Application of sauropus spatulifolius extract in preparation of product for preventing or treating hyperuricemia

Abstract

The invention relates to application of sauropus spatulifolius extract in preparation of a product for preventing or treating hyperuricemia. The sauropus spatulifolius extract is obtained by mixing sauropus spatulifolius and a solvent and extracting. The sauropus spatulifolius extract can reduce the serum uric acid level and the uric acid level in urine of a hyperuricemia rat, reduce reabsorption of GLUT9 and URAT1 transporters in kidney tissue and promote secretion of ABCG2 and OAT1 proteins, the uric acid level in the body is adjusted through two ways, and the liver and the kidney are protected; therefore, the sauropus spatulifolius can be applied to the medicine for preventing or treating the hyperuricemia.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to application of a galangal leaf extract in preparing a product for preventing or treating hyperuricemia. Background Art

[0002] Hyperuricemia (HUA) is a metabolic disease characterized by impaired purine metabolism and is a key clinical stage in the development of gout. Its pathological mechanism is primarily related to abnormal purine metabolism and decreased uric acid excretion, leading to accumulation of urate in the extracellular fluid and supersaturation, resulting in abnormally elevated serum uric acid levels. Studies have shown that hyperuricemia is common among Chinese adults and is associated with various health conditions, including gout, cardiovascular disease, and metabolic disorders. In recent years, the prevalence of hyperuricemia has steadily increased, becoming a major global public health concern and significantly impacting human health. Certain food groups and metabolic processes are associated with elevated serum uric acid levels, highlighting the importance of dietary intervention in controlling hyperuricemia. Fructose is a common monosaccharide found in fruits, honey, and processed foods, particularly high-fructose corn syrup, which is widely used as a sweetener in the food industry. Fructose undergoes a different metabolic pathway than glucose, being metabolized more rapidly and not regulated by insulin. Therefore, excessive fructose intake may lead to metabolic disturbances, subsequently causing hyperuricemia. Fructose metabolism in the liver consumes a significant amount of ATP, leading to intracellular ATP depletion and the generation of AMP. AMP is further degraded into hypoxanthine by adenosine deaminase (ADA), ultimately converting to uric acid, thereby increasing serum uric acid levels. Furthermore, intermediates produced during fructose metabolism, such as fructose-1-phosphate, can activate fructokinase (FK), further accelerating fructose metabolism and leading to even greater ATP consumption and uric acid production.

[0003] Currently, Western medicine treatments for hyperuricemia primarily include xanthine oxidase inhibitors (allopurinol and febuxostat) that inhibit uric acid production; uricosuric drugs (probenecid, pyraclostrobin, and benzbromarone) that enhance uric acid excretion and reduce uric acid concentrations; urate oxidase analogs (prekecil, rasburicase) that catalyze the breakdown of uric acid into allantoin, thereby lowering uric acid levels; and microbial transplantation. However, most Western medications are associated with adverse reactions, including gastrointestinal symptoms, liver and kidney damage, rash, bone marrow suppression, and hypersensitivity reactions. Growing concerns about drug safety and toxic side effects have led to a surge in research exploring the therapeutic potential of natural therapies. Traditional Chinese medicine, as an integral component of traditional medicine, includes extracts from medicinal plants that are naturally safe and highly effective, acting through multiple targets and pathways. These extracts are becoming a research hotspot in the treatment of hyperuricemia.

[0004] Sauropus spatulifolius Beille, a plant of the Euphorbiaceae family, is also known as dragon-benefit leaf, dragon-liver leaf, dragon-snake leaf, dragon-flavor leaf, and cow-ear leaf. It is primarily found in Guangxi, Guangdong, and Hainan provinces of my country and is a uniquely Lingnan-style medicinal and edible plant. The mature leaves are used as medicine. They are sweet, light, and neutral in nature, and enter the lung and stomach meridians. They moisten the lungs, relieve coughs, and promote bowel movements. They are primarily used to treat dry coughs, sore throats, aphonia, and constipation. The main chemical components of Sauropus spatulifolius Beille are sugars and sugar derivatives, phenolic acids, flavonoids, alkaloids, terpenes, phenylpropanoids, steroids, and fatty acids. Modern pharmacological studies have shown that aqueous and ethanolic extracts of Sauropus spatulifolius Beille exhibit antitussive and antiasthmatic, antibacterial, anti-inflammatory, analgesic, antiallergic, antioxidant, and anti-tumor properties.

