Liposome containing natural extract composition as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biological medicines, and particularly discloses a liposome containing a natural extract composition as well as a preparation method and application of the liposome. The method for preparing the liposome containing the natural extract composition comprises the following steps: S1, adding the grape, poria cocos and apple extract, cholesterol and lecithin into an organic solvent, heating, stirring and dissolving to obtain an organic phase; adding the soybean extract and the ginseng extract into ultrapure water, heating and stirring to obtain a water phase; and S2, slowly adding the organic phase into the water phase while stirring, continuing stirring and emulsifying, and removing the organic solvent by reduced pressure distillation to obtain a liposome suspension. And S3, filtering the suspension, adding a freeze-drying protective additive, and freeze-drying to obtain the liposome. The invention also covers the application of the liposome in treating renal failure. In the invention, through the combination of natural extracts and the combination of a specific composition and liposome administration, the components generate a synergistic effect, and the pharmaceutical composition has a remarkable treatment effect on renal failure, especially chronic renal failure.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and particularly relates to liposomes containing natural extract compositions, their preparation methods, and applications. Background Technology

[0002] Chronic renal failure (CRF) is a substantial damage to the kidneys caused by the structural destruction and functional loss of nephrons due to various factors such as diabetes, hypertension, and glomerulonephritis. Characterized by metabolic, water, electrolyte, and acid-base disturbances, and renal atrophy, CRF is a significant public health problem worldwide. When some nephrons die, the afferent arterioles of the remaining nephrons dilate more than the efferent arterioles, leading to increased intravascular pressure (hypertension) and blood flow (hyperperfusion) in the glomerular capillaries. This high-pressure state damages the glomerular basement membrane, causing mesangial cell proliferation and increased matrix, further triggering oxidative stress, inflammatory responses, and cytokine release (especially TGF-β1), ultimately leading to glomerulosclerosis. Once one glomerulus scleroses, the remaining nephrons bear an even heavier burden, creating a vicious cycle that leads to severe renal fibrosis.

[0003] Currently, the core strategies for treating kidney failure mainly include the use of angiotensin-converting enzyme inhibitors (ACEIs) and angiotensin II receptor antagonists (ARBs), strict blood pressure control, low-protein diets, and SGLT2 inhibitors. These methods, through diuresis and antihypertensive effects, aim to delay the deterioration of kidney function and improve kidney prognosis. In addition to the above approaches, some studies have reported that certain plant extracts (such as grape extract, soybean extract, Poria cocos extract, apple extract, and ginseng extract) also have a certain ameliorative effect on some kidney disease models; however, their mechanisms of action differ from those of the target diseases. For example, existing research shows that apple extract binds to uremic toxins and excess phosphorus and potassium in the intestines and excretes them, thereby reducing the kidney's excretory burden and damage caused by toxin accumulation. However, this extract is difficult to directly act on kidney cells and cannot significantly inhibit inflammatory and necrotic pathways. The approach of treating kidney failure with natural extracts is not perfect. Although many natural extracts have been preliminarily verified to have regulatory effects, the results in in vivo experiments have been less than ideal. Currently, there are no natural extract drugs that can effectively treat kidney failure in animals.

[0004] In our previous research, we unexpectedly discovered that while apple extract alone had little effect on improving renal failure, its therapeutic effect was significantly enhanced when combined with extracts from grape, soybean, Poria cocos, and ginseng. This enhancement was particularly pronounced when the proportion of apple extract in the compound system was higher than that of the other components. This suggests that apple extract, at specific compound ratios, may synergistically interact with other components to more effectively inhibit the progression of renal failure. Based on this unexpected finding, we further investigated the impact of different extract ratios on therapeutic efficacy. Furthermore, to optimize drug distribution in vivo and improve therapeutic effects, we attempted to encapsulate the compound extracts in liposomes for delivery. However, the results showed that compared to conventional aqueous solutions, liposome delivery did not consistently demonstrate superior therapeutic effects in all compound ratios; its effectiveness significantly depended on the specific composition of the combination. It is noteworthy that, although existing literature suggests that soybean extract may be one of the most promising natural components for alleviating renal failure among the aforementioned extracts, in this study, its efficacy when administered in liposome form was significantly lower than that of the aqueous solution containing the five extracts. This seems to suggest that the simple combination of "the extract with the best effect on improving renal failure alone + liposome-optimized delivery" does not necessarily produce better results. Therefore, we believe that although liposomes, as a drug delivery system, can enhance drug bioavailability and targeting in most cases, their effect is not universally enhanced when combined with the multi-component plant extract complex system used in this study, and requires systematic evaluation and optimization based on the specific compound ratio. Summary of the Invention

