Lonicera caerulea compound powder as well as preparation method and application thereof

Abstract

The invention discloses lonicera caerulea compound powder as well as a preparation method and application thereof, and relates to lonicera caerulea compound powder as well as a preparation method and application thereof. The objective of the invention is to solve the problem of myopia caused by long-time use of electronic products, few outdoor activities and short eye distance. Every 3 g of the lonicera caerulea compound powder contains 100 mg of lutein ester microcapsule powder with the specification of 10%, 20 mg of zeaxanthin microcapsule powder with the specification of 10%, 450 mg of lycium barbarum polysaccharide with the specification of 80% and 2430 mg of lonicera caerulea freeze-dried powder, and the 2430 mg of lonicera caerulea freeze-dried powder contains 1780 mg of maltodextrin. The lonicera caerulea compound powder is used for dispelling the effects of alcohol, removing DPPH, ABTS and O2-free radicals and resisting lipid peroxidation. The lonicera caerulea compound powder has strong antioxidant capacity, so that good double-effect effects of protecting eyes, protecting liver and dispelling the effects of alcohol are achieved. The invention belongs to the technical field of prevention and control of myopia and hangover alleviating.

Description

Technical Field

[0001] The invention relates to a composite powder of Lonicera edulis and a preparation method and application thereof. Background Art

[0002] Blue honeysuckle (Lonicera caerulea L.), also known as blue honeysuckle, mountain eggplant, etc., is a small berry with a blue-purple color. It is rich in polyphenols and has multiple functional activities such as anti-tumor, antioxidant, liver function improvement, anti-inflammatory and antibacterial, and relief of visual fatigue. Summary of the Invention

[0003] The purpose of the present invention is to solve the technical problem of myopia caused by long-term use of electronic products, lack of outdoor activities, and close eye distance, and provide a caerulea edulis composite powder and its preparation method and application.

[0004] The invention relates to a blue indigo fruit composite powder, wherein each 3 g of the blue indigo fruit composite powder contains 100 mg of 10% lutein ester microcapsule powder, 20 mg of 10% zeaxanthin microcapsule powder, 450 mg of 80% wolfberry polysaccharide, and 2430 mg of blue indigo fruit freeze-dried powder, wherein the 2430 mg of blue indigo fruit freeze-dried powder contains 1780 mg of maltodextrin.

[0005] Preparation method of the composite powder of Indigofera edulis:

[0006] Water and fresh Lonicera edulis fruit were placed in a juicer at a material-liquid ratio of 1 g:3 mL to 1 g:5 mL, and the juice was collected by filtration. 10% to 20% maltodextrin by volume was added as a filler. The juice was freeze-dried in vacuum and then crushed through an 80-mesh sieve to obtain Lonicera edulis freeze-dried powder. The Lonicera edulis freeze-dried powder, Lycium barbarum polysaccharides, lutein esters, and zeaxanthin were mixed in proportion to prepare Lonicera edulis composite powder, in which the addition ratio of Lycium barbarum polysaccharides was 10% to 20%. After optimization, every 3 g of Lonicera edulis composite powder contained 100 mg of 10% lutein ester microcapsule powder, 20 mg of 10% zeaxanthin microcapsule powder, 450 mg of 80% Lycium barbarum polysaccharides, and 2430 mg of Lonicera edulis freeze-dried powder. The 2430 mg of Lonicera edulis freeze-dried powder contained 1780 mg of maltodextrin.

[0007] The blue loquat fruit composite powder is used for sobering up.

[0008] The blue loquat fruit composite powder is used for eye protection.

[0009] The blue loquat composite powder is used to remove DPPH, ABTS, O2 - Free radicals.

[0010] The blue loquat fruit composite powder is used for resisting lipid peroxidation.

[0011] Advantages of the Indigofera edulis composite powder of the present invention:

[0012] The results were significantly higher than those of each individual component (lyophilized powder of blue honeysuckle, wolfberry polysaccharide, lutein ester, and zeaxanthin). Cellular experiments demonstrated that the blue honeysuckle powder composite was non-cytotoxic. The proliferation of ARPE-19 cells treated with the blue honeysuckle powder composite was significantly higher than that of each individual component, indicating a synergistic effect between the components in alleviating retinal damage. Furthermore, the blue honeysuckle powder composite not only effectively alleviated H2O2-induced retinal oxidative stress, but also significantly increased the activities of CAT, SOD, and GSH-px enzymes in ARPE-19 cells, increased GSH content, decreased MDA content, enhanced the antioxidant capacity of ARPE-19 cells, and promoted cell proliferation. By protecting against oxidative stress, the blue honeysuckle powder composite may mitigate retinal damage, alleviate visual fatigue, and reduce the risk of AMD.

