Application of cloves in the preparation of drugs for treating hyperuricemia and gout

Abstract

This invention belongs to the field of traditional Chinese medicine application technology, specifically involving the application of cloves in the preparation of drugs for treating hyperuricemia and gout. The cloves of this invention are the dried flower buds of the clove plant (Eugenia caryophyllata Thunb.) of the Myrtaceae family. It is a traditional Chinese medicine that is both food and medicine, has high safety, and can exert uric acid-lowering effects through multiple targets. It has significant therapeutic effects on hyperuricemia and gout.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine application technology, specifically involving the application of cloves in the preparation of anti-hyperuricemia drugs and anti-gout drugs. Background Technology

[0002] Hyperuricemia is a chronic disease caused by long-term high levels of uric acid in the body. Increased uric acid production, decreased excretion, or the intake of high-purine foods can all lead to an imbalance in purine metabolism, resulting in elevated blood uric acid levels and subsequently gout. Hyperuricemia is also associated with the development of diabetes, hypertension, cardiovascular disease, and kidney disease. It has been reported that aging, obesity, a high-purine diet, and excessive alcohol consumption are the main causes of hyperuricemia.

[0003] Uric acid is a metabolic product of purine compounds in the human body. Disorders of purine metabolism, increased uric acid production, and / or decreased uric acid excretion can lead to a sustained increase in blood uric acid concentration, resulting in hyperuricemia. Excess uric acid in the blood forms tiny needle-like monosodium urate (MSU) crystals, which deposit in joints, kidneys, cartilage, and other areas, causing non-specific inflammatory reactions in soft tissues and ultimately leading to gout. Therefore, hyperuricemia is the biochemical basis for gout.

[0004] Normally, the dynamic balance of serum uric acid levels in the body is mainly related to four uric acid transporters: organic anion transporter 1 (OAT1), glucose transporter 9 (GLUT9), uric acid transporter 1 (URAT1), and ATP-binding protein transporter 2 (ABCG2). Of these four uric acid transporters, GLUT9 and URAT1 are primarily involved in uric acid reabsorption, while OAT1 and ABCG2 are primarily involved in uric acid secretion. Currently, drugs for treating hyperuricemia in clinical practice are mainly divided into two categories: one is xanthine oxidase (XOD) inhibitors, such as allopurinol and febuxostat; the other is uric acid excretion-promoting drugs, such as probenecid and benzbromarone. However, due to the nephrotoxicity of XOD inhibitors such as allopurinol and the hepatotoxicity of benzbromarone, and the fact that these single-target drugs have shown limited efficacy or excessive side effects in clinical applications, their clinical use is limited. Therefore, there is an urgent need to find safer and more effective xanthine oxidase inhibitors to reduce uric acid production in the body, downregulate the expression of urate transporter 1 (URAT1) and glucose transporter 9 (GLUT9) to reduce uric acid reabsorption, and upregulate the expression of ATP-binding protein transporter 2 (ABCG2) and organic anion transporter 1 (OAT1) to promote uric acid excretion.

[0005] Herbal medicines are an important natural treasure trove for finding and developing drugs to treat hyperuricemia and gout. Clove (Caryopteris flos) is the dried flower bud of Eugenia caryophyllata Thunb., a plant in the Myrtaceae family. It is listed as a food and medicine homology in traditional Chinese medicine by the National Health Commission, indicating a relatively high safety profile. It has the effects of warming the middle jiao (spleen and stomach), relieving nausea and vomiting, tonifying the kidneys and strengthening yang. Clinically, it is mainly used to treat spleen and stomach deficiency-cold, hiccups and vomiting, poor appetite and diarrhea, cold pain in the heart and abdomen, and kidney deficiency and impotence. Modern pharmacological studies have confirmed that clove has...