[0005] To solve the above problems, the present invention adopts the following technical solution: The first aspect of this invention provides a method for preparing liposomes containing a natural extract composition, comprising the following steps: S1. Grape extract, Poria cocos extract, apple extract, cholesterol and lecithin are added to an organic solvent, heated and stirred to dissolve, and the organic phase is obtained. Separately, soybean extract and ginseng extract are added to ultrapure water, heated and stirred evenly to obtain an aqueous phase. S2. Add the organic phase to the aqueous phase while stirring. After the addition is complete, continue stirring to emulsify. Remove the organic solvent by vacuum distillation to obtain a liposome suspension. S3. Filter the liposome suspension, add a freeze-drying protectant to the filtrate, and freeze-dry to obtain liposomes containing the natural extract composition. The mass ratio of grape extract, soybean extract, poria cocos extract, apple extract, and ginseng extract is 1:(1~2):(1~2):(1~2):(1~2); the mass ratio of the natural extract composition, lecithin, cholesterol, and freeze-drying protectant is 1:(2.5~5):(0.3~1):(3.2~5), and the mass of the natural extract composition is the sum of the masses of grape extract, soybean extract, poria cocos extract, apple extract, and ginseng extract.

[0006] In some embodiments, the grape extract is Kangyue grape extract, the Poria cocos extract is Jinrun Poria cocos extract, the apple extract is Youtai apple extract, the soybean extract is Yuancai soybean extract, and the ginseng extract is Yuancai ginseng extract. Kangyue grape extract, 10:1 water extraction, extraction source: grape, single product code: sxky-pt, manufacturer: Shaanxi Kangyue Biotechnology Co., Ltd.; Runtai soybean extract, 10:1 water extraction, manufacturer: Shanxi Runtai Biotechnology Co., Ltd.; Jinrun Poria cocos extract, 10:1 water extraction, extraction source: whole Poria cocos plant, model: BC250413, product code: 0413, manufacturer: Shaanxi Jinrun Biotechnology Co., Ltd.; Youtai apple extract, 10:1 water extraction, extraction source: apple fruit, model: XYH-PG, single product code: XYH-PG-000, manufacturer: Shaanxi Xinyanghe Biotechnology Co., Ltd.; Yuancai ginseng extract, 10:1 water extraction, extraction source: ginseng, model: 001, manufacturer: Shaanxi Yuancai Biotechnology Co., Ltd.

[0007] In some methods, the mass ratio of grape extract, soybean extract, poria cocos extract, apple extract, and ginseng extract is 1:1:1:1:2; the mass ratio of the natural extract composition, lecithin, cholesterol, and freeze-drying protectant is 1:2.5:0.5:2.5; the volume ratio of organic solvent to aqueous phase in S2 is 1:(1~2); and a freeze-drying protectant of equal mass to lecithin is added in S3.

[0008] Furthermore, the organic solvent in S1 is any one of ethanol, tetrahydrofuran, acetone or methanol, preferably ethanol; the freeze-drying protectant in S3 is any one of glucose, sucrose or sorbitol, preferably glucose.

[0009] In some methods, in step S2, the reaction temperature for stirring and emulsification is 30~80℃, preferably 40℃, and the reaction time is 1~11h; it can be adjusted according to the degree of emulsification, preferably 5h; in step S3, the liposome suspension is filtered through a filter membrane with a pore size of 0.22μm, and the freeze-drying includes: pre-freezing at -80℃ for 2~6h, followed by freeze-drying at below -15℃ for 1~4h.

[0010] This invention involves dissolving cholesterol, lecithin, grape extract, poria cocos extract, and apple extract in an organic solvent to form an oil phase; and using soybean extract and ginseng extract in water as the aqueous phase. The oil phase is then added dropwise to the aqueous phase, and emulsification is achieved through stirring to form a bilayer liposome structure. In this structure, grape extract, poria cocos extract, and apple extract are encapsulated within the hydrophobic regions of the liposomes, while soybean extract and ginseng extract are encapsulated within the hydrophilic regions. Free grape extract, soybean extract, poria cocos extract, apple extract, ginseng extract, and raw materials are removed by filtration. Finally, a freeze-drying protectant is added, and the mixture is freeze-dried to obtain composite liposomes. Developing these liposomes improves the bioavailability and stability of the grape extract, soybean extract, poria cocos extract, apple extract, and ginseng extract. The liposomes co-encapsulating these extracts exhibit smaller particle size and superior dispersibility, remain stable in aqueous solution, and improve the water solubility of these extracts. Furthermore, the liposomes provide sustained-release effects, prolonging the duration of drug action. The specific amounts of lecithin and cholesterol added can be adjusted according to actual needs and are not specifically limited. In some embodiments, in step S1, an organic solvent is used to uniformly disperse cholesterol, lecithin, grape extract, soybean extract, poria cocos extract, apple extract, and ginseng extract in a system that can emulsify with the aqueous phase. This invention does not specifically limit the type of organic solvent, as long as it can effectively form a homogeneous emulsion. The specific proportions of natural extracts encapsulated in liposomes produce a synergistic effect, significantly enhancing the improvement effect on renal failure.