[0013] The liver and eyes are closely linked, and according to Traditional Chinese Medicine theory, "the liver opens up to the eyes." This study simultaneously investigated the effects of a calyx edulis compound powder on an ethanol-induced HepG2 cell injury model and found that it exhibited significant alcohol detoxification and liver-protecting effects. This suggests that the potent antioxidant capacity of the calyx edulis compound powder provides a dual-effect of eye protection, liver protection, and alcohol detoxification. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is a comparison chart of the single-factor test results for the preparation of the blue loquat fruit composite powder in Experiment 1. In the figure, (A) shows the comparison chart of the effect of the material-liquid ratio on the cell survival rate, (B) shows the comparison chart of the effect of the maltodextrin addition amount on the cell survival rate, and (C) shows the comparison chart of the effect of the wolfberry polysaccharide addition amount on the cell survival rate;

[0015] Figure 2 This is a three-dimensional response surface curve diagram in Experiment 1. (A) shows the response surface analysis and contour map of the effects of material-liquid ratio and maltodextrin addition on cell viability, (B) shows the response surface analysis and contour map of the effects of Lycium barbarum polysaccharide addition and material-liquid ratio on cell viability, and (C) shows the response surface analysis and contour map of the effects of Lycium barbarum polysaccharide addition and maltodextrin addition on cell viability.

[0016] Figure 3 This is a comparison of the antioxidant activity of the blue indigo fruit compound powder and the single component in Experiment 1. In the figure, (A) shows the comparison of the DPPH scavenging rate of the blue indigo fruit compound powder and the single component, (B) shows the ABTS scavenging rate of the blue indigo fruit compound powder and the single component, and (C) shows the superoxide anion free radical (O2 - ·) Comparison of clearance rates. (D) Comparison of clearance rates of Lonicera edulis compound powder and single components on lipid peroxidation.

[0017] Figure 4This is a diagram of the ARPE-19 cell oxidative damage model induced by H2O2 in Experiment 1;

[0018] Figure 5 The figures show the cytotoxicity test and LDH release test of the Indigofera edulis compound powder on ARPE-19 cells in Experiment 1. (A) shows the cytotoxicity test of the Indigofera edulis compound powder on ARPE-19 cells, and (B) shows the LDH release test of the Indigofera edulis compound powder on ARPE-19 cells. Different lowercase letters in the figures indicate significant differences (P<0.05).

[0019] Figure 6 This is a comparative diagram of the effects of the blue Lonicera compound powder on the proliferation of ARPE-19 cells damaged by H2O2 oxidation in Experiment 1. (A) shows the comparative diagram of the blue Lonicera compound powder and its components on the proliferation of ARPE-19 cells damaged by H2O2 oxidation. (B) shows the comparative diagram of the blue Lonicera compound powder on the proliferation of ARPE-19 cells damaged by H2O2 oxidation. Different lowercase letters in the figures indicate significant differences (P<0.05).

[0020] Figure 7 This is a comparative diagram of the effects of the blue indigo fruit compound powder on the CAT, SOD, GSH-px enzyme activities and GSH and MDA contents in ARPE-19 cells damaged by H2O2 oxidation in Experiment 1. In the figure, (A) shows the comparative diagram of the effects of different concentrations of blue indigo fruit compound powder on the CAT enzyme activity in ARPE-19 cells damaged by H2O2 oxidation, (B) shows the comparative diagram of different concentrations of blue indigo fruit compound powder on the SOD enzyme activity in ARPE-19 cells damaged by H2O2 oxidation, (C) shows the comparative diagram of different concentrations of blue indigo fruit compound powder on the GSH-px enzyme activity in ARPE-19 cells damaged by H2O2 oxidation, (D) shows the comparative diagram of different concentrations of blue indigo fruit compound powder on the GSH content in ARPE-19 cells damaged by H2O2 oxidation, (E) shows the comparative diagram of different concentrations of blue indigo fruit compound powder on the MDA content in ARPE-19 cells damaged by H2O2 oxidation, and different lowercase letters in the figures indicate significant differences (P<0.05);

[0021] Figure 8 This is a comparison chart of the effects of the blue loquat compound powder on the proliferation of HepG2 cells damaged by ethanol oxidation in Experiment 1;

[0022] Figure 9 This is a comparison chart of the effects of blue indigo fruit compound powder, blue indigo fruit freeze-dried powder and wolfberry polysaccharide on the proliferation of HepG2 cells damaged by ethanol oxidation in experiment 1. DETAILED DESCRIPTION

[0023] The technical solution of the present invention is not limited to the specific embodiments listed below, but also includes any combination of the specific embodiments.

