Application of ginsenoside CK in preparation of product for reducing TMAO and / or TMA content

Abstract

The invention discloses an application of ginsenoside CK in preparation of a product for reducing the content of TMAO and / or TMA. The ginsenoside CK can regulate and control generation of intestinal flora TMA and / or TMAO, and a theoretical basis is provided for follow-up treatment of TMAO related diseases through GCK.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and in particular relates to the application of ginsenoside CK in preparing products for reducing the content of TMAO and / or TMA. Background Art

[0002] Trimethylamine oxide (TMAO) is a metabolite of choline, betaine, and carnitine. Choline is first metabolized by intestinal flora into trimethylamine (TMA), and then oxidized into TMAO by flavin monooxygenase (FMO3) in the liver. Related studies have shown that TMAO is a risk factor for cardiovascular disease and chronic kidney disease, and TMAO aggravates cardiovascular disease and chronic kidney disease by promoting inflammatory responses. Reducing TMAO levels can effectively alleviate the progression of related diseases. Therefore, inhibiting TMAO production is a potential method for the treatment of cardiovascular disease and chronic kidney disease.

[0003] However, there is little research on products for reducing the content of TMAO and TMA in the related technology. Therefore, there is an urgent need for a safe and effective product for reducing the content of TMAO and TMA. Summary of the invention

[0004] The present invention aims to solve at least one of the technical problems existing in the above-mentioned prior art. To this end, the present invention proposes the use of ginsenoside CK in the preparation of products with reduced TMAO and / or TMA content.

[0005] The invention also provides a product.

[0006] The invention also proposes the application of the product.

[0007] According to one aspect of the present invention, the use of ginsenoside CK in preparing a product with reduced TMAO and / or TMA content is proposed.

[0008] In some embodiments of the present invention, TMAO and TMA are TMAO and TMA produced by choline metabolism.

[0009] According to a second aspect of the present invention, a product is provided, wherein the product contains ginsenoside CK.

[0010] In some embodiments of the invention, the product is a pharmaceutical product.

[0011] In some embodiments of the present invention, the medicine further comprises a pharmaceutical carrier and / or a pharmaceutical excipient.

[0012] In some embodiments of the present invention, the pharmaceutical carrier includes at least one of a diluent, an excipient, a filler, a binder, a disintegrant, an absorption enhancer, a surfactant, an adsorption carrier, a lubricant, a sweetener and a flavoring agent.

[0013] In some embodiments of the invention, the excipient comprises water.

[0014] In some embodiments of the present invention, the filler comprises at least one of starch and sucrose.

[0015] In some embodiments of the present invention, the binder comprises at least one of a cellulose derivative, alginate, gelatin and polyvinyl pyrrolidone.

[0016] In some embodiments of the invention, the humectant comprises glycerin.

[0017] In some embodiments of the present invention, the disintegrant comprises at least one of agar, calcium carbonate and sodium bicarbonate.

[0018] In some embodiments of the invention, the absorption enhancer comprises a quaternary ammonium compound.

[0019] In some embodiments of the invention, the surfactant comprises cetyl alcohol.

[0020] In some embodiments of the present invention, the adsorption carrier includes at least one of kaolin and bentonite.

[0021] In some embodiments of the present invention, the lubricant includes at least one of talc, calcium stearate, magnesium stearate and polyethylene glycol.

[0022] In some embodiments of the present invention, the dosage form of the product is one of powder, tablet and granule.

[0023] According to a third aspect of the present invention, an application of the above product is proposed, wherein the application is an application in preparing a product with reduced TMAO and TMA content.

[0024] In some embodiments of the present invention, the application is application in the preparation of a product for regulating intestinal flora.

[0025] In some embodiments of the present invention, regulating the intestinal flora includes regulating the abundance of intestinal flora to reduce the content of TMAO and / or TMA.

[0026] In some embodiments of the present invention, regulating the abundance of intestinal flora includes reducing the abundance of Firmicutes.

[0027] In some embodiments of the present invention, regulating the abundance of intestinal flora includes increasing the abundance of Bacteroidota or Verrucomicrobiota.

