Application of calycosin glucoside in medicine for treating kidney stone

Abstract

The invention discloses application of calycosin-7-glucoside in a medicine for treating kidney stone, and the calycosin-7-glucoside can prevent / treat kidney stone, so that the calycosin-7-glucoside can be used for preparing the medicine for treating kidney stone and the medicine for preventing kidney stone formation, and a new medicine and a new treatment thought are provided for treating or preventing calcium oxalate kidney stone diseases.

Description

Technical Field

[0001] This invention belongs to the technical field of drugs for treating kidney stones, specifically relating to the use of a salamiflavonoid glucoside in drugs for treating kidney stones. Background Technology

[0002] Kidney stones are a major public health burden worldwide, and their prevalence is on the rise. Kidney stones are a urinary tract disease caused by the abnormal accumulation of crystalline substances such as calcium, oxalate, uric acid, and cysteine ​​in the kidneys, forming solid crystalline masses. Approximately 80% of kidney stones in humans are formed from calcium oxalate. The formation of kidney stones is a complex, multifaceted process, including crystal nucleation, supersaturation, crystal growth and aggregation, preservation of the renal tubular epithelium of the crystal, and continued stone growth on residual crystals.

[0003] In many cases, urinary tract stones can pass spontaneously. However, when they cannot pass spontaneously, medication is generally the first-line treatment, including prescription anti-inflammatory drugs, potassium citrate, calcium channel blockers, and alpha-1-adrenergic receptor antagonists (primarily tamsulosin). However, due to the complex composition and large size of the stones, coupled with the unbearable pain experienced by patients, surgical intervention often becomes necessary. Although minimally invasive surgery has significantly reduced side effects, shortened recovery time, and improved safety, its high cost and risk of postoperative infection limit its effectiveness in treating kidney stones. Furthermore, individual patient differences, environmental factors, and dietary influences contribute to a very high recurrence rate after treatment. Repeated surgeries can easily lead to kidney failure, posing a life-threatening risk. Therefore, it is necessary to find an effective, safe, and compliant oral treatment alternative to minimally invasive surgery to inhibit the recurrence of kidney stones. Summary of the Invention

[0004] In view of the shortcomings of the prior art, the purpose of this invention is to provide the use of verbascoside (CG) in a drug for treating kidney stones.

[0005] The objective of this invention is achieved through the following technical solution.

[0006] Use of verbascoside in medications for treating / relieving kidney stones.

[0007] Uses of Astragalus membranaceus in medications for treating / relieving kidney stones.

[0008] In the above technical solution, when the drug for treating kidney stones / relieving kidney stones is a traditional Chinese medicine composition, Astragalus membranaceus is the principal drug in the drug for treating kidney stones / relieving kidney stones.

[0009] The use of verbascoside and its pharmaceutically acceptable salts or esters in drugs for treating / relieving kidney stones.

[0010] The aforementioned drugs for treating kidney stones / relieving kidney stones contain one or more pharmaceutically acceptable carriers in verbascoside / Astragalus membranaceus; the carriers include diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption promoters, surfactants, adsorbents, and lubricants commonly used in the pharmaceutical field.

[0011] Versicolor isoflavone glucoside / Astragalus membranaceus can be formulated into various forms such as injections, tablets, powders, granules, capsules, oral liquids, ointments, and creams; all of the above dosage forms can be prepared according to conventional methods in the pharmaceutical field.

[0012] This invention demonstrates through experiments that verbascoside isoflavone glucoside can prevent / treat kidney stones, and therefore can be used to prepare drugs for treating kidney stones and preventing kidney stone formation, providing a new drug and treatment approach for the treatment or prevention of calcium oxalate kidney stones. Attached Figure Description

[0013] Figure 1 The isoflavone glucoside component in Example 1 was qualitatively identified;

[0014] Figure 2A The images show Malpighian tubes of Drosophila taken under bright field (first row) and polarized light (first row) in the control group, disease group, positive control group, and Astragalus treatment group in Example 2-1.