[0024] Specific embodiment 1: In this embodiment, the blue indigo fruit composite powder contains 100 mg of lutein ester microcapsule powder with a specification of 10%, 20 mg of zeaxanthin microcapsule powder with a specification of 10%, 450 mg of wolfberry polysaccharide with a specification of 80% and 2430 mg of blue indigo fruit freeze-dried powder, and the 2430 mg of blue indigo fruit freeze-dried powder contains 1780 mg of maltodextrin.

[0025] Specific embodiment 2: This embodiment differs from the specific embodiment 1 in the preparation method of the blue loquat fruit composite powder:

[0026] Water and fresh Lonicera edulis fruit were placed in a juicer at a material-liquid ratio of 1 g:3 mL to 1 g:5 mL, and the juice was collected by filtration. 10% to 20% by volume of maltodextrin was added as a filler. The mixture was freeze-dried under vacuum and then pulverized through an 80-mesh sieve to obtain Lonicera edulis freeze-dried powder. The Lonicera edulis freeze-dried powder, Lycium barbarum polysaccharides, lutein esters, and zeaxanthin were then mixed uniformly in proportion to obtain a Lonicera edulis composite powder, wherein the Lycium barbarum polysaccharide addition ratio was 10% to 20%. After optimization, per 3 g of the Lonicera edulis composite powder, 100 mg of 10% lutein ester microcapsule powder, 20 mg of 10% zeaxanthin microcapsule powder, 450 mg of 80% Lycium barbarum polysaccharides, and 2430 mg of Lonicera edulis freeze-dried powder were contained. The 2430 mg of Lonicera edulis freeze-dried powder contained 1780 mg of maltodextrin. Other ingredients were the same as those in the first embodiment.

[0027] Specific embodiment 3: This embodiment differs from specific embodiment 1 or 2 in that the material-liquid ratio is 1 g:4 mL. Other aspects are the same as specific embodiment 1 or 2.

[0028] Specific embodiment 4: The Indigofera edulis composite powder described in any one of specific embodiments 1 to 3 is used for sobering up.

[0029] Specific embodiment 5: The composite powder of Indigofera edulis described in any one of specific embodiments 1 to 3 is used for eye protection.

[0030] Specific embodiment six: The blue loquat composite powder described in one of the specific embodiments one to three is used to remove DPPH, ABTS, O2 - Free radicals.

[0031] Specific embodiment seven: The composite powder of Indigofera edulis described in any one of specific embodiments one to three is used for resisting lipid peroxidation.

[0032] The following experiments were used to verify the effects of the present invention:

[0033] Experiment 1:

[0034] 1 Materials and Methods

[0035] 1.1 Materials and Reagents

[0036] Blue indigo fruit, wild blue indigo fruit base in Boli County, Heilongjiang Province; 80% wolfberry polysaccharide, Xi'an Xihai Biotechnology Co., Ltd.; 10% lutein ester microcapsule powder, Xi'an Haoyu Kangze Biotechnology Co., Ltd.; 10% zeaxanthin microcapsule powder, Shaanxi Zelang Biotechnology Co., Ltd.; food-grade maltodextrin; ARPE-19 retinal pigment epithelial cells, Cell Center, Shanghai Branch, Chinese Academy of Sciences.

[0037] 1.2 Instruments and Equipment

[0038] BCM-1000 biological purification workbench, Shanghai Glass Instrument Factory; imark-1681130 microplate reader, Biorad, USA; KQ-100DE CNC ultrasonic cleaner, Kunshan Ultrasonic Instrument Co., Ltd.; Thermo-3111 CO2 incubator, Thermo Corporation, USA; DP10 inverted microscope, Olympus, Japan; fluorescence microscope, Nanjing Jiangnan Yongxin Optics Co., Ltd.

[0039] 1.3 Methods

[0040] 1.3.1 Preparation of Indigofera edulis composite powder

[0041] Water and fresh blueberry fruits were placed in a juicer for pulping. The juice was collected by filtration, and maltodextrin was added as a filler. The mixture was freeze-dried under vacuum and then pulverized through an 80-mesh sieve to obtain blueberry freeze-dried powder. The blueberry composite powder is specified to be 3 g. Each 3 g of blueberry composite powder contains 100 mg of 10% lutein ester microcapsule powder, 20 mg of 10% zeaxanthin microcapsule powder, 450 mg of 80% wolfberry polysaccharide, and 2430 mg of blueberry freeze-dried powder. The 2430 mg of blueberry freeze-dried powder contains 1780 mg of maltodextrin. The blueberry freeze-dried powder, wolfberry polysaccharide, lutein ester, and zeaxanthin were mixed uniformly in the appropriate proportion to obtain the blueberry composite powder for oral administration.

