Aging-relieving intestinal flora branched chain amino acid metabolites isobutyric acid and isovaleric acid and application thereof

Abstract

The invention discloses intestinal flora branched chain amino acid metabolites isobutyric acid and isovaleric acid for relieving aging and application of the intestinal flora branched chain amino acid metabolites isobutyric acid and isovaleric acid, and belongs to the technical field of microorganisms and the technical field of medicines. Animal experiments prove that the intestinal flora branched-chain amino acid metabolite isobutyric acid or isovaleric acid has the effects of improving the abundance of key metabolic genes porA of branched-chain amino acids advocated by senescent mice, changing metabolism of nutrient substances by intestinal tracts of the mice, improving the weakness index of the mice, increasing peroxide dismutase in livers of the mice, reducing malondialdehyde, improving the immunity of the mice and improving the immunity of the mice. According to the invention, the effects of increasing the level of glutathione peroxidase (GSH-px), increasing the level of catalase, reducing an inflammatory factor IL-6, reducing an inflammatory factor TNF-alpha, increasing the level of acetylcholine in the brain of a mouse and improving the holding power of the mouse are achieved. The strain disclosed by the invention has a huge application prospect in preparation of products for improving metabolism and relieving senescence.

Description

Technical Field

[0001] The present invention relates to isobutyric acid and isovaleric acid, branched-chain amino acid metabolites of intestinal flora for alleviating aging, and applications thereof, and belongs to the fields of microbial technology and pharmaceutical technology. Background Art

[0002] Aging is a complex process involving changes in cells, tissues, and organs. These changes not only lead to a decline in physiological function but also to an increased risk of various diseases, including neurodegenerative, cardiovascular, metabolic, musculoskeletal, and immune system disorders. The molecular mechanisms of aging are complex and involve the accumulation of DNA damage, telomere shortening, oxidative stress, cellular senescence, and stem cell depletion.

[0003] Interventions for aging are also gaining increasing attention. Studies have shown that the aging process can be slowed down through methods such as calorie restriction, microbiome transplantation, and nutritional intervention. In addition, drug therapy, stem cell therapy, antioxidant and anti-inflammatory therapy, and hormone replacement therapy are also being explored for the treatment of aging-related diseases. Drugs such as rapamycin, metformin, spermidine, NAD+ supplements, and senolytics have shown potential in delaying aging. Among the 12 most recently released markers of aging, the disorder and imbalance of the intestinal microbiome is one of the important markers of aging. The balance of intestinal flora plays an important role in maintaining human metabolic health and delaying the aging process. Methods related to intestinal microorganisms to alleviate aging show great potential.

[0004] Branched-chain amino acids (BCAAs), including leucine, isoleucine, and valine, are essential amino acids for the human body and play a key role in regulating metabolic health and the aging process. Gut microbes participate in BCAA metabolism in various ways. Existing literature suggests that, unlike metabolism in the body and by industrial fermentation bacteria, some gut microbes metabolize BCAAs through a unique, atypical pathway, producing three metabolites: isobutyrate, isovalerate, and 2-methylbutyrate. Both the papers "A Preliminary Study of the Effects of Short-Chain Fatty Acids on Lifespan in Caenorhabditis elegans and Their Molecular Mechanisms" and "Antioxidant Effects of Substituted Short-Chain Fatty Acids on Caenorhabditis elegans" demonstrated that, in oxidative stress experiments, the traditional short-chain fatty acids formate, acetate, propionate, butyrate, and valerate / hexanoate lacked antioxidant activity in vivo and failed to prolong survival under oxidative stress, suggesting they may not have an aging-delaying effect. 2-Methylbutyrate can increase antioxidant activity and extend lifespan in wild-type nematodes, but the improvement in aging is modest and cannot restore optimal health. Therefore, there is an urgent need to screen natural metabolites that can significantly improve the health of aging organisms. Summary of the Invention

[0005] The present invention provides an application of a branched-chain amino acid metabolite in preparing a drug for alleviating aging in mice. The branched-chain amino acid metabolite comprises isobutyrate (ISB) or isovalerate (ISV).

[0006] In one embodiment of the present invention, the chemical formula of the isobutyrate (ISB) is: (CH3)2CHCO2H; the chemical formula of the isovaleric acid (ISV) is: (CH3)2CHCH2COOH;

[0007] The structural formula is:

[0008]

[0009] In one embodiment of the present invention, the drug is used for at least one of (a) to (j):

[0010] (a) Isobutyrate and isovaleric acid increase the abundance of key branched-chain amino acid metabolic genes in the intestinal microbiota of aged mice;

[0011] (b) Changes in the metabolism of nutrients by the intestinal flora of aged mice;

[0012] (c) reducing frailty scores in aging individuals;

[0013] (d) reducing the level of MDA in liver tissue of aging individuals;

[0014] (e) increasing the level of SOD enzyme in liver tissue of aging individuals;

[0015] (f) increasing the level of CAT enzyme in liver tissue of aging individuals;

[0016] (g) increasing the level of GSH-px enzyme in liver tissue of aging individuals;

[0017] (h) increasing acetylcholine levels in brain tissue of aging individuals;

[0018] (i) reducing the expression level of pro-inflammatory factors in the colon tissue of aging individuals; the pro-inflammatory factors include IL-6 or TNF-alpha

[0019] (j) Improve grip strength in aging individuals.

