Beta-lactoglobulin-procyanidine covalent complex as well as preparation method and application thereof
The preparation of β-lactoglobulin-proanthocyanidin covalent complexes by a mild alkaline treatment method solves the problems of low stability and low bioavailability of β-lactoglobulin and proanthocyanidins when used alone, and achieves effective prevention and treatment of non-alcoholic fatty liver disease.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-26
- Publication Date
- 2026-03-13
AI Technical Summary
In existing technologies, β-lactoglobulin and proanthocyanidins, when used alone, suffer from poor stability and low bioavailability, making them difficult to effectively prevent and treat non-alcoholic fatty liver disease (NAFLD) and related lipid metabolism disorders.
A covalent complex of β-lactoglobulin and proanthocyanidins was prepared by a mild alkaline treatment method with a mass ratio of 5:1. The reaction conditions were mild and easy to operate. The resulting complex exhibited excellent anti-NAFLD activity both in vitro and in vivo.
This complex significantly inhibits lipid deposition in hepatocytes in vitro, regulates cholesterol and triglyceride levels, and comprehensively improves the NAFLD model induced by a high-fat diet in vivo, reducing hepatic lipid deposition and serum lipid indicators, with better effects than β-lactoglobulin alone.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of biomedical technology, specifically relating to a β-lactoglobulin-proanthocyanidin covalent complex, its preparation method, and the use of the complex in the prevention and / or treatment of non-alcoholic fatty liver disease (NAFLD) and related lipid metabolism disorders. Background Technology
[0002] Nonalcoholic fatty liver disease (NAFLD) is a chronic liver disease closely related to metabolic abnormalities, characterized by excessive lipid deposition in hepatocytes, excluding other known liver-damaging factors such as alcohol. The NAFLD spectrum includes simple fatty liver, nonalcoholic steatohepatitis, liver fibrosis, and even cirrhosis, seriously threatening public health. Free fatty acid (FFA)-induced hepatocyte lipid deposition models are commonly used in vitro models for studying the pathogenesis of NAFLD and for drug screening, effectively simulating the lipotoxic state during the disease process.
[0003] β-lactoglobulin (BLG) and proanthocyanidins (PC) are both natural bioactive substances with anti-inflammatory, antioxidant, and lipid metabolism-regulating functions, respectively. However, their individual use suffers from poor stability and low bioavailability. Constructing a complex (BLG-PC) through covalent bonding can enhance their physicochemical stability and biological activity, resulting in a synergistic effect.
[0004] Currently, there are no reports on the preparation of covalent complexes of β-lactoglobulin and proanthocyanidins via alkaline treatment and their use in the prevention and treatment of NAFLD. Summary of the Invention
[0005] This invention aims to provide a β-lactoglobulin-proanthocyanidin covalent complex, its preparation method, and its applications. This complex is prepared via a mild alkaline treatment process, exhibits well-defined composition and structural characteristics, and demonstrates excellent anti-NAFLD activity in both in vitro and in vivo models.
[0006] Furthermore, the mass ratio of β-lactoglobulin to proanthocyanidins in the complex is 5:1.
[0007] Furthermore, the complex is prepared by an alkaline treatment method, which has mild reaction conditions and is easy to operate.
[0008] Furthermore, the complex showed no significant inhibitory effect on the growth of AML12 hepatocytes at a concentration of 40 μg / mL (based on β-lactoglobulin), indicating that it has no obvious cytotoxicity within this concentration range and possesses good cell compatibility.
[0009] Furthermore, the complex significantly inhibits lipid deposition in AML12 hepatocytes induced by free fatty acids and reduces intracellular total cholesterol (TC) and triglyceride (TG) levels.
[0010] Furthermore, in a high-fat diet-induced NAFLD model in C57BL / 6 mice, the complex comprehensively improved serum lipid levels and reduced hepatic lipid deposition, demonstrating superior therapeutic efficacy compared to β-lactoglobulin alone. Attached Figure Description
[0011] Figure 1 The fluorescence spectrum of β-lactoglobulin and its complex prepared in Example 1 is shown.
[0012] Figure 2 This is a graph showing the CCK8 assay results of AML12 cell viability after treatment in Example 3.
[0013] Figure 3 This is an Oil Red O staining image of lipid droplets in AML12 cells after treatment in Example 4;
[0014] Figure 4 This is a graph showing the measurement of TC content in AML12 cells after treatment in Example 5;
[0015] Figure 5 This is a graph showing the measurement of TG content in AML12 cells after treatment in Example 5.