[0015] Figure 2B The degree of Malpighian tubule stones in Drosophila from the control group, disease group, positive control group, and Astragalus treatment group in Example 2-1;

[0016] Figure 2C Malpighian tubules of Drosophila taken under bright field (rows 1, 3, and 5) and polarized light (rows 2, 4, and 6) for the Astragalus membranaceus eluent administration group;

[0017] Figure 2D The degree of Marpole tubule stones in the Drosophila group treated with Astragalus membranaceus eluent;

[0018] Figure 3A The diagram shows the reactive oxygen species levels in Malpighian tubules of Drosophila in the control group, disease group, Astragalus treatment group, and Verticillium isoflavone glucoside treatment group in Examples 2-2. The first column is a photograph taken under DHE staining, the second column is a photograph taken under DAPI staining, and the third column is an overlap of the first and second columns.

[0019] Figure 3B This is a quantitative graph showing the levels of reactive oxygen species in Malpighian tubules of Drosophila in the control group, disease group, Astragalus treatment group, and Versicolor isoflavone glucoside treatment group in Examples 2-2.

[0020] Figure 3C This is a comparison of the expression levels of oxidative stress-related genes Sod1 and CAT in Drosophila from the control group, disease group, Astragalus treatment group, and Versicolor isoflavone glucoside treatment group in Example 2-2.

[0021] Figure 4A This is a schematic diagram of in vitro crystallization of the model group, positive control group, Astragalus membranaceus water extract group, and Versicolor isoflavone glucoside group in Examples 2-3;

[0022] Figure 4B This is a quantitative comparison of the in vitro crystallization area of ​​the model group, positive control group, Astragalus water extract group, and Versicolor isoflavone glucoside group in Examples 2-3.

[0023] Figure 4C This is a quantitative comparison of the number of crystals in vitro in the model group, positive control group, Astragalus water extract group, and Versicolor isoflavone glucoside group in Examples 2-3;

[0024] Figure 5A The images show Malpighian tubes of Drosophila in the Astragalus powder treatment group and the Astragalus eluent administration group (1.0 mg / mL 30% ethanol elution (CG)) in Examples 2-4, photographed under bright field (first row) and polarized light (second row).

[0025] Figure 5B The treatment of the degree of Malpighian tubule stones in Drosophila was carried out in the Astragalus powder treatment group and the Astragalus eluent administration group (1.0 mg / mL 30% ethanol elution (CG)) in Examples 2-4.

[0026] Figure 6A Flowchart of mouse experiments;

[0027] Figure 6B The serum creatinine levels in the control group, disease group, verbascoside glucoside treatment group, and citric acid treatment group in Example 3;

[0028] Figure 6C The serum urea nitrogen content of the control group, disease group, verbascoside glucoside treatment group and citric acid treatment group in Example 3;

[0029] Figure 6D Polarized light of paraffin sections of kidney tissue from the control group, disease group, verbascoside glucoside treatment group, and citric acid treatment group in Example 3;

[0030] Figure 6E This is a statistical graph showing the polarization area of ​​paraffin sections of kidney tissue from the control group, disease group, verbascoside glucoside treatment group, and citric acid treatment group in Example 3.

[0031] Figure 6FFor the renal tissue damage in the control group, disease group, calycosin-7-O-β-D-glucoside treatment group, and citric acid treatment group in Example 3;

[0032] Figure 6G It is a statistical chart of the renal injury area in the control group, disease group, calycosin-7-O-β-D-glucoside treatment group, and citric acid treatment group in Example 3. Detailed implementation manners

[0033] The technical solutions of the present invention will be described in detail below in conjunction with specific embodiments.

[0034] Calcium oxalate kidney stones are kidney stone diseases caused by the accumulation of oxalic acid. The animal model of kidney stone disease is prepared with sodium oxalate / glyoxylic acid. In the disease group of Example 2, feeding sodium oxalate leads to excessive exogenous oxalic acid intake and excessive accumulation of oxalic acid in the body, thus forming calcium oxalate stones. In the disease group of Example 3, exogenous glyoxylic acid is metabolized in the liver to produce oxalic acid, increasing the oxalic acid content in the kidney to form calcium oxalate crystals.

[0035] In the following embodiments, the Astragalus membranaceus used is purchased from Hongfa Chinese Herbal Medicine Pieces Co., Ltd., Anguo City.

[0036] HPLC-grade acetonitrile (ACN) and ethanol are purchased from Merck KGaA, Germany.