[0020] In one embodiment of the present invention, the added amount of branched-chain amino acid metabolites in the drug is at least 5 mmol / L or 0.005 mmol / g.

[0021] In one embodiment of the present invention, the drug comprises a branched-chain amino acid metabolite, a drug carrier and / or a pharmaceutical excipient.

[0022] In one embodiment of the present invention, the drug carrier comprises microcapsules, microspheres, nanoparticles and liposomes.

[0023] In one embodiment of the present invention, the pharmaceutical excipient comprises an excipient and an additive.

[0024] In one embodiment of the present invention, the pharmaceutical excipients include anti-adhesive agents, penetration enhancers, buffers, plasticizers, surfactants, defoamers, thickeners, inclusion agents, absorbents, humectants, solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, pH regulators, adhesives, disintegrants, fillers, lubricants, wetting agents, integrators, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, foaming agents, suspending agents, coating materials, fragrances, diluents, flocculants and deflocculants, filter aids and release retardants.

[0025] In one embodiment of the present invention, the additive comprises microcrystalline cellulose, hydroxypropyl methylcellulose or lecithin.

[0026] In one embodiment of the present invention, the dosage form of the drug comprises granules, capsules, tablets, pills or liquid preparations.

[0027] Beneficial effects:

[0028] The present invention provides a branched-chain amino acid metabolite isobutyric acid or isovaleric acid that alleviates aging in mice. Specifically, it is embodied in:

[0029] (1) Isobutyric acid and isovaleric acid increase the abundance of porA, a key branched-chain amino acid metabolic gene, in the intestinal flora of aging mice.

[0030] (2) Isobutyric acid and isovaleric acid change the metabolism of nutrients by the intestinal flora of aged mice. Isobutyric acid and isovaleric acid, the three branched-chain amino acid metabolites of intestinal flora, both change the intestinal protein digestion and absorption pathways of aged mice.

[0031] (3) Isobutyric acid and isovaleric acid significantly reduced the frailty index of aging mice. After oral administration of isobutyric acid, the frailty index decreased by 49.15%, after oral administration of isovaleric acid, the frailty index decreased by 57.26%, after oral administration of 2-methylbutyric acid, the frailty index decreased by 33.33%. Compared with the spermidine drug group, the frailty index of isovaleric acid decreased by 59.00%. The effects of isobutyric acid and isovaleric acid on reducing the frailty index were better than those of 2-methylbutyric acid.

[0032] (4) Isobutyric acid and isovaleric acid significantly increased the level of superoxide dismutase (SOD) in the liver of aging mice. After oral administration of isobutyric acid, SOD increased by 75.21%, and after oral administration of isovaleric acid, SOD increased by 49.28%. Compared with the spermidine drug group, the SOD level of isovaleric acid increased by 1%.

[0033] (5) Isobutyric acid and isovaleric acid significantly reduced the level of malondialdehyde (MDA) in the liver of aging mice. Oral administration of isobutyric acid reduced MDA by 42.03%, oral administration of isovaleric acid reduced MDA by 38.97%, and oral administration of 2-methylbutyric acid reduced MDA by 34.20%. Isobutyric acid and isovaleric acid were more effective than 2-methylbutyric acid in reducing MDA levels in the liver of mice.

[0034] (6) Isobutyric acid and isovaleric acid significantly increased the level of glutathione peroxidase (GSH-px) in the liver of aging mice. After oral administration of isobutyric acid, GSH-px increased by 118.26%, after oral administration of isovaleric acid, GSH-px increased by 112.50%, and after oral administration of 2-methylbutyric acid, GSH-px increased by 83.46%. Isobutyric acid and isovaleric acid increased the level of GSH-px in the liver of mice more than 2-methylbutyric acid. Compared with the spermidine drug group, the level of GSH-px in the liver of mice increased by 4.2% in isobutyric acid.

[0035] (7) Isobutyric acid and isovaleric acid significantly increased the level of catalase (CAT) in the liver of aging mice. After oral administration of isobutyric acid, CAT increased by 33.32%, after oral administration of isovaleric acid, CAT increased by 21.53%, and after oral administration of 2-methylbutyric acid, CAT increased by 23.51%. Isobutyric acid was better than 2-methylbutyric acid in increasing the level of CAT in the liver of mice.

[0036] (8) Isobutyric acid and isovaleric acid reduced the IL-6 level in the colon homogenate of aged mice. After oral administration of isobutyric acid, IL-6 decreased by 38.44%, after oral administration of isovaleric acid, IL-6 decreased by 28.33%, and after oral administration of 2-methylbutyric acid, IL-6 decreased by 35.83%. Isobutyric acid was better than 2-methylbutyric acid in reducing the IL-6 level in mice.

[0037] (9) Isobutyric acid and isovaleric acid reduced the level of TNF-alpha in the colon homogenate of aged mice. After oral administration of isobutyric acid, TNF-alpha decreased by 35.46%, and after oral administration of isovaleric acid, TNF-alpha decreased by 32.03%. Compared with the spermidine drug group, the TNF-alpha level of isobutyric acid decreased by 21.63%, and the TNF-alpha level of isovaleric acid decreased by 9.96%.

[0038] (10) Isobutyric acid and isovaleric acid increased the acetylcholine level in the brains of aging mice. After oral administration of isobutyric acid, acetylcholine increased by 133.94%, after oral administration of isovaleric acid, acetylcholine increased by 215.14%, and after oral administration of 2-methylbutyric acid, acetylcholine increased by 108.50%. Isobutyric acid and isovaleric acid were better than 2-methylbutyric acid in increasing the acetylcholine level in the brain, and better than the spermidine drug group.

