Lactoferrin-apigenin non-covalent complex, and preparation method and application thereof

Abstract

The application discloses a lactoferrin-apigenin non-covalent complex and a preparation method and application thereof, and belongs to the field of food biotechnology and biological medicine technology. An apigenin solution is slowly added into a lactoferrin solution, and the two are stirred and reacted under the conditions of 20-25 DEG C and light shielding, so that the two are self-assembled to form a LF-AP non-covalent complex through non-covalent interaction. Under the preferred preparation conditions, the average particle size of the obtained complex is 99.80+ / -2.30 nm, the PDI is 0.143+ / -0.004, the Zeta potential is 26.01+ / -0.80 mV, the binding rate of the complex to AP is 81.82+ / -0.88%, and the drug loading capacity is 2.95+ / -0.03%. The complex can significantly improve the water phase dispersibility, storage stability and in-vitro biological accessibility of AP, exhibits good safety at a cell level, can cooperatively reduce the lipid accumulation of hepatocytes induced by free fatty acids, and can be used for preparing a medicine or a nutritional preparation for reducing the lipid accumulation of hepatocytes or improving the lipid metabolism of hepatocytes.

Description

Technical Field

[0001] This invention belongs to the fields of food biotechnology and biomedicine, specifically relating to an LF-AP non-covalent complex, its preparation method and application, and particularly to a composite system that utilizes LF as a natural protein carrier to improve the aqueous dispersibility and stability of AP through non-covalent self-assembly, and synergistically reduces lipid accumulation in hepatocytes and its application. Background Technology

[0002] Apogenin (AP) is a natural flavonoid widely found in celery, chamomile, and many other plants. It possesses various biological activities, including antioxidant, anti-inflammatory, and regulatory effects on glucose and lipid metabolism, as well as liver protection, showing promising applications in lipid metabolism disorders, obesity, and metabolic-related fatty liver disease. However, AP is a hydrophobic flavonoid with extremely low solubility in water, easily agglomerating and precipitating. This results in poor dispersibility and low bioavailability in food and biological systems, limiting its further development and application. Therefore, improving the water solubility and stability of AP is a crucial issue that urgently needs to be addressed in its industrial application.

[0003] Lactoferrin (LF) is a natural iron-binding glycoprotein widely found in milk and various bodily fluids, exhibiting good biocompatibility, biosafety, and nutritional function. In recent years, using LF as a carrier to deliver hydrophobic active ingredients has become a research hotspot. The closest existing technology to this invention is the related research on the complexation of lactoferrin with flavonoids. Liu et al. studied the non-covalent interaction between lactoferrin and quercetin, finding that LF and quercetin can spontaneously bind under pH 7.4 conditions. However, this study mainly focused on the interaction mechanism and structural characterization, without reporting key parameters such as the binding rate and drug loading of the complex, nor addressing the improvement of flavonoid water solubility or synergistic enhancement of bioactivity (Liu Y, et al. Non-covalent binding of lactoferrin with quercetin: Structural characterization and functional properties. Food Chemistry, 2023, 405:134851). Other studies have used pulsed electric field treatment to enhance the binding ability of LF to quercetin, but the treatment conditions (electric field strength ≥ 15 kV·cm) are limited. -1This process can lead to disulfide bond crosslinking between LF molecules and involves non-natural treatment methods, which may affect the natural structure and bioactivity of LF (Shan SJ, et al. Pulsed electric field modulates lactoferrin conformation to enhance quercetin binding and complex stability. International Journal of Biological Macromolecules, 2026, 364: 152262). Regarding luteolin, Zhang et al. reported the preparation of luteolin-lactoferrin nanoparticles with an encapsulation efficiency of 71.73%. However, this study used an ultrasound-assisted pH shifting method and a chondroitin sulfate composite carrier, making the preparation process relatively complex (Zhang J, et al. Luteolin-loaded nanocomposites target microglia for sepsis-associated encephalopathy therapy via intranasal delivery. Colloids and Surfaces B: Biointerfaces, 2026, 242: 114248). Another study used a pH-driven method to prepare lactoferrin-luteolin nanocomplexes, achieving an encapsulation efficiency of up to 80% under phosphorylated or succinylated modified LF conditions (Sharmin T, et al. Design, fabrication and molecular dynamics simulations of luteolinPickering emulsion using phosphorylated and succinylated lactoferrin nanocomplex. International Journal of Biological Macromolecules, 2026, 279:135443).Regarding kaempferol, recent studies have confirmed that LF and kaempferol can form a complex through hydrogen bonding and hydrophobic interactions, exhibiting synergistic inhibitory activity against HeLa cell proliferation. However, this study did not address the complex's effects on improving flavonoid water solubility and regulating lipid metabolism (Li X, et al. Structural and in vitro anticancer properties of the kaempferol-lactoferrin complex. Food Science & Nutrition, 2024, 12(11): 9046-9055). Although the aforementioned existing technologies have made some progress in the complexation of LF with polyphenols / flavonoids, the following shortcomings still exist:

[0004] (1) Harsh preparation conditions: The preparation of existing LF-flavonoid complexes often involves relatively harsh conditions such as thermal denaturation, ultrasonic treatment, pulsed electric field, or chemical modification. These treatments may cause irreversible changes in the conformation of LF, affecting its natural biological activity. For example, although sub-denaturing thermal pretreatment (40 °C) can enhance the binding capacity of LF to quercetin, it still involves heating treatment; although pulsed electric field treatment can increase the loading capacity of LF to quercetin by 40.2%, excessively high electric field strength (≥15 kV·cm) is problematic. -1 This leads to disulfide crosslinking between LF molecules (Shan et al., 2026). There are no existing reports of LF-flavonoid complexes with both high binding rates and synergistic biological activity prepared under completely mild conditions (room temperature, no need for ultrasound / heating / electric field, no need for chemical crosslinking agents or modification).

