Veterinary drug composition for treating renal failure of pets and lipidosome and application thereof

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a veterinary medicine composition for treating pet renal failure and a liposome and application thereof. The veterinary drug composition comprises curcumin, emodin and astragalus polysaccharide, and the mass ratio of the curcumin to the emodin to the astragalus polysaccharide is (0.5-2): (1-2): (1-2). The veterinary drug composition provided by the invention can achieve the purpose of synergistically treating renal failure in the aspects of resisting inflammation, inhibiting renal fibrosis, improving glomerular filtration function and the like, and provides a new solution for effectively treating the renal failure of pets. The traditional Chinese medicine composition has a good recovery effect on kidney tissues of the renal failure pets, can effectively reduce urea nitrogen and creatinine levels of blood of the renal failure pets and relieve renal failure symptoms, and has a good treatment effect.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to a veterinary drug composition for treating pet renal failure, and liposomes thereof and applications thereof. Background Art

[0002] With the development of social economy, pet breeding has become a new bright spot of urban residents' consumption, and pet health and welfare are also increasingly valued. Pet renal failure is one of the common urinary system organ diseases in clinical practice, which is manifested by the gradual loss of kidney function, decreased glomerular filtration rate, inability to effectively eliminate waste and excess water in the body, retention of metabolites such as creatinine and urea nitrogen, and disorders of water, electrolytes and acid-base balance. Pets with renal failure usually have symptoms such as anorexia, vomiting, depression, and severe dehydration. The cure rate of renal failure is low and it is a common cause of death in pets.

[0003] At present, there is no specific oral drug for the treatment of renal failure. It is mainly treated by diet and nutritional management to improve the symptoms of renal failure, or by dialysis to delay the disease, which is very harmful to pets, or by drug intervention to maintain the status quo and delay deterioration. Therefore, the development of new and effective drugs for the treatment of pet renal failure is of great clinical significance. Summary of the invention

[0004] In view of the technical problems such as the lack of effective pet renal failure treatment drugs in the above prior art, the present invention provides a veterinary drug composition, which can achieve the purpose of synergistically treating renal failure from the aspects of anti-inflammation, inhibition of renal fibrosis, and improvement of glomerular filtration function, and provides a new solution for effectively treating pet renal failure. In order to improve the bioavailability of the veterinary drug composition, the present invention also provides a liposome containing the veterinary drug composition and a preparation method thereof, and further provides the use of the above veterinary drug composition and its liposome in the preparation of pet kidney treatment drugs.

[0005] To achieve the above object, the present invention is specifically implemented through the following technical solutions:

[0006] The first aspect of the present invention provides a veterinary drug composition for treating pet renal failure, comprising curcumin, emodin and astragalus polysaccharide, wherein the mass ratio of the curcumin, the emodin and the astragalus polysaccharide is 0.5-2:1-2:1-2.

[0007] Furthermore, the mass ratio of the curcumin, the emodin and the astragalus polysaccharide is 0.5-2:1:1.

[0008] Furthermore, the mass ratio of the curcumin, the emodin and the astragalus polysaccharide is 2:1:1.

[0009] The second aspect of the present invention provides a liposome, wherein the raw materials of the liposome include a veterinary drug composition, lecithin, cholesterol and a lyophilization protectant, and the mass ratio of the veterinary drug composition, the lecithin, the cholesterol and the lyophilization protectant is 1:5-8:0.2-2:3-10.

[0010] Furthermore, the mass ratio of the veterinary drug composition, the lecithin, the cholesterol and the lyophilization protectant is 1:6.25:0.5:5.

[0011] Furthermore, the lyophilization protective agent is glucose, sucrose or sorbitol.

[0012] The third aspect of the present invention provides a method for preparing the liposome as described above, comprising the following steps:

[0013] S1, dissolving curcumin, emodin, cholesterol and lecithin in an organic solvent to obtain an organic phase; in addition, dissolving astragalus polysaccharide in water to obtain an aqueous phase;

[0014] S2, adding the organic phase to the aqueous phase, stirring and emulsifying, and then distilling under reduced pressure to remove the organic solvent to obtain a liposome suspension;

[0015] S3, filtering the liposome suspension, adding a lyophilization protective agent to the filtrate, and obtaining liposome lyophilized powder after freeze drying.

