Lactoferricin complex, method for its preparation and use

Abstract

The application discloses a lactoferrin complex and a preparation method and application thereof. The complex is prepared from raw materials including 5-120 parts of lactoferrin, 0.5-20 parts of propolis extract and 0.1-10 parts of chlorogenic acid. The lactoferrin complex has excellent stability, and the immunocompetence, anti-inflammatory and antioxidant functions are significantly enhanced, so that the lactoferrin complex has wide application prospects in the fields of food, health management and pharmaceutical chemical industry.

Description

Technical Field

[0001] This invention belongs to the field of biomaterials technology. More specifically, this invention relates to a lactoferrin complex with enhanced immunity and multiple anti-inflammatory and antioxidant functions, its preparation method, and its application. Background Technology

[0002] Lactoferrin (LF) is an iron-binding protein in the transferrin family. It inhibits the growth of pathogenic microorganisms by chelating iron ions and directly disrupts the cell walls of Gram-negative bacteria, exhibiting broad-spectrum resistance to bacteria, fungi, rotavirus, and adenovirus. Lactoferrin also regulates the balance of Th1 / Th2 cytokines, activates macrophage and NK cell activity, and constitutes the body's innate immune barrier. Responding to various physiological and environmental changes, lactoferrin is considered a key component of the innate defense system. It also possesses anti-inflammatory and anti-cancer activities, inhibits melanin production, and has various enzymatic functions. Lactoferrin plays a crucial role in maintaining cellular iron levels in the body.

[0003] However, lactoferrin is easily denatured and inactivated by temperature, acid and alkali, gastrointestinal enzymatic hydrolysis and other processing or environmental factors. Therefore, maintaining high activity and stability of lactoferrin is crucial in industrial production and application. Summary of the Invention

[0004] Therefore, the purpose of this invention is to maintain the high activity and stability of lactoferrin and to enhance its anti-inflammatory and antioxidant functions.

[0005] The technical solutions for achieving the above-mentioned objectives include the following.

[0006] In a first aspect, the present invention provides a lactoferrin complex, which, by weight, is prepared from a raw material comprising 5 to 120 parts lactoferrin, 0.5 to 20 parts propolis extract and 0.1 to 10 parts chlorogenic acid.

[0007] A second aspect of the present invention provides a method for preparing the above-mentioned lactoferrin complex, comprising the following steps: (1) Dissolve lactoferrin powder in 0.008%~0.012% SDS aqueous solution, heat treat at 98℃~102℃ for 3 minutes~5 minutes, cool on ice for 5 minutes~10 minutes, add NP-40 with a final concentration of 0.8%~1.2%, then add 2 U~10 U of PNGase F enzyme and 45 mM~55 mM sodium phosphate buffer at pH 7.5, incubate at 36℃~38℃ for 12 hours~20 hours, then hydrate at 4℃~8℃ for 8~12 hours to prepare a lactoferrin solution with a mass concentration of 1%~10%, and adjust the pH to 8.0~10.0; (2) Add propolis extract and chlorogenic acid to a 75% to 95% ethanol solution at a mass ratio of 1:2 to 5:1, and stir at room temperature for 30 to 120 minutes to dissolve completely, so as to obtain a mixed solution with a chlorogenic acid concentration of 0.5% to 5.0%. (3) Under conditions of 4℃~25℃, slowly add 1 / 10~1 / 30 of the volume of the mixed solution in step (2) to the lactoferrin solution in step (1) while stirring, stir for 0.5 hours~2 hours until the system is visually homogeneous, sonicate at 5℃~20℃ for 5 minutes~10 minutes, continue stirring for 30 minutes~60 minutes, adjust the pH of the system to 6.0~7.4, let stand, dialyze, freeze dry, and the product is obtained.

[0008] A third aspect of the present invention provides the use of the above-mentioned lactoferrin complex in the preparation of anti-inflammatory and antioxidant products.

[0009] In a fourth aspect, the present invention provides a product made of the above-described lactoferrin complex and excipients.

[0010] The inventors of this invention discovered that by compounding lactoferrin, propolis extract, and chlorogenic acid in a certain mass ratio and controlling the compounding process and reaction parameters, the resulting lactoferrin complex exhibits a zeta potential within -10mV to -30mV and a particle size distribution less than 300 nm in a simulated gastrointestinal pH environment. This effectively maintains the nanoscale particle size and, even after 3 months of accelerated processing, maintains a highly dispersed state, demonstrating excellent stability and high activity.

