A compound apple juice with auxiliary hypolipidemic function based on network pharmacology and a preparation method thereof

Abstract

This invention belongs to the field of food processing technology, specifically relating to a compound apple juice with auxiliary lipid-lowering function based on network pharmacology and its preparation method. The compound apple juice provided by this invention includes five ingredients that are both food and medicine: apple juice, hawthorn puree, ginger juice, kudzu root extract, and mulberry leaf extract. Based on network pharmacology analysis, this invention constructs a product component-hyperlipidemia target-pathway interaction network. Using molecular docking technology, it screens and analyzes the key targets of the compound apple juice's effective components in lowering hyperlipidemia, resulting in a compound apple juice beverage with good lipid-lowering effects and a pleasant taste. This provides a new reference for the application of network pharmacology systems in beverage preparation and has breakthrough significance for the development and industrialization of functional beverages.

Description

Technical Field

[0001] This invention belongs to the field of food processing technology, specifically relating to a compound apple juice based on network pharmacology with auxiliary lipid-lowering function and its preparation method. Background Technology

[0002] With the increasing health awareness of consumers, functional beverages have evolved from meeting basic needs such as replenishing energy and electrolytes to meeting higher-level needs such as precisely improving sub-health and assisting in the regulation of physiological functions. The preparation and research and development of traditional functional beverages mostly rely on single active ingredients, such as screening based on obtaining a certain plant extract, which has limitations such as unclear functional mechanisms and unclear synergistic effects of multiple components.

[0003] Network pharmacology is an emerging research method based on systems biology. Its core is to integrate multi-omics data, including genomics, transcriptomics, and metabolomics, to construct an interaction network of "drug / active ingredient-target-disease / physiological process," thereby elucidating the synergistic regulatory mechanisms of multiple components at the holistic network level. In recent years, network pharmacology has demonstrated significant value in the research and development of traditional Chinese medicine compound formulas and health product development. For example, patents CN117153242A and CN113823364A have used network pharmacology to elucidate the liver cancer protective mechanisms of Huangqin Decoction and galloyl glucose. However, research on its systematic application in beverage preparation is still in its early stages.

[0004] Furthermore, existing research often focuses on bioinformatics analysis of "active ingredients-targets," neglecting the impact of process parameters in beverage preparation on the content of active ingredients and lacking synergistic optimization of "function-taste-stability." In other words, there is an overemphasis on the functional mechanism of beverages. However, as consumer products, beverages need to balance efficacy and taste. If the taste is poor, consumer acceptance will be low. But if the concentration of extracts is reduced in order to improve the taste, although the taste may be improved, the function may be lost due to the low concentration. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a compound apple juice with auxiliary lipid-lowering function based on network pharmacology and its preparation method.

[0006] The first aspect of the present invention is to provide a compound apple juice with the function of assisting in lowering blood lipids, wherein the raw material composition of the compound apple juice is as follows by weight: 80-85 parts apple juice, 7-12 parts hawthorn puree, 2.5-6 parts ginger juice, 1-2 parts mulberry leaf extract, and 2.5-5 parts kudzu root extract.

[0007] A second aspect of the present invention is that a method for preparing the compound apple juice is provided, characterized by comprising the following steps:

[0008] S1 Fresh apples, hawthorns, and ginger are washed, then crushed and juiced at a low temperature of 5~20℃, and centrifuged to obtain apple juice, hawthorn pulp, and ginger juice, respectively.

[0009] S2 Take fresh mulberry leaves, add water at a mass-volume ratio of 1 g: 5~10 mL, crush and juice them at a low temperature of 5~20℃, and obtain mulberry leaf extract after filtration.

[0010] S3 Take kudzu root, add hot water at 80-100℃ at a mass-volume ratio of 1 g: 10-20 mL and perform hot extraction for 20-30 min. After filtration, obtain kudzu root extract.

[0011] S4 mixes, blends, homogenizes, and sterilizes the apple juice, hawthorn pulp, ginger juice, mulberry leaf extract, and kudzu root extract obtained above in proportion to obtain the compound apple juice.

[0012] Furthermore, the compound apple juice prepared by this invention has been verified to have good lipid-lowering effects. Therefore, the compound apple juice can be used in the preparation of lipid-lowering products.

[0013] Before preparing the aforementioned compound apple juice, the inventors first used network pharmacology to analyze and correct the target sites and molecular mechanisms by which the compound apple juice assists in lowering blood lipids. The aforementioned analysis and correction methods are also the key technical content protected by this invention, and specifically include the following steps:

[0014] (1) Obtaining the effective components and targets of compound apple juice

[0015] Using the TCM Systems Pharmacology Database and Analysis Platform (TCMSP) combined with databases such as the Integrated Traditional Chinese Medicine Resources Database (ETCM) and the High-Throughput Experimental and Reference Database of Traditional Chinese Medicine (HERB), the five components of compound apple juice—apple, hawthorn, ginger, kudzu root, and mulberry leaf—we screened, merged, and removed duplicates to obtain the effective components for lowering hyperlipidemia. After further screening and removal of duplicates from the effective components, the effective targets for lowering hyperlipidemia in compound apple juice were obtained.

