Application of flavonols in the preparation of drugs for the prevention and / or treatment of oxidative damage

Abstract

This invention provides the application of flavonols in the preparation of drugs for the prevention and / or treatment of oxidative damage, belonging to the field of biopharmaceutical technology. The flavonols include quercetin and / or kaempferol. Experiments show that quercetin and kaempferol can promote growth by increasing the antioxidant level of the 42-day-old ileum, regulating cecal microbiota, and increasing ileal villus length. This invention provides a new approach to the prevention and treatment of oxidative damage, while also broadening the pharmaceutical applications of flavonols.

Description

Technical Field

[0001] This application belongs to the field of biopharmaceutical technology, specifically relating to the use of flavonols in the preparation of drugs for the prevention and / or treatment of oxidative damage. Background Technology

[0002] Long-term consumption of oxidized oils, fried foods, moldy foods, and high-sugar, high-fat diets introduces a large amount of lipid peroxides and exogenous free radicals. When excessive ROS are generated or the body's own antioxidant capacity is insufficient, the redox homeostasis is disrupted, and free radicals attack cell membranes, proteins, and DNA, ultimately causing oxidative damage, which in turn induces digestive system diseases, cardiovascular diseases, inflammatory or immune-related diseases, and metabolic diseases.

[0003] Currently, common methods for preventing and treating oxidative damage include exogenous supplementation with antioxidants to enhance the body's antioxidant capacity, supplementation with probiotics to regulate the gut microbiota structure and reduce the metabolic toxin production of harmful bacteria, and administration of anti-inflammatory mucosal protectants to suppress local intestinal inflammation. However, these methods only alleviate oxidative damage at a single level. Summary of the Invention

[0004] The purpose of this invention is to provide the application of flavonols in the preparation of drugs for the prevention and / or treatment of oxidative damage, which have good antioxidant capacity, maintain liver and intestinal health, and regulate the balance of intestinal microbiota.

[0005] This invention provides the use of flavonols in the preparation of medicaments for the prevention and / or treatment of oxidative damage.

[0006] Preferably, the flavonols include quercetin and / or kaempferol.

[0007] Preferably, the oxidative damage includes intestinal oxidative damage and / or liver oxidative damage.

[0008] Preferably, the intestinal oxidative damage includes at least one of the following: increased intestinal length, decreased intestinal barrier, activated intestinal inflammatory response, and intestinal flora imbalance.

[0009] Preferably, the liver oxidative damage includes at least one of the following: reduced liver antioxidant capacity, reduced activity of enzyme antioxidant systems, and reduced lipid metabolism capacity.

[0010] Preferably, the oxidative damage includes oxidative damage caused by oxidized lipid uptake.

[0011] Preferably, the oxidized lipids include at least one of the following: oxidized oils, oxidized fats, rancid oils, and peroxidized oils.

[0012] Preferably, drugs containing quercetin have the effect of increasing SOD activity in the ileal mucosa; The kaempferol drug has the effect of increasing the activity of T-AOC and GSH-Px in the ileal mucosa.

[0013] Preferably, the drug comprises at least one of the following dosage forms: powder, tablet, granule, capsule, oral liquid, injectable powder, and injectable solution.

[0014] Preferably, the flavonol content in the drug is 5% to 98% by mass.

[0015] This invention provides the application of flavonols in the preparation of drugs for the prevention and / or treatment of oxidative damage. Quercetin and kaempferol are used as representative flavonols to illustrate their effects in alleviating oxidative damage. In the examples, an oxidative damage model induced by oxidized fats in broilers was used as the subject. The effects of quercetin and kaempferol were analyzed by detecting antioxidant capacity-related indicators and gene expression in blood and organ tissues before and after drug administration. The results showed that quercetin and kaempferol increased the length of ileal villi and decreased the villus-cryptotropic ratio at 42 days of age, and reduced the MDA content in the ileal mucosa at 21 and 42 days of age. Meanwhile, there are also differences in efficacy between the two: quercetin can increase SOD activity in the ileal mucosa of 42-day-old infants, while kaempferol can increase the activities of T-AOC and GSH-Px in the ileal mucosa of 42-day-old infants. Furthermore, both quercetin and kaempferol can upregulate SOD and GPX3 and downregulate the relative expression of NFκb2 gene mRNA in 42-day-old infants, significantly increasing body weight gain and decreasing the feed conversion ratio in this age group. Additionally, at 42 days of age, norank_f_Rikenellaceae and... Alistipes Relative abundance decreased Lactobacillus The relative abundance increased after the addition of quercetin or kaempferol. This suggests that quercetin and kaempferol may improve growth performance by enhancing the antioxidant levels of the 42-day-old ileum, regulating cecal microbiota, and thus increasing ileal villus length. This invention provides a new approach to the prevention and treatment of oxidative damage and expands the medicinal applications of flavonols. Attached Figure Description

[0016] Figure 1 Veen diagram of gut microbiota at 42 days of age; Figure 2 A bar chart showing the relative abundance of gut microbiota species at 42 days of age, where A represents the phylum level and B represents the genus level; Figure 3 PCA analysis was performed to analyze the β-diversity of gut microbiota at 42 days of age; Figure 4 A bar chart showing the Kruskal-Wallis H test at the level of cecal microorganisms; Figure 5 LEfSe analysis diagram of cecal microbiota (42 days old). Detailed Implementation

[0017] This invention provides the use of flavonols in the preparation of medicaments for the prevention and / or treatment of oxidative damage.