[0016] Furthermore, in step S2, the reaction temperature for stirring and emulsifying is 40-80°C, and the reaction time is 3-10h.

[0017] Furthermore, in step S3, freeze drying includes: pre-freezing at -80°C for 5 hours, and then freeze drying at below -15°C.

[0018] The fourth aspect of the present invention provides the use of the above-mentioned veterinary drug composition for treating pet renal failure or the above-mentioned liposome in the preparation of a drug for treating pet renal failure.

[0019] A fifth aspect of the present invention provides a drug for treating pet renal failure, comprising the veterinary drug composition for treating pet renal failure as described above or the liposome as described above.

[0020] The advantages and positive effects of the present invention are:

[0021] In the veterinary drug composition provided by the present invention, curcumin has anti-inflammatory and antioxidant effects, emodin has good diuretic and glomerular filtration rate improvement effects, and astragalus polysaccharide can inhibit renal fibrosis and promote renal cell repair. By combining curcumin, emodin and astragalus polysaccharide, the purpose of synergistic treatment of renal failure can be achieved from the aspects of anti-inflammatory, inhibition of renal fibrosis, and improvement of glomerular filtration function, providing a new solution for the effective treatment of pet renal failure. The veterinary drug composition of the present invention has a good effect on the recovery of renal tissue of pets with renal failure, can effectively reduce the blood urea nitrogen and creatinine levels of pets with renal failure, relieve the symptoms of renal failure, and achieve a good therapeutic effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments.

[0023] Figure 1 This is an H&E staining image of whole kidney sections after one week of treatment of rat renal failure model with liposomes prepared in the examples and comparative examples of the present invention. DETAILED DESCRIPTION

[0024] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with embodiments. The embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0025] Based on the information contained in the present invention, it is easy for those skilled in the art to make various changes to the precise description of the present invention without departing from the spirit and scope of the appended claims. It should be understood that the scope of the present invention is not limited to the defined processes, properties or components, because these embodiments and other descriptions are only for the purpose of illustrating specific aspects of the present invention. In fact, various changes that a person skilled in the art or related fields can obviously make to the embodiments of the present invention are covered within the scope of the appended claims.

[0026] In order to better understand the present invention but not to limit the scope of the present invention, all the numbers and other numerical values ​​used in the present invention to express the amount, percentage, etc. should be understood as modified by the word "about" in all cases. Therefore, unless otherwise specified, the numerical parameters listed in the specification and the appended claims are approximate values, which may be changed according to the different ideal properties to be obtained. Each numerical parameter should at least be regarded as obtained according to the reported significant figures and by conventional rounding methods.

[0027] In addition, it should be noted that, unless otherwise defined, in the context of the present invention, the scientific and technical terms used should have the meanings commonly understood by those of ordinary skill in the art. The meanings of the terms "comprise", "include", "contain", "have" and the like are non-restrictive, that is, other steps and other ingredients that do not affect the results can be added.

[0028] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the present invention is described in detail below.

[0029] An embodiment of the present invention provides a veterinary drug composition for treating pet renal failure, comprising curcumin, emodin and astragalus polysaccharide, wherein the mass ratio of the curcumin, the emodin and the astragalus polysaccharide is 0.5-2:1-2:1-2.

[0030] Since renal failure is accompanied by symptoms such as renal inflammation, renal fibrosis, decreased glomerular filtration function, renal toxin accumulation, and renal cell damage, inhibiting renal inflammation and fibrosis, improving glomerular filtration function, reducing renal toxin accumulation, and repairing damaged renal cells are expected to be effective strategies for the treatment of renal failure.

