Application of paridol saponin B in preparation of drugs for treating or improving hyperuricemia

Abstract

The present application relates to the technical field of biological medicine, and in particular to application of gypsophila oldhamiana saikoside B in preparation of a medicine for treating or improving hyperuricemia. The gypsophila oldhamiana saikoside B can reduce serum uric acid, inhibit xanthine oxidase activity, reduce serum creatinine and urea nitrogen levels, increase excretion of uric acid, creatinine and urea nitrogen in urine, reduce kidney edema and pathological damage effect, etc. The gypsophila oldhamiana saikoside B has a significant therapeutic effect on hyperuricemia and can improve kidney function damage, thereby providing a new medicine selection for treatment of hyperuricemia and having a good clinical application prospect.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically, to the application of Bailiangjin saponin B in the preparation of drugs for treating or improving hyperuricemia. Background Technology

[0002] Gout is a crystalline arthropathy caused by elevated blood uric acid levels due to purine metabolism disorders, leading to the precipitation and deposition of monosodium urate (MSU) crystals in the joints. It is closely related to hyperuricemia (HUA), and the two are different stages of the same disease. In my country, the prevalence of HUA is approximately 13.3%, while the prevalence of gout ranges from 0.03% to 10.47%, showing a trend of increasing annually and affecting younger individuals. Currently, clinical treatments for hyperuricemia mainly fall into two categories: one is drugs that inhibit uric acid production, such as xanthine oxidase inhibitors allopurinol and febuxostat; the other is drugs that promote uric acid excretion, such as benzbromarone and probenecid. However, in the acute phase, colchicine, nonsteroidal anti-inflammatory drugs (NSAIDs), and glucocorticoids are mainly used for anti-inflammatory and analgesic purposes, but these have a narrow therapeutic window and significant gastrointestinal reactions and hepatotoxicity / nephrotoxicity. In the remission phase, uric acid-lowering drugs (allopurinol, febuxostat, benzbromarone) are mainly used, but these have issues such as hypersensitivity reactions, cardiovascular risks, and liver damage.

[0003] Ardisiacrispin B (CAS No. 112766-96-8, molecular formula C) 53 H 86 O 22 (Molecular weight approximately 1075.24) is an oleanane-type pentacyclic triterpenoid saponin, with the aglycone being ciclaminogen A. It contains characteristic groups such as a 13β,28-epoxy ether bridge, a 30-aldehyde group, a 16α-hydroxyl group, and a 3-O-tetrasaccharide chain. It is mainly derived from related plants such as *Ardisia crenata* Sims, *A. crispa*, and *A. mamillata* Hance. Its content is 2.6%–4.5% in the whole herb of *A. crispa*, 3.7%–6.2% in the roots and stems, and reaches 14% in *Ardisia crenata*. Precise quantification can be achieved using HPLC-ELSD. Current research on *A. crispa* saponin B mainly focuses on its cytotoxic and antitumor activities, antifungal effects, and uterine contraction effects; its use in gout, gouty arthritis, and hyperuricemia has not yet been reported. In summary, there are no publicly available reports or records of using Bai Liang Jin saponin B for gout, gouty arthritis, and hyperuricemia in the existing technology.

[0004] Based on this, the present invention aims to explore the anti-hyperuricemia activity of Bailiangjin saponin B, clarify its role in reducing serum uric acid, inhibiting xanthine oxidase activity, promoting uric acid excretion, improving renal function and reducing renal tissue damage, and verify its application value in the preparation of drugs for treating hyperuricemia and related renal damage. Summary of the Invention

[0005] To address at least one of the problems in the background art, the present invention provides the use of bailiangjin saponin B in the preparation of drugs for treating or improving hyperuricemia.

[0006] In a first aspect, the present invention provides the use of bailiangjin saponin B in the preparation of a medicament for treating or improving hyperuricemia.

[0007] Optionally, the drug is a drug for treating or improving kidney damage caused by the hyperuricemia.

[0008] Optionally, the kidney injury includes renal tubular dilatation, glomerular atrophy, renal epithelial cell vacuolar degeneration, and / or renal edema.

