Probiotic abundance regulator and application thereof
Citrus polymethoxyflavones, formulated as a probiotic regulator with CMC-Na, enhance Dubosiella bacteria abundance, addressing the lack of research in this area and improving gut microbiota and metabolic health.
Patent Information
- Application Number
- CN202510292454.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-12
- Publication Date
- 2025-07-15
AI Technical Summary
There is a lack of effective methods in the prior art to regulate the abundance of Durberella in the intestine, and polymethoxyflavonoids are insufficiently studied in improving the abundance of probiotics.
Citrus polymethoxyflavonoids, especially citrus tangerine peel and 3,5,6,7,8,3',4'-heptamethoxyflavonoids, were used as probiotic abundance regulators, and the suspension was formed by mixing it with CMC-Na solution and gavage it, significantly increasing the abundance of the Durberella genus.
It significantly improved the abundance of the genus Durberella, and had no significant weight changes, liver weight changes or liver function in healthy mice, improved the composition of intestinal microbials, and had the advantages of high safety and good effect.
Smart Images
Figure CN120304545A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of microbiology, and particularly relates to an abundance regulator of probiotics and its application. Background Art
[0002] The gut microbiota is considered to be one of the key factors regulating host health, and maintaining the stability and balance of the gut microbiota is of great significance for preventing and intervening in various diseases. Current research has found that the dysregulation of the gut microbiota is related to the onset of various diseases, such as cancer, Alzheimer's disease, obesity, diabetes, autism, etc. Probiotics can play beneficial roles in the body in various ways, including regulating immune function, producing organic acids and antibacterial compounds, improving the integrity of the gut barrier, and interacting with the host and the host's original microbiota. The genus Dubosiella is a poorly studied gut probiotic that can produce a relatively large amount of short-chain fatty acids, and has beneficial effects such as regulating metabolism-related diseases and intervening in the occurrence of colitis. Currently, many natural compounds have been proven to be able to increase the abundance of the genus Dubosiella in the mouse gut, such as wolfberry polysaccharide, apple polyphenol, etc.
[0003] Citrus polymethoxyflavones (PMFs) differ from other flavonoids in that the former have more than one methoxy group (—CH3O), and they have various biological activities, such as antioxidant, anti-inflammatory, anti-cancer, regulating metabolic syndrome and immune system, neuroprotection and skin protection effects. PMFs can intervene in the disease development of various disease model mice by regulating the gut microbiota composition. Currently, there is very little research on polymethoxyflavones for enhancing the abundance of a certain specific probiotic in healthy mice, and there is no research on polymethoxyflavones for regulating the abundance of the gut probiotic genus Dubosiella. Summary of the Invention
[0004] The technical problem to be solved by the present invention is to overcome the deficiencies existing in the prior art, and provide an abundance regulator of probiotics and its application.
[0005] To solve the above technical problem, the present invention provides an abundance regulator of probiotics, comprising citrus polymethoxyflavones.
[0006] For the above-mentioned abundance regulator of probiotics, further, the citrus polymethoxyflavone is one of nobiletin and 3,5,6,7,8,3',4'-heptamethoxyflavone.
[0007] For the above-mentioned abundance regulator of probiotics, further, the concentration of nobiletin is 100 mg / kg.
[0008] For the above-mentioned abundance regulator of probiotics, further, the concentration of 3,5,6,7,8,3',4'-heptamethoxyflavone is 100 mg / kg.
[0009] The above-mentioned abundance regulator of probiotics. Further, the abundance regulator further includes CMC-Na. Since gavage requires a suspension, polymethoxyflavone needs to be dissolved in a CMC-Na solution, and CMC-Na will not affect any indicators for a healthy organism.
[0010] Based on a general inventive concept, the present invention also provides an application of the above-mentioned abundance regulator in the preparation of a functional food for regulating the abundance of the genus Dubosiella.
[0011] Compared with the prior art, the advantages of the present invention are as follows:
[0012] The present invention provides an abundance regulator of probiotics. Under the technical background that the prior art has not disclosed that polymethoxyflavone from citrus can regulate the abundance of the genus Dubosiella, the present application discloses that polymethoxyflavone from citrus performs very excellently in regulating the abundance of the intestinal probiotic genus Dubosiella, and has advantages such as high safety and good effect in increasing the abundance of the genus Dubosiella. Description of the Drawings
[0013] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention.
[0014] Figure 1 Shows the effects of two kinds of polymethoxyflavones on the body weight and liver weight of mice.
[0015] Figure 2 Shows the H&E staining of liver sections of mice with two kinds of polymethoxyflavones.