[0011] The second aspect of the present invention provides liposomes containing natural extract compositions prepared by any of the aforementioned methods.

[0012] Experiments were conducted on rat models, and the results showed that the therapeutic effects of using grape extract, soybean extract, poria cocos extract, apple extract, and ginseng extract alone were all weaker than the combined use of the five drugs of this invention. The combined use of the five drugs of this invention has a good effect on the recovery of kidney tissue in rats with renal failure, can effectively reduce blood urea nitrogen and creatinine levels in rats with renal failure, alleviate symptoms of renal failure, and achieve a good therapeutic effect.

[0013] Studies have found that the therapeutic effect increases with the increase of ginseng extract content in the composition. Therefore, preferably, the mass ratio of grape extract, soybean extract, poria cocos extract, apple extract, and ginseng extract is 1:1:1:1:2. Further increasing the ginseng extract content reduces the therapeutic effect; therefore, there is an optimal value for the ginseng extract content. More preferably, the mass ratio of grape extract, soybean extract, poria cocos extract, apple extract, and ginseng extract is 1:1:1:1:2. Based on the same inventive concept, this invention also provides a liposome, the raw materials of which include the above-mentioned traditional Chinese medicine composition for treating renal failure, and also include lecithin, cholesterol, and a lyophilization protectant. The mass ratio of the traditional Chinese medicine composition, lecithin, cholesterol, and lyophilization protectant is 1:(2.5~5):(0.3~1):(3.2~5). To further improve the therapeutic effect of renal failure, more preferably, the mass ratio of the traditional Chinese medicine composition, lecithin, cholesterol, and lyophilization protectant is 1:2.5:0.5:2.5.

[0014] The third aspect of the present invention provides the use of any of the aforementioned liposomes containing natural extract compositions in the preparation of a medicament for treating renal failure in pets, or is understood as the use of liposomes containing natural extract compositions in the preparation of a veterinary drug for treating renal failure.

[0015] In some embodiments, the drug for treating pet kidney failure is a drug for treating pet chronic kidney failure, and the liposome containing a natural extract composition is primarily capable of significantly improving the treatment of pet chronic kidney failure.

[0016] A fourth aspect of the present invention provides the use of any of the aforementioned compositions containing natural extracts in reducing the severity of renal failure in animal models of renal failure; wherein, reducing the severity of renal failure in animal models of renal failure is any one of the following: A. Reduces the degree of renal tubular dilation; B. Reduces renal tubular epithelial cell necrosis; C. Reduces interstitial inflammatory cell infiltration; D. Reduces kidney atrophy.

[0017] When used in animal models of renal failure, liposomes can be used to control certain variables in the model, such as alleviating renal failure symptoms and assessing the adverse effects of other substances on renal failure. Other usage methods may also be employed as needed for clinical or research purposes.

[0018] In some methods, the animal model of kidney failure is a chronic kidney failure animal model.

[0019] In this disclosure, a combination of natural extracts, administered via liposomes in a specific composition, produces a synergistic effect, demonstrating significant therapeutic efficacy against renal failure, particularly chronic renal failure. Animal models have validated the effectiveness of this liposome composition containing natural extracts in treating chronic renal failure. It significantly improves renal pathological damage in model animals, showing intact glomerular structure, orderly tubular arrangement, and a marked reduction in interstitial inflammation and fibrosis, in stark contrast to control groups. In some cases, no significant necrosis, inflammation, or fibrosis was observed compared to the normal control group, indicating a significant therapeutic effect and demonstrating its clear renal protective and tissue repair effects. Attached Figure Description

[0020] Figure 1 Images of kidneys taken out one week after treatment of a rat model of renal failure with liposomes prepared for the embodiments and comparative examples of the present invention (A, Example 1; B, Example 2; C, Example 3; D, Comparative Example 6).

[0021] Figure 2 shows the H&E staining of whole kidney sections after one week of treatment with liposomes prepared in the embodiments and comparative examples of the present invention on a rat model of renal failure. Detailed Implementation

[0022] The following specific embodiments further illustrate the liposomes containing natural extract compositions disclosed herein, their preparation methods, and applications. This section further illustrates the content of the invention with reference to specific embodiments, but should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the embodiments 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.