[0037] Formic acid is purchased from Beijing百灵威科技有限公司.

[0038] Water is purified using a Milli-Q purification system. The C18HD column is purchased from Huapu Keyi (Beijing) Technology Co., Ltd.

[0039] The photodiode array detector (PDA) is purchased from Waters Corporation, USA. The HPLC-ESI / Q-TOF MS system consists of a 1290 series LC unit of Agilent Technologies, Inc. and a 6540 series time-of-flight mass spectrometer.

[0040] In the following embodiments, the sodium oxalate is produced by Fuchen (Tianjin) Chemical Reagent Co., Ltd.; the positive drug potassium citrate used is produced by Tianjin Jiangtian Chemical Technology Co., Ltd.; the calycosin-7-O-β-D-glucoside used is produced by Chengdu Pus Biotechnology Co., Ltd.

[0041] The structure of calycosin-7-O-β-D-glucoside is:

[0042]

[0043] In the following embodiments, the mice used are 7-week-old SPF-grade C57BL / 6J male mice, purchased from Beijing Xinuo因生物科技有限公司, with the production license number SCXK(Beijing)2022-0006;

[0044] Glyoxylic acid was purchased from Sigma-Aldrich, product number G10601, production batch number WXBD1645V;

[0045] Citric acid was purchased from Beijing Solarbio Technology Co., Ltd., product number C8610, production batch number 425I041;

[0046] DMSO was purchased from Tianjin Berens Biotechnology Co., Ltd.

[0047] Nuclear dye DAPI: CAT product number DA0004; China;

[0048] Diethylhydroacetylene (DHE) probe: CAT product number D1008; China.

[0049] Methyl 4-hydroxybenzoate: purchased from Tianjin Xiens Biochemical Technology Co., Ltd.

[0050] In the attached figures, data are expressed as mean ± SEM; * indicates p < 0.05; ** indicates p < 0.01; *** indicates p < 0.001, and p represents significance.

[0051] In the following implementations, % (w / v) is a mass-volume percentage, where mass w is in g and volume v is in mL; % (v / v) is a volume percentage, where v is in mL.

[0052] The methyl 4-hydroxybenzoate solution is a mixture of methyl 4-hydroxybenzoate and anhydrous ethanol, and the concentration of methyl 4-hydroxybenzoate in the methyl 4-hydroxybenzoate solution is 10.2% w / v.

[0053] Example 1

[0054] (1) Preparation of Astragalus membranaceus water extract stock solution

[0055] Place 100g of Astragalus membranaceus into a round-bottom flask, add 1000mL of distilled water, insert a thermometer and a condenser, and heat the round-bottom flask (voltage 50V) to 100℃ to boiling using an electric heating mantle. Repeat the reflux process three times, refluxing for 2 hours each time, for a total of 6 hours. Cool to room temperature, filter with gauze, collect and combine the filtrates from the three processes, and decoct at 100℃ to concentrate the filtrate until it thickens to 100mL in a beaker, yielding a 1g / mL Astragalus membranaceus water extract stock solution.

[0056] (2) Preparation of Astragalus membranaceus eluate using solid phase extraction (SPE)

[0057] Solid-phase extraction of the Astragalus membranaceus aqueous extract was performed using an RP-C18 column (length × inner diameter: 250 × 30 mm). The eluent was used as the mobile phase, and elution was carried out at a flow rate of 20 mL / min. The eluent was collected, rotary evaporated, and freeze-dried to obtain the Astragalus membranaceus eluent. The eluent was either pure water or an ethanol-water solution. The ethanol-water solution was a mixture of ethanol and pure water. The ethanol content in the ethanol-water solution is shown in Table 1. The Astragalus membranaceus eluent corresponding to the eluent solution is shown in Table 1.

[0058] Table 1

[0059]

[0060] (3) Experimental Results

[0061] The compounds in the Astragalus membranaceus eluent eluted with 30% ethanol were qualitatively identified by liquid chromatography, such as... Figure 1 As shown in the image above, the mass spectrometer is as follows: Figure 1 As shown in the figure below, a unique single peak exists in the range of 4.385-4.538 min, which is identified as salamidoside (CAS No. 20633-67-4).

[0062] HR ESI-MS, m / z: [M+H] + : calculated 447.4041, found 477.1627.