[0016] (2) Acquisition of hyperlipidemia-related targets

[0017] The GeneCards database was searched and deduplicated to obtain hyperlipidemia-related targets. Based on experience, target targets with scores greater than the median were set as potential targets for hyperlipidemia. The DrugBank database was combined to supplement relevant targets. After merging, duplicate values ​​were removed to obtain the relevant targets for hyperlipidemia.

[0018] (3) Screening of targets for the prevention and treatment of hyperlipidemia by compound apple juice and construction of protein-protein interaction networks (PPI).

[0019] Based on the effective targets for lowering high blood lipids selected in (1) and the targets related to high blood lipids in (2), a Venn diagram was drawn and the intersection was taken to obtain the intersection targets of compound apple juice and high blood lipids. The intersection targets were submitted to the interaction gene search tool STRING12.0 platform to obtain the PPI network of compound apple juice and high blood lipids targets.

[0020] (4) Correction of the intersection target of compound apple juice and hyperlipidemia

[0021] The Metascape data platform was used to perform hierarchical clustering analysis on the intersection target of compound apple juice and hyperlipidemia obtained in (3), and the ClueGO plugin in Cytoscape software was used to perform clustering analysis on the potential functions of the target to obtain the top 5 targets with enrichment scores.

[0022] (5) Enrichment analysis of target functions and pathways

[0023] The top 5 targets obtained in (4) were analyzed for signal pathways using the Metascape data platform, and the results were visualized using Origin Lab 2018. The top 20 KEGG pathways were retained based on the scores.

[0024] (6) Construction of a network diagram of compound apple juice components-targets-metabolic pathways

[0025] A network of compound apple juice components, hyperlipidemia targets, and pathways was constructed using CytoScape 3.8.0. The network topology parameters of the compound apple juice treatment for hyperlipidemia were analyzed using the built-in NetworkAnalyzer of CytoScape 3.8.0 to obtain the core components and core targets.

[0026] The beneficial effects of this invention are as follows:

[0027] (1) A compound apple juice beverage with good taste and auxiliary lipid-lowering effect is provided. The compound beverage is composed of ingredients that are both food and medicine, such as apple juice, hawthorn pulp, ginger juice, kudzu root extract, and mulberry leaf extract. The experimental results show that the obtained compound apple juice can significantly reduce the content of cholesterol, triglycerides, low-density and high-density lipoprotein cholesterol and MDA in high-fat zebrafish under the experimental condition of 40 μL / mL, showing a significant lipid-lowering effect.

[0028] (2) Based on network pharmacology analysis, this invention constructs a network of interactions between compound apple juice components, hyperlipidemia targets, and pathways. By using molecular docking technology to screen and analyze the key targets of the compound apple juice components that lower hyperlipidemia, a compound apple juice beverage with good lipid-lowering effects and a suitable taste is obtained. This provides a new reference for the application of network pharmacology systems in the preparation of beverages and has breakthrough significance for the development and industrialization of functional beverages. Attached Figure Description

[0029] Figure 1 This refers to the results of the single-factor experiment in Example 1 of the present invention;

[0030] Figure 2 This is a response surface plot showing the effect of the interaction between various factors on the sensory score of the compound apple juice in Embodiment 1 of the present invention.

[0031] Figure 3 The area of ​​lipid fluorescence in zebrafish after treatment with different concentrations of apple juice in Example 2 of this invention;

[0032] Figure 4 The image shows the actual fluorescence area of ​​lipids in zebrafish in Example 2 of this invention, where A is the blank group, B is the model group, C is the positive drug group, and D is the 40 μL / mL fruit juice administration group.

[0033] Figure 5 The contents of cholesterol a, triglycerides b, low-density lipoprotein cholesterol c, high-density lipoprotein cholesterol d, and malondialdehyde e in zebrafish under different concentrations of apple juice administration conditions in Example 2 of the present invention.

[0034] Figure 6 This is a Venn diagram of the compound apple juice-hyperlipidemia target in Example 3 of the present invention;

[0035] Figure 7 This is a network diagram of the compound apple juice-hyperlipidemia target PPI in Example 3 of the present invention;

[0036] Figure 8 This is a target intersection analysis diagram of compound apple juice and hyperlipidemia in Example 3 of the present invention;

[0037] Figure 9 Enrichment analysis of potential targets of the compound apple juice components in Example 3 of the present invention;

[0038] Figure 10 This is the enrichment pathway for potential targets of the compound apple juice components in Example 3 of the present invention;

[0039] Figure 11 This is a network diagram of the compound apple juice components, hyperlipidemia targets, and pathways in Example 3 of the present invention.

[0040] Figure 12 This is a comparison chart of the lipid-lowering effects of the compound apple juice in each group in Example 4 of the present invention. Detailed Implementation

[0041] To enable those skilled in the art to better understand the present invention, the present invention will now be further described in conjunction with specific embodiments.

[0042] Example 1

[0043] A compound apple juice with auxiliary lipid-lowering function is prepared as follows:

[0044] S1 Fresh apples, hawthorns, and ginger were washed, crushed and juiced at 10℃, and centrifuged to obtain apple juice, hawthorn pulp, and ginger juice, respectively.