[0028] In some embodiments of the present invention, the regulating the abundance of intestinal flora comprises reducing the abundance of at least one of Dubosiella, Clostridium, Faecalibaculum, Lactobacillus, Klebsiella and Escherichia-Shigella.

[0029] In some embodiments of the present invention, regulating the abundance of intestinal flora includes increasing the abundance of at least one of Akkermansia and Parabacteroides.

[0030] In some embodiments of the present invention, the application is application in the preparation of an antibacterial agent.

[0031] In some embodiments of the present invention, the bacteria inhibited by the antibacterial agent are TMA-producing bacteria.

[0032] In some embodiments of the present invention, the application is application in the preparation of drugs for treating cardiovascular diseases and chronic kidney disease.

[0033] According to a fourth aspect of the present invention, a method for inhibiting the production of TMAO and / or TMA is proposed, the method comprising the following steps: introducing ginsenoside CK into an animal body; the method is not intended for the diagnosis or treatment of a disease.

[0034] In some embodiments of the invention, the animal comprises a mammal.

[0035] In some embodiments of the present invention, the introduction method includes oral administration or injection.

[0036] According to some embodiments of the present invention, at least the following beneficial effects are achieved: the scheme of the present invention provides the use of ginsenoside CK in the preparation of products that reduce the content of TMAO and TMA, and it is found that ginsenoside CK can regulate the production of TMA and TMAO in intestinal flora, providing a theoretical basis for the subsequent GCK treatment of TMAO-related diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0038] Figure 1 This is a design idea diagram of the animal experiment in Example 1 of the present invention;

[0039] Figure 2 This is a graph showing the weight changes of mice in Example 1 of the present invention;

[0040] Figure 3The figure is a graph showing the difference in body weight between day 0 and day 35 of each group of mice in Example 1 of the present invention, wherein the figure is mean±SD, n=6, **P is <0.01, ##P is <0.01;

[0041] Figure 4 The change trend of plasma TMAO concentration of each group of mice in Example 1 of the present invention from the 2nd week to the 5th week, where *P<0.05, **P<0.01, ***P<0.001;

[0042] Figure 5 This is a bar graph of TMAO concentrations of mice in each group from week 2 to week 5 in Example 1 of the present invention, where *P<0.05, **P<0.01, ***P<0.001;

[0043] Figure 6 The change trend of plasma TMA concentration of each group of mice in Example 1 of the present invention from the 2nd week to the 5th week, where *P<0.05, **P<0.01, ***P<0.001;

[0044] Figure 7 This is a bar graph of TMA concentrations of mice in each group from week 2 to week 5 in Example 1 of the present invention, where *P<0.05, **P<0.01, ***P<0.001;

[0045] Figure 8 : is a graph of the Alpha diversity detection result in Example 1 of the present invention, wherein A is a Chao1 index graph; B is a Shannon index graph;

[0046] Fig. 9 It is a principal coordinate analysis (PCoA) diagram in Example 1 of the present invention, wherein the horizontal and vertical axes represent the first two principal components, respectively, the percentages in brackets are the percentages of the variables that can be explained by the principal components, and R2, R3, and R4 represent the Control group, the Choline group, the Choline+GCK(H) group, and the Choline+GCK(L) group, respectively;

[0047] Fig.10 The figure is a bar graph showing the distribution of LDA values ​​in Example 1 of the present invention, wherein the LDA scores of the bacterial communities with significant differences among the Choline group, the Choline+GCK(L) group, and the Choline+GCK(H) group (LDA score greater than 4 indicates that the bacteria are differential bacteria);

[0048] Fig.11 The clustering tree diagram in Example 1 of the present invention, wherein the red, green and blue nodes represent bacteria that play an important role in the Choline group, Choline+GCK(H) group and Choline+GCK(L) group, respectively;

[0049] Fig.12 This is a cluster diagram of species abundance at the phylum level of the intestinal flora of mice in the Control group and the Choline group in Example 1 of the present invention, wherein * represents P<0.05, ** represents P<0.01, and *** represents P<0.001;