[0016] Figure 6 These are Oil Red O staining images of lipid droplets in the liver tissue of mice in each group in Example 6;
[0017] Figure 7 The total cholesterol (TC) levels in the serum of mice in each group in Example 6 are shown.
[0018] Figure 8 The values represent the serum triglyceride (TG) levels in each group of mice in Example 6.
[0019] Figure 9 The values represent the serum high-density lipoprotein cholesterol (HDL-c) levels of mice in each group in Example 6.
[0020] Figure 10 The values represent the serum low-density lipoprotein cholesterol (LDL-c) levels of mice in each group in Example 6. Detailed Implementation
[0021] The present invention will be further described below with reference to embodiments and accompanying drawings, but it is not intended to limit the scope of the invention.
[0022] Experimental materials: AML12 mouse hepatocytes were purchased from the Shanghai Cell Bank, Chinese Academy of Sciences; C57BL / 6 mice were purchased from Changchun Institute of Biological Products Co., Ltd. β-lactoglobulin (purity ≥95%) and proanthocyanidins (grape seed extract, purity ≥95%) were purchased from Shanghai Yuanye Biotechnology Co., Ltd. Oleic acid (purity ≥99%), palmitic acid (purity ≥98%), and defatted bovine serum albumin (BSA, fatty acids ≤0.02%) were all purchased from Sigma-Aldrich, USA. The FFA mixture was prepared by dissolving palmitic acid and oleic acid in a 1:2 molar ratio: the two were dissolved separately, mixed, and then combined with the BSA solution. The mixture was filtered through a 0.22 μm filter membrane for sterilization to prepare a 10 mmol / L stock solution, which was stored at -20℃. Before use, it was diluted with complete culture medium to the working concentration (e.g., 250 μM). Oil Red O staining solution was purchased from Beijing Solarbio Science & Technology Co., Ltd.; total cholesterol (TC) and triglyceride (TG) assay kits and CCK-8 cell proliferation assay kits were purchased from Beijing Pulilai Gene Technology Co., Ltd.; high-density lipoprotein cholesterol (HDL-c) and low-density lipoprotein cholesterol (LDL-c) assay kits were purchased from Nanjing Jiancheng Biotechnology Institute. Phosphate-buffered saline (PBS), 0.1 M NaOH solution, and 0.1 M HCl solution were prepared using standard laboratory methods.
[0023] Example 1: Preparation and fluorescence characterization of the complex
[0024] Weigh appropriate amounts of β-lactoglobulin and proanthocyanidins, and dissolve them separately in PBS buffer to achieve a β-lactoglobulin concentration of 1 mg / mL and a proanthocyanidin concentration of 0.2 mg / mL. Adjust the pH of both solutions to 9.0 using 0.1 M NaOH solution. Then, mix the two solutions in equal volumes at a β-lactoglobulin to proanthocyanidin dry weight ratio of 5:1. Place the mixture on a magnetic stirrer and stir for 24 hours at room temperature in the dark. After the reaction, adjust the pH of the reaction solution to 7.0 using 0.1 M HCl solution. Transfer the reaction solution to a dialysis bag with a molecular weight cutoff of 3.5 kDa and dialyze in distilled water for 48 hours, changing the dialysate 6-8 times to remove unreacted small molecules and salt ions. Finally, freeze-dry the dialysate to obtain a lyophilized β-lactoglobulin-proanthocyanidin covalent complex powder (hereinafter referred to as the complex).
[0025] To verify the formation of the complex, fluorescence spectroscopy was used to characterize β-lactoglobulin and its complex. Fluorescence emission spectra in the range of 300-500 nm were scanned at an excitation wavelength of 280 nm. The results are as follows: Figure 1As shown, the fluorescence emission peak intensity of the complex was significantly quenched compared to β-lactoglobulin alone. This indicates a close interaction between proanthocyanidins and β-lactoglobulin, leading to an alteration in the microenvironment of fluorescent groups such as tryptophan in β-lactoglobulin, thus providing evidence for the formation of the covalent complex.
[0026] Example 2: Cell Experiment Grouping and Processing
[0027] AML12 cells were distributed at a rate of 1 × 10⁶ cells per well. 6 Cells were seeded at a density of [number] cells / well in 6-well plates and cultured in DMEM / F12 medium containing 10% fetal bovine serum. The experiment was divided into four groups: normal group (standard medium), model group (treated with 250 μM FFA mixture), treatment group 1 (pretreated with 40 μg / mL β-lactoglobulin for 1 hour, then treated with 250 μM FFA mixture), and treatment group 2 (pretreated with a complex at a final concentration of 40 μg / mL based on β-lactoglobulin for 1 hour, then treated with 250 μM FFA mixture, corresponding to a final proanthocyanidin concentration of 8 μg / mL). Cells were collected from all groups after 48 hours of treatment for subsequent analysis.