[0018] In this invention, the flavonol preferably includes quercetin and / or kaempferol, more preferably kaempferol. The molecular formula of the quercetin is C1. 15 H 10 O7, with a molecular weight of 302.24 and CAS number 117-39-5, has the structural formula shown in Formula I. The molecular formula of the kaempferol is C0. 15 H 10 O6, with a molecular weight of 286.24 and CAS number 520-18-3, has the structural formula shown in Formula II.

[0019] Formula I and Formula II.

[0020] In this invention, the oxidative damage preferably includes intestinal oxidative damage and / or liver oxidative damage. The intestinal oxidative damage preferably includes at least one of the following: increased intestinal length, decreased intestinal barrier, activated intestinal inflammatory response, and intestinal flora imbalance. The increased intestinal length preferably includes an increased relative jejunum length. The decreased intestinal barrier preferably includes villus damage, abnormal expression of ileum development-related genes, and abnormal expression of ileum oxidative damage-related genes. The villus damage preferably includes abnormal ileum tissue morphology, such as shortened ileum villus length, decreased villus-to-crypt ratio, and decreased crypt depth. The ileum development-related genes preferably include at least one of the following genes: MUC2, Lgr5, Bmi1, Notch1, KI67, Caspase3 and BCL2. The ileal oxidative damage-related genes include genes encoding antioxidant-related factors. These antioxidant-related factors preferably include at least one of the following: IL-10, NOX2, Nrf2, CAT, SOD, GPX2, and GPX3. The inflammatory factors involved in the activation of the intestinal inflammatory response preferably include at least one of the following: TNFα, COX2, NFκb2, IL-6, and IL1β.

[0021] In this invention, the liver oxidative damage preferably includes at least one of the following: reduced liver antioxidant capacity, reduced activity of enzyme antioxidant systems, and reduced lipid metabolism capacity. The liver antioxidant capacity includes at least one of the following indicators: malondialdehyde (MDA) content and reactive oxygen species (ROS) content. The enzyme antioxidant system includes at least one of the following indicators: total antioxidant capacity (T-AOC), catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) activity. The lipid metabolism capacity preferably includes serum triglyceride and / or high-density lipoprotein (HDL) content.

[0022] In this invention, the drug preferably comprises at least one of the following dosage forms: powder, tablet, granule, capsule, oral liquid, injectable powder, and injectable solution. The flavonol content in the drug is preferably 5%~98% by mass, but can be 10%~90%, 15%~85%, 20%~80%, further 30%~70%, and even further 40%~60%. The drug preferably also includes pharmaceutically acceptable excipients. The selection and proportioning of the excipients are conventional and depend on the dosage form of the drug. This invention does not impose any special limitations on the preparation method of the drug; any drug preparation method well known in the art can be used.

[0023] In this invention, the oxidative damage preferably includes oxidative damage caused by the intake of oxidized lipids. The oxidized lipids preferably include at least one of the following: oxidized oils, oxidized fats, rancid oils, and peroxidized oils. In this embodiment of the invention, an oxidative damage model in broilers was constructed using oxidized oils as an inducer. The oxidized oils were obtained by heating (90°C) soybean oil to induce oxidation. Depending on the oxidation time, the oils were divided into early-stage and late-stage soybean oils, both of which showed a significant increase in peroxide value. The peroxide value of the soybean oil also increased with increasing heating and aeration time. Compared with the blank control group (CON), the oxidized oil group demonstrated that oxidized oils induced oxidative damage in broilers in terms of intestinal morphology, liver oxidation, intestinal barrier, and intestinal flora, indicating successful model construction.

[0024] In this embodiment of the invention, quercetin and kaempferol were used as drug evaluation objects to verify the drugs in the model group broilers. The results showed that quercetin and kaempferol increased the length of ileal villi and decreased the villi-to-cryptotropic ratio at 42 days of age, and reduced the MDA content in the ileal mucosa at 21 and 42 days of age; quercetin had the effect of increasing the SOD activity of the ileal mucosa; kaempferol had the effect of increasing the T-AOC and GSH-Px activities of the ileal mucosa. Specifically, compared with the oxidized lipid group, the kaempferol group significantly reduced the MDA content in the liver at 21 days of age ( P= 0.001), the quercetin group significantly increased CAT activity in the liver of 21-day-old broilers, while both quercetin and kaempferol reduced MDA content in the liver of 42-day-old broilers. Compared with the oxidized oil group, the quercetin and kaempferol groups significantly increased serum total protein content at 21 days of age, the kaempferol group significantly increased serum albumin at 21 days of age, and the quercetin and kaempferol groups significantly decreased serum albumin-to-globulin ratio at 21 days of age. Compared with the OSO group, the quercetin and kaempferol groups significantly increased serum T-AOC activity at 21 days of age and significantly decreased MDA content at 21 and 42 days of age, while the quercetin group significantly increased serum T-AOC activity at 42 days of age. Compared with the oxidized oil group, the kaempferol group significantly decreased serum triglyceride and high-density lipoprotein levels at 42 days of age. Compared with the oxidized oil group, kaempferol significantly reduced the relative jejunum length and significantly increased breast muscle rate in 42-day-old broilers. Compared with the oxidized oil group, quercetin and kaempferol had no significant effect on the organ indices of liver, spleen, thymus, and bursa of Fabricius in broilers, and thus showed good safety.