[0016] Figure 3 Shows the effects of two kinds of polymethoxyflavones on triglyceride (TG) and total cholesterol (TC) in the plasma and liver of mice.
[0017] Figure 4 Shows the effects of two kinds of polymethoxyflavones on aspartate aminotransferase and alanine aminotransferase (AST and ALT) in the plasma of mice.
[0018] Figure 5 Shows the effects of two kinds of polymethoxyflavones on the abundance of the intestinal flora at the phylum level in mice.
[0019] Figure 6 Shows the effects of two kinds of polymethoxyflavones on the abundance of the intestinal flora at the family level in mice.
[0020] Figure 7 Shows the effects of two kinds of polymethoxyflavones on the abundance of the intestinal flora at the genus level in mice.
[0021] Figure 8Analysis of the differences in the abundance of two polymethoxyflavones at the genus level in the intestinal flora of mice by multiple group comparisons.
[0022] Note: Figures 1 to 8 Among them, there are a blank control group (CON group), a nobiletin group (NBT group), and a 3,5,6,7,8,3',4'-heptamethoxyflavone group (HMF group). Specific implementation manners
[0023] The present invention will be further described below in conjunction with specific preferred embodiments, but the protection scope of the present invention is not limited thereby.
[0024] The materials, reagents, and instruments used in the following embodiments can all be obtained from commercial channels. The experimental methods in the following embodiments are all conventional methods in the art unless otherwise specified.
[0025] The experimental results in the following embodiments are expressed as χ±SD (mean ± standard deviation). The experimental data are statistically analyzed and plotted using GraphPad Prism 10. The between-group difference comparison uses one-way ANOVA test. P < 0.05 indicates a significant difference, denoted by "*"; P < 0.01 indicates a highly significant difference, denoted by "**"; P > 0.05 indicates no significant difference, denoted by "ns".
[0026] The main materials and instruments used in the present invention, and all chemical reagents are of analytical grade. Among them, nobiletin was purchased from Shanghai Yuanye Bio-Technology Co., Ltd., with a purity of ≥95%; 3,5,6,7,8,3',4'-heptamethoxyflavone was purchased from Chengdu Pus Testing and Inspection Co., Ltd., with a purity of ≥98%; the standard feed was purchased from Jiangsu Xietong Pharmaceutical Biotechnology Co., Ltd.; the triglyceride and total cholesterol kits were purchased from Nanjing Jiancheng Bioengineering Research Institute Co., Ltd.
[0027] Forty-five 8-week-old SPF-grade C57BL / 6 male mice, weighing 21 - 25 g, were purchased from Hunan Slack Jingda Experimental Animal Co., Ltd., with the company license number: SYXK (Xiang)-2019-0017, and were raised in Hunan Slack Jingda Experimental Animal Co., Ltd. in an SPF environment (temperature 21 - 23°C, relative humidity 40 - 70%, light-dark cycle of 12 hours). After 2 weeks of adaptive feeding, the experiment began.
[0028] Example 1
[0029] An abundance regulator of a probiotic: nobiletin.
[0030] Example 2
[0031] An abundance regulator of a probiotic: 3,5,6,7,8,3',4'-heptamethoxyflavone.
[0032] Experiment 1: To investigate the effects of nobiletin and 3,5,6,7,8,3',4'-heptamethoxyflavone on the body weight of healthy mice.
[0033] Forty-five male C57BL / 6 mice were fed with standard feed. The mice were randomly divided into 3 groups: blank control group (CON group), nobiletin group (NBT group), 3,5,6,7,8,3',4'-heptamethoxyflavone group (HMF group), with 15 mice in each group.
[0034] CON group: Given 0.5% carboxymethyl cellulose sodium (CMC-Na) solution by gavage daily.
[0035] NBT group: Nobiletin suspension was prepared with 100 mg / kg nobiletin and 0.5% carboxymethyl cellulose sodium, and given by gavage daily for 14 days. On the 15th day, the mice were fasted for 12 h overnight.
[0036] HMF group: 3,5,6,7,8,3',4'-Heptamethoxyflavone suspension was prepared with 100 mg / kg 3,5,6,7,8,3',4'-heptamethoxyflavone and 0.5% carboxymethyl cellulose sodium, and given by gavage daily for 14 days. On the 15th day, the mice were fasted for 12 h overnight.
[0037] The feed eaten by the mice was replaced with new feed every two days, and the body weight of the mice was recorded. After 7 days of feeding, the feces of the mice were collected and stored at -80 °C for testing. After 14 days of feeding, the mice were anesthetized by intraperitoneal injection of 1% pentobarbital, blood was taken from the heart, and part of the liver tissue of the mice was fixed with 4% paraformaldehyde, and the rest was stored at -80 °C for testing.
