Isobutyric acid and isovaleric acid, metabolites of branched-chain amino acid metabolism of anti-aging intestinal flora and application thereof

Abstract

This invention discloses isobutyric acid and isovaleric acid, branched-chain amino acid metabolites of gut microbiota that alleviate aging, and their applications, belonging to the fields of microbial technology and pharmaceutical technology. Animal experiments have demonstrated that isobutyric acid or isovaleric acid, branched-chain amino acid metabolites of gut microbiota, can increase the abundance of the key gene porA in the gut microbiota of aging mice, alter the metabolism of nutrients in the mouse intestine, improve the frailty index in mice, increase liver superoxide dismutase, decrease malondialdehyde, increase glutathione peroxidase (GSH-px) levels, increase catalase levels, decrease the inflammatory factor IL-6, decrease the inflammatory factor TNF-alpha, increase acetylcholine levels in the mouse brain, and improve grip strength in mice. The strains of this invention have great application potential in the preparation of products that improve metabolism and alleviate aging.

Description

Technical Field

[0001] This invention relates to isobutyric acid and isovaleric acid, branched-chain amino acid metabolites of gut microbiota that alleviate aging, and their applications, belonging to the fields of microbial technology and pharmaceutical technology. Background Technology

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

[0003] Interventions for aging are receiving increasing attention. Studies have shown that methods such as calorie restriction, microbiome transplantation, and nutritional intervention can slow down the aging process. Furthermore, drug therapy, stem cell therapy, antioxidant and anti-inflammatory treatments, and hormone replacement therapy are being explored for the treatment of age-related diseases. Drugs such as rapamycin, metformin, spermidine, NAD+ supplements, and senolytics have shown potential for delaying aging. Among the recently released 12 biomarkers of aging, gut microbiome dysbiosis and imbalance are among the important markers of aging. The balance of the gut microbiota plays a crucial role in maintaining metabolic health and slowing down the aging process, and gut microbiota-related methods for alleviating aging show great potential.

[0004] Branched-chain amino acids (BCAAs), including leucine, isoleucine, and valine, are essential amino acids for the human body, playing a crucial role in regulating metabolic health and the aging process. Gut microorganisms participate in the metabolism of BCAAs through various pathways. Existing literature indicates that, unlike the metabolism of the body and industrial fermentation bacteria, some gut microbiota produce three metabolites through specific atypical pathways: isobutyric acid, isovaleric acid, and 2-methylbutyric acid. Studies such as "Preliminary Investigation into the Effects of Short-Chain Fatty Acids on the Lifespan and Molecular Mechanism of *C. elegans*" and "Antioxidant Effects of Substituted Short-Chain Fatty Acids on *C. elegans*" demonstrate that in oxidative stress experiments, traditional short-chain fatty acids such as formic acid, acetic acid, propionic acid, butyric acid, and valerate / hexanoic acid do not possess in vivo antioxidant activity and cannot prolong the survival time of nematodes under oxidative stress conditions, perhaps lacking an anti-aging effect. 2-Methylbutyric acid can increase the antioxidant activity of nematodes and prolong the lifespan of wild-type nematodes; however, the improvement effect of 2-methylbutyric acid on aging organisms is weak, failing to bring aging organisms to an ideal state of health. Therefore, there is an urgent need to screen for natural metabolites that can significantly improve the health of aging organisms. Summary of the Invention

[0005] This invention provides the application of branched-chain amino acid metabolites in the preparation of drugs to alleviate aging in mice, wherein the branched-chain amino acid metabolites include isobutyrate (ISB) or isovalerate (ISV).

[0006] In one embodiment of the present invention, the isobutyric acid (ISB) has the chemical formula (CH3)2CHCO2H; and the isovaleric acid (ISV) has the chemical formula (CH3)2CHCH2COOH.

[0007] The structural formula is:

[0008]

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

[0010] (a) Isobutyric acid and isovaleric acid increase the abundance of key metabolic genes of branched-chain amino acids in the gut microbiota of aging mice.

