Application of terminalia chebulic acid in the preparation of medicines for treating urinary calculi

Abstract

The present invention discloses the use of terminalia chebula acid in the preparation of a drug for treating urinary stones. Terminalia chebula acid is an organic acid containing three carboxyl groups (‑COOH) and three hydroxyl groups (‑OH) in its molecular structure. The coordination bond between the carboxylate ion of terminalia chebula acid and the calcium ion can effectively fix the calcium ion to form a soluble complex. The present invention found that terminalia chebula acid at an appropriate concentration or dose showed a good effect in inhibiting the formation of renal calcium oxalate stones. In addition, terminalia chebula acid at an appropriate dose or concentration can significantly alleviate the damage and resulting apoptosis of renal tubular HK‑2 cells caused by oxalic acid and kidney stones.

Description

Technical Field

[0001] The present invention relates to the field of anti-calculus drugs, and in particular to the application of terminalia chebulic 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 in the kidney. Ureteral stones almost all originate 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 patient's first onset. 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 concentrations of oxalic acid and calcium in the urine are 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] Terminalia chebula has strong antioxidant activity; terminalia chebula is effective in controlling elevated metabolic parameters, oxidative stress, and liver damage, supporting its beneficial role in asthma, diabetes, and liver protection; currently, there are no literature reports on the effect of terminalia chebula 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 chebulic acid in the preparation of a medicament for preventing and / or treating urinary stones.

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

[0006] .

[0007] Preferably, the drug is a drug for preventing and / or treating kidney stones.

[0008] As a further preference, the drug is a drug for preventing and / or treating calcium oxalate, calcium phosphate, calcium carbonate or uric acid stones.

[0009] As a further description of the above scheme: the terminalic acid or a pharmaceutically acceptable salt thereof is used as a pharmaceutical active ingredient.

[0010] As a further description of the above scheme: the pharmaceutically acceptable salt is prepared as follows: the terminalia acid aqueous solution is mixed with an alkali solution, and the pH is adjusted to 6.8-7.5; the alkali solution is generally a sodium or potassium alkali solution.

[0011] As a further description of the above scheme: the terminalic 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.

[0012] As a further description of the above scheme: the terminalia chebulic acid is used alone as an active pharmaceutical ingredient, or is used in combination with existing drugs.

[0013] 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 terminaliacin.

[0014] Terminalia chebula acid is an organic acid that contains 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 groups in the terminalia chebula acid molecule can lose a hydrogen ion (H + ), forming carboxylate ions (-COO - ). Calcium ions (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 ion and the calcium ion of terminal acid can effectively fix the calcium ion to form a soluble complex, preventing the calcium ion 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 kidney 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.

[0015] Compared with the prior art, the present invention has the following beneficial effects: the present invention has found that a suitable concentration or dosage of terminal chebulic acid shows a good effect of inhibiting the formation of renal calcium oxalate stones. In addition, oxalic acid and kidney stones can significantly damage human renal tubular HK-2 cells and cause cell apoptosis. A suitable dose or concentration of terminal chebulic acid can significantly alleviate the damage to renal tubular HK-2 cells caused by oxalic acid and kidney stones and the resulting apoptosis; it can also significantly reduce the production and accumulation of ROS in renal tubular HK-2 cells caused by oxalic acid and kidney stones. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1Figure 3 shows the inhibitory effect of neutral terminal chebulic acid solution containing different concentrations of terminal chebulic acid on stone formation. A is the kidney stone model group without terminal chebulic acid treatment, B is the treatment group containing 2.5 μg / mL terminal chebulic acid, C is the treatment group containing 5.0 μg / mL terminal chebulic acid, D is the treatment group containing 7.5 μg / mL terminal chebulic acid, E is the treatment group containing 10 μg / mL terminal chebulic acid, and F is the treatment group containing 20 μg / mL terminal chebulic acid. G is the statistical results of kidney stone area between the treatment groups containing different doses of terminal chebulic acid and the model (the white spots in Figures A, B, C, D, E, and F are kidney stone crystals, see the enlarged image);

