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 turmeric, rhizoma alismatis and beta-sitosterol. The mass ratio of the rhizoma curcumae longae to the rhizoma alismatis to the beta-sitosterol is (1-2): (1-2): (0.5-2). The veterinary drug composition provided by the invention realizes the purpose of synergistically treating renal failure from the aspects of resisting inflammation, inhibiting renal fibrosis, improving glomerular filtration function, promoting kidney cell repair and the like, and provides a new solution for effectively treating the renal failure of pets. The veterinary drug composition disclosed by the invention has a good recovery effect on kidney tissues of pets with renal failure, can effectively reduce urea nitrogen and creatinine levels of blood of the pets with renal failure, relieves symptoms of renal failure, 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 highlight of urban residents' consumption, and pet health and welfare are increasingly valued. In the process of pet breeding, multiple reasons can easily cause renal toxicity: renal tubular damage can be caused by exogenous toxins, endogenous toxins and iatrogenic toxins. The triggering factors of exogenous renal failure mainly include exogenous drugs: such as antibiotics, antifungal drugs, anthelmintics, analgesics, heavy metals, organic compounds (ethylene glycol, chloroform, carbon tetrachloride, herbicides, pesticides, organic solvents, etc.), chemotherapy drugs, anesthetics, moldy feed, etc. In addition, improper diagnosis and treatment techniques of clinical pet doctors may cause acute renal failure in pets. For example, the use of non-steroidal anti-inflammatory drugs or vasodilators during anesthesia and surgery may cause hypotension and decreased renal perfusion, leading to acute renal failure. In addition, infectious diseases such as (pyelonephritis, leptospirosis, borrelia), urinary tract obstruction, diabetes, etc. can also cause acute renal failure.

[0003] Kidney damage often leads to a decrease in the number of nephrons, a decrease in function, and compensatory changes in the remaining healthy nephrons, including an increase in glomerular filtration rate, an increase in glomerular capillary blood pressure, and an increase in glomerular volume, which can lead to glomerular hyperfiltration, hypertension, and hypertrophy. That is, changes in glomerular morphology ultimately lead to irreversible loss of renal function. With the abnormality of renal function, proteinuria and uremia eventually occur, and the diseased animals die.

[0004] Currently, there is no specific oral drug for the treatment of renal failure. The main treatment methods are to improve the symptoms of renal failure through diet and nutritional management, or to delay the disease through dialysis, which is very harmful to pets. Therefore, the development of new and effective drugs for the treatment of renal failure in pets is of great clinical significance. Summary of the invention

[0005] 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 inhibiting vascular endothelial inflammation, inhibiting renal fibrosis, and improving 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 liposomes of the veterinary drug composition and a preparation method thereof, and further provides the use of the above veterinary drug composition and its liposomes in the preparation of pet kidney treatment drugs.

[0006] To achieve the above object, the present invention is specifically implemented through the following technical solutions:

[0007] In a first aspect, the present invention provides a veterinary drug composition for treating pet renal failure, comprising turmeric, oriental rhizome and β-sitosterol; the mass ratio of turmeric, oriental rhizome and β-sitosterol is (1-2):(1-2):(0.5-2).

[0008] Preferably, the mass ratio of turmeric, oriental rhizome and β-sitosterol is (1-2):1:2.

[0009] Preferably, the mass ratio of turmeric, oriental rhizome and β-sitosterol is 2:1:2.

[0010] In a second aspect, the present invention also provides a liposome, whose raw materials include the veterinary composition for treating pet renal failure, and also include lecithin, cholesterol and a lyophilization protectant. The mass ratio of the veterinary composition, the lecithin, the cholesterol and the lyophilization protectant is 1:(3.75-5):(0.4-1):(3.75-5).

[0011] Preferably, the mass ratio of the veterinary drug composition, the lecithin, the cholesterol and the lyoprotectant is 1:5:0.4:4.

[0012] Preferably, the lyoprotectant includes at least one of glucose, sucrose and sorbitol.

[0013] In a third aspect, the present invention further provides a method for preparing the liposome, comprising the following steps:

[0014] S1, dissolving turmeric, oriental rhizome, β-sitosterol, cholesterol and lecithin in an organic solvent to obtain an organic phase; using water as an aqueous phase;

[0015] 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;

[0016] S3, filtering the liposome suspension, adding a lyophilization protective agent to the filtrate, and obtaining liposome lyophilized powder after freeze drying.