[0050] Fig.13 The relative abundance statistical result graph of Firmicutes, Bacteroidetes, Verrucomicrobia and Proteobacteria in the Control group and Choline group mice in Example 1 of the present invention, wherein, * represents P < 0.05, ** represents P < 0.01, and *** represents P < 0.001;

[0051] Fig.14 This is a cluster diagram of species abundance at the phylum level of the intestinal flora of the mice in the Choline group, Choline+GCK(L) group, and Choline+GCK(H) group in Example 1 of the present invention, wherein * represents P<0.05, ** represents P<0.01, and *** represents P<0.001;

[0052] Fig.15 The relative abundance statistical result graph of Firmicutes, Bacteroidetes, Verrucomicrobia and Proteobacteria in the Choline group, Choline+GCK(L) group and Choline+GCK(H) group of mice in Example 1 of the present invention, wherein, * represents P<0.05, ** represents P<0.01, and *** represents P<0.001;

[0053] Fig.16 This is a cluster diagram of species abundance at the genus level of the intestinal flora of mice in the Control group and the Choline group in Example 1 of the present invention;

[0054] Fig.17 This is a graph showing the relative abundance detection results of Dubosiella, Clostridium, Klebsiella, Faecalibaculum, Lactobacillus, Escherichia, Akkermansia, Bacteroides and Parabacteroides in mice in the Control group and the Choline group in Example 1 of the present invention, wherein * represents P < 0.05, ** represents P < 0.01, and *** represents P < 0.001;

[0055] Fig.18This is a cluster diagram of species abundance at the genus level of the intestinal flora of the Choline group, Choline+GCK(L) group and Choline+GCK(H) group of mice in Example 1 of the present invention;

[0056] Fig.19 This is a graph showing the relative abundance detection results of Dubosiella, Clostridium, Klebsiella, Faecalibaculum, Lactobacillus, Escherichia, Akkermansia, Bacteroides and Parabacteroides in the Choline group, Choline + GCK (L) group and Choline + GCK (H) group of mice in Example 1 of the present invention, wherein * represents P < 0.05, ** represents P < 0.01, and *** represents P < 0.001. DETAILED DESCRIPTION

[0057] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0058] If the specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. If the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

[0059] In the description of the present invention, the terms "include" and "have" and any variations thereof are intended to cover non-exclusive inclusions. For example, a process, method, system, product or apparatus comprising a series of steps or units is not necessarily limited to those steps or units explicitly listed, but may include other steps or units not explicitly listed or inherent to these processes, methods, products or apparatuses.

[0060] When a numerical range is disclosed herein, the above range is considered to be continuous and includes the minimum and maximum values ​​of the range, as well as every value between such minimum and maximum values. Further, when a range refers to an integer, every integer between the minimum and maximum values ​​of the range is included. In addition, when multiple ranges are provided to describe features or characteristics, the ranges can be combined. In other words, unless otherwise indicated, all ranges disclosed herein should be understood to include any and all subranges included therein.

[0061] Unless otherwise specified, "about" in the present invention means that the allowable error is within ±5%.

[0062] Example 1 Application of ginsenoside CK in reducing TMAO and TMA content

[0063] This example provides an experimental study on the effect of ginsenoside CK in reducing the content of TMAO and TMA.

[0064] 1. Effect of GCK on plasma TMA, TMAO and intestinal flora in mice fed with high choline

[0065] Experimental animals: healthy SPF-grade 8-week-old C57BL / 6J male mice, weighing about 20g, and kept under the following conditions: room temperature, relative humidity of about 55%, and 12h of light and dark time per day. Before the formal start of the experiment, all mice were roughly divided into three cages and kept in the same cage for two weeks. The litter and fecal pellets between different groups were exchanged every three days, and new litter was replaced every five days to level the baseline of the intestinal flora of mice. During the feeding period, stable feeding conditions and sufficient feed and drinking water were ensured.