[0038] Figure 1 For the results of the changes in the body weight and liver weight of the mice, A in the figure is the result of the change in the body weight of the mice, and B in the figure is the result of the change in the liver weight of the mice. It can be seen from the figure that nobiletin and 3,5,6,7,8,3',4'-heptamethoxyflavone do not cause significant changes in the body weight growth trend of healthy mice and do not cause significant differences in liver weight.
[0039] Experiment 2: To investigate the effects of nobiletin and 3,5,6,7,8,3',4'-heptamethoxyflavone on the liver of healthy mice.
[0040] By H&E staining method, the fixed tissue samples were dewaxed to water, stained with hematoxylin and eosin, dehydrated and sealed. The stained sections were observed and analyzed by optical microscopy to observe the pathological histological changes.
[0041] Figure 2 For the H&E staining results of the liver sections of the mice in each group, it can be seen from the figure that nobiletin and 3,5,6,7,8,3',4'-heptamethoxyflavone do not cause pathological damage to the liver.
[0042] Experiment 3: To investigate the effects of nobiletin and 3,5,6,7,8,3',4'-heptamethoxyflavone on TC and TG in the serum and liver of healthy mice.
[0043] Blood was collected from the hearts of mice and allowed to stand for 4 hours. Then, it was centrifuged at 5000 rpm for 10 minutes at 4°C, and the serum was taken for testing.
[0044] The weight of the liver tissue was accurately weighed, and 9 times the volume of absolute ethanol was added according to the ratio of weight (g): volume (mL) = 1:9. Mechanical homogenization was carried out under ice-water bath conditions at 2500 rpm / min for 10 min, and the supernatant, i.e., 10% homogenate supernatant, was taken for testing.
[0045] Serum and liver homogenate samples were detected according to the detection instructions of TC and TG kits from Nanjing Jiancheng Bioengineering Research Institute Co., Ltd.
[0046] Figure 3 Shown in the figure are the changes in triglyceride (TG) and total cholesterol (TC) in the plasma and liver of mice. In the figure, A represents the content of triglyceride (TG) in the plasma of mice, B represents the content of triglyceride (TG) in the liver of mice, C represents the content of total cholesterol (TC) in the plasma of mice, and D represents the content of total cholesterol (TC) in the liver of mice. It can be seen from the figure that nobiletin and 3,5,6,7,8,3',4'-heptamethoxyflavone do not cause an increase in TC and TG in the serum and liver.
[0047] Experiment 4: To investigate the effects of nobiletin and 3,5,6,7,8,3',4'-heptamethoxyflavone on the liver function of healthy mice.
[0048] The levels of ALT and AST in the serum of mice were detected using a BS-430 type automatic biochemical analyzer.
[0049] Figure 4 Shown in the figure are the test results of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in the plasma of mice. In the figure, A represents the test result of aspartate aminotransferase (AST) in the plasma of mice, and B represents the test result of alanine aminotransferase (ALT) in the plasma of mice. It can be seen from the figure that nobiletin and 3,5,6,7,8,3',4'-heptamethoxyflavone do not cause an increase in ALT and AST in the serum and do not cause liver function damage.
[0050] Experiment 5: To investigate the effects of nobiletin and 3,5,6,7,8,3',4'-heptamethoxyflavone on intestinal microorganisms.
[0051] The collected fecal samples were sent to Shanghai Majorbio Bio-Pharm Technology Co., Ltd. for total DNA isolation and 16S rRNA high-throughput sequencing technology. The fecal samples were used to extract the total genomic DNA of the microbial community according to the instructions of the FastPure Stool DNA Isolation Kit (MJYH, Shanghai, China). The integrity of the extracted genomic DNA was detected by 1% agarose gel electrophoresis, and the DNA concentration and purity were measured using NanoDrop2000 (Thermo Scientific, USA). The specific steps are as follows:
[0052] 5.1 Using the above-extracted DNA as a template, the V3-V4 variable region of the 16S rRNA gene was amplified by PCR using the upstream primer 338F (5’-ACTCCTACGGGAGGCAGCAG-3’) and the downstream primer 806R (5’-GGACTACHVGGGTWTCTAAT-3’) carrying Barcode sequences. The formal PCR experiment used TransGen AP221-02: TransStart Fastpfu DNA Polymerase, with a 20 μl reaction system and 3 replicates for each sample. The PCR products of the same sample were mixed, and the PCR products were recovered using 2% agarose gel, and the products were purified. The size of the band fragments was detected by 2% agarose gel electrophoresis, and the recovered products were detected and quantified using Synergy HTX (Biotek, USA).
