Application of myrobalaic acid in preparation of medicine for treating urinary calculus

Abstract

The invention discloses an application of myrobalaic acid in preparation of a medicine for treating urinary calculus. The myrobalaic acid is an organic acid, the molecular structure of the myrobalaic acid contains three carboxyl groups (-COOH) and three hydroxyl groups (-OH), and coordination bonds between carboxylate ions of the myrobalaic acid and calcium ions can effectively fix the calcium ions to form a soluble complex. According to the application disclosed by the invention, the myrobalaic acid with proper concentration or dose shows a relatively good effect of inhibiting the kidney calcium oxalate calculi, and in addition, the myrobalaic acid with proper concentration or dose can be used for remarkably relieving the injury of oxalic acid and kidney calculi to renal tubule HK-2 cells and the caused apoptosis.

Description

Technical Field

[0001] The invention relates to the field of anti-calculus drugs, and in particular to the application of terminalia chebula acid in the preparation of drugs for treating urinary calculi. Background Art

[0002] Kidney stones (calculus of kidney) refer to stones that occur in the renal calyces, renal pelvis, and the connection between the renal pelvis and the ureter. The kidney is the main site for stone formation in the urinary system. Stones in any other part of the body can originate from the kidney. Ureteral stones almost all come from the kidney, and kidney stones are more likely to directly damage the kidney than stones in any other part of the body. The recurrence rate of kidney stones is quite high, which is a significant feature of the disease. According to statistics, the recurrence rate can exceed 50% within 5-10 years after the first onset of the disease. Calcium oxalate stones are the most common type of kidney stones, accounting for about 80%. Their formation is closely related to the body's metabolic process, mainly because the concentration of oxalic acid and calcium in the urine is too high, exceeding the solubility of calcium oxalate, and then crystallizing to form stones. Calcium oxalate stones are the most difficult type to treat with drugs. The types and efficacy of current prevention and treatment drugs are very limited.

[0003] Chebulic acid has strong antioxidant activity; it is effective in controlling elevated metabolic parameters, oxidative stress and liver damage, supporting its beneficial role in asthma, diabetes and liver protection; currently there is no literature report on the effect of chebulic acid on kidney stones. Summary of the invention

[0004] In view of the deficiencies of the prior art, the present invention provides the use of terminalia chebula acid in the preparation of a drug for preventing and / or treating urinary calculi.

[0005] In order to solve the above technical problems, the technical solution of the present invention is as follows: use of chebulic acid in the preparation of a drug for preventing and / or treating urinary calculi, wherein the structural formula of the chebulic acid is as follows: .

[0006] Preferably, the drug is a drug for preventing and / or treating kidney stones.

[0007] As further preferred, the drug is a drug for preventing and / or treating calcium oxalate, calcium phosphate, calcium carbonate or uric acid stones.

[0008] As a further description of the above scheme: the terminalia chebulic acid or a pharmaceutically acceptable salt thereof is used as a pharmaceutically active ingredient.

[0009] As a further description of the above scheme: the pharmaceutically acceptable salt preparation process is as follows: the terminalia chebula acid aqueous solution and alkali solution are mixed, and the pH is adjusted to 6.8-7.5; the alkali solution is generally a sodium or potassium alkali solution.

[0010] As a further description of the above scheme: the terminalia chebula acid is used in combination with a pharmaceutically acceptable carrier, adjuvant or excipient to prepare an injection, injection, powder, tablet, oral solution, capsule or granule.

[0011] As a further description of the above scheme: the terminalia chebula acid is used alone as a pharmaceutical active ingredient, or is used in combination with existing drugs.

[0012] As a further description of the above scheme: the concentration of the active ingredient of the drug is 2.5-20 μg / mL, calculated as terminalia chebulic acid.

[0013] Chebulic acid is an organic acid with three carboxyl groups (-COOH) and three hydroxyl groups (-OH) in its molecular structure. These functional groups can react with metal ions to form complexes. In aqueous solution, the carboxyl group in the chebulic acid molecule can lose a hydrogen ion (H + ), forming carboxylate ions (-COO - ). Calcium ion (Ca 2+ ) has an empty d orbital and can accept electron pairs, so it can form a coordination bond with the terminal acid ion. Specifically, calcium ions can react with two or three carboxylate ions in the terminal acid molecule to form a stable complex. Therefore, the mechanism by which terminal acid inhibits the formation and growth of renal calcium oxalate stones is that the coordination bond between the carboxylate ions and calcium ions of terminal acid can effectively fix calcium ions to form a soluble complex, preventing calcium ions from reacting with other substances to form calcium-containing stones (such as calcium oxalate, calcium phosphate and calcium carbonate stones, etc.) and preventing calcium-containing substances from being deposited in the kidneys to form stones, foreign bodies that stimulate and damage the kidneys, and induce cancer. In summary, terminal acid can be used to prepare drugs for the prevention and / or treatment of urinary stones.

