Application of humulus lupulus total flavone extract in preparation of uric acid reducing medicine

Abstract

The invention discloses an application of a humulus lupulus total flavone extract in preparation of a uric acid reducing medicine. According to the invention, a process route of ethanol extraction and macroporous resin impurity removal is adopted to prepare the humulus lupulus total flavone extract, and the uric acid reducing effect of the humulus lupulus total flavone extract is researched and confirmed for the first time, so that the humulus lupulus total flavone extract can be used for preparing uric acid reducing medicines for preventing and treating hyperuricemia related diseases such as hyperuricemia, gout, uric acid nephropathy and the like. The hyperuricemia related diseases are metabolic diseases, high in morbidity, long in intervention period and large in demand on uric acid reducing drugs, and the hops general flavone extract is derived from hops which can be used as food additive raw materials, so that the hops general flavone extract is good in safety and high in acceptability, and has good development and application prospects.

Description

Technical Field

[0001] The present invention relates to the application of total flavonoid extract of hops in the preparation of drugs for reducing uric acid, for preventing and treating hyperuricemia-related diseases such as hyperuricemia, gout, uric acid nephropathy, etc. Background Art

[0002] Uric acid in the human body is the end metabolite of purine compounds. If purine metabolism is disordered, it can lead to an increase in the level of uric acid (UA) in the blood. If a person's fasting blood uric acid level is measured twice on different days under a normal purine diet, for men, it is higher than 420 μmol·L -1 , and for women, it is higher than 360 μmol·L -1 , which is hyperuricemia (High uric acid, HUA). When the body is in a state of hyperuricemia, blood uric acid can form sodium urate crystals and deposit locally in joints, inducing local inflammatory reactions and tissue damage, which is gout; too high blood uric acid can also deposit in the kidneys, causing acute nephropathy, chronic interstitial nephritis or kidney stones, which is called uric acid nephropathy. Existing studies have shown that hyperuricemia-related diseases such as hyperuricemia, gout, uric acid nephropathy, etc. have a high incidence rate, and are independent risk factors for diseases such as chronic kidney disease, hypertension, cardiovascular and cerebrovascular diseases, and diabetes. The harm is serious, and effective intervention is needed in a timely manner.

[0003] At present, for the prevention and treatment of hyperuricemia-related diseases such as hyperuricemia, gout, uric acid nephropathy, etc. clinically, drugs for reducing uric acid need to be used to control the blood uric acid level. At present, commonly used clinical drugs for reducing uric acid include: (1) Drugs that inhibit uric acid synthesis: including allopurinol and febuxostat. Allopurinol is the first-line drug of choice for reducing uric acid, but it may cause allergies, leukopenia, etc. Febuxostat is a new type of drug for reducing uric acid, suitable for the long-term treatment of hyperuricemia in gout patients, but it is not applicable to asymptomatic patients, and there are also reports of adverse reactions such as liver damage, and the price is relatively expensive. (2) Drugs that promote uric acid excretion: mainly include benzbromarone and probenecid. These drugs mainly inhibit the reabsorption of uric acid by the renal tubules and promote uric acid excretion to achieve the purpose of reducing blood uric acid. However, while these drugs promote the excretion of uric acid in the kidneys, they are prone to form urate crystals or uric acid stones in the kidneys, causing kidney damage, and there is a risk of aggravating the existing kidney damage in patients with hyperuricemia-related diseases. In short, there is an urgent need for safe, effective, less side-effect and low-cost drugs for reducing uric acid clinically, for preventing and treating hyperuricemia-related diseases such as hyperuricemia, gout, uric acid nephropathy, etc. And because hyperuricemia-related diseases are metabolic diseases with a high incidence rate and a long intervention period, the demand for products with both drug and food homology is greater and the acceptance is higher.

