Application of compound of colla corii asini and honey in preparation of antioxidant product

Abstract

The invention relates to application of a compound of colla corii asini and honey in preparation of an antioxidant product, and belongs to the technical field of health-care food. The donkey-hide gelatin disclosed by the invention is derived from Dongu donkey-hide gelatin Co., Ltd. The compound of colla corii asini and honey has the effects of reducing oxidative stress, enhancing free radical scavenging capacity and resisting oxidation. After the antioxidant compound is taken for a long time, the elimination capability of mice on lipid peroxide can be improved, the oxidative stress degree of organisms can be slowed down, the reserve of glutathione can be increased, the oxidative damage degree of amino acid can be reduced, and finally the antioxidant effect can be achieved. The invention provides a new component for research and development of health-care food with antioxidant activity, and has important application value.

Description

Technical Field

[0001] This invention relates to the field of health food technology, and in particular to the application of a compound of donkey-hide gelatin and honey in the preparation of antioxidant products. Background Technology

[0002] With the continuous increase in environmental stress and metabolic load, the human body's redox balance system is facing severe challenges. In contemporary society, "sub-health" caused by oxidative stress has become a common phenomenon, with clinical data showing that approximately 68% of adults experience a persistent decline in antioxidant capacity. As a crucial protective mechanism, the antioxidant defense system's imbalance often triggers a chain reaction: excessive generation of reactive oxygen species (ROS) from mitochondria disrupts the integrity of the electron transport chain; abnormal activation of the NADPH oxidase system leads to DNA oxidative damage; and reduced activity of reduced glutathione (GSH) and superoxide dismutase (SOD) directly weakens the cell's ability to scavenge free radicals. This systemic lack of antioxidant capacity results in the accumulation of lipid peroxidation products (MDA), protein carbonylation modification (PCO), and abnormal mitochondrial membrane potential, ultimately forming a vicious cycle of oxidation. When the body is chronically in a state of redox homeostasis imbalance, it will trigger systemic pathological changes. Clinical studies have shown that over 40% of patients with chronic diseases exhibit reduced GSH activity and abnormal expression of thioredoxin reductase (TrxR). This persistent deficiency in antioxidant defense can lead to endocrine disorders, impaired immune surveillance, and cumulative tissue damage. Therefore, based on the multidimensional regulatory needs of redox homeostasis, targeted nutritional intervention is gradually replacing single antioxidant supplementation strategies and becoming a cutting-edge approach to maintaining the body's antioxidant defense system.

[0003] As a traditional and precious Chinese medicine and a modern functional food, donkey-hide gelatin (Asini Corii Colla) is attracting attention due to the potential of its components in regulating antioxidant activity. According to the pharmacopoeia, it is a solid gelatin made from the skin of the donkey (Equus asinus L.) through decoction and concentration. The *Shennong Bencao Jing* (Shennong's Classic of Materia Medica) lists it as a superior medicine, placing it alongside ginseng and deer antler as one of the "Three Treasures of Chinese Medicine." Recent analyses indicate that donkey-hide gelatin contains active components such as hydrolyzed collagen peptides (molecular weight 1-3 kDa), donkey-derived heme iron, and dermatan sulfate polysaccharides. Among these, donkey-specific collagen triple helix structure produced by enzymatic hydrolysis, resulting in donkey-derived oligopeptides (EJOPs), has been shown to possess free radical scavenging capabilities. However, the antioxidant activity of these components is far lower than that of specific antioxidants such as flavonoids, and their mechanism of action is singular (primarily scavenging small amounts of free radicals), making them insufficient to address complex oxidative stress responses.

[0004] Honey is renowned for its sweetness, but this sweetness differs from that of sugar; it's a natural sweetness. As a natural antioxidant, its abundant polyphenols possess significant antioxidant effects. Polyphenols, as natural oxidation inhibitors, can block lipid peroxidation chain reactions by chelating transition metal ions. Flavonoids (such as quercetin and kaempferol) and phenolic acids in honey have strong free radical scavenging capabilities, effectively neutralizing superoxide anions and hydroxyl radicals, and reducing lipid peroxidation levels. Honey's antioxidant effects synergistically interact with its anti-inflammatory and immunomodulatory functions. It can also effectively regulate gut microbiota imbalance and short-chain fatty acid content, thereby improving gut microbiota dysbiosis caused by alcohol damage and regulating oxidative stress from a microbiological perspective. However, the antioxidant activity of honey from different nectar sources varies significantly. This instability makes it difficult to standardize its antioxidant performance, resulting in considerable fluctuations in effectiveness in practical applications. Furthermore, the high sugar content in honey may induce oxidative stress in the body through glycosylation, to some extent counteracting the effects of its own antioxidant components. Studies show that excessive intake of high-sugar honey may actually increase the levels of lipid oxidation products in the body.