[0031] The curcumin selected in the present invention has anti-inflammatory and antioxidant effects, emodin has good diuretic and glomerular filtration rate improvement effects, and astragalus polysaccharide can inhibit renal fibrosis and has the effect of promoting renal cell repair. By combining the pleiotropic effects of curcumin, emodin, and astragalus polysaccharide, the present invention can achieve the purpose of synergistically treating renal failure from the aspects of anti-inflammatory, inhibiting renal fibrosis, and improving glomerular filtration function, and provides a new solution for the treatment of renal failure. Experiments were conducted on rat models, and the results showed that the therapeutic effects of using curcumin, emodin, or astragalus polysaccharide alone, or the combination of the two components were weaker than the combination of the three drugs of the present invention. The combination of the three drugs of the present invention has a good effect on the recovery of renal tissue of pets with renal failure, can effectively reduce the blood urea nitrogen and creatinine levels of pets with renal failure, relieve the symptoms of renal failure, and have a good therapeutic effect.

[0032] Studies have shown that as the content of curcumin in the composition increases, its therapeutic effect increases accordingly. Therefore, preferably, the mass ratio of the curcumin, the emodin and the astragalus polysaccharide is 0.5-2:1:1. More preferably, the mass ratio of the curcumin, the emodin and the astragalus polysaccharide is 2:1:1.

[0033] Another embodiment of the present invention provides liposomes containing the veterinary drug composition as described above, wherein the raw materials of the liposomes include the veterinary drug composition, lecithin, cholesterol and a lyoprotectant, and the mass ratio of the veterinary drug composition, the lecithin, the cholesterol and the lyoprotectant is 1:5-8:0.2-2:3-10.

[0034] To further improve the therapeutic effect of pet renal failure, preferably, the mass ratio of the veterinary drug composition, the lecithin, the cholesterol and the lyophilization protectant is 1:5-7:0.5-1:5-6. More preferably, the mass ratio of the veterinary drug composition, the lecithin, the cholesterol and the lyophilization protectant is 1:6.25:0.5:5.

[0035] Optionally, the lyoprotectant is glucose, sucrose or sorbitol, preferably glucose.

[0036] Another embodiment of the present invention provides a method for preparing the liposome as described above, comprising the following steps:

[0037] S1, dissolving curcumin, emodin, cholesterol and lecithin in an organic solvent to obtain an organic phase; in addition, dissolving astragalus polysaccharide in water to obtain an aqueous phase;

[0038] S2, adding the organic phase to the aqueous phase, stirring and emulsifying, and then distilling under reduced pressure to remove the organic solvent to obtain a liposome suspension;

[0039] S3, filtering the liposome suspension, adding a lyophilization protectant to the filtrate, and freeze-drying to obtain a liposome lyophilized powder containing the veterinary drug composition.

[0040] The present invention dissolves cholesterol, lecithin, curcumin and rheum officinale in an organic solvent to prepare an oil phase, dissolves astragalus polysaccharide in water to prepare an aqueous phase, then drops the oil phase into the aqueous phase, and forms a double-layer liposome structure by stirring and emulsifying, wherein the astragalus polysaccharide is located in the hydrophilic region of the liposome, and the curcumin and rheum officinale are wrapped in the hydrophobic region of the liposome, then removes the free curcumin and rheum officinale raw materials by filtering, finally adds a lyophilization protective agent, and freeze-dries to obtain a composite liposome. By developing into liposomes, it is beneficial to improve the bioavailability and stability of curcumin, rheum officinale and astragalus polysaccharide, the liposomes that co-encapsulate curcumin, rheum officinale and astragalus polysaccharide have a smaller particle size and more excellent dispersibility, can stably exist in an aqueous solution, and are beneficial to improving the water solubility of curcumin and rheum officinale, and the liposomes also have a sustained release effect, and can prolong the drug effect time.

[0041] The dosage of lecithin and cholesterol affects the drug loading and encapsulation efficiency of the active ingredient. Generally speaking, when the amount of drug added is high, the amount of the corresponding liposome matrix material cholesterol and lecithin should also be increased to ensure the encapsulation efficiency of the active ingredient and increase the drug loading. The specific amount added can be adjusted according to actual needs, and the present invention does not make any special restrictions on this.

[0042] In step S1, the organic solvent is used to evenly disperse cholesterol, lecithin, curcumin and rhein in a system, and can be emulsified with the aqueous phase. The present invention does not specifically limit the type of organic solvent, as long as it can effectively form a uniform emulsion. Optionally, in step S1, the organic solvent is one of ethanol, tetrahydrofuran, acetone or methanol, preferably ethanol.