[0039] (11) Isobutyric acid and isovaleric acid improved the gripping strength of aging mice. After oral administration of isobutyric acid, the gripping strength increased by 22.10%, after oral administration of isovaleric acid, the gripping strength increased by 22.37%, and after oral administration of 2-methylbutyric acid, the gripping strength increased by 16.81%. The effects of isobutyric acid and isovaleric acid on improving gripping strength were better than those of 2-methylbutyric acid and the spermidine drug group.

[0040] Therefore, isobutyric acid and isovaleric acid, the intestinal flora metabolites of branched-chain amino acids, have great application prospects in the preparation of drugs to alleviate aging. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 :Abundance of porA, a key gene for branched-chain amino acid metabolism in the intestinal flora of experimental mice in different groups;

[0042] Figure 2 :Enrichment analysis of metabolic pathways in the intestinal flora of experimental mice in different groups;

[0043] Figure 3 : Frailty index of experimental mice in different groups;

[0044] Figure 4 : Superoxide dismutase (SOD) levels in the livers of experimental mice in different groups;

[0045] Figure 5 : Malondialdehyde (MDA) levels in the livers of experimental mice in different groups;

[0046] Figure 6 : Levels of glutathione peroxidase (GSH-px) in the liver of experimental mice in different groups;

[0047] Figure 7 : Catalase (CAT) levels in the livers of experimental mice in different groups;

[0048] Figure 8 : IL-6 levels in colon homogenates of experimental mice in different groups;

[0049] Figure 9 :TNF-alpha levels in the colon of experimental mice in different groups;

[0050] Figure 10 :Acetylcholine levels in the brains of experimental mice in different groups;

[0051] Figure 11 : The grip strength levels of experimental mice in different groups;

[0052] Figure 12 : Flowchart of animal experiments. DETAILED DESCRIPTION

[0053] D-galactose (D-gal) (CAS No.: 59-23-4), isobutyric acid (isobutyrate, ISB, CAS: 79-31-2), isovaleric acid (isovalerate, ISV, CAS: 503-74-2) and 2-methylbutyric acid (2-methylbutyrate, 2-MB, CAS: 116-53-0) involved in the following examples were purchased from Aladdin.

[0054] The preparation method of the D-galactose solution involved in the following embodiments is as follows:

[0055] D-galactose solution: Weigh a certain amount of D-galactose, dissolve it in sterile saline to prepare D-galactose, and inject it subcutaneously at a dose of 700 mg / kg body weight and 1000 mg / kg body weight.

[0056] Example 1: Branched-chain amino acid intestinal flora metabolites isobutyric acid and isovaleric acid increase the abundance of the key gene porA for branched-chain amino acid metabolism in the intestinal flora of mice

[0057] Eight-week-old SPF-grade C57BL / 6J male mice were housed in the Experimental Animal Center of Jiangnan University, fed with ordinary chow, maintained at a constant temperature of 20-26°C, humidity of 40%-70%, noise level less than or equal to 60 dB, animal illumination of 15-20 LX, 12 h light and 12 h dark (all animal experimental procedures were reviewed and approved by the Animal Welfare and Ethics Management Committee of Jiangnan University).

[0058] The experimental design is 20 weeks in total. The specific process is as follows: Figure 10 After entering the housing facility, mice were acclimated for one week. From weeks 2 to 13, an aging model was established using galactose (CAS No. 59-23-4, Aladdin). The control group received a subcutaneous injection of sterile saline, while the other treatment groups received a subcutaneous injection of 700 mg / kg BW / day of D-galactose dissolved in sterile saline. Because mice develop a certain degree of tolerance to D-galactose, treatment was continued with a subcutaneous injection of 1000 mg / kg BW / day from weeks 14 to 20. Each group of mice underwent the corresponding experimental treatment. The control and model groups received daily gavage of 200 μL of sterile saline. The volume of injection fluid was the same for all groups.

[0059] The details are as follows:

[0060] Blank group: After entering the breeding facility, mice were first adapted for one week. From 2 to 13 weeks, mice in the blank group were subcutaneously injected with sterile saline. From 14 to 20 weeks, mice in the blank group were subcutaneously injected with sterile saline and gavaged with 200 μL of sterile saline once a day.

[0061] Model group: After entering the breeding facility, mice were acclimated for one week. From week 2 to week 13, the model group received a subcutaneous injection of 700 mg / kg BW / d of D-galactose dissolved in sterile saline. From week 14 to week 20, mice were subcutaneously injected with 1000 mg / kg BW / d of D-galactose dissolved in sterile saline and gavaged with 200 μL of sterile saline daily. Food and water were available ad libitum.

[0062] Isobutyric acid experimental group (ISB group): After entering the breeding facility, mice were first adapted for one week. From week 2 to 13, the model group was subcutaneously injected with 700 mg / kg BW / d of D-galactose dissolved in sterile saline; from week 14 to 20, the mice were subcutaneously injected with 1000 mg / kg BW / d of D-galactose dissolved in sterile saline, and were gavaged daily with 6.5 mmol / L, 100 μL / 10 g body weight of isobutyric acid. Food and water were available ad libitum.

