and the injection administration route has better curative effect than the gastric lavage administration route.

In order to achieve the above object, the first aspect of the present invention provides a use of diosmetin or a pharmaceutically acceptable salt, solvate thereof for the preparation of a medicament for preventing and/or treating hyperuricemic nephropathy.

Preferably, the medicament is in the form of injection.

Preferably, the medicament further comprises one or more pharmaceutically acceptable carriers.

In a second aspect of the present invention, a pharmaceutical composition is provided, wherein the active ingredients of the pharmaceutical composition are diosmetin and allopurinol.

Preferably, the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers.

Preferably, the pharmaceutical composition is in the form of injection.

In the present invention, the term "pharmaceutical composition" refers to a pharmaceutical composition containing two active ingredients, and the use of the two active ingredients is not limited, and the two active ingredients may be mixed and then prepared into a medicament, or the two active ingredients may be prepared into medicaments respectively and then combined together.

In the above drugs or pharmaceutical compositions, the pharmaceutically acceptable carrier includes diluents, excipients, fillers, emulsifiers, binders, lubricants, absorption enhancers, surfactants, disintegrants, or antioxidants, which are conventional in the pharmaceutical field. The pharmaceutically acceptable carrier may also include flavoring agents, sweetening agents, preservatives or coloring agents.

The pharmaceutically acceptable carrier may be selected from: mannitol, sorbitol, sodium metabisulfite, sodium bisulfite, sodium thiosulfate, cysteine hydrochloride, thioglycolic acid, soybean lecithin, vitamin C, vitamin E, EDTA disodium, calcium sodium EDTA, monovalent alkali metal carbonates, acetates, phosphates or aqueous solutions thereof, hydrochloric acid, acetic acid, sulfuric acid, phosphoric acid, amino acids, sodium chloride, potassium chloride, sodium lactate, ethylparaben, benzoic acid, potassium sorbate, chlorhexidine acetate, xylitol, maltose, glucose, fructose, dextran, starch, sucrose, lactose, mannitol, silicon derivatives, cellulose and derivatives thereof, alginates, gelatin, polyvinylpyrrolidone, glycerol, tween 80, agar, calcium carbonate, calcium bicarbonate, surfactants, polyethylene glycol, cyclodextrin, phospholipid materials, kaolin, talc, calcium stearate or magnesium stearate.

The third aspect of the present invention provides the use of the above pharmaceutical composition in the preparation of a medicament for the prevention and/or treatment of hyperuricemic nephropathy.

Allopurinol is an isomer of hypoxanthine, is a suicide substrate of XOD, and can be competitively combined with the XOD substrate to the active site molybdopterin of XOD so as to prevent the hypoxanthine from being metabolized into uric acid, so that allopurinol becomes a first-line uric acid generation inhibiting drug with wide clinical application. The results of in vitro experiments to determine the influence of diosmetin on xanthine oxidase show that diosmetin can obviously inhibit the activity of xanthine oxidase in vitro, and the half inhibitory concentration is less than that of positive allopurinol, namely that the action of diosmetin in vitro on inhibiting the activity of xanthine oxidase is stronger; the determination of an inhibition type experiment shows that diosmetin is competitive inhibition on the inhibition type of xanthine oxidase. Therefore, diosmetin is a natural inhibitor of high-efficiency xanthine oxidase. In recent years, diosmetin has been reported to improve renal injury after renal ischemia/reperfusion, but no research report on uric acid effect and uric acid nephropathy is found. The research result of the invention shows that after the diosmetin is administrated to the abdominal cavity, the serum uric acid level of a mouse with hyperuricemia nephropathy can be obviously reduced, and the serum urea nitrogen and creatinine levels can be obviously reduced, which shows that the diosmetin can reverse the damage of the mouse kidney function caused by hyperuricemia, thereby protecting the kidney function of the mouse with hyperuricemia. And the geraniin is injected into the abdominal cavity with low and medium dosage groups, so that the increase of the mouse kidney index caused by hyperuricemia can be reduced, the mouse kidney is protected, and meanwhile, the spleen index is increased, which shows that the geraniin has a protection effect on the immune system of the hyperuricemia mouse.

The geraniol and allopurinol are combined, so that the serum uric acid of a hyperuricemia nephropathy mouse is reduced, the serum urea nitrogen and creatinine level of the mouse can be obviously reduced, the rise of the mouse kidney index caused by hyperuricemia can be reduced, and the spleen index is obviously increased. Diosmetin administration has no obvious influence on liver index, thymus index and body weight average of mice, and shows that diosmetin is a compound with low toxicity.