[0042] 1.3.2 Experimental study on the optimization of the formula of the compound powder of Lonicera edulis

[0043] 1.3.2.1 Single-factor experiment

[0044] 5 g of fresh Lonicera edulis fruit was accurately weighed to prepare lyophilized powder of Lonicera edulis. The addition ratio of lutein esters and zeaxanthin was fixed at 5:1 (the total mass fraction of lutein esters and zeaxanthin in the Lonicera edulis composite powder was 4%) to prepare Lonicera edulis composite powder. The survival rate of ARPE-19 cells damaged by H2O2 was evaluated by the composite powder. The effects of different factors including solid-liquid ratio (1 g:1 mL, 1 g:2 mL, 1 g:3 mL, 1 g:4 mL, 1 g:5 mL), maltodextrin addition (mass fraction 10%, 15%, 20%, 25%, 30%) and Lycium barbarum polysaccharide addition (mass fraction 5%, 10%, 15%, 20%, 25%) on the survival rate of ARPE-19 cells damaged by H2O2 were studied.

[0045] 1.3.2.2 Response surface optimization design experiment

[0046] According to the results of the single-factor experiment, the survival rate of ARPE-19 cells damaged by H2O2 was used as the response value to conduct response surface analysis to optimize the formula of the blue loquat fruit composite powder. The response surface test factors and levels are shown in Table 1.

[0047] Table 1 Box-Behnkens design factor level table

[0048]

[0049] 1.3.3 Determination of performance indicators

[0050] 1.3.3.1 Liquidity Testing

[0051] A funnel with a diameter of 5 cm was fixed vertically, with the funnel opening about 10 cm away from the table. A piece of white paper was placed on the table. 10 g of the blue loquat fruit compound powder was added from the funnel opening. The radius and the stacking height formed on the white paper were measured, and the angle of repose was calculated according to formula (1).

[0052]

[0053] 1.3.3.2 Dispersibility and solubility testing

[0054] Weigh 1 g of the Lonicera edulis composite powder into a 250 mL beaker. Add 100 mL of distilled water at 25°C. Stir with a magnetic stirrer until fully dissolved. Record the time it takes for the powder to dissolve. After the powder dissolves, take 25 mL of the solution and dry it in a petri dish at 105°C to constant weight. Calculate the dry matter content of the solution.

[0055] 1.3.3.3 Moisture content and bulk density testing

[0056] The moisture content was determined using the direct drying method specified in GB5009.3-2016, "National Food Safety Standard - Determination of Moisture in Foods." The bulk density is calculated as the mass per unit volume of the edodes fruit compound powder.

[0057] 1.3.4 Antioxidant activity assay

[0058] 1.3.4.1 DPPH free radical scavenging ability assay

[0059] After the reaction between the sample solution and the DPPH reaction solution was completed, the absorbance was measured at a wavelength of 517 nm. An equal volume of deionized water was used instead of the sample solution as a blank control; an equal volume of anhydrous ethanol was used instead of the DPPH solution as a sample control. Ascorbic acid was used as a positive control. The clearance rate was calculated according to the following formula:

[0060]

[0061] 1.3.4.2 Determination of ABTS free radical scavenging ability

[0062] Prepare the ABTS reaction solution so that its absorbance at 734 nm is 0.7 ± 0.02. Add equal volumes of the sample solution and ABTS reaction solution to a 96-well microtiter plate. Incubate in the dark at room temperature for 15 minutes. Measure the absorbance of the mixed solution at 734 nm. Replace the sample solution with an equal volume of deionized water as a blank control; replace the ABTS reaction solution with an equal volume of distilled water as a sample control. Use ascorbic acid as a positive control. Calculate the clearance rate according to the following formula:

[0063]

[0064] 1.3.4.3 Determination of superoxide anion free radical scavenging ability

[0065] The absorbance was measured at a wavelength of 320 nm. An equal volume of distilled water was used instead of the sample solution as a blank control. An equal volume of distilled water was used instead of the pyrogallol solution as a sample control. Ascorbic acid was used as a positive control. The clearance rate was calculated according to the following formula:

[0066]

[0067] 1.3.4.4 Anti-lipid peroxidation activity test

[0068] The absorbance of the reaction supernatant was measured at 532 nm; an equal volume of distilled water was used instead of the sample as a blank control; an equal volume of distilled water was used instead of soybean lecithin as a sample control, and ascorbic acid was used as a positive control.