[0063] ISV experimental group: After entering the breeding facility, mice were first adapted for one week. From week 2 to 13, the model group was subcutaneously injected with 700 mg / kg BW / d of D-galactose dissolved in sterile saline; from week 14 to 20, mice were subcutaneously injected with 1000 mg / kg BW / d of D-galactose dissolved in sterile saline, and were gavaged daily with 5.5 mmol / L, 100 μL / 10 g body weight of isovaleric acid; and food and water were freely available.

[0064] 2-Methylbutyrate control group (2-MB group): After entering the breeding facility, mice were first adapted for one week. From week 2 to 13, the model group was subcutaneously injected with 700 mg / kg BW / d of D-galactose dissolved in sterile saline; from week 14 to 20, the mice were subcutaneously injected with 1000 mg / kg BW / d of D-galactose dissolved in sterile saline, and were gavaged daily with 5.5 mmol / L, 100 μL / 10 g body weight of 2-methylbutyrate; food and water were available ad libitum.

[0065] Before killing mice, feces were collected and total DNA was extracted using the QIAamp DNA Stool Mini Kit (Qiagen, Hilden, Germany). DNA purity and integrity were assessed by 1% agarose gel electrophoresis. Metagenomic sequencing was performed on the DNBSEQ-T7 platform at Beijing Novogene Co., Ltd. Raw sequencing reads were deserialized using Trimmomatic (version 0.39) to remove sequencing adapters and low-quality sequences. Filtered sequences were aligned to the human reference genome using BWA (version 0.7.17), Samtools (version 1.9), and BEDTools (version 2.30.0) to remove host sequences. Quality-controlled and host-deleted sequences were annotated with the MetaCyc database for gut microbial metabolic functions using HUMAnN3 (version 3.8). These sequences were then renamed according to the Uniref 90 database to obtain information on the abundance of gut microbial metabolic gene families.

[0066] Literature reports that the porA gene is a key gene for the metabolism of branched-chain amino acids in the intestinal flora. Knocking out the porA gene will cause the strain to lose the ability to metabolize branched-chain amino acids and produce isobutyric acid, isovaleric acid, and 2-methylbutyric acid. The base sequences of the porA gene reported in the literature were compared with the Uniref 90 database, and base sequences with a base sequence similarity of 85% or more were selected. The Uniref 90 numbers of these genes were matched with the gene family files of the intestinal flora of aging mice. The gene abundance of porA, a key gene for the metabolism of branched-chain amino acids in the intestinal flora of aging mice, was obtained. The results are as follows: Figure 1 As shown:

[0067] The results showed that compared with the model group, the abundance of porA, a key gene for branched-chain amino acid metabolism, in the intestinal microbiota of mice in the isobutyrate and isovalerate groups was significantly increased. This demonstrates that isobutyrate and isovalerate have the ability to enhance branched-chain amino acid metabolism in the intestinal microbiota. The increases in porA abundance by isobutyrate and isovalerate were 1.9 and 1.5 times, respectively, compared to the model group, demonstrating a superior effect to the 2-methylbutyrate group.

[0068] Example 2: Isobutyric acid and isovaleric acid, branched-chain amino acid metabolites of intestinal flora, alter the metabolism of nutrients in the mouse intestine

[0069] Specific embodiments Referring to Example 1, before killing mice, mouse feces were collected. For non-target metabolomics detection, 100 μL of sample was taken and mixed with 400 μL of extraction solution (MeOH:ACN, 1:1 (v / v)), the extraction solution contained a deuterated internal standard, and the mixed solution was vortexed for 30 seconds, ultrasonicated in a 4°C water bath for 10 minutes, and incubated for 1 hour at -40°C to precipitate proteins. The sample was then centrifuged at 12000 rpm (RCF=13800 (×g), R=8.6 cm) for 15 minutes at 4°C. The supernatant was transferred to a new glass vial for analysis. Quality control (QC) samples were prepared by mixing equal amounts of sample supernatants.

[0070] For polar metabolites, MS analysis was performed using a UHPLC system (Vanquish, Thermo Fisher Scientific) using a Waters ACQUITY UPLC BEH Amide (2.1 mm × 50 mm, 1.7 μm) connected to an Orbitrap Exploris 120 mass spectrometer (Orbitrap MS, Thermo). The mobile phase consisted of 25 mmol / L ammonium acetate and 25 mmol / L ammonia in water (pH = 9.75) (A) and acetonitrile (B). The automatic injection temperature was 4°C and the injection volume was 2 μL. The Orbitrap Exploris 120 mass spectrometer was used to acquire MS / MS spectra in information-dependent acquisition (IDA) mode under the control of acquisition software (Xcalibur, Thermo). In this mode, the acquisition software continuously evaluates the full scan mass spectrum. The ESI source conditions were set as follows: sheath gas flow rate of 50 Arb, auxiliary gas flow rate of 15 Arb, capillary temperature of 320 °C, full MS resolution of 60000, MS / MS resolution of 15000, collision energy: SNCE 20 / 30 / 40, and spray voltage of 3.8 kV (positive) or −3.4 kV (negative), respectively.

[0071] KEGG metabolic pathway enrichment analysis was performed on the metabolites, and the results were as follows Figure 2 As shown, both isobutyric acid and isovaleric acid metabolites can change the metabolic pathways of multiple amino acids in the intestines of aging mice, and at the same time change the metabolic pathway of protein digestion and absorption.