[0006] Whether cloves can lower uric acid is unclear and there are few related research reports. Chinese patent CN101953842A discloses the "Application of Syringin in the Preparation of Drugs for Treating Acute Gout," but this syringin is mainly derived from the bark of *Syringa vulgaris* L. (Oleaceae family) and *Acanthopanax Senticosus* (Puper et al.) (Araliaceae family). The structure of plants such as Maxim Harms is not consistent with that of biflorin I and isobiflorin II in clove ([1] Zhao Yongtian, Wang Xing'e, Huang Yu et al. Research progress on chemical composition and bioactivity of clove [J]. Tropical Agricultural Science and Technology, 2022, 45(04): 41-46. [2] Li Shasha, Li Fan, Li Fang et al. Research progress on chemical composition and pharmacological effects of clove [J]. Northwest Pharmaceutical Journal, 2021, 36(05): 863-868. [3] Dan Chun, Jiao Wei. Research on chemical composition of clove [J]. Chinese Medicinal Herbs, 2018, 41(05): 1108-1113.), and this compound is mainly used for the treatment of gouty arthritis, not for gout treatment based on uric acid lowering effect, which is essentially different from this invention. Chinese patent CN106806532A discloses a "medicated wine for treating gout and its preparation method," using the following traditional Chinese medicine formula (by weight): 12 parts Cistanche deserticola, 11 parts Polygala tenuifolia, 12 parts Bombyx mori, 13 parts Lycium barbarum, 12 parts Gynostemma pentaphyllum, 11 parts Aquilaria sinensis, 13 parts Evodia rutaecarpa, 6 parts Ginseng, 6 parts Cordyceps sinensis, 1 part Clove, and 3 parts Rock sugar. It claims to "accelerate blood flow around joints, provide sufficient nutrition to bones, activate and regenerate articular cartilage, gradually restore normal bone metabolism, reduce and eliminate joint swelling, pain, and lameness, increase immunity and kidney function, and prevent hyperuricemia and uric acid crystals." It emphasizes the anti-inflammatory and analgesic effects of this traditional Chinese medicine formula, as well as its ability to increase immunity and kidney function and prevent hyperuricemia and uric acid crystals. It also emphasizes the "warming the middle, warming the kidneys, and lowering adverse qi" effects of Clove. However, the enhancement of kidney function may be related to its renal protective effect, and is not necessarily related to its uric acid-lowering effect. The examples provided are mainly related to the treatment of pain and inflammation in gout, with no evidence of uric acid-lowering effects.

[0007] Therefore, the present invention aims to provide an innovative application that can be used in the preparation of drugs for treating hyperuricemia and gout. Summary of the Invention

[0008] To address the aforementioned problems, this invention proposes the application of cloves, a food and medicine, in the preparation of drugs for treating hyperuricemia and gout. The invention also explores the application of cloves in regulating uric acid levels. Research has shown that cloves can reduce serum uric acid levels in individuals with hyperuricemia, demonstrating promising potential in regulating uric acid.

[0009] The technical solution adopted in this invention is as follows:

[0010] Application of cloves in the preparation of drugs for treating hyperuricemia and gout.

[0011] The cloves mentioned are the dried flower buds of Eugenia caryophyllata Thunb., a plant belonging to the Myrtaceae family.

[0012] The term "anti-hyperuricemia drugs" refers to drugs used to treat, prevent, reduce, and / or alleviate hyperuricemia and diseases related to hyperuricemia; the term "anti-gout drugs" refers to drugs used to treat, prevent, reduce, and / or alleviate gout.

[0013] The diseases related to hyperuricemia refer to other diseases caused by hyperuricemia.

[0014] The clove is any one or more of the following: raw clove, clove extract, or effective parts derived from clove extract.

[0015] The clove extract is any one of clove water extract, alcohol extract, or alcohol-water mixed extract.

[0016] The eugenol extract is a clove ethanol or methanol extract.

[0017] The clove water extract is obtained by using water as a solvent and clove as a solute, and by one or two extraction methods such as decoction or reflux.

[0018] The eugenol-water mixed extract is any one or both of the following: eugenol-water mixed extract and eugenol-water methanol mixed extract.