[0005] (2) Unclear or low binding rate / drug loading: Some existing studies focus on the structural characterization and interaction mechanism analysis of LF-flavonoid complexes, without reporting key parameters such as the binding rate and drug loading of the complexes; while in studies involving encapsulation efficiency, the encapsulation efficiency of luteolin-lactoferrin nanoparticles is 71.73% (Zhang et al., 2026), and the encapsulation efficiency of phosphorylated LF-luteolin complexes can reach 80% under neutral conditions, but chemical modification of LF is required (Sharmin et al., 2026). There are no reports in the existing technology of flavonoid compounds achieving a binding rate of over 80% and a drug loading of close to 3% under natural LF (unmodified and unheated) conditions.

[0006] (3) Insufficient evidence of synergistic lipid-lowering effect: Although both LF and AP have been reported to regulate lipid metabolism, there are no reports in the prior art of any synergistic effect of LF and AP combination in alleviating lipid accumulation in hepatocytes. Existing studies have mostly focused on the structural characterization and antioxidant activity evaluation of LF alone or LF combined with other polyphenols, and none of them have addressed the synergistic alleviating effect of LF-flavonoid complex on FFA-induced lipid accumulation in hepatocytes. According to the applicant's search, as of the date of this application, no literature has been found that directly discloses the formation of a non-covalent complex of natural lactoferrin and apigenin under mild conditions, and simultaneously evaluates its binding rate, drug loading, aqueous dispersibility, bioavailability, and regulatory effect on lipid accumulation in hepatocytes.

[0007] Therefore, developing an LF-AP non-covalent complex with a mild preparation process, requiring no heat treatment or ultrasonic assistance, high binding rate, and the ability to significantly improve AP water solubility and exert a synergistic lipid-lowering effect is of significant scientific and practical value. This invention is proposed based on the aforementioned shortcomings of the prior art. Summary of the Invention

[0008] The purpose of this invention is to provide an LF-AP non-covalent complex, its preparation method, and its application, in order to improve the problems of poor aqueous dispersibility, insufficient storage stability, and low bioavailability of AP; at the same time, through the non-covalent complexation of LF and AP, lipid accumulation is reduced and lipid metabolism is improved at the cellular level, providing a technical solution for the development of related drugs or nutritional preparations.

[0009] To achieve the above objectives, the present invention provides a method for preparing an LF-AP non-covalent complex, comprising the following steps:

[0010] (1) Dissolve LF in water or phosphate buffer to prepare LF stock solution and dilute to obtain LF working solution with a concentration of 1-16 μM;

[0011] (2) Dissolve AP in dimethyl sulfoxide to obtain AP mother liquor;

[0012] (3) Slowly add AP mother liquor to LF working solution to make the molar ratio of LF to AP 1:(5-30), adjust the pH of the system to 3.0-8.5, and stir the reaction at 20-25 ℃ in the dark for 0.5-6 h to make LF and AP form a complex through non-covalent interaction.

[0013] Preferably, the concentration of the LF working solution in step (1) is 2-6 μM.

[0014] Preferably, in step (3), the molar ratio of LF to AP is 1:(5-15), the pH of the system is 6.8-8.0, and the stirring time is 2 h.

[0015] More preferably, the concentration of the LF working solution is 4-4.5 μM, the molar ratio of LF to AP is 1:(10-12), and the pH of the system is 7.0-7.4.

[0016] More preferably, the system has a pH of 7.2, an LF working solution concentration of 4.2 μM, and a molar ratio of LF to AP of 1:11, and is stirred for 2 h at 20-25 °C in the dark.

[0017] This invention also provides an LF-AP non-covalent complex prepared by the above method. The complex has an average particle size of 90-120 nm, a PDI ≤ 0.20, a binding rate of apigenin ≥ 80%, and a drug loading ≥ 2.5%; the zeta potential of the complex is 23-28 mV. The non-covalent interaction between LF and AP includes one or more of hydrophobic interactions, hydrogen bonds, van der Waals forces, and electrostatic interactions.

[0018] The present invention also provides the use of the above-mentioned LF-AP non-covalent complex in the preparation of pharmaceutical or nutritional formulations for reducing lipid accumulation in hepatocytes or improving lipid metabolism in hepatocytes.