[0072] Example 3: Effects of Isobutyric Acid and Isovalerate, Branched-Chain Amino Acid Metabolites of Intestinal Flora, on the Alleviating Effect of Frailty Index in Aging Mice

[0073] DETAILED DESCRIPTION OF THE INVENTION Referring to Example 1, before killing the mice, the frailty of each aging mouse was scored, including the assessment of 13 variables. The frailty index (FI) was calculated based on the scores of the individual variables. The specific scoring criteria are as follows: for each variable, 0 points represent no impairment, 0.5 points represent mild impairment, and 1 point represents severe impairment. The total score is calculated for the assessment, and the total score ranges from 0 to 13.

[0074] Table 1: Mouse weakness index

[0075]

[0076] Depend on Figure 3 It can be seen that the average frailty index of the blank group was 2.06, the average frailty index of the model group was 4.68, the average frailty index of the ISB group was 2.4, the average frailty index of the ISV group was 2, the average frailty index of the 2-MB group was 3.12, and the average frailty index of the spermidine group was 2. Compared with the model group, isobutyric acid and isovaleric acid significantly reduced the frailty index of aging mice. After oral administration of isobutyric acid, the frailty index decreased by 49.15%, after oral use of isovaleric acid, the frailty index decreased by 57.26%, and after oral use of 2-methylbutyric acid, the frailty index decreased by 33.33%. Compared with the spermidine drug group, the frailty index of isovaleric acid decreased by 59.00%. The effects of isobutyric acid and isovaleric acid on reducing the frailty index were better than 2-methylbutyric acid. The above experimental results show that isobutyric acid and isovaleric acid, the metabolites of branched-chain amino acids intestinal flora, can effectively reduce the frailty index of mice and improve the frail state of mice. Isovaleric acid has an effect better than drugs, and isobutyric acid and isovaleric acid have an effect better than 2-methylbutyric acid, a branched-chain amino acid metabolite of other intestinal flora.

[0077] Example 4: Effects of Isobutyric Acid and Isovalerate, Branched-Chain Amino Acid Metabolites of Intestinal Microflora, on the Level of Superoxide Dismutase (SOD) in the Liver of Aging Mice

[0078] Specific implementation method Referring to Example 1, after the experiment, mice in the methylbutyrate control group were killed, the mouse livers were removed and placed in liquid nitrogen, stored in a -80°C refrigerator, and the tissues were rinsed with pre-cooled PBS (0.01M, pH=7.4) to remove residual blood and minced. About 1.2g of fresh tissue was weighed and added to PBS at a weight-to-volume ratio of 1:9. 1mL of universal protease inhibitor (Biyuntian) was added to every 100mL of this PBS. An equal amount of grinding beads was added to each tissue, and a pre-cooled tissue grinder mold was used to grind it thoroughly on ice until no solid matter was seen. Further repeated freezing and thawing was performed to fully lyse the tissue. The homogenate was centrifuged at 5000×g for 5-10 minutes, and the supernatant was taken and divided for use. To measure protein concentration using the BCA assay, add 1.2 ml of a protein standard solution (0.5 g bovine serum albumin, dissolved in distilled water and diluted to 100 ml, to make a 5 mg / ml solution, diluting tenfold as needed) to a tube of protein standard (30 mg BSA). Dissolve thoroughly to make a 25 mg / ml protein standard solution. Take an appropriate amount of the 25 mg / ml protein standard and dilute to a final concentration of 0.5 mg / ml. Based on the number of samples, prepare an appropriate amount of BCA working solution by adding 50 volumes of BCA reagent A (10g BCA (1%), 20g Na2CO3·H2O (2%), 1.6g Na2C4H4O6·2H2O (0.16%), 4g NaOH (0.4%), and 9.5g NaHCO3 (0.95%), and add water to 1L. Adjust the pH to 11.25 with NaOH or solid NaHCO3) to 1 volume of BCA reagent B (2g CuSO4·5H2O (4%), and add distilled water to 50ml) (50:1) and mix thoroughly. Construct a standard curve using a standard concentration ranging from 0-1.5mg / mL. Preliminary sample experiments will determine the appropriate dilution factor. For the final experiment, add 20μL of sample or standard of varying concentrations to each well, along with 200μL of BCA working solution. Incubate at 37°C in the dark for 30 minutes. The absorbance was measured at 562 nm using a microplate reader and the protein concentration of the sample was calculated.

[0079] The superoxide dismutase (SOD) content in mouse liver homogenate was detected according to the instructions of the liver superoxide dismutase (SOD) ELISA kit. The results are as follows: Figure 4 shown.

[0080] The results show:

[0081] Depend on Figure 4The average liver SOD level in the blank group was 27.90 ng / mg protein, the model group was 16.254 ng / mg protein, the ISB group was 28.48 ng / mg protein, the ISV group was 24.26 ng / mg protein, and the spermidine group was 29.51 ng / mg protein. Isobutyric acid and isovaleric acid significantly increased liver SOD levels in aged mice. Oral administration of isobutyric acid increased SOD by 75.21%, and oral administration of isovaleric acid increased SOD by 49.28%. Compared to the spermidine group, isovaleric acid increased SOD by 1%. The above experimental results show that isobutyric acid and isovaleric acid, the metabolites of branched-chain amino acids intestinal flora, can effectively increase the level of superoxide dismutase (SOD) in mouse livers and improve the weakened state of mice, and isovaleric acid has an effect better than drugs.