[0019] The beneficial effects of this invention are as follows: the clove of this invention is a food and medicine with high safety and can significantly improve hyperuricemia or gout.

[0020] The clove of this invention has a multi-target mechanism of action to lower uric acid. In vivo, it can not only downregulate the expression of xanthine oxidase in hyperuricemic mice, reduce its activity, and reduce the production of uric acid in the body, but also downregulate the expression of uric acid transporter 1 (URAT1) and glucose transporter 9 (GLUT9) in hyperuricemic mice, reducing the reabsorption of uric acid, and upregulate the expression of ATP-binding protein transporter 2 (ABCG2) and organic anion transporter 1 (OAT1), promoting the excretion of uric acid, thus playing a very effective role in the prevention and treatment of hyperuricemia and gout. Attached Figure Description

[0021] Figure 1 This is a graph showing the effect of cloves on serum uric acid levels in mice with hyperuricemia;

[0022] Figure 2 This is a graph showing the effects of clove on serum urea nitrogen and creatinine levels in mice with hyperuricemia;

[0023] Figure 3 The effect of cloves on organ indices in mice with hyperuricemia;

[0024] Figure 4 The effects of clove on the activity and protein expression of xanthine oxidase in the serum and liver of mice with hyperuricemia.

[0025] Figure 5 The effect of clove on the expression of ABCG2, OAT1, URAT1 and GLUT9 proteins in hyperuricemic mice;

[0026] Figure 6 The figure shows the effect of clove on renal pathology in mice with hyperuricemia (H&E, 200×). Detailed Implementation

[0027] To more clearly illustrate the present invention, the following description, in conjunction with preferred embodiments, further clarifies the invention. Those skilled in the art should understand that the specific descriptions below are illustrative rather than restrictive, and should not be construed as limiting the scope of protection of the present invention.

[0028] Preparation of clove water extract:

[0029] The whole clove herb was crushed, passed through a No. 4 sieve, and a certain amount was weighed and placed in a round-bottom flask. Eight times the amount of water was added and soaked for 1 hour. The mixture was then decocted and extracted for 2 hours. After filtration, the residue was extracted with eight times the amount of water for 1.5 hours. After filtration, the residue was extracted with eight times the amount of water for another 1 hour. The three filtrates were combined and concentrated into an extract. The extract was then vacuum dried to obtain clove water extract with an extraction rate of 9.93%.

[0030] Clove extract dosage setting basis:

[0031] According to the Chinese Pharmacopoeia 2020, the dosage of clove is 1-3g. We selected low, medium and high doses of 1g, 2g and 4g respectively, and then converted the human dosage to mouse dosage to obtain low, medium and high doses of 11mg / kg, 22mg / kg and 44mg / kg respectively.

[0032] Example 1

[0033] 1. Effects of clove on serum uric acid levels in hyperuricemic mice

[0034] 1.1 Establishment of a mouse model of hyperuricemia:

[0035] Eighty-four male Kunming mice were randomly divided into four groups according to body weight: a normal control group, a model group, an allopurinol group (positive control, 10 mg / kg), a benzbromarone group (positive control, 10 mg / kg), and low, medium, and high dose clove extract groups (10, 20, and 40 mg / kg), with 12 mice in each group. Except for the normal control group, which received intraperitoneal injections and gavage with an equal volume of physiological saline, the other groups received intraperitoneal injections of potassium oxonate 300 mg / kg combined with gavage of hypoxanthine 500 mg / kg at 9:00 AM daily for 7 consecutive days to establish a hyperuricemia model. From day 3 of modeling, mice in each treatment group received the corresponding drug via gavage 1 hour after administration, while the normal control and model groups received an equal volume of physiological saline via gavage, once daily for 5 consecutive days.

[0036] 2.2 Experimental Results

[0037] Figure 1 Effects of cloves on serum uric acid levels in hyperuricemic mice # p < 0.05 ## p < 0.01, ### p < 0.001, compared with the normal control group; * p < 0.05, ** p < 0.01, *** p < 0.001, compared with the model group.