[0019] Compared with the prior art, this application has the following beneficial effects: (1) The preparation process is mild and the conditions are simple. The non-covalent self-assembly of LF and AP is completed under the conditions of room temperature, light protection and mild stirring, without the need for heat treatment, ultrasonic treatment, pulsed electric field, pH shift treatment or chemical cross-linking agent, thus preserving the natural structure and bioactivity of lactoferrin to the greatest extent. Compared with the prior art, which requires heating at 90 °C to prepare lactoferrin nanofibers and using probe ultrasonic treatment or chemical modification of LF, it has significant process advantages. (2) The preparation parameters have controllability on the performance of the complex. Single-factor experiments show that pH, LF concentration, LF / AP molar ratio and stirring time all affect the particle size distribution of the complex and the AP binding effect; under the preferred preparation conditions (pH 7.2, LF concentration 4.2 μM, LF / AP molar ratio 1:11, stirring for 2 h), the binding rate of the LF-AP non-covalent complex reaches 81.82 ± 0.88%, and the drug loading reaches 2.95 ± 0.03% ( Figure 1 ), significantly better than the encapsulation efficiency of LF-flavonoid complexes in the natural state in the prior art (encapsulation efficiency of luteolin-LF is 71.73%, and that of phosphorylated modified LF-luteolin is 80%, but chemical modification is required). (3) Significantly improves the water solubility and dispersion stability of apigenin. The LF-AP non-covalent complex can increase the apparent water solubility of AP to 8.5 times that of free AP ( Figure 5Under light-protected storage conditions at 4 ℃ and 25 ℃, the retention rate of AP in the complex was significantly higher than that in the free AP group, and the AP retention rate remained above 85% during the 14-day storage period. Figure 6 (4) Significantly improved the in vitro bioavailability of apigenin. After simulated in vitro gastrointestinal digestion, the in vitro bioavailability of AP in the LF-AP complex reached 60.89 ± 1.86%, while that of the free AP group was only 31.25 ± 2.13%, and the bioavailability of the complex group was increased by about 1.95 times. Figure 7 (5) LF and AP have a clear synergistic lipid-lowering effect. Using the Chou-Talalay intermediate-effect principle to calculate the combination index (CI), within the concentration range of AP 0.625~1.25 μM (LF 0.057~0.114 μM), the CI values ​​of TG and TC were 0.435~0.700, both less than 0.7, proving that LF and AP have a clear synergistic effect within the above concentration range. Figure 9 E and F). This effect was verified on both TG and TC, two independent lipid indicators, and showed a concentration-dependent trend. (6) Good cell safety. The results of CCK-8 cell viability assay showed that within the concentration range of AP 0.625~2.5 μM (LF 0.057~0.227 μM), the survival rate of AML12 cells treated with LF-AP complex was ≥ 95%, which was not significantly different from the control group (p > 0.05), indicating that the complex had no obvious cytotoxicity within this concentration range. Figure 8 ). Attached Figure Description

[0020] Figure 1 To optimize the binding rate (%) and drug loading (%) of the LF-AP non-covalent complex obtained under the preparation conditions (pH 7.2, LF concentration 4.2 μM, LF / AP molar ratio 1:11, stirring at room temperature in the dark for 2 h), the data are presented as mean ± SD (n = 3).

[0021] Figure 2 To optimize the preparation conditions, the (A) average particle size and polydispersity index (PDI) and (B) zeta potential of the LF-AP non-covalent complex were determined. Data are expressed as mean ± SD (n = 3).

[0022] Figure 3 The fluorescence spectrum of the non-covalent interaction between LF and AP (excitation wavelength 280 nm, scan range 290-450 nm).

[0023] Figure 4 Fourier transform infrared (FTIR) spectrum of the non-covalent interaction between LF and AP;

[0024] Figure 5Comparison of apparent water solubility between free AP and LF-AP nonvalent complexes. Data are expressed as mean ± SD (n = 3);

[0025] Figure 6 The retention rate of free AP and LF-AP nonvalent complexes over time was measured under light-protected storage conditions of 4 ℃ and 25 ℃ for 7 and 14 days. Data are expressed as mean ± SD (n=3).

[0026] Figure 7 Bioaccessibility (%) of free AP and LF-AP nonvalent complexes after in vitro simulated gastrointestinal digestion. Data are expressed as mean ± SD (n = 3);

[0027] Figure 8 The effects of different concentrations of LF, AP, and LF-AP non-covalent complex on the viability of AML12 cells were investigated using the CCK-8 assay. AML12 cells were treated with different concentrations of LF-AP complex (AP 0.625, 1.25, 2.5 μM, corresponding to LF 0.057, 0.114, 0.227 μM) for 48 h. Data are presented as mean ± SD (n = 6). ns indicates no significant difference compared to the control group (p > 0.05).

[0028] Figure 9 To alleviate FFA-induced lipid accumulation in AML12 cells using the LF-AP non-covalent complex. (A) Intracellular TG content; (B) Intracellular TC content; (C) TG inhibition rate; (D) TC inhibition rate; (E) CI values ​​of TG and TC; (F) CI-effect level (Fa) curve. Cells were treated with 250 μM FFA for 48 h to induce a lipid accumulation model. Three concentrations of LF, AP, and the LF-AP complex were set: low, medium, and high (LF: 0.057, 0.114, 0.227 μM; AP: 0.625, 1.25, 2.5 μM). Data are expressed as mean ± SD (n = 3). ** p < 0.001 indicates complex vs. FFA group; ## p < 0.001 indicates the complex versus the same concentration of LF monotherapy; && p < 0.001 indicates the complex versus the same concentration of AP monotherapy. CI < 0.7 indicates a synergistic effect, and CI = 1 (black dashed line) indicates an additive effect. Detailed Implementation

[0029] The following description, in conjunction with embodiments and accompanying drawings, further illustrates the invention but is not intended to limit its scope.

[0030] Experimental materials: LF (purity ≥ 95%, catalog number: S24749) and AP (purity ≥ 98.0%, catalog number: S31423) were purchased from Shanghai Yuanye Biotechnology Co., Ltd. Oleic acid (purity ≥ 99%, catalog number: O1008) and palmitic acid (purity ≥ 98%, catalog number: P9767) were purchased from Sigma-Aldrich, USA. Total cholesterol (TC) and triglyceride (TG) assay kits (catalog numbers: E1013, E1014) were purchased from Beijing Pulilai Gene Technology Co., Ltd.