[0066] The design ideas of the experimental method are as follows Figure 1 As shown, the specific steps are as follows: All 8-week-old C57BL / 6J male mice were housed in the same cage for 2 weeks to level the intestinal flora baseline, and then randomly divided into 7 groups according to body weight after weighing: blank group, choline group, GCK high-dose group (GCK administration amount is 40 mg / kg), GCK low-dose group (GCK administration amount is 20 mg / kg), choline GCK high-dose group (GCK administration amount is 40 mg / kg), choline GCK low-dose group (GCK administration amount is 20 mg / kg), choline pseudo-sterile group. There are 6 mice in each group, all choline groups are fed with feed containing 1.2% choline, all drug-administered groups are gavaged according to body weight, and body weight is measured once a week. The pseudo-sterile group was treated with a quadruple antibiotic, which was administered by gavage according to body weight for the first four days (quadruple antibiotics: ampicillin, neomycin sulfate and metronidazole 100 mg / kg, vancomycin 50 mg / kg), and then the mice were given antibiotic drinking water until the end of the experiment (ampicillin, neomycin and metronidazole: 0.25 g / L; vancomycin: 0.125 g / L). Starting from the second week of the formal experiment, mouse plasma was collected once a week, and TMA and TMAO were detected by HPLC-MS. Fresh feces of mice were collected on the morning of the last day of the animal experiment for 16S rDNA analysis. After the experiment, the required tissues and organs were sampled for research.

[0067] The detailed experimental steps are as follows:

[0068] (1) Collection of mouse plasma samples

[0069] 24 hours before blood collection, the capillary glass tube was extracted and soaked in sodium heparin. About 1 mL of mouse plasma was collected on the 14th, 21st, 28th, and 36th days of the formal experiment (retroorbital venous plexus blood collection method), and the upper serum was separated after centrifugation at 4°C, and pre-treated immediately and detected by HPLC-MS. The remaining serum was packaged and stored in a -80°C refrigerator.

[0070] (2) HPLC-MS method for detecting TMA and TMAO concentrations

[0071] 1) Chromatographic conditions

[0072] Liquid chromatograph: Waters UPLC I-Class; chromatographic column: Waters ACQUITY UPLC BEH HILIK1.7μm (2.1×50mm); online filter: Waters CRITICAL CLEANTM; autosampler temperature: 10°C; column temperature: 40°C; mobile phase: phase A: 10mM ammonium acetate aqueous solution, phase B: acetonitrile; flow rate: 0.3mL / min; injection volume: 5μL. The gradient elution program for the analytical samples is as follows: 0-0.5min, 70%B; 0.5-2.0min, 10%B; 2.4-3.0min, 70%B.

[0073] 2) Mass spectrometry conditions

[0074] The ion source was an electrospray ionization source, the detection was performed in positive ion mode, the signal acquisition mode was MRM, the ion source temperature was 150°C, the capillary voltage was 0.5 kV, the cone voltage was 25 V, the desolvation gas flow rate was 1000 L / hr, and the desolvation gas temperature was 500°C. The mass spectrometry parameters of TMAO, d9-TMAO, TMA, and d9-TMA are shown in Table 1.

[0075] Table 1 Mass spectrometry parameters of TMAO, d9-TMAO, TMA, and d9-TMA

[0076]

[0077]

[0078] 3) Solution preparation and sample processing

[0079] Preparation of reference substance stock solution: Accurately weigh 1 mg of TMA.HCl and TMAO reference substances respectively, transfer them to a volumetric flask, dissolve them to volume with 50% methanol and shake well to obtain 1 mg / mL reference substance stock solutions respectively, and store them in a -20°C refrigerator.

[0080] Preparation of internal standard reference substance stock solution: Accurately weigh 1 mg of D9-TMA.HCl and D9-TMAO reference substances respectively, transfer them to a volumetric flask, dissolve them to volume with 50% methanol and shake well to obtain 1 mg / mL internal standard reference substance stock solutions, and store them in a -20°C refrigerator.

[0081] Preparation of standard curve working solution: Take TMA.HCl and TMAO reference substance stock solutions (concentration of 1 mg / mL), dilute with 50% methanol water, mix TMA.HCl and TMAO reference substances to final concentrations of 2.000 μg / mL and 100.000 μg / mL, respectively, and use them as standard curve working solutions after stepwise dilution.