[0017] Preferably, in step S1, the organic solvent comprises at least one of ethanol, tetrahydrofuran, acetone and methanol;

[0018] In step S2, the volume ratio of the organic solvent to the aqueous phase is 1:(1-2);

[0019] In step S2, the reaction temperature for stirring and emulsifying is 40-80°C, and the reaction time is 3-10h;

[0020] In step S3, freeze drying comprises: pre-freezing at -80°C for 4 to 5 hours, followed by freeze drying at below -15°C for 1 to 2 hours;

[0021] In a fourth aspect, the present invention also provides a veterinary drug composition for treating pet renal failure or use of the liposome in preparing a drug for treating pet renal failure.

[0022] In a fifth aspect, the present invention also provides a drug for treating pet renal failure, comprising the veterinary drug composition for treating pet renal failure or comprising the liposome.

[0023] The veterinary drug composition for treating pet renal failure of the present invention and its liposome and application have the following advantages and positive effects compared with the prior art:

[0024] In the veterinary drug composition provided by the present invention, turmeric has anti-inflammatory and antioxidant effects, Rhizoma Alismatis has good diuretic and glomerular filtration rate improvement effects, and β-sitosterol can inhibit HIF-ɑ, thereby improving the hypoxic microenvironment of the kidney to promote the repair of renal cells. By combining turmeric, Rhizoma Alismatis and β-sitosterol, the purpose of synergistic treatment of renal failure can be achieved from the aspects of anti-inflammatory, inhibition of renal fibrosis, enhancement of glomerular filtration function, and promotion of renal cell repair, 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

[0025] 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.

[0026] 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

[0027] 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.

[0028] 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.

[0029] 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.

[0030] 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.

[0031] 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.

[0032] An embodiment of the present invention provides a veterinary drug composition for treating pet renal failure, comprising turmeric, oriental rhizome and β-sitosterol; the mass ratio of turmeric, oriental rhizome and β-sitosterol is (1-2):(1-2):(0.5-2).

[0033] 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.

[0034] The turmeric selected in the present invention has anti-inflammatory and antioxidant effects, the rhizome of orientalis has good diuretic and glomerular filtration rate improvement effects, and the β-sitosterol (molecular formula is C 29 H 50O) can inhibit HIF-ɑ, thereby improving the hypoxic microenvironment of the kidney and promoting the repair of renal cells. By combining the pleiotropic effects of turmeric, oriental rhizome, and β-sitosterol, the present invention can achieve the purpose of synergistically treating renal failure from the aspects of anti-inflammatory, inhibiting renal fibrosis, improving glomerular filtration function, and repairing renal damaged cells, 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 turmeric, oriental rhizome and β-sitosterol 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.

[0035] Specifically, turmeric contains curcuminoids, among which curcumin is the most important active ingredient. Curcumin has multiple pharmacological effects such as anti-inflammatory and antioxidant. Alisma orientalis mainly contains triterpenoids (such as alismatol A, alismatol B, etc.), sesquiterpenoids, diterpenoids and other ingredients. It has the effect of promoting water and moisture, and is often used in traditional medicine to treat symptoms such as dysuria, edema, diarrhea and oliguria. β-Sitosterol belongs to plant sterols, which is a white flaky or needle-shaped crystal, insoluble in water, and has good solubility in organic solvents. β-Sitosterol has the effect of lowering cholesterol. In addition, it also has anti-inflammatory and antioxidant effects.

[0036] Studies have found that as the content of β-sitosterol in the composition increases, its therapeutic effect increases accordingly. Therefore, preferably, the mass ratio of turmeric, oriental rhizome and β-sitosterol is (1-2):1:2; if the β-sitosterol content is further increased, the therapeutic effect is reduced, so the β-sitosterol content has an optimal value, and more preferably, the mass ratio of turmeric, oriental rhizome and β-sitosterol is 2:1:2.

[0037] Based on the same inventive concept, the present invention also provides a liposome, whose raw materials include the above-mentioned veterinary composition for treating pet renal failure, and also include lecithin, cholesterol and a lyophilization protectant. The mass ratio of the veterinary composition, lecithin, cholesterol and the lyophilization protectant is 1:(3.75-5):(0.4-1):(3.75-5).

[0038] To further improve the therapeutic effect of pet renal failure, more preferably, the mass ratio of the veterinary drug composition, lecithin, cholesterol and lyophilization protectant is 1:5:0.4:4.