[0031] Example 1: Preparation of LF-AP non-covalent complex

[0032] Weigh an appropriate amount of LF powder, dissolve it thoroughly in deionized water to prepare a 25 μM LF stock solution, and store it at 4 °C for later use. Before use, dilute the LF stock solution with deionized water or phosphate buffer to the required concentration to obtain the LF working solution. Weigh an appropriate amount of AP powder, dissolve it thoroughly in dimethyl sulfoxide to prepare a 1 mM AP stock solution, and store it in the dark for later use. Under stirring conditions, slowly add the AP stock solution dropwise to the LF working solution to ensure full contact between LF and AP. Then adjust the pH of the resulting mixture and continue stirring at 20-25 °C in the dark to allow LF and AP to self-assemble into an LF-AP non-covalent complex through one or more non-covalent interactions, such as hydrophobic interactions, hydrogen bonds, van der Waals forces, and electrostatic interactions. After the reaction is complete, the resulting dispersion can be centrifuged, filtered, dialyzed, or freeze-dried as needed to remove unbound free AP or obtain a lyophilized complex powder.

[0033] Example 2: Single-factor optimization of preparation conditions for LF-AP non-covalent complexes

[0034] To obtain an LF-AP non-covalent complex with good stability, small particle size and good AP binding effect, the pH, LF concentration, LF / AP molar ratio and stirring time in the preparation process were optimized by single factors. First, with a fixed LF concentration of 4 μM and an LF / AP molar ratio of 1:10, the system was stirred at room temperature in the dark for 2 h, and the pH of the system was adjusted to 3.0, 5.0, 6.5, 7.4, and 8.5 to prepare LF-AP complexes under different pH conditions. Second, with a fixed pH of 7.4 and an LF / AP molar ratio of 1:10, the system was stirred at room temperature in the dark for 2 h, and the LF concentration was set to 1, 2, 4, 8, and 16 μM to prepare complexes under different LF concentration conditions. Third, with a fixed pH of 7.4 and an LF concentration of 4 μM, the system was stirred at room temperature in the dark for 2 h, and the LF / AP molar ratio was set to 1:5, 1:10, 1:15, 1:20, and 1:30 to prepare complexes under different molar ratio conditions. Finally, with a fixed pH of 7.4, an LF concentration of 4 μM, and an LF / AP molar ratio of 1:10, the stirring time was set to 0.5, 1, 2, 4, and 6 hours. h, complexes were prepared under different stirring time conditions; the particle size, polydispersity index (PDI), zeta potential, binding rate and drug loading of these complexes were determined.

[0035] Table 1. Effects of different preparation conditions on the physicochemical properties, binding rate, and drug loading of LF-AP non-covalent composites.

[0036]

[0037] Note: When examining a single factor, all other preparation parameters remain unchanged. Data are expressed as mean ± standard deviation (n=3).

[0038] The results are shown in Table 1. The preparation conditions significantly affected the particle size distribution, surface potential, and AP binding efficiency of the LF-AP complex. Under pH 3.0 and 8.5 conditions, the particle size of the complex increased to 389.33 and 350.38 nm, respectively, and the PDI exceeded 0.60, indicating that excessively acidic conditions or conditions close to the isoelectric point of LF were not conducive to the formation of a homogeneous and stable complex system. When the LF concentration increased from 1 μM to 4 μM, the particle size and PDI gradually decreased, while the binding rate increased significantly. After further increasing the LF concentration to 8-16 μM, the particle size and PDI increased again. When the LF / AP molar ratio was 1:(5-15), the obtained complex had a particle size of 104.03-112.95 nm and a PDI of 0.134-0.155, showing good dispersion uniformity. When the AP ratio continued to increase to 1:20 and 1:30, the particle size and PDI of the system increased significantly. When stirred for 2 h, the complex exhibited a relatively small particle size, low PDI, and high binding rate. Too short a reaction time may lead to insufficient binding, while further extending the stirring time may promote further particle aggregation. Based on these results, response surface methodology was used to optimize the reaction by selecting pH, LF concentration, and LF / AP molar ratio, with the stirring time fixed at 2 h.

[0039] Example 3: Response surface optimization and validation of LF-AP non-covalent complex

[0040] Based on the single-factor experiments, pH (A), LF concentration (B), and LF / AP molar ratio (C) were selected as independent variables, and the binding rate (Y1), drug loading (Y2), particle size (Y3), and PDI (Y4) of the complex were used as response values. A three-factor, three-level response surface methodology (RSM) optimization experiment was conducted using a Box-Behnken design (Table 2). The single-factor results showed that the overall response values ​​were better when stirring for 2 h. Shortening the reaction time led to insufficient binding, while further extending the time increased the particle size and PDI, and no continuous optimization gain suitable for further fitting was observed. Therefore, the stirring time was fixed at 2 h and not included in the RSM factors to reduce unnecessary experimental combinations and improve the model's ability to analyze key formulation factors.

[0041] Table 2 Factors and Levels

[0042]

[0043] Table 3 Response Surface Experimental Design and Results

[0044]