Additional features and advantages of the invention will be set forth in the detailed description which follows.

Drawings

The above and other objects, features and advantages of the present invention will become more apparent by describing in more detail exemplary embodiments thereof with reference to the attached drawings.

FIG. 1 shows a Lineweaver-Burk double reciprocal equation for diosmetin.

Detailed Description

Preferred embodiments of the present invention will be described in more detail below. While the following describes preferred embodiments of the present invention, it should be understood that the present invention may be embodied in various forms and should not be limited by the embodiments set forth herein.

The present invention was studied using in vitro and in vivo methods.

1. Inhibition of xanthine oxidase by diosmetin in vitro

1.1 materials

1.1.1 drugs and reagents

Diosmetin (purity > 97%), lot number D17081604, from Dierg pharmaceutical science, Inc., Nanjing; dipotassium phosphate (analytically pure) purchased from Tianjin Tianli chemical reagents, Inc.; anhydrous potassium dihydrogen phosphate (analytically pure) purchased from Guangfu Fine chemical research institute, Tianjin; EDTA-2Na (analytically pure) from Biosharp company; xanthine (98% pure, batch No. D1606030), available from Allantin reagents, Inc.; xanthine Oxidase (XOD), batch No.: L04M8Y30480, available from Shanghai-derived leaf Biotech, Inc.; dimethyl sulfoxide (analytical grade) was purchased from chemical reagents of national pharmaceutical group, ltd.

1.1.2 Experimental instruments

AL204 electronic analytical balance (mettler-toledo corporation); XH-C vortex mixer (Tan City medical Instrument factory); pH-3C type acidimeters (Aohaus instruments, Inc.); EnSpire microplate reader (Perkinelmer).

1.2 methods

1.2.1 measurement of inhibition Rate

An improved ultraviolet spectrophotometer method is adopted to determine the inhibition effect of diosmetin on xanthine oxidase activity in vitro (Zhu-deep silver, Zhou-Yuan, Liu Qing mountain and the like. establishment and application of a xanthine oxidase inhibitor high-flux screening model [ J ]. China pharmaceutical journal, 2007, 42(3): 187-190). Diosmetin and allopurinol were dissolved in DMSO, respectively, and diluted with phosphate buffer (pH 7.4) to concentrations of 200 μmol/L, 100 μmol/L, 50 μmol/L, 25 μmol/L, 12.5 μmol/L, 6.25 μmol/L for use. The substrate xanthine was dissolved in 0.1mol/L sodium hydroxide, then adjusted to pH 7.4 with dilute hydrochloric acid and finally diluted to the desired level with phosphate buffer (pH 7.4). Xanthine oxidase was prepared into a solution of the desired concentration using a phosphate buffer solution having a pH of 7.4, and was used.

Adding 100 μ L of sample and blank solution (blank is PBS), 50 μ L of enzyme solution (4U/mL), sequentially into 96-well plate, each concentration is 4-well, and loading in microplate readerIncubation was carried out at 37 ℃ for 3min, and 50. mu.L of the substrate xanthine (0.12. mu. mol/L) was added to initiate the enzymatic reaction, with final concentrations of both diosmetin and allopurinol of 100. mu. mol/L, 50. mu. mol/L, 25. mu. mol/L, 12.5. mu. mol/L, 6.25. mu. mol/L, 3.625. mu. mol/L. The absorbance A was recorded every 20s at an absorbance wavelength of 295nm for 10 min. And performing linear regression by using the absorbance and time to obtain the slope of a regression curve, namely the reaction rate of the xanthine oxidase for oxidizing the xanthine to generate the uric acid. The inhibition rate of the drug on XOD was calculated by the following formula: inhibition ratio (%) ═ 1- (b/a)]× 100%, a is the reaction rate of blank control group, b is the reaction rate of drug group, the inhibition rate and concentration are used as regression curve, and finally the IC is calculated by the software GraphPad Prism 6₅₀。

1.2.2 determination of type of inhibition

Concentration of immobilized enzyme (1U/mL), substrate set up 5 concentration gradients: 1.92. mu. mol/L, 0.96. mu. mol/L, 0.48. mu. mol/L, 0.24. mu. mol/L, 0.12. mu. mol/L. The reaction rates of diosmetin (50. mu. mol/L, 3.625. mu. mol/L) at different substrate concentrations were determined in the same manner as in 1.2.1, respectively. And (4) performing Lineweaver-Burk double reciprocal equation mapping according to the substrate concentration and the reaction rate to judge the inhibition type of diosmetin.