[0045] S2 Take fresh mulberry leaves, add water at a mass-volume ratio of 1 g: 8 mL, crush and juice them at 10℃, and filter to obtain mulberry leaf extract;

[0046] S3. Take kudzu root, add hot water at 85℃ at a mass-volume ratio of 1 g: 15 mL, and extract for 30 min. After filtration, obtain kudzu root extract.

[0047] S4 mixes, blends, homogenizes, and sterilizes the apple juice, hawthorn pulp, ginger juice, mulberry leaf extract, and kudzu root extract obtained above in proportion to obtain the compound apple juice.

[0048] In this embodiment, the proportions of each raw material are determined based on the sensory scores of the obtained compound apple juice. Ten people are randomly selected to form a sensory evaluation group to conduct sensory evaluation of the compound apple juice product according to the standards in Table 1 below.

[0049] Table 1 Sensory Rating Table

[0050] 1.1 Single-factor experiment

[0051] Figure 1 In Figures a through e, the effects of the amounts of apple juice, hawthorn puree, ginger juice, mulberry leaf extract, and kudzu root extract added on the sensory quality of the compound apple juice are respectively.

[0052] Depend on Figure 1 As can be seen from a, the amount of apple juice added has a significant impact on the sensory quality of apple-infused fruit juice. If the amount of apple juice added is too low, the juice will taste too sour and not smooth, and will not have an apple aroma. If the amount of apple juice added is too high, the juice will taste too sweet and have an overpowering apple flavor. The best amount of apple juice added is 84%, which results in a juice with a moderately sweet and sour taste, a uniform and bright color, and a harmonious fruit aroma.

[0053] Figure 1The results showed that if the amount of hawthorn puree added was too low, the juice would taste too sweet and have a strong apple flavor. If the amount added was too high, the juice would have uneven color, flocculation, and an overly sour taste. The best addition amount in the sensory evaluation was 8%, which resulted in a juice with a moderate sweet and sour taste, a uniform pale yellow color, and a harmonious fruity aroma.

[0054] Figure 1 The results showed that when the amount of ginger juice added was 3%, the juice exhibited the best sensory characteristics (including flavor harmony, aroma intensity and taste balance), indicating that this range is the optimal addition amount.

[0055] Figure 1 The results showed that the mulberry leaf extract has a darker color, and its addition amount will affect the color and flavor of the compound apple juice. The unique aroma brought by the mulberry leaf extract blends with the aroma of the apple compound juice to form a new aroma feature, and the optimal addition amount is 2%.

[0056] Figure 1 The results show that kudzu root extract is a light yellow liquid with the characteristic aroma of kudzu root. Kudzu root extract can affect the original color of apple blended juice and add some of the unique herbal fragrance of kudzu root. At the same time, the bitterness and astringency of kudzu root extract can affect the taste of apple blended juice to some extent. The optimal amount of kudzu root extract added is 3.5%.

[0057] 1.2 Response Surface Experiment

[0058] Based on the results of the single-factor experiments in section 1.1, and using sensory score (Y) as the response value, five factors were selected for process optimization: apple juice addition (A), hawthorn puree (B), ginger juice (C), kudzu root extract (D), and mulberry leaf extract (E). A 5-factor, 3-level response surface optimization experiment was designed. The optimal process parameters for the compound apple juice were optimized using the response surface experiment. The factor and level design table is shown in Table 2, the experimental results are shown in Table 3, and the regression model variance analysis is shown in Table 4.

[0059] Table 2 Box-Behnken Experimental Factor Level Design

[0060] Table 3. Response surface methodology and results for optimizing fruit juice formulation conditions. Serial Number A B C D E Sensory rating 1 1 0 -1 0 0 84 2 0 0 0 1 -1 82 3 0 -1 0 1 0 81 4 -1 0 -1 0 0 76 5 0 0 -1 0 0 80 6 1 0 0 0 -1 82 7 0 -1 0 0 -1 79 8 0 0 0 0 0 75 9 -1 0 0 1 0 78 10 1 -1 0 0 0 88 11 0 0 0 -1 -1 77 12 -1 0 0 0 1 69 13 0 0 -1 0 1 75 14 0 0 0 0 0 73 15 0 0 -1 -1 0 74 16 -1 0 1 0 0 71 17 0 1 0 0 1 69 18 -1 0 0 0 -1 73 19 0 -1 1 0 0 73 20 0 1 0 -1 0 69 21 1 0 0 0 1 71 22 0 1 0 1 0 71 23 1 0 -1 0 1 78 24 0 0 -1 0 -1 79 25 0 0 0 0 0 77 26 0 0 0 0 0 75 27 -1 -1 0 0 0 73 28 0 1 -1 0 0 75 29 -1 1 0 0 0 69 30 0 0 1 1 0 71 31 1 0 0 -1 0 71 32 0 1 0 0 -1 66 33 0 1 0 0 1 67 34 0 0 0 1 1 71 35 0 0 1 0 -1 77 36 -1 0 0 -1 0 65 37 0 -1 0 0 1 81 38 0 0 0 0 0 80 39 1 0 1 0 0 74 40 1 1 0 0 0 74 41 0 1 1 0 0 68 42 0 0 -1 1 0 81 43 0 -1 0 -1 0 79 44 0 0 1 -1 0 61 45 0 0 0 0 0 72 46 0 0 1 0 1 65