[0039] In some embodiments, the lyoprotectant comprises at least one of glucose, sucrose, and sorbitol; preferably glucose.

[0040] Based on the same inventive concept, the present invention also provides a method for preparing the above-mentioned liposome, comprising the following steps:

[0041] S1, dissolving turmeric, oriental rhizome, β-sitosterol, cholesterol and lecithin in an organic solvent to obtain an organic phase; using water as an aqueous phase;

[0042] 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;

[0043] S3, filtering the liposome suspension, adding a lyophilization protective agent to the filtrate, and obtaining liposome lyophilized powder after freeze drying.

[0044] The invention dissolves cholesterol, lecithin, turmeric, oriental rhizome and beta-sitosterol in an organic solvent to prepare an oil phase; uses water as the water phase; drops the oil phase into the water phase, and forms a double-layer liposome structure by stirring and emulsifying, wherein turmeric, oriental rhizome and beta-sitosterol are encapsulated in a hydrophobic region of the liposome, and then free turmeric, oriental rhizome and beta-sitosterol raw materials are removed by filtering, and finally a freeze-drying protective agent is added, and freeze-dried to obtain a composite liposome; the liposome is developed into a liposome, which is beneficial to improving the bioavailability and stability of turmeric, oriental rhizome and beta-sitosterol; the liposome co-encapsulating turmeric, oriental rhizome and beta-sitosterol has a smaller particle size and more excellent dispersibility, can exist stably in an aqueous solution, and is beneficial to improving the water solubility of turmeric, oriental rhizome and beta-sitosterol; the liposome also has a sustained release effect, and can prolong the drug effect time.

[0045] The dosage of lecithin and cholesterol affects the drug loading and encapsulation rate of the active ingredients. Generally speaking, when the amount of drug added is high, the amount of cholesterol and lecithin, the corresponding liposome matrix material, should also be increased to ensure the encapsulation rate of the active ingredients and increase the drug loading. The specific amount added can be adjusted according to actual needs and is not specifically limited.

[0046] In some embodiments, in step S1, an organic solvent is used to evenly disperse cholesterol, lecithin, turmeric, oriental rhizome and β-sitosterol in a system, and can be emulsified with an 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 at least one of ethanol, tetrahydrofuran, acetone or methanol, preferably ethanol.

[0047] In some embodiments, in step S2, the volumetric dosage of the organic solvent and water affects the mass concentration of cholesterol, lecithin, turmeric, oriental rhizome, and β-sitosterol 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 aqueous phase is 1:(1-2).

[0048] In some embodiments, 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.

[0049] In some embodiments, in step S3, freeze drying comprises: pre-freezing at -80°C for 4 to 5 hours, followed by freeze drying at below -15°C for 1 to 2 hours.

[0050] In some embodiments, the mass volume ratio of turmeric, Alisma orientalis, β-sitosterol, cholesterol, lecithin, lyoprotectant, organic solvent, and aqueous phase is (20-40) mg:20 mg:(20-40) mg:(40-60) mg:(300-500) mg:(300-400) mg:20 mL:20 mL.

[0051] Based on the same inventive concept, the present invention also provides a use of the above-mentioned veterinary drug composition for treating pet renal failure or the above-mentioned liposome in preparing a drug for treating pet renal failure.

[0052] 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.

[0053] Based on the same inventive concept, the present invention also provides a drug for treating pet renal failure, comprising the above-mentioned veterinary drug composition for treating pet renal failure or comprising the above-mentioned liposome.

[0054] The following further illustrates the veterinary composition for treating pet renal failure of the present application and its liposome and application with specific examples. This section further illustrates the content of the present invention in conjunction with specific examples, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means adopted in the examples are conventional means well known to those skilled in the art. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0055] The turmeric and orientalis used in the following examples are commercially available products, β-sitosterol (CAS No.: 83-46-5) was purchased from MacLean Reagent, item No. S817963-100g; ethanol, lecithin, cholesterol, and glucose were purchased from Sinopharm Chemical Reagent Co., Ltd.; all raw materials used were analytical grade unless otherwise specified.

