Pharmaceutical composition of mulberroside C and 7-hydroxywarfarin and application of pharmaceutical composition in acute kidney injury

Abstract

The invention discloses a medicine composition of mulberroside C and 7-hydroxywarfarin and application of the medicine composition in acute kidney injury, and belongs to the technical field of medicine development. The mulberroside C and the 7-hydroxywarfarin are combined according to the mass ratio of 1: 1, so that multiple physiological and biochemical indexes related to the acute kidney injury can be remarkably improved, and the kidney tissue structure change can be repaired; compared with single use of mulberroside C or 7-hydroxywarfarin, the combination of mulberroside C and 7-hydroxywarfarin shows an obvious synergistic effect. The pharmaceutical composition disclosed by the invention can effectively relieve acute kidney injury induced by various factors, improve kidney function indexes and reduce the injury degree of kidney tissues, so that the pharmaceutical composition has a good application prospect in prevention and treatment of the acute kidney injury, and a new treatment strategy and a potential target spot are provided for intervention of the acute kidney injury.

Description

Technical Field

[0001] The present invention relates to a pharmaceutical combination of mulberroside C and 7-hydroxywarfarin and its application in acute kidney injury, belonging to the technical field of drug development. Background Art

[0002] Acute Kidney Injury (AKI) is a common and severe complication in clinical practice, characterized by high incidence, high mortality, and poor prognosis. It is widely seen in clinical scenarios such as severe infections, tumor chemotherapy, cardiovascular surgery, and ischemia-reperfusion. Studies have shown that AKI can cause tubular epithelial cell injury, inflammatory response, apoptosis, and necrosis through multiple mechanisms, thereby leading to a sharp decline in renal function.

[0003] Current treatment methods for AKI mainly focus on supportive treatments such as fluid management, correction of electrolyte disorders, and alternative renal therapies. There is no specific drug that can significantly reverse the process of AKI. Therefore, the development of effective, safe, and multi-target acting mechanism drugs has become a hot and difficult point in AKI research.

[0004] Mulberroside C is a flavonoid compound extracted from mulberry bark, which has good antioxidant, anti-inflammatory, and anti-apoptotic activities. Related studies have shown its potential protective effects in diseases such as liver injury, neurodegenerative diseases, and metabolic syndrome, but there is still little research in the field of kidney injury.

[0005] 7-hydroxycoumarin, also known as umbelliferone, is a natural coumarin compound with various biological activities such as scavenging reactive oxygen species, regulating oxidative stress, inhibiting inflammation, and apoptosis. It has been applied in liver and neuroprotection research. Existing studies have suggested that it may also have certain protective potential for the kidneys, but its application value in acute kidney injury has not been systematically studied.

[0006] Therefore, developing a pharmaceutical combination based on mulberroside C and 7-hydroxycoumarin to improve AKI injury at the cellular and animal levels has extremely high practical and economic value, providing a new direction and strategy for the treatment of clinical AKI. Summary of the Invention

[0007] To solve the above problems, the present invention combines mulberroside C and 7-hydroxywarfarin at equal concentrations and finds that mulberroside C and 7-hydroxywarfarin have a synergistic effect in the treatment of acute kidney injury.

[0008] The first object of the present invention is to provide an application of a composition in the preparation of a drug for preventing and / or treating acute kidney injury, wherein the composition contains mulberroside C and 7-hydroxywarfarin.

[0009] In one embodiment, the mass ratio of morusin C to 7-hydroxywarfarin is 0.8 to 1.2:0.8 to 1.2.

[0010] In one embodiment, the mass ratio of morusin C to 7-hydroxywarfarin is 1:1.

[0011] In one embodiment, the composition contains pharmaceutically acceptable pharmaceutical excipients, and the pharmaceutical excipients refer to conventional drug carriers in the pharmaceutical field.

[0012] In one embodiment, the excipients include one or more of the following: binders such as cellulose derivatives, alginates, gelatin, and polyvinylpyrrolidone; diluents such as starch, pregelatinized starch, dextrin, sucrose, lactose, mannitol; fillers such as starch, sucrose; wetting agents such as glycerol; disintegrants such as sodium carboxymethyl starch, crosslinked polyvinylpyrrolidone, and dry starch; absorption promoters such as quaternary ammonium compounds; surfactants such as polysorbate, sorbitan fatty acid esters, and fatty acid glycerides; colorants such as titanium dioxide, sunset yellow, methylene blue, medicinal iron oxide red, etc.; lubricants such as hydrogenated vegetable oil, talc powder, and polyethylene glycol; coating materials such as acrylic resin, hypromellose, polyvinylpyrrolidone, and cellulose acetate phthalate; additionally, other adjuvants such as flavoring agents and sweetening agents can be added to the composition.