1.3 results

1.3.1 inhibition of XOD by diosmetin

As shown in Table 1, the inhibition rates of geraniol and allopurinol were gradually increased and XOD activity was gradually decreased as the concentrations of geraniol and allopurinol were increased. IC of diosmetin and allopurinol₅₀The concentration is respectively 5.83 mu mol/L and 49.60 mu mol/L, which shows that the inhibition effect of diosmetin on XOD is obviously stronger than that of allopurinol.

TABLE 1 inhibition of XOD by diosmetin

3.2 analysis of the XOD inhibition types by diosmetin

As shown in fig. 1, according to the Lineweaver-Burk reciprocal double equation 1/V ═ Km/(Vmax [ S ]) +1/Vmax mapping, the Lineweaver-Burk reciprocal double curves of different concentrations of diosmetin and blank groups intersect almost at the same point on the Y axis, indicating that Vmax is unchanged, i.e. the presence of diosmetin does not cause a change in the maximum reaction rate Vmax of xanthine oxidase; the slope of the curve increases with the increase of the diosmetin concentration, indicating that the michaelis constant Km increases, showing that the type of inhibition of xanthine oxidase by diosmetin is competitive inhibition.

2. Protective effect of diosmetin on hyperuricemic renal injury mice

2.1 materials of the experiment

2.1.1 Experimental animals

SPF-level Kunming mice, male, with a weight of 20-22 g, provided by the Experimental animal center of Huazhong university of science and technology. Animal production license number: SCXK (jaw) 2016-.

2.1.2 drugs and reagents

Allopurinol (batch No. Y27O8C46913), available from Shanghai-derived leaf Biotech, Inc.; diosmetin (purity not less than 97%, batch No. D17081604) available from south kyo dielg pharmaceutical science ltd; potassium oxonate (batch No. P137112), available from Shanghai Allantin Biotech, Inc.; yeast extract (lot 20170802), available from biotechnology responsibility of Obo Star, Beijing; a uric acid determination kit (batch No. 20181017), a xanthine oxidase test kit (batch No. 20180920), a urea nitrogen kit (batch No. 20181024), a creatinine kit (batch No. 20181022) and a Coomassie brilliant blue kit (batch No. 20180921) are all purchased from Nanjing to build a bioengineering institute; DMSO (batch No. 20161109) was purchased from national pharmaceutical group Chemicals, Inc.

2.1.3 Experimental instruments

AL204 electronic analytical balance (mettler-toledo corporation); SPS2001F electronic balance (aohaus corporation); TGL-16C bench centrifuge (Shanghai' an pavilion scientific instruments factory); UV-2000 ultraviolet spectrophotometer (ewnikov); FSH-2A Adjustable high speed homogenizer (Instrument works, gold Tan).

2.2 methods

2.2.1 animal grouping and administration

80 male Kunming mice were divided into 8 groups of 10 mice each by weight after adaptive feeding for 2 days. The group was divided into a normal group, a model group, an allopurinol group (5mg/kg), a diosmetin intraperitoneal injection low dose group, a medium dose group, a high dose group (10mg/kg, 20mg/kg, 40mg/kg), a diosmetin lavage group (20mg/kg), diosmetin (20mg/kg) + allopurinol (5 mg/kg). Beginning to perform intraperitoneal injection administration in morning, injecting physiological saline into a normal group and a model group, injecting corresponding drugs into the abdominal cavity of a diosmetin low-dose group and a diosmetin high-dose group, respectively administering the corresponding drugs, continuously administering 15 days, performing intragastric administration for 0.5% CMC-Na 6 days before the allopurinol group, and finally performing intragastric administration for 9 days to obtain the corresponding allopurinol dose. The volume of the medicine for intraperitoneal injection is 0.15ml/10g, and the volume of the medicine for intragastric administration is 0.2ml/10 g.