[0004] Chinese medicinal hops are the female inflorescences (immature flower spikes with fruits) of Humulus lupulus L. of the genus Humulus in the family Cannabaceae, which have the effects of strengthening the stomach and promoting digestion, calming the nerves and promoting diuresis, and can be used to treat indigestion, abdominal distension, pulmonary tuberculosis, cystitis, neurasthenia, and insomnia. There are many reports on the chemical constituents of hops. The flavonoid components represented by xanthohumol and isoxanthohumol have received more attention and have effects such as antioxidant, anti-osteoporosis, and hypoglycemic effects.

[0005] The inventors have been engaged in the development of hops resources and pharmacological research for a long time, especially focusing on the research and development of new uses based on translational medicine. It was first discovered and confirmed that xanthohumol, a representative flavonoid component contained in hops, has a significant uric acid-lowering effect. However, due to the low content of xanthohumol in hops, the extraction and preparation process is relatively complex, and the large-scale production cost is high. To overcome this drawback, the inventors further extracted and prepared the total flavonoid extract of hops, and further studied and confirmed that this total flavonoid extract also has a good uric acid-lowering effect, and thus can be used to prepare uric acid-lowering drugs for the prevention and treatment of hyperuricemia-related diseases such as hyperuricemia, gout, and uric acid nephropathy. And because the preparation process of the total flavonoid extract of hops is simple, the cost is lower, it is more suitable for industrialization, and the value of transformation and application is greater. Since hyperuricemia-related diseases are metabolic diseases with a high incidence and a long intervention period, there is a large demand for uric acid-lowering drugs. In addition, this total flavonoid extract of hops is derived from hops that can be used as food additive raw materials, with good safety and high acceptance, so it has good development and application prospects. Summary of the Invention

[0006] According to the embodiments, the present invention hopes to propose the application of the total flavonoid extract of hops in the preparation of uric acid-lowering drugs for the prevention and treatment of hyperuricemia-related diseases such as hyperuricemia, gout, and uric acid nephropathy.

[0007] The inventors have been engaged in the development of hop resources and pharmacological research for a long time. Previously, they have discovered and confirmed that xanthohumol, a representative flavonoid component contained in hops, has a significant uric acid-lowering effect, and have applied for Chinese Patent CN202410245325.X and published academic papers (1. Han, J., Xia, T., Jiang, Y., Fan, Q., Li, K., Zhao, Z., Xin, H. Regulation of serum uric acid level and bone metabolism by xanthohumol in hyperuricemic rats. Acta Academiae Medicinae Militaris Tertiae, 2024, 45(3): 260-267; 2. Han, J., Xia, T., Jiang, Y., et al. Effect of Xanthohumol from Humulus lupulus L. Against Gouty Bone Damage in Arthritis of Rats Induced by Mono-sodium Urate. Cell Biochem Biophys (2024). https: / / doi.org / 10.1007 / s12013-024-01429-z).

[0008] However, since xanthohumol is a monomer component and its content in hops is not high, the extraction and preparation process is relatively complex and the large-scale production cost is high. To overcome this drawback, the inventors further extracted and prepared the total flavonoid extract of hops containing xanthohumol, and further studied and confirmed that it also has a good uric acid-lowering effect. At present, there are many research reports on the preparation process of the total flavonoid extract of hops, and many patents have been involved and disclosed (application numbers CN201410460743.7, CN201510027779.0, CN201310024052.8, CN201510220128.3). Most of them use ethanol with a concentration greater than 50% for extraction, and then use non-polar macroporous resins (there are various models of macroporous resins available for optimized selection in the market, such as D300L, AB-8, ADS-7, D101, HPD826, S-8) for impurity removal (to obtain the total flavonoid extract). In recent years, the present inventors have also adopted the process route of "ethanol extraction + macroporous resin impurity removal" to prepare the total flavonoid extract of hops, and then studied and confirmed its uric acid-lowering effect, which can be used for the preparation of uric acid-lowering drugs for the prevention and treatment of hyperuricemia-related diseases.

[0009] Subsequent examples will prove that the total flavonoid extract of hops has a significant uric acid-lowering effect and can be used for the preparation of uric acid-lowering drugs.

