Baicalin yeast microcapsule with detoxification function as well as preparation method and application of baicalin yeast microcapsule

Abstract

The invention belongs to the field of biological pharmacy, and particularly relates to a baicalin yeast microcapsule with a detoxifying function as well as a preparation method and application of the baicalin yeast microcapsule. The baicalin-loaded cationic liposome and the yeast cell wall are prepared through film hydration and acid-base-organic preparation, and the baicalin-loaded cationic liposome and the yeast cell wall are subjected to electrostatic self-deposition to form the baicalin yeast microcapsule. Experiments show that the baicalin yeast microcapsule provided by the invention can improve the damage of toxins to the body by means of improving renal fibrosis and body inflammatory response, promoting toxin discharge and the like. The invention provides a nano-carrier improvement strategy for low bioavailability of the baicalin, and provides a scientific basis for improving the renal injury caused by ochratoxin A by the baicalin.

Description

Technical Field

[0001] The present invention relates to the field of drug delivery system development, and specifically relates to a baicalin yeast microcapsule with detoxification efficacy, a preparation method and an application thereof. Background Art

[0002] Ochratoxin A (OTA) is one of the most widespread mycotoxins globally, second only to aflatoxin in its harmful effects. It affects cereals such as wheat and corn, beans and bean products, oilseed crops, spices, nuts, tea, grapes and wine, beer, cocoa and its products, and coffee. OTA also has multiple harmful effects on the human body, including nephrotoxicity, hepatotoxicity, neurotoxicity, and immunotoxicity. The kidneys are its primary target organ and metabolic site. OTA is considered one of the most potent renal carcinogenic mycotoxins known and is closely associated with the development of Balkan nephropathy.

[0003] Baicalin (BA) is a natural flavonoid compound. In recent years, research on baicalin has become increasingly intense in the medical field. Studies have shown that baicalin exhibits multiple biological activities, including antioxidant, anti-inflammatory, anti-tumor, immunomodulatory, antibacterial, and antidepressant properties. Furthermore, baicalin has demonstrated pharmacological protective effects on various organs and tissues, including the liver, kidneys, and nerves. Research on baicalin provides important scientific support for the further development and utilization of the traditional Chinese medicine Scutellaria baicalensis, and also provides a crucial theoretical foundation for its drug development and clinical application.

[0004] Liposomes are nanoscale vesicles composed of a phospholipid bilayer. Due to their unique biocompatibility, degradability, and low immunogenicity, they have become an important research direction for drug delivery systems. In the 1990s, the first liposome drug Liposomes containing doxorubicin (doxorubicin liposomes) received FDA approval for use in cancer treatment. This breakthrough laid the foundation for the clinical application of liposomes as drug carriers. Since then, liposomes have been widely used in anticancer, antifungal, vaccine delivery, and gene therapy. The development of various liposome types, including conventional liposomes, long-circulating liposomes, targeted liposomes, immunoliposomes, and stimulus-responsive liposomes, has significantly enhanced their potential for drug delivery.

[0005] While liposomes offer numerous advantages in drug delivery, their lack of digestion resistance has limited current research on liposome-based drug delivery to intraperitoneal or intravenous injection, hindering large-scale deployment. However, the yeast cell wall, with its unique porous structure and digestion stability, offers unique advantages for oral drug delivery, opening up the possibility of oral delivery for non-digestion-resistant liposomes. Furthermore, β-1,3-glucan, a major component of the yeast cell wall, can activate the immune system and enhance drug absorption. Mannans and mannan proteins on the yeast cell wall surface interact with liposomes, and the porous structure allows liposomes to penetrate the yeast cell wall through pores through electrostatic adsorption and self-deposition, both of which increase drug encapsulation efficiency and enhance drug delivery. As an emerging drug delivery strategy, yeast cell wall-encapsulated liposomes combine biocompatibility, cost-effectiveness, safety, and digestion resistance, providing strong support for the development of novel precision delivery systems, particularly for oral delivery. Summary of the Invention

[0006] The purpose of the present invention is to provide a yeast microcapsule with fungal toxin detoxification effect, which has good toxicity barrier performance in in vivo experimental verification and can be used in the medical clinical field.

[0007] The purpose of the present invention is achieved through the following technical solutions:

[0008] The first aspect of the present invention provides a baicalin yeast microcapsule, which comprises baicalin cationic liposomes and yeast cell walls; wherein the mass ratio of the baicalin cationic liposomes to the yeast cell walls is 8 to 12:1; and the molar ratio of lecithin to baicalin in the baicalin cationic liposomes is 3 to 5:1.

[0009] Furthermore, the mass ratio of the baicalin cationic liposomes to the yeast cell wall is 10:1; and the molar ratio of lecithin to baicalin in the baicalin cationic liposomes is 4:1.

[0010] The second aspect of the present invention provides a method for preparing the above-mentioned baicalin yeast microcapsules, comprising the following steps:

[0011] (1) Preparing a baicalin lipid material mixture: dissolving lecithin, cholesterol, trimethyl-2,3-dioleoyloxypropylammonium bromide (DOTAP), and baicalin in a round-bottom flask filled with chloroform to obtain a baicalin lipid material mixture;

[0012] (2) rotary evaporating the organic solvent of the baicalin lipid material mixed liquid obtained in step (1) at 37° C. to form a thin film;

[0013] (3) After removing the O2 in the bottle with N2, ultrapure water was added and shaken thoroughly to hydrate; the liposomes were disrupted by a cell disruptor to make the particle size distribution uniform, and finally the liposome emulsion was filtered through a filter membrane to obtain the liposome emulsion, which was stored in a refrigerator at 4°C until use;

[0014] (4) removing yeast cytoplasm using an acid-base-organic reagent method and freeze-vacuum drying to obtain yeast cell walls;

[0015] (5) suspending the dried yeast cell walls obtained in step (4) in a carbonate buffer solution and incubating the suspension, and then adding the baicalin cationic liposome emulsion obtained in step (3); after incubation, centrifuging and collecting the liposome-loaded yeast cell walls; the obtained product is the yeast microcapsule.