[0043] In step S2, the volumetric dosage of the organic solvent and water affects the mass concentrations of cholesterol, lecithin, curcumin, rhamnolide and astragalus polysaccharide in the emulsified reaction system, but has little effect on the formation of the liposome structure, and can be adjusted according to actual needs. In a preferred embodiment, the volume ratio of the organic solvent to the water is 1:1-2.

[0044] Optionally, in step S2, the reaction temperature for stirring and emulsifying is 40-80°C, preferably 50°C, and the reaction time is 3-10h; it can be adaptively adjusted according to the degree of emulsification, preferably 4h.

[0045] Optionally, in step S3, freeze drying comprises: pre-freezing at -80°C for 5 hours, and then freeze drying at below -15°C.

[0046] Another embodiment of the present invention provides the use of the above-mentioned veterinary composition for treating pet renal failure or liposomes containing the above-mentioned veterinary composition for treating pet renal failure in the preparation of a drug for treating pet renal failure.

[0047] The use of the veterinary composition for treating pet renal failure or the liposome containing the veterinary composition for treating pet renal failure in preparing a drug for treating pet renal failure has the same advantages as the veterinary composition for treating pet renal failure described above over the prior art, which will not be repeated here.

[0048] Based on the same inventive concept, another embodiment of the present invention provides a drug for treating pet renal failure, including the veterinary drug composition for treating pet renal failure as described above or liposomes containing the veterinary drug composition for treating pet renal failure as described above.

[0049] The present invention is further described below in conjunction with specific examples. The experimental methods in the following examples without specifying specific conditions are generally carried out according to the conditions recommended by the manufacturer.

[0050] Curcumin (CAS No.: 458-37-7) used in the following examples was purchased from Aladdin Reagent, item number C400271, rhubarb (CAS No.: 518-82-1) was purchased from Aladdin Reagent, item number E106692, and astragalus polysaccharide (CAS No.: 89250-26-0) was purchased from Aladdin Reagent, item number A708486; the HPLC column C18 column was purchased from Agilent, item number 993967-902. Ethanol, lecithin, cholesterol, and glucose were purchased from Sinopharm Chemical Reagent Co., Ltd. All raw materials used were analytically pure unless otherwise specified.

[0051] Example 1-Example 3

[0052] The liposomes of this embodiment contain curcumin, emodin and astragalus polysaccharide as medicinal ingredients, and the preparation method thereof comprises the following steps:

[0053] S1. Add curcumin, emodin, cholesterol and lecithin to 20 mL of anhydrous ethanol, heat to 50°C and stir to dissolve, to obtain an organic phase; in addition, add astragalus polysaccharide to 20 mL of ultrapure water, heat to 50°C and dissolve, to obtain an aqueous phase;

[0054] S2. Add the organic phase to the aqueous phase under a 50° C. water bath condition while stirring. After the addition is complete, continue stirring for 4 hours to emulsify, then remove ethanol by distillation under reduced pressure to obtain a liposome suspension;

[0055] S3. Filter the liposome suspension through a 0.22 μm filter membrane, add glucose to the filtrate, pre-freeze at -80°C for 5 hours, and then freeze-dry at below -15°C to obtain liposomes containing the veterinary drug composition of the present invention (curcumin, emodin and astragalus polysaccharide).

[0056] In Example 1, the mass ratio of curcumin, emodin and astragalus polysaccharide in the veterinary drug composition is 1:1:1, and the mass ratio of the veterinary drug composition, lecithin, cholesterol and lyoprotectant is 1:5:1:5.

[0057] In Example 2, the mass ratio of curcumin, emodin and astragalus polysaccharide in the veterinary drug composition is 0.5:1:1, and the mass ratio of the veterinary drug composition, lecithin, cholesterol and lyophilization protective agent is 1:6:0.8:6.

[0058] In Example 3, the mass ratio of curcumin, emodin and astragalus polysaccharide in the veterinary drug composition is 2:1:1, and the mass ratio of the veterinary drug composition, lecithin, cholesterol and lyoprotectant is 1:6.25:0.5:5.