[0082] Example 5: Effect of Isobutyric Acid and Isovalerate, Branched-Chain Amino Acid Metabolites of Intestinal Microflora, on the Reduction of Malondialdehyde (MDA) Levels in the Liver of Aging Mice

[0083] Specific embodiments Referring to the examples, the malondialdehyde (MDA) content in the mouse liver homogenate was detected according to the instructions of the liver malondialdehyde (MDA) level ELISA kit. The results are as follows: Figure 5 shown.

[0084] The results show:

[0085] Depend on Figure 5 The results showed that the average liver MDA value in the blank group was 21.95 nmol / mg protein, the average liver MDA value in the model group was 34.71 nmol / mg protein, the average liver MDA value in the ISB group was 20.12 nmol / mg protein, the average liver MDA value in the ISV group was 21.18 nmol / mg protein, the average liver MDA value in the 2-MB group was 22.84 nmol / mg protein, and the average liver MDA value in the spermidine group was 44.38 nmol / mg protein. Isobutyric acid and isovaleric acid significantly reduced the liver MDA level in aging mice. Oral administration of isobutyric acid reduced MDA by 42.03%, oral administration of isovaleric acid reduced MDA by 38.97%, and oral administration of 2-methylbutyric acid reduced MDA by 34.20%. Isobutyric acid and isovaleric acid were more effective than 2-methylbutyric acid in reducing MDA levels in mouse liver.

[0086] The above experimental results show that isobutyric acid and isovaleric acid, the metabolites of branched-chain amino acids intestinal flora, can effectively reduce the level of malondialdehyde (MDA) in the liver of mice, improve the frail state of mice, and achieve an effect that is better than drugs and other products such as 2-methylbutyric acid.

[0087] Example 6: Effect of branched-chain amino acid metabolites from intestinal flora on the elevation of glutathione peroxidase (GSH-px) levels in the liver of aging mice

[0088] Specific embodiments Referring to Example 4, the content of glutathione peroxidase (GSH-px) in mouse liver homogenate was detected according to the instructions of the liver glutathione peroxidase (GSH-px) ELISA kit. The results are as follows: Figure 6 shown.

[0089] The results show:

[0090] Depend on Figure 6 The results showed that the average liver glutathione peroxidase (GSH-px) value of the blank group was 180.25 U / mg protein, the average liver glutathione peroxidase (GSH-px) value of the model group was 77.07 U / mg protein, the average liver glutathione peroxidase (GSH-px) value of the ISB group was 168.21 U / mg protein, the average liver glutathione peroxidase (GSH-px) value of the ISV group was 163.77 U / mg protein, and the average liver glutathione peroxidase (GSH-px) value of the 2-MB group was 141.38 U / mg protein. In the spermidine group, the average liver glutathione peroxidase (GSH-px) level was 161.38 U / mg protein. Compared with the model group, isobutyric acid and isovaleric acid significantly increased liver glutathione peroxidase (GSH-px) levels in aged mice. Oral administration of isobutyric acid increased GSH-px by 118.26%, isovaleric acid by 112.50%, and 2-methylbutyric acid by 83.46%. Isobutyric acid and isovaleric acid increased liver GSH-px levels more than 2-methylbutyric acid. Compared with the spermidine group, isobutyric acid increased liver GSH-px levels by 4.2%.

[0091] The above experimental results show that isobutyric acid and isovaleric acid, the branched-chain amino acid metabolites of intestinal flora, can effectively increase the level of glutathione peroxidase (GSH-px) in mouse liver and improve the frail state of mice. Among them, isobutyric acid is more effective than drugs, and isobutyric acid and isovaleric acid are more effective than 2-methylbutyric acid, another product of intestinal flora branched-chain amino acids.

[0092] Example 7: Effect of branched-chain amino acid metabolites of intestinal flora on the elevation of catalase (CAT) levels in the liver of aging mice

[0093] Specific implementation method Referring to Example 4, the catalase (CAT) content in mouse liver homogenate was detected according to the instructions of the catalase (CAT) level ELISA kit. The results are as follows: Figure 7 shown.

[0094] The results show:

[0095] Depend on Figure 7 It can be seen that the average liver catalase (CAT) value of the blank group was 539.19 pg / mg protein, the average liver catalase (CAT) value of the model group was 258.53 pg / mg protein, the average liver catalase (CAT) value of the ISB group was 344.67 pg / mg protein, the average liver catalase (CAT) value of the ISV group was 314.20 pg / mg protein, the average liver catalase (CAT) value of the 2-MB group was 319.31 pg / mg protein, and the average liver catalase (CAT) value of the spermidine drug group was 575.71 pg / mg protein. Compared with the model group, isobutyric acid and isovaleric acid significantly increased liver catalase (CAT) levels in aged mice. Oral administration of isobutyric acid increased CAT by 33.32%, oral administration of isovaleric acid increased CAT by 21.53%, and oral administration of 2-methylbutyric acid increased CAT by 23.51%. Isobutyric acid was more effective than 2-methylbutyric acid in increasing liver CAT levels in mice. These experimental results indicate that branched-chain amino acid metabolites from the gut microbiota can effectively increase liver catalase (CAT) levels in mice and improve their frailty, with isobutyric acid being more effective than 2-methylbutyric acid, another branched-chain amino acid product from the gut microbiota.