[0025] The following examples illustrate the application of the flavonols provided by the present invention in the preparation of medicaments for the prevention and / or treatment of intestinal oxidative damage, but these examples should not be construed as limiting the scope of protection of the present invention.

[0026] Example 1 1. Materials and Methods 1.1 Experimental Animals and Experimental Design A total of 192 one-day-old Ross 308 male chicks were selected for the experiment and divided into four treatments, with six replicates per treatment and eight chicks per replicate. The treatments were: Control Group (CON): fed a basal diet; Oxidized Oil Group (OSO): fed a basal diet containing oxidized oils; Quercetin Group (Q): fed a basal diet containing oxidized oils and administered quercetin; Kaempferol Group (Ka): fed a basal diet containing oxidized oils and administered kaempferol. The experiment lasted 42 days, divided into two phases: an early phase (0-21 days) and a later phase (22-42 days). Chicks were housed in multi-layered, overlapping cages according to broiler rearing management methods, with free access to feed and water, and routine immunizations. Production performance was measured at 21 and 42 days of age, and blood and organ tissues were collected from slaughtered chicks for analysis.

[0027] 1.2 Reagent preparation and diet formulation Quercetin (98%, purchased from Shaanxi Senfu Group Co., Ltd., Xi'an) and kaempferol (98%, purchased from Xirun Biotechnology Co., Ltd., Xi'an) were mixed into the basic feed in stages. The dosage of quercetin was 400 mg / kg and the dosage of kaempferol was 378.82 mg / kg (equimolar mass of quercetin).

[0028] Preparation of oxidized oil: Fresh soybean oil without added antioxidants was purchased, heated to 90℃ and kept warm while continuously purging air at 64 L / min. The oil treatment time for the initial diet was 80 h, and for the subsequent diet, it was 160 h. The oil was then cooled to room temperature for later use. A corn-soybean meal diet was formulated according to the chicken feeding standard (NY / T 33-2004), with an oil content of 40 g / kg. The composition and nutrient levels of the basal diet are shown in Table 1.

[0029] Table 1. Composition and nutrient levels of the experimental basal diet

[0030] Note: 1. The blank control group was supplemented with fresh soybean oil without added antioxidants, and the oxidized oil treatment group was supplemented with the same proportion of oxidized oil; 2. Sand was used as the additive carrier; all nutrient levels in the table are calculated values.

[0031] 1.3 Test Instruments Mindray fully automated biochemical analyzer (Hitachi 7160, Hitachi High Technology Co., Ltd., Tokyo, Japan); constant temperature high-speed centrifuge (Xiangli Scientific Instruments Co., Ltd., China, TG16-WS); full-function microplate analyzer (Biotek, USA, Synergy H1); ice maker (Panasonic PHCBI, Japan, sim-f140ay65); optical microscope (Nikon Eclipse E100, Nikon Corporation, Tokyo, Japan).

[0032] 1.4 Detection Indicators and Methods 1.4.1 Growth performance On the mornings of days 21 and 42 of the experiment, broilers were weighed on an empty stomach, and the weight of each group of broilers was recorded. Feed consumption and feed intake (FI) were recorded during days 0-21 and 21-42, and body weight gain (BWG) and feed conversion ratio (FCR) were calculated for each stage.

[0033] 1.4.2 Sample Collection and Preservation On days 21 and 42, after a 12-hour fast, blood and tissue samples were collected from one broiler chicken of near-average weight in each replicate. Blood was collected via the wing vein into 10 mL anticoagulant-free vacuum tubes and allowed to stand at room temperature for 1 hour. The supernatant was then separated by centrifugation at 3500 rpm for 15 minutes and stored at -80°C for further analysis. After blood collection, the broilers were slaughtered and dissected, and the liver, spleen, thymus, and bursa of Fabricius were weighed for organ index calculation. Approximately 1 cm segments of the jejunum and ileum were collected and incubated in 4% paraformaldehyde solution for subsequent section analysis. Additionally, liver, intestinal tissue samples, and cecal chyme were collected and stored at -80°C for analysis.

[0034] 1.4.3 Organ Index The organ indices of the collected liver, spleen, thymus, and bursa of Fabricius were calculated according to Formula I: Organ index = organ weight (g) / broiler live weight (g) × 100% Formula I; The collected intestines were calculated according to Formula II: Relative intestinal length (cm / Kg) = Intestinal length (cm) / Broiler live weight (Kg) Formula II.