[0059] Comparative Example 1-Comparative Example 6

[0060] The preparation methods of the comparative examples are basically the same as those of the embodiments, except that the medicinal components are different. Comparative examples 1-6 lack one or two of the veterinary drug compositions of the present invention, respectively, to explore the synergistic effect of the veterinary drug compositions of the present invention.

[0061] The raw material compositions and their proportions of the examples of the present invention and the comparative examples are shown in Table 1.

[0062] Table 1 Liposome raw material composition and content of Examples 1-3 and Comparative Examples 1-6

[0063] Curcumin Rhein Astragalus polysaccharide Lecithin cholesterol glucose Total (mg) Example 1 20 20 20 300 60 300 720 Example 2 10 20 20 300 40 300 690 Example 3 40 20 20 500 40 400 1020 Comparative Example 1 20 / / 400 50 400 870 Comparative Example 2 / 20 / 400 50 400 870 Comparative Example 3 / / 20 400 50 400 870 Comparative Example 4 20 20 / 400 50 400 890 Comparative Example 5 20 / 20 400 50 400 890 Comparative Example 6 / 20 20 400 50 400 890

[0064] Note: “ / ” means not added.

[0065] Test Case

[0066] This example is intended to evaluate the performance of the liposomes or single drug component liposomes prepared in the examples and comparative examples.

[0067] 1. Drug loading and encapsulation efficiency of composite liposomes

[0068] The contents of curcumin, emodin and astragalus polysaccharide were determined by high performance liquid chromatography. The chromatographic column used was a C18 column, the mobile phase was a mixture of acetonitrile and water, the volume ratio of acetonitrile to water was 80:20, the injection volume was 10 μL, the detection wavelength of curcumin was 400 nm, the detection wavelength of emodin was 360 nm, and the detection wavelength of astragalus polysaccharide was 205 nm. Drug loading = drug mass in liposome freeze-dried powder / liposome feed mass × 100%; encapsulation efficiency = drug mass in liposome freeze-dried powder / drug feed mass × 100%; the corresponding results are shown in Table 2.

[0069] Table 2 Liposome or drug loading and encapsulation efficiency determination results

[0070]

[0071]

[0072] It can be seen from Table 2 that the drug of the present invention has a high encapsulation rate and drug loading. It should be noted that due to the loss of filtrate during filtration, there is a slight loss of astragalus polysaccharide, so the encapsulation rate of astragalus polysaccharide is not 100%.

[0073] 2. Particle size and potential test

[0074] The liposomes prepared in the examples and comparative examples were dispersed in ultrapure water to prepare a 10 mg / mL aqueous solution, and the particle size and potential of the liposomes were measured by dynamic light scattering. Each sample was measured three times and the average value was calculated. The corresponding results are shown in Table 3.

[0075] Table 3 Liposome particle size and potential measurement results

[0076] sample Particle size (nm) Potential (mV) Polydispersity index Example 1 142 -2.6 0.215 Example 2 157 -2.4 0.252 Example 3 165 -2.1 0.292 Comparative Example 1 175 -3.2 0.301 Comparative Example 2 189 -2.5 0.320 Comparative Example 3 171 -1.9 0.295 Comparative Example 4 180 -2.1 0.283 Comparative Example 5 175 -2.2 0.281 Comparative Example 6 176 -2.0 0.292

[0077] As can be seen from Table 3, the average particle size of the liposomes of the present invention is in the range of 100-210nm, the potential is negative, and the polydispersity index is less than 0.3, indicating that the prepared liposomes have a narrower particle size distribution and a higher dispersion stability. The liposome particle size is related to the drug loading. Generally speaking, the liposomes with low drug loading have a smaller particle size, and the liposomes with high drug loading have a larger particle size. In comparison, the liposome particle size prepared by the embodiment of the present invention has a higher drug loading than Comparative Examples 1-6, but it has a smaller particle size, which may be due to the formation of a hydrophobic cross-linked structure between emodin and curcumin, thereby reducing the particle size of the liposome.

