NL2032836A

NL2032836A - Extract for treating hyperuricemia, and preparation method and application thereof

Info

Publication number: NL2032836A
Application number: NL2032836A
Authority: NL
Inventors: Li Yiping; Liu Chao; Sun Jinyue; Huang Yuhong; Sun Wenlong; Song Xinhua
Original assignee: Univ Shandong Technology; Shandong Qingyu Jiangxing Biotechnology Co Ltd
Priority date: 2022-04-01
Filing date: 2022-08-23
Publication date: 2023-10-24
Also published as: CN114515299A

Abstract

The present invention relates to the technical ﬁeld of traditional Chinese medicines, and speciﬁcally relates to an extract for treating hyperuricemia, and a preparation method and application thereof. The preparation method includes the following steps: @ mixing dry and ground pomegranate peels With an organic solvent aqueous solution at a volume concentration of 0-90%‚ extracting, and ﬁltering to obtain an extract solution I, @ placing the extract solution I onto a rotatory evaporator at 50-70°C to remove water and the organic solvent so as to obtain an oily mixture; @ mixing the oily mixture with an organic solvent; and @ centrifuging to remove the organic solvent, and drying to obtain a plant extract. The extract of the present invention has a good effect of reducing uric acid activity in hyperuricemia, can quickly and effectively lower a serum uric acid level; and can effectively improve renal function indicators.

Description

EXTRACT FOR TREATING HYPERURICEMIA, AND PREPARATION

METHOD AND APPLICATION THEREOF

TECHNICAL FIELD

[OI] The present invention relates to the technical field of traditional Chinese medicines, and particularly relates to a plant extract, and a preparation method and use thereof.

BACKGROUND ART

[02] Hyperuricemia (HUA) is caused by increased production and decreased excretion of uric acid in the human body, and is associated with abnormal metabolism of purine nucleotides in the body. The accumulation of uric acid in an HUA patient to a certain extent will cause a series of metabolic diseases in the body. For example, gouty arthritis will be caused. If the uric acid level in the blood rises, and the concentration of urate reaches saturation, crystals will be formed and accumulated in soft tissues to cause lesions and inflammatory responses, thereby producing joint deformities and tophi. For another example, uric acid nephropathy will be caused. Saturated serum urate will form crystals that are deposited in the kidney, causing kidney damage and even leading to renal insufficiency in severe cases. At present, new drugs for treating

HUA are constantly updated, traditional Chinese medicines can overcome the defects of strong side effects of western medicines to some extent, and thus it is urgent to develop a new traditional Chinese medicine for treatment.

[03] Gout is the most widely known complication caused by HUA. In the early stage, patients will experience joint redness and swelling. As the disease progresses, the pain will gradually intensify, and patients with severe conditions will develop tophi.

Tophi are clearly visible under the skin around patients auricle, joints, tendons, and soft tissues. Tophi formed in various parts of the body, especially the limbs, not only seriously affects the shape of the limbs, but also leads to joint deformities, dysfunction, pinched nerve, skin ulceration, and prolonged sinus tract unhealing, requiring surgical treatment.

[04] HUA is closely associated with multiple risk factors for cardiovascular disease.

Inflammation caused by uric acid will gradually lead to tissue and even systemic damage, and is a risk factor for hypertension, myocardial ischemia, stroke, and atherosclerosis. HUA is also closely associated with the occurrence, progression, prognosis of hypertension, and there are many common risk factors for the pathogenesis of the two diseases, such as obesity, dyslipidemia, and impaired glucose regulation.

[05] HUA will lead to kidney damage and lithangiuria. Damage caused by HUA, as an independent risk factor for chronic kidney disease, to renal endothelial functions is an important factor causing kidney damage. At present, kidney stones or inflammatory damage in renal interstitium caused by increased serum uric acid and urate crystal deposition, as well as high incidence of lithangiuria are major symptoms of HUA.

[06] Studies found that with the increasing in the national economy level and the changes in the dietary structure of modern people, the incidence of HUA is increasing year by year worldwide, especially in young and middle-aged males. According to new

HUA-related epidemiological data given in Expert Consensus for the Diagnosis and

Treatment of Patient with Hyperuricemia and High Cardiovascular Risk: 2021 update, patients with serum uric acid levels of greater than 6.0 mg/dl accounted for 32.3% of the total population, with 49.5% of males and 16.4% of females. HUA is the fourth high following the “three highs” (i.e. high blood pressure, high cholesterol, and high blood sugars), which is a chronic metabolic disease and is constantly affecting people’s life health and safety.