[0013] In one embodiment, the dosage forms of the drug include, but are not limited to, oral dosage forms, injection dosage forms, and inhalation dosage forms.

[0014] In one embodiment, the oral dosage forms include tablets, capsules, granules, oral liquids, and oral suspensions;

[0015] The injection dosage forms include injection solutions and injection powder injections;

[0016] The inhalation dosage forms include aerosols and powder aerosols.

[0017] In one embodiment, the drug is used for at least one of (a) to (d):

[0018] (a) To increase cell viability in a hypoxia-reoxygenation cell model (Hypoxia / Reoxygenation, H / R), reduce the release of lactate dehydrogenase, and inhibit Caspase-3 activity to alleviate apoptosis;

[0019] (b) To reduce the levels of serum urea nitrogen, creatinine, and cystatin C in an animal model;

[0020] (c) To down-regulate the mRNA expression of kidney injury markers KIM-1 and NGAL in kidney tissues;

[0021] (d) To improve the pathological manifestations of kidney tissues and reduce the renal tubular injury score.

[0022] The second object of the present invention is to provide a composition containing sangpicoside C and 7-hydroxywarfarin, and the mass ratio of sangpicoside C to 7-hydroxywarfarin is 0.8-1.2:0.8-1.2.

[0023] In one embodiment, the mass ratio of sangpicoside C to 7-hydroxywarfarin is 1:1.

[0024] In one embodiment, the composition contains pharmaceutically acceptable pharmaceutical excipients, and the pharmaceutical excipients refer to conventional drug carriers in the pharmaceutical field.

[0025] In one embodiment, the excipients include one or more of the following: binders such as cellulose derivatives, alginates, gelatin and polyvinylpyrrolidone; diluents such as starch, pregelatinized starch, dextrin, sucrose, lactose, mannitol; fillers such as starch, sucrose; wetting agents such as glycerol; disintegrants such as sodium carboxymethyl starch, cross-linked polyvinylpyrrolidone and dry starch; absorption promoters such as quaternary ammonium compounds; surfactants such as polysorbate, sorbitan fatty acid esters and fatty acid glycerides; coloring agents such as titanium dioxide, sunset yellow, methylene blue, medicinal iron oxide red, etc.; lubricants such as hydrogenated vegetable oil, talc powder and polyethylene glycol; coating materials such as acrylic resin, hypromellose, polyvinylpyrrolidone, cellulose acetate phthalate; additionally, other adjuvants such as flavoring agents and sweetening agents can also be added to the composition.

[0026] In one embodiment, the dosage forms of the drug include, but are not limited to, oral dosage forms, injection dosage forms, inhalation dosage forms.

[0027] In one embodiment, the oral dosage forms include tablets, capsules, granules, oral liquids, oral suspensions;

[0028] The injection dosage forms include injection solutions, injection powder injections;

[0029] The inhalation dosage forms include aerosols, powder aerosols.

[0030] Advantages of the present invention

[0031] In the present invention, sangpicoside C and 7-hydroxywarfarin are used in combination at a ratio of 1:1, and it is found that sangpicoside C and 7-hydroxywarfarin have a synergistic effect in the treatment of acute kidney injury.

[0032] Specifically:

[0033] (1) In cell experiments, at the same concentration, when sangpicoside C and 7-hydroxywarfarin are administered in combination at a ratio of 1:1 (the total administration concentration is 5 μM), the cell viability, cell membrane integrity and anti-apoptosis of H / R cells can be significantly improved, and the effect is better than that of only administering sangpicoside C and 7-hydroxywarfarin;

[0034] (2) In animal experiments, at the same concentration, when sangpicoside C and 7-hydroxy warfarin were co-administered at a ratio of 1:1 (total administration concentration was 10 mg / kg / day), it could significantly improve renal injury in AKI mice (improve BUN, Scr, and Cystatin C indicators), inhibit the gene expression levels of early markers of acute kidney injury (KIM-1 and NGAL), and reduce the Tubular Injury Score in renal tissue morphology. Description of the Drawings

[0035] Figure 1 Effects of sangpicoside C and 7-hydroxy warfarin on the H / R cell model; among them, A is the detection of cell viability, B is the LDH release level, and C is the detection of Caspase-3 activity.

[0036] Figure 2 Effects of sangpicoside C and 7-hydroxy warfarin on the renal function of AKI mice; among them, A is the detection result of BUN level, B is the detection result of BUN level, and C is the detection result of Cystatin C level.