[0061] Table 4. Analysis of Variance of Regression Model Source of variance sum of squares Degrees of freedom Mean Square F value p-value Significance Model 1163.99 20 58.20 5.14 <0.0001 Significant AA 144.00 1 144.00 10.36 0.0015 BB 351.56 1 351.56 11.23 <0.0001 CC 256.00 1 256.00 3.81 <0.0001 DD 169.00 1 169.00 12.04 0.0007 EE 138.06 1 138.06 1.77 0.0018 AB 25.00 1 25.00 0.1287 0.1500 AC 6.25 1 6.25 0.3022 0.4646 AD 9.00 1 9.00 0.0246 0.3814 AE 12.25 1 12.25 0.2054 0.3085 BC 0.0000 1 0.0000 0.5627 1.0000 BD 1.0000 1 1.0000 0.0611 0.7689 BE 0.2500 1 0.2500 0.1445 0.8831 CD 2.25 1 2.25 2.97 0.6597 CE 16.00 1 16.00 0.1946 0.2459 DE 0.2500 1 0.2500 2.43 0.8831 <![CDATA[A 2 ]]> 0.4583 1 0.4583 0.4715 0.8422 <![CDATA[B 2 ]]> 0.0038 1 0.0038 1.12 0.9856 <![CDATA[C 2 ]]> 5.19 1 5.19 0.6864 0.5050 <![CDATA[D 2 ]]> 22.46 1 22.46 4.19 0.1715 <![CDATA[E 2 ]]> 6.37 1 6.37 0.5618 0.4605 residual 283.33 25 11.33 Missing item 242.00 20 12.10 1.46 0.3585 Not significant Pure error 41.33 5 8.27 sum 1447.33 45

[0062] Using Design-Expert software, a quadratic regression equation was fitted to the data in Table 4 to obtain the multiple quadratic regression equation for sensory scores (Y) on the amount of apple juice added (A), hawthorn puree added (B), ginger juice added (C), mulberry leaf extract added (D), and kudzu root extract added (E): Y = 75.33 + 3A - 4.69B - 4C + 3.25D - 2.94E - 2.50AB - 1.25AC - 1.50AD - 1.75AE + 0.5BD + 0.25BE + 0.75CD - 2CE - 0.25DE + 0.2292A 2 +0.0208B²-0.7708C²-1.60D²-0.8542E².

[0063] As shown in Table 4, the regression model is highly significant (P < 0.0001), and the lack-of-fit term is not significant (P = 0.3585 > 0.05), indicating that the model fit is good. R 2 =0.8042, adjusted coefficient of determination R 2 Adj=0.6476 indicates that 64% of the change in the response value comes from the selected independent variables, and this model can be used for optimization analysis and prediction of apple compound juice formulation. Among them, the linear terms A, B, C, D, and E have a highly significant impact on the results (P<0.01), while the other terms have no significant impact on the results (P>0.05). According to the F-value, the order of influence of the amount of each component added on the sensory score is: hawthorn puree (B) > ginger juice (C) > mulberry leaf extract (D) > apple juice (A) > kudzu root extract (E).

[0064] The response surface of the interaction between various factors on the sensory rating is shown in Figure 2 .

[0065] Analysis using Design-Expert software revealed the optimal proportions for the compound apple juice as follows: 81.96% apple juice, 8.07% hawthorn puree, 3.43% ginger juice, 1.47% mulberry leaf extract, and 3.00% kudzu root extract. Under these proportions, the predicted sensory score for the resulting compound apple juice was 90.596.

[0066] Considering the requirements of actual processes, the optimal ratio was modified to: 82% apple juice, 8% hawthorn pulp, 3% ginger juice, 2% mulberry leaf extract, and 3% kudzu root extract. The optimal ratio was used to process apple compound juice for verification experiments. The actual sensory score of the apple compound juice was 89.128 points, which is basically consistent with the predicted value, indicating that this model can be used to optimize the formulation conditions of compound apple juice.

[0067] 1.3 The compound apple juice was prepared according to the method in 1.1, and its physicochemical properties were determined. The proportions of the raw materials for the compound apple juice were as follows: apple juice 82%, hawthorn pulp 8%, ginger juice 3%, mulberry leaf extract 2%, and kudzu root extract 3%. The results of the physicochemical properties of the compound apple juice are shown in Table 5.

[0068] Table 5 Physicochemical Results of Compound Apple Juice project pH value Total acidity (g / kg) Soluble solids (%) Measured values 3.89±0.01 3.58±0.03 13.5±0.35

[0069] Table 5 shows that the compound apple juice has a pH of 3.89, a total acid of 3.58 g / kg, and a soluble solids content of 13.5%, making it a sweet and sour juice. The reason the compound juice tastes more sweet and sour after blending may be that apple juice is quite sweet and doesn't taste good on its own; while hawthorn pulp is quite sour, and ginger concentrate is spicy and doesn't taste good on its own. Blending it with other juices makes it more palatable and enjoyable.

[0070] The polyphenol content in apple juice and compound apple juice is shown in Table 6.