[07] At present, drugs for treating HUA on the market are mainly divided into drugs for inhibiting uric acid synthesis such as allopurinol and febuxostat, drugs for promoting excretion of uric acid such as benzbromarone and probenecid, drugs for breakdown of uric acid such as uricase, and drugs for treating acute gouty arthritis such as colchicine, non-steroidal anti-inflammatory drugs, IL-IR antagonists, and glucocorticoids. However, these drugs have more or less deficiencies in the treatment of HUA. Allopurinol will cause rash, nausea, vomiting, abdominal pain, diarrhea, fatigue, stabbing pain, etc., and febuxostat has common adverse effects of abnormal liver function, diarrhea, headache, nausea, rash, etc. Benzbromarone has an anti-gout effect similar to that of probenecid, but the two drugs have the risk of causing urinary stones and causing kidney disease, and may induce fulminant hepatitis, so the two drugs have certain side effects. The presence of uricase has the risk of readily inducing acute gouty, and this drug has antigenicity, which readily leads to hypersensitivity and drug tolerance in patients. The drugs for treating acute gouty arthritis can quickly alleviate symptoms of patients and control acute gout attack, but these drugs generally do not have an effect of lowering serum uric acid levels in patients Although there are many western medicines for treating HUA in clinical practice, there is a serious conflict between efficacy and side effects, and HUA is easy to relapse, so it is urgent to develop a traditional Chinese medicine with strong stability.

[08] According to traditional Chinese medical science, gout is a kind of bi syndrome, and a certain treatment effect can be achieved by traditional Chinese medicines administrated through the kidney channel, spleen channel or liver channel based on the treatment principle of invigorating the spleen and resolving turbidity, invigorating kidney and removing dampness, and clearing away heat and eliminating dampness. In the treatment of HUA and gout with traditional Chinese medicines, the traditional Chinese medicines reduce uric acid by multiple mechanisms, supplement each other, work together to achieve a treatment effect, and can overcome the defects of strong side effects of western medicines. Therefore, the Chinese medicine therapeutic regimen is more popular. Traditional Chinese medicine regulates “multiple targets” of a disease by “multiple ingredients”, which not only prevents the occurrence of gout, but also improves the body quality and delay or reduce lesions of important organs such as heart, brain, liver, and kidney, thereby treating the disease by lowering the uric acid level. China has a long history of treating HUA with traditional Chinese medicines. On the basis of accurate differentiation, the traditional Chinese medicines achieve a significant treatment effect and has unique clinical advantages. However,

patients need individualized treatment and traditional Chinese medicines are complex, active ingredients for treating HUA need to be explored, and thus application of traditional Chinese medicines in prevention and treatment of HUA and gout is limited.

[09] Pomegranate peel is the dry peel of pomegranate, which is irregular flaky or scooped, 1.5 to 3 mm thick, reddish-brown, yellow-brown or dark red on the outer surface, and slightly shiny, and has brown dots and many warts. In daily life and the production process of pomegranate juice, a pomegranate peel is usually considered as industrial waste, which accounts for about 30-40% of the mass of a fresh pomegranate fruit. The peel contains tannin, resin, mannitol, gum, gallic acid, malic acid, etc. It has the effects of astringing intestines, checking spermatorrhea, stopping bleeding, checking vaginal discharge, de-worming, and astringing sores, and is mainly used to treat prolonged diarrhea and dysentery, efflux diarrhea and spermatorrhea, intestinal wind bleeding, metrorrhagia and leukorrhea, worm accumulation abdominal pain, scabies, sores, and scalds. Therefore, it is usually used in food sterilization and preservation. Although pomegranate has many beneficial properties, so far, there is no relevant study, and it is necessary to further explore the pharmacological action of pomegranate so as to widely apply pomegranate to the treatment of human and animal diseases in clinic practice.

[10] Pomegranate peel has radical scavenging activity, so it has strong antioxidant and antibacterial effects. A pomegranate peel extract contains abundant tannins, which can coagulate or precipitate proteins when binding to proteins so as to promote local wound healing and avoid irritation. From a toxicological standpoint, pomegranate and its extracts are widely considered safe.

SUMMARY

[11] An objective of the present invention is to provide an extract for treating HUA, and a preparation method and application thereof in order to solve the problem that existing drugs for treating HUA have strong side effects. The extract has uric acid-reducing activity, can effectively interfere with kidney damage caused by high uric acid level, and effectively relieve disease symptoms caused by HUA.

[12] In order to solve the above technical problem, the present invention provides the following technical solutions.

[13] A preparation method of an extract for treating HUA includes the following 5 steps.

[14] (OD Mixing dry and ground pomegranate peels with an organic solvent aqueous solution at a volume concentration of 0-90% according to a mass-to-volume ratio of 1: (3-50) g/mL, extracting, and filtering to obtain an extract solution I,

[15] wherein the organic solvent is selected from at least one of methanol, ethanol, acetone, ethyl acetate, n-propanol, isopropanol, isobutanol, and butanediol,

[16] the extraction method is one or more of

[17] a. Aqueous extraction: in a water bath at 40-70°C for 120-240 min;

[18] b. Organic solvent aqueous solution extraction: with 70-80% ethanol at 40-70°C by Soxhlet extraction for 3-15 h;

[19] c. Ultrasonic extraction: with a 70-80% organic solvent at 40-70°C and 1-20 W by ultrasound for 30-120 min;

[20] ©) Placing the extract solution I onto a rotatory evaporator at 50-70°C to remove water and the organic solvent so as to obtain an oily mixture;

[21] © Mixing the oily mixture with an organic solvent according to a volume ratio of 1: (3-500), wherein the organic solvent is selected from petroleum ether and n-hexane; and the organic solvent is added to the oily mixture to remove impurities in the oily mixture;

[22] @ Centrifuging to remove the organic solvent, and drying to obtain a pomegranate peel extract containing a main ingredient of polyphenol.