[0014] Compared with the prior art, the present invention has the following beneficial effects: the present invention finds that appropriate concentrations or doses of terminal chebulic acid show a good effect of inhibiting kidney calcium oxalate stones. In addition, oxalic acid and kidney stones can significantly damage human renal tubular HK-2 cells and cause cell apoptosis. Appropriate doses or concentrations of terminal chebulic acid can significantly alleviate the damage of oxalic acid and kidney stones to renal tubular HK-2 cells and the resulting apoptosis; and can also significantly reduce the generation and accumulation of ROS in renal tubular HK-2 cells caused by oxalic acid and kidney stones. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1The inhibitory effect of different concentrations of terminalia chebula acid in neutral terminalia chebula acid solution on stones, A is the kidney stone model group without terminalia chebula acid treatment, B is the treatment group containing 2.5ug / mL terminalia chebula acid, C is the treatment group containing 5.0ug / mL terminalia chebula acid, D is the treatment group containing 7.5ug / mL terminalia chebula acid, E is the treatment group containing 10ug / mL terminalia chebula acid, F is the treatment group containing 20ug / mL terminalia chebula acid, G is the statistical results of kidney stone area compared with the treatment groups containing different doses of terminalia chebula acid and the model (the white spots in A, B, C, D, E, and F are kidney stone crystals, see the enlarged picture); Figure 2 The effect of different concentrations of chebulic acid in neutral chebulic acid solution on the survival rate of renal tubular cells; Figure 3 The neutral chebula acid salt solution containing different concentrations of chebula acid inhibits cell apoptosis caused by oxalic acid and kidney stones. A is the apoptosis of human kidney HK-2 cells under normal culture, B is the apoptosis of renal tubular cells in the presence of oxalic acid and kidney stones, C is the neutral chebula acid salt solution (containing 5μg / mL chebula acid) relieving renal tubular cell apoptosis, D is the neutral chebula acid salt solution (containing 7.5μg / mL chebula acid) relieving renal tubular cell apoptosis, and E is the statistical results of renal tubular cell apoptosis in four groups; Figure 4 The cells under the microscope are shown in which neutral chebula acid salt solution containing different concentrations of chebula acid alleviates the damage of oxalic acid and kidney stones to renal cells. A shows that the cells grow well and are closely arranged under normal conditions. B shows that the cells grow poorly under the stimulation of oxalic acid and kidney stone crystals, with a large number of damage and apoptosis. C shows that the cell growth is alleviated under neutral chebula acid salt solution 1 (containing 5 μg / mL chebula acid), and the damage and apoptosis are reduced. D shows that the cell growth is significantly improved under neutral chebula acid salt solution 2 (containing 7.5 μg / mL chebula acid), and the damage and apoptosis are significantly reduced (the red arrow is the kidney stone crystal). Figure 5 The results show that neutral terminal chebula acid solution containing different concentrations of terminal chebula acid significantly reduced the production and accumulation of ROS, a marker of renal cell damage. A is the ROS situation of human kidney HK-2 cells under normal culture, B is the ROS situation of renal tubular cells in the presence of oxalic acid and kidney stones, C is the neutral terminal chebula acid solution (containing terminal chebula acid 5μg / mL) relieving the ROS situation of renal tubular cells, D is the neutral terminal chebula acid solution (containing terminal chebula acid 7.5μg / mL) relieving the ROS situation of renal tubular cells, and E is the statistical results of the ROS situation of renal tubular cells and renal cell damage markers in four groups; Figure 6 The effects of low, medium and high doses of neutral chebula salt solution on inhibiting stone formation in a mouse kidney stone model; Figure 7The neutral terminal chebula acid solution contains different concentrations of terminal chebula acid to directly inhibit oxalate stones in vitro without cells. A is the oxalate stone model group without terminal chebula acid treatment, B is the treatment group containing 2.5ug / mL terminal chebula acid, C is the treatment group containing 5.0ug / mL terminal chebula acid, D is the treatment group containing 7.5ug / mL terminal chebula acid, E is the treatment group containing 10ug / mL terminal chebula acid, F is the treatment group containing 12.5ug / mL terminal chebula acid, G is the treatment group containing 15ug / mL terminal chebula acid, H is the comparison between the treatment groups containing different doses of terminal chebula acid and the model group, and the statistical results of oxalate stone area (the white spots in A, B, C, D, E, F, and G are stone crystals); Figure 8 The neutral terminal chebula acid salt solution contains different concentrations of terminal chebula acid to inhibit uric acid stone formation; A is the uric acid stone model group without terminal chebula acid treatment, B is the treatment group containing 2.5ug / mL terminal chebula acid, C is the treatment group containing 5.0ug / mL terminal chebula acid, D is the treatment group containing 7.5ug / mL terminal chebula acid, E is the treatment group containing 10ug / mL terminal chebula acid, and F is the statistical results of uric acid stone area between the treatment groups containing different doses of terminal chebula acid and the model group (the black spots in A, B, C, D, and E are stone crystals); Fig. 9 The figure shows the direct inhibitory effect of different concentrations of terminalia chebula acid solution on oxalate stone formation in vitro without cells. A is the oxalate stone model group without terminalia chebula acid treatment, B is the treatment group with 2.5ug / mL terminalia chebula acid, C is the treatment group with 5.0ug / mL terminalia chebula acid, D is the treatment group with 7.5ug / mL terminalia chebula acid, E is the treatment group with 10ug / mL terminalia chebula acid, F is the treatment group with 12.5ug / mL terminalia chebula acid, G is the treatment group with 15ug / mL terminalia chebula acid, H is the comparison between the treatment groups with different doses of terminalia chebula acid and the model group, and the statistical results of oxalate stone area (the white spots in A, B, C, D, E, F, and G are stone crystals); Fig.10 The results of different concentrations of terminalia chebula acid solution in inhibiting uric acid calculi in vitro. A is the uric acid calculi model group without terminalia chebula acid treatment, B is the treatment group with 2.5ug / mL terminalia chebula acid, C is the treatment group with 5.0ug / mL terminalia chebula acid, D is the treatment group with 7.5ug / mL terminalia chebula acid, E is the treatment group with 10ug / mL terminalia chebula acid, and F is the statistical results of uric acid calculi area between the treatment groups with different doses of terminalia chebula acid and the model group (the black spots in A, B, C, D, and E are stone crystals); Fig.11This is a comparison of the effects of terminalia chebula acid salt, terminalia chebula and ten-flavor terminalia chebula pills in inhibiting stone formation in a mouse kidney stone model. A is the calcium oxalate stone model group without drug treatment, B is the treatment group given 75 mg / kg terminalia chebula acid, C is the treatment group given 780 mg / kg terminalia chebula, D is the treatment group given 780 mg / kg ten-flavor terminalia chebula pills, and E is the statistical results of calcium oxalate stone area compared between the three treatment groups and the model group (the white spots in A, B, C, and D are stone crystals). DETAILED DESCRIPTION