[0071] Table 6. Comparison of polyphenol content in apple juice and compound apple juice Types and content of polyphenols (mg / L) Apple juice Apple compound juice Catechin content 20.58±0.75 8.42±0.86 chlorogenic acid content 133.89±5.16 118.62±1.58 caffeic acid content 0.18±0.07 0.02±0.01 Ferulic acid content 25.12±1.27 15.46±2.29 Rutin content 0.00±0.00 0.52±0.05 Quercetin content 0.66±0.68 13.31±6.78 Phlorizin content 5.95±0.29 4.14±0.16

[0072] As shown in Table 6, the rutin and quercetin content in the compound apple juice were higher than those in the original apple juice, reaching 0.52±0.05 mg / L and 13.31±6.78 mg / L, respectively.

[0073] Example 2: Pharmacodynamic evaluation of compound apple juice in lowering blood lipids

[0074] A zebrafish hyperlipidemia model was established by using common AB strain 5 dpf zebrafish juveniles and inducing the development of hyperlipidemia in zebrafish with 0.1% egg yolk powder aqueous solution for 72 h.

[0075] Grouping: The test juice group (the compound apple juice prepared in Example 1.3) and the blank group were set up, with 3 replicates in each group and 50 zebrafish juveniles in each replicate.

[0076] Test juice gradient: The test juice groups were set with concentration gradients of 50 μg / mL, 100 μg / mL, 200 μg / mL, 400 μg / mL, 600 μg / mL, 800 μg / mL, and 1000 μg / mL.

[0077] The zebrafish were treated with the test juice for 72 hours, and the mortality rate was observed to calculate the maximum tolerance.

[0078] Evaluation of the lipid-lowering effect of compound apple juice:

[0079] Test juice treatment: Based on the modeling time and test juice dosage determined in the above experiment, a test juice group and a positive drug group (lovastatin 0.24 μg / mL) were set up. The test juice group was set up with three gradient dosage groups of low, medium and high, and the test juice was treated for 72 hours. Each group had 3 replicates, and each replicate had 50 zebrafish.

[0080] The effects of different concentrations of the test fruit juice (10-40 μL / mL) on lipid accumulation in high-fat zebrafish juveniles were analyzed using fluorescence imaging. The results are as follows: Figure 3 , Figure 4 As shown, where, Figure 3 The fluorescence area of ​​lipids in zebrafish under different concentrations of tested apple juice treatment was measured (###P<0.001, comparison between model group and blank group, ***P<0.001, all comparisons were made with the zebrafish hyperlipidemia model group, the same below). Figure 4 The images show the actual fluorescence area of ​​lipids in zebrafish, where (A) is the blank group, (B) is the model group, (C) is the positive drug group, and (D) is the 40 μL / mL fruit juice administration group.

[0081] Figures 3-4 The results showed that the lipid fluorescence area in the blank control group ranged from 37,036 to 74,680, while the lipid fluorescence area in the hyperlipidemia model group significantly increased to 171,223 to 194,868, indicating that the hyperlipidemia model was successfully established. The lipid fluorescence area in the positive control group decreased to 55,164 to 74,497, and the lipid fluorescence area in the 40 μL / mL test juice treatment group decreased to 53,932 to 64,713 (the lowest value was 53,932, indicating the most significant lipid-lowering effect); the lipid fluorescence area in the 20 μL / mL test juice treatment group ranged from 72,496 to 88,687; and the lipid fluorescence area in the 10 μL / mL treatment group ranged from 81,786 to 106,757.

[0082] Comprehensive data analysis showed that the tested fruit juices significantly reduced lipid accumulation in juvenile zebrafish within the concentration range of 10–40 μL / mL (P<0.001), with the 40 μL / mL group showing the best effect, approaching the level of positive drugs.

[0083] Table 7 and Figure 5 Figures a through e show the levels of cholesterol, triglycerides, low-density lipoprotein, high-density lipoprotein, and MDA in zebrafish under different concentrations of fruit juice administration.

[0084] Table 7. Effects of different concentrations of fruit juice administration on hyperlipidemia in zebrafish. Experimental group (nmol / mg tissue) Blank group Model group Yang medicine group cholesterol 0.02085~0.02821 0.03925~0.04620 0.02658~0.03271 Triglycerides 0.008362~0.01194 0.01398~0.01863 0.008362~0.01194 Low-density lipoprotein 0.02832~0.04592 0.05321~0.06838 0.02934~0.05832 High-density lipoprotein 0.002288~0.003315 0.0009910~0.001694 0.001964~0.003532 MDA 1.2401~1.4928 1.4855~1.9855 1.1449~1.4710 Experimental group (nmol / mg tissue) 10 μL / mL 20 μL / mL 40 μL / mL cholesterol 0.02658~0.03434 0.02126~0.02821 0.02126~0.02576 Triglycerides 0.01183~0.01469 0.01003~0.01386 0.008242~0.01254 Low-density lipoprotein 0.04330~0.06272 0.04188~0.05058 0.03196~0.05058 High-density lipoprotein 0.0007207~0.002667 0.0009639~0.002613 0.001423~0.003099 MDA 1.2391~1.4783 1.1739~1.4783 1.2101~1.4493

[0085] The above experimental results show that each concentration group, especially the 40 μL / mL compound apple juice, can significantly reduce the levels of cholesterol, triglycerides, low-density and high-density lipoprotein cholesterol, and MDA in high-fat zebrafish, demonstrating a significant lipid-lowering effect.