[0096] Example 8: Effect of branched-chain amino acid metabolites from intestinal flora on reducing IL-6 levels in colon homogenates of aged mice

[0097] Specific implementation method: Refer to Example 5. After killing the mouse, remove the mouse colon and place it in liquid nitrogen, store it in a -80°C refrigerator, rinse the tissue with pre-cooled PBS (0.01M, pH=7.4), remove residual blood, and cut it into pieces. Weigh about 1.2g of fresh tissue, add PBS at a weight-to-volume ratio of 1:9, add 1mL of universal protease inhibitor (Biyuntian) to every 100mL of this PBS, add an equal amount of grinding beads to each tissue, use a pre-cooled tissue grinder mold, and grind it thoroughly on ice until no solid matter is completely gone. Further freeze and thaw repeatedly to fully lyse. Centrifuge the homogenate at 5000×g for 5-10 minutes, take the supernatant and divide it into portions for use. To measure protein concentration using the BCA method, add 1.2 ml of a protein standard solution (0.5 g bovine serum albumin, dissolved in distilled water and diluted to 100 ml, to make a 5 mg / ml solution, diluting tenfold as needed) to a tube of protein standard (30 mg BSA). Dissolve thoroughly to make a 25 mg / ml protein standard solution. Take an appropriate amount of the 25 mg / ml protein standard and dilute to a final concentration of 0.5 mg / ml. Based on the number of samples, prepare an appropriate amount of BCA working solution by adding 50 volumes of BCA reagent A (weigh 10g BCA (1%), 20g Na2CO3·H2O (2%), 1.6g Na2C4H4O6·2H2O (0.16%), 4g NaOH (0.4%), and 9.5g NaHCO3 (0.95%), add water to 1L, and adjust the pH to 11.25 with NaOH or solid NaHCO3) to 1 volume of BCA reagent B (take 2g CuSO4·5H2O (4%) and add distilled water to 50ml) (50:1) and mix thoroughly. Construct a standard curve using a standard concentration between 0 and 1.5 mg / mL. Preliminary sample experiments will determine the appropriate dilution factor. For the final experiment, add 20μL of sample or standard of varying concentrations to each well. Add 200μL of BCA working solution to each well and incubate at 37°C in the dark for 30 minutes. The absorbance was measured at 562 nm using a microplate reader and the protein concentration of the sample was calculated.

[0098] The IL-6 content in the mouse colon homogenate was detected according to the instructions of the IL-6 ELISA kit. Figure 8 shown.

[0099] The results show:

[0100] Depend on Figure 8The average IL-6 level in colon homogenate of the blank group was 306.95 pg / mg protein, the average IL-6 level in colon homogenate of the model group was 366.24 pg / mg protein, the average IL-6 level in colon homogenate of the ISB group was 225.47 pg / mg protein, the average IL-6 level in colon homogenate of the ISV group was 262.49 pg / mg protein, the average IL-6 level in colon homogenate of the 2-MB group was 235.02 pg / mg protein, and the average IL-6 level in colon homogenate of the spermidine group was 312.41 pg / mg protein. Compared with the model group, isobutyric acid and isovaleric acid reduced the level of IL-6 in colon homogenate of aged mice. After oral administration of isobutyric acid, IL-6 decreased by 38.44%, after oral administration of isovaleric acid, IL-6 decreased by 28.33%, and after oral administration of 2-methylbutyric acid, IL-6 decreased by 35.83%. Isobutyric acid was superior to 2-methylbutyric acid in reducing IL-6 levels in mice.

[0101] The above experimental results show that the branched-chain amino acid metabolites of intestinal flora can effectively reduce the IL-6 in the colon homogenate of mice and improve the frail state of mice. Among them, isobutyric acid is more effective than 2-methylbutyric acid, another metabolite of branched-chain amino acids of intestinal flora.

[0102] Example 9: Effect of branched-chain amino acid metabolites from intestinal flora on reducing TNF-alpha levels in colon homogenates of aged mice

[0103] Specific implementation method Referring to Example 5, the TNF-alpha content in the mouse colon homogenate was detected according to the instructions of the TNF-alpha ELISA kit. The results are as follows: Figure 9 shown.

[0104] The results show:

[0105] Depend on Figure 9It can be seen that the average TNF-alpha content in the colon homogenate of the blank group was 281.00 pg / mg protein, the average TNF-alpha content in the colon homogenate of the model group was 324.56 pg / mg protein, the average TNF-alpha content in the colon homogenate of the ISB group was 209.48 pg / mg protein, the average TNF-alpha content in the colon homogenate of the ISV group was 220.61 pg / mg protein, and the average TNF-alpha content in the colon homogenate of the spermidine drug group was 267.28 pg / mg protein. Compared with the model group, isobutyric acid and isovaleric acid reduced the level of TNF-alpha in the colon homogenate of aging mice. After oral administration of isobutyric acid, TNF-alpha decreased by 35.46%, and after oral administration of isovaleric acid, TNF-alpha decreased by 32.03%. Compared with the spermidine drug group, the TNF-alpha level of isobutyric acid decreased by 21.63% and the TNF-alpha level of isovaleric acid decreased by 9.96%. The above experimental results show that the branched-chain amino acid metabolites of intestinal flora can effectively reduce TNF-alpha in the colon homogenate of mice and improve the frail state of mice. The effects of isobutyric acid and isovaleric acid are better than those of spermidine drugs.