[23] The pomegranate peel extract prepared by the above preparation method is used as a main medicinal ingredient.

[24] The pomegranate peels used in the present invention are dry peels of Punica granatum L. Pomegranate peel is sour, astringent, and warm, returns to the large intestine channel, has the effects of astringing intestines and checking diarrhea,

stopping bleeding, and de-worming, and is mainly used to treat prolonged diarrhea and dysentery, hemafecia, rectocele, metrorrhagia, leukorrhea, and worm accumulation abdominal pain. The research team of the present invention, in the process of exploring fast, efficient and low-toxic drugs for improving HUA, found that a pomegranate peel extract, i.e. pomegranate peel polyphenol, has a good effect of improving HUA, and thus completed the technical solutions of the present invention on this basis.

[25] The drug extract of the present invention can be prepared into different pharmaceutical forms as needed by conventional preparation methods known in the medical field. For example, the drug extract can be prepared into tablets including ordinary tablets, film-coated tablets, enteric coated tablets, etc. Drug extract dry powder is mixed with an appropriate amount of diluent selected from starch, dextrin, mannitol, and microcrystalline cellulose, an appropriate amount of adhesive selected from water, ethanol, cellulose, starch, and gelatin, an appropriate amount of disintegrant selected from sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, and sodium alginate, and a suitable lubricant selected from magnesium stearate, talc, and polyethylene glycol, a sweetener selected from D-xylose, xylitol, maltitol, stevioside, and aspartame is added, the mixture is prepared into grains by the conventional wet method, the whole grains are pressed into tablets after being dried, or the mixture is prepared into grains by the dry method and then pressed into tablets such as film-coated tablets, and the tablets are coated with a film-forming material selected from cellulose and polyethylene glycol by the conventional method, and sub-packaged into airtight bottles or aluminum-plastic panels. The drug extract can be prepared into capsules including ordinary capsules, enteric coated capsules, etc.

Drug extract dry powder is mixed with a suitable excipient selected from calcium carbonate, mannitol, magnesium oxide, and micronized silica gel, a suitable lubricant selected from talc, magnesium stearate, gylcol ester, and silicones, and a suitable adhesive selected from mineral oil and cooking oil, a suitable sweetener selected from

D-xylose, xylitol, maltitol, stevioside, and aspartame is added, and the mixture is prepared into dry powder or grains, filled into capsules, and sub-packaged into airtight bottles or aluminum-plastic panels.

[26] The drug extract of the present invention can be orally administrated to patients in need.

[27] Another objective of the present invention is to provide application of any one of the above drug extracts in preparation of a drug for treating HUA.

[28] According to the above application of the present invention, the extract is preferably used for preparing a drug for treating HUA and reducing uric acid activity.

[29] Compared with the prior art, the extract for treating HUA, and the preparation method and application thereof of the present invention at least have the following beneficial effects.

[30] The pomegranate peel extract of the present invention has a good effect of improving HUA, which is mainly manifested in quickly and effectively reducing the serum uric acid concentration, alleviate kidney damage caused by high uric acid concentration, and better lowering serum creatinine levels in HUA mice. [BI] The extract for treating HUA, and the preparation method and application thereof of the present invention will be further described below with reference to the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

[32] FIG. 11s a diagram showing measured uric acid levels in mice in experimental groups according to Example 5, wherein CON: blank group; HUA: model group;

Pun-L: low-dose pomegranate peel group; and Pun-H: high-dose pomegranate peel group.

[33] FIG. 2 is a diagram showing measured creatinine levels in the mice in the experimental groups according to Example 6, wherein CON: blank group; HUA: model group; Pun-L: low-dose pomegranate peel group; and Pun-H: high-dose pomegranate peel group.

[34] FIG. 3 is a diagram showing measured changes in body weight of the mice in the experimental groups according to Example 7, wherein CON: blank group; HUA:

model group, Pun-L: low-dose pomegranate peel group; and Pun-H: high-dose pomegranate peel group.

[35] FIG. 4 is a diagram showing measured morphology of kidney tissues of the mice in the experimental groups according to Example 8, wherein Al and A2: CON group; BI and B2: HUA group; Cl and C2: Pun-L group; and D1 and D2: Pun-H group.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[36] The following non-restrictive examples may help those of ordinary skill in the art more comprehensively understand the present invention, and are not intended to limit the present invention in any form.

[37] Pomegranate peels used in the present invention are purchased from different places of origin, and mainly from Anhui and Hebei.

[38] The grinder used in the present invention is a DFT-200 portable high-speed grinder for traditional Chinese medicines purchased from Wenling Linda Machinery

Co., Ltd.

[39] Example | Preparation of a pomegranate peel extract

[40] (OD Air-dried pomegranate peels were ground into powder by using a grinder, the powder was sieved by using a 60-mesh sieve, and 100 g of powder was weighed and wrapped in filter paper to obtain a medicine package.