[0009] Optionally, the drug is a drug that inhibits xanthine oxidase activity.

[0010] Optionally, the Hundred-Taels Gold Saponin B is extracted from plants of the genus Ardisia.

[0011] Optionally, the *Ardisia* species include *Ardisia crenata*, *Ardisia crenata*, or *Ardisia crenata*.

[0012] Secondly, the present invention provides a method for preparing the above-mentioned saponin B of *Cinnamomum cassia*, *Cinnamomum cassia* or *Eriocaulon rubrum* through alcohol extraction, macroporous resin adsorption, column separation and gel chromatography.

[0013] Optionally, the dosage form of the drug includes powder, granules, tablets, pills, capsules, or injections.

[0014] Thirdly, the present invention provides a traditional Chinese medicine composition comprising the above-mentioned Zhu Bailiangjin saponin B and a pharmaceutically acceptable carrier, wherein the Zhu Bailiangjin saponin B is the active ingredient.

[0015] Optionally, the carrier is a solid or semi-solid.

[0016] The present invention has the following beneficial effects:

[0017] In the preparation of drugs for treating or improving hyperuricemia, bailiangjin saponin B can lower serum uric acid, inhibit xanthine oxidase activity, reduce serum creatinine and urea nitrogen levels, increase urinary excretion of uric acid, creatinine, and urea nitrogen, and alleviate renal edema and pathological damage. Bailiangjin saponin B has a significant therapeutic effect on hyperuricemia and can improve renal function, providing a new drug option for the treatment of hyperuricemia. Attached Figure Description

[0018] Figure 1 The HPLC chromatogram of Hundred-Tael Gold Saponin B;

[0019] Figure 2 The appearance of the kidneys of mice in each group;

[0020] Figure 3 Liver index of mice in each group;

[0021] Figure 4 Kidney index for each group of mice;

[0022] Figure 5 H&E staining of kidney tissue from mice in each group;

[0023] Figure 6 Serum uric acid levels in each group of mice;

[0024] Figure 7 Serum creatinine levels in each group of mice;

[0025] Figure 8 The serum urea nitrogen level in each group of mice;

[0026] Figure 9 The uric acid level in the urine of mice in each group;

[0027] Figure 10 The creatinine level in the urine of mice in each group;

[0028] Figure 11 The blood urea nitrogen level in the urine of mice in each group;

[0029] Figure 12 XOD activity in the serum of mice in each group. Detailed Implementation

[0030] The invention will now be discussed with reference to several exemplary embodiments. It should be understood that these embodiments are described merely to enable those skilled in the art to better understand and thus implement the invention, and are not intended to imply any limitation on the scope of the invention.

[0031] As used herein, the term "comprising" and its variations are to be interpreted as open-ended terms meaning "including but not limited to". The terms "one embodiment" and "an embodiment" are to be interpreted as "at least one embodiment". The term "another embodiment" is to be interpreted as "at least one other embodiment". These terms are primarily used to better describe this application and its embodiments and are not intended to limit the indicated components to having a particular orientation, or being constructed and operated in a particular orientation. Those skilled in the art can understand the specific meaning of the above terms in this application according to the specific circumstances. Furthermore, the terms "first", "second", etc., are primarily used to distinguish different components (the specific types may be the same or different) and are not intended to indicate or imply the relative importance and number of the indicated components. Unless otherwise stated, "a plurality of" means two or more.

[0032] Example 1

[0033] 1. Materials

[0034] 1.1 Reagents and Consumables

[0035] Hypoxanthine and potassium oxonate were purchased from Shanghai Yuanye Co., Ltd., 4% paraformaldehyde was purchased from Beijing Lanjieke Technology Co., Ltd., allopurinol was purchased from Maclean Co., Ltd., the hematoxylin-eosin staining kit was purchased from Beijing Solarbio Technology Co., Ltd., creatinine (CRE), blood urea nitrogen (BUN) and uric acid (UA) were purchased from Nanjing Jiancheng Bioengineering Institute, and the xanthine oxidase (XOD) enzyme-linked immunosorbent assay kit was purchased from Shanghai Zhuocai Biotechnology Co., Ltd.