[0017] Figure 2 The effect of different concentrations of chebulic acid in neutral chebulic acid solution on the survival rate of renal tubular cells;

[0018] Figure 3 Figure 3: Neutral terminal chebula salt solution containing different concentrations of terminal chebula acid inhibits cell apoptosis caused by oxalic acid and kidney stones. A shows the apoptosis of human kidney HK-2 cells under normal culture. B shows the apoptosis of renal tubular cells in the presence of oxalic acid and kidney stones. C shows the apoptosis of renal tubular cells alleviated by neutral terminal chebula salt solution (containing 5 μg / mL terminal chebula acid). D shows the apoptosis of renal tubular cells alleviated by neutral terminal chebula salt solution (containing 7.5 μg / mL terminal chebula acid). E shows the statistical results of renal tubular cell apoptosis in the four groups.

[0019] Figure 4 Figure 3: Neutral chebulic acid solution containing different concentrations of chebulic acid alleviates renal cell damage caused by oxalic acid and kidney stones. Cells in A show good and tightly packed growth under normal conditions. Cells in B show poor growth under stimulation by oxalic acid and kidney stone crystals, with extensive damage and apoptosis. Cells in C show relief from growth and reduced damage and apoptosis in neutral chebulic acid solution 1 (containing 5 μg / mL of chebulic acid). Cells in D show significant improvement in growth and a marked reduction in damage and apoptosis in neutral chebulic acid solution 2 (containing 7.5 μg / mL of chebulic acid) (red arrows indicate kidney stone crystals).

[0020] Figure 5 The results show that neutral terminal chebula salt solution containing different concentrations of terminal chebula acid significantly reduced the production and accumulation of ROS, a marker of renal cell damage. A shows the ROS situation in normal culture of human kidney HK-2 cells, B shows the ROS situation in renal tubular cells in the presence of oxalic acid and kidney stones, C shows the situation of neutral terminal chebula salt solution (containing terminal chebula acid 5 μg / mL) alleviating ROS in renal tubular cells, D shows the situation of neutral terminal chebula salt solution (containing terminal chebula acid 7.5 μg / mL) alleviating ROS in renal tubular cells, and E shows the statistical results of ROS, a marker of renal cell damage, in four groups of renal tubular cells.

[0021] Figure 6The effects of low, medium and high doses of neutral chebulic acid salt solution on the inhibition of stone formation in a mouse kidney stone model were investigated.

[0022] Figure 7 Figure 3: Neutral terminal chebulic acid solution containing different concentrations of terminal chebulic acid directly inhibits oxalate stone formation in vitro in a cell-free environment. A is the oxalate stone model group without terminal chebulic acid treatment, B is the treatment group containing 2.5 μg / mL terminal chebulic acid, C is the treatment group containing 5.0 μg / mL terminal chebulic acid, D is the treatment group containing 7.5 μg / mL terminal chebulic acid, E is the treatment group containing 10 μg / mL terminal chebulic acid, F is the treatment group containing 12.5 μg / mL terminal chebulic acid, and G is the treatment group containing 15 μg / mL terminal chebulic acid. H is the comparison between the treatment groups containing different doses of terminal chebulic acid and the model group. Statistical results of oxalate stone area (white spots in Figures A, B, C, D, E, F, and G are stone crystals).

[0023] Figure 8 Figure 3: Neutral terminal chebulic acid solution containing different concentrations of terminal chebulic acid inhibiting uric acid stone formation. A is the uric acid stone model group without terminal chebulic acid treatment, B is the treatment group containing 2.5 μg / mL terminal chebulic acid, C is the treatment group containing 5.0 μg / mL terminal chebulic acid, D is the treatment group containing 7.5 μg / mL terminal chebulic acid, and E is the treatment group containing 10 μg / mL terminal chebulic acid. F is the statistical results of uric acid stone area between the treatment groups containing different doses of terminal chebulic acid and the model group (the black spots in Figures A, B, C, D, and E are stone crystals).