[0082] Preparation of internal standard working solution: Take D9-TMA.HCl and D9-TMAO internal standard reference substance stock solutions (concentration is 1 mg / mL), mix them with acetonitrile to obtain internal standard working solutions containing 50.000 ng / mL and 800.000 ng / mL of D9-TMAO and D9-TMA.HCl, respectively.

[0083] Take 50 μL of the sample to be tested, add 150 μL of internal standard working solution, oscillate for 30 s, centrifuge (18400×g, 6 min), take 50 μL of the supernatant, add 250 μL of ultrapure water, vortex to mix, and inject 5 μL.

[0084] (2) Collection of mouse fecal samples

[0085] Fecal sample collection: On the morning of the 36th day after the formal experiment began, prepare a transparent plastic bucket and wipe it clean. Place clean sterilized filter paper on the bottom of the bucket, transfer the mice from the breeding cage, and put only one mouse in a plastic bucket. After the mouse defecates, use sterilized tweezers to collect fresh feces into a cryopreservation tube and immediately put it into a liquid nitrogen tank within 10 minutes. After collection, remove the cryopreservation tube from the liquid nitrogen and transfer it to a -80℃ refrigerator for subsequent 16S rDNA analysis.

[0086] (3) Fecal 16S rDNA analysis

[0087] A sequencing

[0088] (1) The steps of DNA extraction and PCR amplification are as follows: First, DNA is extracted from the stool sample using the QIAamp DNA Stool Mini kit. The DNA content is then determined by agarose gel electrophoresis. An appropriate amount of DNA is placed in a centrifuge tube and diluted to 1 ng / μl with sterile water. Next, specific primers with barcodes are selected and (Phusion) uses a method combining GC Buffer and High-Fidelity PCR Mix to ensure fast and accurate PCR. According to the type of microorganism to be identified, the following primers can be selected for amplification: 16S V4 region primers - identification of bacteria; 18S V4 region primers - identification of eukaryotic microorganisms; ITS1 region primers - identification of fungi.

[0089] (2) PCR product mixing and purification: First, the results of the PCR reaction were determined by 2% agarose gel electrophoresis. The product results after the PCR reaction were evenly mixed according to the different proportions of the PCR reaction. Then the desired bands were determined by 2% agarose electrophoresis. Gel Extraction Kit is used to purify specific fragments.

[0090] (3) Library construction and sequencing: Use the database construction toolkit to build a database ( Ultra3.0 II DNA Library Pretreatment Kit) was used, and the established database was quantified using Qubit and Q-PCR. After the library passed the qualification check, the NovaSeq6000 was used to start the computer for sequencing.

[0091] B Information Analysis

[0092] 1) Analyze and process sequencing data: Separate the data of each sample from the offline data according to the barcode and PCR amplification primers, and splice them using FLASH software to obtain Raw Tags; use fastp software to perform quality control on Raw Tags to obtain high-quality Clean Tags. Finally, use Vsearch software to compare with the database, remove chimeras, and obtain Effective Tags, which is the final effective data.

[0093] 2) Denoise ASVs and annotate species: For Effective Tags, use the DADA2 module or deblur of the QIIME2 software to denoise and filter out sequences with abundance less than 5 to obtain ASVs and feature tables. Then, use the classify-sklearn module of the QIIME2 software to compare the ASVs with the database to obtain the species information of each ASV.

[0094] In general, the Silva138.1 database was used to annotate the 16S and 18S regions, and the UNITEv8.2 database was used to annotate the ITS region.

[0095] 3) Analysis of sample complexity (Alpha Diversity): QIIME2 software was used to calculate the Alpha Diversity of the test samples, including Shannon, Simpson, Chao1 and Pielou_e indexes, etc., and dilution curves and species accumulation box plots were also drawn. If there were groupings, the inter-group differences in Alpha diversity were further analyzed.

[0096] 4) Comparative analysis of different samples (Beta Diversity): Unifrac distance was calculated using QIIME2 software, and dimensionality reduction plots (PCA, PCoA, and NMDS) were drawn using R software. Next, the significance of community structure differences between groups was analyzed using adonis and anosim functions. Finally, LEfSe or R software was used to analyze species with significant differences between groups. LEfSe was completed using LEfSe software, and MetaStat and T tests were implemented using R software.