[0023] I. Product Preparation Raw materials: Grape extract (Kangyue, 10:1 water extraction, extraction source: grape, single product code: sxky-pt, manufacturer: Shaanxi Kangyue Biotechnology Co., Ltd.), soybean extract (Runtai, 10:1 water extraction, manufacturer: Shanxi Runtai Biotechnology Co., Ltd.), Poria cocos extract (Jinrun, 10:1 water extraction, extraction source: whole Poria cocos plant, model: BC250413, product code: 0413, manufacturer: Shaanxi Jinrun Biotechnology Co., Ltd.), apple extract (Youtai, 10:1 water extraction, extraction source: apple fruit, model: XYH-PG, single product code: XYH-PG-000, manufacturer: Shaanxi Xinyanghe Biotechnology Co., Ltd.), ginseng extract (Yuancai, 10:1 water extraction, extraction source: ginseng, model: 001, manufacturer: Shaanxi Yuancai Biotechnology Co., Ltd.).

[0024] This embodiment provides a method for preparing liposomes containing grape extract, soybean extract, poria cocos extract, apple extract, and ginseng extract as pharmacologically active ingredients, including the following steps: S1. Add 20 mg of grape extract, 20 mg of Poria cocos extract, 20 mg of apple extract, 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 the organic phase; Separately, add 40 mg of soybean extract and 20 mg of ginseng extract to 20 mL of ultrapure water, heat to 50 °C and stir until homogeneous to obtain the aqueous phase; S2. Under 50℃ water bath conditions, the organic phase is added to the aqueous phase while stirring. After the addition is complete, stirring is continued for 4 hours to emulsify. Then, the ethanol is removed by vacuum distillation to obtain the liposome suspension. 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, freeze-dry, and obtain liposomes containing the traditional Chinese medicine composition of the present invention (grape extract, soybean extract, poria extract, apple extract, ginseng extract).

[0025] In Example 1, the mass ratio of grape extract, soybean extract, poria cocos extract, apple extract, and ginseng extract in the traditional Chinese medicine composition is 1:2:1:1:1, and the mass ratio of the traditional Chinese medicine composition, lecithin, cholesterol, and freeze-drying protectant is 1:2.5:1:2.5.

[0026] Example 2 This embodiment provides a method for preparing liposomes containing grape extract, soybean extract, poria cocos extract, apple extract, and ginseng extract as pharmacologically active ingredients, including the following steps: S1. Add 20 mg of grape extract, 20 mg of Poria cocos extract, 40 mg of apple extract, 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 the organic phase; Separately, dissolve 20 mg of soybean extract and 20 mg of ginseng extract in 20 mL of ultrapure water, heat to 50 °C, and set as the aqueous phase; S2. Under 50℃ water bath conditions, the organic phase is added to the aqueous phase while stirring. After the addition is complete, stirring is continued for 4 hours to emulsify. Then, the ethanol is removed by vacuum distillation to obtain the liposome suspension. 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, freeze-dry, and obtain liposomes containing the traditional Chinese medicine composition of the present invention (grape extract, soybean extract, poria extract, apple extract, ginseng extract).

[0027] Example 3 This embodiment provides a method for preparing liposomes containing grape extract, soybean extract, poria cocos extract, apple extract, and ginseng extract as pharmacologically active ingredients, including the following steps: S1. Add 20 mg of grape extract, 20 mg of apple extract, 20 mg of Poria cocos extract, 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 the organic phase; Separately, take 20 mg of soybean extract, 40 mg of ginseng extract, and 20 mL of ultrapure water, heat to 50 °C, and set as the aqueous phase; S2. Under 50℃ water bath conditions, the organic phase is added to the aqueous phase while stirring. After the addition is complete, stirring is continued for 4 hours to emulsify. Then, the ethanol is removed by vacuum distillation to obtain the liposome suspension. 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, freeze-dry, and obtain liposomes containing the traditional Chinese medicine composition of the present invention (grape extract, soybean extract, poria extract, apple extract, ginseng extract).

[0028] In Example 3, the mass ratio of grape extract, soybean extract, poria cocos extract, apple extract, and ginseng extract in the traditional Chinese medicine composition is 1:1:1:1:2, and the mass ratio of the traditional Chinese medicine composition, lecithin, cholesterol, and freeze-drying protectant is 1:2.5:0.5:2.5.

[0029] Comparative Example 1 This comparative example provides a method for preparing liposomes containing grape extract as a pharmacologically active ingredient, including the following steps: S1. Add 120 mg of grape extract, 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 the organic phase; Separately, take 20 mL of ultrapure water and heat to 50 °C to prepare the aqueous phase. S2. Under 50℃ water bath conditions, the organic phase is added to the aqueous phase while stirring. After the addition is complete, stirring is continued for 4 hours to emulsify. Then, the ethanol is removed by vacuum distillation to obtain the liposome suspension. S3. The liposome suspension was filtered through a 0.22 μm pore size filter membrane. 300 mg of glucose was added to the filtrate. After freeze-drying, liposomes containing grape extract as the active ingredient were obtained.