[0016] Furthermore, in step (1), the molar ratio of lecithin, cholesterol, trimethyl-2,3-dioleoyloxypropylammonium bromide (DOTAP) and baicalin is 3-5:2:3-5:1; preferably, the molar ratio is 4:2:4:1.

[0017] Furthermore, the temperature of the rotary evaporation in step (2) is 35-40°C, preferably 37°C;

[0018] Furthermore, the operation of step (4) is as follows: suspending the yeast cell pellet collected by centrifugation in NaOH, and heating the resulting suspension at 75-85°C for 0.5-1.5h, centrifuging at 2000-2500g for 10-20min to collect a sample, rinsing it twice with deionized water, and then dispersing it in a pH 4.5 HCl solution, and incubating it at 50-60°C for 0.5-1.5h; collecting the yeast sample after centrifugation, and washing it thoroughly with deionized water; then, rinsing the obtained sample with isopropanol 3-5 times, and then washing it with acetone twice, collecting the yeast sample after washing with deionized water, and freeze-drying it in a vacuum to obtain yeast cell walls;

[0019] Furthermore, in the step (5), the amount of baicalin cationic liposome emulsion added is such that the mass of the cationic liposomes contained therein is 8 to 12:1 of the yeast cell wall; wherein, the solution to which the baicalin cationic liposome emulsion is added is incubated at 35 to 40° C. for 10 to 14 hours; preferably, the amount of baicalin cationic liposome emulsion added is such that the mass of the cationic liposomes contained therein is 10:1 of the yeast cell wall; wherein, the solution to which the baicalin cationic liposome emulsion is added is incubated at 37° C. for 12 hours.

[0020] The third aspect of the present invention provides the use of the above-mentioned baicalin yeast microcapsules in the preparation of a drug for treating body damage caused by ochratoxin A.

[0021] Furthermore, the body damage caused by ochratoxin A includes kidney damage and / or liver damage.

[0022] The fourth aspect of the present invention is to provide use of the baicalin yeast microcapsules described in the first aspect in the preparation of a drug delivery system.

[0023] Furthermore, the drugs include small molecule chemical drugs, protein drugs, and nucleic acid drugs.

[0024] Compared with the prior art, the present invention has the following advantages:

[0025] 1. The thin-film hydration method used in this study to prepare liposomes offers advantages over other liposome preparation methods, including high encapsulation efficiency, ease of operation, and wide applicability. Different ratios of phosphatidylcholine to baicalin were used to prepare cationic liposomes, and the optimal ratio was determined by dialysis and liquid phase encapsulation efficiency measurements. Furthermore, by measuring the potential of cationic liposomes in different aqueous phase systems, the optimal liposome storage and subsequent yeast microcapsule synthesis system were determined, which is beneficial for improving the encapsulation efficiency of subsequent yeast microcapsules.

[0026] 2. The present invention utilizes electrostatic self-deposition of cationic liposomes onto yeast cell walls to create a drug delivery system with digestive stability and high bioavailability. Compared to the digestive resistance of liposomes alone, this bilayer drug delivery system overcomes the shortcomings of liposomes. Furthermore, the biodegradability, biocompatibility, digestive stability, scalability, and culturability of yeast cell walls make yeast microencapsulated baicalin delivery possible.

[0027] 3. Baicalin has multiple biological activities such as anti-inflammatory, anti-tumor, and antioxidant. It significantly improves the pathological process of acute kidney injury and renal fibrosis by regulating oxidative stress, inflammation, apoptosis, fibrosis, and metabolic abnormalities through the synergistic effects of multiple targets and pathways. Its multiple regulatory mechanisms provide new strategies and research directions for the prevention and treatment of acute kidney injury and chronic kidney disease. The target organ of ochratoxin A is the kidney, and excessive exposure to ochratoxin A can induce severe acute kidney injury in the body. Baicalin, with the help of yeast microcapsules, demonstrates efficient detoxification performance, providing a new scientific basis for the quality of mycotoxins. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 is the TEM image of liposomes;

[0029] Figure 2 is the liposome particle size and zeta potential;

[0030] Figure 3 FTIR spectra of baicalin, empty liposomes and liposomes loaded with baicalin;

[0031] Figure 4 is the XRD pattern of liposomes loaded with baicalin;

[0032] Figure 5 is the dialysis time and encapsulation efficiency of liposomes;

[0033] Figure 6 The effect of different placement systems on the liposome potential;

[0034] Figure 7 Yeast cells in different states;

[0035] Figure 8 Preparation and optimization of yeast microcapsules;

[0036] Figure 9 TEM images of yeast cell wall and yeast microcapsules;

[0037] Figure 10 Results of free fatty acid release from yeast microcapsules and liposomes simulating gastrointestinal digestion;

[0038] Figure 11 Effects of empty yeast microcapsules, baicalin, liposomes loaded with baicalin, and baicalin yeast microcapsules on body weight, food intake, and organ indexes of mice induced by ochratoxin A;

[0039] Figure 12 H&E pathological examination of kidney of mice infected with ochratoxin A by empty yeast microcapsules, baicalin, liposomes loaded with baicalin, and yeast microcapsules loaded with baicalin;

[0040] Figure 13 Immunohistochemical examination of Kim-1 in the kidneys of mice infected with ochratoxin A by empty yeast microcapsules, baicalin, liposomes loaded with baicalin, and baicalin yeast microcapsules;

[0041] Figure 14 To test the key genes of renal fibrosis and Masson pathology in mice induced by ochratoxin A by empty yeast microcapsules, baicalin, liposomes loaded with baicalin, and baicalin yeast microcapsules;

[0042] Figure 15 This study examined the effects of empty yeast microcapsules, baicalin, liposomes loaded with baicalin, and baicalin yeast microcapsules on key inflammatory genes in mice induced by ochratoxin A and conducted F4 / 80 pathological tests. DETAILED DESCRIPTION

[0043] The following is a further description of the concept of the present invention and the technical effects produced in conjunction with specific embodiments, so as to fully understand the purpose, features and effects of the present invention. The methods described are all conventional methods unless otherwise specified. The materials described can be obtained from public commercial channels unless otherwise specified. The illustrative embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute undue limitations of the present invention. It should be noted that, unless there is a conflict, the embodiments of the present invention and the features in the embodiments can be combined with each other.