[0024] Figure 9 Figure 3: 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 2.5 μg / mL terminalia chebula acid, C is the treatment group with 5.0 μg / mL terminalia chebula acid, D is the treatment group with 7.5 μg / mL terminalia chebula acid, E is the treatment group with 10 μg / mL terminalia chebula acid, F is the treatment group with 12.5 μg / mL terminalia chebula acid, and G is the treatment group with 15 μg / mL terminalia chebula acid. H is the comparison between the treatment groups with different doses of terminalia chebula acid and the model group. Statistical results of oxalate stone area (white spots in Figures A, B, C, D, E, F, and G are stone crystals).

[0025] Figure 10Figure 3 shows the in vitro inhibitory effect of different concentrations of terminalia chebula acid solution on uric acid stone formation. A shows the uric acid stone model group without terminalia chebula acid treatment, B shows the group treated with 2.5 μg / mL terminalia chebula acid, C shows the group treated with 5.0 μg / mL terminalia chebula acid, D shows the group treated with 7.5 μg / mL terminalia chebula acid, and E shows the group treated with 10 μg / mL terminalia chebula acid. F shows the statistical results of uric acid stone area between the treatment groups with different doses of terminalia chebula acid and the model group (the black spots in Figures A, B, C, D, and E are stone crystals).

[0026] Figure 11 Figure 3 compares 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, and D is the treatment group given 780 mg / kg ten-flavor terminalia chebula pills. E is the statistical results of calcium oxalate stone area compared between the three treatment groups and the model group (the white spots in Figures A, B, C, and D are stone crystals). DETAILED DESCRIPTION

[0027] The technical solutions of the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, but the present invention is not limited to the following technical solutions.

[0028] Sources of reagents: MEM powder culture medium was purchased from Gibco, fetal bovine serum was purchased from ExCell, sodium oxalate and terminal chebulic acid were purchased from MCE; the culture medium used in the following examples was DMEM culture medium purchased from Gibco.

[0029] Example 1 Cellular Test Verification of the Inhibition of Kidney Stones by Terminalia Chebula

[0030] 1. The effect of terminalia chebula salt on inhibiting kidney stones

[0031] Experimental groups

[0032] Group 1: HK-2 cells were cultured normally in culture medium;

[0033] Group 2: HK-2 cells were treated with 1 mmol / L sodium oxalate solution;

[0034] 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);

[0035] Group 4: HK-2 cells were co-treated with 1 mmol / L sodium oxalate and neutral chebulic acid solution 2 (containing 5.0 μg / mL chebulic acid);

[0036] Group 5: HK-2 cells were co-treated with 1 mmol / L sodium oxalate and neutral chebulic acid solution 3 (containing 7.5 μg / mL chebulic acid);

[0037] 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);

[0038] Group 7: HK-2 cells were co-treated with 1 mmol / L sodium oxalate and neutral chebulic acid solution 5 (containing chebulic acid 20 μg / mL);

[0039] The preparation process of neutral terminal chebulic acid salt solutions of different concentrations is as follows:

[0040] Preparation of neutral terminal chebulic acid solution: Dissolve 201.6 mg of terminal chebulic acid powder in 1.2 mL of DMSO. Ultrasonicate until the drug is completely dissolved. Add 10.8 mL of 20% SBE-β-CD saline solution and mix thoroughly to obtain a 16.8 mg / mL terminal chebulic acid solution. Prior to use, adjust the pH to neutral (pH 6.8-7.5) with a strong base (0.1 M NaOH).

[0041] Calculate the amount of terminal chebulic acid solution that needs to be added based on the total volume of complete culture medium added to each well of the culture plate to obtain groups with different concentrations of terminal chebulic acid. For example, if the total volume of complete culture 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 chebulic acid, i.e., 2.5μg / mL, 5.0μg / mL, 7.5μg / mL, 10μg / mL, 20μg / mL, and A is the volume of terminalia chebulic acid solution required to be added corresponding to different concentrations of terminalia chebulic acid.