[0030] Comparative Example 2 This comparative example provides a method for preparing liposomes containing soybean extract as a pharmacologically active ingredient, including the following steps: S1. Add 60 mg cholesterol and 300 mg lecithin to 20 mL anhydrous ethanol, heat to 50 °C and stir to dissolve, obtaining the organic phase; Separately, dissolve 120 mg soybean extract in 20 mL ultrapure water, heat to 50 °C, and set as the aqueous phase; S2. Under 50℃ water bath conditions, the organic phase is added to the aqueous phase while stirring. After the addition is complete, stirring is continued for 4 hours to emulsify. Then, the ethanol is removed by vacuum distillation to obtain the liposome suspension. S3. The liposome suspension was filtered through a 0.22 μm pore size filter membrane. 300 mg of glucose was added to the filtrate. After freeze-drying, liposomes containing soybean extract as the active ingredient were obtained.

[0031] Comparative Example 3 This comparative example provides a method for preparing liposomes containing Poria cocos extract as the active ingredient, including the following steps: S1. Add 120 mg of Poria cocos extract, 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 the organic phase; Separately, take 20 mL of ultrapure water and heat to 50 °C to prepare the aqueous phase. S2. Under 50℃ water bath conditions, the organic phase is added to the aqueous phase while stirring. After the addition is complete, stirring is continued for 4 hours to emulsify. Then, the ethanol is removed by vacuum distillation to obtain the liposome suspension. S3. The liposome suspension was filtered through a 0.22 μm pore size filter membrane. 300 mg of glucose was added to the filtrate. After freeze-drying, liposomes containing Poria cocos extract as the active ingredient were obtained.

[0032] Comparative Example 4 This comparative example provides a method for preparing liposomes containing apple extract as a pharmaceutically active ingredient, including the following steps: S1. Add 120 mg of apple extract, 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 the organic phase; Separately, take 20 mL of ultrapure water and heat to 50 °C to prepare the aqueous phase. S2. Under 50℃ water bath conditions, the organic phase is added to the aqueous phase while stirring. After the addition is complete, stirring is continued for 4 hours to emulsify. Then, the ethanol is removed by vacuum distillation to obtain the liposome suspension. S3. The liposome suspension was filtered through a 0.22 μm pore size filter membrane. 300 mg of glucose was added to the filtrate. After freeze-drying, liposomes containing apple extract as the active ingredient were obtained.

[0033] Comparative Example 5 This comparative example provides a method for preparing liposomes containing ginseng extract as a pharmacologically active ingredient, including the following steps: S1. Add 60 mg cholesterol and 300 mg lecithin to 20 mL anhydrous ethanol, heat to 50 °C and stir to dissolve, obtaining the organic phase; Separately, dissolve 120 mg ginseng extract in 20 mL ultrapure water, heat to 50 °C, and set as the aqueous phase; S2. Under 50℃ water bath conditions, the organic phase is added to the aqueous phase while stirring. After the addition is complete, stirring is continued for 4 hours to emulsify. Then, the ethanol is removed by vacuum distillation to obtain the liposome suspension. S3. The liposome suspension was filtered through a 0.22 μm pore size filter membrane. 300 mg of glucose was added to the filtrate. After freeze-drying, liposomes containing ginseng extract as the active ingredient were obtained.

[0034] Comparative Example 6 This comparative example provides a method for preparing a mixture of grape extract, apple extract, ginseng extract, soybean extract, and poria cocos extract, comprising the following steps: mixing 20 mg of grape extract, 20 mg of poria cocos extract, 20 mg of apple extract, 60 mg of cholesterol, 300 mg of lecithin, 20 mg of soybean extract, 40 mg of ginseng extract, and 300 mg of glucose.

[0035] Table 1 shows the raw material composition and content of Examples 1-3 and Comparative Examples 1-6 of the present invention.

[0036] Table 1. Composition and content of liposome raw materials in Examples 1-3, Comparative Examples 1-5, and the mixture in Comparative Example 6. Note: " / " indicates that it has not been added.

[0037] II. Performance Testing This test case aims to evaluate the performance of liposomes or single-drug liposomes prepared in the examples and comparative examples.

[0038] 1. Drug loading and encapsulation efficiency of composite liposomes The content of the active ingredient pamoate was determined by high performance liquid chromatography (HPLC). A C18 column (purchased from Agilent Technologies, catalog number 993967-902) was used as the mobile phase, with a volume ratio of acetonitrile and water of 80:20. The injection volume was 10 μL, and the detection wavelength for pamoate was 210 nm. Drug loading was calculated as (mass of drug in lyophilized liposome powder / mass of compound lyophilized liposome powder) × 100%; encapsulation efficiency was calculated as (mass of drug in lyophilized liposome powder / mass of drug fed) × 100%. The corresponding results are shown in Table 2.