[0044] Example 1: Preparation, Optimization and Characterization of Baicalin Cationic Liposomes

[0045] 1. Experimental methods

[0046] (1) Lecithin, cholesterol, trimethyl-2,3-dioleoyloxypropylammonium bromide (DOTAP), and baicalin were dissolved in a round-bottom flask filled with chloroform at a molar ratio of 4:2:4:1 to obtain a baicalin lipid material mixture.

[0047] (2) The obtained mixed liquid was subjected to rotary evaporation of the organic solvent at 37°C to form a thin film.

[0048] (3) After removing the O2 in the bottle with N2, add ultrapure water and shake thoroughly to hydrate. Disrupt the liposomes using a cell disruptor to achieve a uniform particle size distribution, and finally filter through a filter membrane to obtain a liposome emulsion.

[0049] (4) Only the ratio of lecithin to baicalin was changed, and the molar ratios of lecithin to baicalin were selected as 4:1, 5:1, and 6:1 to prepare baicalin cationic liposomes. The encapsulation efficiency of baicalin was measured to determine the optimal molar ratio of lecithin to baicalin for the preparation of baicalin liposomes.

[0050] (5) The obtained liposomes were placed in pure water, 1× PBS aqueous solution, and carbonate buffer (0.1 mol / L) to measure the particle size, PDI dispersion coefficient, and potential of the liposomes to determine the placement system of the liposomes. Generally speaking, the larger the absolute value of the potential, the more stable the liposomes.

[0051] (6) The optimized liposomes were subjected to particle size, PDI, Zeta potential, TEM, Fourier transform infrared and X-ray diffraction measurements.

[0052] 2. Experimental results

[0053] (1) Cationic liposomes with uniform dispersion and particle size of about 200 nm were prepared by thin film hydration method. The morphology of liposomes was analyzed by Malvern particle size analyzer and transmission electron microscopy (TEM). Figure 1 TEM images and Figure 2 The particle size, PDI and Zeta potential of the liposomes were shown in the TEM images. The average particle size of the empty liposomes was 228.2 nm, the PDI was 0.171 and the Zeta potential was 25.5 Mv ( Figure 2 A). After entrapment of baicalin, the particle size of the liposomes slightly increased to 233.4 nm, the PDI was 0.181, and the Zeta potential was 24.1 mV, which was basically the same as that of the empty liposomes ( Figure 2 B). Figure 3 The FTIR spectra of BA, FCL, and FCL-BA are shown. The baicalin BA spectrum exhibits characteristic peaks at 3388.317 cm-1 (OH stretch) and 1726.46 cm-1 (C=O stretch). Liposome FCL exhibits characteristic peaks at 2931.27 cm-1 and 2857.988 cm-1 (CH stretch bands of long fatty acid chains), and 1742.37 cm-1 (C=O stretch). Liposome FCL-BA encapsulating baicalin exhibits characteristic peaks at 2933.198 cm-1 and 2857.024 cm-1 (CH stretch bands of long fatty acid chains), and at 3415.797 cm-1 (OH stretch). Compared to empty liposomes, the characteristic peaks of baicalin are shifted, indicating weak interactions between baicalin and liposomes, such as hydrogen bonding or hydrophobic interactions. Figure 4 The XRD pattern of the liposomes is shown. The diffraction peaks at low angles may indicate that the liposomes have a lamellar liquid crystal structure, a common liposome structure that indicates an orderly arrangement of lipid molecules within the membrane. Furthermore, the narrow diffraction peaks indicate a high degree of order in the liposomes.

[0054] (2) The optimal liposome encapsulation efficiency was obtained by changing the phosphorus-drug ratio and storage system. Figure 5 The dialysis time for measuring the encapsulation efficiency was determined. Long dialysis time can easily lead to rupture of liposomes, causing the substances encapsulated in the liposomes to be released, thus reducing the encapsulation efficiency. Therefore, the appropriate dialysis time is the key factor to ensure the accuracy of the encapsulation efficiency. Figure 5 As shown in A, the encapsulation efficiency decreased after dialysis for more than 9 h, which may be because the stirring time was too long, causing the liposomes to rupture and the baicalin encapsulated in the liposomes to be released. Therefore, the more stable 3 h was selected as the optimal dialysis time. Figure 5 B shows that different ratios of lecithin: baicalin have a great influence on the encapsulation efficiency of baicalin. When the ratio of lecithin: baicalin = 4:1, the encapsulation efficiency is the highest, reaching 92.29%; as the ratio further increases, the encapsulation efficiency shows a downward trend. Figure 6The results show that different storage systems have a significant impact on the potential of liposomes. However, because studies have shown that the greater the absolute value of the potential, the more stable the liposomes are, we chose carbonate buffer as the storage solution for liposomes and the preparation system for subsequent materials.

[0055] Example 2: Preparation, Optimization and Characterization of Baicalin Yeast Microcapsules

[0056] 1. Experimental content and methods

[0057] (1) Yeast cell culture

[0058] The yeast cell culture process involves preparing the culture medium, inoculating, and culturing. Take 50g of YPD culture medium powder and add it to 1L of pure water. Sterilize at 121°C for 15 minutes to ensure sterility. Remove 1g of Saccharomyces cerevisiae powder and inoculate the yeast into the cooled YPD culture medium using a sterile technique (e.g., an inoculating loop). Incubate the culture medium on a shaker at 30°C for 12 hours. Separate the yeast cells from the culture medium by centrifugation (3000g, 10 minutes) to prepare for subsequent cell wall treatment.