[0011] (b) Altering the metabolism of nutrients by the gut microbiota in aging mice;

[0012] (c) Reduce the frailty score index of aging individuals;

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

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

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

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

[0017] (h) Increase the level of acetylcholine in the brain tissue of aging individuals;

[0018] (i) Reduce the expression levels of pro-inflammatory factors in the colonic tissue of aging individuals; said pro-inflammatory factors include IL-6 or TNF-alpha.

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

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

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

[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 excipients and additives.

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

[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 includes granules, capsules, tablets, pills, or liquid preparations.

[0027] Beneficial effects:

[0028] This invention provides isobutyric acid or isovaleric acid, branched-chain amino acid metabolites that can alleviate aging in mice. Specifically, it includes:

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

[0030] (2) Isobutyric acid and isovaleric acid alter the metabolism of nutrients by the gut microbiota of aged mice. Isobutyric acid and isovaleric acid, the branched-chain amino acid metabolites of the three gut microbiota, altered the digestion and absorption pathways of intestinal proteins in 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%, and after oral administration of 2-methylbutyric acid, the frailty index decreased by 33.33%. Compared with the spermidine drug group, isovaleric acid reduced the frailty index by 59.00%. Isobutyric acid and isovaleric acid were more effective than 2-methylbutyric acid in reducing the frailty index.

[0032] (4) Isobutyric acid and isovaleric acid significantly increased the level of superoxide dismutase (SOD) in the liver of aged 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. After oral administration of isobutyric acid, MDA decreased by 42.03%, after oral administration of isovaleric acid, MDA decreased by 38.97%, and after oral administration of 2-methylbutyric acid, MDA decreased by 34.20%. Isobutyric acid and isovaleric acid were more effective than 2-methylbutyric acid in reducing the level of MDA 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 2-methylbutyric acid, GSH-px increased by 83.46%. The increase in GSH-px level in the liver of mice by isobutyric acid and isovaleric acid was higher than that by 2-methylbutyric acid. Compared with the spermidine drug group, the level of GSH-px in the liver of mice increased by 4.2% with isobutyric acid.

[0035] (7) Isobutyric acid and isovaleric acid significantly increased the level of catalase (CAT) in the liver of aged 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 increased the level of CAT in the liver of mice better than 2-methylbutyric acid.

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

[0037] (9) Isobutyric acid and isovaleric acid reduced the TNF-alpha level in 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 level of acetylcholine in the brain 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 more effective than 2-methylbutyric acid in increasing the level of acetylcholine in the brain, and were also better than spermidine drug group.

[0039] (11) Isobutyric acid and isovaleric acid improved the grip strength of aging mice. After oral administration of isobutyric acid, the grip strength increased by 22.10%, after oral administration of isovaleric acid, the grip strength increased by 22.37%, and after oral administration of 2-methylbutyric acid, the grip strength increased by 16.81%. The grip strength improvement effect of isobutyric acid and isovaleric acid was better than that of 2-methylbutyric acid and better than that of spermidine drug group.

[0040] Therefore, isobutyric acid and isovaleric acid, which are gut microbiota metabolites of branched-chain amino acids, have great application potential in the preparation of drugs to alleviate aging. Attached Figure Description

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

[0042] Figure 2 Enrichment analysis of gut microbiota metabolic pathways in different groups of experimental mice;

[0043] Figure 3 The frailty index of mice in different experimental groups;

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

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

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

[0047] Figure 7 : Hepatic catalase (CAT) levels in mice from different experimental groups;

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

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

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

[0051] Figure 11 Grasping strength levels of mice in different experimental groups;

[0052] Figure 12 Animal experiment flowchart. Detailed Implementation

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

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

[0055] D-galactose solution: Weigh a certain amount of D-galactose, dissolve it in sterile physiological saline to prepare D-galactose, and administer subcutaneous injections of 700 mg / kg body weight and 1000 mg / kg body weight.