[0005] Therefore, there is an urgent need to provide a compound that can be mixed according to a certain scientific formula in order to achieve complementary advantages and synergistic effects. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides the application of a compound of donkey-hide gelatin and honey in the preparation of antioxidant products. Donkey-hide gelatin is a commonly used traditional Chinese medicine. According to the 2015 edition of the Chinese Pharmacopoeia, it is made from donkey skin through decoction and concentration to produce a solid gelatin. The main components of donkey-hide gelatin include proteins, amino acids, polypeptides, polysaccharides, trace elements, vitamins, and fatty acids. Studies have shown that many components of donkey-hide gelatin, such as small molecule peptides, polysaccharides, and certain amino acids (such as glycine), possess significant antioxidant activity. They can scavenge free radicals in the body, reduce oxidative stress damage, and enhance the activity of antioxidant enzymes (such as SOD and GSH-Px). These physiological activities are considered one of the important material bases for donkey-hide gelatin's functions of nourishing blood, beautifying the skin, and regulating immunity. This invention provides a new application of a compound of donkey-hide gelatin and honey in the field of health foods. Specifically, it describes the application of the compound of donkey-hide gelatin and honey as an antioxidant active ingredient in the preparation of products that enhance the body's antioxidant capacity.

[0007] This invention is achieved through the following technical solution:

[0008] The purpose of this invention is to provide the application of a compound of donkey-hide gelatin and honey in the preparation of antioxidant products, wherein the mass ratio of donkey-hide gelatin and honey in the compound is 1:2-1:4.

[0009] In one embodiment of the present invention, the mass ratio of donkey-hide gelatin and honey in the compound is 1:4.

[0010] In one embodiment of the present invention, the antioxidant activity is the ability to alleviate oxidative damage and scavenge free radicals in the body.

[0011] In one embodiment of the present invention, the donkey-hide gelatin is a powder made from the dried or fresh skin of the donkey (Equus asinus L.) through decoction and concentration.

[0012] In one embodiment of the present invention, the donkey-hide gelatin is produced by Dong-E-E-Jiao Co., Ltd.

[0013] In one embodiment of the invention, the honey is Manuka flower honey; a unique honey originating from New Zealand and Australia, made by bees collecting nectar from the Manuka tree (Leptospermum scoparium).

[0014] In one embodiment of the present invention, the product is a food product.

[0015] In one embodiment of the present invention, the food is one or more of beverages, pastes, preserved fruits, and pastries.

[0016] In one embodiment of the present invention, the product is a health supplement.

[0017] In one embodiment of the present invention, excipients for health products are also included.

[0018] In one embodiment of the present invention, the dosage form of the health product is selected from one or more of tablets, capsules, soft capsules, granules, pills and oral liquids.

[0019] In one embodiment of the present invention, the product is a pharmaceutical product.

[0020] In one embodiment of the present invention, the pharmaceutical product further includes excipients; the excipients include one or more of excipients, colorants, flavoring agents, disintegrants, and lubricants.

[0021] The present invention also provides a method for processing feed prepared for animal experiments, comprising the following steps:

[0022] Weigh different weights of donkey-hide gelatin and / or honey, and mix them evenly with ordinary feed according to the proportions of each experimental group.

[0023] In one embodiment of the present invention, the model group is constructed using an ethanol-induced oxidative stress model.

[0024] The present invention also provides a method for evaluating the antioxidant activity of a compound of donkey-hide gelatin and honey from the perspective of gut microbiota.

[0025] This invention also provides a mechanism from the perspective of signaling pathways to explain the mechanism by which the compound of donkey-hide gelatin and honey enhances the ability to scavenge free radicals.

[0026] The present invention also provides a product with antioxidant activity, comprising a compound of donkey-hide gelatin and honey; wherein the mass ratio of donkey-hide gelatin to honey in the compound is 1:2-1:4.