2.2.2 establishment of hyperuricemia Kidney Damage animal model

A mouse hyperuricemia kidney damage model is established by adopting yeast paste intragastric administration and oteracil potassium intraperitoneal injection. Except for the normal group, the mice in other groups were intragastrically injected with 30g/kg of yeast extract every day for 15 days continuously, and finally injected with 300mg/kg of Potassium Oxonate in the abdominal cavity for 1 day, except for the allopurinol group, the intragastrically injected volume of the other groups was 0.2ml/10g, the intraabdominal injection volume was 0.15ml/10g, and the intragastrically injected volume of the allopurinol group was 0.1ml/10 g. 1h after the intraperitoneal injection of diosmetin, the gastric lavage yeast solution is injected into other groups except the normal group, and finally the oteracil potassium solution is injected into the abdominal cavity after the 1d of yeast extract gastric lavage.

2.2.3 preparation of pharmaceutical solutions

Diosmetin was dissolved in DMSO and diluted with physiological saline to prepare a diosmetin suspension (containing 5% DMSO) at 0.67mg/mL, 1.33mg/mL, and 2.67 mg/mL.

0.5 percent of CMC-Na is used as a solvent to prepare 0.5mg/mL allopurinol suspension, 3g/mL yeast extract suspension, 1.5g/mL yeast suspension and 20mg/mL oteracil potassium suspension respectively.

2.2.4 Biochemical index determination

The mice were observed and weighed daily, and the relative change rate (%) of the body weight per mouse was calculated as (weight per weighed body weight/body weight at the start of the experiment) × 100. And (3) taking blood from eyeballs of mice in normal groups, model groups, allopurinol groups and diosmetin groups 1h after the administration of the drug at noon, centrifuging at 3500r/min for 10min, taking supernatant, freezing and storing at-20 ℃ for measuring serum uric acid, BUN and Cr levels, carrying out cervical dislocation to kill the mice, weighing the whole kidney, liver, thymus and spleen, and calculating the organ index.

2.3 results

2.3.1 Effect of diosmetin on Kidney function of mice with hyperuricemia

The results are shown in Table 2. As can be seen from Table 2, compared with the normal group, the levels of serum uric acid, urea nitrogen and creatinine of the mice in the model group are remarkably increased (P is less than 0.01), which indicates that the hyperuricemia of the mice can be caused by combining yeast extract with oteracil potassium and the kidney is damaged; compared with a model group, the serum uric acid level of a mouse in the positive medicine group is remarkably reduced (P is less than 0.01), which shows that allopurinol can reduce the serum uric acid of the mouse, but the serum urea nitrogen and creatinine levels of the mouse have no statistical significance (P is more than 0.05), which shows that allopurinol has no protective effect on the kidney; the mouse serum uric acid can be remarkably reduced (P is less than 0.01) by injecting the diosmetin in the abdominal cavity at low dose, the mouse serum uric acid level can be remarkably reduced (P is less than 0.05) by injecting the diosmetin in the abdominal cavity at medium dose and high dose, and the mouse serum urea nitrogen and creatinine water level can be remarkably reduced (P is less than 0.01) by injecting the diosmetin in the abdominal cavity at the same time, which shows that the mouse serum uric acid with hyperuricemia can be reduced and the kidney of the mouse serum uric acid can be protected to a certain extent by injecting the diosmetin in the abdominal cavity; the mouse serum uric acid can be reduced remarkably by administering diosmetin through gavage (P is less than 0.01), but the average of mouse serum urea nitrogen and creatinine water has no statistical significance (P is more than 0.05), which indicates that the mouse serum uric acid with hyperuricemia can be reduced by the gavage administration, but the kidney is not protected; the combination of the diosmetin and the allopurinol has the advantages that the average of serum uric acid and urea nitrogen level is remarkably reduced (P is less than 0.01), and the serum creatinine level is remarkably reduced (P is less than 0.05), so that the combination of the diosmetin and the allopurinol can reduce mouse uric acid and simultaneously has a certain protection effect on the kidney of the mouse.

Table 2 influence of diosmetin on serum uric acid, urea nitrogen, creatinine levels in hyperuricemia mice (n ═ 10)

In comparison with the normal group,^#P<0.05，^##P<0.01; in comparison with the set of models,^*P<0.05，^**P<0.01. ig: performing intragastric administration; ip: and (5) carrying out intraperitoneal injection.