[0056] Example 1: Isobutyric acid and isovaleric acid, metabolites of branched-chain amino acids in gut microbiota, increase the abundance of porA, a key gene in branched-chain amino acid metabolism, in mouse gut microbiota.

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

[0058] The experiment was designed to last 20 weeks, and the specific process is as follows: Figure 10 After mice were introduced into the breeding facility, they were allowed to acclimatize for one week. From weeks 2 to 13, an aging model was established using galactose (CAS No. 59-23-4, Aladdin). Control group mice were subcutaneously injected with sterile saline, while other treatment groups were subcutaneously injected with 700 mg / kg BW / d of D-galactose dissolved in sterile saline. Because mice develop some resistance to D-galactose, from weeks 14 to 20, subcutaneous injections of 1000 mg / kg BW / d were continued. Simultaneously, each group of mice underwent corresponding experimental treatments. The control and model groups were administered 200 μL of sterile saline via gavage daily. The volume of fluid injected was the same for all groups.

[0059] Specifically as follows:

[0060] Control group: After mice entered the breeding facility, they were acclimatized for one week. From 2 to 13 weeks, mice in the control group were injected subcutaneously with sterile saline. From 14 to 20 weeks, they were injected subcutaneously with sterile saline and 200 μL of sterile saline was administered by gavage once a day.

[0061] Model group: After entering the breeding facility, the mice were acclimatized 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 physiological saline. From week 14 to 20, the mice were subcutaneously injected with 1000 mg / kg BW / d of D-galactose dissolved in sterile physiological saline and were gavaged with 200 μL of sterile physiological saline daily. They had free access to food and water.

[0062] Isobutyric acid experimental group (ISB group): After mice entered the breeding facility, they were acclimatized for one week. From weeks 2 to 13, the model group was subcutaneously injected with 700 mg / kg BW / d of D-galactose dissolved in sterile physiological saline. From weeks 14 to 20, mice were subcutaneously injected with 1000 mg / kg BW / d of D-galactose dissolved in sterile physiological saline, and were also administered 6.5 mmol / L, 100 μL / 10g body weight of isobutyric acid by gavage daily. They had free access to food and water.

[0063] Isovalerate experimental group (ISV group): After mice entered the breeding facility, they were acclimatized for one week. From weeks 2 to 13, the model group was subcutaneously injected with 700 mg / kg BW / d of D-galactose dissolved in sterile physiological saline. From weeks 14 to 20, mice were subcutaneously injected with 1000 mg / kg BW / d of D-galactose dissolved in sterile physiological saline, and were also administered 5.5 mmol / L, 100 μL / 10g body weight of isovaleric acid by gavage daily. They had free access to food and water.

[0064] 2-Methylbutyric acid control group (2-MB group): After mice entered the breeding facility, they were acclimatized for one week. From weeks 2 to 13, the model group was subcutaneously injected with 700 mg / kg BW / d of D-galactose dissolved in sterile physiological saline. From weeks 14 to 20, mice were subcutaneously injected with 1000 mg / kg BW / d of D-galactose dissolved in sterile physiological saline, and were also administered 5.5 mmol / L, 100 μL / 10g body weight of 2-methylbutyric acid by gavage daily. They had free access to food and water.

[0065] Before euthanizing mice, fecal samples were collected, and total fecal DNA was extracted using the QIAamp DNA Stool Mini Kit (Qiagen, Hilden, Germany). DNA purity and integrity were assessed using 1% agarose gel electrophoresis. Metagenomic sequencing was performed on the DNBSEQ-T7 platform at Novogene Co., Ltd. in Beijing. Original sequencing reads were processed using Trimmomatic (version 0.39) to remove sequencing adapters and low-quality sequences. The 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. The quality-controlled and host-removed sequences were annotated with gut microbiota metabolic functions based on the MetaCyc database using HUMAnN3 (version 3.8), and then renamed according to the Uniref 90 database to obtain the abundance information of gut microbiota metabolic gene families.