[0016] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments, but the present invention is not limited to the following technical solution.

[0017] Sources of reagents: MEM powder culture medium was purchased from Gibco, fetal bovine serum was purchased from ExCell, sodium oxalate and terminalia chebulic acid were purchased from MCE; the culture medium used in the following examples was DMEM culture medium purchased from Gibco.

[0018] Example 1 Cell test verification of the inhibition of kidney stones by terminalia chebula acid salt 1. The effect of terminalia chebula acid salt on inhibiting kidney stones

[0019] Experimental Grouping Group 1: HK-2 cells were cultured normally in culture medium; Group 2: HK-2 cells were treated with 1 mmol / L sodium oxalate solution; Group 3: HK-2 cells were treated with 1 mmol / L sodium oxalate and neutral chebulic acid solution 1 (containing 2.5 μg / mL chebulic acid); Group 4: HK-2 cells were treated with 1 mmol / L sodium oxalate and neutral chebulic acid solution 2 (containing 5.0 μg / mL chebulic acid); Group 5: HK-2 cells were treated with 1 mmol / L sodium oxalate and neutral chebulic acid solution 3 (containing 7.5 μg / mL chebulic acid); Group 6: HK-2 cells were treated with 1 mmol / L sodium oxalate and neutral chebulic acid solution 4 (containing 10 μg / mL chebulic acid); Group 7: HK-2 cells were treated with 1 mmol / L sodium oxalate and neutral chebulic acid solution 5 (containing 20 μg / mL chebulic acid); The preparation process of neutral chebulic acid salt solutions of different concentrations is as follows: Preparation of neutral terminal chebulic acid solution: Weigh 201.6 mg terminal chebulic acid powder and dissolve it in 1.2 mL DMSO solvent. After ultrasonic dissolution until the drug is completely dissolved, add 10.8 mL 20% SBE-β-CD saline solution and mix thoroughly to obtain 16.8 mg / mL terminal chebulic acid solution. Before use, use a strong base (0.1 M NaOH) solution to adjust the pH to neutral (pH 6.8-7.5).

[0020] According to the total volume of complete medium added to each well of the culture plate, calculate the amount of terminal chebulic acid solution that needs to be added to obtain different concentrations of terminal chebulic acid groups. For example, if the total volume of complete medium added to each well is 1 mL, the calculation formula is X μg / mL = (16.8 × 10 3 μg / mL×AmL)÷(1mL+AmL), X is the required concentration of terminalia chebula acid, i.e., 2.5μg / mL, 5.0μg / mL, 7.5μg / mL, 10μg / mL, 20μg / mL, and A is the volume of terminalia chebula acid solution required to be added corresponding to different concentrations of terminalia chebula acid.