[0063] Example 2: Experiment on the inhibition of calcium oxalate kidney stone formation in Drosophila by Astragalus / Verbena isoflavone glucoside

[0064] Example 2-1

[0065] (1) Preparation of fruit flies

[0066] The fruit flies used were wild female Canton.S. fruit flies, 1-2 days after emergence. All fruit flies were obtained from Professor Jianzhen Zhang's lab at Shanxi University and were reared according to a standard protocol: the fruit flies were kept in a 25℃ incubator with a relative humidity maintained at 55.3%, using a 12-hour light-12-hour dark cycle. The fruit flies were allowed to mate and reproduce naturally in a standard culture medium (standard culture medium: 1L of pure water with 50g corn flour, 10g agar, 30g brown sugar, 7.25g sucrose, and 24.5g dry yeast, boiled, cooled, and then 0.3% (v / v) propionic acid and 1.5% (v / v) antibiotics were added; the antibiotics and propionic acid prevented microbial growth and bacterial contamination). The standard culture medium was changed every 3 days. The fruit fly larvae were allowed to emerge as adults, and female fruit flies 1-2 days after emergence were selected for the experiment.

[0067] (2) Set up the following experimental groups:

[0068] a. Control group: Thirty female fruit flies that had emerged 1-2 days prior were randomly transferred to a blank culture medium. The fruit flies were fed in the blank culture medium for 7 days, with the blank culture medium being changed every 3 days. During the test, 20 fruit flies were randomly selected from each blank culture medium. The number and size of calcium oxalate crystals in the Malpighian tubules of the fruit flies were analyzed under a polarized light microscope. The blank culture medium included: 2.17% (w / v) yeast, 1.31% (w / v) sucrose, 1% (w / v) agar, 6.67% (w / v) corn syrup, 0.06% (v / v) propionic acid, 0.1% (v / v) methyl 4-hydroxybenzoate solution and distilled water.

[0069] b. Disease Group: The disease group was essentially the same as the control group, except that the blank culture medium was replaced with the first culture medium, which consisted of: 2.17% (w / v) yeast, 1.31% (w / v) sucrose, 1% (w / v) agar, 6.67% (w / v) corn syrup, 0.06% (v / v) propionic acid, 0.1% (v / v) methyl 4-hydroxybenzoate solution, 0.1% (w / v) sodium oxalate, and distilled water. The final sodium oxalate content in the first culture medium was 0.1% (w / v), for example, 0.1g of sodium oxalate was added to 100mL of the first culture medium.

[0070] c. Astragalus treatment group: The Astragalus treatment group was basically the same as the control group, except that the blank culture medium was replaced with the second culture medium, which included: 2.17% (w / v) yeast, 1.31% (w / v) sucrose, 1% (w / v) agar, 6.67% (w / v) corn syrup, 0.06% (v / v) propionic acid, 0.1% (v / v) methyl 4-hydroxybenzoate solution, 0.1% (w / v) sodium oxalate, Astragalus water extract stock solution and distilled water. The concentration of Astragalus water extract stock solution in the second culture medium was 0.5 g / mL.

[0071] d. Astragalus eluent administration group: The Astragalus eluent administration group was basically the same as the control group, except that the blank culture medium was replaced with the third culture medium, which consisted of: 2.17% (w / v) yeast, 1.31% (w / v) sucrose, 1% (w / v) agar, 6.67% (w / v) corn syrup, 0.06% (v / v) propionic acid, 0.1% (v / v) methyl 4-hydroxybenzoate solution, 0.1% (w / v) sodium oxalate, Astragalus eluent, and distilled water. The group numbers, the Astragalus eluent in the third culture medium, and its concentration are shown in Table 2.

[0072] Table 2

[0073]

[0074]

[0075] e. Positive control group: The positive control group is basically the same as the control group, except that the blank culture medium is replaced with the fourth culture medium, which includes: 2.17% (w / v) yeast, 1.31% (w / v) sucrose, 1% (w / v) agar, 6.67% (w / v) corn syrup, 0.06% (v / v) propionic acid, 0.1% (v / v) methyl 4-hydroxybenzoate solution, 0.1% (w / v) sodium oxalate, 5% (w / v) potassium citrate and distilled water.