[0011] In preparing the lactoferrin complex, this invention adjusts the pH of the lactoferrin solution to 6.0-7.4 and strictly controls the temperature, allowing lactoferrin to carry a positive charge under near-physiological pH conditions. This enables it to electrostatically attract the negatively charged groups of the polyphenolic structure of propolis extract and chlorogenic acid, promoting their binding. On the one hand, this effectively improves the problems of precipitation, stratification, oxidative degradation, and inactivation caused by compatibility issues of lactoferrin and phenolic acid components. On the other hand, it helps maintain the natural conformation and iron-binding capacity of lactoferrin. Ultimately, the prepared lactoferrin complex can exert a synergistic effect of lactoferrin, propolis extract, and chlorogenic acid, significantly enhancing immunity, anti-inflammatory, and antioxidant functions, and has broad application prospects in the food, health management, and pharmaceutical chemical fields. Attached Figure Description

[0012] Figure 1 This is a particle size distribution diagram of the lactoferrin complex in Example 2.

[0013] Figure 2 This is a zeta potential diagram of the lactoferrin complex in Example 2.

[0014] Figure 3This is a particle size distribution diagram of the lactoferrin complex in Example 2 after 3 months of accelerated processing.

[0015] Figure 4 The image shows the Zeta potential of the lactoferrin complex from Example 2 after 3 months of accelerated processing.

[0016] Figure 5 This is a comparison of the inhibition of IL-6, an inflammatory cytokine, in LPS-induced mouse peritoneal macrophages by different experimental groups.

[0017] Figure 6 The results show the inhibition of proliferation of mouse peritoneal macrophages by different experimental groups. Detailed Implementation

[0018] To facilitate understanding of the present invention, a more complete description will be provided below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0019] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention. The term "and / or" as used in this invention includes any and all combinations of one or more of the associated listed items.

[0020] Unless otherwise specified, experimental methods in the following examples were performed under standard conditions, such as those described in Green and Sambrook et al., *Molecular Cloning: A Laboratory Manual* (2013), or as recommended by the manufacturer. All commonly used chemical reagents used in the examples are commercially available products.

[0021] In some embodiments of the present invention, a lactoferrin complex is disclosed, which is prepared by means of a raw material comprising 5 to 120 parts lactoferrin, 0.5 to 20 parts propolis extract and 0.1 to 10 parts chlorogenic acid.

[0022] In one embodiment, the lactoferrin complex is prepared from a raw material comprising 1 to 10 parts lactoferrin, 0.04 to 1 part propolis extract, and 0.01 to 0.5 parts chlorogenic acid.

[0023] In one embodiment, the lactoferrin complex is prepared from a raw material comprising 1 to 2 parts lactoferrin, 0.04 to 0.08 parts propolis extract, and 0.01 to 0.02 parts chlorogenic acid.

[0024] In one embodiment, the lactoferrin complex further includes 0.001 to 1 part of an antioxidant.

[0025] In one embodiment, the lactoferrin complex further includes 0.005 to 0.5 parts of an antioxidant.

[0026] In one embodiment, the antioxidant is selected from at least one of ascorbic acid, vitamin C, vitamin E, sodium metabisulfite, sodium metabisulfite, and L-cysteine ​​salt.

[0027] In one embodiment, the lactoferrin complex further includes 5 to 500 parts of wall material.

[0028] In one embodiment, the lactoferrin complex further includes 30 to 250 parts of wall material.

[0029] In one embodiment, the wall material is selected from at least one of maltodextrin, β-cyclodextrin, gum arabic, soy protein, and porous starch.