[0021] Cell treatment and detection: HK-2 cells were digested with 0.25% trypsin to collect the cells, the supernatant was removed, and the cells were resuspended in 1 mL of complete culture medium.

[0022] Cell seeding: 2×10 per well 4 The cells were seeded in 24-well plates at a density of 10 cells / well, with 3 replicates per group.

[0023] When the cell confluence reached about 80%, group 2 was treated with 1 mmol / L sodium oxalate for 48 hours, and groups 3-7 were treated with 1 mmol / L sodium oxalate and corresponding concentrations of terminalia chebulic acid for 48 hours.

[0024] Detection: Samples were provided in 24-well plates and observed and photographed using a polarized light microscope.

[0025] Experimental results: Figure 1 As shown in the results, compared with other concentrations, the stone area was greatly reduced at a concentration of 7.5ug / mL of chebulic acid, showing a good inhibitory effect on calcium oxalate stones (p < 0.0001), with an inhibition rate of about 80%. The high concentration of 20ug / mL promoted the formation of kidney stones.

[0026] 2. Effect of chebulic acid salt on the survival rate of renal tubular cells Experimental Grouping Group 1: HK-2 cells were cultured normally in culture medium; Group 2: HK-2 cells were co-cultured with neutral chebulic acid solution a (containing chebulic acid 2.5 μg / mL); Group 3: HK-2 cells were co-cultured with neutral chebulic acid solution b (containing chebulic acid 5.0 μg / mL); Group 4: HK-2 cells were co-cultured with neutral chebulic acid solution c (containing chebulic acid 7.5 μg / mL); Group 5: HK-2 cells were co-cultured with neutral chebulic acid solution d (containing chebulic acid 10 μg / mL); Group 6: HK-2 cells were co-cultured with neutral chebulic acid solution e (containing chebulic acid 20 μg / mL); Cell seeding: 5×10 3 The cells were seeded in 96-well plates, with 5 replicates per group.

[0027] When the cell confluence reached about 80%, groups 2 to 6 were treated with corresponding different concentrations of chebulic acid for 48 hours.

[0028] After 48 hours of treatment with terminalia chebula acid, the CCK8 detection method (CCK8 reagent purchased from targetmol) was used for detection: 1. Pipette 100 μL of basal medium from the 96-well plate according to the time point and group; 2. Set up a blank control well and add 100 μL of basal culture medium to the other 3 wells; 3. Add 10 μL CCK8 solution to each well (be careful not to create bubbles in the well); 4. Place the plate back in the incubator and incubate for 2 hours away from light. Use an ELISA reader to measure the OD value of each well in the 96-well plate at 450 nm. Cell viability %=(OD of experimental well-OD of blank well) / (OD of control well-OD of blank well)×100%.

[0029] Experimental results: Figure 2 It can be seen that compared with the normal group, the neutral terminal chebulic acid solution containing 2.5 μg / mL, 5 μg / mL, 7.5 μg / mL, 10 μg / mL and 20 μg / mL terminal chebulic acid doses did not affect the survival rate of human renal tubular cells HK-2, that is, the dose within the effective range would not damage human renal tubular cells.

[0030] 3. Chebulic acid can inhibit the damage of renal tubular cells caused by oxalic acid and kidney stones Experimental groups: (1) HK-2 cells were cultured normally in culture medium; (2) HK-2 cells + 1 mmol / L sodium oxalate solution; (3) HK-2 cells + sodium oxalate + neutral chebulic acid solution 1 (containing chebulic acid 5 μg / mL); (4) HK-2 cells + sodium oxalate + neutral chebulic acid solution 2 (containing chebulic acid 7.5 μg / mL); HK-2 cells were digested with 0.25% trypsin and collected. After removing the supernatant, the cells were resuspended in 1 mL of complete medium and inoculated at 5 × 10 cells per well. 4 The cells were inoculated in 6-well plates, with 3 replicates in each group. When the cell confluence reached about 80%, 1 mmol / L sodium oxalate was added to group 2 for 48 h, and 1 mmol / L sodium oxalate and corresponding concentrations of terminalia chebulic acid were added to groups 3 and 4 for 48 h.