[0069]

[0070] 1.3.5 ARPE-19 cell culture

[0071] ARPE-19 cells were cultured in a DMEM / F12 basal medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin double-antibody complete medium at 37°C in a constant temperature incubator with 5% CO2.

[0072] 1.3.6 Establishment of H2O2-induced ARPE-19 cell injury model and grouping

[0073] ARPE-19 human retinal pigment epithelial cells were treated with hydrogen peroxide (H2O2) at concentrations of 100, 250, 500, 750, and 1000 μmol / L (μM) for 24 h to establish an oxidative stress injury model.

[0074] This study was divided into four groups: control group, H2O2 group, sample group and co-incubation group.

[0075] (1) Control group: cells were cultured normally with DMEM / F12 complete medium without any intervention.

[0076] (2) H2O2 group: H2O2 with a final concentration of 500 μM was added to the cell culture medium and cultured for 24 h.

[0077] (3) Sample group: Sample solutions with different final concentrations were added to the cell culture medium for pretreatment for 24 h, and then H2O2 with a final concentration of 500 μM was added for 24 h.

[0078] (4) Sample + H2O2 co-incubation group: Sample solution with a final concentration of 800 μg / mL and H2O2 with a final concentration of 500 μM were added to the cell culture medium and co-cultured for 24 h.

[0079] 1.3.7 Toxicity test of Lonicera edulis compound powder on ARPE-19 cells

[0080] When the cells grew to 80%-90%, they were prepared into single cell suspension and the density of ARPE-19 cell suspension was adjusted to 1×10 5 The ARPE-19 cell suspension was pipetted evenly and then added to a 96-well plate at a volume of 100 μL per well. The plate was cultured in a 37°C, 5% CO2 incubator. Once the cells were fully adhered and in good condition, the old culture medium was discarded and complete culture medium containing sample solutions at different final concentrations was added to continue the culture. After the culture was completed, the cell viability was determined using the LDH and CCK-8 assays, and the cell viability was calculated as follows:

[0081]

[0082] 1.3.8 Effect of Indigofera edulis compound powder on H2O2-induced oxidative damage model in ARPE-19 cells

[0083] 1.3.8.1 Cell proliferation assay

[0084] 1×10 5 ARPE-19 cells were seeded into 96-well plates at a concentration of 100 cells / mL and cultured in a constant temperature incubator at 37°C and 5% CO2. After the cells were observed to be completely adherent and in good condition under a microscope, the old culture medium was discarded and the corresponding culture medium was added to the control group, H2O2 group, and sample group respectively. The culture was continued and the cell survival rate was determined and calculated using the CCK-8 method.

[0085] 1.3.8.3 Determination of SOD, CAT, GSH-px enzyme activities and GSH and MDA contents

[0086] 1×10 5 ARPE-19 cells were repeatedly pipetted evenly at a concentration of 10 cells / mL and seeded into 6-well plates at a volume of 2 mL per well. The plates were then cultured in a constant temperature incubator. After complete cell attachment, the old culture medium was discarded and the corresponding culture medium was added to the control, H2O2, sample, and co-incubation groups. After the incubation period, the culture medium was discarded, and the cells were digested with trypsin and collected by centrifugation. The cells were processed and assayed according to the methods provided in the instructions for the superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-px), glutathione (GSH), and malondialdehyde (MDA) detection kits.

[0087] 1.3.9 Effect of Indigofera edulis compound powder on ethanol-induced oxidative damage model of HepG2 cells

[0088] In the laboratory, an ethanol-induced oxidative damage model for HepG2 cells was established, resulting in an IC50 of 500 mmol / L ethanol. Subsequently, a proliferation assay was conducted on HepG2 cells using a 3g edible indigo fruit powder (per 3g of edible indigo fruit powder, containing 100 mg of 10% lutein ester microcapsules, 20 mg of 10% zeaxanthin microcapsules, 450 mg of 80% lycium barbarum polysaccharides, and 2430 mg of edible indigo fruit freeze-dried powder, with 1780 mg of maltodextrin in the 2430 mg of edible indigo fruit freeze-dried powder). The results were published online in the journal Cell Biology. Since both edible indigo fruit and lycium barbarum polysaccharides have been previously studied for their alcohol-detoxifying effects, a comparative study was conducted using the edible indigo fruit powder and the edible indigo fruit freeze-dried powder plus lycium barbarum polysaccharides. The edible indigo fruit freeze-dried powder and lycium barbarum polysaccharides were prepared in the same proportions as in the edible indigo fruit powder.