[0005] The inventor's research team conducted extensive research on the leaves of the dragon's tongue, isolating components such as flavonoids, alkaloids, phenolic acids, and carbohydrate compounds. The dragon's tongue leaf has anti-acute lung injury effects, significantly reducing the wet-to-dry weight ratio of the lung and the total protein concentration in the alveolar lavage fluid, significantly improving pulmonary edema, and significantly reducing the level of inflammatory factors. Some compounds in the dragon's tongue leaf have anti-pulmonary fibrosis activity. Using the maximum tolerated dose method, 12 8-week-old Balb / c mice, half male and half female, were gavaged with the water extract of the dragon's tongue leaf at a dose of 5000 mg / kg. After 14 consecutive days of observation, no obvious symptoms of poisoning were observed and no deaths occurred. The LD500 of the oral acute toxicity test in mice was estimated to be 0. 50 Above 5000mg / kg, dragon tongue leaf is non-toxic.

[0006] There is no report on whether the leaf of the Chinese dragon has an anti-hyperuricemia effect. The present invention finds that the water extract of the leaf of the Chinese dragon has an anti-hyperuricemia effect. Therefore, the leaf of the Chinese dragon can be used in the preparation of drugs or health foods or foods related to the prevention or treatment of hyperuricemia. Summary of the Invention

[0007] In order to solve the above technical problems, the present invention provides the use of a Polygonum multiflorum leaf extract in the preparation of a product for preventing or treating hyperuricemia.

[0008] The present invention is achieved through the following technical solutions:

[0009] The present invention aims to provide a use of an extract of galangal leaves in preparing a product for preventing or treating hyperuricemia, wherein the extract of galangal leaves is prepared by the following method:

[0010] The galangal leaves are mixed with a solvent for extraction, and the obtained extract is concentrated and dried to obtain the galangal leaves extract.

[0011] Furthermore, the solvent is selected from water and / or ethanol.

[0012] Furthermore, the longifolia leaf extract includes flavonoids, polyphenols and polysaccharides; wherein the total flavonoid content is ≥36.96 mg / g; the total polyphenol content is ≥10.49 mg / g; and the total polysaccharide content is ≥214.62 mg / g; preferably, the total flavonoid content is ≥110.89 mg / g; the total polyphenol content is ≥31.48 mg / g; and the total polysaccharide content is ≥643.85 mg / g.

[0013] Furthermore, the product includes medicine, health food or food.

[0014] Furthermore, the dosage form of the drug is selected from tablets, capsules, granules, oral liquids, emulsions, dry suspensions, dry extracts or injections.

[0015] Furthermore, the medicine also includes a pharmaceutically or pharmacologically acceptable carrier and / or salt.

[0016] Furthermore, the carrier is selected from one or more of a disintegrant, a diluent, a lubricant, a binder, a wetting agent, a flavoring agent, a filler, a suspending agent, a surfactant and a preservative.

[0017] Furthermore, the filler is selected from one or more of starch, sucrose and lactose; the wetting agent includes glycerin; and the surfactant includes cetyl alcohol.

[0018] Furthermore, the binder is selected from one or more of cellulose derivatives, alginate, gelatin and polyvinyl pyrrolidone.

[0019] Furthermore, the disintegrant is selected from one or more of agar, calcium carbonate and sodium bicarbonate.

[0020] Furthermore, the pharmaceutically or pharmacologically acceptable salt is selected from inorganic acid salts and / or organic acid salts; the organic acid salt is selected from alkyl sulfonates and / or aryl sulfonates.

[0021] The above technical solution of the present invention has the following advantages over the prior art:

[0022] The present invention provides the use of a galangal leaf extract in preparing a product for preventing or treating hyperuricemia. The galangal leaf extract reduces serum uric acid and uric acid levels in hyperuricemic rats, reduces liver FK and ADA activities in hyperuricemic rats, and inhibits fructose metabolism to produce high uric acid.