[0066] Literature reports that the porA gene is a key gene for branched-chain amino acids in gut microbiota metabolism. Knocking out the porA gene results in strains losing the ability to metabolize branched-chain amino acids and produce isobutyric acid, isovaleric acid, and 2-methylbutyric acid. The reported porA gene base sequences were compared with the Uniref 90 database. Sequences with a similarity of 85% or higher were selected, and their Uniref 90 numbers were matched with gene family files of gut microbiota in aged mice. The gene abundance of porA, a key gene for branched-chain amino acids in gut microbiota metabolism in aged mice, was obtained, and 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 in the branched-chain amino acid metabolism of the gut microbiota, was significantly increased in the isobutyric acid group and the isovaleric acid group. This demonstrates that isobutyric acid and isovaleric acid have the ability to improve the branched-chain amino acid metabolism of the gut microbiota. The increase in porA gene abundance by isobutyric acid and isovaleric acid was 1.9 and 1.5 times that of the model group, respectively, which was superior to that of the 2-methylbutyric acid group.

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

[0069] For specific implementation details, refer to Example 1. Before euthanizing the mice, collect their feces. For non-target metabolomics analysis, take 100 μL of sample and mix with 400 μL of extraction solution (MeOH:ACN, 1:1 (v / v)). The extraction solution contains a deuterated internal standard. Vortex the mixture for 30 seconds, sonicate in a 4°C water bath for 10 minutes, and incubate at -40°C for 1 hour to precipitate proteins. Then, centrifuge the sample at 12000 rpm (RCF = 13800 (×g), R = 8.6 cm) for 15 minutes at 4°C. Transfer the supernatant to a new glass vial for analysis. Prepare quality control (QC) samples by mixing equal volumes of the sample supernatant.

[0070] For polar metabolites, MS analysis was performed using a UHPLC system (Vanquish, Thermo Fisher Scientific) 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 autosampler was set at 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 evaluated the full-scan mass spectra. The ESI source conditions are set as follows: sheath gas flow rate of 50 Arb, auxiliary gas flow rate of 15 Arb, capillary temperature of 320℃, full MS resolution of 60000, MS / MS resolution of 15000, collision energy of SNCE 20 / 30 / 40, and spray voltage of 3.8kV (positive) or -3.4kV (negative).

[0071] KEGG pathway enrichment analysis was performed on the metabolites, and the results are as follows: Figure 2 As shown, both isobutyric acid and isovaleric acid metabolites can alter multiple amino acid metabolic pathways in the intestines of aging mice, and also alter the metabolic pathway of protein digestion and absorption.

[0072] Example 3: The alleviating effect of isobutyric acid and isovaleric acid, branched-chain amino acid metabolites of intestinal flora, on the frailty index of aging mice.

[0073] For specific implementation details, refer to Example 1. Before euthanizing the mice, the vulnerability of each aged mouse was scored, including an assessment of 13 variables. A frailty index (FI) was calculated based on the scores of individual variables, with the following scoring criteria: for each variable, 0 points represented no defect, 0.5 points represented mild defect, and 1 point represented severe defect. A total score was calculated for evaluation, ranging from 0 to 13.

[0074] Table 1: Mouse Weakness Index

[0075]

[0076] Depend on Figure 3 It can be seen that the average weakening index of the blank group was 2.06, the average weakening index of the model group was 4.68, the average weakening index of the ISB group was 2.4, the average weakening index of the ISV group was 2, the average weakening index of the 2-MB group was 3.12, and the average weakening 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 administration of isovaleric acid, the frailty index decreased by 57.26%, and after oral administration of 2-methylbutyric acid, the frailty index decreased by 33.33%. Compared with the spermidine drug group, isovaleric acid reduced the frailty index by 59.00%. The effects of isobutyric acid and isovaleric acid in reducing the frailty index were better than those of 2-methylbutyric acid. The above experimental results indicate that isobutyric acid and isovaleric acid, which are branched-chain amino acid metabolites of intestinal flora, can effectively reduce the frailty index of mice and improve their frailty state. Isovalleric acid achieved a better effect than drugs, and isobutyric acid and isovaleric acid achieved a better effect than other branched-chain amino acid metabolites of intestinal flora, such as 2-methylbutyric acid.