[0036] 1.2 Experimental Apparatus

[0037] The following equipment was used: a variosska multi-functional microplate reader, a fresco 17 low-temperature high-speed centrifuge, a JB-L8 paraffin embedding machine and an HM340E paraffin microtome (Thermo Fisher Scientific, USA), a BS-460 veterinary biochemical analyzer (Shenzhen Mindray Bio-Medical Electronics Co., Ltd.), a JT-12S automatic dehydrator and a JRS-14C tissue staining machine (Wuhan Junjie Electronics Co., Ltd.), an Easy Scan digital slide scanner (Macody Industrial Group Co., Ltd.), and an MX204 / A electronic balance (Mettler-Toledo International Ltd.).

[0038] 2. Experimental Methods

[0039] 2.1 Preparation of Hundred-Tael Gold Saponin B

[0040] Dried *Ardisia crenata* root was extracted 2-4 times by reflux with 30-95% ethanol at a material-to-liquid ratio of 1:6-1:12, each time for 1-4 hours. The extracts were combined, and the ethanol was recovered under reduced pressure. The extract was then dried to obtain a total extract. The total extract was suspended in water and loaded onto a pretreated D101 macroporous resin. A gradient elution was performed sequentially with water, 70% ethanol, and 95% ethanol. The 70% ethanol eluent was collected. This fraction was subjected to normal-phase silica gel column chromatography with a gradient elution using a dichloromethane-methanol system. Similar fractions were combined by TLC monitoring, and the mixture was appropriately concentrated to precipitate a white solid component. This solid component was washed with methanol to remove impurities, yielding a purified intermediate. The intermediate was further purified using conventional saponin purification methods such as gel chromatography, preparative chromatography, or recrystallization to obtain high-purity Ardisiacrispin B monomer. Ardisiacrispin B has the following structural formula:

[0041]

[0042] The HPCL spectrum of Hundred-Tael Gold Saponin B is as follows: Figure 1 as shown in the figure.

[0043] 2.2 Test animals

[0044] Male Kunming (KM) mice (18-22g) were purchased from Wuhan Hualianke Biotechnology Co., Ltd., with the production license number: SCXK (E) 2023-0033, and the experimental unit license number: SYXK (Gui) 2023-0002. The animals were raised and managed by dedicated personnel, with free access to food, and the feeding environment was controlled with a 12h / 12h day-night cycle. The feed was provided by Beijing Huanyu Zhongke Biotechnology Co., Ltd., with the production license number: Yu Si Zheng (2008) 25540. The laboratory has sufficient light, good ventilation and air conditioning equipment, a temperature of 20~26°C, and a relative humidity of 40~70%. The laboratory is regularly disinfected according to conventional procedures.

[0045] 2.3 Grouping, model establishment and drug administration

[0046] After one week of adaptive feeding, the mice were randomly divided into 6 groups according to body weight, namely the blank group, the model group, the allopurinol group (5mg / kg), and the low, medium and high dose groups of Ardisiasaponin B (10mg / kg, 20mg / kg, 40mg / kg), with 6 mice in each group. The mice were intraperitoneally injected with potassium oxonate (100mg / kg / day) and intragastrically administered hypoxanthine (500mg / kg) for 7 consecutive days. The normal group was injected with normal saline and intragastrically administered 0.5% sodium carboxymethyl cellulose in the same manner. One hour after daily model establishment, the administration groups were given a single intragastric administration at 10mL / kg, and the blank control group and the model group were intragastrically administered 0.5% sodium carboxymethyl cellulose.

[0047] 2.4 Sample collection

[0048] After administration on the 6th day, mouse urine was collected using metabolic cages for the determination of uric acid, creatinine and urea nitrogen. One hour after the last administration on the 7th day, blood was collected by eyeball removal (the animals were fasted for 12 hours before blood collection with free access to water). After being placed at room temperature for 1 hour, the blood was centrifuged at 3000rpm for 10 minutes at 4°C. The supernatant was aliquoted and stored at -80°C for subsequent detection of uric acid, creatinine, urea nitrogen levels and xanthine oxidase activity. After blood collection, the liver and kidney tissues were quickly separated and weighed. Meanwhile, both kidneys were photographed and then fixed in 4% paraformaldehyde for pathological examination.