[0023] Longgan leaf is a medicinal and edible plant unique to Lingnan. The inventors' previous acute toxicity tests showed it to be non-toxic. Therefore, Longgan leaf extract can be used in health foods or specialty foods to prevent the development of hyperuricemia. Longgan leaf extract can lower serum and urinary uric acid levels in rats with hyperuricemia, reduce the reabsorption of GLUT9 and URAT1 transporters in renal tissue, and promote the secretion of ABCG2 and OAT1 proteins, regulating uric acid levels in the body through two pathways. Furthermore, it protects the liver and kidneys. Therefore, Longgan leaf extract can be used in medications to prevent or treat hyperuricemia. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below based on specific embodiments of the present invention in conjunction with the accompanying drawings, wherein:

[0025] Figure 1 The present invention is the effect of the leaves of the dragon's purse on the serum uric acid level in hyperuricemia rats; wherein, compared with the blank group, ## P<0.01, #### P < 0.0001; compared with the model group, *P < 0.05, ****P < 0.0001; (n = 3);

[0026] Figure 2 The effect of the galangal leaves of the present invention on uric acid in the urine of hyperuricemic rats; compared with the blank group, ## P<0.01; compared with the model group, *P<0.05; (n=3);

[0027] Figure 3 The present invention relates to the effect of the leaves of Polygonum multiflorum on the expression of URAT1, GLUT9, ABCG2 and OAT1 mRNA in the kidneys of hyperuricemia rats; compared with the blank group, ## P<0.01, ### P<0.001, #### P < 0.0001; compared with the model group, *P < 0.05, **P < 0.01, ***P < 0.001; (n = 3);

[0028] Figure 4 The changes in liver tissue of hyperuricemia rats in the present invention (H&E, 200×); wherein, A: Control; B: Model; C: LLY; D: Positive;

[0029] Figure 5 The changes in kidney tissue of hyperuricemia rats in the present invention (H&E, 200×); wherein, A: Control; B: Model; C: LLY; D: Positive;

[0030] Figure 6 The effect of the galangal leaves of the present invention on FK and ADA in the liver of hyperuricemia rats; compared with the blank group, #### P<0.0001; compared with the model group, ***P<0.001, ****P<0.0001; (n=3). DETAILED DESCRIPTION

[0031] The present invention will be further described below with reference to the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it. However, the embodiments are not intended to limit the present invention.

[0032] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the materials and reagents used are all commercially available unless otherwise specified.

[0033] Example 1: Preparation of Longtan Leaf Sample

[0034] Weigh 500g of dried Polygonum multiflorum leaves, add about 8L of deionized water, boil for 1h, cool and filter to obtain a water extract, concentrate the water extract to obtain 186mL of Polygonum multiflorum leaf concentrate, and after drying, obtain 130g of extract.

[0035] Example 2: Determination of the Contents of Total Sugar, Total Flavonoids and Total Polyphenols in the Water Extract of Longtan Leaves

[0036] 2.1 Content of total flavonoids in the water extract of Glechoma longituba leaves

[0037] Accurately weigh 20.00 mg of rutin standard and dissolve it in 50 mL of anhydrous ethanol to prepare a 0.40 mg / mL reference solution. Pipette 0.00 mL, 1.00 mL, 2.00 mL, 3.00 mL, 4.00 mL, and 5.00 mL of the reference solution respectively; add 5 mL of deionized water and place it in a 25 mL volumetric flask; add 1.00 mL of 5% sodium nitrite, shake well, and let it stand for 6 minutes; add 1.00 mL of 10% aluminum nitrate solution, shake well, and let it stand for 6 minutes; add 10.00 mL of 4% sodium hydroxide test solution, shake well, let it stand for 15 minutes, and dilute to the scale with deionized water; measure the absorbance at 511 nm, measure three times in parallel, and draw a standard curve y = 6.1046x + 0.0293, R 2 =0.9981. The test sample was prepared into a 0.031 mg / mL aqueous solution. 10 μL of the test sample aqueous solution was accurately aspirated and the total flavonoid content in the sample was determined according to the standard curve method. The total flavonoid content in the water extract of the leaves of the Chinese dragon fruit was 110.89 mg / g.