[0059] (2) Preparation of yeast cell wall

[0060] Yeast samples were suspended in 1 mol / L NaOH using the acid-base-organic reagent method. The resulting suspension was heated at 80°C for 1 hour and then centrifuged at 2200g for 15 minutes. The sample was then rinsed twice with deionized water and dispersed in a pH 4.5 HCl solution and incubated at 55°C for 1 hour. After centrifugation, the yeast sample was collected and thoroughly washed with deionized water. Subsequently, the sample was rinsed four times with 40 mL of isopropanol and twice with acetone. After washing with deionized water, the yeast sample was collected and freeze-dried in a vacuum oven to obtain the yeast cell wall.

[0061] (3) Preparation of baicalin yeast microcapsules

[0062] Dried yeast cell walls were suspended in carbonate buffer (pH 9.2) and incubated at 37°C. An optimized baicalin cationic liposome emulsion was then added. The amount of baicalin cationic liposome solution added was such that the mass ratio of cationic liposomes to yeast cell walls was 10:1, resulting in a cationic liposome solution. After incubation at 37°C, the liposome-loaded yeast cell walls were collected by centrifugation. The resulting product was yeast microcapsules.

[0063] (4) Optimization of baicalin yeast microcapsules

[0064] The encapsulation efficiency, which refers to the ratio of the encapsulated substance in the yeast microcapsules to the total substance in the added liposomes, is an important indicator for evaluating yeast microcapsule quality. The yeast cell wall to liposome mass ratio and incubation time were varied, with yeast cell wall to liposome ratios of 1:10, 1:5, and 1:1 selected. Yeast microcapsules were prepared at 4, 6, 8, 10, 12, and 14 hours. The baicalin concentration in the supernatant of the incubation supernatant at different times and ratios was measured to determine the optimal preparation time and yeast cell wall to liposome ratio for Scutellaria yeast microcapsules.

[0065] (5) Characterization of baicalin yeast microcapsules

[0066] The morphology of the yeast microcapsules was observed using a transmission electron microscope. The specific operation was as follows: 2 μL of the prepared sample was dropped onto a silicon wafer, dried in an oven, and then the morphology of the yeast microcapsules was observed using a transmission electron microscope.

[0067] 2. Experimental results

[0068] (1) Preparation of yeast cell wall

[0069] In this study, the acid-base-organic reagent method was used to prepare yeast cell walls. Figure 7 A shows the normal morphology of yeast cells. The cells are oval or ovoid, with obvious cell walls and cytoplasm, and may contain granules or vesicles inside, which is a common phenomenon in yeast cells during normal growth. Figure 7 Figure B shows the partial removal of cytoplasm after treatment with an acid-base-organic reagent. Although the cell morphology retains some integrity, the intracellular contents are significantly reduced. The cell wall remains intact and retains its original shape, but cavities or irregular structures may appear within the cell, indicating that the cytoplasm has been partially removed. Figure 7 C shows the state after the cytoplasm is completely removed. The cell wall remains intact, but there is no longer any visible cytoplasm inside. The cell wall presents a more hollow structure, indicating that after the cytoplasm is removed, only the cell wall remains in the cell.

[0070] (2) Preparation and optimization of baicalin yeast microcapsules

[0071] Figure 8 A is the potential of the yeast cell wall in carbonate buffer and the cationic liposomes loaded with baicalin. The yeast cell wall is negatively charged at -10.09±0.91 mV, and the cationic liposomes are positively charged at 27.03±0.87 mV. The absolute value of the potential difference between the two is 37 mV, which indicates that the liposomes can pass through the pores of the yeast cell wall through electrostatic adsorption and enter the cell wall to form yeast microcapsules.

[0072] Figure 8Figure B shows the encapsulation efficiency of yeast microcapsules prepared with different incubation times and different mass ratios of liposomes to yeast cell walls. When incubated for longer than 12 hours, the encapsulation efficiency remained essentially unchanged, so subsequent experiments used a 12-hour, 37°C temperature to prepare yeast microcapsules. A higher proportion of yeast cell wall resulted in higher encapsulation efficiencies, but with increasing incubation time, the encapsulation efficiencies remained essentially the same across different ratios. Therefore, all subsequent experiments used a yeast cell wall:liposome ratio of 1:10.

[0073] (3) Characterization of baicalin yeast microcapsules

[0074] The yeast microcapsule suspension encapsulating liposomes was prepared by using the optimized yeast cell wall to liposome mass ratio and the optimal incubation time. The morphological characteristics of yeast microcapsules and yeast cell walls were characterized by transmission electron microscopy (TEM). Figure 9 As shown. Figure 9 As can be observed in A, the yeast cell wall after cytoplasm removal appears gray under TEM imaging, indicating that the high-energy electron beam can penetrate the sample, proving that the cytoplasm has been basically removed. Figure 9 In B, after adding liposomes, the sample appears dark black, indicating that the high-energy electron beam cannot penetrate the structure, indicating that the liposomes have successfully entered and filled the interior of the yeast cell wall. Figure 9 Compared with A, Figure 9 The surface of the yeast microcapsule in B changed from a smooth surface to a rough surface, indicating that some liposomes were electrostatically adsorbed on the periphery of the yeast cells. However, because the liposomes were positively charged, they were more likely to interact with biological barriers such as the skin, thereby enhancing the biological function of the yeast microcapsule.