[0049] 2.5 Detection of liver and kidney organ indices in rats

[0050] The body weight of the rats was weighed, their liver and kidneys were isolated, and the organ weights were weighed. The corresponding organ indices were calculated according to the formula: organ index = organ weight (mg) / body weight (g).

[0051] 2.6 Detection of uric acid, creatinine and urea nitrogen

[0052] Following the instructions of the corresponding assay kits for uric acid, creatinine, and blood urea nitrogen, the levels of uric acid, creatinine, and blood urea nitrogen in mouse serum and urine were measured, respectively.

[0053] 2.7 Detection of xanthine oxidase activity in serum

[0054] Following the instructions of the corresponding assay kits for uric acid, creatinine, and blood urea nitrogen, the levels of uric acid, creatinine, and blood urea nitrogen in mouse serum and urine were measured, respectively.

[0055] 2.8 Pathological examination of mouse kidneys

[0056] Mouse kidney tissue was fixed for 24 hours, then dehydrated, embedded in paraffin, cut into 3-4 μm thin sections, dewaxed, stained with hematoxylin and eosin (HE), mounted with neutral resin solution, and the pathological changes of the kidney tissue were observed and photographed under a microscope.

[0057] 2.9 Statistical Analysis

[0058] Experimental data were statistically analyzed using SPSS 26.0 and plotted using Graphpad Prism software. The experimental data are presented as follows: The data indicates that one-way ANOVA was used for comparisons among multiple groups, and independent samples t-tests were used to analyze differences between two groups. A p-value < 0.05 was considered statistically significant.

[0059] 3. Test Results

[0060] 3.1 Gross examination of the kidneys

[0061] The kidneys of mice in the blank control group were reddish-brown and shiny. Compared with the blank control group, the kidneys of mice in the model group were pale red or even grayish-white, and larger in volume, exhibiting irregular shape and obvious edema. Compared with the model group, treatment with Bai Liang Jin saponin B alleviated the above symptoms, as evidenced by improved kidney color and reduced edema. Figure 2 As shown.

[0062] 3.2 Effects of Bai Liang Jin saponin B on liver and kidney indices in hyperuricemic mice

[0063] Liver and kidney indices of mice in each group are as follows: Figure 3 and Figure 4 As shown ( Compared with the blank control group, ### P < 0.001; compared with the model group, ** P<0.01, ***P<0.001. Compared with the blank control group, there was no significant change in the liver index of mice in the model group and each drug-treated group (P>0.05), indicating that the modeling conditions and drug dosages in this experiment had no significant effect on the liver weight of mice. Compared with the blank control group, the kidney index of mice in the model group was significantly increased (P<0.001); compared with the model group, the kidney index of each drug-treated group was significantly decreased (P<0.01).

[0064] 3.3 Effects of Bai Liang Jin saponin B on pathological changes in mouse liver tissue

[0065] Representative results of H&E staining (×400) of kidney tissue from each group of mice are shown below. Figure 5 As shown in the figure, red arrows indicate renal tubular dilation, black arrows indicate glomerular atrophy and increased glomerular lumen volume, and blue arrows indicate vacuoles and edema within renal epithelial cells. The figure shows that the kidney tissue of the control group mice maintained normal surface and cellular structure, with clear and orderly glomerular boundaries, intact tubular structure, and no pathological phenomena such as renal epithelial cell edema. Compared with the control group, the kidney tissue of the model group mice showed pathological conditions such as renal tubular lumen dilation, epithelial cell vacuoles, edematous cell shedding and blurred boundaries, glomerular atrophy, and increased glomerular lumen volume. After drug intervention, the kidney tissue structure of hyperuricemic mice was significantly improved. Combined with the kidney index results, this indicates that Bai Liang Jin saponin B has a protective effect on the kidney tissue of hyperuricemic mice.