[41] & An appropriate amount (enough to immerse the traditional Chinese medicine package in an extraction cylinder, and about 1/3 of the liquid remained in an extraction bottle) of petroleum ether was placed into a Soxhlet extractor, the medicine package was extracted at 40°C until the liquid in the extraction cylinder containing the medicine package became colorless and transparent to remove redundant grease from the pomegranate peels.

[42] ©) After grease was removed from the medicine package, the medicine package was air-dried and placed into a breaker, 700 mL of 75% ethanol aqueous solution was poured into the breaker, the breaker was sealed, and the mixture was placed overnight.

[43] @ A Soxhlet extraction device was assembled in sequence, the medicine package and a 75% ethanol aqueous solution were poured into the extractor at the same time, a heater was turned on, the medicine package was extracted at 70°C for 8 h until the 75% ethanol aqueous solution in the extractor became colorless and transparent, and an extraction solution was collected.

[44] ©) The extraction solution obtained at step © was placed onto a rotatory evaporator and subjected to rotary evaporation at 60°C until no ethanol remained in the extraction bottle, and the remaining extraction solution was collected and dried in a vacuum drier to remove redundant water so as to obtain dry powder.

[45] © The dry powder obtained at step ©) was dissolved in 75% ethanol, petroleum ether was added according to a ratio of 1: 1, the mixture was shaken up and down for 10 min, the petroleum ether layer was removed, and this step was repeated until no oil droplets appeared at the boundary between the ethanol and the petroleum ether to remove redundant grease so as to obtain an oil-free pomegranate peel extraction solution.

[46] (© The extraction solution obtained at step © was placed onto the rotatory evaporator and subjected to rotary evaporation at 60°C until no ethanol remained in the extraction bottle, and the remaining extraction solution was collected and dried in the vacuum drier to remove redundant water so as to obtain dry pomegranate peel extract powder.

[47] Example 2 Preparation of a pomegranate peel extract oral preparation 1

[48] 50 mg of pomegranate peel extract powder of Example 1 was accurately weighed and added to 10 mL of 0.5% sodium carboxymethyl cellulose (CMC-Na) aqueous solution, the mixture was uniformly mixed in a vortex shaker and subjected to ultrasonic treatment for 3 min to obtain a uniform suspension in which the final concentration of the pomegranate peel extract was 50 mg/mL.

[49] Example 3 Preparation of a pomegranate peel extract oral preparation 2

[50] 200 mg of pomegranate peel extract powder of Example | was accurately weighed and added to 10 mL of 0.5% CMC-Na aqueous solution, the mixture was uniformly mixed in a vortex shaker and subjected to ultrasonic treatment for 3 min to obtain a uniform suspension in which the final concentration of the pomegranate peel extract was 200 mg/mL.

[5] Comparative Example 1 Preparation of a benzbromarone oral preparation 1

[52] 20 mg of benzbromarone powder was accurately weighed and added to 10 mL of 0.5% CMC-Na aqueous solution, the mixture was uniformly mixed in a vortex shaker and subjected to ultrasonic treatment for 3 min to obtain a uniform suspension in which the final concentration of the benzbromarone suspension was 2 mg/mL.

[53] Example 4

[54] Properties of the drugs of Example 2, Example 3, and Comparative Example 1 were tested as follows.

[55] (1) Effect of the drug extract of Example 2 on the uric acid activity in zebrafish models of HUA

[56] Sexually mature wild-type 3-to-6-month-old zebrafishes were purchased from

China Zebrafish Resource Center. 10 female fishes and 20 male fishes were fed separately. After 2 weeks of acclimation, the fishes bred. Obtained fish eggs were transferred into embryo culture media and cultured for 5 d until juvenile fishes were obtained. The obtained juvenile zebrafishes were placed into a 6-well cell culture plate (30 fishes/well). Potassium oxonate (PO) and xanthine sodium salt (XSS) that served as drugs for increasing uric acid were added. After samples were added, an embryo culture medium for fish eggs was added to make up the volume of the liquid in each well to 15 mL, and the juvenile fishes were fed at a constant temperature of 28.5°C for 1d.

[57] The drug was added as follows:

[58] blank group: 30 zebrafishes were added to 15 mL of embryo culture medium and cultured in an incubator at 28.5°C for 24 h;

[59] model group: 30 zebrafishes were added to 30 uL of 100 mM PO and 30 pL of 5 mM XSS, an embryo culture medium was added to make up the volume of the liquid to 15 mL, and the zebrafishes were cultured in the incubator at 28.5°C for 24 h;

[60] pomegranate peel extract group 1 (50% pomegranate peel extract at a concentration of 10 pg/mL): 30 zebrafishes were added to 30 uL of 100 mM PO and 30 uL of 5 mM XSS, 3 uL of 50% pomegranate peel extract at a concentration of 50 mg/mL was added, an embryo culture medium was added to make up the volume of the liquid to 15 mL, and the zebrafishes were cultured in the incubator at 28.5°C for 24 h;