[0076] (3) Experimental methods

[0077] To comprehensively and systematically test the therapeutic effect of verbascoside on calcium oxalate kidney stones in Drosophila, a semi-quantitative method was employed. Methods: Drosophila treated for 7 days in each experimental group were anesthetized on a carbon dioxide operating table and placed in a dissecting dish containing PBS (pH=7.2). Malpighian tubules (equivalent to human kidneys) were dissected using a stereomicroscope. 20 μL of PBS was added to a slide using a pipette, and the Malpighian tubules were placed on the slide and covered with a coverslip. The quantity and size of calcium oxalate crystals within the Malpighian tubules were analyzed under a polarized light microscope.

[0078] The severity of calcium oxalate stones was categorized into four levels based on the number and size of the stones: no stones (no stone accumulation in the main Malpighian tubules of the fruit flies); slight stones (small, granular, unformed fragments of stones in the main Malpighian tubules); moderate stones (a few large, blocky stones in the main Malpighian tubules, with no more than 10 blocky stones per tubule); and severe stones (a large number of large, blocky stones in the main Malpighian tubules, with more than 10 blocky stones per tubule). Fruit flies from each experimental group (20 flies per group) were dissected, and the severity of calcium oxalate stones was assessed, statistically analyzed, and quantified.

[0079] (4) Experimental Results

[0080] like Figure 2A As shown in the figure, under a polarized light microscope, a large number of blocky calcium oxalate crystals were observed in the Malpighian tubules of the disease group of Drosophila, indicating that the Drosophila calcium oxalate kidney stone model was successfully established. In the positive control group, after co-treatment with potassium citrate and sodium oxalate, there were virtually no stones in the Malpighian tubules of the Drosophila. The degree of stones in the Malpighian tubules of Drosophila in the control group, disease group, positive control group, and Astragalus treatment group (n=20 per group) was statistically analyzed, and the results are as follows: Figure 2B As shown in the bar chart, the differences in stone severity between the groups are illustrated. No stones were deposited in the Malpighian tubules of the fly after treatment with Astragalus membranaceus, demonstrating the significant therapeutic effect of Astragalus membranaceus aqueous extract on calcium oxalate stones.

[0081] like Figure 2Cand Figure 2D As shown, with Figure 2A and 2B Compared to the disease group, the Astragalus membranaceus eluent group showed a significant reduction in the degree of Malpighian tubule calculi in Drosophila, with the "1.0 mg / mL 30% ethanol elution (CG)" showing the best reduction in Malpighian tubule calculi. Figure 1 It can be seen that the Astragalus aqueous extract eluent after elution with 30% (v / v) ethanol aqueous solution contains only verbascoside, so it can be inferred that it is the core substance. The subsequent experiment used verbascoside, and its concentration was consistent with the optimal concentration (1.0 mg / mL) of the Astragalus eluent eluted with 30% ethanol for the treatment of Drosophila kidney stones.

[0082] Example 2-2 Astragalus / Verbena isoflavone glucoside inhibits oxidative stress in fruit flies

[0083] (1) Set up the following experimental groups:

[0084] Control group: The control group in Example 2-2 was the same as the control group in Example 2-1;

[0085] Disease group: The disease group in Example 2-2 is the same as that in Example 2-1;

[0086] Astragalus treatment group: The Astragalus treatment group in Example 2-2 is the same as the Astragalus treatment group in Example 2-1;

[0087] Versicolor isoflavone glucoside treatment group: The versicolor isoflavone glucoside treatment group was basically the same as the control group in Example 2-1, except that the blank culture medium was replaced with the fifth culture medium, which included: 2.17% (w / v) yeast, 1.31% (w / v) sucrose, 1% (w / v) agar, 6.67% (w / v) corn syrup, 0.06% (v / v) propionic acid, 0.1% (v / v) methyl 4-hydroxybenzoate solution, 0.1% (w / v) sodium oxalate, purchased versicolor isoflavone glucoside (CG) and distilled water. The concentration of versicolor isoflavone glucoside (CG) in the fifth culture medium was 1.0 mg / mL.