[0077] Example 4: The effect of intestinal microbiota branched-chain amino acid metabolites isobutyric acid and isovaleric acid on increasing the level of superoxide dismutase (SOD) in the liver of aging mice.

[0078] For specific implementation details, refer to Example 1. After the experiment, mice in the methylbutyric acid control group were sacrificed, and their livers were harvested and placed in liquid nitrogen, stored at -80°C. The tissues were rinsed with pre-chilled PBS (0.01M, pH=7.4) to remove residual blood and then minced. Approximately 1.2g of fresh tissue was weighed and added to PBS at a weight-to-volume ratio of 1:9. 1mL of a universal protease inhibitor (Beyotime) was added to every 100mL of PBS. An equal amount of grinding beads was added to each tissue sample, and the tissue was thoroughly ground on ice using a pre-chilled tissue homogenizer mold until no solids were visible. Further freeze-thaw cycles were performed to ensure complete lysis. The homogenate was centrifuged at 5000×g for 5–10 minutes, and the supernatant was aliquoted for later use. To determine protein concentration using the BCA method, take 1.2 ml of protein standard preparation solution (0.5 g bovine serum albumin dissolved in distilled water and diluted to 100 ml to prepare a 5 mg / ml solution, diluted tenfold before use) and add it to one tube of protein standard (30 mg BSA). After thorough dissolution, prepare 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 mixing 50 volumes of BCA reagent A (weighing 10g BCA (1%), 20g Na2CO3·H2O (2%), 1.6g Na2C4H4O6·2H2O (0.16%), 4g NaOH (0.4%), and 9.5g NaHCO3 (0.95%), adding water to 1L, and adjusting the pH to 11.25 with NaOH or solid NaHCO3) with 1 volume of BCA reagent B (2g CuSO4·5H2O (4%), adding distilled water to 50ml) (50:1). Mix thoroughly. Select a suitable concentration within the standard concentration range of 0-1.5 mg / mL to construct a standard curve. Preliminary experiments with samples will determine the appropriate dilution factor. In the formal experiment, add 20μL of sample or standards of different concentrations to each well, and add 200μL of BCA working solution to each well. Incubate at 37℃ in the dark for 30 minutes. The absorbance was measured at 562 nm using an ELISA reader. 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 As shown.

[0080] The results show:

[0081] Depend on Figure 4The results showed that the average SOD level in the liver of the control group was 27.90 ng / mg protein, the average SOD level in the model group was 16.254 ng / mg protein, the average SOD level in the ISB group was 28.48 ng / mg protein, the average SOD level in the ISV group was 24.26 ng / mg protein, and the average SOD level in the spermidine drug group was 29.51 ng / mg protein. Isobutyric acid and isovaleric acid significantly increased the SOD level 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 increased by 1% with isovaleric acid. The above experimental results indicate that isobutyric acid and isovaleric acid, metabolites of branched-chain amino acids from intestinal flora, can effectively increase the level of superoxide dismutase (SOD) in mouse liver and improve the weakened state of mice. Iovaleric acid achieves a better effect than drugs.

[0082] Example 5: The effect of isobutyric acid and isovaleric acid, branched-chain amino acid metabolites of gut microbiota, on reducing malondialdehyde (MDA) levels in the liver of aging mice.

[0083] For specific implementation details, refer to the example. The malondialdehyde (MDA) level in mouse liver homogenate was detected according to the instructions of the ELISA kit for liver malondialdehyde (MDA) levels. The results are as follows: Figure 5 As shown.

[0084] The results show:

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

[0086] The above experimental results indicate that isobutyric acid and isovaleric acid, metabolites of branched-chain amino acids from gut microbiota, can effectively reduce the level of malondialdehyde (MDA) in mouse liver and improve the weakened state of mice, achieving effects superior to drugs and other products such as 2-methylbutyric acid.