[0056] Example 1

[0057] This embodiment provides a method for preparing liposomes containing turmeric, oriental rhizome and β-sitosterol as medicinal ingredients, comprising the following steps:

[0058] S1. Add 20 mg of turmeric (crushed before use to obtain turmeric powder), 20 mg of oriental rhizome (crushed before use to obtain oriental rhizome powder), 20 mg of β-sitosterol, 60 mg of cholesterol and 300 mg of lecithin to 20 mL of anhydrous ethanol, heat to 50° C., and stir to dissolve to obtain an organic phase; in addition, take 20 mL of ultrapure water, heat to 50° C., and set it as an aqueous phase;

[0059] 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;

[0060] S3. Filter the liposome suspension through a filter membrane with a pore size of 0.22 μm, add 300 mg of glucose to the filtrate, and freeze-dry to obtain liposomes containing the veterinary drug composition of the present invention (turmeric, oriental rhizome and β-sitosterol).

[0061] In Example 1, the mass ratio of turmeric, oriental rhizome and β-sitosterol 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.

[0062] Example 2

[0063] This embodiment provides a method for preparing liposomes containing turmeric, oriental rhizome and β-sitosterol as medicinal ingredients, comprising the following steps:

[0064] S1. Add 20 mg of turmeric (crushed before use to obtain turmeric powder), 20 mg of oriental rhizome (crushed before use to obtain oriental rhizome powder), 40 mg of β-sitosterol, 40 mg of cholesterol and 300 mg of lecithin to 20 mL of anhydrous ethanol, heat to 50° C., and stir to dissolve to obtain an organic phase; in addition, take 20 mL of ultrapure water, heat to 50° C., and set it as an aqueous phase;

[0065] 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;

[0066] S3. Filter the liposome suspension through a filter membrane with a pore size of 0.22 μm, add 300 mg of glucose to the filtrate, and freeze-dry to obtain liposomes containing the veterinary drug composition of the present invention (turmeric, oriental rhizome and β-sitosterol).

[0067] In Example 2, the mass ratio of turmeric, oriental rhizome and β-sitosterol in the veterinary drug composition is 1:1:2, and the mass ratio of the veterinary drug composition, lecithin, cholesterol and lyoprotectant is 1:3.75:0.5:3.75.

[0068] Example 3

[0069] This embodiment provides a method for preparing liposomes containing turmeric, oriental rhizome and β-sitosterol as medicinal ingredients, comprising the following steps:

[0070] S1. Add 40 mg of turmeric (crushed before use to obtain turmeric powder), 20 mg of oriental rhizome (crushed before use to obtain oriental rhizome powder), 40 mg of β-sitosterol, 40 mg of cholesterol and 500 mg of lecithin to 20 mL of anhydrous ethanol, heat to 50° C., and stir to dissolve to obtain an organic phase; in addition, take 20 mL of ultrapure water, heat to 50° C., and set it as an aqueous phase;

[0071] 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;

[0072] S3. Filter the liposome suspension through a filter membrane with a pore size of 0.22 μm, add 400 mg of glucose to the filtrate, and freeze-dry to obtain liposomes containing the veterinary drug composition of the present invention (turmeric, oriental rhizome and β-sitosterol).

[0073] In Example 3, the mass ratio of turmeric, oriental rhizome and β-sitosterol in the veterinary drug composition is 2:1:2, and the mass ratio of the veterinary drug composition, lecithin, cholesterol and lyoprotectant is 1:5:0.4:4.

[0074] Comparative Example 1

[0075] This comparative example provides a method for preparing liposomes containing turmeric as a medicinal ingredient, comprising the following steps:

[0076] S1. Add 20 mg of turmeric (crushed to obtain turmeric powder before use), 50 mg of cholesterol and 400 mg of lecithin to 20 mL of anhydrous ethanol, heat to 50° C., and stir to dissolve to obtain an organic phase; in addition, take 20 mL of ultrapure water, heat to 50° C., and set it as an aqueous phase;

[0077] 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;

[0078] S3. Filter the liposome suspension through a filter membrane with a pore size of 0.22 μm, add 400 mg of glucose to the filtrate, and freeze-dry to obtain liposomes containing turmeric.