[0038] 2.2 Content of total polyphenols in the water extract of Glechoma longituba leaves

[0039] Accurately weigh 10 mg of gallic acid standard, dissolve it in 50 mL of anhydrous ethanol to prepare a 0.20 mg / mL reference solution. In a 15.00 mL centrifuge tube, add different volumes of 0.00 mL, 0.20 mL, 0.40 mL, 0.60 mL, 1.20 mL, 1.40 mL, and 2.00 mL of 0.20 mg / mL gallic acid-ethanol reference solution, respectively, and replenish the solvent to 2.00 mL; add 3 mL of 1N Folin phenol reagent, and after 8 minutes, add 3.00 mL of 10% Na2CO3 solution, shake well, and adjust the volume to 15.00 mL with solvent. Let it stand at 25°C for 1 hour, then centrifuge (3500 rpm, 6 minutes); measure its absorbance value at a wavelength of 765 nm with an enzyme reader, repeat three times, and draw a standard curve y=1.8195x+0.1763, R 2 =0.9870. The test sample was prepared into a 0.031 mg / mL aqueous solution. 10 μL of the test sample solution was accurately pipetted and made up to 2 mL with deionized water. The total polyphenol content in the sample was determined using the standard curve method. The total polyphenol content in the water extract of the leaves of the Chinese dragon fruit was 31.48 mg / g.

[0040] 2.3 Content of total polysaccharides in the water extract of Glechoma longituba leaves

[0041] Prepare a series of glucose reference aqueous solutions with concentrations of 10 mg / L, 20 mg / L, 40 mg / L, 60 mg / L, 80 mg / L, and 100 mg / L. Take 1 mL of each reference solution, sample solution, and blank (deionized water is used as blank), add 1.00 mL of 5% phenol, shake well, and quickly add 3.00 mL of concentrated sulfuric acid vertically, shake well, and boil in a water bath for 15 minutes. Remove and cool quickly. Measure the absorbance value at a wavelength of 490 nm using an enzyme reader. Repeat three times to draw a standard curve y = 0.0058x + 0.0794, R 2 =0.9927. The test sample was prepared as a 0.031 mg / mL aqueous solution. 1 μL of the test sample solution was accurately pipetted and added to a 10,000-fold volume of the test sample solution in deionized water. The total polysaccharide content in the sample was determined using the standard curve method. The total polysaccharide content in the water extract of the leaves of the Chinese dragon was 643.85 mg / g.

[0042] Example 3: Experiment on hyperuricemia rats

[0043] Statistical analysis was performed in this example. The following data were expressed as mean ± standard deviation (mean ± SD). Graphs and data were analyzed using GraphPad Prism 8. One-way analysis of variance (ANOVA) was used to compare and evaluate the significant differences between the groups. P < 0.05 was considered statistically significant.

[0044] 3.1 Experimental Animal Grouping

[0045] SPF male Wistar rats, weighing 180-200 g, were purchased from Beijing Huafukang Biotechnology Co., Ltd. with an animal production license of SCXK (Beijing) 2024-0003. The experimental animals were housed in an environment with a relative temperature of 24±1°C and a relative humidity of 40%-60%, with free access to food and water. Ordinary feed was purchased from Suzhou Shuangshi Experimental Animal Feed Technology Co., Ltd. Wistar rats were adaptively raised for 7 days, and the uric acid content in their serum and 24-hour urine was measured, and samples with large differences were eliminated. According to the uric acid level, they were divided into 4 groups: blank group (Control), model group (Model), allopurinol group (Positive), and longifolia leaf group (LLY), with 3 rats in each group.