[0027] The technical solution of the present invention has the following advantages compared with the prior art:

[0028] (1) This invention provides the application of a compound of donkey-hide gelatin and honey in the preparation of antioxidant products. This invention aims to provide a safe ingredient with high nutritional value, antioxidant effects, and enhanced immunity, offering a new option for the research of antioxidant formulation products. Based on existing antioxidant-related indicators, such as lipid peroxidation products (MDA), protein carbonylation level (PCO), superoxide dismutase (SOD), and reduced glutathione (GSH), this invention can scientifically and rationally demonstrate that the compound of donkey-hide gelatin and honey contains components that can enhance the antioxidant capacity of mice, thereby enhancing the ability of mice to scavenge free radicals and achieving the effect of improving antioxidant activity.

[0029] (2) This invention provides insights into the mechanism of antioxidant capacity from the perspective of gut microbiota, such as regulating gut metabolism, promoting polysaccharide absorption, and improving gut flora. It scientifically and rationally proves that the compound of donkey-hide gelatin and honey contains components that can enhance the antioxidant capacity of mice.

[0030] (3) This invention analyzes the mechanism of the product’s antioxidant capacity from the perspective of gene signaling pathways, such as promoting antioxidant pathways, upregulating dominant genes, and alleviating metabolic disorders. It precisely and meticulously elucidates the corresponding pathways in the compound of donkey-hide gelatin and honey that can enhance the antioxidant capacity of mice.

[0031] (4) The compound of donkey-hide gelatin and honey has the effects of reducing oxidative stress, enhancing free radical scavenging ability, and antioxidation. Long-term consumption of the antioxidant food or health product ingredients of this invention can enhance the elimination of lipid peroxides in mice, reduce the degree of oxidative stress in the body, increase glutathione reserves, and reduce the degree of oxidative damage to amino acids, ultimately achieving an antioxidant effect. This invention provides a new ingredient for the development of health foods with antioxidant activity and has significant application value. Attached Figure Description

[0032] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, wherein:

[0033] Figure 1 This is a graph showing the MDA content of different groups in Example 2 of the present invention;

[0034] Figure 2 This is a graph showing the SOD content of different groups in Example 2 of the present invention;

[0035] Figure 3 This is a graph showing the GSH content of different groups in Example 2 of the present invention;

[0036] Figure 4 This is a graph showing the PCO content of different groups in Example 2 of the present invention;

[0037] Figure 5 This is the Alpha diversity index diagram in Embodiment 3 of the present invention; where A is ace analysis; B is chao1 analysis; C is observed analysis; and D is shannon analysis.

[0038] Figure 6 This is an analysis of the differences in the quantity and abundance of microbial communities in Example 3 of the present invention; wherein, A is a stacked bar chart of the relative abundance of species at the phylum level; B is a Venn diagram based on OTUs;

[0039] Figure 7 This is the LEfSe analysis chart in Embodiment 3 of the present invention; wherein, A is the LEfSe analysis chart of group M and group FAD; B is the LEfSe analysis chart of group M and group FAG;

[0040] Figure 8 This is the differential gene MA diagram in Example 4 of the present invention; wherein, the vertical axis represents the log2 FC value, which represents the logarithm of the difference (log2 Fold_Change); the horizontal axis represents the average gene expression level in the two groups of samples; red dots represent gene expression upregulation, blue dots represent gene expression downregulation, and gray dots represent no significant difference in gene expression;

[0041] Figure 9 This is a heatmap of differential gene clustering in Embodiment 4 of the present invention; wherein, the horizontal axis represents the sample name and hierarchical clustering results, and the vertical axis represents the differential genes and hierarchical clustering results; red indicates high expression, and blue indicates low expression;

[0042] Figure 10 This is a bar chart of differentially expressed KEGG enrichment in the FAD group in Example 4 of this invention;

[0043] Figure 11 This is a bar chart of KEGG enrichment of differentially expressed genes in the FAG group in Example 4 of this invention;

[0044] Figure 12It is the KEGG enrichment bar chart of the differential genes in Group A in Example 4 of the present invention;

[0045] Figure 13 It is the KEGG enrichment bar chart of the differential genes in Group FD in Example 4 of the present invention;

[0046] Figure 14 It is the KEGG enrichment bar chart of the differential genes in Group FG in Example 4 of the present invention;

[0047] Figure 15 It is the KEGG enrichment bar chart of the differential genes in Group M in Example 4 of the present invention;

[0048] Among them, Figure 1-15 In it, D represents the blank control group, M represents the model control group, A represents the donkey-hide gelatin group, FD represents the low-dose honey group, FG represents the high-dose honey group, FAD represents the low-dose compound group of donkey-hide gelatin and honey, and FAG represents the high-dose compound group of donkey-hide gelatin and honey; * indicates significant difference compared with the model group (P<0.05). Specific implementation manners

[0049] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, so that those skilled in the art can better understand the present invention and be able to implement it, but the embodiments cited are not intended to limit the present invention.