[61] pomegranate peel extract group 2 (50% pomegranate peel extract at a concentration of 50 pg/mL). 30 zebrafishes were added to 30 uL of 100 mM PO and 30 pL of 5 mM XSS, 15 uL of 50% pomegranate peel extract at a concentration of 50 mg/mL was added, an embryo culture medium was added to make up the volume of the liquid to 15 mL, and the zebrafishes were cultured in the incubator at 28.5°C for 24 h;

[62] pomegranate peel extract group 3 (50% pomegranate peel extract at a concentration of 100 pg/mL): 30 zebrafishes were added to 30 uL of 100 mM PO and 30 uL of 5 mM XSS, 30 pL of 50% pomegranate peel extract at a concentration of 50 mg/mL was added, an embryo culture medium was added to make up the volume of the liquid to 15 mL, and the zebrafishes were cultured in the incubator at 28.5°C for 24 h; and

[63] different concentrations between the above concentrations were set to determine the optimum concentration.

[64] Results are shown in the table below.

Experimental groups UA (pg/mL)

Blank group 6.51+0.56

Model group 9.69+0.66 10 ng/mL pomegranate peel extract 4.32+0.57 pg/mL pomegranate peel extract 4.16+0.83 100 ug/mL pomegranate peel extract 6.50+0.52

[65] The results show that the pomegranate peel extract can reduce the uric acid activity in the zebrafish models of HUA to a certain extent. Furthermore, there is a significant statistical difference between the pomegranate peel extract group 2 (pomegranate peel extract at a concentration of 50 pg/mL) and the model group, which indicates that the pomegranate peel extract at a concentration of 50 pg/mL has an obvious effect of reducing the uric acid activity.

[66] (2) Effect of the drug of Comparative Example 1 on uric acid levels in mouse models of HUA

[67] 40 SPF male Kunming mice (18-22 g) purchased from Shandong Animal

Experiment Center were normally fed at a temperature of 21-25°C, a relative humidity of 55-65%, and a photoperiod of 12 h, and had free access to water and food. After one week of acclimation, models were constructed. During 5 weeks of modelling, the dosage of PO was gradually increased from 250 mg/kg on the first day to S00 mg/kg at a daily increase rate of 50 mg/kg. Mice injected with PO according to a dosage of 500 mg/kg and freely drinking 5% fructose were taken as mouse models of HUA.

[68] 5% fructose: 50 g of fructose was dissolved in 1 L of water to obtain 5% fructose water.

[69] PO is insoluble in water. Therefore, PO was added to 0.9% normal saline to obtain a suspension at a concentration of 50 mg/mL, and then the suspension was intraperitoneally injected into the mice.

[70] The drug was added as follows:

[71] blank group: each mouse was injected with 0.2 mL of 0.9% normal saline and normally ate food and drank water;

[72] model group: each mouse was injected with a PO solution according to a dosage of 500 mg/kg, freely drank 5% fructose water, and normally ate food; and

[73] benzbromarone group: each mouse was injected with a PO solution according to a dosage of 500 mg/kg, freely drank 5% fructose water, intragastrically administered with benzbromarone according to a dosage of 20 mg/kg, and normally ate food.

[74] After 5 weeks of continuous administration, the mice were fasted for 12 h, urine was collected, eyeballs were taken, blood was collected, and plasma was separated out for biochemical indicator analysis. The mice were killed by decapitation, liver tissues were quickly taken out and washed three times with a 0.9% sodium chloride solution, and liver and kidney tissue samples were preserved in liquid nitrogen or formalin for later use. After 5 weeks of continuous administration, the mice were fasted for 12 h, urine was collected, eyeballs were taken, blood was collected, and plasma was separated out for biochemical indicator analysis. Uric acid levels in the mice were measured by using uric acid (UA) assay kits.

[75] Results are shown in the table below.

Groups Serum uric acid SUA (mmol/L)

Blank group 69.39+11.89

Model group 118.24+23.10

Benzbromarone group 60.72+£27.71

[76] The results show that there is an extremely significant difference between the benzbromarone group and the HUA mouse model group, which indicates that benzbromarone effectively reduces the uric acid activity in the mouse models of HUA, and has an obvious effect of reducing the uric acid activity.

[77] (3) Effect of the drug of Example 3 on biochemical indicators of mouse models of HUA

[78] 40 SPF male Kunming mice (18-22 g) purchased from Shandong Animal

[79] 5% fructose: 50 g of fructose was dissolved in 1 L of water to obtain 5% fructose water.

[80] PO is insoluble in water. Therefore, PO was added to 0.9% normal saline to obtain a suspension at a concentration of 50 mg/mL, and then the suspension was intraperitoneally injected into the mice.

[81] Grouping and administration were performed as follows:

[82] A group (Blank group CON): each mouse was injected with 0.2 mL of 0.9% normal saline and normally ate food and drank water;

[83] B group (model group HUA): each mouse was injected with a PO solution according to a dosage of 500 mg/kg, freely drank 5% fructose water, and normally ate food;

[84] C group (low-dose pomegranate peel group Pun-L): each mouse was injected with a PO solution according to a dosage of 250 mg/kg, freely drank 5% fructose water, intragastrically administered with the pomegranate peel extract according to a dosage of 50 mg/kg, and normally ate food; and

[85] D group (high-dose pomegranate peel group Pun-H): each mouse was injected with a PO solution according to a dosage of 250 mg/kg, freely drank 5% fructose water, intragastrically administered with the pomegranate peel extract according to a dosage of 200 mg/kg, and normally ate food.