[0039] Table 2 - Results of Drug Loading and Encapsulation Efficiency Measurement As can be seen from Table 2, the liposomes prepared in Example 2 of this invention have a higher pachymic acid encapsulation efficiency than those in Example 1, Example 3, and Comparative Example 3.

[0040] 2. Particle size and potential testing The liposomes prepared in the examples and comparative examples were dispersed in ultrapure water to prepare an aqueous solution of 10 mg / mL. The particle size and potential of the liposomes were determined by dynamic light scattering method. Each sample was measured three times, and the average value was calculated. The corresponding results are shown in Table 3.

[0041] Table 3 - Results of liposome particle size and potential determination As shown in Table 3, the average particle size of the liposomes prepared in this invention is in the range of 100-200 nm, the potential is negative, and the polydispersity index is less than 0.3, indicating that the prepared liposomes have a narrow particle size distribution and high dispersion stability. Among them, the liposomes prepared in Examples 1-3 have smaller particle sizes, which may be due to the interaction between various drug components, resulting in a more compact liposome structure.

[0042] 3. In vitro drug release behavior Preparation of pH 7.4 release solution: Weigh 0.26 g potassium dihydrogen phosphate and 2.16 g disodium hydrogen phosphate dodecahydrate, add them to a beaker, add 1 liter of ultrapure water and 5 g Tween-80, stir to dissolve, and then adjust the pH to 7.4.

[0043] In vitro drug release experiment: The liposomes prepared in Examples 1-3 and Comparative Example 3 were dispersed in ultrapure water to prepare an aqueous solution of 50 mg / mL. 1 mL of each solution was added to a dialysis bag, which was then immersed in 50 mL of the prepared pH 7.4 release solution. The bag was placed in a constant-temperature shaker at 37°C and 180 rpm. 3 mL of the release solution was collected 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. The release experiment was performed in triplicate for each release solution, and the average value was calculated. The concentration of the active ingredient in the collected release solutions was determined by high-performance liquid chromatography (HPLC), and the average cumulative release was calculated as: Cumulative release = (Mass of drug in the release solution / Mass of drug in the added liposomes) × 100%. The results are shown in Table 4.

[0044] Table 4 - Results of cumulative release (%) of pachymic acid in liposomes As shown in Table 4, the liposomes exhibited a sustained-release effect on pachymic acid, with the cumulative release amount remaining below 60% after 48 hours. Overall, the liposomes used in combination with the three pharmacodynamic components in Examples 1-3 demonstrated a superior sustained-release effect on pachymic acid compared to the liposomes prepared in Comparative Example 3.

[0045] 4. Cell uptake assay Test solution preparation: The liposomes prepared in Examples 1-3 and Comparative Example 3 were dispersed in ultrapure water to prepare an aqueous solution with a total pachymic acid concentration of 5 mg / mL. Each liposome test solution was diluted with RPMI 1640 medium to a total pachymic acid concentration of 20 μg / mL.

[0046] Cell uptake assay: 3T3 cells were seeded at a density of 100,000 cells per well in 12-well plates and cultured for 48 h. Then, the cells were incubated with the above-prepared test solutions for 4 h. The cells were lysed and the content of pamoate acid was determined by HPLC. Three parallel groups were tested for each sample, and the average uptake was calculated. The corresponding results are shown in Table 5.

[0047] Table 5 - Uptake of pamoate in 3T3 cells via liposomes (ug / 10) 5 cell) As shown in Table 5, the liposomes prepared in Examples 1-3 of the present invention have a higher cellular uptake than those in Comparative Example 3. This may be because the liposomes prepared in Examples 1-3 of the present invention have smaller particle sizes and are more easily phagocytosed by cells.

[0048] 5. Experimental Treatment of Kidney Failure Establishment of a renal failure model: 1g of adenine was dissolved in 40mL of physiological saline to prepare a suspension with a concentration of 2.5%. The suspension was administered to rats by gavage at a dose of 200mg / (kg·d) daily. Rats had free access to normal feed and water. The modeling period was 30 days. On the evening of day 29, rats were fasted for 12 hours. On the morning of day 30, 5mL of blood was collected, centrifuged, and serum was separated to determine serum creatinine and blood urea nitrogen levels.