[0038] The results are as follows Figure 1 As shown, compared with the normal control group, the serum uric acid level in the model group mice was significantly increased (p < 0.001), indicating that the hyperuricemia mouse model was successfully established. Compared with the model group, the serum uric acid level in the allopurinol group, benzbromarone group, and clove-treated groups was significantly decreased (p < 0.001), indicating that clove has a significant uric acid-lowering effect.

[0039] Example 2

[0040] 2. Effects of clove on serum creatinine and blood urea nitrogen levels in hyperuricemic mice

[0041] 2.1 Experimental Methods

[0042] After the experimental procedure in Example 1 was completed, about 1.5 mL of blood was collected from the mice and allowed to settle naturally for 2 hours. Then, the mice were centrifuged at 3000 r / min for 10 min at 4°C. The supernatant was collected and the serum urea nitrogen and creatinine levels were measured according to the instructions of the urea nitrogen and creatinine kit.

[0043] 2.2 Experimental Results

[0044] Figure 2 Effects of clove on serum urea nitrogen and creatinine levels in hyperuricemic mice # p < 0.05 ## p < 0.01, ### p < 0.001, compared with the normal control group; * p < 0.05, ** p < 0.01, *** p < 0.001, compared with the model group.

[0045] The results are as follows Figure 2 As shown, compared with the normal control group, the serum creatinine and urea nitrogen levels in the model group mice were significantly increased (p < 0.05, p < 0.01). Conversely, compared with the model group, the serum creatinine and urea nitrogen levels in the allopurinol group mice were significantly increased (p < 0.05), indicating that allopurinol significantly worsened renal function in hyperuricemic mice. Compared with the model group, there were no significant changes in serum urea nitrogen levels in any of the clove-treated groups (p > 0.05), but the serum creatinine levels in the medium and high dose clove groups were significantly decreased (p < 0.05). These results indicate that clove has a certain ameliorative effect on renal function in hyperuricemic mice.

[0046] Example 3

[0047] 3. Effects of clove on organ indices in hyperuricemic mice

[0048] 3.1 Experimental Methods

[0049] After the experimental method of Example 1 was completed, the body weight, liver, kidney and spleen of the mice were weighed and the organ index of the mice was calculated according to the formula: organ coefficient = organ mass / mouse body weight × 100% to investigate the effect of clove on organ index of hyperuricemic mice.

[0050] 3.2 Experimental Results

[0051] Figure 3 Effects of clove on organ indices in hyperuricemic mice # p < 0.05 ## p < 0.01, ###p < 0.001, compared with the normal control group; * p < 0.05, ** p < 0.01, *** p < 0.001, compared with the model group.

[0052] like Figure 3 As shown, compared with the normal control group, the body weight of mice in the model group was significantly reduced (p < 0.05). Compared with the model group, the body weight of mice in the allopurinol group was significantly reduced on day 7 after administration (p < 0.01), indicating that allopurinol can significantly inhibit the natural weight gain of mice. However, there were no significant changes in body weight in the other administration groups (p > 0.05). Clove had no significant effect on mouse body weight gain, liver index, or spleen index (p > 0.05), indicating that clove had no significant effect on the liver and spleen of mice. Compared with the normal control group, the kidney index of mice in both the model group and the allopurinol group was significantly increased (p < 0.001), indicating that the modeling agents potassium oxonate, hypoxanthine, and the positive control drug allopurinol had toxic side effects on the kidneys of mice. Compared with the model group, the kidney index of mice in all clove dosage groups was reduced, indicating that clove can alleviate kidney damage induced by the modeling agents.

[0053] Example 4

[0054] 4. Effects of clove on xanthine oxidase protein expression in the liver of hyperuricemic mice and XOD activity in serum and liver tissue.