[0031] (1) Cell apoptosis detection method (cell apoptosis detection kit was purchased from Bio-Tech Company) 1. After adding 0.25% trypsin, cover the lid and return to the incubator to digest for about 1-2 minutes. When the cells are found to shrink into a spherical shape or a quicksand-like shape, 0.5 mL of fresh complete medium should be added immediately to terminate the digestion; 2. Transfer the cell suspension to a new 1.5 mL centrifuge tube and centrifuge at 1000 rpm for 3 min to collect the cells; 3. Discard the supernatant, gently resuspend the cells with 0.5 mL PBS, and centrifuge at 1000 rpm for 3 min to collect the cells; 4. Add 100 μL 1×Banding Buffer to resuspend the cells and separate into 3 tubes for blank control, AnnexinV / PE single positive control and 7-AAD single positive control respectively; 5. Except for the blank, AnnexinV / PE, and 7-AAD controls (5μL AnnexinV / PE and 10μL 7-AAD were added to the AnnexinV / PE and 7-AAD single positive controls, respectively), add 5μL AnnexinV / PE and 10μL 7-AAD to each tube, mix gently, and incubate in the dark for 15 minutes; 6. Add 400 μL 1×Banding Buffer, mix well, and detect on flow cytometry.

[0032] Experimental results: Figure 3 The neutral chebulic acid solution contains different concentrations of chebulic acid to inhibit cell apoptosis caused by oxalic acid and kidney stones. Figure 4 The neutral chebulic acid solution contains different concentrations of chebulic acid to relieve the damage of oxalic acid and kidney stones to renal cells under the microscope. Figure 3 and Figure 4It can be seen that oxalic acid and kidney stones can significantly damage human renal tubular HK-2 cells and cause cell apoptosis. Neutral terminal chebula acid solution (containing 5 μg / mL and 7.5 μg / mL terminal chebula acid, respectively) can significantly alleviate the damage and apoptosis of terminal chebula HK-2 cells caused by oxalic acid and kidney stones, and the terminal chebula acid containing 7.5 μg / mL has a better effect.

[0033] (2) Neutral terminal chebula acid solution relieves renal cell damage caused by oxalic acid and kidney stones Detection of reactive oxygen species, a marker of renal cell damage (detection kit purchased from Solebao): 1. Dilute DHE fluorescent dye 1:1000 in serum-free medium to a final concentration of 5 μM.

[0034] 2. Remove the cell culture medium and add an appropriate volume of diluted DHE. The volume added should be sufficient to cover the cells. Usually, add no less than 1 mL of diluted DHE to one well of a 6-well plate.

[0035] 3. The positive control was added at a ratio of 1:1000.

[0036] 4. Incubate in a 37°C cell culture incubator for 30-40 min.

[0037] 5. Remove the staining solution and wash the cells twice with serum-free cell culture medium.

[0038] 6. Streaming on-machine testing.

[0039] Experimental results: Figure 5 The results show that the neutral terminal chebula acid solution containing different concentrations of terminal chebula acid significantly reduced the production and accumulation of ROS, a marker of renal cell damage. Figure 5 It can be seen that oxalic acid and kidney stones can significantly promote the production of reactive oxygen species (ROS), a damage marker of human renal tubular HK-2 cells. Neutral terminal chebula acid solution (i.e., containing 5 μg / mL and 7.5 μg / mL terminal chebula acid) can significantly reduce the damage of renal tubular HK-2 cells caused by oxalic acid and kidney stones (i.e., the production and accumulation of damage marker ROS are significantly reduced), among which the terminal chebula acid with a content of 7.5 μg / mL has a better effect.

[0040] Example 2 Animal test verification of the inhibition of kidney stones by terminalia chebula acid salt The kidney stone model was established by intraperitoneally injecting 40 mg / kg sodium oxalate solution every day for 7 consecutive days to obtain a kidney stone animal model.

[0041] 1. Experimental Grouping (1) Control group: blank control (2) Model group: kidney stone model (3) Low-dose group: Model + low-dose neutral chebulic acid solution (i.e., the chebulic acid content administered was 37.5 mg / kg) (4) Medium-dose group: Model + medium-dose neutral chebulic acid solution (i.e., the chebulic acid content administered was 75 mg / kg) (5) High-dose group: model + high-dose neutral chebulic acid solution (i.e., the chebulic acid content administered was 150 mg / kg) 2. Reagent preparation

[0042] (1) Preparation of sodium oxalate solution: Weigh 40 mg of sodium oxalate powder and dissolve it in physiological saline to a volume of 5 mL. Mix thoroughly and dissolve with ultrasound to prepare an 8 mg / mL sodium oxalate solution. Then calculate the injection volume based on the weight of each mouse.

[0043] (2) Preparation of neutral terminal chebulic acid salt solution: Terminal chebulic acid (37.5 mg / kg): weigh 63 mg powder, dissolve in pure water, and dilute to 15 ml. Prepare a 7-day supply. Terminal chebulic acid (75 mg / kg): weigh 126 mg powder, dissolve in pure water, and dilute to 15 ml. Prepare a 7-day supply. Terminal chebulic acid (150 mg / kg): weigh 252 mg powder, dissolve in pure water, and dilute to 15 ml. Prepare a 7-day supply. Before use, adjust the pH to neutral (pH 6.8-7.5) using a strong base (0.1 M NaOH) solution.