[0079] Comparative Example 2

[0080] This comparative example provides a method for preparing liposomes containing Rhizoma Alismatis as a medicinal ingredient, comprising the following steps:

[0081] S1. Add 20 mg of Rhizoma Alismatis (crushed to obtain Rhizoma Alismatis powder before use), 50 mg of cholesterol and 400 mg of lecithin to 20 mL of anhydrous ethanol, heat to 50° C., and stir to dissolve to obtain an organic phase; in addition, take 20 mL of ultrapure water, heat to 50° C., and set it as an aqueous phase;

[0082] 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;

[0083] S3. Filter the liposome suspension through a filter membrane with a pore size of 0.22 μm, add 400 mg of glucose to the filtrate, and freeze-dry to obtain liposomes containing Alisma orientalis.

[0084] Comparative Example 3

[0085] This comparative example provides a method for preparing liposomes containing β-sitosterol as a pharmaceutical ingredient, comprising the following steps:

[0086] S1. Add 20 mg of β-sitosterol, 50 mg of cholesterol and 400 mg of lecithin to 20 mL of anhydrous ethanol, heat to 50°C and stir to dissolve to obtain an organic phase; in addition, take 20 mL of ultrapure water, heat to 50°C and set it as an aqueous phase;

[0087] 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;

[0088] S3. Filter the liposome suspension through a filter membrane with a pore size of 0.22 μm, add 400 mg of glucose to the filtrate, and freeze-dry to obtain liposomes containing β-sitosterol.

[0089] Comparative Example 4

[0090] This comparative example provides a method for preparing liposomes containing turmeric and orientalis as medicinal ingredients, comprising the following steps:

[0091] S1. Add 20 mg of turmeric (crushed before use to obtain turmeric powder), 20 mg of oriental rhizome (crushed before use to obtain oriental rhizome powder), 50 mg of cholesterol and 400 mg of lecithin to 20 mL of anhydrous ethanol, heat to 50° C., and stir to dissolve to obtain an organic phase; in addition, take 20 mL of ultrapure water, heat to 50° C., and set it as an aqueous phase;

[0092] 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;

[0093] S3. Filter the liposome suspension through a filter membrane with a pore size of 0.22 μm, add 400 mg of glucose to the filtrate, and freeze-dry to obtain liposomes containing turmeric and orientalis.

[0094] Comparative Example 5

[0095] This comparative example provides a method for preparing liposomes containing turmeric and β-sitosterol as medicinal ingredients, comprising the following steps:

[0096] S1. Add 20 mg of turmeric (crushed to obtain turmeric powder before use), 20 mg of β-sitosterol, 50 mg of cholesterol and 400 mg of lecithin to 20 mL of anhydrous ethanol, heat to 50° C., and stir to dissolve to obtain an organic phase; in addition, take 20 mL of ultrapure water, heat to 50° C., and set it as an aqueous phase;

[0097] 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;

[0098] S3. Filter the liposome suspension through a filter membrane with a pore size of 0.22 μm, add 400 mg of glucose to the filtrate, and freeze-dry to obtain liposomes containing turmeric and β-sitosterol.

[0099] Comparative Example 6

[0100] This comparative example provides a method for preparing liposomes containing orientalis and β-sitosterol as medicinal ingredients, comprising the following steps:

[0101] S1. Add 20 mg of Rhizoma Alismatis (crushed to obtain Rhizoma Alismatis powder before use), 20 mg of β-sitosterol, 50 mg of cholesterol and 400 mg of lecithin to 20 mL of anhydrous ethanol, heat to 50°C, and stir to dissolve to obtain an organic phase; in addition, take 20 mL of ultrapure water, heat to 50°C, and set it as an aqueous phase;

[0102] 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;

[0103] S3. Filter the liposome suspension through a filter membrane with a pore size of 0.22 μm, add 400 mg of glucose to the filtrate, and freeze-dry to obtain liposomes containing oriental alisma and β-sitosterol.

[0104] Specifically, Table 1 shows the raw material compositions and contents of Examples 1 to 3 and Comparative Examples 1 to 6 of the present invention.

[0105] Table 1 Liposome raw material composition and content of Examples 1-3 and Comparative Examples 1-6

[0106] turmeric Alisma orientalis β-Sitosterol Lecithin cholesterol glucose Total (mg) Example 1 20 20 20 300 60 300 720 Example 2 20 20 40 300 40 300 720 Example 3 40 20 40 500 40 400 1040 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

[0107] Note: “ / ” means not added.

[0108] Test Case

[0109] This test example is intended to evaluate the performance of the liposomes or single drug component liposomes prepared in the examples and comparative examples.