[0055] 4.1 Determination of xanthine oxidase activity in serum and liver:

[0056] After the experimental procedure in Example 1 was completed, approximately 1.5 mL of blood was collected from mice and allowed to settle naturally for 2 hours. The collected blood was then centrifuged at 3000 rpm for 10 minutes at 4°C, and the supernatant was used to determine XOD activity. Separately, 100 mg of liver tissue from each group of mice was accurately weighed and added to 0.9 mL of physiological saline. A 10% homogenate was prepared in an ice-water bath, centrifuged at 3000 rpm for 10 minutes, and the supernatant was collected. XOD activity was then measured according to the XOD kit instructions.

[0057] 4.2 Experimental Results

[0058] Figure 4 Effects of clove on serum xanthine oxidase activity (A), liver tissue xanthine oxidase activity (B), and protein expression (C / D) in hyperuricemic mice # p < 0.05 ## p < 0.01, ### p < 0.001, compared with the normal control group; * p < 0.05, ** p < 0.01, ***p < 0.001, compared with the model group.

[0059] The results are as follows Figure 4 As shown, compared with the normal control group, the XOD activity in serum and liver tissue of mice in the model group and the XOD protein expression in liver tissue were significantly increased (p < 0.05, p < 0.01). Compared with the model group, the XOD activity and protein expression in serum and liver tissue of mice in the allopurinol group and each dose group of clove were significantly decreased (p < 0.01), indicating that clove can reduce the uric acid level in hyperuricemic mice by inhibiting XOD activity and protein expression in serum and liver tissue of hyperuricemic model mice.

[0060] Example 5

[0061] 5. Effects of clove on the expression of urate transporter 1 (URAT1), glucose transporter 9 (GLUT9), ATP-binding protein transporter 2 (ABCG2), and organic anion transporter 1 (OAT1) in the kidney tissue of hyperuricemic mice.

[0062] 5.1 Experimental Methods

[0063] After the experimental procedure in Example 1 was completed, blood was collected from the mice and kidney tissue was quickly removed on an ice tray. 100 mg of kidney tissue from a fixed location was weighed, rinsed with pre-cooled physiological saline to remove surface blood and wiped dry with filter paper, RIPA cell lysis buffer was added, and the mixture was placed in an ice-water bath for lysis (20 min), followed by centrifugation (12000 r / min, 10 min); protein concentration was measured using the BCA method. An equal amount of protein (45 μg) was subjected to 10% SDS-PAGE electrophoresis. After transfer and blocking using standard methods, primary antibodies URAT1 (1:3000), GLUT9 (1:5000), ABCG2 (1:2000), and OAT1 (1:2000) were added. The membrane was incubated overnight at 4°C and then at room temperature for 1 h. After washing with TBST buffer (10 min × 3 times), horseradish peroxidase-labeled goat anti-rabbit antibody (secondary antibody) was added, and the membrane was incubated at room temperature for another 1 h. After washing with TBST buffer (10 min × 3 times), the membrane was developed using enhanced chemiluminescence. The optical density values ​​of each band were analyzed using image analysis software. Semi-quantitative analysis was performed by the ratio of the optical density of the target band to that of the internal control (GAPDH), and statistical analysis was conducted.

[0064] 5.2 Experimental Results

[0065] Figure 5 Effects of clove on the expression of ABCG2, OAT1, URAT1 and GLUT9 proteins in hyperuricemic mice # p < 0.05 ## p < 0.01, ###p < 0.001, compared with the normal control group; * p < 0.05, ** p < 0.01, *** p < 0.001, compared with the model group.

[0066] like Figure 5 As shown, compared with the normal control group, the relative expression levels of URAT1 and GLUT9 proteins in the kidney tissue of mice in the model group were significantly upregulated (p < 0.001), while the relative expression levels of ABCG2 and OAT1 proteins were significantly downregulated (p < 0.01). Compared with the model group, the relative expression levels of URAT1 and GLUT9 proteins in the kidney tissue of mice in each clove dosage group were significantly downregulated (p < 0.001), while the relative expression levels of ABCG2 and OAT1 proteins were significantly upregulated (p < 0.01). These results indicate that the mechanism of clove's anti-HUA effect may be related to inhibiting uric acid reabsorption and promoting uric acid excretion.