[0042] Cell treatment and detection: HK-2 cells were harvested by digesting with 0.25% trypsin. The supernatant was removed and the cells were resuspended in 1 mL of complete culture medium.

[0043] Cell seeding: 2×10 cells per well 4 The cells were seeded in 24-well plates at a density of 10 cells / well, with 3 replicates per group.

[0044] 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.

[0045] Detection: Samples were provided in 24-well plates and observed and photographed using a polarized light microscope.

[0046] Experimental results: Figure 1 As shown, compared with other concentrations, the 7.5 μg / mL concentration of chebulic acid significantly reduced stone area, demonstrating a significant inhibitory effect on calcium oxalate renal stone formation (p < 0.0001), with an inhibition rate of approximately 80%. A high concentration of 20 μg / mL promoted kidney stone formation.

[0047] 2. Effect of terminalia chebula on the survival rate of renal tubular cells

[0048] Experimental groups

[0049] Group 1: HK-2 cells were cultured normally in culture medium;

[0050] Group 2: HK-2 cells were co-cultured with neutral chebulic acid solution a (containing chebulic acid 2.5 μg / mL);

[0051] Group 3: HK-2 cells were co-cultured with neutral chebulic acid solution b (containing chebulic acid 5.0 μg / mL);

[0052] Group 4: HK-2 cells were co-cultured with neutral chebulic acid solution c (containing chebulic acid 7.5 μg / mL);

[0053] Group 5: HK-2 cells were co-cultured with neutral chebulic acid solution d (containing chebulic acid 10 μg / mL);

[0054] Group 6: HK-2 cells were co-cultured with neutral terminal chebulic acid solution (containing terminal chebulic acid 20 μg / mL);

[0055] Cell seeding: 5×10 cells per well 3 The cells were seeded in 96-well plates, with 5 replicates per group.

[0056] When the cell confluence reached about 80%, groups 2 to 6 were treated with corresponding concentrations of chebulic acid for 48 hours.

[0057] After 48 hours of treatment with terminalia chebulic acid, the CCK8 assay was used (CCK8 reagent purchased from targetmol):

[0058] 1. Pipette 100 μL of basal culture medium from a 96-well plate according to the time point and group;

[0059] 2. Set up blank control wells and add 100 μL of basal culture medium to the other three wells;

[0060] 3. Add 10 μL of CCK8 solution to each well (be careful not to create bubbles in the wells);

[0061] 4. Place the plate back in the incubator and incubate in the dark for 2 hours. Measure the OD value of each well in the 96-well plate at 450 nm using a microplate reader.

[0062] Cell viability % = (OD of experimental well - OD of blank well) / (OD of control well - OD of blank well) × 100%.

[0063] 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.

[0064] 3. Terminalia chebula salt can inhibit the damage of renal oxalic acid and kidney stones to renal tubular cells

[0065] Experimental groups:

[0066] (1) HK-2 cells were cultured normally in culture medium;

[0067] (2) HK-2 cells + 1 mmol / L sodium oxalate solution;

[0068] (3) HK-2 cells + sodium oxalate + neutral chebulic acid solution 1 (containing chebulic acid 5 μg / mL);

[0069] (4) HK-2 cells + sodium oxalate + neutral chebulic acid solution 2 (containing chebulic acid 7.5 μg / mL);

[0070] 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 into each well at a rate of 5 × 10 4 The cells were seeded in 6-well plates, with 3 replicates per group. When the cell confluence reached about 80%, group 2 was treated with 1 mmol / L sodium oxalate for 48 h, and groups 3 and 4 were treated with 1 mmol / L sodium oxalate and corresponding concentrations of terminalia chebulic acid for 48 h.

[0071] (1) Apoptosis detection method (apoptosis detection kit was purchased from Beyotime)

[0072] 1. Add 0.25% trypsin, cover the lid and return to the incubator to digest for about 1-2 minutes. If the cells shrink into a ball or become a quicksand, immediately add 0.5 mL of fresh complete medium to terminate the digestion.