[0106] Example 10: Effect of branched-chain amino acid metabolites from intestinal flora on the elevation of acetylcholine levels in brain homogenates of aged mice

[0107] Specific implementation method Referring to Example 5, the acetylcholine content in the mouse brain homogenate was detected according to the instructions of the acetylcholine kit. The results are as follows: Figure 10 shown.

[0108] The results show:

[0109] Depend on Figure 10It can be seen that the average acetylcholine content in the brain homogenate of the blank group was 842.99 μg / mg protein, the average acetylcholine content in the brain homogenate of the model group was 440.88 μg / mg protein, the average acetylcholine content in the brain homogenate of the ISB group was 1031.40 μg / mg protein, the average acetylcholine content in the brain homogenate of the ISV group was 1389.40 μg / mg protein, the average acetylcholine content in the brain homogenate of the 2-MB group was 919.26 μg / mg protein, and the average acetylcholine content in the brain homogenate of the spermidine drug group was 401.33 μg / mg protein. Compared with the model group, isobutyric acid and isovaleric acid increased acetylcholine levels in the brains of aging mice. Oral administration of isobutyric acid increased acetylcholine by 133.94%, oral administration of isovaleric acid increased acetylcholine by 215.14%, and oral administration of 2-methylbutyric acid increased acetylcholine by 108.50%. Isobutyric acid and isovaleric acid were more effective in increasing brain acetylcholine levels than 2-methylbutyric acid and spermidine. These experimental results demonstrate that branched-chain amino acid metabolites from gut microbiota can effectively elevate acetylcholine levels in mouse brain homogenates and improve frailty in mice, achieving an effect superior to that achieved by drugs and 2-methylbutyric acid, another metabolite of branched-chain amino acids from gut microbiota.

[0110] Example 11: Effect of branched-chain amino acid metabolites from intestinal flora on the grip strength of aging mice

[0111] Specific implementation method Referring to Example 4, before killing the mouse, the mouse was placed flat on the T-frame of the grip strength meter (Jinan Yiyan Technology Co., Ltd.), the tail of the mouse was grasped and slowly pulled backward, and the data was read. The results were as follows: Figure 11 shown.

[0112] The results show:

[0113] Depend on Figure 11 The average grip strength of mice in the blank group was 101.26g, that of mice in the model group was 80.54g, that of mice in the ISB group was 98.34g, that of mice in the ISV group was 98.56g, that of mice in the ISB group was 94.08g, and that of mice in the spermidine group was 68.27g. Compared with the model group, isobutyric acid and isovaleric acid improved the grip strength of aging mice. Oral administration of isobutyric acid increased grip strength by 22.10%, that of isovaleric acid by 22.37%, and that of 2-methylbutyric acid by 16.81%. Isobutyric acid and isovaleric acid were more effective in improving grip strength than 2-methylbutyric acid and the spermidine group. These experimental results indicate that branched-chain amino acid metabolites from gut microbiota can effectively increase grip strength and improve frailty in mice, with isobutyric acid and isovaleric acid being more effective than drugs and 2-methylbutyric acid, another branched-chain amino acid metabolite from gut microbiota.

[0114] Although the present invention has been disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. Use of branched-chain amino acid metabolites in the preparation of drugs for alleviating aging, characterized in that: The branched chain amino acid metabolites include isobutyric acid or isovaleric acid.

2. The use according to claim 1, characterized in that: The aging alleviation comprises at least one of (a) to (j): (a) Increase the abundance of porA, a key branched-chain amino acid metabolic gene, in the intestinal flora of aged mice; (b) Changes in the metabolism of nutrients by the intestinal flora of aged mice; (c) reducing the frailty score index of aging individuals; (d) reducing the level of MDA in liver tissue of aging individuals; (e) increasing the level of SOD enzyme in liver tissue of aging individuals; (f) increasing the level of CAT enzyme in liver tissue of aging individuals; (g) increasing the level of GSH-px enzyme in liver tissue of aging individuals; (h) increasing the level of acetylcholine in the brain tissue of aging individuals; (i) reducing the expression level of pro-inflammatory factors in colon tissue of aging individuals; the pro-inflammatory factors include IL-6 or TNF-alpha; (j) Improving grip strength in aging individuals.

3. The use according to claim 2, characterized in that: In the drug, the added amount of branched-chain amino acid metabolites is at least 5 mmol / L or 0.005 mmol / g.

4. The use according to claim 3, characterized in that: The drug comprises branched-chain amino acid metabolites, drug carriers and / or pharmaceutical excipients.

5. The use according to claim 4, characterized in that: The drug carriers include microcapsules, microspheres, nanoparticles and liposomes.

6. The use according to claim 5, characterized in that: The pharmaceutical excipients include excipients and / or additives.

7. The use according to claim 5, characterized in that: The pharmaceutical excipients include anti-adhesive agents, penetration enhancers, buffers, plasticizers, surfactants, defoamers, thickeners, inclusion agents, absorbents, humectants, solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, pH regulators, adhesives, disintegrants, fillers, lubricants, wetting agents, integrators, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, foaming agents, suspending agents, coating materials, fragrances, diluents, flocculants and deflocculating agents, filter aids and release retardants.

8. The use according to claim 6, characterized in that: The additional agent comprises microcrystalline cellulose, hydroxypropyl methylcellulose or lecithin.

9. The use according to any one of claims 1 to 8, characterized in that: The dosage form of the drug includes granules, capsules, tablets, pills or liquid preparations.

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