[0030] In other embodiments of the present invention, a method for preparing the above-mentioned lactoferrin complex is disclosed, comprising the following steps: (1) Dissolve lactoferrin powder in 0.008%~0.012% SDS aqueous solution, heat treat at 98℃~102℃ for 3 minutes~5 minutes, cool on ice for 5 minutes~10 minutes, add NP-40 with a final concentration of 0.8%~1.2%, then add 2 U~10 U of PNGase F enzyme and 45 mM~55 mM sodium phosphate buffer at pH 7.5, incubate at 36℃~38℃ for 12 hours~20 hours, then hydrate at 4℃~8℃ for 8~12 hours to prepare a lactoferrin solution with a mass concentration of 1%~10%, and adjust the pH to 8.0~10.0; (2) Add propolis extract and chlorogenic acid to a 75% to 95% ethanol solution at a mass ratio of 1:2 to 5:1, and stir at room temperature for 30 to 120 minutes to dissolve completely, so as to obtain a mixed solution with a chlorogenic acid concentration of 0.5% to 5.0%. (3) Under conditions of 4℃~25℃, slowly add 1 / 10~1 / 30 of the volume of the mixed solution in step (2) to the lactoferrin solution in step (1) while stirring, stir for 0.5 hours~2 hours until the system is visually homogeneous, sonicate at 5℃~20℃ for 5 minutes~10 minutes, continue stirring for 30 minutes~60 minutes, adjust the pH of the system to 6.0~7.4, let stand, dialyze, freeze dry, and the product is obtained.

[0031] In one embodiment, the settling time in step (3) is 8 to 12 hours.

[0032] In one embodiment, the dialysis conditions in step (3) are: dialysis in a 3500Da~7000Da dialysis bag for 46 to 50 hours, with distilled water replaced every 5 to 6 hours.

[0033] In other embodiments of the present invention, the application of the above-mentioned lactoferrin complex in the preparation of anti-inflammatory and antioxidant products is disclosed. The lactoferrin complex can effectively inhibit the activity of Staphylococcus aureus, promote lymphocyte proliferation, and has anti-inflammatory and antioxidant activity. It is stable in quality for 3 months under accelerated conditions.

[0034] In other embodiments of the invention, a product is disclosed, which is made from the above-described lactoferrin complex and excipients. The product may be in solution, solid, or semi-solid form.

[0035] In one embodiment, the excipients include one or more of fillers, disintegrants, flow aids, flavoring agents, surfactants, emulsifiers, and pH flavoring agents.

[0036] In one embodiment, the product is a pharmaceutical or skincare product.

[0037] In one embodiment, the dosage form of the drug is a gel, ointment, powder, granules, tablet, or capsule.

[0038] In one embodiment, the skincare product is a lotion, cream, or serum. The skincare product has anti-inflammatory, antioxidant, whitening, and spot-fading effects.

[0039] In one embodiment, the lactoferrin complex is used in the skin care product at an amount of 0.01wt% to 10wt%.

[0040] In one embodiment, the lactoferrin complex is used in the skin care product at an amount of 0.05wt% to 8wt%.

[0041] In the following embodiments of the present invention, the sources of the reagents or raw materials used are shown in Table 1.

[0042]

[0043] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0044] Example 1: Comparison of properties of lactoferrin complex solutions with different mass ratios In this embodiment, lactoferrin complex solutions were prepared using different mass ratios of lactoferrin (LF), propolis extract (PE), and chlorogenic acid (CA), and the turbidity, particle size distribution, and zeta potential of different lactoferrin complexes were investigated.

[0045] Turbidity: Compare the turbidity with the formazan standard suspension in a turbidity meter (since the suspension is prone to particle sedimentation, it is recommended to prepare the samples one by one and test them as soon as possible after preparation to minimize interference, and shake the samples thoroughly before measurement), and record the turbidity ratio.

[0046] Particle size distribution and Zeta potential: The particle size distribution, Pdl and Zeta potential of the lactoferrin complex solution were determined using a Malvern Zetasizer (ZEN3690) particle size potentiometer.

[0047] The preparation method of lactoferrin complex solution includes the following steps: 1. Preparation of lactoferrin stock solution (LF solution) Lactoferrin was dissolved in a phosphate buffer solution at pH 6.8 and gently stirred at low temperature to prepare a homogeneous LF solution with a concentration of 1.0% (w / v) for later use.

[0048] 2. Preparation of propolis extract stock solution (PE solution) Dissolve an appropriate amount of propolis extract in 95% ethanol and stir thoroughly to obtain a homogeneous PE solution with a solid content of 1.0% (w / v). Seal the solution for later use.

[0049] 3. Preparation of chlorogenic acid stock solution (CA solution) Dissolve an appropriate amount of chlorogenic acid in 95% ethanol and stir thoroughly to obtain a homogeneous CA solution with a solid content of 1.0% (w / v). Seal the solution for later use.