[0010] When the hop total flavonoids extract is used to prepare uric acid-lowering drugs, it can be directly suspended, dissolved, diluted, or prepared into a suitable preparation form with pharmaceutically acceptable pharmaceutical excipients. The pharmaceutically acceptable carrier refers to conventional drug carriers in the pharmaceutical field, such as: diluents, excipients such as water, fillers such as starch, sucrose, etc.; binders such as cellulose derivatives, alginates, gelatin and polyvinyl pyrrolidone; wetting agents such as glycerol; disintegrants such as agar, calcium carbonate and sodium bicarbonate; absorption promoters such as quaternary ammonium compounds; surfactants such as hexadecanol; adsorption carriers such as kaolin and soap clay; lubricants such as talc, calcium and magnesium stearate, and polyethylene glycol, etc. In addition, other adjuvants such as flavoring agents, sweeteners, etc. can also be added to the composition. The suitable preparation form of the present invention means that, for oral administration, it can be made into conventional solid preparations such as tablets, powders, granules, capsules, etc., or liquid preparations such as water or oil suspensions, or other liquid preparations such as syrups, elixirs, etc.; for parenteral administration, it can be made into solutions for injection, water or oil suspensions, etc. Preferred forms are tablets (including coated tablets, flash-release tablets), capsules, granules, and oral solutions.

[0011] In short, the hop total flavonoid extract is derived from hops for both medicinal and edible purposes, has good safety, high acceptance, and is very suitable and feasible for being made into functional foods or food additives, such as various forms of beverages, oral liquids, compressed candies, jellies, biscuits, coffee, etc. In view of the high incidence, great harm, long intervention cycle, and great demand for uric acid-lowering drugs of hyperuricemia-related diseases in modern society, the hop total flavonoid extract of the present invention has good development and application prospects, and contains huge social and economic benefits. DETAILED DESCRIPTION

[0012] The present invention will be further described below in conjunction with specific embodiments. These embodiments should be understood to be only used to illustrate the present invention and not to limit the scope of protection of the present invention. After reading the contents of the present invention, those skilled in the art may make various changes or modifications to the present invention, and these equivalent changes and modifications also fall within the scope defined by the claims of the present invention.

[0013] Example 1 Preparation of total flavonoids extract from hops

[0014] 400 g of Humulus lupulus medicinal materials were used, and 12 times the 70% ethanol aqueous solution (V / V) was used as the solvent. The extraction was carried out by refluxing 3 times, each time for 1 hour. The extract was filtered while it was hot, and the extracts were combined. The solvent was recovered under reduced pressure until there was no alcohol smell. The relative density was adjusted to 1.05, and the supernatant was obtained by centrifugation. It was adsorbed on a D101 macroporous resin column (the ratio of diameter to height was 1:7.5), washed with 5 BV of water to remove impurities, and then eluted with 50% ethanol aqueous solution. The eluate was collected, concentrated and dried to obtain the total flavonoid extract. By comparing and identifying this total flavonoid extract with HPLC and LC-MS methods, it was found that this total flavonoid extract contained flavonoid components such as xanthohumol, quercetin, and kaempferol. The total flavonoid content was determined by the sodium nitrite-aluminum nitrate colorimetric method to be 64.30%. (The process parameters of the macroporous resin were set with reference to the literature: Zhu Jia, Hu Yueyue, Ge Liang. Study on the process of enriching total flavonoids from Humulus lupulus in Xinjiang by macroporous resin. Chinese Journal of Ethnomedicine and Ethnopharmacy, 2017, 26(4): 20-22)

[0015] Example 2 Pharmacological Experiment on the Hypouricemic Effect of Total Flavonoid Extract from Humulus lupulus

[0016] I. Materials, Reagents and Instruments

[0017] (I) Materials and Reagents

[0018] The materials and reagents used in this experiment are shown in Table 1.

