Application of carbon dioxide humulus lupulus extract in preparation of uric acid reducing medicine

Abstract

The invention discloses an application of a carbon dioxide humulus lupulus extract in preparation of a medicament for reducing uric acid. The invention finds and confirms that the carbon dioxide humulus lupulus extract has a remarkable uric acid reducing effect for the first time, and can be used for preparing the uric acid reducing medicine for preventing and treating hyperuricemia related diseases such as hyperuricemia, gout, uric acid nephropathy and the like. The hyperuricemia related diseases are metabolic diseases, the incidence rate is high, the intervention period is long, the demand on uric acid reducing drugs is large, the carbon dioxide humulus lupulus extract is a food additive, the safety is good, the acceptability is high, and the development and application prospects are good.

Description

Technical Field

[0001] The present invention relates to the application of carbon dioxide hop extract 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, and it is higher than 420 μmol·L -1 for men and higher than 360 μmol·L -1 for women, it is hyperuricemia (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] Currently, for the prevention and treatment of hyperuricemia-related diseases such as hyperuricemia, gout, uric acid nephropathy, etc. in clinical practice, drugs for reducing uric acid need to be used to control the blood uric acid level. Currently, 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 in clinical practice for safe, effective, less side-effect, and low-cost drugs for reducing uric acid, 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 that are both medicine and food is greater and the acceptance is higher.

[0004] The traditional Chinese medicine 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 inducing diuresis, and can be used to treat indigestion, abdominal distension, pulmonary tuberculosis, cystitis, neurasthenia, and insomnia. There have been many reports on the chemical constituents of hops. The resinous constituents represented by humulone (α-acid) and lupulone (β-acid) contained therein have received more attention and have effects such as antioxidation and anti-osteoporosis, but there have been no reports on their uric acid-lowering effects.

[0005] Hops are also an essential additive in beer brewing. The specific application forms can be compressed hops, hop pellets, or an extract prepared by carbon dioxide extraction of the active ingredients contained in compressed hops or hop pellets - carbon dioxide hop extract. The main components contained therein are resinous constituents, and α-acid can be regarded as its medicinal ingredient and the index ingredient for quality control. SUMMARY OF THE INVENTION

[0006] According to the embodiments, the present invention aims to provide the application of carbon dioxide hop extract in the preparation of uric acid-lowering drugs for the prevention and treatment of hyperuricemia-related diseases such as hyperuricemia, gout, uric acid nephropathy, etc.

[0007] The inventors have been engaged in the development of hop resources and pharmacological research for a long time, and have particularly focused on the research and development of new uses based on translational medicine. The inventors have previously successfully established a process technology for preparing hop extract by supercritical carbon dioxide fluid extraction and applied for a Chinese patent with the application number CN201811186942.8. On this basis, the inventors further studied the uric acid-lowering effect of commercial carbon dioxide hop extract and for the first time discovered and confirmed that it has a significant uric acid-lowering effect. Moreover, since hyperuricemia, gout, and uric acid nephropathy are metabolic diseases with a long intervention period and a large demand for uric acid-lowering drugs, carbon dioxide hop extract itself is a food additive with good safety and high acceptance, and has good development and application prospects.

[0008] Currently, there is a supply of commercial carbon dioxide hop extract on the market (the quality should comply with the Chinese national standard GB / T 20369-2006 and the Chinese industry standard NY / T 2973-2016). Based on the discovery of the new use of this commercial carbon dioxide hop extract, it is easier to achieve translational application. The subsequent embodiments will prove that this carbon dioxide hop extract has a significant uric acid-lowering effect and can be used to prepare uric acid-lowering drugs for the prevention and treatment of hyperuricemia-related diseases such as hyperuricemia, gout, uric acid nephropathy, etc.