[0110] 1. Drug loading and encapsulation efficiency of composite liposomes

[0111] The content of the active ingredient β-sitosterol was determined by high performance liquid chromatography, the chromatographic column used was a C18 column (high performance liquid chromatography column C18 column purchased from Agilent, item number 993967-902), the mobile phase was a mixture of acetonitrile and water, wherein the volume ratio of acetonitrile to water was 80:20, the injection volume was 10 μL, and the β-sitosterol detection wavelength was 205 nm. Drug loading = drug mass in liposome freeze-dried powder / composite liposome freeze-dried powder mass × 100%; encapsulation efficiency = drug mass in liposome freeze-dried powder / drug feed mass × 100%; the corresponding results are shown in Table 2.

[0112] Table 2 - Drug loading and encapsulation efficiency test results

[0113]

[0114]

[0115] It can be seen from Table 2 that the drug of the present invention has a high encapsulation efficiency and drug loading. It should be noted that due to the loss of filtrate during filtration, there is a slight loss of β-sitosterol, so the encapsulation efficiency of β-sitosterol is not 100%.

[0116] 2. Particle size and potential test

[0117] 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.

[0118] Table 3 - Liposome particle size and potential measurement results

[0119] Example Particle size (nm) Potential (mV) Polydispersity index Example 1 151 -2.2 0.225 Example 2 152 -2.3 0.242 Example 3 168 -2.4 0.261 Comparative Example 1 171 -3.1 0.285 Comparative Example 2 190 -2.3 0.314 Comparative Example 3 173 -2.1 0.293 Comparative Example 4 182 -2.2 0.281 Comparative Example 5 177 -2.1 0.285 Comparative Example 6 179 -2.3 0.296

[0120] As can be seen from Table 3, the average particle size of the liposomes of the present invention is in the range of 100-200nm, 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 drug loading is low, the particle size is smaller, and the drug loading is high, and the particle size is larger. 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 that a hydrophobic cross-linking agent structure is formed between turmeric, oriental rhizome and beta-sitosterol, thereby reducing the particle size of the liposome.

[0121] 3. In vitro drug release behavior

[0122] 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.

[0123] In vitro drug release experiment: The liposomes prepared in Examples 1 to 3 and Comparative Examples 3, 5 to 6 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 1h, 2h, 4h, 8h, 12h, 24h, and 48h, and then 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 Table 4.

[0124] Table 4 - Results of the measurement of the cumulative release of β-sitosterol in liposomes (%)

[0125] Time (h) Example 1 Example 2 Example 3 Comparative Example 3 Comparative Example 5 Comparative Example 6 1 7.62 8.86 8.62 9.86 10.76 10.25 2 11.24 12.65 12.34 14.86 15.21 14.65 4 16.38 19.56 18.73 22.69 22.89 21.84 8 21.76 25.67 24.52 27.85 27.62 26.63 12 26.43 35.82 33.62 39.16 38.63 38.52 24 34.82 41.36 39.29 47.72 44.67 46.34 48 45.26 48.76 47.46 55.23 53.54 54.31

[0126] As shown in Table 4, the liposomes have a sustained release effect on β-sitosterol, and the cumulative release amount in 48 hours is within 60%. In general, the liposomes prepared by combining the three active ingredients in Examples 1-3 have a better sustained release effect on β-sitosterol than the liposomes prepared in Comparative Examples 3, 5-6.

[0127] 4. Cellular Uptake Assay

[0128] Test solution preparation: The liposomes prepared in Examples 1 to 3 and Comparative Examples 3, 5 to 6 were dispersed in ultrapure water to prepare an aqueous solution with a total β-sitosterol concentration of 5 mg / mL, wherein each liposome test solution was diluted with RPMI1640 culture medium to a total β-sitosterol concentration of 20 μg / mL, and the same concentration was prepared with free β-sitosterol as a control.

[0129] 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 5.

[0130] Table 5 - Uptake of β-sitosterol in liposomes in 3T3 cells (%)

[0131] Example β-Sitosterol Example 1 3.32 Example 2 3.58 Example 3 3.69 Comparative Example 3 3.28 Comparative Example 5 3.19 Comparative Example 6 3.25

[0132] As shown in Table 5, the cellular uptake rates of turmeric, orientalis, and β-sitosterol in the drug-encapsulated liposomes of all the embodiments and comparative examples were between 3.0% and 4.0%.

[0133] 5. Kidney failure treatment experiment

[0134] 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 + 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 on the left abdomen slightly below 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.