[0086] Example 3

[0087] The target and molecular mechanism of the compound apple juice prepared in this invention for assisting in lowering blood lipids were analyzed and corrected based on network pharmacology. The specific steps are as follows:

[0088] (1) Obtaining the effective components and targets of compound apple juice

[0089] The natural product components of the five components of the compound apple juice (apple, hawthorn, ginger, kudzu root, and mulberry leaf) were retrieved and compared using the TCMSP, ETCM, and HERB databases. Based on two ADME attribute values ​​(oral bioavailability ≥30% and drug-likeness ≥0.18) from the TCMSP database, preliminary screening of active ingredients was conducted to obtain active compounds and their protein targets. Known targets of unpredicted active compounds were supplemented based on published literature from CNKI. After screening, to standardize protein target information, the protein targets of the compounds were standardized in the Uniprot protein database, resulting in 88 potential active ingredients for the prevention and treatment of hyperlipidemia, including kaempferol, quercetin, naringenin, and luteolin. Furthermore, analysis revealed that most active ingredients interacted with only 1-2 targets, with a median of 3. Based on experience, 33 active ingredients with more than the median number of targets were selected as candidate ingredients for the product's prevention and treatment of hyperlipidemia. Specific parameter information is shown in Table 8.

[0090] Table 8 Main Components of Compound Apple Juice Raw Materials

[0091] As shown in Table 8, there are 5 common components among various raw materials; 2 unique components of apple; 5 unique components of hawthorn; 2 unique components of ginger; 3 unique components of kudzu root; and 15 unique components of mulberry leaf. After merging and removing duplicate values, a total of 247 target points were obtained.

[0092] (2) Acquisition of hyperlipidemia-related targets

[0093] A search of the GeneCards database yielded 3162 targets for hyperlipidemia after deduplication. Based on experience, targets with scores greater than the median were defined as potential targets for hyperlipidemia. The maximum score for hyperlipidemia targets obtained from GeneCards was 191.08, the minimum was 0.15, and the median was 0.60. Therefore, targets with a score > 0.60 were defined as potential targets for hyperlipidemia. Further investigation was conducted using the DrugBank database to supplement relevant targets. After merging and removing duplicates, 1598 hyperlipidemia-related targets were finally obtained.

[0094] (3) Screening of targets for the prevention and treatment of hyperlipidemia by compound apple juice and construction of PPI network

[0095] A Venn diagram was drawn between the 247 potential targets of product active ingredients screened in (1) and the 1598 targets of hyperlipidemia obtained in (2), and the intersection was taken as follows: Figure 6 As shown, 97 common targets of compound apple juice and hyperlipidemia were obtained, and the specific target information is shown in Table 9.

[0096] Next, the target was submitted to the STRING 12.0 platform to obtain the product target PPI network, see... Figure 7 The total number of edges and the average node degree in the target PPI network are 272 and 4.61, respectively.

[0097] Table 9 Information on potential protein targets NO. Target Name NO. Target Name NO. Target Name NO. Target Name NO. Target Name 1 ABCA1 21 CRP 41 GLB1 61 MPO 81 SELE 2 ABCG1 22 CRTC2 42 HAS2 62 MTTP 82 SELP 3 ACACA 23 CTSD 43 HMOX1 63 NCOA1 83 SERPINE1 4 ACE 24 CXCL2 44 HSPB1 64 NFKBIA 84 SLC2A4 5 ACHE 25 CXCL8 45 ICAM1 65 NOS2 85 SLPI 6 ADRA2A 26 CYP19A1 46 IFNG 66 NOS3 86 SOD1 7 ADRB1 27 CYP1A1 47 IGFBP3 67 NR1I2 87 SPP1 8 ADRB2 28 CYP3A4 48 IL10 68 NR3C2 88 STAT1 9 AKR1B1 29 DGAT2 49 IL1A 69 PECAM1 89 TGFB1 10 AKT1 30 EGF 50 IL1B 70 PLAT 90 SLC6A2 11 ALOX5 31 EGFR 51 IL2 71 PON1 91 TNF 12 APOB 32 ERBB2 52 IL4 72 PPARA 92 TNFRSF1A 13 CAV1 33 ESR1 53 IL6 73 PPARD 93 TP53 14 CCL2 34 ESR2 54 INSR 74 PPARG 94 VCAM1 15 CCND1 35 F10 55 KCNH2 75 PTEN 95 VEGFA 16 CD40LG 36 F2 56 MAPK14 76 RELA 96 XDH 17 CDK4 37 F3 57 MDM2 77 RUNX2 97 XIAP 18 CDKN2A 38 F7 58 MMP1 78 RXRA 19 CETP 39 FASN 59 MMP3 79 RXRG 20 COL3A1 40 G6PD 60 MMP9 80 SCN5A

[0098] In the complex PPI network, some high-density regions are called communities or modules. The networks within a module are potential subnetworks of the PPI network. These subnetworks have high connection density, while some regions have fewer connections. Therefore, a module is considered a biologically significant set, with two meanings: one is a protein complex, where multiple proteins work together to exert biological effects; the other is a functional module, such as proteins located in the same pathway, whose interactions are more intimate. Therefore, to more accurately analyze the mechanism of action of a product in treating hyperlipidemia, it is necessary to further identify its internal modules after obtaining the product's PPI network.