[0049] Drug treatment: After modeling, liposomes prepared in the examples and comparative examples were administered via gavage at a total dose of 50 mg / kg. The combined drug treatment group had a total dose of 50 mg / kg for all drugs. Five mice were in each group (n=5). One week after administration, blood urea nitrogen and creatinine levels in rats were measured using a urea nitrogen assay kit (Nanchang Baxter Biotechnology Co., Ltd.) and a creatinine assay kit (Wuhan Shengzhiyuan Biotechnology Co., Ltd.). The average percentage reduction in blood urea nitrogen and creatinine levels in each group was calculated as follows: Average percentage reduction = (Saline group level - Drug treatment group level) × 100% / Saline group level; the corresponding results are shown in Table 6.

[0050] Table 6 - Decrease in blood urea nitrogen and creatinine levels in rats after liposome administration Compared with the single-component liposomes of grape extract, apple extract, soybean extract, ginseng extract, and poria cocos extract prepared in Comparative Examples 1-5, the five-component combination liposomes of grape extract, apple extract, soybean extract, ginseng extract, and poria cocos extract prepared in Examples 1-3 showed more significant therapeutic effects, confirming that the combined use of the five-component combination of grape extract, apple extract, soybean extract, ginseng extract, and poria cocos extract can synergistically exert a better therapeutic effect. Although the mixture of grape extract, apple extract, soybean extract, ginseng extract, and poria cocos extract in Comparative Example 6 also showed some therapeutic effect on renal failure, the effect was significantly lower than that of the liposomes composed of grape extract, apple extract, soybean extract, ginseng extract, and poria cocos extract, indicating that the liposomes have a superior therapeutic effect. By optimizing the proportion of each drug component, the optimal mass ratio of grape extract, soybean extract, poria cocos extract, apple extract, and ginseng extract was found to be 1:1:1:1:2; in addition, rat kidneys were used. Figure 1 The images show rat kidneys after treatment in the normal group, the renal failure model group, Example 1 (A), Example 2 (B), and Example 3 (C). The results also showed that the kidneys treated with the combined liposomes of grape extract, apple extract, soybean extract, ginseng extract, and Poria cocos extract exhibited good morphology, indicating effective inhibition of renal cell necrosis. This was consistent with the results of blood urea nitrogen and creatinine levels, demonstrating a better therapeutic effect on renal failure than Comparative Example 6 (D), showcasing the superiority of liposomes.

[0051] In addition, rat kidneys were harvested and H&E sections were stained. Figure 2 The images sequentially show H&E staining images of whole kidney sections from rats after treatment in the normal group, the renal failure model group, Examples 1, 2, 3, Comparative Examples 1, 2, 3, 4, 5, and 6. The results also showed that the composite liposomes of grape extract, apple extract, soybean extract, ginseng extract, and poria cocos extract exhibited excellent renal integrity after treatment, and the effect was significantly stronger than the mixture of grape extract, apple extract, soybean extract, ginseng extract, and poria cocos extract. The composite liposomes effectively inhibited renal cell necrosis, consistent with the results of blood urea nitrogen and creatinine, demonstrating a better therapeutic effect on renal failure. While the single-component grape extract, apple extract, soybean extract, ginseng extract, and poria cocos extract showed therapeutic effects, they were not significant. This further indicates that the components in the composite liposomes can exert a better synergistic therapeutic effect.

[0052] like Figure 2 As shown in Table 7, Examples 1-3 all demonstrated significant efficacy in the treatment of renal failure. Compared with the renal failure model group, the experimental group showed significant improvement in renal histopathology, specifically a significant reduction in renal tubular dilation, decreased renal tubular epithelial cell necrosis, and an increased number of structurally intact renal tubules. Interstitial inflammatory cell infiltration was significantly reduced, and tissue edema was largely resolved. Although some traces of damage, such as flattening of renal tubular epithelial cells, were still visible, the overall renal structure was effectively repaired. In contrast, the treatment effects of Comparative Examples 1-6 were significantly inferior to those of the Example group. Although Comparative Example 1 showed some improvement, such as a reduction in renal tubular damage and interstitial inflammation compared to the model group, the degree and thoroughness of the improvement were not as good as those of the Example group, with many dilated renal tubules or residual inflammatory foci remaining. The treatment effects of Comparative Examples 2-6 were weak or insignificant, and the renal pathological changes were similar to those of the renal failure model group, with obvious renal tubular dilation, epithelial cell necrosis, and interstitial fibrosis / inflammation still visible, which contrasted sharply with the Example group. Furthermore, we can see that Example 3 has the most significant effect, even comparable to the normal group, with a higher content of ginseng extract compared to the other four, which seems to indicate that ginseng extract plays a more important role in improving renal failure. However, Comparative Example 5 shows the opposite result, with its effect being significantly inferior to that of Comparative Examples 1 and 2. This seems to indicate that only when combined with the other four extracts and the content of ginseng extract is higher than that of the other components can it have a more significant effect in alleviating renal failure.