[0097] 5) Functional annotation: PICRUSt2 is a method for predicting bacterial metabolic functions. Based on the ASV tree and ASV gene information in the Greengene database, it infers the gene function spectrum of its common ancestor and predicts the gene functions of the entire spectrum of the archaea and bacteria domains. Finally, the bacterial composition obtained by sequencing is "mapped" to the database to predict the metabolic function of the bacterial community.

[0098] 6) Correlation analysis of environmental factors: When performing Spearman correlation analysis, the functions in the psych package of R software were used to calculate the correlation coefficient and test the significance, and the pheatmap package was used for visualization. Manteel test used the vegan package in R to transform the species and environmental factor data matrix into a distance matrix, and used the mantel function to perform spearman correlation analysis. CCA, RDA, and VIF analyses were implemented using the functions in the vegan package, and the envfit function was used to calculate the significance of the impact of environmental factors on species distribution. The bioenv function was used to screen the environmental factors or combinations with the greatest correlation with the species matrix, and the vif.cca function was used to screen the environmental factors with redundant constraints.

[0099] 7) When the number of samples is greater than or equal to 24, microPITA can be used for metagenomic sequencing sample screening, which provides multiple screening modes, including 3 unsupervised and 2 supervised methods, to screen representative and interesting samples. The main screening criteria include samples with the largest α diversity (Diverse), representative samples that reflect overall differences based on β diversity (Representative), samples with the largest differences based on β diversity (Extreme), samples with the largest β diversity distance between groups (Distinct, samples within a group must be greater than 6), and samples in the center of each group (Discriminant, samples within a group must be greater than 6).

[0100] Table 2 Body weight of mice in each group (unit: g) (mean ± SD)

[0101]

[0102] The experimental results are shown in Table 2 and Figure 2-15 As shown, from Figure 2-3 As can be seen from Table 2, the weight of mice was measured every 7 days from the beginning of the formal experiment. The mice were not restricted in diet and drinking water. The weight was measured and recorded at 8:30 am. After the experiment, the weight line graph and weight difference graph were drawn. The results showed that compared with the Control group, the weight of the GCK (L) and GCK (H) groups tended to decrease, and the higher the dose, the more significant the trend; Choline + GCK (L) and Choline + GCK (H) also tended to decrease compared with the Choline group. Compared with the Control group, the weight of the Choline + GCK (H) group was significantly reduced (P < 0.01), and compared with the Choline group, the Choline + GCK (H) group also had the same significant difference (P < 0.01). The experimental observation also found that the weight loss caused by the Abx group was accompanied by diarrhea in mice, but the weight loss caused by high-dose GCK did not cause diarrhea in mice, indicating that GCK would not cause intestinal flora disorder and destroy intestinal flora.

[0103] from Figure 4-5It can be seen that the plasma TMAO levels of mice in the Control, GCK (H), and GCK (L) groups have been relatively stable and at a low level. The TMAO level in the Choline group began to increase from the second week, and by the fifth week, the TMAO concentration was 1271ng / ml. The TMAO levels in the Choline+GCK (H) and Choline+GCK (L) groups showed a downward trend from the second week, and the concentrations were 834.5 and 728ng / ml, respectively, in the fifth week, among which the TMAO concentration in the Choline+GCK (H) group was significantly reduced (P<0.01). At the same time, in choline-fed mice, GCK greatly reduced the TMAO level and showed a dose-dependent pattern, while for non-choline-fed mice, the effect of GCK on TMAO levels was not significant. The Choline+Abx group fed antibiotic-treated pseudo-sterile mice with choline feed, that is, the pseudo-sterile group. The TMAO concentration of mice in the pseudo-sterile group remained at a low level, and in the fifth week, the TMAO level increased slightly, which may be related to the stabilization of the intestinal flora structure after long-term use of Abx. In addition, the TMA level in the pseudo-sterile group showed the same trend as that in the Choline+GCK(L) group and the Choline+GCK(H) group: TMAO level decreased from the third week and slightly increased in the fifth week. Compared with the Choline group, the TMA level in the pseudo-sterile group was significantly lower from the second week to the fifth week (the third and fifth weeks, P<0.01; the second and fourth weeks, P<0.001).