[0044] 3. Experimental methods Fifty SPF C57 male mice, 6-8 weeks old, 18-22g. They were randomly divided into 5 groups: control group, model group, low-dose group, medium-dose group and high-dose group. The model group was intraperitoneally injected with 40 mg / kg sodium oxalate solution every day for 7 consecutive days (to establish a kidney stone model). In addition to intraperitoneal injection of 40 mg / kg sodium oxalate solution every day to induce kidney stone formation, the low-dose group, medium-dose group and high-dose group were gavaged with low, medium and high doses of neutral chebulic acid solution every day to intervene in kidney stone formation (i.e., the amount of chebulic acid administered was 37.5 mg / kg, 75 mg / kg, and 150 mg / kg, respectively), and the control group was gavaged with normal saline for 7 consecutive days.

[0045] 4. Experimental results After 7 days, the kidneys of the mice were sliced ​​and stained with HE. After observation under a polarizing microscope and elimination of samples for which modeling was not successful, photos were taken and statistics were obtained. Figure 6 The effects of low, medium and high doses of chebulic acid salt on the inhibition of stone formation in a mouse kidney stone model were investigated. Figure 6 The white spots in the picture are kidney stones). Figure 6The results showed that compared with the kidney stone model group, neutral chebulic acid salt (i.e., 37.5 mg / kg, 75 mg / kg, and 150 mg / kg of chebulic acid respectively) could significantly inhibit the formation of kidney stones, and the medium dose group (administered 75 mg / kg of chebulic acid) had the best effect ( p < 0.0001).

[0046] Example 3 Neutral terminal chebula acid salt inhibits the formation of calcium oxalate stones in vitro Experimental Grouping Group A: DMEM medium was treated with 1 mmol / L sodium oxalate solution; Group B: DMEM medium was treated with 1 mmol / L sodium oxalate and neutral chebulic acid solution 1 (containing 2.5 μg / mL chebulic acid); Group C: DMEM medium was treated with 1 mmol / L sodium oxalate and neutral chebulic acid solution 2 (containing 5.0 μg / mL chebulic acid); Group D: DMEM medium was treated with 1 mmol / L sodium oxalate and neutral chebulic acid solution 3 (containing 7.5 μg / mL chebulic acid); Group E: DMEM medium was treated with 1 mmol / L sodium oxalate and neutral chebulic acid solution 4 (containing 10 μg / mL chebulic acid); Group F: DMEM medium was treated with 1 mmol / L sodium oxalate and neutral chebulic acid solution 5 (containing 12.5 μg / mL chebulic acid); Group G: DMEM medium was treated with 1 mmol / L sodium oxalate and neutral chebulic acid solution 6 (containing 15 μg / mL chebulic acid); The preparation of neutral chebulic acid salt was referred to Example 1. Groups A to G were placed in a 37° C. cell culture incubator using 24-well plates for 48 hours and then the stone status was measured.

[0047] Experimental results: Figure 7 As shown, compared with the control group (group A) oxalate stone model, the neutral chebulic acid solution containing 2.5 μg / mL, 5 μg / mL, 7.5 μg / mL, 10 μg / mL, 12.5 μg / mL and 15 μg / mL chebulic acid could significantly inhibit the formation of oxalate stones ( p < 0.0001), but large-diameter calcium oxalate stones were formed at 12.5 μg / mL and 15 μg / mL of chebulic acid, indicating that excessive concentration of chebulic acid may promote the formation of large-diameter calcium oxalate stones. The above results show that neutral chebulic acid salt solution can directly inhibit oxalate stone formation in the absence of cells, but the concentration should not be too high.

[0048] Example 4: Inhibition of Uric Acid Stone Formation by Chebulic Acid Salt in Vitro Experimental Grouping Group A: Complete medium (DMEM medium) was treated with 10 mmol / L uric acid; Group B: Complete medium was treated with 10 mmol / L uric acid and neutral chebulic acid solution 1 (containing 2.5 μg / mL chebulic acid); Group C: complete medium was treated with 10 mmol / L uric acid and neutral chebulic acid solution 2 (containing 5.0 μg / mL chebulic acid); Group D: complete medium was treated with 10 mmol / L uric acid and neutral chebulic acid solution 3 (containing 7.5 μg / mL chebulic acid); Group E: complete medium was treated with 10 mmol / L uric acid and neutral chebulic acid solution 4 (containing 10 μg / mL chebulic acid); The present application found in Examples 1 and 3 that the test results were mainly related to the calcium ions in the culture medium, and whether or not cell culture was added did not affect the test results, so cell culture was not added in this example. Groups A to E were placed in a 37°C cell culture incubator using 24-well plates for 48 hours and then the stone status was measured.