[0028] Example 3: CCK8 assay to detect the effect of the complex on cell viability
[0029] To assess the cellular safety of the complex, cell viability was determined using the CCK8 assay. AML12 cells were seeded in 96-well plates (1 × 10⁶ cells per well). 4 Cells were treated with different concentrations (0, 10, 20, 40, 80, 160 μg / mL, based on β-lactoglobulin) of the complex for 48 hours. After treatment, 10 μL of CCK8 solution was added to each well, and the cells were cultured for another 2 hours. The absorbance (OD value) at 450 nm was measured using a microplate reader, and the cell viability was calculated.
[0030] The results are as follows Figure 2 As shown, at concentrations of 40 μg / mL and below, the complex had no significant effect on the survival rate of AML12 cells, indicating that the complex had no obvious cytotoxicity within this concentration range and possessed good biosafety.
[0031] Example 4: Oil Red O staining of lipid droplets in AML12 cells
[0032] AML12 cells were seeded in 24-well plates (2 × 10⁶ cells per well). 5(Number of cells), discard the culture medium, and gently wash twice with PBS. Fix cells in each well with 4% paraformaldehyde for 30 minutes. After washing with PBS, infiltrate cells with 60% isopropanol for 1 minute. Discard the isopropanol and stain with freshly prepared Oil Red O working solution for 30 minutes. Then differentiate with 60% isopropanol until the background is clean, and counterstain the nuclei with hematoxylin. After washing with PBS, observe and photograph under an optical microscope (×200x).
[0033] The results are as follows Figure 3 As shown in the figure, the cells in the normal control group had normal cell morphology and almost no red lipid droplets were observed. In the model group, a large number of bright red lipid droplets were visible in the cells, indicating that FFA successfully induced lipid deposition in hepatocytes. The number of lipid droplets in the β-lactoglobulin treatment group was reduced. The number and volume of red lipid droplets in the cells of the complex treatment group were significantly less than those in the model group and the β-lactoglobulin treatment group, indicating that the complex was more effective than β-lactoglobulin alone in inhibiting lipid deposition in hepatocytes.
[0034] Example 5: Determination of TC and TG levels in AML12 cells
[0035] Collect cells from each group, lyse them, centrifuge to collect the supernatant, strictly follow the instructions of the TC and TG detection kit, use an ELISA reader to measure the absorbance value, and calculate the intracellular TC and TG content based on the standard curve.
[0036] The results are as follows Figure 4 , Figure 5 As shown in the figure, the intracellular TC and TG levels in the model group were significantly increased compared to the normal group. Notably, the TC and TG levels in the complex treatment group were significantly lower than those in the β-lactoglobulin treatment group, further demonstrating that the complex is superior to β-lactoglobulin alone in regulating hepatocyte lipid metabolism.
[0037] Example 6: Animal model establishment, drug administration, and sample collection
[0038] Twenty-four healthy male C57BL / 6 mice aged 6 weeks were selected and, after one week of acclimatization, randomly divided into four groups of six mice each: a normal control group (fed normal diet and administered physiological saline by gavage), a model group (fed high-fat diet (HFD) and administered physiological saline by gavage), a β-lactoglobulin group (fed HFD and administered 200 mg / kg β-lactoglobulin by gavage), and a complex group (fed HFD and administered 200 mg / kg complex by gavage, calculated as β-lactoglobulin), for a total of 8 weeks. After the experiment, the mice were fasted for 12 hours, sacrificed, and blood and liver tissue were collected and stored at -80℃ or used immediately for subsequent analysis (Examples 7-9).
[0039] Example 7: Oil Red O staining of mouse liver tissue
[0040] Frozen sections of mouse liver were fixed with 4% paraformaldehyde at room temperature for 30 min, then washed three times with PBS for 5 min each time. Next, the sections were immersed in 60% isopropanol for 3 s to dehydrate, and then incubated in Oil Red O working solution for 30 min. Subsequently, the sections were washed three more times with PBS for 5 min each time. Next, the sections were incubated in hematoxylin staining solution for 15 min to stain the cell nuclei. These sections were then washed three more times with PBS for 5 min each time. Finally, the stained sections were observed and imaged under an Olympus IX71 microscope.