2.3.2 Effect of Geraniin on the index of visceral organs in hyperuricemia Kidney-compromised mice

The results are shown in Table 3. As shown in Table 3, the renal index of the model group is significantly increased (P <0.05) compared with that of the normal group, which indicates that the kidney of the hyperuricemia mice is increased, and the modeling mode has certain damage to the kidney. Compared with the model group, the kidney index of the mice in the positive medicine group has no statistical difference (P is more than 0.05), which shows that allopurinol has no protective effect on the kidney injury of the mice with hyperuricemia; the kidney index of mice in a diosmetin low-dose group is obviously reduced (P <0.05), and the kidney index of mice in a dose group in diosmetin is extremely obviously reduced (P <0.01), which indicates that diosmetin has a certain protective effect on kidney damage of mice with hyperuricemia. When the diosmetin and the allopurinol are combined, the mouse kidney index is remarkably reduced (P is less than 0.01), which indicates that the combined use of the two medicines can play a role in protecting the kidney of a hyperuricemia mouse. Compared with the normal group, the renal indexes of the diosmetin low dose, the diosmetin medium dose and the combination of the diosmetin and allopurinol have no statistical difference (P >0.05), which indicates that the diosmetin low dose, the diosmetin medium dose and the combination of the diosmetin can restore the uric acid induced increase of the renal index of mice to normal mouse levels. Compared with the normal group, the spleen index of the model group is obviously reduced (P <0.05), which indicates that the modeling mode has damage to the immune system of the mouse. Compared with the model group, the spleen index of the diosmetin at low dose and high dose is remarkably increased (P <0.01), and the spleen index of diosmetin at medium dose is remarkably increased (P <0.05), which indicates that diosmetin plays a protective role on the immune system of a hyperuricemia mouse; the geraniol and the allopurinol are combined, so that the spleen index of a mouse is remarkably increased (P is less than 0.01), and the protective effect of the combination of the geraniol and the allopurinol on the immune system of the mouse with the hyperuricemia is shown; compared with the normal group, the spleen indexes of the diosmetin low, medium and high dose groups and the combination of diosmetin and allopurinol have no statistical difference (P >0.05), which indicates that diosmetin can restore the spleen index of hyperuricemia kidney-damaged mice to the normal level of the mice. Compared with a model group, the kidney index and the spleen index of diosmetin gavage have no statistical difference (P is more than 0.05), which indicates that gavage administration has no protective effect on the kidney damage of mice with hyperuricemia and has no effect on the immune system of mice with hyperuricemia.

TABLE 3 Effect of diosmetin on mouse organ index (n ═ 10)

In comparison with the normal group,^#P<0.05，^##P<0.01; in comparison with the set of models,^*P<0.05，^**P<0.01. ig: performing intragastric administration; ip: and (5) carrying out intraperitoneal injection.

2.3.3 Effect of Geraniin on XOD Activity in hyperuricemia Kidney-compromised mice

The results are shown in Table 4. As can be seen from Table 4, the XOD activity was significantly increased in the model group (P <0.05) compared to the normal group, indicating that the hyperuricemia mice were able to increase the XOD activity. Compared with the model group, the XOD activity of the positive medicine group, the abdominal cavity injection geraniol low, medium and high dose group, the enema geraniol group and the combination group of geraniol and allopurinol are all reduced, but the statistical difference is not generated (P is more than 0.05).

TABLE 4 Effect of diosmetin on xanthine oxidase activity in mouse liver (n ═ 10)

In comparison with the normal group,^#P<0.05. ig: performing intragastric administration; ip: and (5) carrying out intraperitoneal injection.

2.3.4 Effect of Geraniin on the body weight of hyperuricemia Kidney-compromised mice

As can be seen from Table 5, the relative body weight of the model group mice decreased in comparison with the normal group mice, but the difference was not statistically significant (P > 0.05). The relative weights of mice in the diosmetin low, medium and high dose groups, the diosmetin gavage group and the diosmetin + allopurinol combined group are compared with the model group, and the difference has no statistical significance (P is more than 0.05).

TABLE 5 Effect of diosmetin on the body weight of mice with hyperuricemia (n ═ 10)

ig: performing intragastric administration; ip: and (5) carrying out intraperitoneal injection.

Having described embodiments of the present invention, the foregoing description is intended to be exemplary, not exhaustive, and not limited to the embodiments disclosed. Many modifications and variations will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments.

Claims (3)

1. A pharmaceutical composition comprises diosmetin and allopurinol as active ingredients; the dosage form of the pharmaceutical composition is injection.

2. The pharmaceutical composition of claim 1, further comprising one or more pharmaceutically acceptable carriers.

3. Use of the pharmaceutical composition according to any one of claims 1-2 for the preparation of a medicament for the prevention and/or treatment of hyperuricemic nephropathy.

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