[0050] 4. Preparation of lactoferrin complex solution Under water bath conditions of 10℃±2℃, slowly add the target amount of PE solution and CA solution to a 1% (w / v) LF solution while stirring. Stir the reaction at 20℃±2℃ for 1 hour until the system is visually homogeneous. Adjust the pH of the mixed solution to 6.8 to obtain the final product.

[0051] The results of mass ratio, turbidity, particle size distribution and zeta potential comparison are shown in Table 1.

[0052]

[0053] As shown in Table 1, when the amount of LF is fixed and the amounts of PE and CA are changed, the turbidity ratio of the lactoferrin complex shows a non-linear change with the change of the PE / CA ratio. The turbidity is lower when the PE / CA ratio is low, and a soluble complex is formed. The particle size tends to increase with the increase of the PE / CA ratio. When the PE content is high, the absolute value of the Zeta potential is higher, indicating better ion stability.

[0054] Based on the combined results of particle size and Zeta potential, the optimal mass ratio of LF:PE:CA is within the range of 1~10:0.05~1:0.01~0.5. When PE:CA=1:2~5:1, the particle size is <1000nm, the absolute value of Zeta potential is above 10mV, and the stability of the lactoferrin complex is even better.

[0055] Example 2 Preparation of lactoferrin complex powder This embodiment explores a method for preparing a lactoferrin complex with a mass ratio of LF:PE:CA = 1:0.04:0.01. The method includes the following steps: 1. Preparation of lactoferrin solution Lactoferrin powder was dissolved in 0.01% SDS aqueous solution, denatured at 100℃ for 5 minutes, cooled on ice for 8 minutes, PNGase F enzyme and reaction buffer were added, and incubated at 37℃ for 16 hours. Then, it was hydrated at 6±2℃ for 10 hours to prepare a 1.0% (w / v) lactoferrin stock solution. The pH was adjusted to 8.5 using 2N NaOH solution for later use.

[0056] 2. Preparation of a mixed solution of propolis extract and chlorogenic acid Propolis extract and chlorogenic acid were added to a 75% ethanol solution at a ratio of 4:1 (w / w). The solution was stirred at room temperature for 60 minutes to ensure complete dissolution. The volume was then increased to obtain a mixed solution with a concentration of approximately 0.1% (w / v) based on chlorogenic acid. The solution was then sealed for later use.

[0057] 3. Preparation of lactoferrin complex At 8℃±2℃, a mixture of propolis extract and chlorogenic acid was slowly added to a 1.0% (w / v) lactoferrin stock solution while stirring. The mixture was stirred for 1 hour until the system was visually homogeneous. Then, 0.001% ascorbic acid was added to the system, and the mixture was sonicated for 5 minutes at 8℃±2℃, followed by stirring for another 60 minutes.

[0058] Adjust the pH of the mixed solution to 6.8, let it stand for 12 hours, dialyze it in a 6000Da dialysis bag for 48 hours (replacing distilled water every 6 hours), and freeze-dry it to obtain lactoferrin complex powder.

[0059] Example 3: Topical gel containing lactoferrin complex The preparation method is as follows: 1. Weigh 1.0 g of carbomer 940 and slowly disperse it into a 50 g glycerol-water (3.5:46.5, w / w) mixed solution to avoid clumping. After it has swollen sufficiently, slowly add triethanolamine to adjust the pH to 7.0.

[0060] 2. Add 0.5 g of the lactoferrin complex powder prepared in Example 2, stir and disperse evenly, add 0.02 g of ethylparaben, mix well, add water to 100 g, stir and mix evenly, let stand to defoam, and obtain a gel preparation containing 0.5% lactoferrin complex.

[0061] The gel prepared in this embodiment has the advantages of being refreshing, easy to apply, non-greasy, and biocompatible, and also has anti-inflammatory and antioxidant effects.

[0062] Comparative Example 1 This comparative example uses lactoferrin and propolis extract to prepare a lactoferrin complex. The preparation method includes the following steps: 1. Preparation of lactoferrin solution Same as Example 2.

[0063] 2. Preparation of propolis extract solution Slowly add an appropriate amount of propolis extract to a 75% ethanol solution while stirring. Stir magnetically at room temperature for 60 minutes to ensure complete dissolution, thus preparing a 0.4% (w / v) propolis extract solution. Seal the solution for later use.