[0035] 1.4.4 Serum Biochemistry The levels of total cholesterol (TC), triglycerides (TG), low-density lipoprotein (LDL), and high-density lipoprotein (HDL) in the serum of 21-day-old infants were analyzed using an automated biochemical analyzer (Hitachi 7160, Hitachi High Technology Co., Ltd., Tokyo, Japan). The levels of TC (catalog number: A111-1-1), TG (catalog number: A110-1-1), LDL (catalog number: A113-1-1), and HDL (catalog number: A112-1-1) in the serum of 42-day-old infants were analyzed using a kit (Nanjing Jiancheng Biological Research Institute, Nanjing, China) according to the manufacturer's instructions.

[0036] 1.4.5 Antioxidant properties The peroxide value of oils and fats was determined according to the national standard GB T5532-2008. Following the instructions, the total antioxidant capacity (T-AOC) (kit number: A015-1-2), catalase (CAT) (kit number: A007-1-1), superoxide dismutase (SOD) (kit number: A001-1-2), glutathione peroxidase (GSH-Px) (kit number: A005-1-2), malondialdehyde (MDA) (kit number: A003-1-2), and ROS content (kit number: E004-1-1) in tissue and serum samples were detected using a kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

[0037] 1.4.6 Intestinal tissue section Partial liver sections were fixed in 4% paraformaldehyde solution for 48 hours, then dehydrated for 12 hours, embedded in paraffin, and cut into 3 μm thick sections. These sections were stained with hematoxylin and eosin (H&E) and observed under an optical microscope (Nikon Eclipse E100, Nikon Corporation, Tokyo, Japan). Images were acquired. Each section was observed under a 40x microscope, and villus length and crypt depth were measured.

[0038] 1.4.7 Gene expression in ileum tissue Ileal tissue mRNA was reverse transcribed using a reverse transcription kit purchased from Takara to obtain cDNA. Quantitative real-time PCR was performed using Takara's SYBR® Premix Ex Taq™ II (TliRNAase H Plus) kit (catalog number RR802A). The qPCR reaction mixture consisted of 20 μL: 10 μL SYBR Premix Ex Taq II (Tli RNase H Plus) (2 ×), 0.8 μL forward primer (10 μM), 0.8 μL reverse primer (10 μM), 0.4 μg ROX Reference Dye (50 ×), 2 μL cDNA, and 6 μL ddH2O. Primer design and synthesis were performed by Shanghai Sangon Biotech Co., Ltd., and primer information is shown in Table 2. The qPCR reaction procedure was as follows: pre-denaturation at 95℃ for 30 s, followed by polymerase chain reaction at 95℃ for 5 s and 60℃ for 30 s, for a total of 45 cycles. The melting curve conditions were: 95℃ for 15 s, 60℃ for 1 min, with the temperature increasing from 60℃ to 95℃ at a rate of 0.5℃ / 10 s. Finally, 2... -△△CT The relative expression level of genes can be calculated.

[0039] Table 2 Primer sequences and parameters

[0040] 1.4.8 16S rRNA sequencing analysis of cecal chyme Total genomic DNA was extracted from the microbial community according to the EZNA® soil DNA kit (Omega Bio-tek, Norcross, GA, US). The quality of the extracted genomic DNA was detected by 1% agarose gel electrophoresis, and the DNA concentration and purity were determined using a NanoDrop2000 (Thermo Scientific, USA). Using the extracted DNA as a template, PCR amplification of the V3-V4 variable region of the 16S rRNA gene was performed using upstream primer 338F (5'-ACTCCTACGGGAGGCAGCAG-3', SEQ ID NO:49) and downstream primer 806R (5'-GGACTACHVGGGTWTCTAAT-3', SEQ ID NO:50) carrying the barcode sequence. The PCR reaction system was as follows: 4 μL of 5×TransStart FastPfu buffer, 2 μL of 2.5 mM dNTPs, 0.8 μL of upstream primer (5 uM), 0.8 μL of downstream primer (5 uM), 0.4 μL of TransStart FastPfu DNA polymerase, 10 ng of template DNA, and brought to a final volume of 20 μL. The amplification program was as follows: pre-denaturation at 95℃ for 3 min, 27 cycles (denaturation at 95℃ for 30 s, annealing at 55℃ for 30 s, extension at 72℃ for 30 s), followed by a stable extension at 72℃ for 10 min, and finally storage at 4℃ (PCR instrument: ABI GeneAmp® 9700). PCR products were recovered using a 2% agarose gel, purified using a DNA gel purification kit (PCR Clean-Up Kit, China Yuhua), and quantified using a Qubit 4.0 (Thermo Fisher Scientific, USA).