[0099] Therefore, after obtaining the PPI network, the interaction relationship was analyzed using the MCODE plugin in CytoScape 3.8.0 through a molecular complex detection algorithm.

[0100] (4) Correction of the intersection target of compound apple juice and hyperlipidemia

[0101] Considering the multi-system pathological changes caused by hyperlipidemia in this study and the wide range of potential active ingredients in food-medicine homology products, the traditional single method of analyzing pathway correlations using enrichment scores is prone to bias. Therefore, based on the hierarchical clustering results of Metascape, combined with the ClueGO plugin in Cytoscape software, we performed clustering analysis on the potential functions of the targets to reduce the research bias of network proximity analysis. Colors from light to dark represent enrichment scores from low to high (e.g., ...). Figure 8 As shown in the table). The results show that the functions of multiple targets are closely related to the occurrence of dyslipidemia. The information of the top 5 targets with enrichment scores (-log10 (P-Value)) is detailed in Table 10.

[0102] Table 10 Characteristic parameters of the target network nodes of the main active ingredients in compound apple juice target Connectivity Medium Tightness IL6 83 441.1871892 0.009174312 TNF 82 467.7679061 0.009090909 AKT1 80 475.1389356 0.008928571 IL1B 78 468.7185423 0.00877193

[0103] (5) Enrichment analysis of target functions and pathways

[0104] The Metascape data platform was used to analyze the signaling pathways of targets related to the treatment of hyperlipidemia by compound apple juice, and the results were visualized using Origin Lab 2018. After enrichment analysis based on the Gene Prioritization by Evidence Count (GPEC) computational model, 694 GO items with P-values ​​<0.01 were obtained. These GO items belonged to three categories: 526 biological processes (BP), 51 cellular components (CC), and 117 molecular functions (MF). Figure 9 As shown in the figure, orange represents GO-BP analysis; blue represents GO-CC analysis; and green represents GO-MF analysis.

[0105] The biological processes involved in compound apple juice are related to the body's response to external stimuli and intracellular transcriptional regulation. These include positive regulation of gene expression, cellular response to lipopolysaccharide, positive regulation of DNA template transcription, and positive regulation of transcription by RNA polymerase II. Furthermore, it is related to cell differentiation processes such as positive regulation of cell migration, inflammatory responses, and negative regulation of apoptosis. This also corroborates the pathogenesis and pathological mechanisms of hyperlipidemia. The functional sites of the targets regulating dyslipidemia in cells are mainly concentrated in the extracellular space, on the outer side of the plasma membrane, and in the perinuclear region of the cytoplasm. The function of these targets in regulating hyperlipidemia is also closely related to this, mainly enriched in molecular functions such as enzyme binding, nuclear receptor activity, cytokine activity, and protein binding.

[0106] The top 20 KEGG Pathways are retained based on their ratings. Figure 10It presents functional descriptions of major biological processes involved by potential targets, including lipids and atherosclerosis, the role of the AGE-RAGE signaling pathway in diabetic complications, fluid shear stress and atherosclerosis, and cancer pathways.

[0107] (6) Construction of a network diagram of compound apple juice components-targets-metabolic pathways

[0108] A complex apple juice component-hyperlipidemia target-pathway network was constructed using CytoScape 3.8.0. The network topology parameters for the product's treatment of hyperlipidemia were analyzed using CytoScape 3.8.0's built-in NetworkAnalyzer to identify the core components and key targets. Figure 11 As shown, circular nodes represent active ingredients of different categories, rectangles represent target points, and triangles represent pathways. The area and color transparency of the nodes represent degrees, and the area is proportional to its enrichment level.

[0109] Figure 11 The network showed that quercetin had a connectivity of 66, followed by luteolin, kaempferol, and arachidonic acid, with connectivity of 25, 23, and 17, respectively. These compounds are predicted to be the main components of compound apple juice for treating hyperlipidemia. Other potential active ingredients include epicatechin, tetramethoxyluteolin, stigmasterol, and others. RELA, MAPK14, and PPARG had connectivity of 22, 20, and 19, respectively. AKT1, TNF, IL-6, ESR1, and IL-1β, all inflammation-related targets, were also relatively core targets. Furthermore, lipids and atherosclerosis, the role of the AGE-RAGE signaling pathway in diabetic complications, and fluid shear stress and atherosclerosis showed the most significant enrichment in the network diagram.

[0110] This invention introduces a network pharmacology model to analyze the active ingredients, targets, and mechanisms of the combined products in preventing and treating hyperlipidemia and oxidative stress damage. The analysis revealed that different types of medicinal and edible ingredients have different numbers and types of targets, and the same target also corresponds to different numbers and types of active ingredients. This demonstrates that the products achieve the effect of preventing and treating hyperlipidemia and oxidative stress damage through the overall synergy of multiple components and multiple targets.

[0111] Example 4

[0112] The lipid-lowering efficacy of the compound apple juice formulation of this invention was evaluated and verified in vitro based on network pharmacology. The specific steps are as follows:

[0113] The test juice experimental group (the compound apple juice prepared in Example 1.3) and the control group (one component was removed from the formula, namely No. 1 - no apple, No. 2 - no hawthorn, No. 3 - no ginger, No. 4 - no mulberry leaf, No. 5 - no kudzu root) were set up to evaluate the bile salt binding capacity of different formulas.