[0009] The carbon dioxide hop extract described in the present invention is used for preparing a drug for reducing uric acid, and can be directly suspended, dissolved, and diluted for application, or can be prepared into a suitable preparation form with pharmaceutically acceptable pharmaceutical excipients for application. The pharmaceutically acceptable carrier mentioned refers to the 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 polyvinylpyrrolidone; wetting agents such as glycerol; disintegrants such as agar, calcium carbonate, and sodium bicarbonate; absorption promoters such as quaternary ammonium compounds; surfactants such as cetyl alcohol; adsorption carriers such as kaolin and soap clay; lubricants such as talc, calcium stearate, magnesium stearate, and polyethylene glycol, etc. In addition, other adjuvants such as flavoring agents and sweetening agents can also be added to the composition. The suitable preparation forms described in the present invention refer to: when used for oral administration, it can be made into conventional solid preparations such as tablets, powders, granules, capsules, etc., or can be made into liquid preparations such as water or oil suspensions, or other liquid preparations such as syrups, elixirs, etc.; when used for parenteral administration, it can be made into injection solutions, water or oil suspensions, etc. The preferred forms are tablets (including coated tablets, immediate release tablets), capsules, granules, and oral liquids.

[0010] Since the carbon dioxide hop extract itself can be used as an additive in beer brewing, it has high safety and good acceptance. In addition, the intervention period for hyperuricemia-related diseases is long, so it is very suitable and feasible to make it into a functional food or use it as a food additive, such as various forms of beverages, oral liquids, chewable candies, jellies, biscuits, coffee, etc. In view of the fact that in modern society, the incidence of hyperuricemia-related diseases is high, the harm is great, and the intervention period is long, the demand for drugs for reducing uric acid is large. The carbon dioxide hop extract described in the present invention has good development and application prospects, and contains huge social and economic benefits. Detailed implementation manners

[0011] The following further elaborates the present invention in conjunction with specific embodiments. These embodiments should be understood as only used to illustrate the present invention and not used to limit the protection scope of the present invention. After reading the content recorded in the present invention, those skilled in the art can 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.

[0012] Example - Pharmacological experimental study on the uric acid-lowering effect of carbon dioxide hop extract

[0013] I. Materials, reagents and instruments

[0014] (I) Materials, reagents

[0015] The materials and reagents used in this experiment are shown in Table 1.

[0016] Table 1 Information table of materials and reagents

[0017]

[0018] (2) Instruments

[0019] The main instruments used in this experiment are shown in Table 2.

[0020] Table 2 Instrument Information Table

[0021]

[0022] II. Experimental Animals

[0023] Forty SPF-grade male Wistar rats, with a body weight of (150 ± 10) g, were purchased from Shanghai Slack Experimental Animal Co., Ltd. The certificate number is: 20220004024235, and the animal production license number is 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.

[0024] III. Experimental Methods

[0025] (1) Preparation of Drugs and Reagents

[0026] 1. 0.5% CMC-Na Suspension

[0027] 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 until evenly dispersed, and make up the volume to 100 mL to obtain 0.5% CMC-Na suspension.

[0028] 2. Suspension for Modeling

[0029] Weigh 1 g of potassium oxonate (OAPS) powder and 1.25 g of hypoxanthine (HX) powder, grind them thoroughly and evenly in a mortar, transfer them to 50 mL of 0.5% CMC-Na, and vortex and ultrasonically mix well to obtain the suspension for modeling (OAPS 20 mg / mL + HX 25 mg / mL).

[0030] 3. ALLO Suspension

[0031] 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 ALLO suspension (2 mg / mL).

[0032] 4. Humulus lupulus Extract Suspension

[0033] Weigh 25 mg and 75 mg of carbon dioxide hop extract (provided by Xinjiang Changji City Landscape Hop Co., Ltd., with quality meeting Chinese national standard GB / T 20369-2006 and Chinese industry standard NY / T 2973-2016, batch number: 20221024) respectively, and transfer them to 50 mL of 0.5% CMC-Na. Vortex and ultrasonically mix well to obtain carbon dioxide hop extract suspensions with different concentrations (hereinafter also referred to as hop extract 0.5 mg / mL and 1.5 mg / mL).