[0049] Experimental results: Figure 8 As shown in the figure, compared with the control group (group A) uric acid stone model, the neutral chebulic acid solution containing 2.5 μg / mL, 5 μg / mL and 7.5 μg / mL chebulic acid can significantly inhibit the formation of uric acid stones ( p < 0.05).

[0050] Example 5: Chebulic acid inhibits the formation of calcium oxalate stones in vitro Experimental Grouping Group A: DMEM medium was treated with 1 mmol / L sodium oxalate solution; Group B: DMEM medium was treated with 1 mmol / L sodium oxalate and terminalia chebulic acid solution 1 (termia chebulic acid 2.5 μg / mL); Group C: DMEM medium was treated with 1 mmol / L sodium oxalate and terminalia chebulic acid solution 2 (termia chebulic acid 5.0 μg / mL); Group D: DMEM medium was treated with 1 mmol / L sodium oxalate and terminalia carbolic acid solution 3 (termia carbolic acid 7.5 μg / mL); Group E: DMEM medium was treated with 1 mmol / L sodium oxalate and terminalia chebulic acid solution 4 (termica chebulic acid 10 μg / mL); Group F: DMEM medium was treated with 1 mmol / L sodium oxalate and terminalia chebulic acid solution 5 (termia chebulic acid 12.5 μg / mL); Group G: DMEM medium was treated with 1 mmol / L sodium oxalate and terminalia chebulic acid solution 6 (termia chebulic acid 15 μg / mL); Experimental results: Fig. 9 As shown, compared with the control group (Group A) oxalate stone model, 2.5 μg / mL, 5 μg / mL, 7.5 μg / mL, 10 μg / mL, 12.5 μg / mL and 15 μg / mL of terminalia chebula acid solution could significantly inhibit oxalate stone formation (p < 0.0001), indicating that terminalia chebula acid solution can directly inhibit oxalate stone formation.

[0051] Example 6: Terminalia chebula acid inhibits the formation of uric acid stones in vitro

[0052] Experimental Grouping Group A: complete medium was treated with 10 mmol / L uric acid; Group B: Complete medium was treated with 10 mmol / L uric acid and terminalia chebulic acid solution 1 (termia chebulic acid 2.5 μg / mL); Group C: complete medium was treated with 10 mmol / L uric acid and terminalia carbolic acid solution 2 (termia carbolic acid 5.0 μg / mL); Group D: complete medium was treated with 10 mmol / L uric acid and terminalia carbolic acid solution 3 (termia carbolic acid 7.5 μg / mL); Group E: complete medium was treated with 10 mmol / L uric acid and terminalia carbolic acid solution 4 (termicalic acid 10 μg / mL); Experimental results: Fig.10 As shown, compared with the control group (Group A) in the uric acid stone model, the terminalia chebula acid solution containing 5 μg / mL terminalia chebula acid could significantly inhibit the formation of uric acid stones (p < 0.05).

[0053] Example 7 Animal test verification of the effect of terminalia chebula acid salt in inhibiting kidney stones is better than that of terminalia chebula and ten-flavor terminalia chebula pills The kidney stone model was established by intraperitoneally injecting 40 mg / kg sodium oxalate solution every day for 7 consecutive days to obtain a kidney stone animal model.

[0054] 1. Experimental Grouping (1) Model group: kidney stone model (2) Terminalia chebula acid group: model + neutral terminalia chebula acid solution (i.e. terminalia chebula acid content of 75 mg / kg) (3) Terminalia chebula group: model + terminalia chebula powder solution (i.e., the terminalia chebula content administered was 780 mg / kg) (4) Shiwei Chebula Pill Group: Model + Shiwei Chebula Pill Solution (i.e., the Shiwei Chebula Pill content administered was 780 mg / kg) 2. Reagent preparation (1) Preparation of sodium oxalate solution: Weigh 40 mg of sodium oxalate powder and dissolve it in physiological saline to a volume of 5 mL. Mix thoroughly and dissolve with ultrasound to prepare an 8 mg / mL sodium oxalate solution. Then calculate the injection volume based on the weight of each mouse.

[0055] (2) Preparation of terminalia chebula acid solution: Weigh 201.6 mg terminalia chebula acid powder and dissolve it in 1.2 ml DMSO solvent. After ultrasonic dissolution until the drug is completely dissolved, add 10.8 ml 20% SBE-β-CD saline solution and mix thoroughly to obtain a 16.8 mg / ml terminalia chebula acid solution. Before use, use a strong base (0.1 M NaOH) solution to adjust the pH to neutral (pH 6.8-7.5).