[0050] Unless otherwise specified, the experimental methods used in the following embodiments are all conventional methods, and the materials, reagents, etc. used, unless otherwise specified, can be obtained from commercial channels.

[0051] (1) The sources of the materials used in this embodiment are as follows:

[0052] SPF-grade male BALB / c mice were provided by Beijing Spearf Bio-tech Co., Ltd. (license number: SCXK(Beijing)2,022-0,006). [[ID=३३]]

[0053] The donkey-hide gelatin is from Dong'e E-Jiao Co., Ltd.

[0054] Manuka honey is from Richelland (Shanghai) Food Technology Co., Ltd.

[0055] (2) 16S rRNA sequencing method:

[0056] 1.1 Extraction and PCR amplification of genomic DNA

[0057] The genomic DNA of the sample was extracted by the CTAB or SDS method, and then the purity and concentration of the DNA were detected by agarose gel electrophoresis. An appropriate amount of sample DNA was taken into a centrifuge tube, and the sample was diluted to 1 ng / μL with sterile water.

[0058] Using diluted genomic DNA as a template, and based on the selected sequencing region, specific primers with barcodes were used from New England Biolabs. High-Fidelity PCRMaster Mix with GC Buffer and high-efficiency, high-fidelity enzymes are used for PCR to ensure amplification efficiency and accuracy.

[0059] Primer corresponding region:

[0060] 16S V4 region primers (515F and 806R): for identifying bacterial diversity;

[0061] 18S V4 region primers (528F and 706R): for identifying eukaryotic microbial diversity;

[0062] ITS1 primers (ITS5-1737F and ITS2-2043R): for identifying fungal diversity;

[0063] In addition, the amplified regions also include: 16SV3-V4 / 16SV4-V5 / 16SV5-V7; archaea 16SV4-V5 / archaea 16SV8; 18SV9 and ITS2 regions.

[0064] 1.2 Mixing and purification of PCR products

[0065] PCR products were detected by electrophoresis on a 2% agarose gel. Qualified PCR products were purified by magnetic beads, quantified by enzyme labeling, and mixed in equal volumes according to the PCR product concentration. After thorough mixing, the PCR products were detected by electrophoresis on a 2% agarose gel. The target band was recovered using a gel recovery kit provided by Qiagen.

[0066] 1.3 Library construction and sequencing

[0067] use The DNAPCR-FreeSample Preparation Kit was used to construct the library. The constructed library was quantified by Qubit and Q-PCR. After the library was qualified, it was sequenced using NovaSeq6000.

[0068] 2. Transcriptome Sequencing Analysis

[0069] The experimental workflow for transcriptome sequencing includes RNA extraction and detection, library construction, library quality control, and sequencing.

[0070] 2.1 RNA extraction and detection

[0071] High-quality RNA is fundamental to the success of the entire project. To ensure RNA quality, samples are tested using the following methods, and library construction can only proceed after the RNA has passed the tests.

[0072] (1) Qubit 4.0 fluorometer / MD microplate reader: high-precision measurement of RNA concentration;

[0073] (2) Qsep400 Bioanalyzer: Accurately detects RNA integrity.

[0074] 2.2 Library Construction

[0075] Eukaryotic transcriptome mRNA is obtained by utilizing the structural feature of polyA tails found in most eukaryotic mRNAs. PolyA-tailed mRNAs are enriched using Oligo(dT) magnetic beads. Fragmentation buffer is then added to break the RNA into short fragments. Using these short fragments as templates, first-stranded cDNA is synthesized using six-base random hexamers. Second-stranded cDNA is then synthesized using buffer, dNTPs (dTTP, dATP, dGTP, and dCTP), and DNA polymerase. The double-stranded cDNA is then purified using DNA purification beads. The purified double-stranded cDNA undergoes end repair, A-tailing, and ligation with sequencing adapters. Fragment size selection is then performed using DNA purification beads, and finally, PCR enrichment is conducted to obtain the final cDNA library.

[0076] 2.3 Document Quality Inspection

[0077] After the library is constructed, its quality is checked. Only after the check results meet the requirements can sequencing be performed. The check method is as follows:

[0078] (1) Preliminary quantification was performed using the Qubit dye method;

[0079] (2) Use a fragment analyzer to check the insert size of the library. Only after the insert size meets the expectations can the next step of the experiment be carried out.