[0088] (2) Experimental methods:

[0089] To determine ROS, Malpighian tubules from Drosophila were isolated from the control group, disease group, Astragalus treatment group, and Versicolor isoflavone glucoside treatment group according to the experimental method in Example 2-1. The isolated Malpighian tubules were stained with a 45 μM ethidium dihydrogen ion (DHE) probe in dimethyl sulfoxide (DMSO) for 5 min. They were then washed three times with 1×PBS for 5 min each time, followed by staining with the nuclear dye DAPI for 5 min, and then washed three times with PBST buffer (pH=4) for 15 min each time. The Drosophila Malpighian tubule specimens were then mounted on microscope slides. Images were acquired using a NIKON fluorescence microscope (SMZ 1270). Image J was used to quantify the intensity of DHE.

[0090] (3) Experimental Results

[0091] like Figure 3A and 3B The ROS level in the disease group was significantly higher than that in the control group, while the ROS levels in the Astragalus treatment group and the Versicolor isoflavone glucoside treatment group were both reduced.

[0092] Fruit flies from the control group, disease group, astragalus treatment group, and isoflavone glucoside treatment group were crushed, and the expression of the genes Sod1 and Cat, which encode antioxidant enzymes, in the cultured fruit flies was detected. Figure 3C As shown, the expression of these genes was significantly downregulated in both the Astragalus membranaceus (Aqua regia) and verbascoside (Isoflavone glucoside) treatment groups. These results suggest that Astragalus membranaceus aqueous extract and verbascoside may alleviate kidney stones by reducing ROS production and oxidative stress levels in Drosophila Malpighian tubules.

[0093] Example 2-3 In vitro crystallization test

[0094] (1) Set up the following experimental groups:

[0095] Model group: Dissolve 0.15M sodium chloride, 0.56mM calcium chloride and 0.56mM sodium oxalate (Na2C2O4) in distilled water to obtain a mixed aqueous solution.

[0096] Positive control group: 0.15M sodium chloride, 0.56mM calcium chloride, 0.56mM Na2C2O4 and citric acid were dissolved in distilled water to obtain a mixed aqueous solution with a citric acid concentration of 50 mg / mL.

[0097] Astragalus aqueous extract group: 0.15M sodium chloride, 0.56mM calcium chloride, 0.56mM Na2C2O4 and Astragalus aqueous extract stock solution (filtered through a 0.22μm membrane before use) were dissolved in distilled water to obtain a mixed aqueous solution. The group number of Astragalus aqueous extract and the concentration of Astragalus aqueous extract stock solution in the mixed aqueous solution are shown in Table 3.

[0098] Table 3

[0099]

[0100] Versicolor isoflavone glucoside group: Since versicolor isoflavone glucoside is insoluble in water, it was replaced by the Astragalus membranaceus eluent eluted with 30% ethanol. 0.15M sodium chloride, 0.56mM calcium chloride, 0.56mM Na2C2O4 and the Astragalus membranaceus eluent eluted with 30% ethanol were dissolved in distilled water to obtain a mixed aqueous solution. The numbering of the versicolor isoflavone glucoside group and the concentration of the Astragalus membranaceus eluent eluted with 30% ethanol in the mixed aqueous solution are shown in Table 4.

[0101] Table 4

[0102]

[0103] (2) Experimental methods:

[0104] At room temperature, place a clean coverslip at the bottom of a 25 mL beaker, add the mixed aqueous solution to the beaker, and place the beaker on a heating plate (37°C) for 3 days. Gently remove the coverslip from the beaker; crystals are attached to the surface of the coverslip, and their morphology is characterized using a bright-field optical microscope.

[0105] (3) Experimental Results

[0106] The effects of different concentrations of Astragalus membranaceus aqueous extract / Astragalus membranaceus eluent on the inhibition of crystal size and number were evaluated. The experimental results are as follows: Figures 4A-4C As shown, by Figure 4A , 4B As shown in 4C, the crystals formed in the model group were regular hexagonal crystals. In the positive control group, the crystal morphology changed significantly, with both size and number significantly reduced, indicating that the model was successfully established. The crystal morphology of the Astragalus aqueous extract group and the verbascoside group changed significantly, with a significant decrease in crystal area. Furthermore, the number of crystals formed was also significantly reduced, suggesting that Astragalus aqueous extract and verbascoside may inhibit stone formation by inhibiting in vitro crystallization.