[0089] 1×10 5 ARPE-19 cells were seeded into 96-well plates at a concentration of 100 cells / mL and cultured in a constant temperature incubator at 37°C and 5% CO2. After the cells were observed to be completely attached and in good condition under a microscope, the old culture medium was discarded and the corresponding culture medium was added to the control group, ethanol group, and sample group respectively. The culture was continued and the cell survival rate was determined and calculated using the CCK-8 method.

[0090] 1.4 Statistical Analysis

[0091] Statistical analysis was performed using SPSS 26.0 software, and one-way ANOVA was performed using Duncan's multiple significant difference test (P ≤ 0.05). All data were based on three independent replicates, and the data were recorded as mean ± SD. Graphpad Prism 8.0.2 was used to draw graphs.

[0092] 2 Results and Discussion

[0093] 2.1 Single-factor test results

[0094] The appropriate material-liquid ratio can most effectively complete the extraction of blue indigo fruit polyphenols, and the appropriate amount of maltodextrin added plays a vital role in the shaping and biological activity of blue indigo fruit freeze-dried powder. Figure 1 A shows that when the material-liquid ratio is 1:4, the survival rate of ARPE-19 cells treated with the blue loquat composite powder reaches a peak value of 127.80%. Figure 1 B shows that with the increase of maltodextrin addition, the cell survival rate first increases and then decreases. When the maltodextrin addition amount is 15%, the cell survival rate is the highest. Figure 1C shows that the survival rate of ARPE-19 cells gradually increased when the addition amount of Lycium barbarum polysaccharides was less than 15%, and gradually decreased when the addition amount was greater than 15%. Therefore, the response surface optimization experiment was carried out under the conditions of solid-liquid ratio of 1:3-1:4, maltodextrin addition of 10-20%, and Lycium barbarum polysaccharide addition of 10-20%.

[0095] 2.2 Response surface optimization of the formula of Lonicera edulis composite powder

[0096] To determine the optimal formula of Lonicera edulis compound powder, a response surface optimization experiment was designed using Design-Expert 8.0.6, with the survival rate of ARPE-19 cells as the response value.

[0097] 2.2.2 Response surface analysis and optimization

[0098] The three-dimensional response surface diagram is shown in Figure 2 The three-dimensional graph shows the interrelationships between factors and displays the optimal parameters for each. The effects of the three factors, material-to-liquid ratio, maltodextrin addition, and Lycium barbarum polysaccharide addition, on ARPE-19 cell viability are as follows: material-to-liquid ratio (A) > maltodextrin addition (B) > Lycium barbarum polysaccharide addition (C).

[0099] 2.2.3 Box-Behnken regression model validation

[0100] Based on regression model analysis, the optimal process parameters for the formulation of the blue Lonicera edulis composite powder were determined to be: a solid-liquid ratio of 1:4.08 (g / mL), a maltodextrin addition of 15.44%, and a wolfberry polysaccharide addition of 15.19%. Under these conditions, the predicted cell viability was 126.16%. Considering actual production, the three parameters were adjusted to a solid-liquid ratio of 1:4 (g / mL), a maltodextrin addition of 15%, and a wolfberry polysaccharide addition of 15%. Three independent parallel experiments were conducted. Under these conditions, the cell viability was (126.65 ± 0.81)%, close to the predicted value, demonstrating the feasibility of response surface analysis for optimizing the formulation of the blue Lonicera edulis composite powder.

[0101] 2.3 Performance indicators of blue indigo fruit compound powder

[0102] The performance indicators of the edulis fruit composite powder are shown in Table 2. The angle of repose of the edulis fruit composite powder was 35.57°, which is less than 40°, indicating good flowability. The test results also showed that the dissolution time of the edulis fruit composite powder was 31.04 s, and the dry matter content of the solution reached 97.49%, indicating good dispersibility and solubility. The test results showed that the moisture content of the edulis fruit composite powder was 2.98%, meeting the standard specified in GB / T 29602-2013. The powder bulk density was 0.53 g / mL. The edulis fruit composite powder had fine and uniform particles, and no significant sedimentation was observed after 3 days of dissolution, indicating good stability.

[0103] Table 2 Performance index results of blue loquat fruit composite powder

[0104]

[0105] 2.4 Antioxidant activity of Lonicera edulis composite powder

[0106] The blue indigo fruit composite powder (each 3 g of blue indigo fruit composite powder contains 100 mg of 10% lutein ester microcapsule powder, 20 mg of 10% zeaxanthin microcapsule powder, 450 mg of 80% wolfberry polysaccharide and 2430 mg of blue indigo fruit freeze-dried powder, and 2430 mg of blue indigo fruit freeze-dried powder contains 1780 mg of maltodextrin) was dissolved in PBS and then gradiently diluted with culture medium to final concentrations of solutions with different concentrations to obtain blue indigo fruit composite powder solutions with different concentrations.