[0073] 2. Transfer the cell suspension to a new 1.5 mL centrifuge tube and centrifuge at 1000 rpm for 3 minutes to collect the cells;

[0074] 3. Discard the supernatant, gently resuspend the cells with 0.5 mL of PBS, and centrifuge at 1000 rpm for 3 minutes to collect the cells;

[0075] 4. Add 100 μL 1× Banding Buffer to resuspend the cells and separate into 3 tubes for blank control, Annexin V / PE single positive control, and 7-AAD single positive control respectively;

[0076] 5. Add 5 μL of Annexin V / PE and 10 μL of 7-AAD to each tube, except for the blank, Annexin V / PE, and 7-AAD controls (add 5 μL of Annexin V / PE and 10 μL of 7-AAD to the Annexin V / PE and 7-AAD single-positive controls, respectively), mix gently, and incubate in the dark for 15 minutes.

[0077] 6. Add 400 μL 1× Banding Buffer, mix well, and detect on flow cytometry.

[0078] Experimental results: Figure 3 The neutral terminal chebulic acid solution contains different concentrations of terminal chebulic acid to inhibit cell apoptosis caused by oxalic acid and kidney stones. Figure 4 The microscopic images of cells showing that neutral terminal chebulic acid solution containing different concentrations of terminal chebulic acid alleviates the damage of renal cells caused by oxalic acid and kidney stones. Figure 3 and Figure 4 Oxalic acid and kidney stones can significantly damage human renal tubular HK-2 cells and induce apoptosis. Neutral chebulic acid solutions (containing 5 μg / mL and 7.5 μg / mL of chebulic acid, respectively) significantly alleviated the damage and apoptosis induced by oxalic acid and kidney stones in renal tubular HK-2 cells, with the 7.5 μg / mL dose of chebulic acid showing the greatest effect.

[0079] (2) Neutral terminal chebulic acid solution alleviates the damage to renal cells caused by oxalic acid and kidney stones

[0080] Detection of reactive oxygen species, a marker of renal cell damage (detection kit purchased from Solebo Company):

[0081] 1. Dilute DHE fluorescent dye 1:1000 in serum-free medium to a final concentration of 5 μM.

[0082] 2. Remove the cell culture medium and add an appropriate volume of diluted DHE. The volume added should be sufficient to fully cover the cells. Usually, add no less than 1 mL of diluted DHE to one well of a 6-well plate.

[0083] 3. The positive control was added at a ratio of 1:1000.

[0084] Incubate in a 37°C cell culture incubator for 30-40 minutes.

[0085] 5. Remove the staining solution and wash the cells twice with serum-free cell culture medium.

[0086] 6. Streaming on-machine testing.

[0087] Experimental results: Figure 5 The results show that the neutral terminal chebulic acid solution containing different concentrations of terminal chebulic acid significantly reduced the production and accumulation of ROS, a marker of renal cell damage. Figure 5 Oxalic acid and kidney stones significantly promote the production of reactive oxygen species (ROS), a marker of renal tubular damage in human HK-2 cells. Neutral terminal salt solutions (i.e., containing 5 μg / mL and 7.5 μg / mL terminal salts) significantly reduced oxalic acid- and kidney stone-induced damage in HK-2 cells (i.e., significantly reduced the production and accumulation of ROS, a marker of damage), with the terminal salt containing 7.5 μg / mL being more effective.

[0088] Example 2 Animal Experimental Verification of the Inhibition of Kidney Stones by Terminalia Chebula

[0089] 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.