[0078] 3. In vitro drug release behavior

[0079] Preparation of pH 7.4 release solution: weigh 0.26 g potassium dihydrogen phosphate and 2.16 g disodium hydrogen phosphate dodecahydrate, add them into a beaker, add 1 liter ultrapure water and 5 g Tween-80, stir to dissolve, and then adjust the pH to 7.4.

[0080] In vitro drug release experiment: The liposomes prepared in Example 1 and Comparative Example 1 were dispersed in ultrapure water to obtain a 50 mg / mL aqueous solution. 1 mL of each solution was added to a dialysis bag, and then the dialysis bag was immersed in 50 mL of the pH 7.4 release solution prepared above, and the bag was placed in a constant temperature shaker for shaking, with the temperature set to 37°C and the speed set to 180 rpm. 3 mL of release solution was taken out at 1 h, 2 h, 4 h, 8 h, 12 h, 24 h, and 48 h, and 3 mL of blank release solution was added. Three groups of release experiments were performed in parallel in each release solution, and the average value was calculated. The concentration of the active ingredient in the released solution was detected by high performance liquid chromatography, and the average cumulative release amount was calculated, and the cumulative release amount = the mass of the drug in the release solution / the mass of the drug in the added liposomes × 100%; the corresponding results are shown in Tables 4-6.

[0081] Table 4 Cumulative release of curcumin in liposomes

[0082] Time (h) Example 1 Example 2 Example 3 Comparative Example 1 Comparative Example 4 Comparative Example 5 1 8.2 9.4 8.9 12.2 11.3 10.1 2 12.1 13.1 13.0 15.4 13.2 13.2 4 17.2 20.2 18.8 23.6 21.5 20.5 8 22.4 26.5 24.1 28.7 26.2 25.7 12 27.5 36.3 32.4 38.8 34.5 34.4 24 35.5 42.1 38.7 46.3 41.3 41.7 48 45.7 49.8 46.5 54.2 50.4 50.6

[0083] Table 5 Results of the cumulative release of emodin in liposomes

[0084] Time (h) Example 1 Example 2 Example 3 Comparative Example 2 Comparative Example 4 Comparative Example 6 1 6.6 8.6 8.9 12.5 11.1 10.2 2 10.1 12.5 12.1 15.1 12.7 12.7 4 14.2 17.5 16.3 22.2 20.2 19.5 8 18.3 23.1 22.7 27.3 24.5 25.6 12 22.6 32.6 31.2 36.2 32.7 34.4 24 30.6 37.2 36.2 44.5 41.2 43.1 48 36.3 49.2 45.5 52.3 50.2 51.6

[0085] Table 6 Determination results of cumulative release of astragalus polysaccharide in liposomes

[0086] Time (h) Example 1 Example 2 Example 3 Comparative Example 3 Comparative Example 5 Comparative Example 6 1 10. 12.2 13.1 17.4 16.2 15.3 2 14.5 15.4 16.0 19.2 21.4 20.6 4 18.4 20.2 21.7 24.7 25.6 23.5 8 23.1 27.4 29.1 34.7 34.7 33.4 12 28.3 38.3 39.6 42.4 41.8 43.5 24 36.4 44.5 46.5 49.7 48.3 47.1 48 46.9 51.8 52.5 55.2 50.4 48.6

[0087] As shown in Tables 4-6, curcumin, emodin and astragalus polysaccharide liposomes have sustained release effects, and the cumulative release amounts in 48 hours are all within 60%. In general, the drug release characteristics of the liposomes prepared by combining the three active ingredients in Examples 1-3 and Comparative Examples 1-6 are similar.

[0088] 4. Cellular Uptake Assay

[0089] Test solution preparation: The liposomes prepared in the examples and comparative examples were dispersed in ultrapure water to prepare an aqueous solution with a total concentration of curcumin, rhein and astragalus polysaccharide of 5 mg / mL, wherein astragalus polysaccharide was labeled with FITC coupling, and curcumin and rhein have ultraviolet light absorption, and no coupling detection label is required, and then diluted with RPMI1640 culture medium to a total concentration of curcumin, rhein and astragalus polysaccharide of 20 μg / mL of each liposome test solution, and free curcumin, rhein and astragalus polysaccharide were prepared with the same concentration as a control.