[0019] Table 1 Information Table of Materials and Reagents

[0020]

[0021] (II) Instruments

[0022] The main instruments used in this experiment are shown in Table 2.

[0023] Table 2 Information Table of Instruments

[0024]

[0025] II. Experimental Animals

[0026] 40 SPF-grade male Wistar rats, with a body weight of (150±10) g, were purchased from Shanghai Slac Laboratory Animal Co., Ltd., certificate number: 20220004024235, and the animal production license number was SCXK (Shanghai) 2022-0004. During the research period, they were housed in the Experimental Animal Center of the School of Pharmacy, Naval Medical University. All animal studies complied with the ethical and usage principles of NMU animal experiments.

[0027] III. Experimental Methods

[0028] (I) Preparation of Drugs and Reagents

[0029] 1. 0.5% CMC-Na suspension

[0030] Weigh 500 mg of sodium carboxymethyl cellulose (CMC-Na) powder, add an appropriate amount of distilled water under a water bath at about 75 °C, stir well to disperse evenly, and make up the volume to 100 mL to obtain a 0.5% CMC-Na suspension.

[0031] 2. Suspension for modeling

[0032] Weigh 1 g of potassium oxonate (OAPS) powder and 1.25 g of hypoxanthine (HX) powder, grind them thoroughly and disperse evenly in a mortar, transfer them to 50 mL of 0.5% CMC-Na, and vortex and ultrasonically mix well to obtain a suspension for modeling (OAPS 20 mg / mL + HX 25 mg / mL).

[0033] 3. ALLO suspension

[0034] Weigh 100 mg of allopurinol (ALLO) powder, transfer it to 50 mL of 0.5% CMC-Na, and vortex and ultrasonically mix well to obtain an ALLO suspension (2 mg / mL).

[0035] 4. Suspension of total flavonoid extract from Humulus lupulus

[0036] Weigh 50 mg and 150 mg of the total flavonoid extract from Humulus lupulus (prepared in Example 1 of the present invention) respectively, transfer them to 50 mL of 0.5% CMC-Na respectively, and vortex and ultrasonically mix well to prepare suspensions of total flavonoid extract from Humulus lupulus at different concentrations (hereinafter also referred to as total flavonoids from Humulus lupulus 1 mg / mL and 3 mg / mL).

[0037] (II) Experimental grouping and treatment

[0038] The rats were adaptively fed for 1 week and randomly divided into 5 groups of 8 rats each. Potassium oxonate (200 mg·kg -1 ·d -1 ) and hypoxanthine (250 mg·kg -1 ·d -1 ) were used in combination to establish a HUA model. After 2 hours of modeling, the corresponding drug suspensions were given respectively (ALLO 20 mg·kg -1 ·d -1 , low-dose total flavonoids from Humulus lupulus 15 mg·kg -1 ·d -1 , high-dose total flavonoids from Humulus lupulus 45 mg·kg -1 ·d -1) The dosage and treatment plan were mainly set according to the literature (Zhou H, Li X, Li Y, Zhu X, Zhang L, Li J. Synthesis and bioevaluation of 1-phenylimidazole-4-carboxylic acid derivatives as novel xanthine oxidoreductase inhibitors. Eur J Med Chem. 2020, 186: 111883. doi: 10.1016 / j.ejmech.2019.111883.) and the results of preliminary experiments. The intervention period was 14 days, as shown in Table 3 specifically.

[0039] Table 3 Treatment plan

[0040]

[0041] (III) Sampling and detection

[0042] Blood was collected from the orbits of rats on the 3rd, 7th, 10th, and 14th days. After 2 h, the supernatant serum was obtained by centrifugation at 5810×g for 10 min, and the indexes such as UA, CRE, BUN, and XOD in the serum of rats in each group were detected.