[0090] 1. Experimental Grouping

[0091] (1) Control group: blank control

[0092] (2) Model group: kidney stone model

[0093] (3) Low-dose group: Model + low-dose neutral chebulic acid solution (i.e., the chebulic acid content administered is 37.5 mg / kg)

[0094] (4) Medium-dose group: Model + medium-dose neutral chebulic acid solution (i.e., the chebulic acid content administered is 75 mg / kg)

[0095] (5) High-dose group: Model + high-dose neutral chebulic acid solution (i.e., the chebulic acid content administered is 150 mg / kg)

[0096] 2. Reagent preparation

[0097] (1) Preparation of sodium oxalate solution: Weigh 40 mg of sodium oxalate powder and dissolve it in normal saline. The volume is adjusted to 5 mL. Mix thoroughly and ultrasonically dissolve it to prepare an 8 mg / mL sodium oxalate solution. Then calculate the injection volume based on the weight of each mouse.

[0098] (2) Preparation of neutral terminal chebulic acid solution: Terminal chebulic acid (37.5 mg / kg): weigh 63 mg of powder, dissolve in pure water, and dilute to 15 ml. This is a 7-day supply. Terminal chebulic acid (75 mg / kg): weigh 126 mg of powder, dissolve in pure water, and dilute to 15 ml. This is a 7-day supply. Terminal chebulic acid (150 mg / kg): weigh 252 mg of powder, dissolve in pure water, and dilute to 15 ml. This is 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.

[0099] 3. Experimental methods

[0100] Fifty SPF C57 male mice, 6-8 weeks old, weighing 18-22 g, were randomly divided into five groups: control, model, low-dose, medium-dose, and high-dose. The model group received a daily intraperitoneal injection of 40 mg / kg sodium oxalate solution for 7 consecutive days to establish a kidney stone model. In addition to daily intraperitoneal injections of 40 mg / kg sodium oxalate solution to induce kidney stone formation, the low-dose, medium-dose, and high-dose groups were also gavaged daily with low, medium, and high-dose neutral chebulic acid solutions (i.e., 37.5 mg / kg, 75 mg / kg, and 150 mg / kg, respectively) to prevent kidney stone formation. The control group received normal saline by gavage for 7 consecutive days.

[0101] 4. Experimental results

[0102] After 7 days, the mouse kidneys were sliced ​​and stained with HE. After observation under a polarizing microscope and elimination of samples that failed modeling, photos were taken and counted. 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 are presented. Figure 6 The white spots in the picture are kidney stones). Figure 6 The results showed that compared with the kidney stone model group, neutral terminal chebulic acid salt (i.e., terminal chebulic acid at doses of 37.5 mg / kg, 75 mg / kg, and 150 mg / kg) could significantly inhibit the formation of kidney stones, and the medium-dose group (administered 75 mg / kg terminal chebulic acid) had the best effect ( p < 0.0001).

[0103] Example 3 Neutral Terminalia Chebula Salt Inhibits Calcium Oxalate Stone Formation in Vitro

[0104] Experimental groups

[0105] Group A: DMEM medium was treated with 1 mmol / L sodium oxalate solution;

[0106] Group B: DMEM medium was added with 1 mmol / L sodium oxalate and treated with neutral chebulic acid solution 1 (containing 2.5 μg / mL chebulic acid);

[0107] 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);

[0108] Group D: DMEM medium was added with 1 mmol / L sodium oxalate and neutral chebulic acid solution 3 (containing 7.5 μg / mL chebulic acid) for co-treatment;

[0109] Group E: DMEM medium was added with 1 mmol / L sodium oxalate and treated with neutral chebulic acid solution 4 (containing 10 μg / mL chebulic acid);

[0110] 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);

[0111] Group G: DMEM medium was treated with 1 mmol / L sodium oxalate and neutral chebulic acid solution 6 (containing 15 μg / mL chebulic acid);

[0112] The preparation of neutral chebulic acid salt was referred to Example 1. Groups A to G were placed in a 24-well plate in a cell culture incubator at 37° C. for 48 hours and then the stone formation was measured.

[0113] Experimental results: Figure 7 As shown in the results, compared with the control group (Group A) in the 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 of chebulic acid significantly inhibited the formation of oxalate stones ( p < 0.0001), but large-diameter calcium oxalate stones formed at doses of 12.5 μg / mL and 15 μg / mL, suggesting that excessively high concentrations of chebulic acid may promote the formation of large-diameter calcium oxalate stones. These results demonstrate that neutral chebulic acid salt solutions can directly inhibit oxalate stone formation in the absence of cells, but that excessive concentrations are not recommended.