[0041] Library construction was performed on purified PCR products using the NEXTFLEX Rapid DNA-Seq Kit. Sequencing was conducted using the Illumina Nextseq 2000 platform (Shanghai Meiji Biotechnology Co., Ltd.). Raw data were uploaded to the NCBIO BioProject database (Submission ID: SUB15184425; BioProject ID: PRJNA1238495). Quality control of the paired-end raw sequencing sequences was performed using fastp (https: / / github.com / OpenGene / fastp, version 0.19.6), and assembly was performed using FLASH (http: / / www.cbcb.umd.edu / software / flash, version 1.2.11). Based on default parameters, noise reduction was performed on the optimized sequences after quality control assembly using the DADA2 plugin (or Deblur plugin) in the Qiime2 workflow. Bioinformatics analysis was performed using the Meiji BioCloud platform at https: / / cloud.majorbio.com / page / project / overview.html.

[0042] 1.4.9 Statistical Analysis All data were analyzed using SPSS 27.0. One-way ANOVA was used for statistical analysis, and Tukey multiple comparison analysis was used to analyze differences between groups. P <0.05 indicates a significant difference.

[0043] 2 Results of oxidative damage in broilers induced by oxidized oils 2.1 Growth performance and organ index As shown in Table 3, compared with the blank control group, the intake of oxidized oils significantly reduced feed intake and feed conversion ratio in infants aged 0-21 days. P = 0.034、 P = 0.017), significantly reducing feed intake and body weight gain in 21-42 day old infants. P = 0.042、 P <0.001, significantly increasing the feed conversion ratio (FCR) in the 21-42 day old age group ( P <0.001).

[0044] Table 3. Effects of oxidized oil intake on broiler growth performance

[0045] Note: n=6, different lowercase letters in the superscript of the same data indicate significant differences.

[0046] As shown in Table 4, compared with the blank control group, the intake of oxidized oils significantly increased the liver organ index of broilers aged 0-42 days ( P = 0.026), significantly reducing the spleen organ index in broilers aged 0-42 days ( P = 0.030).

[0047] Table 4. Effects of oxidized oil intake on organ index in broilers (%)

[0048] Note: n=6, different lowercase letters in the superscript of the same data indicate significant differences.

[0049] As shown in Table 5, compared with the blank control group, the intake of oxidized oils significantly reduced the relative length of the duodenum and jejunum in 42-day-old broilers. P = 0.016、 P <0.001, significantly increased the relative length of the jejunum in 42-day-old broilers ( P <0.001).

[0050] Table 5. Effects of oxidized oil intake on the relative intestinal length of broilers (cm / kg)

[0051] Note: n=6, different lowercase letters in the superscript of the same data indicate significant differences.

[0052] 2.2 Liver oxidation and health indicators As shown in Table 6, compared with the blank control group, the intake of oxidized lipids significantly increased the MDA content in the liver of 21-day-old infants ( P =0.012) and GSH-PX activity ( P = 0.024), significantly reducing liver T-AOC in 42-day-old infants ( P = 0.002), significantly increased the liver MDA content of 42-day-old infants ( P = 0.029) and GSH-PX activity ( P = 0.009).

[0053] Table 6. Effects of oxidized oil intake on the antioxidant status of broiler livers

[0054] Note: n=6, different lowercase letters in the superscript of the same data indicate significant differences.

[0055] As shown in Table 7, compared with the blank control group, the intake of oxidized lipids significantly increased serum ALT levels in 21-day-old infants. P =0.007), AST ( P<0.001) and TP content ( P <0.001), significantly increased serum AST ( ) in 42-day-old infants. P = 0.005), TP ( P <0.001), ALB ( P <0.001) and the content of GLB ( P = 0.001).

[0056] Table 7. Effects of oxidized oil intake on liver health in broilers.

[0057] Note: n=6, different lowercase letters in the superscript of the same data indicate significant differences.

[0058] As shown in Table 8, compared with the blank control group, the intake of oxidized lipids was significantly reduced at 21 days of age ( P <0.001) and 42 days old ( P <0.001) chicken serum T-AOC significantly increased in 21-day-old chickens ( P <0.001) and serum MDA content in 42-day-old chickens ( P =0.002).

[0059] Table 8. Effects of oxidized oil intake on antioxidant status in broiler serum.

[0060] Note: n=6, different lowercase letters in the superscript of the same data indicate significant differences.

[0061] 2.3 Intestinal barrier function As shown in Table 9, compared with the blank control group, the intake of oxidized lipids significantly reduced the ileochorionic villus ratio at 42 days of age. P =0.024).

[0062] Table 9. Effects of oxidized oil intake on ileal tissue morphology in 42-day-old broilers

[0063] Note: n=6, different lowercase letters in the superscript of the same data indicate significant differences.

[0064] As shown in Table 10, compared with the blank control group, the intake of oxidized lipids significantly reduced the T-AOC in the ileal mucosa of 21-day-old infants (T-AOC). P< 0.001), significantly increased the MDA content in the ileal mucosa of 21-day-old infants ( P = 0.002); the intake of oxidized lipids significantly reduced the T-AOC of the ileal mucosa in 42-day-old infants ( P = 0.004), CAT ( P= 0.003), SOD ( P< 0.001) and GSH-PX ( P = 0.001), significantly increased the MDA content in the ileal mucosa of 21-day-old infants ( P = 0.008).