[0090] Cell uptake test: 3T3 cells were seeded in a 12-well plate at a density of 100,000 cells per well and cultured for 48 h. The cells were then incubated with the above-prepared test solutions for 4 h. The uptake was detected by flow cytometry. Three parallel groups were tested for each sample, and the average uptake was calculated. The corresponding results are shown in Table 7.

[0091] Table 7 Uptake of curcumin, emodin and astragalus polysaccharide in liposomes in 3T3 cells

[0092] sample Curcumin Rhein Astragalus polysaccharide Example 1 3.2 3.5 3.0 Example 2 2.2 3.4 3.4 Example 3 4.8 2.5 2.6 Comparative Example 1 3.1 / / Comparative Example 2 / 3.0 / Comparative Example 3 / / 2.8 Comparative Example 4 3.0 2.9 / Comparative Example 5 3.2 / 3.1 Comparative Example 6 / 3.4 3.2

[0093] As shown in Table 7, curcumin, emodin, and astragalus polysaccharide liposomes can promote the effective absorption of curcumin, emodin, and astragalus polysaccharide; under the same amount of added active ingredients, the three-drug combination of the present invention has a cellular uptake amount equivalent to that of single curcumin, emodin, and astragalus polysaccharide. The cellular uptake rates of curcumin, emodin, and astragalus polysaccharide in the drug-encapsulated liposomes of all embodiments and comparative examples are between 2.5% and 5.0%.

[0094] 5. Kidney failure treatment experiment

[0095] Establishment of renal failure model: The rat renal failure model was established by unilateral ureteral ligation. Preoperative instruments and items were prepared (all items were sterilized and ready for use), including dry cotton balls, alcohol cotton balls, iodine tincture + cotton swabs, 10% chloral hydrate + syringes, conventional surgical instruments, microsurgical instruments, suture packs, etc. Rats were fasted from food and water for 12 hours. Anesthesia treatment, 10% chloral hydrate was injected intraperitoneally at a dose of 330 μL / 100 g; after anesthesia, the rat was fixed on the dissection board in a supine position, the neck was prepared and disinfected; unilateral ureteral ligation, the hair on the left abdomen of the rat was cut with a wire scissor, the abdomen was disinfected with medical iodine, and a surgical incision of about 2 cm was made slightly below the left abdomen with a scalpel, and the abdominal tissue was separated layer by layer until the abdominal cavity, and the intestinal tube covering the left ureter was gently opened with a medical cotton swab, and the left iliac blood vessel was used as a mark to find the left ureter crossing the left iliac blood vessel, and the ureter was freed upward to the left renal pelvis, and the ureter was freed and tied at the junction of the left ureter and renal pelvis and about 1 cm downward with No. 0 silk thread, and the intestinal tube was reset, and the abdominal cavity was closed by suture layer by layer. Put it back in the breeding cage and keep warm.

[0096] Drug treatment: The liposomes prepared in the examples and comparative examples were administered by oral gavage before and after surgery, with a total dose of 50 mg / kg. The combined drug group had a total of 50 mg / kg of various drugs, with 5 mice in each group (n=5).

[0097] One week after administration, the blood urea nitrogen and creatinine levels of rats were measured using a urea nitrogen detection kit (Nanchang Better Biotech Co., Ltd.) and a creatinine detection kit (Wuhan Shengzhiyuan Biotechnology Co., Ltd.), and the average reduction percentages of blood urea nitrogen and creatinine in each group were calculated. The average reduction percentage = (normal saline group level - drug treatment group level) × 100% / normal saline group level; the corresponding results are shown in Table 8.