[0087] Example 6: Effect of gut microbiota branched-chain amino acid metabolites on increasing glutathione peroxidase (GSH-px) levels in the liver of aging mice

[0088] For specific implementation details, refer to Example 4. The glutathione peroxidase (GSH-px) level in mouse liver homogenate was detected according to the instructions of the ELISA kit for liver glutathione peroxidase (GSH-px). The results are as follows: Figure 6 As shown.

[0089] The results show:

[0090] Depend on Figure 6 The results showed that the average glutathione peroxidase (GSH-px) level in the liver was 180.25 U / mg protein in the control group, 77.07 U / mg protein in the model group, 168.21 U / mg protein in the ISB group, 163.77 U / mg protein in the ISV group, and 141.38 U / mg protein in the 2-MB group. The average glutathione peroxidase (GSH-px) level in the liver of the spermidine drug group was 161.38 U / mg protein. Compared with the model group, 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 2-methylbutyric acid, GSH-px increased by 83.46%. The increase in GSH-px level in the liver of mice by isobutyric acid and isovaleric acid was higher than that by 2-methylbutyric acid. Compared with the spermidine drug group, the level of GSH-px in the liver of mice increased by 4.2%.

[0091] The above experimental results indicate that isobutyric acid and isovaleric acid, metabolites of branched-chain amino acids from gut microbiota, can effectively increase the level of glutathione peroxidase (GSH-px) in mouse liver and improve the weakened 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 branched-chain amino acids from gut microbiota.

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

[0093] For specific implementation details, refer to Example 4. The catalase (CAT) level ELISA kit instructions were followed to detect the catalase (CAT) content in mouse liver homogenate. The results are as follows: Figure 7 As shown.

[0094] The results show:

[0095] Depend on Figure 7 The results showed that the average catalase (CAT) value in the liver was 539.19 pg / mg protein in the blank group, 258.53 pg / mg protein in the model group, 344.67 pg / mg protein in the ISB group, 314.20 pg / mg protein in the ISV group, 319.31 pg / mg protein in the 2-MB group, and 575.71 pg / mg protein in the spermidine drug group. Compared with the model group, isobutyric acid and isovaleric acid significantly increased the level of catalase (CAT) in the liver of aging mice. Oral administration of isobutyric acid increased CAT by 33.32%, isovaleric acid by 21.53%, and 2-methylbutyric acid by 23.51%. Isobutyric acid significantly increased CAT levels in the liver of mice compared to 2-methylbutyric acid. These experimental results indicate that branched-chain amino acid metabolites from gut microbiota can effectively increase the level of catalase (CAT) in the liver of mice and improve their debilitating condition, with isobutyric acid showing a superior effect compared to 2-methylbutyric acid, another branched-chain amino acid metabolite from gut microbiota.

[0096] Example 8: Effect of gut microbiota branched-chain amino acid metabolites on the reduction of IL-6 levels in colon homogenate of aging mice