[0034] (II) Experimental grouping and treatment

[0035] The rats were adaptively fed for 1 week and randomly divided into 5 groups with 8 rats in each group. The HUA model was established by the combined use of potassium oxonate (200 mg·kg -1 ·d -1 ) and hypoxanthine (250 mg·kg -1 ·d -1 ). After 2 h of modeling, the corresponding drug suspensions (ALLO 20 mg·kg -1 ·d -1 , low-dose hop extract 7.5 mg·kg -1 ·d -1 , high-dose hop extract 22.5 mg·kg -1 ·d -1 ) were given respectively. The drug 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.

[0036] Table 3 Treatment plan

[0037]

[0038] (III) Sampling and detection

[0039] Blood was taken from the rat orbital cavities on the 3rd, 7th, 10th, and 14th days. After 2 h, the supernatant serum was taken by centrifuging 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.

[0040] (4) Data statistical analysis

[0041] All data are expressed as . With the help of the software GraphPad Prism 9.0, Shapiro-Wilk normality test and F-test for homogeneity of variance are carried out. If the variances are homogeneous, one-way analysis of variance (One-Way ANOVA) and Newman-Keuls are used for multiple comparisons; if the variances are not homogeneous, variable transformation is first performed to meet the homogeneity of variance test, and then the transformed data is further statistically analyzed. The test level α is 0.05.

[0042] IV. Experimental results

[0043] (1) Effects of hop extract on serum UA in rats

[0044] The results showed that the serum UA level in the MOD group was always higher than that in the CON group from day 3 to day 14 (P<0.01), indicating that the HUA model was successfully constructed. The UA levels in the low-dose and high-dose hop extract groups and the ALLO group were always lower than those in the MOD group (all P<0.01), indicating that hop extract has a good effect on reducing serum UA, as shown in Table 4.

[0045] Table 4 Effects of hop extract on serum UA level in rats (μmol·L -1 , )

[0046]

[0047] ** P<0.01 vs CON group; ΔΔ P<0.01 vs MOD group.

[0048] (2) Effects of hop extract on XOD activity in rats.

[0049] 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 more UA was produced. Compared with the MOD group, the XOD activities in the ALLO group and each dose group of hop extract decreased (all P<0.01), indicating that hop extract can reduce XOD activity, thereby inhibiting UA synthesis and playing a role in reducing UA, as shown in Table 5.

[0050] Table 5 Effects of hop extract on XOD activity in rats (U·L -1 , )

[0051]

[0052] **P < 0.01 vs CON group; ΔΔ P < 0.01 vs MOD group.

[0053] (3) Effects of hop extract on renal function in rats.

[0054] 1. Serum CRE

[0055] 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 hop extract decreased (all P < 0.01), indicating that hop extract could effectively reduce the CRE level, improve the renal injury induced by HUA, and had a renal protective effect, as shown in Table 6.

[0056] Table 6 Effects of hop extract on serum CRE level (μmol·L -1 , )

[0057]

[0058] ** P < 0.01 vs CON group; ΔΔ P < 0.01 vs MOD group.

[0059] 2. Serum BUN

[0060] 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 each dose group of hop extract decreased to varying degrees (P < 0.01, P < 0.05), indicating that hop extract could effectively reduce the BUN level and improve the renal injury induced by HUA, as shown in Table 7. ·

[0061] Table 7 Effects of hop extract on serum BUN level (μmol·L -1 , )

[0062]

[0063] * P < 0.05, ** P < 0.01 vs CON group; Δ P < 0.05, ΔΔ P < 0.01 vs MOD group.

Claims

1. Use of carbon dioxide hop extract in the preparation of a drug for reducing uric acid.

2. Use of carbon dioxide hop extract 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, characterized in that, 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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