[0065] Table 10 Effects of oxidized oil intake on the antioxidant status of ileal mucosa in broilers

[0066] Note: n=6, different lowercase letters in the superscript of the same data indicate significant differences.

[0067] As shown in Table 11, compared with the blank control group, at 21 days of age, the intake of oxidized lipids significantly downregulated the expression of the KI67 gene ( P <0.05).

[0068] Table 11 Effects of oxidized oil intake on the relative expression levels of genes related to ileal development in 21-day-old broilers

[0069] Note: n=6, different lowercase letters in the superscript of the same data indicate significant differences.

[0070] As shown in Table 12, compared with the blank control group, at 42 days of age, the intake of oxidized lipids significantly downregulated the expression of Claudin2, Lgr5, Bmi1, and Notch1 genes. P <0.05, significantly upregulated the expression of the Caspase3 gene ( P <0.05).

[0071] Table 12 Effects of oxidized oil intake on the relative expression levels of genes related to ileal development in 42-day-old broilers

[0072] Note: n=6, different lowercase letters in the superscript of the same data indicate significant differences.

[0073] As shown in Table 13, compared with the blank control group, at 21 days of age, the intake of oxidized lipids significantly upregulated the expression of COX2 and Nrf2 genes. P <0.05, significantly downregulated the expression levels of IL-10 and NOX2 genes ( P <0.05).

[0074] Table 13 Effects of oxidized oil intake on the relative expression levels of genes related to oxidative damage in the ileum of 21-day-old broilers

[0075] Note: n=6, different lowercase letters in the superscript of the same data indicate significant differences.

[0076] As shown in Table 14, compared with the blank control group, at 42 days of age, the intake of oxidized lipids significantly upregulated the expression of TNFα, NFκb2, NOX2, Nrf2, and CAT genes. P <0.05, significantly downregulated the expression of COX2, IL-6, ILIβ, IL-10, GPX2, and GPX3 genes ( P <0.05).

[0077] Table 14 Effects of oxidized oil intake on the relative expression levels of genes related to oxidative damage in the ileum of 42-day-old broilers

[0078] Note: n=6, different lowercase letters in the superscript of the same data indicate significant differences.

[0079] As shown in Table 15, compared with the blank control group, at 42 days of age, the intake of oxidized oils had no significant effect on the α-diversity index of cecal microorganisms in broilers. P >0.05).

[0080] Table 15 Effects of oxidized oil intake on the α-diversity index of cecal microbiota in 42-day-old broilers

[0081] Note: n=6, different lowercase letters in the superscript of the same data indicate significant differences.

[0082] 3. Results of administration of quercetin and kaempferol on alleviating oxidative damage induced by oxidized lipids in broilers. 3.1 Growth performance and organ index As shown in Table 16, compared with the oxidized oil group, the addition of quercetin and kaempferol significantly increased the body weight at 21 and 42 days of age and the body weight gain from 0 to 21 days of age. P <0.001、 P = 0.002), significantly reducing the feed conversion ratio (FCR) in 0-21 day old animals. P <0.001).

[0083] Table 16 Effects of quercetin and kaempferol on growth performance of broilers consuming oxidized oils

[0084] Note: n=6, different lowercase letters in the superscript of the same data indicate significant differences.

[0085] As shown in Table 17, compared with the oxidized oil group, quercetin and kaempferol had no significant effect on the organ indices of liver, spleen, thymus, and bursa of Fabricius in broilers.P >0.05), kaempferol significantly increased the breast muscle percentage of 42-day-old broilers ( P = 0.018).

[0086] Table 17 Effects of quercetin and kaempferol on organ index of broilers consuming oxidized oil (%)

[0087] Note: n=6, different lowercase letters in the superscript of the same data indicate significant differences.

[0088] As shown in Table 18, compared with the oxidized oil group, quercetin significantly reduced the relative length of the jejunum in 42-day-old broilers. P =0.029).

[0089] Table 18 Effects of quercetin and kaempferol on the relative intestinal length of broilers that ingest oxidized oil (cm / kg)

[0090] Note: n=6, different lowercase letters in the superscript of the same data indicate significant differences.

[0091] 2.2 Liver oxidation and health indicators As shown in Table 19, compared with the oxidized lipid group, the kaempferol group significantly reduced the MDA content in the liver of 21-day-old infants. P <0.001), both quercetin and kaempferol reduced the MDA content in the liver of 42-day-old broilers. P = 0.021).

[0092] Table 19 Effects of quercetin and kaempferol on the antioxidant status of broiler livers after ingesting oxidized oils

[0093] Note: n=6, different lowercase letters in the superscript of the same data indicate significant differences.

[0094] As shown in Table 20, compared with the oxidized oil group, the serum total protein content of the quercetin and kaempferol groups was significantly increased at 21 days of age. P = 0.001), serum albumin levels were significantly increased in the kaempferol group at 21 days of age ( P = 0.045), the serum albumin-to-globulin ratio was significantly lower in the quercetin and kaempferol groups at 21 days of age ( P = 0.011).

[0095] Table 20 Effects of quercetin and kaempferol on liver health in broilers consuming oxidized oils

[0096] Note: n=6, different lowercase letters in the superscript of the same data indicate significant differences.