[0064] 3. Preparation of lactoferrin complex At 8℃±2℃, slowly add about 1 / 10 volume of propolis extract solution to 1.0% (w / v) lactoferrin stock solution while stirring. Stir for 1 hour until the system is visually homogeneous. Add 0.001% ascorbic acid to the system, sonicate for 5 minutes at 8℃±2℃, and continue stirring for 60 minutes.

[0065] Adjust the pH of the mixed solution to 7.0, let the mixed solution stand for 12 hours, dialyze it in a 6000Da dialysis bag for 48 hours (replace distilled water every 6 hours), and freeze dry to obtain lactoferrin complex powder.

[0066] Comparative Example 2 In this comparative example, a lactoferrin complex was prepared using lactoferrin and chlorogenic acid. The preparation method included the following steps: 1. Preparation of lactoferrin solution Same as Example 2.

[0067] 2. Preparation of chlorogenic acid solution Add an appropriate amount of chlorogenic acid to a 75% ethanol solution while stirring at room temperature. Stir magnetically for 60 minutes at room temperature to ensure complete dissolution, resulting in an ethanol solution containing 0.1% (w / v) chlorogenic acid. Seal the solution for later use.

[0068] 3. Preparation of lactoferrin complex At 8℃±2℃, slowly add about 1 / 10 volume of chlorogenic acid solution to 1.0% (w / v) lactoferrin stock solution while stirring. Stir for 1 hour until the system is visually homogeneous. Add 0.001% ascorbic acid to the system, sonicate for 5 minutes at 8℃±2℃, and continue stirring for 60 minutes.

[0069] Adjust the pH of the mixed solution to 7.0, let the mixed solution stand for 12 hours, dialyze it in a 6000Da dialysis bag for 48 hours (replace distilled water every 6 hours), and freeze dry to obtain lactoferrin complex powder.

[0070] Particle size, zeta potential characterization, and preliminary stability study of the lactoferrin complex in Example 1 and Example 2. An appropriate amount of the lactoferrin complex powder from Example 2 was dissolved in phosphate buffer at pH 6.8 to prepare a 1% (w / v) lactoferrin complex stock solution. Samples were taken to detect the particle size distribution and zeta potential. The results are shown in Table 2. Figures 1-2 .

[0071] The lactoferrin complex powder from Example 2 was placed under accelerated conditions (40±2℃, 75%±5%RH) for 3 months. Samples were then dissolved in phosphate buffer at pH 6.8, and particle size and zeta potential were measured. The results were compared with data from day 0. (See attached table). Figures 3-4 .

[0072] Take an appropriate amount of lactoferrin complex stock solution and adjust the pH of the solution to 5.0 and 3.0 respectively with 0.1N HCl solution and 2N NaOH solution. Immediately detect the turbidity, particle size distribution and zeta potential of the solution. The results are shown in Table 2.

[0073]

[0074] From Table 2 and Figures 1-4 It can be seen that the lactoferrin complex powder prepared in Example 2 of the present invention exhibits fluctuations in zeta potential in a simulated gastrointestinal pH environment, but all remain within -10mV to -30mV. The particle size is slightly aggregated, but the particle size distribution is less than 300nm, indicating that it can effectively maintain the nanoscale particle size. Even after 3 months of acceleration, it still maintains a highly dispersed state, indicating excellent stability and high activity.

[0075] Experimental Example 2: In vitro anti-inflammatory efficacy test Lactoferrin powder, lactoferrin complex powder of Example 2, lactoferrin complex powder of Comparative Example 1 and lactoferrin complex powder of Comparative Example 2 were taken separately and systematically evaluated in a stratified control design to evaluate their synergistic effect and mechanism in a macrophage inflammation model. The experimental groups are shown in Table 3.