[0080] 2.4 Sequencing

[0081] After the libraries pass the library inspection, different libraries are pooled according to the target amount of data to be sequenced, and then sequenced using the Illumina platform.

[0082] Example 1: Preparation of a compound of donkey-hide gelatin and honey

[0083] This embodiment provides a method for preparing a compound of donkey-hide gelatin and honey, including the following steps:

[0084] 20g of donkey-hide gelatin was poured into 40g and 80g of honey respectively, water was added and diluted until dissolved and transparent, and then evaporated and concentrated: the liquid was boiled and evaporated, concentrated to 1 / 4 of the original volume, and then freeze-dried using a freeze dryer. After freeze-drying, the freeze-dried powder was stored and preserved to obtain a compound with a mass ratio of donkey-hide gelatin and honey of 1:2 and a compound with a mass ratio of donkey-hide gelatin and honey of 1:4 respectively.

[0085] Example 2: The effect of a compound of donkey-hide gelatin and honey on alleviating oxidative stress in mice in an ethanol-induced oxidative damage model.

[0086] Experimental Methods: SPF-grade male BALB / c mice were used in the experiment. After being pre-fed in a barrier facility quarantine room for 5 days, the animals were randomly divided into four groups according to their body weight: a blank control group, a model control group (ethanol injury group), a donkey-hide gelatin group (0.5 g / kg BW), a low-dose honey group (1.0 g / kg BW), a high-dose honey group (2.0 g / kg BW), and a low-dose donkey-hide gelatin and honey compound group (1.0 g / kg BW): using the donkey-hide gelatin and honey compound with a mass ratio of 1:2 as described in Example 1; and a high-dose donkey-hide gelatin and honey compound group (2.0 g / kg BW): using the donkey-hide gelatin and honey compound with a mass ratio of 1:4 as described in Example 1. Ten animals were in each group and were orally fed the corresponding amounts of donkey-hide gelatin and honey once a day for 30 days. After the last feeding, except for the blank control group, the other groups were fasted for 16 hours and then given 50% ethanol solution at a dose of 12 mL / kg BW to induce acute oxidative stress. Six hours after modeling, the animals were euthanized under CO2 anesthesia, and liver tissue was harvested under aseptic conditions. MDA content, PCO content, GSH content, and SOD activity were measured in the mouse liver.

[0087] In this invention, MDA, as a terminal decomposition product of lipid peroxidation, can objectively reflect the degree of damage to cell membrane structural integrity through its tissue concentration. Experimental data show ( Figure 1 The model control group showed a significantly higher MDA content than the blank control group (P<0.05, P=0.001), confirming the successful construction of the antioxidant damage model. After intervention, the MDA levels in all experimental groups were significantly lower than those in the model control group (P<0.05), and the effect of reducing MDA content showed a clear dose-dependent increase with increasing honey concentration. Notably, there was a statistically significant difference between the donkey-hide gelatin single-ingredient intervention group and the high-dose compound group (P<0.05, P=0.003), with the donkey-hide gelatin-honey synergistic intervention strategy showing the strongest antioxidant efficacy. Both donkey-hide gelatin and honey, used alone and in combination, showed significant antioxidant effects, inhibiting lipid peroxidation in a dose-dependent manner, and their synergistic effect produced a stronger antioxidant effect.

[0088] This invention relates to SOD, a key antioxidant enzyme for scavenging excess free radicals in the body, whose activity level directly reflects the body's oxidative stress state. Experimental results show ( Figure 2 The SOD activity in the model control group was significantly lower than that in the blank control group (P<0.05, P=0.001), further validating the effectiveness of the antioxidant damage model. After intervention, the SOD activity in all experimental groups significantly increased compared to the model group (P<0.05). There was no significant difference between the low- and high-dose honey groups (P>0.05, P=0.053), while the single-dose donkey-hide gelatin intervention group showed a significant upward trend (P<0.05, P=0.007). For the compound groups, the high-dose compound group showed a more significant increase compared to the donkey-hide gelatin group, indicating that donkey-hide gelatin, honey, and the combination of donkey-hide gelatin and honey have an effect on increasing SOD levels in the body. The effect is more significant with increasing honey concentration after compounding. Donkey-hide gelatin and honey can positively regulate SOD expression through independent or synergistic effects, and honey concentration is positively correlated with antioxidant effect.