[0114] Example 4: Terminalia chebula salt inhibits the formation of uric acid stones in vitro

[0115] Experimental groups

[0116] Group A: Complete medium (DMEM medium) was treated with 10 mmol / L uric acid;

[0117] 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);

[0118] 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);

[0119] 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);

[0120] Group E: Complete medium was treated with 10 mmol / L uric acid and neutral chebulic acid solution 4 (containing 10 μg / mL chebulic acid);

[0121] In Examples 1 and 3, the present application found that the test results were primarily related to the calcium ion content of the culture medium. The addition of cell culture did not affect the test results, so cell culture was not added in this example. Groups A-E were placed in a 37°C cell culture incubator using 24-well plates for 48 hours and then the stone status was measured.

[0122] Experimental results: Figure 8 As shown in the results, 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 significantly inhibited the formation of uric acid stones ( p < 0.05).

[0123] Example 5: Terminalia chebula acid inhibits the formation of calcium oxalate stones in vitro

[0124] Experimental groups

[0125] Group A: DMEM medium was treated with 1 mmol / L sodium oxalate solution;

[0126] Group B: DMEM medium was added with 1 mmol / L sodium oxalate and treated with terminalic acid solution 1 (terminalic acid 2.5 μg / mL);

[0127] Group C: DMEM medium was added with 1 mmol / L sodium oxalate and treated with terminalic acid solution 2 (terminalic acid 5.0 μg / mL);

[0128] Group D: DMEM medium was added with 1 mmol / L sodium oxalate and treated with terminalia acid solution 3 (terminalia acid 7.5 μg / mL);

[0129] Group E: DMEM medium was added with 1 mmol / L sodium oxalate and treated with terminalic acid solution 4 (terminalic acid 10 μg / mL);

[0130] Group F: DMEM medium was added with 1 mmol / L sodium oxalate and treated with terminal chebulic acid solution 5 (terminal chebulic acid 12.5 μg / mL);

[0131] Group G: DMEM medium was added with 1 mmol / L sodium oxalate and treated with terminal chebulic acid solution 6 (terminal chebulic acid 15 μg / mL);

[0132] Experimental results: Figure 9 As shown in the data, 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.

[0133] Example 6: Terminalia chebula acid inhibits the formation of uric acid stones in vitro

[0134] Experimental groups

[0135] Group A: complete culture medium was treated with 10 mmol / L uric acid;

[0136] Group B: Complete medium was treated with 10 mmol / L uric acid and terminalia acid solution 1 (2.5 μg / mL terminalia acid);

[0137] Group C: Complete medium was treated with 10 mmol / L uric acid and terminalia acid solution 2 (terminalia acid 5.0 μg / mL);

[0138] Group D: Complete medium was treated with 10 mmol / L uric acid and terminalia acid solution 3 (terminalia acid 7.5 μg / mL);

[0139] Group E: Complete medium was treated with 10 mmol / L uric acid and terminalia acid solution 4 (terminalia acid 10 μg / mL);

[0140] Experimental results: Figure 10 As shown in the results, compared with the control group (Group A) in the uric acid stone model, the 5 μg / mL chebulic acid solution could significantly inhibit the formation of uric acid stones (p < 0.05).

[0141] Example 7 Animal Experimental Verification of the Inhibitory Effect of Terminalia Chebula Salt on Kidney Stones as Better Than Terminalia Chebula and Ten-flavor Terminalia Chebula Pills

[0142] 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.