[0045] As shown in Table 5, significant differences exist in the response values ​​under different experimental conditions. Within the scope of the study, the binding rate ranged from 70.28% to 94.80%, the drug loading from 1.39% to 3.93%, the particle size from 103.03 to 296.93 nm, and the PDI from 0.133 to 0.453. Among these, the complexes obtained under experimental conditions 1, 3, 5, 7, and 13–17 exhibited smaller particle sizes and lower PDIs, demonstrating better dispersion uniformity. While conditions 9 and 10 showed higher binding rates (93.67% and 94.80%), the corresponding particle sizes were 215.83 nm and 241.84 nm, respectively, with PDIs of 0.230 and 0.242, indicating that while an excessively high AP ratio can improve binding rate, it significantly increases particle size and dispersion inhomogeneity. The response values ​​in the center point repeat experiments (13–17) showed relatively small fluctuations, indicating good experimental reproducibility, which can be used for subsequent regression model establishment and analysis. The experimental data were fitted using Design-Expert software via multiple regression, yielding a quadratic polynomial regression equation for each response value on the independent variable: Y1 = 85.01 - 3.86*A + 1.15*B - 8.17*C - 0.86*AB - 0.3*AC + 0.46*BC - 6.4*A 2 - 3.06*B 2 + 3.21*C 2 ; Y2 = 2.8 -0.14*A + 0.04*B + 1.09*C - 0.03*AB - 0.09*AC + 0.03*BC - 0.18*A 2 - 0.12*B 2 -0.03*C 2 ; Y3 = 106.78 + 56.45*A + 13.59*B - 4.44*C + 21.33*AB + 9.72*AC -0.71*BC + 26.5*A 2 + 69.91*B 2 + 45.66*C 2 ; Y4 = 0.14 + 0.09*A + 0.01*B + 0.03*C+ 0.02*AB + 0.005*AC + 0.005*BC + 0.11*A 2 + 0.06*B 2 + 0.05*C 2 .

[0046] Table 4. Analysis of variance (ANOVA) results for each response value model.

[0047]

[0048] The above results show that: the p-values ​​of all four response value models are < 0.0001, indicating that each regression model is highly significant; the p-values ​​of the lack-of-fit terms of each model are > 0.05, indicating that the lack-of-fit terms are not significant, suggesting that each model has a good fit and small experimental error; the R-values ​​of each model are... 2 All > 0.98, R 2 The adj values ​​are all greater than 0.97, indicating that each model can explain more than 97% of the response value changes; the signal-to-noise ratio of each model is all greater than 4 (actually between 25.37 and 82.10), indicating that each model has sufficient predictive signal strength and can be used for prediction and optimization of preparation conditions.

[0049] The optimal preparation conditions predicted by the regression model were: pH 7.174, LF concentration 4.209 μM, and LF / AP molar ratio 1:10.844. Considering practical ease of operation, the optimal conditions were modified to pH 7.2, LF concentration 4.2 μM, and LF / AP molar ratio 1:11. A verification experiment (n = 3) was conducted under these conditions, and the results are shown in Table 3.

[0050] Table 5. Predicted values ​​and validation results under optimized conditions

[0051]

[0052] The LF-AP nonvalent complex was prepared repeatedly under the above-mentioned preferred conditions in the formal experiment. The results are as follows: Figure 1 As shown, the binding rate of the obtained complex was 81.82 ± 0.88%, and the drug loading was 2.95 ± 0.03%. Figure 2 As shown in (A, B), the obtained composite exhibits good dispersibility, with an average particle size of 99.80 ± 2.30 nm, a PDI of 0.143 ± 0.004, and a Zeta potential of 26.01 ± 0.80 mV. These results indicate that the composite prepared under these conditions has a small particle size, uniform distribution, and good colloidal stability.

[0053] Example 4: Spectroscopic characterization of the LF-AP non-covalent complex

[0054] The interaction between LF and AP was analyzed using fluorescence spectroscopy. Fluorescence emission spectra in the range of 290-450 nm were scanned using 280 nm as the excitation wavelength. The results are as follows: Figure 3 As shown, compared with LF alone, the intrinsic fluorescence intensity of the LF-AP non-covalent complex was significantly quenched, suggesting that AP interacts with LF and changes the microenvironment of fluorescent groups such as tryptophan and tyrosine in LF.

[0055] Fourier transform infrared spectroscopy results are as follows Figure 4As shown, the characteristic absorption peaks and protein amide bands of the LF-AP non-covalent complex change to some extent, indicating that there may be non-covalent interactions between LF and AP, such as hydrophobic interactions, hydrogen bonds, van der Waals forces, and electrostatic interactions.

[0056] Example 5: The effect of LF-AP non-covalent complex on improving the water solubility and storage stability of AP

[0057] To evaluate the effect of the LF complex on improving the water solubility of AP, free AP and LF-AP non-covalent complex groups were prepared. After thorough mixing and centrifugation, the supernatant was collected, diluted with methanol, or extracted, and the AP content was determined. Results are as follows: Figure 5 As shown, free AP exhibits poor dispersibility in the aqueous system, with a saturated solubility of 5.36 ± 0.15 μM (i.e., 1.45 ± 0.04 μg / mL). In contrast, the LF-AP non-covalent complex forms a homogeneous dispersion, significantly increasing the apparent solubility of AP to 45.46 ± 0.41 μM (i.e., 12.29 ± 0.11 μg / mL), approximately 8.5 times that of free AP. This indicates that the LF complex can significantly improve the aqueous solubility and dispersion of AP.

[0058] To evaluate storage stability, the LF-AP nonvalent complex dispersion was stored in the dark at 4 ℃ and 25 ℃, respectively. Samples were taken at 7 and 14 days to determine the AP retention rate. The results are as follows: Figure 6 As shown, during storage, the AP retention rate of the LF-AP non-covalent complex was higher than that of the free AP group, indicating that the LF complex can improve the aqueous dispersion stability and storage stability of AP.

[0059] Example 6: Evaluation of bioaccessibility of LF-AP non-covalent complex in simulated gastrointestinal digestion

[0060] The bioavailability of the LF-AP nonvalent complex was evaluated using an in vitro simulated gastrointestinal digestion model. After intestinal digestion, the digestive fluid was centrifuged at high speed, and the supernatant was collected as the micelle phase. The AP content in the micelle phase was measured, and the in vitro bioavailability was calculated. The results are as follows: Figure 7 As shown, the LF-AP non-covalent complex group exhibited higher in vitro bioavailability compared to the free AP group. Specifically, the in vitro bioavailability of the free AP group was 31.25 ± 2.13%, while that of the complex group was 60.89 ± 1.86%.