[0097] For specific implementation details, refer to Example 5. After euthanizing the mice, the colon was removed and placed in liquid nitrogen, stored at -80°C. The tissue was rinsed with pre-chilled PBS (0.01M, pH=7.4) to remove residual blood and then minced. Approximately 1.2g of fresh tissue was weighed and added to PBS at a weight-to-volume ratio of 1:9. 1mL of a universal protease inhibitor (Beyotime) was added to every 100mL of PBS. An equal amount of grinding beads was added to each tissue sample. Using a pre-chilled tissue homogenizer mold, the sample was thoroughly homogenized on ice until no solids were visible. Further freeze-thaw cycles were performed to fully lyse the homogenate. The homogenate was centrifuged at 5000×g for 5–10 minutes, and the supernatant was aliquoted for later use. To determine protein concentration using the BCA method, take 1.2 ml of protein standard preparation solution (0.5 g bovine serum albumin dissolved in distilled water and brought to a final volume of 100 ml to prepare a 5 mg / ml solution, which should be diluted tenfold before use) and add it to one tube of protein standard (30 mg BSA). After thorough dissolution, prepare 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 mixing 50 volumes of BCA reagent A (weighing 10g BCA (1%), 20g Na2CO3·H2O (2%), 1.6g Na2C4H4O6·2H2O (0.16%), 4g NaOH (0.4%), and 9.5g NaHCO3 (0.95%), adding water to 1L, and adjusting the pH to 11.25 with NaOH or solid NaHCO3) with 1 volume of BCA reagent B (2g CuSO4·5H2O (4%), adding distilled water to 50ml) (50:1). Mix thoroughly. Select a suitable concentration within the standard concentration range of 0-1.5 mg / mL to construct a standard curve. Preliminary experiments with samples will determine the appropriate dilution factor. In the formal experiment, add 20μL of sample or different concentrations of standard to each well, and add 200μL of BCA working solution to each well. Incubate at 37℃ in the dark for 30 minutes. The absorbance was measured at 562 nm using an ELISA reader. The protein concentration of the sample was calculated.

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

[0099] The results show:

[0100] Depend on Figure 8The results showed that the average IL-6 level in the colon homogenate of the blank group was 306.95 pg / mg protein, the average IL-6 level in the model group was 366.24 pg / mg protein, the average IL-6 level in the ISB group was 225.47 pg / mg protein, the average IL-6 level in the ISV group was 262.49 pg / mg protein, the average IL-6 level in the 2-MB group was 235.02 pg / mg protein, and the average IL-6 level in the spermidine drug group was 312.41 pg / mg protein. Compared with the model group, isobutyric acid and isovaleric acid reduced the IL-6 level in the colon homogenate of aging 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 reduced the IL-6 level in mice better than 2-methylbutyric acid.

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

[0102] Example 9: Effect of gut microbiota branched-chain amino acid metabolites on the reduction of TNF-alpha levels in colon homogenate of aging mice

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

[0104] The results show:

[0105] Depend on Figure 9The results showed that the average TNF-alpha in the colon homogenate of the blank group was 281.00 pg / mg protein, the average TNF-alpha in the colon homogenate of the model group was 324.56 pg / mg protein, the average TNF-alpha in the colon homogenate of the ISB group was 209.48 pg / mg protein, the average TNF-alpha in the colon homogenate of the ISV group was 220.61 pg / mg protein, and the average TNF-alpha 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 TNF-alpha level 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 more than 9.96%. The experimental results indicate that the branched-chain amino acid metabolites of intestinal flora can effectively reduce TNF-alpha in the colon homogenate of mice and improve the debilitated state of mice. Isobutyric acid and isovaleric acid are more effective than spermidine drugs.

[0106] Example 10: Effect of gut microbiota branched-chain amino acid metabolites on increasing acetylcholine levels in brain homogenate of aging mice

[0107] For specific implementation details, refer to Example 5. The acetylcholine content in mouse brain homogenate was detected according to the instructions of the acetylcholine reagent kit. The results are as follows: Figure 10 As shown.

[0108] The results show:

[0109] Depend on Figure 10The results showed that the average acetylcholine level in the brain homogenate of the blank group was 842.99 μg / mg protein, the average acetylcholine level in the brain homogenate of the model group was 440.88 μg / mg protein, the average acetylcholine level in the brain homogenate of the ISB group was 1031.40 μg / mg protein, the average acetylcholine level in the brain homogenate of the ISV group was 1389.40 μg / mg protein, the average acetylcholine level in the brain homogenate of the 2-MB group was 919.26 μg / mg protein, and the average acetylcholine level 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 the level of acetylcholine 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 more effective than 2-methylbutyric acid in increasing brain acetylcholine levels, and also more effective than the spermidine drug group. These experimental results indicate that branched-chain amino acid metabolites from gut microbiota can effectively increase the level of acetylcholine in mouse brain homogenate, improve the weakened state of mice, and achieve effects superior to drugs and another branched-chain amino acid metabolite from gut microbiota, 2-methylbutyric acid.