[0135] 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).

[0136] One week after administration, the urea nitrogen detection kit (Nanchang Biotech Co., Ltd.) and the creatinine detection kit (Wuhan Shengzhiyuan Biotechnology Co., Ltd.) were used to measure the blood urea nitrogen and creatinine levels of the rats, 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 6.

[0137] Table 6 - Reduction of blood urea nitrogen and creatinine in rats after liposome administration

[0138]

[0139] Compared with the single-drug component liposomes of turmeric, oriental rhizome, and beta-sitosterol of comparative examples 1-3, the two-component combination liposomes of turmeric, oriental rhizome, and beta-sitosterol prepared in comparative examples 4-6 have a more significant therapeutic effect, indicating that the two components have played a synergistic effect of inhibiting renal failure. On the other hand, the results show that the therapeutic effect of the composite liposomes of oriental rhizome and beta-sitosterol is significantly better than that of turmeric, oriental rhizome composite liposomes and turmeric, beta-sitosterol composite liposomes, indicating that the composite liposomes of oriental rhizome and beta-sitosterol have better therapeutic effect. The therapeutic effect of the three-component composite liposomes of turmeric, oriental rhizome, and beta-sitosterol of embodiment 1-3 is significantly better than that of the two-component composite liposomes, confirming that the three-drug component combination of turmeric, oriental rhizome, and beta-sitosterol of the present invention can synergistically exert better therapeutic effect. By increasing the composite ratio of β-sitosterol (i.e., the ratio of turmeric: oriental water chestnut: β-sitosterol in Example 1 is increased from 1:1:1 to 2:1:2 in Example 3), it is found that as the mass ratio of β-sitosterol increases, the therapeutic effect of the three-component composite liposome increases accordingly, indicating that β-sitosterol has a more important therapeutic effect on renal failure; when the composite ratio of β-sitosterol is further increased (i.e., the ratio of turmeric: oriental water chestnut: β-sitosterol in Example 3 is increased from 2:1:2 to 1:1:2 in Example 2), the therapeutic effect of the three-component composite liposome decreases accordingly, therefore, the preferred ratio of turmeric: oriental water chestnut: β-sitosterol is 2:1:2.

[0140] In addition, rat kidneys were obtained and sectioned for H&E staining. Figure 1 The H&E staining images of whole kidney sections of rats after treatment in normal, renal failure model group, Example 1, Example 2, Example 3, Comparative Example 1, Comparative Example 2, Comparative Example 3, Comparative Example 4, Comparative Example 5, and Comparative Example 6 are shown in sequence. The results also show that the turmeric, oriental rhizome, and β-sitosterol composite liposomes have excellent renal integrity after treatment, effectively inhibit renal cell necrosis, which is consistent with the results of blood urea nitrogen and creatinine, and has a better therapeutic effect for renal failure. Although the single component turmeric or β-sitosterol has a therapeutic effect, it is not significant.

[0141] 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 turmeric, oriental rhizome and beta-sitosterol; the mass ratio of turmeric, oriental rhizome and beta-sitosterol is (1-2):(1-2):(0.5-2).

2. The veterinary drug composition for treating pet renal failure according to claim 1, characterized in that: The mass ratio of turmeric, oriental rhizome and beta-sitosterol is (1-2):1:

2.

3. The veterinary drug composition for treating pet renal failure according to claim 2, characterized in that: The mass ratio of turmeric, oriental rhizome and beta-sitosterol is 2:1:

2.

4. A liposome, characterized in that The raw materials include the veterinary 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 composition, the lecithin, the cholesterol and the lyophilization protectant is 1:(3.75-5):(0.4-1):(3.75-5).

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 protectant is 1:5:0.4:

4.

6. The liposome according to claim 4, characterized in that The lyophilization protective agent includes at least one of glucose, sucrose and sorbitol.

7. A method for preparing liposomes according to any one of claims 4 to 6, characterized in that: The following steps are involved: S1, dissolving turmeric, oriental rhizome, β-sitosterol, cholesterol and lecithin in an organic solvent to obtain an organic phase; using water as 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 S1, the organic solvent includes at least one of ethanol, tetrahydrofuran, acetone, and methanol; In step S2, the volume ratio of the organic solvent to the aqueous phase is 1:(1-2); 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 4 to 5 hours, and then freeze drying at below -15°C for 1 to 2 hours.

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

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