[0067] Example 6

[0068] 6. Effects of clove on renal pathology in hyperuricemic mice

[0069] 6.1 Experimental Methods

[0070] After the experimental procedure in Example 1 was completed, mouse kidneys were quickly removed from an ice tray, washed with pre-cooled PBS to remove blood, and then blotted dry on filter paper. The right mouse kidney was fixed in 4% paraformaldehyde, dehydrated with ethanol, embedded in paraffin, sectioned, stained with hematoxylin and eosin, and the pathological changes of the kidney tissue were observed under an upright optical microscope.

[0071] 6.2 Experimental Results

[0072] Figure 6 Effects of clove on renal pathology in hyperuricemic mice (H&E, 200×). (A, Normal control group; B, Model group; C, Allopurinol group; D, Benzbromarone group; E, Low-dose clove group; F, Medium-dose clove group; G, High-dose clove group)

[0073] The results are as follows Figure 6 As shown, in the normal control group, the renal tubules of mice had clear boundaries and neatly arranged epithelial cells; in the model group, the renal tubular epithelial cells were damaged and sloughed off, characterized by mild tubular dilation and mild glomerular atrophy accompanied by basement membrane thickening; and in the allopurinol group, the mice showed severe renal edema and severe tubular dilation. Compared with the model group, the renal tubular dilation of mice in all clove dosage groups was reduced, and the high-dose clove group had recovered to normal levels, indicating that clove improved the renal injury status of hyperuricemia model mice to varying degrees.

[0074] It should be noted that all data were statistically analyzed using SPSS 25.0 software, and the data are presented in [data format]. This indicates that one-way ANOVA was used for comparisons among multiple groups, and LSD-t test was used for pairwise comparisons between groups. The significance level was set at α = 0.05.

[0075] Conclusion: The above activity test results indicate that clove can reduce the serum uric acid content in hyperuricemia model mice by inhibiting the activity and protein expression of xanthine oxidase in serum and liver. Serum urea nitrogen and creatinine levels, as well as kidney pathological section results, all indicate that clove is safe and has no side effects. In vivo, clove not only downregulates the expression and activity of xanthine oxidase in hyperuricemic mice, reducing uric acid production, but also downregulates the expression of urate transporter 1 (URAT1) and glucose transporter 9 (GLUT9) in hyperuricemic mice, reducing uric acid reabsorption, and upregulates the expression of ATP-binding protein transporter 2 (ABCG2) and organic anion transporter 1 (OAT1), promoting uric acid excretion. Therefore, it is very effective in the prevention and treatment of hyperuricemia and gout. In summary, clove can be effectively used to regulate uric acid levels, treat diseases related to hyperuricemia, or prepare drugs for treating hyperuricemia-related diseases by inhibiting uric acid production and promoting uric acid excretion.

[0076] Finally, it should be noted that the above embodiments are merely representative examples of the present invention. Obviously, the technical solution of the present invention is not limited to the above embodiments, and many variations are possible. All variations that can be directly derived or conceived by those skilled in the art from the content disclosed in this invention should be considered within the scope of protection of this invention.

Claims

1. The application of clove in the preparation of anti-hyperuricemia drugs, wherein the clove is the dried flower bud of Eugenia caryophyllata Thunb., a plant of the Myrtaceae family; and the clove is the sole source of the active ingredient.

2. The application according to claim 1, characterized in that, The clove mentioned is either a water extract or an alcohol extract of clove.

3. The application according to claim 2, characterized in that, The eugenol extract is a clove ethanol or methanol extract.

4. The application according to claim 2, characterized in that, The clove water extract is obtained by using water as a solvent and clove as a solute, and by either decoction or reflux extraction.

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