[0143] 1. Experimental Grouping

[0144] (1) Model group: kidney stone model

[0145] (2) Terminalia chebula acid group: model + neutral terminalia chebula acid solution (i.e., terminalia chebula acid content of 75 mg / kg)

[0146] (3) Terminalia chebula group: model + Terminalia chebula powder solution (i.e., the Terminalia chebula content administered was 780 mg / kg)

[0147] (4) Ten-flavor Terminalia chebula pill group: model + Ten-flavor Terminalia chebula pill solution (i.e., the content of Ten-flavor Terminalia chebula pill administered was 780 mg / kg)

[0148] 2. Reagent preparation

[0149] (1) Preparation of sodium oxalate solution: Weigh 40 mg of sodium oxalate powder and dissolve it in normal saline. The volume is adjusted to 5 mL. Mix thoroughly and ultrasonically dissolve it to prepare an 8 mg / mL sodium oxalate solution. Then calculate the injection volume based on the weight of each mouse.

[0150] (2) Preparation of Terminalia chebula acid solution: Weigh 201.6 mg of terminalia chebula acid powder and dissolve it in 1.2 ml of DMSO. Ultrasonicate until the drug is completely dissolved. Then add 10.8 ml of 20% SBE-β-CD saline solution and mix thoroughly to obtain a 16.8 mg / ml terminalia chebula acid solution. Before use, adjust the pH to neutral (pH 6.8-7.5) using a strong base (0.1 M NaOH) solution.

[0151] (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 Terminalia chebula powder solution of 78 mg / ml (Terminalia chebula dry powder produced in Yunnan).

[0152] (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.).

[0153] 3. Experimental methods

[0154] Twenty-four SPF C57 male mice, 6-8 weeks old, weighing 18-22 g, were randomly divided into four groups: a model group, a terminalia acid group, a terminalia chebula group, and a ten-flavor terminalia chebula pill group. The model group received a daily intraperitoneal injection of 40 mg / kg sodium oxalate solution for 7 consecutive days to establish a kidney stone model. In addition to receiving daily intraperitoneal injections of 40 mg / kg sodium oxalate solution to induce kidney stone formation, the terminalia acid, terminalia chebula, and ten-flavor terminalia chebula pill groups were also gavaged daily for 7 consecutive days with a neutral terminalia acid solution, a terminalia chebula powder solution, and a ten-flavor terminalia chebula pill solution to prevent kidney stone formation (i.e., terminalia acid at 75 mg / kg, terminalia chebula at 780 mg / kg, and terminalia chebula at 780 mg / kg, respectively).

[0155] 4. Experimental results

[0156] After 7 days, the mouse kidneys were sliced ​​and stained with HE. After observation under a polarizing microscope and elimination of samples that failed modeling, photos were taken and counted. Figure 11 The inhibitory effects of terminalia chebula salt, terminalia chebula and ten-flavor terminalia chebula pills on stone formation in a mouse kidney stone model were investigated. Figure 11 The white spots in the picture are kidney stones). Figure 11 The results showed that compared with the kidney stone model group, terminalia chebula salt, terminalia chebula and ten-flavor terminalia chebula pills (terminalia 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 kidney stone formation, and the terminalia chebula acid group had the best effect (p < 0.0001).

[0157] The above are only preferred embodiments of the present invention. The scope of protection of the present invention is not limited to the above embodiments. All technical solutions based on the concept of the present invention are within the scope of protection of the present invention. It should be pointed out that for those skilled in the art, any modification, equivalent replacement or improvement without departing from the principle of the present invention should be considered as within the scope of protection of the present invention.

Claims

1. Use of terminalia chebulic acid or a pharmaceutically acceptable salt thereof as a pharmaceutical active ingredient in the preparation of a drug for preventing and / or treating calcium oxalate stones or uric acid stones, characterized in that: The structural formula of the terminaliacetic acid is as follows: ； The active ingredient of the medicine is calculated as terminalia chebulic acid, and the concentration is 2.5-10 μg / mL.

2. The use according to claim 1, characterized in that The preparation process of the pharmaceutically acceptable salt of terminalia chebulic acid is as follows: an aqueous solution of terminalia chebulic acid and an alkali solution are mixed and the pH is adjusted to 6.8-7.5.

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