[0097] As shown in Table 21, compared with the OSO group, the quercetin group and the kaempferol group showed significantly increased serum T-AOC activity at 21 days of age and significantly decreased MDA content at 21 days and 42 days of age. P <0.05%, the quercetin group showed a significant increase in T-AOC activity at 42 days of age ( P <0.001).

[0098] Table 21 Effects of quercetin and kaempferol on the antioxidant status of serum in broilers that have ingested oxidized oils

[0099] Note: n=6, different lowercase letters in the superscript of the same data indicate significant differences.

[0100] As shown in Table 22, compared with the oxidized lipid group, the serum triglyceride and high-density lipoprotein levels in the kaempferol group were significantly lower at 42 days of age. P = 0.003、 P = 0.029).

[0101] Table 22 Effects of quercetin and kaempferol on serum lipid metabolism in broilers that have ingested oxidized oils

[0102] Note: n=6, different lowercase letters in the superscript of the same data indicate significant differences.

[0103] 2.3 Intestinal barrier function As shown in Table 23, compared with the oxidized lipid group, the quercetin group and the kaempferol group showed significantly increased ileal villus length and villus-to-cryptotropy ratio at 42 days of age. P <0.001, P = 0.001), the crypt depth was significantly reduced in the quercetin group ( P = 0.009) Table 23 Effects of quercetin and kaempferol on ileal tissue morphology in 42-day-old broilers that have ingested oxidized oil

[0104] Note: n=6, different lowercase letters in the superscript of the same data indicate significant differences.

[0105] As shown in Table 24, compared with the oxidized lipid group, quercetin and kaempferol significantly reduced the MDA content in the ileal mucosa of 21-day-old and 42-day-old infants. P = 0.012、 P = 0.001), quercetin significantly increased SOD activity in the ileal mucosa of 42-day-old infants ( P= 0.004), Kaempferol significantly increased the activity of T-AOC and GSH-Px in the ileal mucosa of 42-day-old infants ( P = 0.010、 P = 0.047).

[0106] Table 24 Effects of quercetin and kaempferol on the antioxidant status of ileal mucosa in broilers that ingest oxidized oils

[0107] Note: n=6, different lowercase letters in the superscript of the same data indicate significant differences.

[0108] As shown in Table 25, quercetin and kaempferol did not significantly alter genes related to ileal development at 21 days of age. P >0.05).

[0109] Table 25 Effects of quercetin and kaempferol on the relative expression levels of genes related to ileal development in 21-day-old broilers that have ingested oxidized oil.

[0110] Note: n=6, different lowercase letters in the superscript of the same data indicate significant differences.

[0111] As shown in Table 26, at 42 days of age, quercetin upregulated the expression of MUC2, Lgr5, Bmi1, Notch1, and other genes. P <0.05), downregulated the expression of ZO-1, KI67, and Caspase3 genes ( P <0.05), kaempferol upregulated the expression of MUC2, BCL2, Lgr5, Bmi1, and Notch1 genes. P <0.05, downregulated the expression of KI67 and Caspase3 genes ( P <0.05).

[0112] Table 26 Effects of quercetin and kaempferol on the relative expression levels of genes related to ileal development in 42-day-old broilers that have ingested oxidized oil

[0113] Note: n=6, different lowercase letters in the superscript of the same data indicate significant differences.

[0114] As shown in Table 27, kaempferol significantly downregulated the expression of NFκb2 and upregulated the expression of IL-10 and Nrf2 genes. P <0.05%, quercetin upregulated the expression levels of NOX2 and Nrf2 genes ( P <0.05).

[0115] Table 27 Effects of quercetin and kaempferol on the relative expression levels of genes related to oxidative damage in the ileum of 21-day-old broilers that have ingested oxidized oil.

[0116] Note: n=6, different lowercase letters in the superscript of the same data indicate significant differences.

[0117] As shown in Table 28, at 42 days of age, quercetin upregulated the expression of ILIβ, IL-10, CAT, SOD, and GPX3 genes. P <0.05, downregulating the expression of NFκb2, NOX2, and Nrf2 genes ( P <0.05%, kaempferol upregulated the expression of IL-6, ILIβ, Nrf2, SOD, GPX2, and GPX3 genes. P <0.05, downregulated the expression of TNFα, NFκb2, and NOX2 genes ( P <0.05).

[0118] Table 28 Effects of quercetin and kaempferol on the relative expression levels of genes related to oxidative damage in the ileum of 42-day-old broilers that have ingested oxidized oil.

[0119] Note: n=6, different lowercase letters in the superscript of the same data indicate significant differences.

[0120] As shown in Table 29, at 42 days of age, quercetin or kaempferol had no significant effect on the α-diversity index of cecal microorganisms in broilers after oxidized oil treatment. P >0.05).

[0121] Table 29 Effects of quercetin and kaempferol on the α-diversity index of cecal microorganisms in 42-day-old broilers after oxidized oil treatment

[0122] Note: n=6, different lowercase letters in the superscript of the same data indicate significant differences.