[0041] The results are as follows Figure 6 As shown, a high-fat diet can significantly increase lipid deposition in mouse hepatocytes. Compared with the model group, both the β-lactoglobulin group and the complex group can reduce lipid deposition in the liver, and the effect of the complex group is more obvious.
[0042] Example 8: Determination of TC and TG content in mouse liver
[0043] Approximately 50 mg of mouse liver tissue was weighed using the weight reduction method. 20 μL of TG lysis buffer or 10 μL of TC lysis buffer was added to 1 mg of liver tissue. The liver tissue was diced as finely as possible, and then the appropriate volume of lysis buffer was added as required, followed by thorough grinding. After lysis, the supernatant was collected by centrifugation. The absorbance was measured using a microplate reader, strictly following the instructions for the TC and TG assay kits. The TC and TG contents were calculated based on the standard curve.
[0044] The results are as follows Figure 7 , Figure 8 As shown in the figure, the levels of TC and TG in the liver tissue of the model group were significantly increased compared with the normal group. Notably, the TC and TG levels in the complex treatment group were lower than those in the β-lactoglobulin treatment group, further demonstrating that the complex has a superior effect on improving hepatic lipid metabolism compared with the single component.
[0045] Example 9: Determination of HDL-c and LDL-c levels in mouse serum
[0046] According to the kit instructions, the levels of HDL-c and LDL-c in mouse serum obtained in Example 6 were measured.
[0047] The results are as follows Figure 9 , Figure 10 As shown in the figure, compared with the normal control group, the serum HDL-c level of mice in the model group was significantly reduced, while the LDL-c level was increased. Both were improved in the treatment group, and the complex was more effective than β-lactoglobulin alone.
[0048] in conclusion
[0049] This invention prepares a β-lactoglobulin-proanthocyanidin covalent complex using a mild alkaline treatment method. This complex demonstrates good preventative and therapeutic effects against non-alcoholic fatty liver disease (NAFLD) at both cellular and animal levels: in vitro, it safely and effectively inhibits lipid deposition in hepatocytes and regulates cholesterol and triglyceride metabolism; in vivo, it comprehensively improves liver function damage and dyslipidemia induced by a high-fat diet in C57BL / 6 mice, significantly reducing TC, TG, and LDL-c while also increasing beneficial HDL-c levels. The complex is derived from natural sources, has a simple preparation process, and exhibits clear and comprehensive effects, superior to single components, possessing great potential for development into novel drugs for the prevention and / or treatment of NAFLD and related lipid metabolism disorders.
Claims
1. A method for preparing a β-lactoglobulin-proanthocyanidin covalent complex, characterized in that, Includes the following steps: (1) Dissolve β-lactoglobulin and proanthocyanidins separately in PBS buffer and hydrate at 4-25℃ for 1-4 hours; (2) Adjust the pH of the two solutions to 8.5-9.5 respectively using alkaline solutions; (3) Mix the pH-adjusted β-lactoglobulin solution and proanthocyanidin solution in equal volumes at a mass ratio of (4-6):1; (4) Stir the mixture at room temperature and in the dark for 18-30 hours; (5) After the reaction is complete, adjust the pH of the reaction solution to 6.5-7.5 with an acid solution; (6) The reaction solution obtained in step (5) is purified by dialysis and then freeze-dried to obtain the final product.
2. The preparation method according to claim 1, characterized in that, The alkaline solution mentioned in step (2) is a 0.1 M NaOH solution.
3. The preparation method according to claim 1, characterized in that, In step (3), the concentration of the β-lactoglobulin solution is 1 mg / mL, and the concentration of the proanthocyanidin solution is 0.2 mg / mL.
4. The preparation method according to claim 1, characterized in that, The acid solution mentioned in step (5) is a 0.1 M HCl solution.
5. The preparation method according to claim 1, characterized in that, The dialysis conditions described in step (6) are as follows: using a dialysis bag with a molecular weight cutoff of 3.5 kDa, dialyzing with distilled water at 4°C for 48 hours, during which the dialysis fluid is changed 6-8 times.
6. A β-lactoglobulin-proanthocyanidin covalent complex, characterized in that, It is prepared by the method described in any one of claims 1-5.
7. The use of the β-lactoglobulin-proanthocyanidin covalent complex of claim 6 in the preparation of a medicament for the prevention and / or treatment of non-alcoholic fatty liver disease.
8. The application according to claim 7, characterized in that, The drug is also used to regulate blood lipids, including lowering serum total cholesterol (TC), triglycerides (TG), and low-density lipoprotein cholesterol (LDL-c) levels, and raising high-density lipoprotein cholesterol (HDL-c) levels.