[0046] 3.2 Hyperuricemia rat model and drug treatment

[0047] An appropriate amount of fructose was weighed and dissolved in deionized water to prepare a 10% fructose solution. Except for the blank control group, which drank deionized water, the other groups drank 10% fructose for 8 consecutive weeks, and the water was changed every two days. Four weeks after modeling, treatment was given. According to the adult dose conversion, the allopurinol group was gavaged with a dose of 10 mg / kg, and the dragonfly leaf group was gavaged with 0.35 g / kg of extract for 4 consecutive weeks. The uric acid content in the serum was tested at the same time every week. The day before treatment, the rats were placed in metabolic cages for 24 hours, and their urine was collected and the uric acid concentration was measured. A portion of the liver and kidney tissues was placed in 4% paraformaldehyde for fixation and sectioning.

[0048] Effects of Longifolia leaves on serum uric acid levels in hyperuricemic rats Figure 1 As shown in the figure, before modeling, there was no significant difference in uric acid levels among the rats in each group. Four weeks after modeling, except for the blank group, the uric acid concentration in the serum of rats in all groups increased significantly. After the start of drug administration, the uric acid concentration in the rats in the Longtan leaf group showed a certain degree of decline in the seventh week, indicating that Longtan leaf can reduce serum uric acid levels.

[0049] like Figure 2 As shown in the data, compared with the blank group, the urinary uric acid in the model group was significantly increased (P<0.01), and compared with the model group, the urinary uric acid in the Longtan leaf group was reduced (P<0.05). Longtan leaf can reduce the level of uric acid in urine.

[0050] 3.3 Reverse Transcription-Quantitative Polymerase Chain Reaction (RT-qPCR) detection

[0051] Kidney tissue homogenate was added with 1 ml of Trizol reagent (purchased from Wuhan Sevier Biotechnology Co., Ltd., catalog number G3013), ground, and centrifuged at 12,000 rpm for 10 minutes. The supernatant was removed, 250 μL of chloroform was added, and the mixture was mixed. The mixture was centrifuged at 12,000 rpm for 15 minutes at 4°C. The supernatant was removed, 0.8 volumes of isopropanol were added, and the mixture was mixed. The mixture was incubated at -20°C for 20 minutes, and centrifuged at 12,000 rpm for 10 minutes at 4°C. The white precipitate at the bottom of the tube was RNA. The precipitate was washed with 1 ml of 75% ethanol, centrifuged at 12,000 rpm for 5 minutes at 4°C, and washed once more. The RNA was dissolved in 10 μL of RNase-free water. The cells were incubated at 55°C for 5 min, and total RNA was extracted. The cells were reverse transcribed into cDNA and used as template for amplification to detect changes in the mRNA expression of glucose transporter 9 (GLUT9), organic anion transporter 1 (OAT1), ATP-binding cassette transporter G2 (ABCG2), and urate transporter 1 (URAT1). The primer sequences are shown in Table 1.

[0052] Table 1 Transporter Primer Sequence List

[0053]

[0054] The results of the effect of Longifolia leaves on URAT1, GLUT9, ABCG2, and OAT1 mRNA in the kidneys of hyperuricemic rats are as follows Figure 3 As shown, compared with the blank group, the model group showed increased mRNA expression of GLUT9 (P < 0.001) and URAT1 (P < 0.01) in the renal tissue of rats in the model group, while decreased mRNA expression of ABCG2 (P < 0.001) and OAT1 (P < 0.0001). Compared with the model group, the expression of GLUT9 mRNA in the renal tissue of rats in the Longliu leaf group was decreased (P < 0.05). Although the expression of URAT1 mRNA in the renal tissue of rats in the Longliu leaf group was decreased, the difference was not statistically significant. Compared with the model group, the expression of ABCG2 (P < 0.01) and OAT1 (P < 0.05) mRNA in the renal tissue of rats in the Longliu leaf group was increased. These results suggest that Longliu leaf reduces the reabsorption of GLUT9 and URAT1 transporters and promotes the secretion of ABCG2 and OAT1 proteins, regulating uric acid levels in the body through two pathways.

[0055] 3.3 HE staining of liver and kidney sections

[0056] The liver and kidney tissues were dehydrated with graded alcohol, embedded, sectioned, stained with HE, dewaxed, stained with hematoxylin for nuclei, stained with eosin for cytoplasm, dehydrated and mounted, examined under a microscope, and image acquisition and analysis were performed.