[0061] Example 7: Cellular safety of the LF-AP non-covalent complex

[0062] The cellular safety of the LF-AP complex was evaluated using AML12 mouse hepatocytes. AML12 cells were seeded in 96-well plates, and after cell attachment, they were treated with LF, free AP, and the LF-AP complex for 48 h, respectively. The LF-AP complex was diluted according to AP equivalent concentrations, with final AP concentrations of 0.625, 1.25, and 2.5 μM, corresponding to final LF concentrations of approximately 0.057, 0.114, and 0.227 μM, respectively. The LF-only treatment group and the free AP treatment group were set with the same LF and AP concentrations, respectively. The results are shown below. Figure 8 As shown, within the experimental concentration range (AP 0.625–2.5 μM, LF 0.057–0.227 μM), the LF-AP complex showed no significant cytotoxicity against AML12 cells. At low concentrations of the LF-AP complex (LF-AP-L, AP 0.625 μM, LF 0.057 μM), cell viability was 98.23 ± 1.12%; at medium concentrations (LF-AP-M, AP 1.25 μM, LF 0.114 μM), cell viability was 97.56 ± 0.98%; and at high concentrations (LF-AP-H, AP 2.5 μM, LF 0.227 μM), cell viability was 96.87 ± 1.05%. Cell viability was ≥ 95% at all concentrations, with no significant difference compared to the control group (100%) (p > 0.05), indicating that the LF-AP complex has good cell safety within the above concentration range.

[0063] Example 8: LF-AP non-covalent complex alleviates FFA-induced lipid accumulation in AML12 cells

[0064] AML12 cells were divided into a control group, an FFA model group, an FFA + LF single-drug group (LF concentrations of 0.057, 0.114, and 0.227 μM, corresponding to low, medium, and high concentrations), an FFA + AP single-drug group (AP concentrations of 0.625, 1.25, and 2.5 μM, corresponding to low, medium, and high concentrations), and an FFA + LF-AP complex group (LF / AP concentrations in the complex corresponded to the low, medium, and high concentrations mentioned above, with a molar ratio of 1:11). Except for the control group, all other groups were treated with 250 μM FFA mixture for 48 h to establish a hepatocyte lipid accumulation model. After treatment, intracellular TC and TG levels were detected using a kit, the inhibition rate of each treatment group was calculated, and the CI and CI-effect level (Fa) relationship curves were calculated based on the Chou-Talalay intermediate-efficacy principle.

[0065] (1) Evaluation of FFA-induced hepatocyte lipid accumulation model

[0066] After treatment with 250 μM FFA for 48 h, the intracellular TG and TC levels in the FFA model group were significantly higher than those in the control group, indicating that FFA successfully induced lipid accumulation in hepatocytes. Results are as follows. Figure 9 As shown in (A and B), the intracellular TG content in the FFA model group significantly increased from 6.50 ± 0.42 μM / g in the control group to 40.33 ± 1.12 μM / g (a 6.2-fold increase), and the TC content significantly increased from 5.80 ± 0.35 μM / g in the control group to 32.47 ± 1.54 μM / g (a 5.6-fold increase). These results indicate that the FFA-induced hepatocyte lipid accumulation model was successfully established, and the model window is sufficient, providing a reliable dynamic range for subsequent inhibition rate evaluation.

[0067] (2) Inhibitory effect of LF-AP complex on FFA-induced lipid accumulation in AML12 cells

[0068] The results are as follows Figure 9As shown in (A and B), compared with the FFA model group, LF monotherapy, AP monotherapy, and LF-AP complex all reduced intracellular TG and TC levels to varying degrees at different concentrations, and all showed a concentration-dependent effect. Among them, the LF-AP complex had the most significant lipid-lowering effect, and was superior to LF monotherapy or AP monotherapy at the same concentration at all concentrations. At low concentrations, the TG level in the LF monotherapy group (0.057 μM) was 24.69 ± 0.86 μM / g, and the TC level was 32.40 ± 1.08 μM / g; the TG level in the AP monotherapy group (0.625 μM) was 25.27 ± 0.87 μM / g, and the TC level was 36.34 ± 2.16 μM / g. At medium concentrations, the TG content of the LF monotherapy group (0.114 μM) was 21.13 ± 0.53 μM / g, and the TC content was 28.15 ± 0.46 μM / g; the TG content of the AP monotherapy group (1.25 μM) was 22.13 ± 0.62 μM / g, and the TC content was 29.61 ± 0.94 μM / g. At high concentrations, the TG content of the LF monotherapy group (0.227 μM) was 16.56 ± 0.28 μM / g, and the TC content was 22.25 ± 0.50 μM / g; the TG content of the AP monotherapy group (2.5 μM) was 18.30 ± 0.88 μM / g, and the TC content was 24.09 ± 0.28 μM / g. At all concentrations, the intracellular TG and TC levels in the LF-AP complex group were significantly lower than those in the LF monotherapy and AP monotherapy groups at the same concentrations (p < 0.001). Specifically, at low concentrations (AP 0.625 μM / LF 0.057 μM in the complex), the TG level in the complex group was 15.89 ± 1.08 μM / g, and the TC level was 21.81 ± 1.04 μM / g; at medium concentrations (AP 1.25 μM / LF 0.114 μM), the TG level was 11.71 ± 0.28 μM / g, and the TC level was 16.77 ± 0.68 μM / g; and at high concentrations (AP 2.5 μM / LF 0.227 μM), the TG level was 9.73 ± 0.84 μM / g, and the TC level was 15.19 ± 0.84 μM / g. The above results indicate that the LF-AP complex has a significant alleviating effect on FFA-induced lipid accumulation in hepatocytes, and its effect is better than that of LF or AP alone at the same concentration.