[0043] (IV) Data statistical analysis

[0044] All data are expressed as . With the help of the software GraphPad Prism 9.0, Shapiro-Wilk normality test and F-test homogeneity of variance test were carried out. If the variances were homogeneous, one-way analysis of variance (One-Way ANOVA) and Newman-Keuls were used for multiple comparisons; if the variances were not homogeneous, variable transformation was first performed to meet the homogeneity of variance test, and then the transformed data were further statistically analyzed. The test level α was 0.05.

[0045] IV. Experimental results

[0046] (I) Effect of total flavonoids from Humulus lupulus on UA in rat serum

[0047] The results showed that the serum UA level in the MOD group was always higher than that in the CON group from the 3rd to 14th days (P < 0.01), indicating that the HUA model was successfully constructed. The UA levels in the low-dose and high-dose groups of total flavonoids from Humulus lupulus and the ALLO group were always lower than those in the MOD group (all P < 0.01), indicating that total flavonoids from Humulus lupulus had a good effect on reducing blood UA, as shown in Table 4.

[0048] Table 4 Effect of total flavonoids from Humulus lupulus on UA level in rat serum (μmol·L -1 , )

[0049]

[0050] ** P < 0.01 vs CON group; ΔΔ P < 0.01 vs MOD group.

[0051] (2) Effects of total flavonoids from Humulus lupulus on XOD activity in rats

[0052] The results showed that the XOD activity in the MOD group was higher than that in the CON group from day 3 to day 14 (P < 0.01), indicating that the XOD activity in HUA rats was significantly increased and the production of UA was increased. Compared with the MOD group, the XOD activities in the ALLO group and each dose group of total flavonoids from Humulus lupulus decreased (both P < 0.01), indicating that total flavonoids from Humulus lupulus could reduce the XOD activity, thereby inhibiting UA synthesis and playing a role in reducing UA, as shown in Table 5.

[0053] Table 5 Effects of total flavonoids from Humulus lupulus on XOD activity in rats (U·L -1 , )

[0054]

[0055] ** P < 0.01 vs CON group; ΔΔ P < 0.01 vs MOD group.

[0056] (3) Effects of total flavonoids from Humulus lupulus on renal function in rats

[0057] 1. Serum CRE

[0058] The results showed that the CRE level in the MOD group was higher than that in the CON group from day 3 to day 14 (P < 0.01), indicating that HUA rats were accompanied by renal insufficiency. Compared with the MOD group, the CRE levels in the ALLO group and each dose group of total flavonoids from Humulus lupulus decreased (both P < 0.01), indicating that total flavonoids from Humulus lupulus could effectively reduce the CRE level, improve renal injury induced by HUA, and have a renal protective effect, as shown in Table 6.

[0059] Table 6 Effects of total flavonoids from Humulus lupulus on serum CRE level (μmol·L -1 , )

[0060]

[0061] ** P < 0.01 vs CON group; ΔΔ P < 0.01 vs MOD group.

[0062] 2. Serum BUN

[0063] The results showed that the BUN level in the MOD group was higher than that in the CON group from day 3 to day 14 (P<0.01), indicating that HUA rats were accompanied by renal insufficiency. Compared with the MOD group, the BUN levels in the ALLO group and the total flavonoids of hops groups at each dose decreased to varying degrees (P<0.01, P<0.05), indicating that the total flavonoids of hops could effectively reduce the BUN level and improve the renal injury induced by HUA, as shown in Table 7.

[0064] Table 7 Effects of total flavonoids of hops on serum BUN level (μmol·L -1 , )

[0065]

[0066] ** P<0.01 vs CON group; Δ P<0.05, ΔΔ P<0.01 vs MOD group.

Claims

1. Use of total flavonoid extract of hops in the preparation of a drug for reducing uric acid.

2. Use of total flavonoid extract of hops in the preparation of a drug for preventing and treating hyperuricemia-related diseases.

3. The use according to claim 2, characterized in that, The hyperuricemia-related disease is hyperuricemia.

4. The use according to claim 2, wherein The hyperuricemia-related disease is gout.

5. The use according to claim 2, characterized in that, The hyperuricemia-related disease is uric acid nephropathy.

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