[0075] Example 3: Anti-digestion stability of baicalin yeast microcapsules

[0076] 1. Experimental content and methods

[0077] (1) Drawing of the standard curve for free fatty acid determination

[0078] Prepare a 100 μM palmitic acid standard solution by adding 20 μL of palmitic acid standard solution (1 mM) to 180 μL of assay buffer. Add 0, 5, 10, 20, 30, 40, and 50 μL of each solution to a 96-well plate and add assay buffer to the final volume of 50 μL. Add 2 μL of enzyme mix B to each well, mix thoroughly, and incubate at 30°C in the dark for 30 minutes. Then, add 50 μL of the free fatty acid assay working solution (44 μL of assay buffer, 2 μL of Amplex Red, 2 μL of enzyme mix A, and 2 μL of enhancer), mix thoroughly, and incubate at 37°C in the dark for 30 minutes. Measure the absorbance at 570 nm and plot a standard curve.

[0079] (2) Simulated gastrointestinal digestion stability test

[0080] Monitoring the free fatty acid content directly reflects liposome membrane disruption, thereby assessing membrane integrity and stability. Samples (10 μL) were added to 190 μL of simulated gastric fluid (SGF, containing 2 mg / mL NaCl, 3.2 mg / mL pepsin, pH = 1.5) and incubated at 37°C for 2 h. Samples were removed and analyzed at different time points (0, 5, 15, 30, 60, and 120 min). Following SGF digestion, the digested samples were added to simulated intestinal fluid (SIF, containing 6.8 mg / mL K₂HPO₃, 0.2 mg / mL bile salts, 8.8 mg / mL NaCl, 3.2 mg / mL pancreatin, pH = 7.4) at a volume ratio of 1:19 and stirred at 37°C for 4 h. The free fatty acid content of the samples was measured at predetermined time intervals (0, 10, 30, 60, 120, 180, and 240 min) to assess the gastrointestinal digestion stability of the samples.

[0081] 2. Experimental results

[0082] According to the Amplex Red free fatty acid kit steps, palmitic acid standard, free fatty acid buffer and working solution were added in sequence. The sample absorbance was measured at a wavelength of 570 nm. The free fatty acid concentration was used as the horizontal axis and the absorbance as the vertical axis. The free fatty acid standard curve was drawn by linear regression, as shown in the following figure: Figure 10 As shown in A, the obtained free fatty acid standard curve equation is: y = 0.0105x + 0.1334, and the correlation coefficient R 2 =0.9936, indicating that baicalin has good linearity in the concentration range of 0-100 μM, and this equation can be used to calculate the content of free fatty acids.

[0083] Liposomes and yeast microcapsules were digested at 37°C for 0, 5, 15, 30, 60, and 120 min, and the digestion stability of the samples was determined by measuring the free fatty acid content in the samples. Figure 10 As shown in Figure B, in the early stage of simulated gastric digestion, the free fatty acid content released by yeast microcapsules and liposomes was relatively low. After 30 minutes, the free fatty acid content of both increased significantly, but the free fatty acid content of the yeast microcapsule group was significantly lower than that of the liposome group. This also confirms that the yeast cell wall has good digestion stability and improves the anti-digestion ability of the encapsulated substance.

[0084] Next, the digestive fluid after gastric digestion was added to the simulated intestinal fluid, and the liposomes and yeast microcapsules were digested at 37°C for 10, 30, 60, 120, 180, and 240 minutes. The free fatty acid content in the sample was determined as follows: Figure 10As shown in Figure C, in the early stage of simulated intestinal digestion, the free fatty acid content increased significantly compared to that in the stomach, which was mainly attributed to the interference of bile salts on the bilayer structure of liposomes, resulting in reduced liposome stability. In addition, the action of digestive enzymes such as pancreatic lipase accelerated the degradation of liposomes, making the encapsulated active substances easier to release. However, the free fatty acid content of yeast microcapsules was higher than that of liposomes overall, and after 180 minutes, the free fatty acid content of the two tended to be consistent. It is worth noting that although the digestibility of yeast microcapsules in intestinal fluid is lower than that in the gastric fluid environment, its "stomach stability-intestinal release" digestion mode can effectively prevent the premature degradation of baicalin in the stomach and reduce the drug's irritation to the stomach. This result further confirms the protective effect of the yeast cell wall on liposomes in the early stage of intestinal digestion, making the sustained release of baicalin in the intestinal environment possible.

[0085] Example 4: Restorative effect of baicalin yeast microcapsules on OTA-induced mouse phenotype

[0086] 1. Experimental content and methods

[0087] (1) Experimental animal grouping and treatment

[0088] Before the formal experiment began, the mice were fed with sterile water for one week. After one week, the mice were randomly divided into six groups, with six mice in each group, and gavage was performed for seven consecutive days. The specific grouping and dosing were as follows:

[0089] ① Blank control group (CK): 6 rats, fed with maintenance feed, normal drinking water, and gavage with sterile water at a volume of 0.1 mL / 10 g each time;

[0090] ② Model group (OTA): 6 mice were fed a maintenance diet and given OTA at a dose of 4 mg / kg, once a day at a volume of 0.1 mL / 10 g.

[0091] ③Baicalin group (BA): 6 mice were fed with maintenance diet and gavage with OTA at a dose of 4 mg / kg and baicalin at a concentration of 20 mg / kg, respectively, at a volume of 0.1 mL / 10 g, once a day;

[0092] ④ Baicalin-liposome group (F-BA): 6 mice were fed a maintenance diet and given OTA at a dose of 4 mg / kg by gavage. The concentration of baicalin in the liposomes encapsulating baicalin was 20 mg / kg. The mice were gavaged once a day at a volume of 0.1 mL / 10 g.

[0093] ⑤ Baicalin-yeast microcapsule group (YF-BA): 6 mice were fed a maintenance diet and administered OTA at a dose of 4 mg / kg. The concentration of baicalin in the yeast cell wall encapsulated by liposomes was 20 mg / kg. The mice were administered OTA at a volume of 0.1 mL / 10 g once daily.