[0056] (3) Preparation of Terminalia chebula powder solution: Weigh 117 mg of Terminalia chebula powder and dissolve it in 1.5 ml of pure water. Ultrasonic dissolution was performed until the drug was completely dissolved to obtain a 78 mg / ml Terminalia chebula powder solution (Termia chebula dry powder produced in Yunnan).

[0057] (4) Preparation of Ten-flavor Terminalia chebula pill solution: Weigh 117 mg of Ten-flavor Terminalia chebula pill and dissolve it in 1.5 ml of pure water. Ultrasonic dissolution was performed until the drug was completely dissolved to obtain a 78 mg / ml Ten-flavor Terminalia chebula pill solution (Ten-flavor Terminalia chebula pill produced by Jinhe Tibetan Medicine Co., Ltd.).

[0058] 3. Experimental methods Twenty-four SPF C57 male mice, 6-8 weeks old, 18-22g, were randomly divided into four groups: model group, terminalia acid group, terminalia chebula group, and ten-flavor terminalia chebula pill group. The model group was intraperitoneally injected with 40 mg / kg sodium oxalate solution every day for 7 consecutive days (to establish a kidney stone model). In addition to intraperitoneal injection of 40 mg / kg sodium oxalate solution every day to induce kidney stone formation, the terminalia acid group, terminalia chebula group, and ten-flavor terminalia chebula pill group were intragastrically administered with neutral terminalia chebula salt solution, terminalia chebula powder solution, and ten-flavor terminalia chebula pill solution every day to intervene in kidney stone formation (i.e., the terminalia chebula acid content was 75 mg / kg, terminalia chebula content was 780 mg / kg, and ten-flavor terminalia chebula pill content was 780 mg / kg, respectively), for 7 consecutive days.

[0059] 4. Experimental results After 7 days, the kidneys of the mice were sliced ​​and stained with HE. After observation under a polarizing microscope and elimination of samples that failed to model, photos were taken and statistics were obtained. Fig.11The inhibitory effects of terminalia chebula salt, terminalia chebula and ten-flavor terminalia chebula pills on the formation of kidney stones in mice model. Fig.11 The white spots in the picture are kidney stones). Fig.11 The results showed that compared with the kidney stone model group, terminalia chebula salt, terminalia chebula and ten-flavor terminalia chebula pills (termia chebula acid content of 75 mg / kg, terminalia chebula content of 780 mg / kg, and ten-flavor terminalia chebula pills content of 780 mg / kg, respectively) could significantly inhibit the formation of kidney stones, and the terminalia chebula acid group had the best effect (p < 0.0001).

[0060] The above are only preferred embodiments of the present invention, and the protection scope of the present invention is not limited to the above embodiments. All technical solutions under the concept of the present invention belong to the protection scope of the present invention. It should be pointed out that for ordinary technicians in this technical field, any modification, equivalent replacement or improvement without departing from the principle of the present invention should be regarded as the protection scope of the present invention.

Claims

1. Use of terminalia chebula acid in the preparation of a drug for preventing and / or treating urinary calculi, characterized in that: The structural formula of the terminalia carbolic acid is as follows: .

2. The use of terminalia chebula acid according to claim 1 in the preparation of a drug for preventing and / or treating urinary calculi, characterized in that: The medicine is a medicine for preventing and / or treating kidney stones.

3. The use of terminalia chebula acid according to claim 1 in the preparation of a drug for preventing and / or treating urinary calculi, characterized in that: The medicine is a medicine for preventing and / or treating calcium oxalate, calcium phosphate, calcium carbonate or uric acid stones.

4. The use of terminalia chebula acid according to claim 1 in the preparation of a drug for preventing and / or treating urinary calculi, characterized in that: The terminal acid or a pharmaceutically acceptable salt thereof is used as a pharmaceutically active ingredient.

5. The use of terminalia chebula acid according to claim 4 in the preparation of a drug for preventing and / or treating urinary calculi, characterized in that: The pharmaceutically acceptable salt preparation process is as follows: the terminalia acid aqueous solution and alkaline solution are mixed, and the pH is adjusted to 6.8-7.

5.

6. Use of the terminalic acid according to claim 1 in the preparation of a drug for preventing and / or treating urinary calculi, characterized in that: The terminalia chebula acid is used in combination with a pharmaceutically acceptable carrier, adjuvant or excipient to prepare an injection, an injection, a powder, a tablet, an oral solution, a capsule or a granule.

7. Use of the terminalic acid according to claim 1 in the preparation of a drug for preventing and / or treating urinary calculi, characterized in that: The terminalia chebula acid is used alone as a pharmaceutical active ingredient or in combination with existing drugs.

8. Use of the terminalic acid according to claim 4 or 7 in the preparation of a drug for preventing and / or treating urinary calculi, characterized in that: The concentration of the active ingredient of the medicine is 2.5-20 μg / mL, calculated as terminalia chebulic acid.

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