[0089] The GSH of this invention is composed of glutamic acid, cysteine, and glycine. Its inherent sulfhydryl groups can neutralize free radicals and decompose hydrogen peroxide and lipid peroxides, making it a core component of an important endogenous antioxidant defense system within cells. Experimental data show ( Figure 3 The GSH content in the model control group was significantly lower than that in the blank control group (P<0.05, P=0.001), meeting the criteria for successful model establishment. Comparison of inter-group differences showed no statistically significant differences in GSH content among the donkey-hide gelatin group, the low- and high-dose honey groups, and the low-dose honey-donkey-hide gelatin compound group (P>0.05), but there was a significant difference compared to the high-dose honey-donkey-hide gelatin group (P<0.05, P=0.012). Honey concentration exhibited a threshold effect of 1.0-2.0 g / kg in the compound system. When the critical concentration was exceeded, it could activate the activity of the rate-limiting enzyme in GSH synthesis (γ-glutamylcysteine ​​ligase), synergistically enhancing the effect of donkey-hide gelatin polypeptide components. Under the compound intervention mode, honey concentration was positively correlated with GSH synthesis efficiency, suggesting a synergistic mechanism based on substrate complementarity.

[0090] This invention utilizes PCO as a marker of protein covalent modification induced by oxidative stress, and its level can accurately reflect the degree of oxidative damage to amino acid residues. Experimental data show that ( Figure 4The PCO content in the model control group was significantly higher than that in the blank control group (P<0.05, P=0.001), meeting the criteria for establishing a protein oxidative damage model. Compared with the model group, the PCO levels in the high-dose honey group and the high-dose compound group were significantly lower (P<0.05, P=0.037), while the differences in other intervention groups (low-dose honey, donkey-hide gelatin group, and low-dose honey-donkey-hide gelatin compound group) were not significant (P>0.05). This indicates that when the honey concentration is maintained at 2.0 g / kg, it can effectively inhibit the formation of protein carbonyl adducts through conformational stability regulation, and exhibits a dose-dependent synergistic protective effect when combined with donkey-hide gelatin.

[0091] Example 3: Regulation of intestinal flora in mice in an ethanol-induced oxidative damage model by a compound of donkey-hide gelatin and honey.

[0092] Experimental Methods: SPF-grade male BALB / c mice were used in the experiment. After being pre-fed in a barrier facility quarantine room for 5 days, the animals were randomly divided into four groups according to their body weight: a blank control group, a model control group (ethanol injury group), a donkey-hide gelatin group (0.5 g / kg BW), a honey group (low: 1.0 g / kg BW, high: 2.0 g / kg BW), and a donkey-hide gelatin (0.5 g / kg BW) + honey group (low: 1.0 g / kg BW, high: 2.0 g / kg BW). Each group consisted of 10 animals. The animals were orally fed the corresponding prepared feed once daily for 30 days. After the last feeding, except for the blank control group, the other groups were fasted for 16 hours and then given 50% ethanol solution at a dose of 12 mL / kg BW to induce acute oxidative stress. Six hours after modeling, the animals were euthanized under CO2 anesthesia, and mouse feces were collected aseptically. Fecal samples were analyzed, with microbial 16S rRNA sequencing being the key indicator.

[0093] Alpha diversity index inter-group difference analysis results showed that box plots can intuitively characterize the diversity distribution characteristics of microbial communities within different experimental groups. This study used ace, chao1, observed species, and Shannon indices to test inter-group differences. Figure 5Data showed that compared with the model group, there were no significant changes in the single donkey-hide gelatin or honey groups, while the honey-donkey-hide gelatin compound exhibited dose-dependent changes in the alpha diversity index: the high-dose compound group showed statistically significant decreases in the abundance indices of ace, chao1, and observed species (P<0.05), at 19%, 23%, and 9%, respectively. This suggests that the compound maintains the abundance of gut microbiota species while optimizing the microbiota structure to achieve diversity homeostasis. The reduction in relative deviation also demonstrates improved evenness, thus forming a stable proportion of dominant microbiota. The Shannon diversity index showed no statistically significant difference between groups (P>0.05), reflecting the similarity in the distribution of microbiota species among the experimental groups. These findings confirm that the donkey-hide gelatin-honey compound significantly enhances the structural stability of the gut microbiota ecosystem by regulating the alpha diversity index, and its mechanism of action may be closely related to the regulation of the relative abundance of specific bacterial genera.