[0037] Figure 3 Effects of sangpicoside C and 7-hydroxy warfarin on the mRNA expression of renal injury markers KIM-1 and NGAL in AKI mice; among them, A is the expression level of KIM-1 mRNA, and B is the expression level of NGAL mRNA.

[0038] Figure 4 Effects of sangpicoside C and 7-hydroxy warfarin on the renal tubules of AKI mice; among them, A is the representative image of the HE-stained pathological section of renal tissue (scale = 50 μm), and B is the statistical analysis of the renal tubule injury score.

[0039] Data in the drawings are expressed as mean ± standard deviation (Mean ± SD) (n = 6), and one-way ANOVA was used. The significance of differences between groups was indicated by *P < 0.05 compared with the Con group, indicating comparison with the H / R group, indicating comparison with the sangpicoside C group, and #P < 0.05 indicating comparison with the 7-hydroxy warfarin group. Detailed Embodiments

[0040] The following are descriptions of the preferred embodiments of the present invention. It should be understood that the embodiments are for better explaining the present invention and are not used to limit the present invention.

[0041] Raw materials used in the embodiments:

[0042] Sangpicoside C, CAS number: 102841-43-0, purchased from MCE Company;

[0043] 7-Hydroxywarfarin, CAS No.: 17834-03-6, purchased from MCE.

[0044] Test methods:

[0045] 1. Cell viability:

[0046] Add 10 μL of CCK-8 reagent to each well of the cell culture plate, continue to incubate for 2 hours, and measure the absorbance value (OD value) at a wavelength of 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader. Cell viability is expressed as the ratio of the OD value of each group to that of the blank control group.

[0047] 2. Cell membrane integrity:

[0048] Collect the cell culture supernatant, and operate according to the instructions of the LDH release detection kit. Read the absorbance value at a wavelength of 490 nm using an ELISA reader. The LDH release rate is expressed as the ratio of the experimental group to the positive control group.

[0049] 3. Cell apoptosis:

[0050] Use the Caspase-3 activity detection kit to detect the activity of apoptosis-related enzymes. Collect the treated cells, add the substrate Ac-DEVD-pNA after extracting the total protein, incubate at 37 °C for 1 hour, and then read the absorbance value at a wavelength of 405 nm. The Caspase-3 activity is positively correlated with the degree of cell apoptosis.

[0051] 4. mRNA expression detection:

[0052] Take mouse kidney tissue, extract total RNA and perform real-time fluorescence quantitative PCR detection to analyze the mRNA expression levels of the early markers of acute kidney injury, kim-1 and ngal.

[0053] 5. Serum index detection:

[0054] BUN: Use the BUN colorimetric assay kit to measure at a wavelength of 630 nm; Scr: Use the enzymatic assay kit to measure at 520 nm or the appropriate wavelength; Cystatin C: Use the ELISA method to calculate the concentration according to the standard curve.

[0055] 6. HE staining:

[0056] Take mouse kidney tissue, fix it in 10% neutral buffered formaldehyde solution for 24 hours, embed it in paraffin, section (4 μm), and perform routine hematoxylin-eosin (HE) staining. Observe the pathological changes such as swelling, degeneration, lumen dilation, cell shedding, and cast formation of renal tubular epithelial cells under a microscope.

[0057] 7. Tubular Injury Score:

[0058] Six visual fields were randomly selected for each animal, and the degree of renal tubular injury was scored according to the following criteria: 0 point: no pathological changes; 1 point: <10% renal tubular injury; 2 points: 10 - 25%; 3 points: 26 - 50%; 4 points: 51 - 75%; 5 points: >75%.

[0059] Example 1: Application of the drug combination of mulberroside C and 7-hydroxywarfarin in improving acute kidney injury

[0060] 1. Cell experiment

[0061] Construct an acute kidney injury cell model:

[0062] An hypoxia-reoxygenation model was constructed using human renal proximal tubular epithelial cells (HK-2), and the acute kidney injury microenvironment was simulated by hypoxia for 12 hours under 1% O2 followed by reoxygenation for 12 hours. The experimental groups were as follows:

[0063] Control group (Control): Cultured under normoxic conditions, without hypoxia and drug treatment;

[0064] Model group (H / R): Hypoxia for 12 hours + reoxygenation for 12 hours, without drugs;

[0065] Mulberroside C group (H / R+MulberrosideC): Hypoxia for 12 hours + reoxygenation for 12 hours, mulberroside C was added 1 hour before hypoxia, with a final concentration of 5 μM;

[0066] 7-hydroxycoumarin group (H / R+7-hydroxycoumarin): Hypoxia for 12 hours + reoxygenation for 12 hours, 7-hydroxycoumarin was added 1 hour before hypoxia, with a final concentration of 5 μM;

[0067] Combined group (H / R+Mixture): Mulberroside C and 7-hydroxycoumarin were jointly added 1 hour before hypoxia, with a final concentration of 2.5 μM each and a total concentration of 5 μM.