[0110] Example 11: Effect of gut microbiota branched-chain amino acid metabolites on increasing grip strength in aging mice

[0111] For a detailed implementation method, refer to Example 4. Before euthanizing the mouse, place the mouse flat on the T-frame of the gripping device (Jinan Yiyan Technology Co., Ltd.), grasp the mouse's tail and slowly pull it backward, and read the data. The results are as follows. Figure 11 As shown.

[0112] The results show:

[0113] Depend on Figure 11 The results showed that the average grip strength of mice in the blank control group was 101.26g, the average grip strength of mice in the model group was 80.54g, the average grip strength of mice in the ISB group was 98.34g, the average grip strength of mice in the ISV group was 98.56g, the average grip strength of mice in the ISB group was 94.08g, and the average grip strength of mice in the spermidine drug group was 68.27g. Compared with the model group, isobutyric acid and isovaleric acid improved the grip strength of aging mice. After oral administration of isobutyric acid, the grip strength increased by 22.10%, after oral administration of isovaleric acid, the grip strength increased by 22.37%, and after oral administration of 2-methylbutyric acid, the grip strength increased by 16.81%. The grip strength improvement effect of isobutyric acid and isovaleric acid was better than that of 2-methylbutyric acid, and better than that of the spermidine drug group. The above experimental results indicate that the branched-chain amino acid metabolites of intestinal flora can effectively increase the grip strength level of mice and improve the debilitated state of mice. Isobutyric acid and isovaleric acid are more effective than drugs and another branched-chain amino acid metabolite of intestinal flora, 2-methylbutyric acid.

[0114] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. The application of branched-chain amino acid metabolites in the preparation of drugs to alleviate aging, characterized in that, The branched-chain amino acid metabolites are isobutyric acid or isovaleric acid.

2. The application according to claim 1, characterized in that, The aging relief includes at least one aspect of (a) to (j): (a) Increase the abundance of porA, a key gene for branched-chain amino acid metabolism, in the gut microbiota of aging mice; (b) Altering the metabolism of nutrients by the gut microbiota in aging mice; (c) Reduce the frailty score index in aging individuals; (d) Reduce the level of MDA in the liver tissue of aging individuals; (e) Increase the level of SOD enzyme in the liver tissue of aging individuals; (f) Increase the level of CAT enzyme in the liver tissue of aging individuals; (g) Increase the level of GSH-px enzyme in the liver tissue of aging individuals; (h) Increase the level of acetylcholine in the brain tissue of aging individuals; (i) Reduce the expression levels of pro-inflammatory factors in the colon tissue of aging individuals; said pro-inflammatory factors include IL-6 or TNF-alpha; (j) Improve the grip strength of aging individuals.

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

4. The application according to claim 3, characterized in that, The drug comprises branched-chain amino acid metabolites and a drug carrier, wherein the drug carrier includes microcapsules, microspheres, nanoparticles, and liposomes.

5. The application according to claim 4, characterized in that, The drug also includes pharmaceutical excipients.

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

7. The application according to claim 5, characterized in that, The pharmaceutical excipients include anti-adhesives, penetration enhancers, buffers, plasticizers, defoamers, thickeners, encapsulating agents, humectants, solvents, propellants, solubilizers, co-solvents, emulsifiers, colorants, pH adjusters, binders, disintegrants, fillers, lubricants, wetting agents, integrators, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, foaming agents, suspending agents, coating materials, fragrances, flocculants and anti-flocculation agents, filter aids, and release inhibitors.

8. The application according to claim 6, characterized in that, The additives include microcrystalline cellulose, hydroxypropyl methylcellulose, or lecithin.

9. The application according to any one of claims 1 to 8, characterized in that, The dosage forms of the drug include granules, capsules, tablets, pills, or liquid preparations.

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