[0094] ⑥ Empty group (YF): 6 mice, fed with maintenance diet, gavage with OTA at a dose of 4 mg / kg, yeast cell walls wrapped in empty liposomes, at a volume of 0.1 mL / 10 g, once a day;

[0095] (2) Body weight measurement

[0096] During the experiment, the body weight and average food intake of mice were measured every day, and the body weight and food intake change curves were drawn based on this.

[0097] (3) Organ index measurement

[0098] After blood was collected from the eye sockets, the mice were killed by cervical dislocation and autopsied. The kidneys and livers of the mice were removed and weighed.

[0099] 2. Experimental results

[0100] like Figure 11 As shown in Figure A, the weight changes of mice in the different treatment groups showed different trends. Baicalin, baicalin-liposomes, and baicalin-yeast microcapsules all alleviated OTA-induced weight loss to a certain extent, and the differences were statistically significant (p < 0.001). This result suggests that baicalin may partially counteract the growth inhibitory effects of OTA by improving metabolism or enhancing adaptive responses.

[0101] like Figure 11 As shown in Figure B, OTA intake did not significantly affect the food intake of mice, indicating that OTA-induced weight loss may be mainly attributed to nutrient metabolism disorders or organ dysfunction rather than the effects of feeding behavior. Figure 11 As shown in Figure C, OTA significantly reduced the renal coefficient of mice (p<0.05), while the intake of baicalin could effectively alleviate this phenomenon, suggesting that baicalin may have a certain renal protective effect. Figure 11 As shown in Figure D, OTA treatment significantly increased the liver coefficient of mice (p < 0.05), while baicalin intake effectively inhibited this change. This result may be related to the OTA-induced liver inflammatory response, and baicalin may exert liver protection through anti-inflammatory or detoxification mechanisms. In addition, Figure 11 As shown in Figure E, OTA treatment led to a significant increase in the kidney / liver coefficient of mice (p<0.05), while the intake of baicalin alleviated this change to some extent, further supporting its potential protective role in OTA-induced organ damage.

[0102] In summary, baicalin may alleviate OTA-induced weight loss and liver and kidney damage in mice by regulating metabolic balance, reducing organ damage or improving nutrient utilization efficiency.

[0103] Example 5: Effects of baicalin yeast microcapsules on OTA-induced tissue pathology

[0104] 1. Experimental content and methods

[0105] (1) Hematoxylin and Eosin (H&E) staining

[0106] The liver and kidney fixed with 4% paraformaldehyde were dehydrated, embedded, paraffin-sectioned, and stained with hematoxylin-eosin. The tissue sections were observed under a microscope (Laica, Germany, DM2500).

[0107] (2) Immunohistochemical staining

[0108] Immunofluorescence staining of signaling molecules in cells is performed to visually display their expression levels and tissue localization. The specific steps of the process are as follows

[0109] 1. Dewaxing: The sections were treated with xylene, anhydrous ethanol, and graded alcohol in sequence and then washed with distilled water.

[0110] 2. Antigen retrieval: Place the sections in citric acid buffer and microwave-repair them at medium heat for 8 minutes, then stop at medium heat for 8 minutes and then at medium-low heat for 7 minutes. After cooling, wash with PBS.

[0111] 3. Blocking: Incubate with 3% hydrogen peroxide solution for 25 minutes, then wash with PBS.

[0112] 4. Circle drawing: Shake dry and slice, circle the tissue with a tissue pen.

[0113] 5. Serum blocking: add 3% BSA and incubate at room temperature for 30 minutes.

[0114] 6. Add primary antibody: shake off the blocking solution, add primary antibody, and incubate in a humidified box at 4°C overnight.

[0115] 7. Add secondary antibody: After washing with PBS, add secondary antibody and incubate in the dark for 50 minutes.

[0116] 8. DAB color development and counterstaining: After washing with PBS, add DAB color developer, control the color development, wash with water, counterstain with hematoxylin, differentiate with hydrochloric acid and alcohol, and turn blue with ammonia water.

[0117] 9. Dehydration and sealing: After treatment with gradient alcohol and xylene, seal the slides with neutral gum and observe and photograph under a microscope.

[0118] 2. Experimental results

[0119] In order to more significantly observe the damage of OTA to mouse organs and the restorative effect of baicalin and the drug delivery system on the body, H&E staining and Kim-1 immunohistochemistry were further performed to visually reflect the changes in the kidneys. Figure 12 As shown in H&E pathological sections, significant pathological changes were observed in the renal tissue of mice treated with OTA. These changes included partial glomerular collapse, capillary plexus atrophy, and basement membrane thickening. Tubular epithelial cells showed varying degrees of degeneration and necrosis, with marked tubular luminal dilation and the formation of hyaline casts. Furthermore, interstitial inflammatory cell infiltration was prominent, accompanied by early features of fibrosis. These histological abnormalities indicate that OTA induces severe renal damage, primarily manifested by impaired glomerular function and tubular lesions. Further analysis revealed that, compared with the OTA-treated group, the BA, FCL-BA, and YF-BA-treated groups all exhibited varying degrees of relief from pathological damage, with the FCL-BA and YF-BA groups exhibiting particularly significant protective effects. In both groups, glomerular structural integrity was significantly restored, tubular necrosis was significantly reduced, and interstitial inflammatory cell infiltration was reduced, resulting in histological manifestations more similar to those of normal CK. These results suggest that FCL-BA and YF-BA may possess a more potent renal protective effect than BA and YF, effectively alleviating OTA-induced renal damage. H&E staining directly reflects the overall changes in the structure of renal tissue. Immunohistochemistry further reflects the expression of proteins characteristic of renal injury. For example, Kim-1 is a transmembrane glycoprotein with extremely low expression in healthy renal tissue. However, its expression level is significantly upregulated when proximal renal tubular epithelial cells are damaged. Therefore, the effect of baicalin and drug delivery system on the alleviation of OTA-induced renal injury can be evaluated from the perspective of Kim-1 immunohistochemistry. Figure 13 As shown, OTA caused a significant increase in Kim-1 in the mouse kidneys, while intervention with BA, FCL-BA, and YF-BA reduced Kim-1 in the kidneys. Furthermore, YF-mediated treatment did not alleviate the increase in Kim-1 content caused by OTA. This suggests that baicalin plays a role in alleviating OTA-induced kidney damage, and the drug delivery system is merely a "booster" for baicalin's effects.