[0068] Cell viability, cell membrane integrity and apoptosis rate were detected, and the results are as Figure 1 shown as follows:

[0069] Cell viability is as shown in A of Figure 1 . Compared with the H / R group, the combined treatment of mulberroside C and 7-hydroxycoumarin significantly increased cell viability, which was better than that of any single drug treatment group;

[0070] Cell membrane integrity is as shown in B of Figure 1 . H / R treatment led to an increase in LDH release, and the combined drug treatment could significantly reduce the LDH release caused by cell membrane damage, which was better than that of any single drug treatment group;

[0071] The apoptosis rate is as shown inFigure 1 As shown in C in , the combined use of drugs significantly inhibited Caspase-3 activity, had a good anti-apoptotic effect, and was superior to any single-drug treatment group.

[0072] 2. Animal experiments

[0073] Male C57BL / 6J mice were selected to establish a mouse model of acute kidney injury (AKI), and the grouping was as follows:

[0074] Normal control group (Control): Intraperitoneally injected with an equal volume of 0.9% sodium chloride solution (normal saline), without cisplatin and drug treatment;

[0075] Model group (Cisplatin): Intraperitoneally injected with cisplatin (20 mg / kg) to establish a model for 10 consecutive days;

[0076] Mulberroside C group (Cisplatin+Mulberroside C): Intraperitoneally injected with cisplatin (20 mg / kg) to establish a model for 10 consecutive days. From the 8th day of model establishment, mulberroside C (10 mg / kg / day) was orally administered continuously for 3 days (i.e., days 8-10), and the interval between cisplatin injection and oral administration was 6 h;

[0077] 7-Hydroxycoumarin group (Cisplatin+7-hydroxycoumarin): Intraperitoneally injected with cisplatin (20 mg / kg) to establish a model for 10 consecutive days. From the 8th day of model establishment, 7-hydroxycoumarin (10 mg / kg / day) was orally administered continuously for 3 days (i.e., days 8-10), and the interval between cisplatin injection and oral administration was 6 h;

[0078] Combined use group (Cisplatin+Mixture): Intraperitoneally injected with cisplatin (20 mg / kg) to establish a model for 10 consecutive days. From the 8th day of model establishment, mulberroside C (5 mg / kg / day) and 7-hydroxycoumarin (5 mg / kg / day) were orally administered continuously for 3 days (i.e., days 8-10), and the interval between cisplatin injection and oral administration was 6 h.

[0079] Blood was collected and the animals were sacrificed 24 hours after the last administration, and the kidney tissues were separated for further detection. Blood was collected from the mouse orbital cavity or by cardiac puncture, centrifuged at 3500 rpm for 10 minutes, and the serum was separated.

[0080] (1) Serum indexes

[0081] The serum BUN, Scr and cystatin C (Cystatin C) of the mice were detected, and the detection results were as Figure 2 shown, specifically as follows:

[0082] The BUN level was as Figure 2As shown in A of [Figure 0], the serum BUN level of cisplatin-induced AKI mice (Cisplatin group) was significantly increased, indicating impaired renal function; both mulberroside C and 7-hydroxycoumarin alone could partially reduce the BUN level, and the combined treatment could significantly inhibit the increase of BUN, improve renal function, and the effect was better than that of any single drug treatment group;

[0083] The Scr level was as Figure 2 shown in B of [Figure 0], the serum Scr level of cisplatin-induced AKI mice (Cisplatin group) increased; the combined treatment could significantly reduce the Scr level, and the effect was better than that of any single drug treatment group, indicating that the glomerular filtration function was restored;

[0084] The cystatin C level was as Figure 2 shown in C of [Figure 0], as a sensitive biomarker of renal injury, Cystatin C was significantly increased in the Cisplatin group, and the combined treatment group could significantly reverse its increasing trend, showing better renal function protection, and the effect was better than that of any single drug treatment group.

[0085] (2) Biomarker expression

[0086] The mRNA expression levels of early biomarkers of acute kidney injury (KIM-1 and NGAL) in mouse kidney tissues were detected, and the detection results were as Figure 3 shown as follows:

[0087] The mRNA expression level of KIM-1 was as Figure 3 shown in A of [Figure 1], compared with the control group (Control), the expression of KIM-1 mRNA in the kidney tissues of AKI mice (Cisplatin group) was significantly increased; the single drug treatment groups (Cisplatin+MulberrosideC, Cisplatin+7-hydroxycoumarin) could partially reduce its expression, while the combined treatment group (Cisplatin+Mixture) significantly inhibited the up-regulation of KIM-1, and the effect was better than that of any single drug treatment group.