[0066] 3.4 Effects of Bai Liang Jin saponin B on serum and urine uric acid, blood urea nitrogen and creatinine levels in hyperuricemic mice

[0067] The serum uric acid, creatinine, and blood urea nitrogen levels in mice are as follows: Figure 6 , 7 As shown in Figure 8 Compared with the blank control group, ### P < 0.001; compared with the model group, * P<0.05, ** P<0.01, *** P<0.01), the levels of uric acid, creatinine, and urea nitrogen in mouse urine were as follows: Figure 9 , 10 As shown in Figure 11 ( Compared with the blank control group, ### P < 0.001; compared with the model group, * P<0.05, ** P<0.01, ***(P<0.01). Serum measurements showed that, compared with the blank control group, the serum levels of uric acid, creatinine, and urea nitrogen in the model group mice were significantly increased (P<0.01), indicating that the hyperuricemia model was successfully established. Compared with the model group, the serum levels of uric acid, creatinine, and urea nitrogen in each treatment group mice were significantly decreased (P<0.05). Urine measurements showed that, compared with the blank control group, the urine levels of uric acid, creatinine, and urea nitrogen in the model group mice were significantly decreased (P<0.01), indicating that the modeling conditions in this experiment, in addition to increasing the levels of uric acid and metabolic waste in serum, also inhibited the excretion of uric acid and metabolites. Compared with the model group, the urine levels of uric acid, creatinine, and urea nitrogen in each treatment group mice were significantly increased (P<0.05, P<0.01). The results indicate that Bai Liang Jin saponin B has certain anti-hyperuricemia activity.

[0068] Effects of 3,5-Liangjin saponin B on serum XOD in hyperuricemic mice

[0069] Xanthine oxidase (XOD) is a rate-limiting enzyme that converts hypoxanthine to xanthine, and then xanthine to uric acid. Its activity is directly related to the production of uric acid in the body. XOD activity in mouse serum is as follows... Figure 12 As shown ( Compared with the blank control group, ### P < 0.001; compared with the model group, * P<0.05, ** P<0.01, *** (P<0.01). Compared with the blank control group, the serum XOD activity level of mice in the model group was significantly increased (P<0.001), indicating that the significant increase in serum uric acid in the hyperuricemia model in this study may be mainly related to the significant increase in XOD activity. The serum XOD activity level of mice in each treatment group was significantly decreased (P<0.05), and the decreasing trend was basically consistent with the serum uric acid level. The above data suggest that Bailiangjin saponin B may exert its anti-hyperuricemia activity by inhibiting XOD activity.

[0070] The above experimental results show that:

[0071] (1) Bailiangjin saponin B significantly reduced serum uric acid levels: Compared with the model group, the serum uric acid levels of mice in each dose group of Bailiangjin saponin B (10, 20, 40 mg / kg) were significantly reduced (low dose group P<0.05, medium dose group P<0.01, high dose group P<0.001), showing a good dose-response relationship. The uric acid-lowering effect of the high dose group was comparable to that of the positive control drug allopurinol (5 mg / kg).

[0072] (2) Bailiangjin saponin B significantly inhibited xanthine oxidase activity: Compared with the model group, the serum xanthine oxidase activity in each dose group of Bailiangjin saponin B was significantly reduced (P<0.05~0.001), and the decreasing trend was consistent with the serum uric acid level, suggesting that inhibiting uric acid production is its important mechanism.

[0073] (3) Bailiangjin saponin B significantly promotes the excretion of uric acid and metabolic waste: Compared with the model group, the levels of uric acid, creatinine and urea nitrogen in the urine of mice in the medium and high dose groups of Bailiangjin saponin B were significantly increased (P<0.05-0.01), indicating that it can effectively promote the excretion of uric acid and metabolic waste through the kidneys.