[0076]

[0077] The test method is as follows: 1. Mouse peritoneal macrophages were seeded into cell culture dishes, and DMEM high glucose culture medium (containing 10% BFS + 1% penicillin antibody) was added. The cells were incubated overnight (12-16 hours, 37°C, 5% CO2). Under a microscope, the cells were observed to be adherent cells, and the viability of cells stained with trypan blue was over 95%. 2. Take the above adherent cells and prepare 2~5 x 10⁻⁵ cells. 4 Cell suspension at 100 μL per well was seeded into 96-well plates, grouped as described above, with a density of 2–5 x 10⁶ cells / mL. 3 Cells / well, 6 replicates per group, 2 culture plates prepared in parallel, and cultured at 37℃, 5% CO2 for 12-24 hours; 3. Before adding LPS, all experimental groups should first add the solution of each test sample, incubate at 37°C for 2 hours, then add LPS solution with a final concentration of 0.5 μg / mL, and continue culturing for 6 to 24 hours. 4. Cell culture supernatant was collected at 6 h and 24 h of incubation, respectively. Following the ELISA kit instructions, the IL-6 secretion level of each group was detected. Using the model group as a reference, the relative expression level of IL-6 in each group was calculated. Results are shown below. Figure 5 Cell viability was assessed using the CCK-8 assay. 10 μL of CCK-8 reagent was added to each well in three replicates of each group, and incubation was continued for 1 hour. The absorbance at 450 nm was measured using a microplate reader to calculate cell viability and evaluate the effect of LPS modeling agent on cell survival, excluding cytotoxic interference. Results are shown below. Figure 6 .

[0078] from Figure 6 It can be seen that the cell viability of the model group (Group B, 0.5 μg / mL LPS), Group H (0.01% (w / v) of Example 2) and the blank control group (Group A) is comparable, indicating that the concentration of the selected modeling agent and the concentration of the test sample have no effect on the cell viability of mouse peritoneal macrophages and the toxic side effects are negligible.

[0079] Figure 5The results showed that after adding 0.5 μg / mL LPS to the model group (group B), the relative expression level of IL-6 in the cell supernatant was high, and the inflammation model was successfully established. The expression of IL-6 in the cell supernatant of group H (0.01% (w / v) of Example 2) was comparable to that of the blank control group (group A), indicating that the selected culture medium and the concentration of the test sample did not have a stimulating effect on mouse peritoneal macrophages. The expression levels of IL-6 in experimental groups C through G were reduced to varying degrees. Among them, group G (positive control drug) < group D (0.01% (w / v) Example 2) < group F (0.01% (w / v) Comparative Example 2) < group E (0.01% (w / v) Comparative Example 1) < group C (0.01% (w / v) LF), indicating that lactoferrin has antioxidant activity. The antioxidant activity is significantly enhanced when combined with propolis extract or chlorogenic acid. The lactoferrin complex prepared by combining lactoferrin, propolis extract, and chlorogenic acid is significantly better than the single or compound formulations, has antioxidant activity comparable to the positive control drug, and has high safety performance.

[0080] Test Example 3: In vitro antibacterial test The lactoferrin complex powder from Example 2 was used to investigate its inhibitory activity against Staphylococcus aureus using a micro-broth dilution method. The specific steps included: 1. Preparation of bacterial suspension: Staphylococcus aureus ATCC 25923 was inoculated into tryptic soy peptone medium and incubated at 30℃~35℃ for 18~24 hours. The suspension was then diluted to 10 mM sterile PBS solution. 6 CFU / ml bacterial suspension; 2. Sample addition and incubation: Perform the procedure in a sterile 96-well plate according to Table 4, with 3 replicates for each concentration; seal the 96-well plate with sealing film after sample addition and incubate at 37℃ for 24 hours.

[0081] The preparation method of the test sample in the table is as follows: take the lactoferrin complex of Example 2, dissolve it in 10mM sterile PBS solution to prepare a 1mg / mL stock solution, and then dilute it with 10mM sterile PBS solution to a series of concentrations.

[0082] 3. OD 600 Measurement: After the culture is completed, gently shake to mix the liquid in the wells, and use an ELISA reader to measure the absorbance value of each well at a wavelength of 600 nm.

[0083] 4. Data Processing and Analysis: Calculate the mean OD of each experimental group and the control group. 600 The value is set at 90% growth inhibition rate as the threshold for determining complete bacterial inhibition.

[0084] The experimental results are shown in Table 5.