[0107] DPPH and ABTS are two commonly used methods to measure antioxidant activity. Superoxide anion radical (O2 - ·) is a common free radical in the eye, excessive O2 - It can cause lipid peroxidation in retinal cells, which is an important sign of oxidative stress. Figure 3 The results showed that the blue loquat powder solution had good DPPH, ABTS, O2 - Free radical scavenging ability and anti-lipid peroxidation effect, and as the concentration increases, the antioxidant capacity becomes stronger. When the blue loquat fruit compound powder is 2 mg / mL, DPPH, ABTS, O2 - The ABTS free radical scavenging capacity of the blueberry powder was no different from that of ascorbic acid, and its anti-lipid peroxidation capacity was higher than that of ascorbic acid. Furthermore, the antioxidant capacity of the blueberry powder was significantly higher than that of any individual component, indicating a synergistic antioxidant effect between the components of the powder.

[0108] 2.5 Cell assay

[0109] 2.5.1 Establishment of H2O2-induced ARPE-19 cell injury model

[0110] To further study the eye protection function of blue loquat fruit compound powder, human retinal pigment epithelial cells (ARPE-19) were selected for cell-level experiments. ARPE-19 cells were induced by H2O2 to establish an oxidative damage model. In order to determine the optimal concentration of H2O2 for modeling,

[0111] Cells were treated with different concentrations of H2O2 (100, 250, 500, 750, 1000 μmol / L) for 24 h, and cell viability was determined using the CCK-8 assay. Figure 4 After dose-response curve analysis, H2O2 reached IC50 at 502.2 μmol / L, and the modeling conditions for subsequent cell experiments were determined to be 500 μmol / L H2O2 for 24 h.

[0112] 2.5.2 Toxicity test of Lonicera edulis compound powder on ARPE-19 cells

[0113] To test the cytotoxicity of the blue loquat powder, the survival rate of ARPE-19 cells was determined by CCK-8 assay, and the degree of cell damage was evaluated by measuring the LDH content in the culture medium. Figure 5 . The test results showed that after 24 hours of treatment with different concentrations of blue indigo fruit compound powder solution, the cell survival rate showed a gradual upward trend with the increase of concentration. The cell survival rate was 105.85% at 50 μg / mL and reached 126.92% at 1000 μg / mL. In addition, within the concentration range of 200-800 μg / mL, the cell survival rate increased significantly with the increase of concentration, and there was a significant trend of cell proliferation. The LDH release of the blue indigo fruit compound powder solution in the concentration range of 50-1000 μg / mL was not significantly different from that of the control group, indicating that the cell membrane integrity was good. Combined with the results of cell survival rate and LDH release, it can be seen that the blue indigo fruit compound powder solution has no cytotoxicity in the concentration range of 50-1000 μg / mL.

[0114] 2.5.3 Protective effect of Lonicera edulis composite powder on H2O2-induced ARPE-19 cell damage

[0115] 2.5.3.1 Cell proliferation assay

[0116] To study the effect of blue loquat powder on the proliferation of ARPE-19 cells damaged by H2O2, a cell proliferation test was conducted using CCK-8 method. The results are shown in Figure 6 . Figure 6 A shows that the blue indigo fruit compound powder significantly improved the proliferation ability of ARPE-19 cells compared with the single components of blue indigo fruit powder, wolfberry polysaccharide, lutein ester and zeaxanthin at the same concentration. This is because the synergistic effect between different substances makes the blue indigo fruit compound powder have good cell proliferation ability. Figure 6 As shown in Figure 2, increasing the concentration of the blueberry compound powder alleviated the oxidative damage caused by H2O2 to ARPE-19 cells. At a concentration of 200 μg / mL, the cell survival rate reached 100.25%, and within the concentration range of 200-800 μg / mL, a significant trend of promoting cell proliferation was observed. Therefore, three concentrations of 200 μg / mL, 400 μg / mL, and 800 μg / mL were selected to further explore the protective effect of the blueberry compound powder on H2O2-induced ARPE-19 cell damage.