[0123] Figure 1 Species analysis at the genus level of the samples showed that there were differences in populations among the groups.

[0124] Microbial composition analysis was performed on three groups of samples at each phylum and genus. Figure 2 According to the lowest selected classification level, unclassified species are grouped into the "others" category.

[0125] At the phylum level, the dominant bacterial groups in the OSO and Q groups were Firmicutes and Bacteroidota (both accounting for more than 96% in total), while the dominant bacterial groups in the Ka group were Firmicutes, Bacteroidota, and Proteobacteria (accounting for more than 97% in total). Compared with the OSO group, the relative abundance of Firmicutes increased in the Q and Ka groups, and the relative abundance of Proteobacteria increased in the Ka group.

[0126] At the genus level, the dominant genera in the OSO group are: norank_f_Rikenellaceae , Alistipes , Lactobacillus , Christensenellaceae_R-7_group and norank_o_Clostridia_UCG-014 The percentages were 17.99%, 14.63%, 6.49%, 5.97%, and 5.78%, respectively. The dominant bacterial genus in group Q was... Lactobacillus , treptococcus , norank_f_Rikenellaceae , Bacillus and Alistipes They accounted for 34.29%, 9.70%, 9.61%, 6.53%, and 5.22% respectively, with the dominant genera in group Ka being... Lactobacillus , Alistipes and Streptococcus They accounted for 34.17%, 6.47% and 5.18% respectively.

[0127] At the genus level, there were significant differences in the relative abundance of dominant species among different groups. norank_f_ Rikenellaceae (OSO vs Q vs Ka = 17.99% vs 9.61% vs 1.70%) and Alistipes (OSO vs Q vs Ka = 17.99% vs 5.22% vs 6.47%) The relative abundance decreased after the addition of quercetin or kaempferol; Lactobacillus (OSO vs Q vs Ka = 6.49% vs 34.29% vs 34.17%) The relative abundance increased after the addition of quercetin or kaempferol, and the dominant bacterial species in the Q and Ka groups changed.

[0128] Figure 3 The results of PCA analysis based on the Bray-Curtis distance algorithm show that at 42 days of age, the OSO group was not significantly distinguished from the Q and Ka groups.

[0129] Figure 4 This study presents the differences in average relative abundance of the same species among different groups at the genus level, and marks significant differences. The results show that, compared to group CON, LactobacillusThe abundance increased significantly in groups Q and Ka. [[ID= , ​ , ​ The abundance was significantly reduced in the Q and Ka groups, among which ​ It is unique to the Ka group.

[0130] ​ For LEfSe multilevel species difference discriminant analysis, difference tests were performed at multiple levels from phylum to genus to analyze the differences among species at multiple levels. At 4.5 days of age, compared with the Q and Ka groups, the OSO group showed significantly higher LDA. ​ , ​ , ​ , ​ , ​ , ​ , ​ , ​ , ​ , ​ , ​ Enrichment ( P <0.05), while group Q ​ , ​ Enrichment ( P <0.05), in the Ka group, ​ , ​ , ​ Enrichment ( P <0.05).

[0131] The results of the above examples show that quercetin and kaempferol mainly exert their effects in the later stages of culture, increasing ileal villus length and upregulating the expression of ileal mucin 2 and proliferation-related genes in 42-day-old ileum; kaempferol may exert its effects by downregulating... ​ Upward ​ The expression of [something] increases the antioxidant level of the ileum in 42-day-old infants and reduces [something]. ​ Increase the abundance of harmful bacteria genera. ​ Relative abundance improves growth performance.

[0132] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The use of flavonols in the preparation of drugs for the prevention and / or treatment of oxidative damage.

2. The application according to claim 1, characterized in that, The flavonols include quercetin and / or kaempferol.

3. The application according to claim 1, characterized in that, The oxidative damage includes intestinal oxidative damage and / or liver oxidative damage.

4. The application according to claim 3, characterized in that, The intestinal oxidative damage includes at least one of the following: increased intestinal length, decreased intestinal barrier, activation of intestinal inflammatory response, and intestinal flora imbalance.

5. The application according to claim 3, characterized in that, The liver oxidative damage includes at least one of the following: reduced liver antioxidant capacity, reduced activity of enzyme antioxidant systems, and reduced lipid metabolism capacity.

6. The application according to claim 3, characterized in that, The oxidative damage includes oxidative damage resulting from the intake of oxidized lipids.

7. The application according to claim 6, characterized in that, The oxidized lipids include at least one of the following: oxidized oils, oxidized fats, rancid oils, and peroxidized oils.

8. The application according to claim 2, characterized in that, Drugs containing quercetin have the effect of increasing the activity of SOD in the ileal mucosa; The kaempferol drug has the effect of increasing the activity of T-AOC and GSH-Px in the ileal mucosa.

9. The application according to any one of claims 1 to 8, characterized in that, The drug includes at least one of the following dosage forms: powder, tablet, granule, capsule, oral liquid, injectable powder, and injectable solution.

10. The application according to claim 9, characterized in that, The flavonol content in the drug is 5% to 98% by mass.

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