[0057] Effects of Longifolia leaves on the liver of hyperuricemic rats Figure 4 As shown, the liver tissue structure of rats in the blank group was normal, with abundant hepatocytes, plump cytoplasm, clear nuclei, and no degeneration or necrosis. The hepatic cords were tight, the sinusoids were not dilated, and there was no inflammatory cell infiltration. Compared with the blank group, the liver tissue structure of rats in the model group was moderately abnormal, with abundant hepatocytes, numerous hepatocytes with edema and vacuolation, and obvious focal inflammatory cell infiltration. Compared with the model group, the liver tissue structure of rats in the Longguliye group was mildly abnormal, with a small amount of hepatocyte edema. The liver structure of rats in the positive group was mildly abnormal, with abundant hepatocytes, numerous hepatocytes with edema and vacuolation, and no inflammatory cell infiltration.

[0058] Effects of Longifolia leaves on the kidneys of hyperuricemic rats Figure 5 As shown, the renal tissue structure of rats in the blank group was normal, with clear and intact glomeruli and abundant mesangial cells. The renal tubular epithelial cells were tightly arranged without desquamation or necrosis, and there was no inflammatory cell infiltration. Compared with the blank group, the renal tissue structure of rats in the model group was moderately abnormal, with clear and intact glomeruli, edema and vacuolation of a small number of renal tubular epithelial cells, and a large amount of protein mucus in some tubular lumens. There was no inflammatory cell infiltration. Compared with the model group, the renal tissue structure of rats in the Longifolia leaf group was mildly abnormal, with some tubular epithelial cells desquamating. The renal tissue structure of rats in the positive group was mildly abnormal, with edema and vacuolation of a small number of renal tubular epithelial cells, and a small amount of protein mucus in some tubular lumens.

[0059] 3.4 Determination of liver fructokinase (FK) and adenosine dehydrogenase (ADA) activity levels

[0060] The supernatant of rat liver tissue homogenate was used to determine the activity of ADA and FK in the liver by enzyme activity colorimetric assay. The results were as follows: Figure 6 Compared with the blank group, the activities of FA and ADA in the liver of rats in the model group were significantly increased (P<0.0001); compared with the model group, the ADA activity of rats in the Longtan leaf group was significantly decreased (P<0.001); and the FK activity in the Longtan leaf group was extremely significantly decreased (P<0.0001).

[0061] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will appreciate that other variations or modifications can be made based on the above description. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. Use of an extract of Polygonum multiflorum leaf in preparing a product for preventing or treating hyperuricemia, characterized in that: The galangal leaf extract is obtained by mixing the galangal leaf with a solvent for extraction.

2. The use according to claim 1, characterized in that The solvent is selected from water and / or ethanol.

3. The use according to claim 1, characterized in that The products include medicines, health foods or foods.

4. The use according to claim 2, characterized in that The dosage form of the medicine is selected from tablets, capsules, granules, oral liquids, emulsions, dry suspensions, dry extracts or injections.

5. The use according to claim 2, characterized in that The medicine further includes a pharmaceutically or pharmacologically acceptable carrier and / or salt.

6. The use according to claim 5, characterized in that The carrier is selected from one or more of a disintegrant, a diluent, a lubricant, a binder, a wetting agent, a flavoring agent, a filler, a suspending agent, a surfactant and a preservative.

7. The use according to claim 6, characterized in that The filler is selected from one or more of starch, sucrose and lactose; the humectant includes glycerin; and the surfactant includes cetyl alcohol.

8. The use according to claim 6, characterized in that The binder is selected from one or more of cellulose derivatives, alginate, gelatin and polyvinyl pyrrolidone.

9. The use according to claim 6, characterized in that The disintegrant is selected from one or more of agar, calcium carbonate and sodium bicarbonate.

10. The use according to claim 5, characterized in that The pharmaceutically or pharmacologically acceptable salt is selected from inorganic acid salts and / or organic acid salts; the organic acid salt is selected from alkyl sulfonates and / or aryl sulfonates.

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