[0120] Example 6: Effects of baicalin yeast microcapsules on OTA-induced renal fibrosis

[0121] 1. Experimental content and methods

[0122] (1) Extraction of kidney RNA

[0123] Kidney tissue samples were removed from -80°C. 0.05 g of mouse liver tissue was placed in a sterilized, RNase-free tube soaked in DEPC water. 600 μL of Trizol solution was added and the tissue was homogenized using a grinder (70 Hz, 90 s). After grinding, an additional 400 μL of Trizol solution was added. RNA was then extracted by adding chloroform in a 5:1 ratio of Trizol to chloroform. The mixture was centrifuged at 12,000 g and 4°C for 15 min with vigorous shaking. The supernatant was transferred to a new EP tube, and isopropanol was added in a 1:1 ratio. The mixture was thoroughly mixed, allowed to stand at -20°C for 100 min, and then centrifuged at 12,000 g at 4°C for 15 min. The supernatant was discarded, and the EP tube was inverted on clean toilet paper and allowed to stand for 1 min. Add 600 μL of 0.1% DEPC-containing ethanol to an EP tube, mix thoroughly, and centrifuge at 12,000 g and 4°C for 15 minutes. Remove the supernatant and dry the remaining residue. Add an appropriate amount of RNase-free water preheated in a 60°C metal bath, pipette thoroughly, and store in a -80°C freezer.

[0124] (2) Reverse transcription and fluorescence quantitative PCR

[0125] RNA concentration was determined using Nanodrop, and RNA quality was considered good when the 260 / 280 value was between 1.8 and 2.0. cDNA was processed using the HiScript IV RT SuperMix for qPCR reverse transcription kit. The mRNA level of the target gene was detected using the FastKing One-Step RT-PCR Kit. GAPDH was selected as the internal reference gene for normalization, and 2 -△△ The relative gene expression levels were calculated using the Ct method. The primer sequences used in this study were obtained from the official website of the primer library (https: / / pga.mgh.harvard.edu / Parabiosys / ) and are shown in Table 1.

[0126] Table 1 RT-qPCR sequences

[0127]

[0128] (3) Masson's staining of tissue

[0129] Masson's Trichrome Stain (Masson) staining is suitable for the differential analysis of collagen and smooth muscle and is widely used to assess the degree of renal fibrosis. In this study, paraffin sections of renal tissue were dewaxed and stained using the Masson's Trichrome staining kit, and photographed under a pathological microscope.

[0130] 2. Experimental results

[0131] After acute kidney injury (AKI), if tissue repair mechanisms are impaired or damage persists, the kidneys may undergo a fibrotic process, ultimately leading to a progressive decline in renal function. Even if renal function recovers temporarily, fibrosis may still affect renal function after long-term intervention. In the study of renal fibrosis, α-SMA, E-cad, and FN are key marker genes used to assess myofibroblast activation, epithelial-mesenchymal transition (EMT), and extracellular matrix (ECM) deposition, respectively, thereby revealing the pathogenesis of fibrosis. α-SMA is a characteristic protein of myofibroblasts, and its expression is significantly upregulated during EMT, marking the progression of fibrosis. E-cad, an epithelial cell-specific adhesion protein, is downregulated during EMT, indicating a loss of epithelial cell polarity and a transition to a mesenchymal phenotype, thereby promoting fibrosis. FN is a major component of the ECM, and its upregulation reflects excessive ECM accumulation, further driving fibrosis. Changes in the expression of these three genes are interrelated and can serve as molecular indicators of fibrosis progression, providing a key basis for systematically assessing the severity of renal fibrosis and its underlying mechanisms.

[0132] like Figure 14 As shown in Figures AC, the expression of fibrosis marker genes in the renal tissues of mice treated with OTA showed significant changes compared to those in the CK group (p < 0.001), suggesting that OTA induces renal fibrosis. However, the expression levels of α-SMA and FN in the YF, BA, FCL-BA, and YF-BA treatment groups were not significantly different from those in the CK group, indicating that these treatments alleviated OTA-induced renal fibrosis to some extent, with the YF-BA group showing a particularly significant alleviating effect. Furthermore, although E-cad expression remained somewhat different in the YF, BA, and FCL-BA treatment groups compared to the CK group (p < 0.05), its expression levels recovered compared to the OTA group, further suggesting that these treatments have a regulatory effect on the EMT process, with the YF-BA group showing the most significant effect.

[0133] In addition, Figure 14 The results showed that OTA-induced renal fibrosis in mice was significant, and administration of the drug delivery system YF alone did not significantly improve the degree of fibrosis. However, under the mediation of BA, FCL-BA, and YF-BA, renal fibrosis was improved to varying degrees.

[0134] In summary, baicalin and its drug delivery system treatment may not only have a strong renal protective effect, but also is superior to the baicalin alone group in alleviating OTA-induced renal fibrosis, showing a more significant anti-fibrotic potential.

[0135] Example 7: Effect of baicalin yeast microcapsules on OTA-induced renal inflammatory response

[0136] 1. Experimental content and methods

[0137] The method used in this example to determine relative gene expression levels was the same as in Example 6, and the immunohistochemistry method for F4 / 80 was the same as the Kim-1 immunohistochemistry method in Example 5. The primer sequences used in this study are shown in Table 2.