[0088] The mRNA expression level of NGAL was as Figure 3 shown in B of [Figure 1], compared with the normal control group (Control), the combined treatment group (Cisplatin+Mixture) significantly reduced the mRNA expression level of NGAL in kidney tissues, and the effect was better than that of any single drug treatment group. It can be seen that the combination of mulberroside C and 7-hydroxycoumarin has excellent gene-level intervention effects on acute kidney injury.

[0089] (3) Tissue morphology

[0090] The morphology of mouse kidney tissues was evaluated by HE staining and Tubular Injury Score was performed, and the results were as Figure 4As shown, the results are as follows:

[0091] The HE staining pathological sections are as Figure 4 shown in A of the figure. In the normal control group (Control), the renal tubular structure was intact and the cells were arranged orderly. In the AKI mice (Cisplatin group), severe pathological changes such as necrosis, exfoliation of renal tubular epithelial cells, and lumen dilation were observed. The single-drug groups (Cisplatin+Mulberroside C, Cisplatin+7-hydroxycoumarin) showed a certain degree of improvement. In the combination group (Cisplatin+Mixture), the renal tissue injury was significantly reduced, the structure tended to be normal, and the effect was better than that of any single-drug treatment group.

[0092] In summary, the results indicate that mulberroside C and 7-hydroxycoumarin have a synergistic effect in the treatment of acute kidney injury. At the same dosage, the combined use has a significantly better effect than only administering mulberroside C or only administering 7-hydroxycoumarin.

[0093] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person familiar with this technology can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. Use of a composition in the preparation of a medicament for preventing and / or treating acute kidney injury, characterized in that, The composition contains morusin C and 7-hydroxywarfarin.

2. The application according to claim 1, wherein The mass ratio of morusin C to 7-hydroxywarfarin is 0.8 to 1.2: 0.8 to 1.

2.

3. The application according to claim 1, characterized in that The mass ratio of morusin C to 7-hydroxywarfarin is 1:

1.

4. The application according to claim 1, characterized in that The composition contains pharmaceutically acceptable medicinal excipients, and the medicinal excipients refer to conventional drug carriers in the pharmaceutical field.

5. The application according to claim 4, wherein The excipients include one or more of the following: binders such as cellulose derivatives, alginates, gelatin and polyvinylpyrrolidone; diluents such as starch, pregelatinized starch, dextrin, sucrose, lactose, mannitol; fillers such as starch, sucrose; wetting agents such as glycerol; disintegrants such as sodium carboxymethyl starch, cross-linked polyvinylpyrrolidone and dry starch; absorption promoters such as quaternary ammonium compounds; surfactants such as polysorbate, sorbitan fatty acid esters and glycerol fatty acid esters; coloring agents such as titanium dioxide, sunset yellow, methylene blue, medicinal iron oxide red; lubricants such as hydrogenated vegetable oil, talc powder and polyethylene glycol; coating materials such as acrylic resin, hypromellose, povidone, cellulose acetate phthalate; additionally, other adjuvants such as flavoring agents and sweetening agents can be added to the composition.

6. The application according to claim 1, characterized in that, The dosage forms of the drug include, but are not limited to, oral dosage forms, injection dosage forms, and inhalation dosage forms.

7. The application according to claim 6, wherein The oral dosage forms include tablets, capsules, granules, oral liquids, oral suspensions; The injection dosage forms include injection solutions, injection powder for injection; The inhalation dosage forms include aerosols, powder aerosols.

8. The application according to claim 1, characterized in that, The drug is used for at least one of (a) to (d): (a) Improving cell viability in an hypoxia-reoxygenation cell model, reducing the release of lactate dehydrogenase, and inhibiting Caspase-3 activity to alleviate cell apoptosis; (b) Lowering the levels of serum urea nitrogen, creatinine, and cystatin C in an animal model; (c) Downregulating the mRNA expression of renal injury markers KIM-1 and NGAL in renal tissues; (d) Improving the pathological manifestations of renal tissues and reducing the renal tubular injury score.

9. A composition, characterized in that, The composition contains morusin C and 7-hydroxywarfarin, and the mass ratio of morusin C to 7-hydroxywarfarin is 0.8 to 1.2: 0.8 to 1.

2.

10. The composition according to claim 8, wherein, The mass ratio of morusin C to 7-hydroxywarfarin is 1:1.

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