[0107] Examples 2-4: Comparative Experiments on the Inhibition of Malpighian Tube Stones in Drosophila by Astragalus aqueous extract powder and Versicolor isoflavone glucoside at the Same Concentration

[0108] (1) Experimental group:

[0109] The astragalus water extract stock solution was placed in a freeze dryer at -58℃ for 4 hours to obtain astragalus water extract powder. The following experimental groups were set up:

[0110] Astragalus membranaceus dry powder treatment group: 30 female fruit flies羽化1 - 2 days later were randomly transferred to the sixth culture medium. The fruit flies were fed in the sixth culture medium for 7 days, and the sixth culture medium was changed every 3 days. When testing, 20 fruit flies were randomly selected from each sixth culture medium. Among them, the sixth culture medium included: 2.17% (w / v) yeast, 1.31% (w / v) sucrose, 1% (w / v) agar, 6.67% (w / v) corn syrup, 0.06% (v / v) propionic acid, 0.1% (v / v) methyl 4 - hydroxybenzoate solution, 0.1% (w / v) sodium oxalate, Astragalus membranaceus water extract powder and distilled water. The concentration of Astragalus membranaceus water extract powder in the sixth culture medium was 1.0 mg / mL.

[0111] Astragalus membranaceus eluate administration group: The Astragalus membranaceus eluate administration groups in Examples 2 - 4 were the same as the Astragalus membranaceus eluate administration group in Example 2 - 1. Among them, the Astragalus membranaceus eluate in the third culture medium was the Astragalus membranaceus eluate eluted with 30% ethanol, and the concentration of the Astragalus membranaceus eluate in the third culture medium was 1.0 mg / mL.

[0112] (2) Experimental methods and experimental results:

[0113] Dissect the Malpighian tubules of fruit flies according to the experimental method in Example 2 - 1. The results are as Figure 5A and 5B shown. Under a polarized light microscope, when the concentration of Astragalus membranaceus water extract powder is the same as the optimal concentration (1.0 mg / mL) of the Astragalus membranaceus eluate eluted with 30% ethanol for treating fruit fly kidney stones, the effect of Astragalus membranaceus water extract powder in inhibiting fruit fly kidney stones is not obvious. It can be obtained from this that the effect of the Astragalus membranaceus eluate eluted with 30% ethanol is much higher than that of Astragalus membranaceus water extract powder at the same concentration, indicating that the Astragalus membranaceus eluate eluted with 30% ethanol (i.e., calycosin - 7 - O - glucoside) is the segment where the main active substance for treating kidney stones in Astragalus membranaceus is located.

[0114] Example 3: Therapeutic experiment of calycosin - 7 - O - glucoside on kidney stone model mice

[0115] (1) Preparation of experimental model mice

[0116] C57BL / 6J male mice were strictly raised in the laboratory mouse house according to the feeding standards. The experimental license number is SYXK(津)2019 - 0001: The temperature in the laboratory mouse house was maintained at 23 ± 3 °C, the relative humidity was 55 ± 5%, and the environment was relatively quiet. After the mice were purchased, they were adaptively fed in the laboratory mouse house for one week. After the mice's conditions were stable, subsequent experiments were carried out.

[0117] (2) Experimental grouping and treatment

[0118] a. Control group: Four male C57BL / 6J mice that had been acclimatized for one week were fed normally and injected intraperitoneally with physiological saline at 12-hour intervals in the morning and evening. The dosage was 200 μL of physiological saline per 30g of mouse body weight.

[0119] b. Disease group: Four male C57BL / 6J mice that had been acclimatized for one week were injected intraperitoneally with glyoxylic acid saline solution daily. The dosage was 200 μL of glyoxylic acid saline solution per 30g of mouse body weight. After 12 hours, the same volume of saline solution was injected intraperitoneally. This was continued for 6 days. The amount of glyoxylic acid in the injected glyoxylic acid saline solution was determined according to the weight of the mouse. The amount of glyoxylic acid in the injected glyoxylic acid saline solution per 1kg of mouse body weight was 75mg.