[0053] Table 7 - Quantitative data of H&E staining of whole kidney sections after one week of treatment with liposomes prepared in the examples and comparative examples in a rat model of renal failure. In summary, while single plant extracts may possess some renal protective effects, the results of Comparative Examples 1-5 indicate that the efficacy of a single component remains limited even at high doses. Example 3, employing a specific formulation (relatively high ginseng extract content, with other extracts balanced), combined with liposome technology, demonstrated significantly superior therapeutic effects compared to single components or simple physical mixtures. Notably, Example 3 exhibited the most significant improvement in a renal failure model, with kidney tissue section results virtually indistinguishable from the normal group. The comparison between Example 3 and Comparative Example 6 further highlights the crucial role of formulation technology (i.e., liposome encapsulation). This technology not only enhances the bioavailability of the active ingredient but may also improve overall efficacy through a synergistic effect. Therefore, specific component ratios and liposome formulations may be the core factors achieving this remarkable effect, and its specific mechanism of action requires further in-depth research.

[0054] Those skilled in the art will appreciate that various modifications can be made to the above embodiments without departing from the overall spirit and concept of the present invention. For any aspects not detailed herein, reference can be made to the prior art. All such modifications fall within the protection scope of the present invention. The protection scheme of the present invention is defined by the appended claims.

Claims

1. A method for preparing liposomes containing a natural extract composition, characterized in that, Includes the following steps: S1. Grape extract, Poria cocos extract, apple extract, cholesterol and lecithin are added to an organic solvent, heated and stirred to dissolve, and the organic phase is obtained. Separately, soybean extract and ginseng extract are added to ultrapure water, heated and stirred evenly to obtain an aqueous phase. S2. Add the organic phase to the aqueous phase while stirring. After the addition is complete, continue stirring to emulsify. Remove the organic solvent by vacuum distillation to obtain a liposome suspension. S3. Filter the liposome suspension, add a freeze-drying protectant to the filtrate, and freeze-dry to obtain liposomes containing the natural extract composition. The mass ratio of grape extract, soybean extract, poria cocos extract, apple extract, and ginseng extract is 1:(1~2):(1~2):(1~2):(1~2); the mass ratio of the natural extract composition, lecithin, cholesterol, and freeze-drying protectant is 1:(2.5~5):(0.3~1):(3.2~5), and the mass of the natural extract composition is the sum of the masses of grape extract, soybean extract, poria cocos extract, apple extract, and ginseng extract.

2. The method for preparing liposomes containing a natural extract composition according to claim 1, characterized in that, The grape extract is Kangyue grape extract, the poria extract is Jinrun poria extract, the apple extract is Youtai apple extract, the soybean extract is Yuancai soybean extract, and the ginseng extract is Yuancai ginseng extract.

3. The method for preparing liposomes containing a natural extract composition according to claim 1, characterized in that, The mass ratio of grape extract, soybean extract, poria cocos extract, apple extract, and ginseng extract is 1:1:1:1:2; the mass ratio of the natural extract composition, lecithin, cholesterol, and freeze-drying protectant is 1:2.5:0.5:2.5; the volume ratio of organic solvent to aqueous phase in S2 is 1:(1~2); and a freeze-drying protectant of equal mass to lecithin is added in S3.

4. The method for preparing liposomes containing a natural extract composition according to claim 3, characterized in that, The organic solvent in S1 is any one of ethanol, tetrahydrofuran, acetone or methanol, preferably ethanol; the freeze-drying protectant in S3 is any one of glucose, sucrose or sorbitol, preferably glucose.

5. The method for preparing liposomes containing a natural extract composition according to claim 1, characterized in that, In step S2, the reaction temperature for stirring and emulsification is 30~80℃, preferably 40℃, and the reaction time is 1~11h; it can be adjusted according to the degree of emulsification, preferably 5h; in step S3, the freeze-drying includes: pre-freezing at -80℃ for 2~6h, followed by freeze-drying at below -15℃ for 1~4h, and filtering the liposome suspension through a filter membrane with a pore size of 0.22μm.

6. Liposomes containing natural extracts prepared by the method for preparing liposomes containing natural extract compositions according to any one of claims 1 to 5.

7. Application of liposomes containing natural extract compositions in the preparation of drugs for treating renal failure in pets.

8. The application according to claim 7; wherein, The medication mentioned is for treating chronic kidney failure in pets.

9. Application of liposomes containing natural extracts in reducing the severity of renal failure in animal models; among which, To reduce the severity of renal failure in animal models of renal failure, any of the following can be achieved: Reduce the degree of renal tubular dilation; Reduce renal tubular epithelial cell necrosis; Reduce interstitial inflammatory cell infiltration; Reduce kidney atrophy.

10. The application according to claim 9; wherein, The animal model of renal failure is a chronic renal failure animal model.

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