[0117] 2.5.3.3 Antioxidant enzyme activity and GSH and MDA contents

[0118] CAT, SOD, GSH-px, GSH and MDA are key indicators for evaluating cellular antioxidant capacity and the degree of oxidative stress damage, and are closely related to the ROS level in ARPE-19 cells. Figure 7 Compared with the control group, the H2O2 model group showed significantly decreased CAT, SOD, and GSH-px enzyme activities, significantly decreased GSH content, and significantly increased MDA content in ARPE-19 cells. Compared with the model group, the Lonicera edulis composite powder-treated group showed significantly increased CAT, SOD, and GSH-px enzyme activities, significantly increased GSH content, and significantly decreased MDA content in a concentration-dependent manner. The 800 μg / mL co-incubation group also showed significant effects, but compared with the 800 μg / mL pretreatment group, all other indicators showed significant differences, except for MDA content. Compared with the 400 μg / mL pretreatment group, in addition to CAT and SOD enzyme activities, GSH-px enzyme activity and GSH content were significantly increased, while MDA content was significantly decreased. The group pretreated with 800 μg / mL Lonicera edulis composite powder solution had the most significant effect in alleviating H2O2 oxidative stress damage and most effectively enhanced the antioxidant capacity of ARPE-19 cells. Compared with the control group, the activities of CAT, SOD and GSH-px enzymes increased by 53.53%, 119.23% and 124.53%, respectively, the GSH content increased by 46.67%, and the MDA content decreased by 20.06%.

[0119] 2.6 Effect of Indigofera edulis compound powder on ethanol-induced oxidative damage model of HepG2 cells

[0120] Figure 8The results showed that as the concentration of blue loquat fruit composite powder increased, it could effectively alleviate the oxidative damage of HepG2 cells induced by ethanol, promote cell proliferation, and have the effect of sobering up. Figure 9 The results showed that the cell survival rate of the edodes fruit powder was significantly higher in HepG2 cells exposed to ethanol-induced oxidative damage than the combination of edodes fruit freeze-dried powder and wolfberry polysaccharides. This suggests that the edodes fruit powder, with its added lutein and zeaxanthin, significantly improves its alcohol-resolving and liver-protecting properties compared to the original alcohol-resolving combination of edodes fruit freeze-dried powder and wolfberry polysaccharides. In line with Traditional Chinese Medicine theory, the liver opens to the eyes, and the liver and eyes are closely connected. The potent antioxidant effect of the edodes fruit powder on ARPE-19 cells was also effective against ethanol-induced oxidative damage in HepG2 cells, achieving even greater alcohol-resolving and liver-protecting effects.

Claims

1. A composite powder of Lonicera edulis, characterized in that Every 3g of the blue honeysuckle composite powder contains 100mg of 10% lutein ester microcapsule powder, 20mg of 10% zeaxanthin microcapsule powder, 450mg of 80% wolfberry polysaccharide and 2430mg of blue honeysuckle freeze-dried powder, and the 2430mg of blue honeysuckle freeze-dried powder contains 1780mg of maltodextrin.

2. The method for preparing the composite powder of Lonicera edulis according to claim 1, wherein Preparation method of the composite powder of Indigofera edulis: Water and fresh Lonicera edulis fruit were placed in a juicer at a material-liquid ratio of 1 g:3 mL to 1 g:5 mL, and the juice was collected by filtration. 10% to 20% maltodextrin by volume was added as a filler. The juice was freeze-dried in vacuum and then crushed through an 80-mesh sieve to obtain Lonicera edulis freeze-dried powder. The Lonicera edulis freeze-dried powder, Lycium barbarum polysaccharides, lutein esters, and zeaxanthin were mixed in proportion to prepare Lonicera edulis composite powder, in which the addition ratio of Lycium barbarum polysaccharides was 10% to 20%. After optimization, every 3 g of Lonicera edulis composite powder contained 100 mg of 10% lutein ester microcapsule powder, 20 mg of 10% zeaxanthin microcapsule powder, 450 mg of 80% Lycium barbarum polysaccharides, and 2430 mg of Lonicera edulis freeze-dried powder. The 2430 mg of Lonicera edulis freeze-dried powder contained 1780 mg of maltodextrin.

3. The method for preparing the composite powder of Lonicera edulis according to claim 2, wherein The material-liquid ratio is 1g:4mL.

4. Application of the composite powder of Lonicera edulis, characterized in that The blue loquat fruit composite powder is used for sobering up.

5. Application of the composite powder of Lonicera edulis, characterized in that The blue loquat fruit composite powder is used for eye protection.

6. Application of the composite powder of Lonicera edulis, characterized in that The blue loquat composite powder is used to remove DPPH, ABTS, O2 - Free radicals.

7. Application of the composite powder of Lonicera edulis, characterized in that The blue loquat fruit composite powder is used for resisting lipid peroxidation.

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