[0114] Experimental results are as follows Figure 12 As shown in the figure, the experimental group had the highest binding rates of sodium taurocholate and glycocholate. In the control group, due to the absence of any single component, the binding capacity of both bile salts decreased significantly. Furthermore, the degree of impact of different component absences on binding capacity varied. These results further demonstrate that the five components—apple, hawthorn, ginger, mulberry leaf, and kudzu root—do not act independently, but rather work synergistically to enhance bile salt binding capacity. The integrity of the formula is key to achieving optimal lipid-lowering effects; the absence of a single component disrupts this synergistic effect, significantly reducing the health benefits of the compound apple juice.

Claims

1. A compound apple juice with auxiliary function of lowering blood lipids, characterized in that, The raw material composition of the compound apple juice, by weight, is as follows: 80-85 parts apple juice, 7-12 parts hawthorn puree, 2.5-6 parts ginger juice, 1-2 parts mulberry leaf extract, and 2.5-5 parts kudzu root extract.

2. The compound apple juice as described in claim 1, characterized in that, The raw material composition of the compound apple juice, by weight, is as follows: 82-85 parts apple juice, 8-10 parts hawthorn puree, 2.5-4 parts ginger juice, 1.5-2 parts mulberry leaf extract, and 3-4 parts kudzu root extract.

3. The compound apple juice as described in any one of claims 1 to 2, characterized in that, Based on the volume of the compound apple juice, it contains 8-9 mg of catechin, 100-125 mg of chlorogenic acid, 0.01-0.05 mg of caffeic acid, 10-20 mg of ferulic acid, 0.2-0.8 mg of rutin, 10-15 mg of quercetin, and 2-5 mg of phlorizin.

4. The method for preparing the compound apple juice according to any one of claims 1 to 3, characterized in that, The steps include the following: S1 Fresh apples, hawthorns, and ginger are washed, then crushed and juiced at a low temperature of 5~20℃, and centrifuged to obtain apple juice, hawthorn pulp, and ginger juice, respectively. S2 Take fresh mulberry leaves, add water at a mass-volume ratio of 1 g: 5~10 mL, crush and juice them at a low temperature of 5~20℃, and obtain mulberry leaf extract after filtration. S3 Take kudzu root, add hot water at 80-100℃ at a mass-volume ratio of 1 g: 10-20 mL and perform hot extraction for 20-30 min. After filtration, obtain kudzu root extract. S4 mixes, blends, homogenizes, and sterilizes the apple juice, hawthorn pulp, ginger juice, mulberry leaf extract, and kudzu root extract obtained above in proportion to obtain the compound apple juice.

5. The use of the compound apple juice as described in any one of claims 1 to 3, or the compound apple juice prepared by the method described in claim 4, in the preparation of products that assist in lowering blood lipids.

6. A method for analyzing and correcting the target and molecular mechanism of the compound apple juice in lowering blood lipids according to any one of claims 1 to 3 based on network pharmacology, characterized in that, Includes the following steps: (1) Obtaining the effective components and targets of compound apple juice Using the TCM Systems Pharmacology Database and Analysis Platform (TCMSP) combined with the ETCM and HERB databases, the five components of compound apple juice—apple, hawthorn, ginger, kudzu root, and mulberry leaf—were screened, combined, and deduplicated to obtain the effective components for lowering hyperlipidemia. After further screening and removal of duplicates from the effective components, the effective targets for lowering hyperlipidemia in compound apple juice were obtained. (2) Acquisition of hyperlipidemia-related targets The GeneCards database was searched and deduplicated to obtain hyperlipidemia-related targets. Based on experience, target targets with scores greater than the median were set as potential targets for hyperlipidemia. The DrugBank database was combined to supplement relevant targets. After merging and deleting duplicate values, the relevant targets for hyperlipidemia were obtained. (3) Screening of targets for the prevention and treatment of hyperlipidemia by compound apple juice and construction of PPI network Based on the effective targets for lowering high blood lipids selected in (1) and the targets related to high blood lipids in (2), a Venn diagram was drawn and the intersection was taken to obtain the intersection targets of compound apple juice and high blood lipids. The intersection targets were submitted to the interaction gene search tool STRING12.0 platform to obtain the PPI network of compound apple juice and high blood lipids targets. (4) Correction of the intersection target of compound apple juice and hyperlipidemia Based on the hierarchical clustering results of Metascape, and combined with the ClueGO plugin in Cytoscape software, cluster analysis was performed on the potential functions of the targets to obtain the top 5 targets with enrichment scores. (5) Enrichment analysis of target functions and pathways The top 5 targets for treating hyperlipidemia obtained in (4) were analyzed for signal pathways using the Metascape data platform, and the results were visualized using Origin Lab 2018. The top 20 KEGG pathways were retained based on the scores. (6) Construction of a network diagram of compound apple juice components-targets-metabolic pathways A network of compound apple juice components, hyperlipidemia targets, and pathways was constructed using CytoScape 3.8.

0. The network topology parameters of the compound apple juice treatment for hyperlipidemia were analyzed using the built-in NetworkAnalyzer of CytoScape 3.8.0 to obtain the core components and core targets.

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