[86] After 5 weeks of continuous administration, the mice were fasted for 12 h, urine was collected, eyeballs were taken, blood was collected, and plasma was separated out for biochemical indicator analysis. The mice were killed by decapitation, liver tissues were quickly taken out and washed three times with a 0.9% sodium chloride solution, and liver and kidney tissue samples were preserved in liquid nitrogen or formalin for later use. Serum uric acid levels and urine uric acid levels in the mice were measured by using uric acid (UA) assay kits so as to determine an effect of the pomegranate peel extract on reduction of the uric acid activity in the mice.

Serum creatinine levels and urine creatinine levels in the mice were measured by using creatinine (Cr) ELISA assay kits and blood urea nitrogen (BUN) ELISA assay kits.

[87] Table 1 Comparison of the serum uric acid levels in the mice in the groups after treatment with the pomegranate peel extract

Experimental groups SUA (mmol/L)

Blank group 69.31+11.66

Model group 118. 16422. 964##

Low-dose pomegranate peel group 83.23+24.94%

High-dose pomegranate peel group 57.35+15.09%**$

[88] #P<0.05, ##p<0.01, ###p<0.001 (vs blank group)

[89] *P<0.05, **p<0.01, ***p<0.001 (vs model group)

[90] $P<0.05, $$p<0.01, $$$p<0.001 (vs low-dose pomegranate peel group)

[91] Comparison results of the serum uric acid levels in the mice in the groups after treatment with the pomegranate peel extract are shown in Table 1. The results show that serum uric acid (SUA) values of the mice in the model group are 118.16+22.96 umol/L, and there is an extremely significant statistical difference (p<0.001) between the model group and the blank group, which indicates that the SUA levels in the mice in the model group obviously raise. There is a statistical difference (p<0.05) between the low-dose pomegranate peel group and the model group, and there is an extremely significant statistical difference (p<0.001) between the high-dose pomegranate peel group and the model group, which indicates that SUA values of the mice in the low-dose pomegranate peel group and the high-dose pomegranate peel group are decreased, and the SUA values of the mice in the high-dose pomegranate peel group are extremely obviously decreased. Furthermore, there is a statistical difference (p<0.05) between the low-dose pomegranate peel group and the high-dose pomegranate peel group, which indicates that the SUA levels in the mice in the low-dose pomegranate peel group are controlled, and the SUA levels in the mice in the high-dose pomegranate peel group are better controlled.

[92] Table 2 Comparison of the uric acid levels in the mice in the groups after treatment with the pomegranate peel extract

Groups UUA (mmol/L)

Blank group 50.97+10.05

Model group 104.61+8.02##

Low-dose pomegranate peel group 66.1845.48**

High-dose pomegranate peel group 17.72+4 2#*** $$

[93] #P<0.05, ##p=<0.01, ###p<0.001 (vs blank group)

[94] *P<0.05, **p<0.01, ***p<0.001 (vs model group)

[95] $P<0.05, $$p<0.01, $$$p<0.001 (vs low-dose pomegranate peel group)

[96] Comparison results of the urine uric acid levels in the mice in the groups after treatment with the pomegranate peel extract are shown in Table 2. The results show that urine uric acid (UUA) values of the mice in the model group are 104.61+8.02 umol/L, and there is a significant statistical difference (p<0.01) between the model group and the blank group, which indicates that the UUA levels in the mice in the model group obviously raise. There is a statistical difference (p<0.05) between the high-dose pomegranate peel group and the blank group, there is a significant statistical difference (p<0.01) between the model group and the low-dose pomegranate peel group, and there is an extremely significant statistical difference (p<0.001) between the model group and the high-dose pomegranate peel group, which indicates that the UUA levels in the mice in the low-dose pomegranate peel group and the high-dose pomegranate peel group are obviously lowered, and the UUA levels in the mice in the high-dose pomegranate peel group are extremely obviously lowered. There is a significant statistical difference (p<0.01) between the low-dose pomegranate peel group and the high-dose pomegranate peel group, which indicates that the UUA levels in the mice in the low-dose pomegranate peel group are controlled, and the UUA levels in the mice in the high-dose pomegranate peel group are better controlled.

[97] It can be seen from the measurement results of the uric acid levels shown in

Table 1 and Table 2 that there is a significant statistical difference or an extremely significant statistical difference between the model group and the blank group, and there are also different degrees of difference between the model group and the low-dose pomegranate peel group, and between the model group and the high-dose pomegranate peel group, which indicates that the uric acid activity in the mice in the low-dose pomegranate peel group is controlled, and the uric acid activity in the mice in the high-dose pomegranate peel group is better controlled.