[0138] Table 2 RT-qPCR sequences

[0139]

[0140]

[0141] 2. Experimental results

[0142] The inflammatory response is a physiological process designed to protect the body from acute harmful stimuli. In the ochratoxin (OTA)-induced toxin model, immune cells in the kidney, such as resident dendritic cells and macrophages, respond to OTA-induced kidney damage by actively secreting pro-inflammatory cytokines such as IL-6 and IL-1β. These cytokines can act on nearby blood vessels to recruit more white blood cells to the damaged area, help clear possible pathogens, repair damaged kidney tissue, and attempt to restore kidney homeostasis. However, the persistent inflammatory response caused by OTA may lead to further damage to kidney function and aggravate long-term kidney damage and fibrosis.

[0143] like Figure 15 As shown in AD, the expression of inflammatory marker genes in the renal tissue of mice in the OTA-treated group changed significantly compared with that in the CK group, suggesting that toxin exposure led to an inflammatory response in the kidneys. However, in the BA, FCL-BA and YF-BA treatment groups, the differences in the expression levels of TNF-α, IL-6, IL-1β, and IL-8 compared with the CK group and the differences between the OTA group and the CK group were all reduced, indicating that these treatments alleviated the renal inflammatory response caused by OTA to a certain extent. Among them, the alleviating effect of the yeast microcapsule (YF-BA) group was particularly significant. The expression levels of the four genes TNF-α, IL-6, IL-1β, and IL-8 in this group all returned to the levels of the CK group, while the expression levels of the four inflammatory marker genes in the empty liposome-yeast microcapsule (YF) group were significantly different from those in the CK group. This suggests that baicalin, rather than the liposomes and yeast cell walls that encapsulate baicalin, plays a major role in the mechanism of alleviating renal injury. However, comparing the results of the BA group with those of the F-BA and YF-BA groups, it can be seen that the alleviating effect was better after the addition of the drug delivery system, which suggests that although the delivery carrier alone will not significantly alleviate the inflammatory response, the "1+1>2" effect is achieved after being loaded with baicalin.

[0144] In addition, the effects of baicalin and drug delivery system on OTA-induced renal inflammation were evaluated from the perspective of immunohistochemistry in pathological sections (e.g. Figure 15 E) OTA significantly increased F4 / 80-positive cells in the mouse kidneys, whereas BA, FCL-BA, and YF-BA treatments reduced F4 / 80-positive cells in the kidneys. Furthermore, YF-mediated treatment did not alleviate the OTA-induced increase in macrophages. These results suggest that baicalin, rather than the drug delivery system alone, is the primary alleviating factor in OTA-induced renal inflammation.

[0145] In summary, existing evidence supports the potential of baicalin for renal protection, as well as the use of liposomes and yeast cell walls for drug delivery. However, direct evidence for the specific technology combination described by the user was not found. This technology may represent an innovative drug delivery system that combines the pharmacological effects of baicalin with the targeting properties of microcapsules. Future studies are needed to further validate its feasibility and efficacy.

[0146] The embodiments described above are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work shall fall within the scope of protection of the present invention.

Claims

1. A baicalin yeast microcapsule, characterized in that: The liposome comprises baicalin cationic liposomes and yeast cell walls; the mass ratio of the baicalin cationic liposomes to the yeast cell walls is 8-12:1; and the molar ratio of lecithin to baicalin in the baicalin cationic liposomes is 3-5:

1.

2. The baicalin yeast microcapsule according to claim 1, characterized in that The mass ratio of the baicalin cationic liposomes to the yeast cell wall is 10:1; and the molar ratio of lecithin to baicalin in the baicalin cationic liposomes is 4:

1.

3. A method for preparing the baicalin yeast microcapsules according to claim 1 or 2, characterized in that: The method comprises the following steps: (1) Preparing a baicalin lipid material mixture: dissolving lecithin, cholesterol, trimethyl-2,3-dioleoyloxypropylammonium bromide (DOTAP), and baicalin in a round-bottom flask filled with chloroform to obtain a baicalin lipid material mixture; (2) rotary evaporating the organic solvent from the baicalin lipid material mixed liquid obtained in step (1) to form a thin film; (3) After removing the O2 in the bottle with N2, ultrapure water was added and shaken thoroughly to hydrate; the liposomes were disrupted by a cell disruptor to make the particle size distribution uniform, and finally the liposome emulsion was filtered through a filter membrane to obtain the liposome emulsion, which was stored in a refrigerator at 4°C until use; (4) removing yeast cytoplasm using an acid-base-organic reagent method and freeze-vacuum drying to obtain yeast cell walls; (5) suspending the dried yeast cell walls obtained in step (4) in a carbonate buffer solution and incubating the suspension, and then adding the baicalin cationic liposome emulsion obtained in step (3); after incubation, centrifuging and collecting the liposome-loaded yeast cell walls; the obtained product is the yeast microcapsule.

4. The method according to claim 3, characterized in that in, In step (1), the molar ratio of lecithin, cholesterol, trimethyl-2,3-dioleoyloxypropylammonium bromide (DOTAP) and baicalin is 3-5:2:3-5:

1.

5. The method according to claim 3, characterized in that The temperature of the rotary evaporation in step (2) is 35-40°C.

6. The method according to claim 3, characterized in that The amount of baicalin cationic liposome emulsion added in step (5) is such that the mass of the cationic liposome contained therein is 8-12:1 of the mass of the yeast cell wall; and the solution to which the baicalin cationic liposome emulsion is added is incubated at 35-40° C. for 10-14 hours.

7. Use of the baicalin yeast microcapsules according to claim 1 or 2 or the baicalin yeast microcapsules prepared by the method according to any one of claims 3 to 6 in the preparation of a drug for treating body damage caused by ochratoxin A.

8. The use according to claim 7, characterized in that The body damage caused by ochratoxin A includes kidney damage and / or liver damage.

9. Use of the baicalin yeast microcapsules according to claim 1 or 2 or the baicalin yeast microcapsules prepared by the method according to any one of claims 3 to 6 in preparing a drug delivery system.

10. The use according to claim 9, characterized in that The drugs include small molecule chemical drugs, protein drugs, and nucleic acid drugs.

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