[0094] Species composition analysis at the phylum level showed that the 12 major phyla included unannotated groups (labeled as unidentified) and low-abundance groups (classified as other). For example... Figure 6 As shown in Figure A, the dominant bacterial groups (relative abundance >1%) were, in descending order: Bacteroides, Firmicutes, Actinobacteriota, Proteobacteria, and Desulfobacterota. The relative abundance of Bacteroides in the model group was lower than that in the control and experimental groups, suggesting that this genus may be involved in antioxidant-related metabolic pathways, which is beneficial for the secretion and metabolism of antioxidant-related substances. The abnormal proliferation of Firmicutes and Actinobacteria confirmed that the intestinal flora dysbiosis was successfully induced during the modeling process. Compared to the model control group, the donkey-hide gelatin group and the honey group had less impact on the distribution ratio of the bacterial flora, and different doses of honey did not significantly affect the abundance. The addition of the compound of donkey-hide gelatin and honey caused changes in the distribution of the intestinal flora; with the increase of the dosage of the compound of donkey-hide gelatin and honey, the relative abundance of Bacteroides gradually increased, while the relative abundance of Firmicutes gradually decreased. The regulatory gradient was significantly negatively correlated with host metabolic indicators, suggesting that the compound may optimize intestinal metabolic homeostasis by reshaping the proportion of core phyla, and its mechanism of action is related to the dose-response relationship of the host-microbe co-metabolic regulatory network.

[0095] To further investigate the effects of the donkey-hide gelatin-honey compound on the microbial community structure, corresponding analyses of bacterial counts were performed on the blank control group, the model control group, and the two compound dosage groups. Microbial community structure analysis based on OTU clustering showed ( Figure 6In section B), the four groups shared a total of 55 core OTUs. The blank control group, model control group, low-dose group, and high-dose group showed specific OTUs of 137, 52, 186, and 191, respectively. Bioinformatics analysis showed a significant positive correlation between the number of OTUs and the gut microbiota α diversity index. In this experiment, the high-dose group with donkey-hide gelatin and honey had the highest number of OTUs. Feeding mice with low and high doses of the donkey-hide gelatin and honey compound showed a trend of increased abundance and diversity of gut microbiota, which corresponds to the gut microbiota abundance stacking plot.

[0096] The LEfSe (LDA Effect Size) algorithm is a statistical biomarker mining tool specifically designed to identify significantly different microbial groups or functional units between different biological groups (such as experimental and control groups). To further compare the effects of different compound dosages on the gut microbiota, analysis of gut microbiota differential characteristics based on the LEfSe algorithm showed (…). Figure 7 (A) Among them, four OTUs served as characteristic biomarkers in the model group, and 12 OTUs specifically drove the microbial community remodeling in the high-dose group. *Lactobacillus rhamnosus* and *G. g. streptococci* showed significantly higher abundance in the low-dose honey-donkey-hide gelatin compound group, while bacteria such as *Marinifilaceae* and *Odoribacter*, which influence inflammatory factors, showed significantly higher abundance in the control group. Meanwhile, from... Figure 7 As shown in B, the high-dose group of honey and donkey-hide gelatin compound was significantly enriched with Lactobacillus, Lactobacillus, Bacillus, Lactobacillus rhamnosus, and g-streptococcus, which corresponded to the composition of the low-dose group of honey and donkey-hide gelatin compound. This suggests that the compound achieves the reconstruction of intestinal microenvironment homeostasis by regulating the ecological niche competition of key functional flora.

[0097] Example 4: Regulation of antioxidant pathways in mice in an ethanol-induced oxidative damage model by a compound of donkey-hide gelatin and honey.

[0098] Experimental Methods: SPF-grade male BALB / c mice were used in the experiment. After 5 days of pre-feeding in a barrier-controlled quarantine room, the animals were randomly divided into four groups according to their body weight: a blank control group, a model control group (ethanol injury group), a donkey-hide gelatin group (0.5 g / kg BW), a honey group (low: 1.0 g / kg BW, high: 2.0 g / kg BW), and a donkey-hide gelatin (0.5 g / kg BW) + honey group (low: 1.0 g / kg BW, high: 2.0 g / kg BW). Each group consisted of 10 animals. The animals were orally fed the corresponding prepared feed once daily for 30 days. After the last feeding, except for the blank control group, the other groups were fasted for 16 hours and then given 50% ethanol solution at a dose of 12 mL / kg BW to induce acute oxidative stress. Six hours after model establishment, the animals were euthanized under CO2 anesthesia, and liver tissue was harvested under aseptic conditions. Transcriptome sequencing was performed on the liver samples.