[0120] c and d, verbascoside glucoside treatment group, and citric acid treatment group: Eight male C57BL / 6J mice that had been acclimatized for one week were administered medication one day before modeling, via intraperitoneal injection. Twelve hours after injection, glyoxylate saline solution was injected intraperitoneally for seven days. The dosage was 200 μL of glyoxylate saline solution per 30g of mouse body weight. The glyoxylate content in the injected glyoxylate saline solution was determined based on the mouse's body weight, with 75 mg of glyoxylate injected per kg of mouse body weight. The eight mice were randomly divided into two groups (n=4 per group). The medication administered to the two groups and the grouping details are as follows:

[0121] c. Vernacoside glucoside treatment group: The mice in the vernacoside glucoside treatment group were injected with a mixture of vernacoside glucoside and DMSO. Vernacoside glucoside and DMSO were mixed to prepare a stock solution with a vernacoside glucoside concentration of 4.4 mg / mL. Each mouse was injected with a stock solution containing 30 mg of vernacoside glucoside per kg of body weight.

[0122] d. Citric acid treatment group: The mice in the citric acid treatment group were injected with a mixture of citric acid and water, that is, citric acid and water were mixed to prepare a stock solution with a citric acid concentration of 7.4 mg / mL. Each mouse was injected with a stock solution containing 50 mg of citric acid (positive drug) per kg of body weight.

[0123] (3) Experimental methods

[0124] To comprehensively evaluate the therapeutic effect of verbascoside on renal calculi in mice, serum and histological markers related to renal calculi were measured. Daily changes in mouse body weight were recorded during the experiment. The mouse experimental flowchart is shown below. Figure 6AAs shown, after 7 days of treatment, blood was drawn from the eyeballs to collect serum samples. The mice were then euthanized by cervical dislocation, and kidney tissue samples were collected.

[0125] At the serum level, the levels of creatinine and blood urea nitrogen in mouse serum were measured to assess the filtration function of mouse kidneys. Simultaneously, paraffin sections of mouse kidney tissue were prepared, and the distribution of stones in the mouse kidneys was observed using polarized light imaging. Kidney damage was also observed using periodic acid-Schiff (PAS) staining.

[0126] (4) Experimental Results

[0127] Figure 6B and 6C The serum creatinine and blood urea nitrogen levels were measured in the control group, disease group, verbenafil glucoside treatment group, and citric acid treatment group in Example 3, respectively. The results showed that in the verbenafil glucoside treatment group, mice treated with verbenafil glucoside experienced some recovery, with decreased serum creatinine and blood urea nitrogen levels and restored renal filtration capacity.

[0128] Figure 6D Polarized light images of paraffin sections of kidney tissue from the control group, disease group, verbascoside treatment group, and citric acid treatment group. Figure 6E This is a statistical graph showing the polarization area of ​​paraffin sections of kidney tissue from the control group, disease group, verbascoside glucoside treatment group, and citric acid treatment group. From... Figure 6D and Figure 6E It can be seen that a large number of stones accumulated in the kidneys of mice in the disease group, and the number of stones in the kidneys of mice decreased after treatment with isoflavone glucoside.

[0129] Figure 6F The kidney tissue damage in the control group, disease group, verbascoside treatment group, and citric acid treatment group in Example 3 is illustrated below. Figure 6G This is a statistical chart showing the area of ​​renal injury in the control group, disease group, verbascoside glucoside treatment group, and citrate treatment group. Combined with... Figure 6F and Figure 6G It was found that the control group mice exhibited renal damage lesions such as renal tubular dilation and brush border detachment. In the treatment group with isoflavone glucoside, the degree of renal tubular dilation in the mice was reduced, and the renal damage was alleviated.

[0130] The present invention has been described above by way of example. It should be noted that any simple modifications, alterations or other equivalent substitutions that can be made by those skilled in the art without creative effort without departing from the core of the present invention fall within the protection scope of the present invention.

Claims

1. Use of calycosin-7-glucoside in treating kidney stone medicine / relieving kidney stone medicine.

2. Use of Astragalus in treating kidney stone medicine / relieving kidney stone medicine.

3. Use according to claim 2, characterized in that, When the kidney stone treating / relieving medicine is a traditional Chinese medicine composition, Astragalus is used as the monarch drug in the kidney stone treating / relieving medicine.

4. Use of calycosin-7-glucoside and pharmaceutically acceptable salts or esters thereof in treating kidney stone medicine / relieving kidney stone medicine.

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