[0085]

[0086] Table 5 shows that there was no significant difference in OD values ​​between the solvent control group and the growth control group, indicating that the solvent used had no effect on the growth of Staphylococcus aureus. 1 µg / mL gentamicin sulfate completely inhibited bacterial growth, and the OD value was close to that of the blank control, indicating that the experimental system was reliable. Within the concentration range of 256 µg / mL to 2 µg / mL, the OD values ​​of each well were... 600 The OD values ​​were not statistically different from those of the blank control group, and the growth inhibition rates were all >98%, indicating that bacterial growth was completely inhibited. At 1 µg / mL, the OD value increased significantly, and the inhibition rate was only 28.9%, indicating that the antibacterial effect was greatly weakened. At 0.5 µg / mL, the OD value was close to that of the growth control group, with almost no inhibitory effect.

[0087] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0088] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A lactoferrin complex, characterized in that, It is prepared from raw materials comprising 5 to 120 parts lactoferrin, 0.5 to 20 parts propolis extract and 0.1 to 10 parts chlorogenic acid by weight.

2. The lactoferrin complex according to claim 1, characterized in that, It is prepared from raw materials including 1 to 10 parts lactoferrin, 0.04 to 1 part propolis extract and 0.01 to 0.5 parts chlorogenic acid; preferably, it is prepared from raw materials including 1 to 2 parts lactoferrin, 0.04 to 0.08 parts propolis extract and 0.01 to 0.02 parts chlorogenic acid.

3. The lactoferrin complex according to claim 1 or 2, characterized in that, It also includes 0.001 to 1 part of antioxidant, preferably 0.005 to 0.5 parts of antioxidant.

4. The lactoferrin complex according to claim 3, characterized in that, The antioxidant is selected from at least one of ascorbic acid, vitamin C, vitamin E, sodium metabisulfite, sodium metabisulfite, and L-cysteine ​​salt.

5. The lactoferrin complex according to claim 1 or 2, characterized in that, The lactoferrin complex further includes 5 to 500 parts of wall material, preferably 30 to 250 parts of wall material.

6. The lactoferrin complex according to claim 5, characterized in that, The wall material is selected from at least one of maltodextrin, β-cyclodextrin, gum arabic, soy protein, and porous starch.

7. The method for preparing the lactoferrin complex according to any one of claims 1 to 6, characterized in that, Includes the following steps: (1) Dissolve lactoferrin powder in 0.008%~0.012% SDS aqueous solution, heat treat at 98℃~102℃ for 3 minutes~5 minutes, cool on ice for 5 minutes~10 minutes, add NP-40 with a final concentration of 0.8%~1.2%, then add 2 U~10 U of PNGase F enzyme and 45 mM~55 mM sodium phosphate buffer at pH 7.5, incubate at 36℃~38℃ for 12 hours~20 hours, then hydrate at 4℃~8℃ for 8~12 hours to prepare a lactoferrin solution with a mass concentration of 1%~10%, and adjust the pH to 8.0~10.0; (2) Add propolis extract and chlorogenic acid to a 75% to 95% ethanol solution at a mass ratio of 1:2 to 5:1, and stir at room temperature for 30 to 120 minutes to dissolve completely, so as to obtain a mixed solution with a chlorogenic acid concentration of 0.5% to 5.0%. (3) Under conditions of 4℃~25℃, slowly add 1 / 10~1 / 30 of the volume of the mixed solution in step (2) to the lactoferrin solution in step (1) while stirring, stir for 0.5 hours~2 hours until the system is visually homogeneous, sonicate at 5℃~20℃ for 5 minutes~10 minutes, continue stirring for 30 minutes~60 minutes, adjust the pH of the system to 6.0~7.4, let stand, dialyze, freeze dry, and the product is obtained.

8. The use of the lactoferrin complex according to any one of claims 1 to 6 in the preparation of anti-inflammatory and antioxidant products.

9. A product characterized in that, It is made from the lactoferrin complex and excipients as described in any one of claims 1 to 6.

10. The product according to claim 9, characterized in that, The excipients include one or more of the following: fillers, disintegrants, flow aids, flavoring agents, surfactants, emulsifiers, and pH flavoring agents; And / or, the product is a pharmaceutical or skincare product; Preferably, the dosage form of the medicine is a gel, ointment, powder, granules, tablet or capsule; Preferably, the skincare product is a lotion, cream, or serum; Preferably, the lactoferrin complex is used in the skin care product at an amount of 0.01wt% to 10wt%, more preferably 0.05wt% to 8wt%.

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