[98] Table 3 Comparison of the serum creatinine levels in the mice in the groups after treatment with the pomegranate peel extract

Experimental groups SCr (mmol/L)

Blank group 67.98+4.167

Model group 81.40+19.01

Low-dose pomegranate peel group 63.62+3.93*

High-dose pomegranate peel group 66.23+8.66

[99] _ #P<0.05, ##p<0.01, ###p<0.001 (vs blank group)

[100] *P<0.05, **p<0.01, ***p<0.001 (vs model group)

[101] $P<0.05, $$p<0.01, $$$p<0.001 (vs low-dose pomegranate peel group)

[102] It can be seen from the measurement results of Scr shown in Table 3 that the

Scr levels in the mice in the model group, the low-dose pomegranate peel group, and the high-dose pomegranate peel group are similar to those in the mice in the blank group, and there is no significant difference (p>0.05). There is a statistical difference (p<0.05) between the low-dose pomegranate peel group and the model group. The above results show that the Scr levels in the mice in the low-dose pomegranate peel group are effectively lowered.

[103] Table 4 Comparison of the urine creatinine levels in the mice in the groups after treatment with the pomegranate peel extract

Experimental groups UCr (mmol/L)

Blank group 849.97+4.81

Model group 426.68+10.06###

Low-dose pomegranate peel group 808.85+21.26*

High-dose pomegranate peel group 885.10+66.36*

[104] #P<0.05, ##p<0.01, ###p<0.001 (vs blank group)

[105] *P<0.05, **p<0.01, ***p<0.001 (vs model group)

[106] $P<0.05, $$p<0.01, $$$p<0.001 (vs low-dose pomegranate peel group)

[107] Measurement values of the urine creatinine levels in the mice in the groups after treatment with the drug are shown in Table 4. The results show that there is an extremely significant statistical difference (p<0.001) between the model group and the blank group. There are statistical differences (p<0.05) between the low-dose pomegranate peel group and the model group, and between the high-dose pomegranate peel group and the model group, which indicates that the UCr levels in the mice in the low-dose pomegranate peel group and the high-dose pomegranate peel group have a tendency to raise. It indicates that the UCr levels in the mice in the low-dose pomegranate peel group and the high-dose pomegranate peel group are affected.

[108] It can be seen from the measurement results of the creatinine levels shown in

Table 3 and Table 4 that there is a large difference between the model group and the blank group in the creatinine level, after treatment with the drug, the creatinine levels in the mice in the administration groups approach those in the mice in the blank group, and there is a statistic difference (P<0.05) between the low-dose pomegranate peel group and the model group. Compared with the model group, the SCr levels in the mice in the low-dose pomegranate peel group and the high-dose pomegranate peel group are lowered, and the UCr levels in the mice raise, which indicates that the creatinine levels in the mice in the low-dose pomegranate peel group and the high-dose pomegranate peel group are effectively controlled and normalized.

[109] Table 5 Comparison of the body weight of the mice in the groups after treatment with the pomegranate peel extract

Experimental Body weight (g) 3079239 an

[110] Weekly comparison results of the body weight of the mice in the groups after treatment with the drug are shown in Table 5. It can be seen from the measurement results that with the increase of modelling time, there is a statistic difference (p<0.05) between the model group and the blank group, and there is no difference among other groups.

[111] Example 5 Effect of the drug extract on uric acid levels in mice in experimental groups

[112] The mice in the administration groups used for the drug property test (3) of

Example 4 were administrated with drug for continuous 5 weeks, blood was collected from the eye, placed into a centrifuge tube containing an anticoagulant, and centrifuged at 2,000 rpm and 4°C for 10 min, and upper serum was taken for biochemical indicator measurement.

[113] The obtained serum was treated in accordance with the instruction of the uric acid assay kit (purchased from Beijing Solarbio Co., Ltd.), and the measurement principle was as follows: uricase catalyzed the breakdown of uric acid to allantoin,

CO:, and H,0,, H20: oxidized Fe?" in potassium ferrocyanide to produce Fe’, Fe’* further reacted with 4-aminoantipyrine and phenol to produce red quinones, a characteristic absorption peak appeared at 505 nm, and an uric acid level was calculated by measuring absorbance at 505 nm. Results are shown in FIG. 1.

[114] There is a significant statistical difference or an extremely significant difference between the model group and the blank group, and there are different degrees of difference between the model group and the low-dose pomegranate peel group, and between the model group and the high-dose pomegranate peel group, which indicates that the uric acid activity in the mice in the low-dose pomegranate peel group is controlled, and the uric acid activity in the mice in the high-dose pomegranate peel group is better controlled

[115] Example 6 Effect of the drug extract on creatinine levels in mice in experimental groups

[116] The serum obtained in Example 4 was treated in accordance with the instruction of the mouse creatinine (Cr) ELISA assay kit (purchased from Beijing

Solarbio Co., Ltd.). Experimental results are shown in FIG. 2.

[117] There is a large difference between the model group and the blank group in the creatinine level, after treatment with the drug, the creatinine levels in the mice in the administration groups approach those in the mice in the blank group, and there is a statistical difference (P<0.05) between the low-dose pomegranate peel group and the model group. Compared with the model group, the SCr levels in the mice in the low-dose pomegranate peel group and the high-dose pomegranate peel group are lowered, and the UCr levels in the mice raise, which indicates that the creatinine levels in the mice in the low-dose pomegranate peel group and the high-dose pomegranate peel group are effectively controlled and normalized.