[0069] (3) Inhibition rate of LF-AP complex on TG and TC

[0070] The inhibition rates of each treatment group were calculated based on the TG and TC contents mentioned above, and the results are shown in Figures 9 (C and D). The inhibition rates of TG and TC by LF monotherapy, AP monotherapy, and LF-AP complex all increased in a gradient with increasing concentration, showing a clear dose-response relationship; among them, the inhibition rate of LF-AP complex was significantly higher than that of LF monotherapy or AP monotherapy at the same concentration at all concentrations. At low concentrations (AP 0.625 μM, LF 0.057 μM), the TG inhibition rate of the LF monotherapy group was 41.86%, and the TC inhibition rate was 37.87%; the TG inhibition rate of the AP monotherapy group was 39.87%, and the TC inhibition rate was 25.42%; the TG inhibition rate of the LF-AP complex group was 73.56%, and the TC inhibition rate was 71.30%. At medium concentrations (AP 1.25 μM, LF 0.114 μM), the TG inhibition rate was 54.71% and the TC inhibition rate was 51.32% in the LF monotherapy group; the TG inhibition rate was 51.09% and the TC inhibition rate was 46.66% in the AP monotherapy group; and the TG inhibition rate was 88.55% and the TC inhibition rate was 87.33% in the LF-AP complex group. At high concentrations (AP 2.5 μM, LF 0.227 μM), the TG inhibition rate was 71.16% and the TC inhibition rate was 70.06% in the LF monotherapy group; the TG inhibition rate was 64.91% and the TC inhibition rate was 64.26% in the AP monotherapy group; and the TG inhibition rate was 95.64% and the TC inhibition rate was 92.19% in the LF-AP complex group. At all concentrations, the TG and TC inhibition rates of the LF-AP complex group were significantly higher than those of the LF monotherapy group and the AP monotherapy group at the same concentration (p < 0.001), further demonstrating the synergistic effect of LF and AP combined.

[0071] (4) Quantitative evaluation of the synergistic effect between LF and AP

[0072] To further quantitatively evaluate the synergistic effect of LF and AP, based on the Chou-Talalay moderate effect principle, CI was used as the evaluation index (CI < 0.7 for synergy, CI = 1 for addition). The CI values ​​of TG and TC at each concentration were calculated, and the results are shown in Figure 9E. At low and medium concentrations, the CI values ​​were all less than 0.7, indicating that LF and AP have a clear synergistic effect over a wide concentration range. However, as the concentration increases to the saturation plateau, the synergistic effect gradually weakens and tends to be simply additive, showing a typical concentration-dependent trend. At low concentrations (AP 0.625 μM, LF 0.057 μM), the CI value of TG was 0.435 ± 0.019, and the CI value of TC was 0.462 ± 0.040; at medium concentrations (AP 1.25 μM, LF 0.114 μM), the CI value of TG was 0.636 ± 0.048, and the CI value of TC was 0.700 ± 0.028. At the above concentrations, the CI values ​​for both TG and TC were less than 0.7, indicating a clear synergistic effect between LF and AP in the low and medium concentration ranges. At high concentrations (AP 2.5 μM, LF 0.227 μM), the CI value for TG was 1.132 ± 0.115, and for TC it was 1.302 ± 0.114. The CI values ​​approached or slightly exceeded 1.0, suggesting that the combined effect reached a saturation plateau at high concentrations, and the synergistic effect tended to be simply additive. To further verify the concentration-dependent trend of the synergistic effect, a CI-Fa relationship curve was plotted with the effect level (Fa, i.e., inhibition rate / 100) on the x-axis and CI on the y-axis. The results are shown below. Figure 9 As shown in Figure F, at low and medium effect levels (Fa ≈ 0.71–0.89, corresponding to AP concentrations of 0.625–1.25 μM), the CI values ​​for TG and TC were both less than 1.0, and increased with increasing Fa. When the effect level increased to Fa ≈ 0.92–0.96 (corresponding to an AP concentration of 2.5 μM), the CI value approached or slightly exceeded 1.0. This curve fully demonstrates the optimal synergistic window of the complex (Fa ≈ 0.71–0.89), further confirming the synergistic effect of LF and AP within the core concentration range and its concentration dependence. These results indicate that the LF-AP non-covalent complex can alleviate FFA-induced lipid accumulation in hepatocytes within the non-toxic concentration range (cell viability ≥ 95%) through the synergistic effect of LF and AP. Its effect in reducing intracellular TG and TC levels is significantly better than that of LF or AP alone at the same concentration. The CI value and CI-Fa curve quantitatively verify the existence of the synergistic effect and its concentration-dependent trend.