[0098] Table 8 Reduction of blood urea nitrogen and creatinine in rats after liposome administration

[0099]

[0100] Compared with the single-pharmacological component liposomes of curcumin, emodin, and astragalus polysaccharide in comparative examples 1-3, the curcumin, emodin, and astragalus polysaccharide dual-component combination liposomes prepared in comparative examples 4-6 have a more significant therapeutic effect, indicating that the dual components have a synergistic effect in inhibiting renal failure. On the other hand, the results show that the therapeutic effect of the composite liposomes of curcumin and emodin is significantly better than that of the composite liposomes of emodin and astragalus polysaccharide and the composite liposomes of curcumin and astragalus polysaccharide, indicating that the composite liposomes of curcumin and emodin have better therapeutic effects. The therapeutic effect of the three-component composite liposomes of curcumin, emodin, and astragalus polysaccharide in Examples 1-3 is significantly better than that of the two-component composite liposomes, confirming that the combination of the three pharmacological components of curcumin, emodin, and astragalus polysaccharide of the present invention can synergistically exert a better therapeutic effect. By increasing the composite ratio of curcumin (the ratio of curcumin: emodin: astragalus polysaccharide is 0.5:1:1 to 2:1:1), it was found that as the mass ratio of curcumin increased, the therapeutic effect of the three-component composite liposome increased accordingly, indicating that curcumin has a more important therapeutic effect on renal failure. Therefore, the preferred ratio of curcumin: emodin: astragalus polysaccharide is 2:1:1.

[0101] In addition, rat kidneys were obtained and sectioned for H&E staining. Figure 1 From left to right, the H&E staining images of rat whole kidney sections after liposome treatment of Example 1, Example 2, Comparative Example 1, Comparative Example 2 and Comparative Example 4 are shown in turn. The results also show that the kidney integrity after treatment with curcumin, emodin, and astragalus polysaccharide composite liposomes is better, and renal cell necrosis is effectively inhibited, which is consistent with the results of blood urea nitrogen and creatinine, and has a better therapeutic effect for renal failure. Although single-component curcumin or astragalus polysaccharide has a therapeutic effect, it is not significant.

[0102] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A veterinary drug composition for treating renal failure of pets, characterized in that: The invention comprises curcumin, rhein and astragalus polysaccharide, wherein the mass ratio of the curcumin, rhein and astragalus polysaccharide is 0.5-2:1-2:1-2.

2. The veterinary drug composition for treating pet renal failure according to claim 1, characterized in that: The mass ratio of the curcumin, the emodin and the astragalus polysaccharide is 0.5-2:1:

1.

3. The veterinary drug composition for treating pet renal failure according to claim 2, characterized in that: The mass ratio of the curcumin, the emodin and the astragalus polysaccharide is 2:1:

1.

4. A liposome, characterized in that The raw materials include the veterinary drug composition for treating pet renal failure as described in any one of claims 1 to 3, and also include lecithin, cholesterol and a lyophilization protectant. The mass ratio of the veterinary drug composition, the lecithin, the cholesterol and the lyophilization protectant is 1:5-8:0.2-2:3-10.

5. The liposome according to claim 4, characterized in that The mass ratio of the veterinary drug composition, the lecithin, the cholesterol and the lyophilization protective agent is 1:6.25:0.5:

5.

6. The liposome according to claim 4, characterized in that The freeze-drying protective agent is glucose, sucrose or sorbitol.

7. A method for preparing liposomes, characterized in that: For preparing the liposome according to any one of claims 4 to 6, the preparation method comprises the following steps: S1, dissolving curcumin, emodin, cholesterol and lecithin in an organic solvent to obtain an organic phase; in addition, dissolving astragalus polysaccharide in water to obtain an aqueous phase; S2, adding the organic phase to the aqueous phase, stirring and emulsifying, and then distilling under reduced pressure to remove the organic solvent to obtain a liposome suspension; S3, filtering the liposome suspension, adding a lyophilization protective agent to the filtrate, and obtaining liposome lyophilized powder after freeze drying.

8. The method for preparing liposomes according to claim 7, characterized in that: In step S2, the reaction temperature for stirring and emulsifying is 40-80°C, and the reaction time is 3-10h; In step S3, freeze drying includes: pre-freezing at -80°C for 5 hours, and then freeze drying at below -15°C.

9. Use of the veterinary composition for treating pet renal failure as claimed in claims 1 to 3 or the liposome as claimed in any one of claims 4 to 6 in preparing a drug for treating pet renal failure.

10. A drug for treating renal failure in pets, characterized in that: The veterinary composition for treating pet renal failure as described in claims 1-3 or the liposome as described in any one of claims 4-6.

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