[0099] MA plots can visually display the overall distribution of gene expression levels and fold changes, such as Figure 8 As shown in the figure. The results indicated that the high-dose group of donkey-hide gelatin honey significantly upregulated the PI3 / AKT gene pathway and glutathione metabolism genes, while downregulating factors related to oxidative stress and inflammatory responses.

[0100] Differentially expressed genes were normalized using Z-scores, and cluster heatmaps of differentially expressed genes in the union of all comparison groups and cluster heatmaps for each differentially expressed group were generated. Figure 9 According to the differential gene clustering heatmap, genes such as ENSMUSG00000031963 were significantly upregulated in the model group. High expression of genes such as ENSMUSG00000025889 and ENSMUSG00000005547 was observed in the high-dose honey group, which may be related to mitochondrial metabolism or antioxidant defense. Low expression of genes such as ENSMUSG00000064367 and ENSMUSG00000035697 suggests that this treatment may inhibit inflammatory pathways or apoptosis. The results show that the high-dose donkey-hide gelatin honey group significantly promoted cell survival, repair, and antioxidant defense by activating the PI3K / AKT pathway, with better effects than other experimental groups.

[0101] Pathway significant enrichment analysis was performed using pathways in the KEGG database as units. Hypergeometric tests were applied to identify pathways that were significantly enriched in differentially expressed genes compared to the overall genomic background. Based on... Figures 10-15The 50 KEGG pathways with the lowest qvalues ​​in the enrichment analysis results were selected, and a bar chart of enrichment entries was plotted. Differentially enriched genes between the control and model groups were mainly concentrated in glycolysis / gluconeogenesis, carbon metabolism, amino acid biosynthesis, pyruvate metabolism, and fructose and mannose metabolism. Significantly enriched pathways were mainly concentrated in the metabolic domain (such as glucose metabolism and amino acid metabolism), especially glycolysis / gluconeogenesis and carbon metabolism pathways, which showed extremely high significance (P<0.001). These results suggest that energy metabolism may be the core biological change under experimental conditions. Through comprehensive analysis of the six KEGG pathway enrichment bar charts, the high-dose donkey-hide gelatin honey group and the high-dose honey group showed significant characteristics in gene pathway regulation compared to the control group. The high-dose group of donkey-hide gelatin and honey showed significant enrichment in glycolysis / gluconeogenesis (14.29%) and carbon metabolism (14.29%) pathways, and activation of fatty acid metabolism (6.12%) and pyruvate metabolism (8.16%) in the high-dose honey group, indicating a shift in energy metabolism towards glucose utilization and lipid oxidation. The high-dose groups of donkey-hide gelatin and honey also showed enrichment in glutathione metabolism (6.12%) and cytochrome P450-mediated drug metabolism (4.08%) pathways, suggesting that the synergistic intervention of donkey-hide gelatin and honey alleviates oxidative stress through scavenging reactive oxygen species (ROS) and detoxification of exogenous substances. This confirms that the synergistic effect of donkey-hide gelatin and honey activates the antioxidant system and alleviates oxidative stress and liver damage.

[0102] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. Use of a combination of donkey-hide gelatin and honey for the preparation of an antioxidant product, characterized in that, The mass ratio of Ejiao and honey in the compound is 1:2-1:

4.

2. Use according to claim 1, characterized in that, The mass ratio of Ejiao and honey in the compound is 1:

4.

3. Use according to claim 1, characterized in that, The Ejiao is a powder prepared by decocting and concentrating the dried or fresh skin of Equus asinus L.

4. Use according to claim 1, characterized in that, The Ejiao is produced by Donge Ejiao Co., Ltd.

5. The use according to claim 1, characterized in that, The product is a food.

6. Use according to claim 5, characterized in that, The food is one or more of a drink, a paste food, a preserved fruit food, and a cake food.

7. Use according to claim 1, characterized in that, The product is a health care product.

8. Use according to claim 7, characterized in that, The dosage form of the health care product is selected from one or more of a tablet, a capsule, a soft capsule, a granule, a pill, and an oral liquid.

9. The use according to claim 1, characterized in that, The product is a medicine.

10. The use according to claim 1, characterized in that, The medicine further comprises an auxiliary material; the auxiliary material comprises one or more of an excipient, a pigment, a flavoring agent, a disintegrating agent, and a lubricant.

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