[0074] (4) Bailiangjin saponin B significantly improves renal function: Compared with the model group, the serum creatinine and urea nitrogen levels in each dose group of Bailiangjin saponin B were significantly reduced (P<0.01 in the medium and high dose groups), suggesting that the drug can effectively improve renal function damage caused by hyperuricemia.

[0075] (5) Bailiangjin saponin B significantly reduced kidney pathological damage: Compared with the model group, the kidney index of each dose group of Bailiangjin saponin B was significantly reduced (P<0.01 in the low dose group, P<0.001 in the medium and high dose groups), and the gross morphology of the kidney was significantly improved (red color and reduced edema); the pathological examination of kidney tissue showed that Bailiangjin saponin B could significantly reduce pathological changes such as renal tubular dilation, glomerular atrophy, and vacuolar degeneration of renal epithelial cells, and the kidney tissue morphology of the high dose group was close to that of the normal control group.

[0076] (6) Good safety of Bailiangjin saponin B: There was no significant difference in liver index among the groups of mice during the experiment (P>0.05), indicating that Bailiangjin saponin B has no obvious liver toxicity in the dose range of 10~40mg / kg and has good safety.

[0077] In summary, Bai Liang Jin saponin B reduces uric acid production by inhibiting xanthine oxidase activity while promoting uric acid excretion, exhibiting a dual mechanism of action. It can also significantly improve renal function and reduce renal tissue damage, demonstrating significant anti-hyperuricemia and renal protective activity, and can be used to prepare related drugs.

[0078] The above description is merely a preferred embodiment of this application and an explanation of the technical principles employed. Those skilled in the art should understand that the scope of the invention involved in this application is not limited to technical solutions formed by specific combinations of the above-described technical features, but should also cover other technical solutions formed by arbitrary combinations of the above-described technical features or their equivalents without departing from the inventive concept. For example, technical solutions formed by substituting the above features with (but not limited to) technical features with similar functions disclosed in this application.

[0079] It should be understood that the sequence number of each step in the invention and embodiments of this application does not absolutely imply the order of execution. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application. The foregoing description of the implementation of this application has been given for illustrative and descriptive purposes. The foregoing description is not exhaustive and is not intended to limit this application to the exact form disclosed. Various modifications and variations may exist based on the above teachings, or various modifications and variations may be derived from the practice of this application. These embodiments were chosen and described to illustrate the principles of this application and its practical application, so that those skilled in the art can utilize this application in various implementations and modifications to suit the specific purpose of the concept. Those skilled in the art will understand that the above-described embodiments are specific examples of implementing this application, and in practical applications, various changes in form and detail may be made without departing from the spirit and scope of this application.

Claims

1. Application of Bailiangjin saponin B in the preparation of drugs for treating or improving hyperuricemia.

2. Use according to claim 1, characterized in that, The drug is a medication for treating or improving kidney damage caused by the hyperuricemia.

3. Use according to claim 1, characterized in that, The drug is a drug that promotes the excretion of uric acid, creatinine and / or blood urea nitrogen.

4. Use according to claim 1, characterized in that, The drug is a drug that inhibits xanthine oxidase activity.

5. Use according to any one of claims 1 to 4, characterized in that, The Hundred-Taels Gold Saponin B was extracted from plants of the genus Ardisia.

6. The use according to claim 5, characterized in that, The plants in the genus Ardisia include Ardisia crenata, Ardisia crenata var. rubra, or Ardisia crenata var. rubra.

7. Use according to any one of claims 1 to 4, characterized in that, The preparation method of the saponin B of the Hundred-Taels-of-Gold includes alcohol extraction of cinnamon root, Hundred-Taels-of-Gold or Red Hairy Horse Fetus, adsorption with macroporous resin, column separation and gel chromatography.

8. The application according to claims 1-4, characterized in that, The dosage forms of the drug include powders, granules, tablets, pills, capsules, or injections.

9. A traditional Chinese medicine composition, characterized in that, It includes the saponin B of any one of claims 1-4 and a pharmaceutically acceptable carrier, wherein the saponin B is the active ingredient.

10. The traditional Chinese medicine composition according to claim 9, characterized in that, The carrier is a solid or semi-solid.

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