[118] Example 7 Effect of the drug extract on the body weight of mice in experimental groups

[119] Experimental results are shown in FIG. 3. With the increase of modelling time, there is a statistical difference (p<0.05) between the model group and the blank group, and there is no difference among other groups.

[120] Example 8 Effect of the drug extract on the morphology of kidney tissues of mice in experimental groups

[121] Kidney tissues of the mice used for the drug property test (3) of Example 4 were histomorphologically sectioned. Section results are shown in FIG. 4.

[122] HE staining results show that:

[123] CON group: the renal tubular structure is normal, there is no abnormality, and there is no accumulation of crystals; the renal cortical structure is normal, the wall of the proximal convoluted tubule is relatively thick, the lumen surface has visible brush border, the cells in the distal convoluted tubule cells are arranged regularly, and there are no inflammatory cells in the interstitium;

[124] HUA group: mesangial matrices of the glomerulus are slightly increased and have an atrophic structure; the wall of the renal tubule is slightly thickened, the wall of the renal tubule is incomplete, the cells in the proximal convoluted tubule are vacuolated and partially exfoliated, the cells in the distal convoluted tubule are separated and arranged irregularly, the blood vessels in the interstitium are reduced, and there is inflammatory cells infiltration.

[125] Pun-L and Pun-H groups: the lesions of the renal tubule and glomerulus are reduced, the cytoplasm is clear, the swelling of the renal tubule is reduced, the border of the adjacent tubules is obvious, and necrotic proximal tubules become less. It indicates that the drug extract reduces kidney damage to different extents.

[126] Example 8 Study on acute toxicity of the pomegranate peel extract to mice

[127] 20 Kunming male mice (18-22 g purchased from Shandong Animal

Experiment Center) were randomly divided into two groups (10 mice/group) and administrated with the drug as follows:

[128] the first group: the pomegranate peel extract was intragastrically administrated to each mouse once a day according to a dosage of 50 mg/(kg*d) for 7 d; and

[129] the second group: the pomegranate peel extract was intragastrically administrated to each mouse every 8 h a day according to a dosage of 200 mg/(kg*d) for 28 d.

[130] The body weight of each mouse was recorded and the mental state of each mouse was observed throughout the experiment. After the experiment was completed, the experimental mice were killed for pathological analysis.

[131] Results show that throughout the experiment, all the mice are in good mental state and develop normally, none of the mice died, and no toxicity is observed.

[132] The above examples are only preferred implementation modes of the present invention, and are not intended to limit the scope of the present invention. Various changes and improvements made by those of ordinary skill in the art without departing from the design spirit of the present invention shall fall within the scope of protection defined by the claims of the present invention.

Claims

Conclusions

1. Method of preparing an extract for treating hyperuricemia, characterized in that the method comprises the following steps: (D mixing dry and ground pomegranate peels with an aqueous solution of an organic solvent with a volume concentration of 0-90%, extracting and filtering it to obtain an extract solution I; @ placing the extract solution I in a rotary evaporator at 50-70°C to remove water and the organic solvent and obtain an oily mixture; ©) mixing the oily mixture with an organic solvent; @ centrifugation to remove the organic solvent, and drying to obtain a plant extract.

A method of preparing an extract for treating hyperuricemia according to claim 1, characterized in that in step (1) the pomegranate peels are mixed with the aqueous solution of the organic solvent according to a mass-to-volume ratio of 1: (3 -50) g/mL.

Method of preparing an extract for treating hyperuricemia according to claim 2, characterized in that in step (15) the organic solvent is selected from at least one of methanol, ethanol, acetone, ethyl acetate, n-propanol, isopropanol, isobutanol and butanediol .

A method of preparing an extract for treating hyperuricemia according to claim 3, characterized in that in step {1} the extraction method is one or more of the following:

a. an aqueous extraction: in a water bath at 40-70 °C for 120-240 minutes;

b. an extraction with an aqueous solution of organic solvent: with 70-80% ethanol at 40-70 °C by Soxhlet extraction for 3-15 hours; and c. ultrasonic extraction: with an organic solvent of 70-80% at 40-70 °C and 1-20

W by ultrasound for 30-120 minutes.

A method of preparing an extract for treating hyperuricemia according to claim 4, characterized in that in step (3) the oily mixture is mixed with the organic solvent in a volume ratio of 1:(3-500).

A method of preparing an extract for treating hyperuricemia according to claim 5, characterized in that in step 3 the organic solvent is selected from petroleum ether and n-hexane.

7. Extract prepared by the preparation method according to any one of claims

1-6.

Pharmaceutical composition characterized in that it comprises the extract according to claim 7 and a pharmaceutically acceptable carrier and/or excipient.

Pharmaceutical composition according to claim 8 characterized in that pharmaceutical forms of the pharmaceutical composition include tablets, capsules, drop pills and particles

Use of the preparation method according to any one of claims 1-6 or the extract according to claim 7 for the preparation of a medicament for treating hyperuricemia.