[0073] Unrestricted by specific theories, the synergistic effect of LF and AP may originate simultaneously from delivery effects and the complementary regulation of lipid metabolism by the two components. First, experimental results from this invention show that the LF combination can increase the apparent water solubility of AP by approximately 8.5 times and its in vitro bioavailability from 31.25% to 60.89%, indicating that LF, as a natural protein carrier, can reduce AP aggregation in the aqueous phase and improve its effective dispersion and usable proportion, thereby potentially increasing the effective AP accessible to cells. Second, previous studies have shown that LF can participate in the regulation of hepatic lipid homeostasis, and AP can also inhibit hepatocyte lipid accumulation through AMPK-related signaling, lipid droplet turnover, and mitochondrial function. The combination of the two may produce a higher effect than individual treatments through "carrier-improved delivery—active ingredient exerts its effect" and complementary regulation of different lipid metabolism pathways. Lactoferrin also has the potential for receptor-mediated endocytosis, and thus may further promote cellular uptake of the combined system; however, this application has not verified specific receptors or uptake pathways, and this possible mechanism does not constitute a limitation on the scope of protection of this invention. The synergistic effect of this application is directly based on the results of TG and TC reduction and CI values ​​at the same dosage.

[0074] Comparative Example: Comparison with Existing Techniques

[0075] Following the preparation method of luteolin-lactoferrin-chondroitin sulfate nanoparticles reported by Zhang et al., the LF-AP complex was prepared using an ultrasound-assisted pH shift method: LF was dissolved in deionized water to prepare a 4.2 μM LF working solution, and the pH was adjusted to 9.0 with 0.1 M NaOH. The solution was then treated with ultrasound at 50 W for 60 s under ice bath conditions. Subsequently, the pH was adjusted back to 7.2 with 0.1 M HCl, and the AP stock solution was slowly added dropwise to the LF working solution (LF / AP molar ratio 1:11) under stirring. The reaction was continued for 2 h under room temperature and in the dark, with the remaining conditions the same as in Example 1 of this invention. The results are shown in Table 6.

[0076] Table 6 Performance Comparison between the Method of the Present Invention and the Reference Method

[0077]

[0078] The above results show that the complex prepared by existing technology has significantly inferior binding rate, drug loading and particle size distribution compared with the present invention (different letters in the same column indicate extremely significant differences, p < 0.001), proving that the present invention can obtain a high-performance LF-AP complex.

[0079] in conclusion

[0080] This invention involves slowly adding apigenin solution dropwise to lactoferrin solution and stirring the mixture under light-protected conditions at 20-25 °C, allowing the two to self-assemble through non-covalent interactions to form an LF-AP non-covalent complex. Through single-factor experiments and response surface methodology optimization, the optimal preparation conditions were determined to be: system pH 7.2, LF working solution concentration 4.2 μM, LF / AP molar ratio 1:11, and stirring for 2 h. The resulting complex had an average particle size of 99.80 ± 2.30 nm, a PDI of 0.143 ± 0.004, a Zeta potential of 26.01 ± 0.80 mV, an AP binding rate of 81.82 ± 0.88%, and a drug loading of 2.95 ± 0.03%. This complex increased the apparent water solubility of AP to 8.5 times that of free AP, maintained an AP retention rate of over 85% during a 14-day storage period, and achieved an in vitro bioavailability of 60.89%. Cellular experiments showed that the complex exhibited good safety within the AP concentration range of 0.625–2.5 μM; within the AP concentration range of 0.625–1.25 μM (LF 0.057–0.114 μM), the CI values ​​for TG and TC were 0.435–0.700, indicating that LF and AP have a clear synergistic effect in reducing hepatocyte lipid accumulation. Therefore, the LF-AP non-covalent complex can be used to prepare drugs or nutritional agents that reduce hepatocyte lipid accumulation or improve hepatocyte lipid metabolism.

Claims

1. A method for preparing a lactoferrin-apigenin non-covalent complex, characterized in that, Includes the following steps: (1) Dissolve lactoferrin (LF) in water or phosphate buffer to prepare an LF working solution with a concentration of 2-6 μM; (2) Dissolve apigenin (AP) in dimethyl sulfoxide to prepare AP stock solution; (3) Under stirring conditions, the AP mother liquor obtained in step (2) is slowly added dropwise to the LF working solution obtained in step (1), and the molar ratio of LF to AP is controlled to be 1:(5-15). The pH of the mixed system is adjusted to 6.5-8.0, and the mixture is stirred and reacted for 1-4 h under the dark at 20-25 °C, so that lactoferrin and apigenin can self-assemble into a complex through non-covalent interaction; wherein the final concentration of dimethyl sulfoxide in the system is not higher than 1.0%v / v.

2. The preparation method according to claim 1, characterized in that, The concentration of the LF working solution in step (1) is 4 to 4.5 μM; the molar ratio of LF to AP in step (3) is 1:(10 to 12), the pH of the mixed system is 7.0 to 7.4, and the stirring reaction time is 2 h.

3. The preparation method according to claim 2, characterized in that, The pH of the mixed system was 7.2, the concentration of the LF working solution was 4.2 μM, and the molar ratio of LF to AP was 1:

11. The mixture was stirred and reacted for 2 h at 20-25 °C in the dark.

4. A lactoferrin-apigenin non-covalent complex, characterized in that, It is prepared by the preparation method according to any one of claims 1-3.

5. The lactoferrin-apigenin non-covalent complex according to claim 4, characterized in that, The average particle size of the complex is 90–120 nm, the particle size distribution index (PDI) is ≤ 0.20, the binding rate of apigenin is ≥ 80%, and the drug loading is ≥ 2.5%.

6. The lactoferrin-apigenin non-covalent complex according to claim 4 or 5, characterized in that, The zeta potential of the complex is 23–28 mV; lactoferrin and apigenin are bound together through one or more non-covalent interactions, including hydrophobic interactions, hydrogen bonds, van der Waals forces, and electrostatic interactions.

7. The use of the lactoferrin-apigenin non-covalent complex according to any one of claims 4 to 6 in the preparation of a drug or nutritional preparation for reducing lipid accumulation in hepatocytes or improving lipid metabolism in hepatocytes.

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