[0053] 5.2 The purified PCR products were used to construct a library using the NEXTFLEX Rapid DNA-Seq Kit (Bioo Scientific, Austin, Texas, USA):
[0054] (1) Ligation of adapters;
[0055] (2) Screening and removal of self-ligated adapter fragments using magnetic beads;
[0056] (3) Enrichment of the library template by PCR amplification;
[0057] (4) The final library was obtained by magnetic bead recovery of the PCR products. Sequencing was performed using the Illumina NextSeq 2000 PE300 platform (Shanghai Majorbio Bio-Pharm Technology Co., Ltd.).
[0058] 5.3. Use the fastp (https: / / github.com / OpenGene / fastp, version 0.19.6) software to perform quality control on the paired-end raw sequencing sequences, and use the FLASH (http: / / www.cbcb.umd.edu / software / flash, version 1.2.11) software for splicing:
[0059] (1) Filter the bases with a quality value below 20 at the tail of the reads. Set a window of 50 bp. If the average quality value within the window is lower than 20, truncate the trailing bases from the start of the window. Filter the reads shorter than 50 bp after quality control, and remove the reads containing N bases;
[0060] (2) According to the overlap relationship between PE reads, splice (merge) the paired reads into one sequence, with the minimum overlap length being 10 bp;
[0061] (3) The maximum mismatch ratio allowed in the overlap region of the spliced sequence is 0.2, and the sequences that do not meet the requirements are screened;
[0062] (4) Distinguish samples according to the barcodes and primers at both ends of the sequence, and adjust the sequence direction. The allowed number of mismatches for the barcode is 0, and the maximum number of primer mismatches is 2. Based on the default parameters, use the DADA2 plugin in the Qiime2 pipeline to perform denoising on the optimized sequences after quality control and splicing. The sequences after DADA2 denoising are usually called ASVs (i.e., amplicon sequence variants). To minimize the impact of sequencing depth on subsequent Alpha diversity and Beta diversity data analysis, the number of sequences in all samples is rarefied to 20,000. After rarefaction, the average sequence coverage (Good’s coverage) of each sample can still reach 99.09%. Based on the Sliva 16S rRNA gene database (v 138), use the Naivebayes classifier in Qiime2 to perform taxonomic analysis of ASVs.
[0063] All data analysis was performed on the Majorbio Cloud Platform (https: / / cloud.majorbio.com).
[0064] Figures 5 to 7 They are the effects of two polymethoxyflavones on the abundances of the phylum, family, and genus levels of the mouse gut microbiota, respectively. Figure 8 It is the multi-group comparison and difference analysis of the abundances of the genus level of the mouse gut microbiota by two polymethoxyflavones.
[0065] Figures 5 to 7The results show that nobiletin and 3,5,6,7,8,3',4'-heptamethoxyflavone can affect the composition of gut microbiota. At the genus level, the relative abundance of Dubosiella in the CON group was 0.017547, that in the NBT group was 0.076885, which was 4.38 times that of the CON group, and that in the HMF group was 0.2482359, which was 14.14 times that of the CON group.
[0066] Figure 8 The results show that nobiletin and 3,5,6,7,8,3',4'-heptamethoxyflavone can increase the abundance of Dubosiella, showing a significant difference from the blank control group.
[0067] As mentioned above, it is only a preferred embodiment of the present invention and does not impose any formal restrictions on the present invention. Although the present invention has been disclosed as above with the preferred embodiment, it is not intended to limit the present invention. Any person skilled in the art can make many possible changes and modifications to the technical solution of the present invention by using the methods and technical contents disclosed above, or modify it into equivalent embodiments with equivalent changes. Therefore, any simple modification, equivalent replacement, equivalent change and modification made to the above embodiments based on the technical essence of the present invention without departing from the technical solution of the present invention still fall within the scope of protection of the technical solution of the present invention.
Claims
1. An abundance regulator for probiotics, characterized in that, It includes citrus polymethoxyflavones.
2. The abundance regulator according to claim 1, characterized in that, The citrus polymethoxyflavone is one of nobiletin and 3,5,6,7,8,3',4'-heptamethoxyflavone.
3. The abundance regulator according to claim 2, characterized in that, The concentration of nobiletin is 100 mg / kg.
4. The abundance regulator according to claim 2, characterized in that, The concentration of 3,5,6,7,8,3',4'-heptamethoxyflavone is 100 mg / kg.
5. The abundance regulator according to any one of claims 1 to 4, characterized in that, The abundance regulator also includes CMC-Na.
6. Use of the abundance regulator according to any one of claims 1 to 5 in the preparation of a functional food for regulating the abundance of the genus Dubosiella.