[0104] from Figure 6-7 It can be seen that the TMA level in the Choline group began to increase from the second week, and by the fifth week, the TMA concentration was 3363ng / ml. The TMA levels in the Choline+GCK(H) and Choline+GCK(L) groups showed a downward trend from the second week and were dose-dependent, among which the TMA concentration in the Choline+GCK(H) group was significantly reduced (P<0.01). At the fifth week, the TMA concentrations in the Choline+GCK(H) and Choline+GCK(L) groups were 1807.5ng / ml and 1295.3ng / ml, respectively. The plasma TMA levels of mice in the Control, GCK(H), and GCK(L) groups were always at a low level. The Choline+Abx group was the pseudo-sterile group. The changes in the TMA concentration of pseudo-sterile mice were the same as those of TMAO. They remained at a low level from the second week, and the TMA level increased slightly in the fifth week.

[0105] The above results show that GCK can reduce the levels of TMA and TMAO in choline-fed mice. In order to further explore whether GCK regulates the production of TMA through intestinal flora, the feces of mice in the Control group, Choline group, Choline+GCK (H) group and Choline+GCK (L) group were collected for 16S rDNA sequencing analysis and marked as R1, R2, R3 and R4 respectively.

[0106] The results of Alpha diversity analysis are as follows Figure 8-15 Display, from Figure 8-9 It can be seen that compared with the Choline group, the Alpha diversity of the intestinal flora of mice increased after GCK administration, and the Chao1 index and Shannon index increased significantly (* represents P < 0.05, ** represents P < 0.01, compared with the Choline group), indicating that GCK administration increased the richness and diversity of the intestinal flora community. Beta polymorphism compared the composition of different intestinal microbial communities. The PCoA analysis based on weighted_unifrac showed that the flora structure of mice fed with high choline changed significantly after GCK administration;

[0107] from Figure 10-11 It can be seen that the present invention uses LEfSe to analyze the bacterial communities with significant differences between different groups, and sets the linear discriminant analysis (LDA) value to evaluate the impact of the significantly different bacterial communities. The LDA value distribution bar graph and cluster tree diagram show that Firmicutes is the main bacterial phylum in the Choline group, and the level from the phylum to the genus level has increased significantly. Compared with the Choline group, the LDA diagram ( Fig.10 ) showed that Bacteroidota (Bacteroidetes) and Desulfovibrionales (Desulfovibrio) were the dominant bacterial phyla in the Choline+GCK(L) group. The cluster tree diagram ( Fig.11 ) showed that they increased significantly from phylum to family level. In the Choline+GCK(H) group, Enterobacterales increased significantly from class to genus level;

[0108] from Figure 12-15As can be seen in the figure, the species abundance clustering shows that at the phylum level, the main dominant bacteria in the intestinal flora of the control group mice are Firmicutes and Bacteroidetes, accounting for 38.98% and 42.05% of the total intestinal bacteria, respectively, followed by Verrucomicrobia and Proteobacteria, accounting for 8.15% and 6.18%, respectively. After choline feeding, the abundance of Firmicutes in the intestinal flora of mice increased significantly (accounting for 62.30%), and the abundance of Bacteroidota, Verrucomicrobiota and Proteobacteria decreased (27.33%, 2.94% and 1.77%, respectively). After GCK administration, the abundance of Bacteroidota, Verrucomicrobiota, and Proteobacteria increased in the Choline+GCK(L) and Choline+GCK(H) groups compared with the Choline group, and the abundance of Firmicutes decreased significantly in the Choline+GCK(L) and Choline+GCK(H) groups (accounting for 30.48% and 30.19%, respectively). Therefore, GCK may reduce the production of TMA and TMAO by significantly reducing the abundance of Firmicutes.

[0109] from Figure 16-19It can be seen that at the genus level, the main intestinal bacterial genera in the Control group are: Dubosiella, Akkermansia, Bacteroides, Parabacteroides, Faecalibaculum, Escherichia-Shigella, Lactobacillus, Clostridium and Klebsiella. Compared with the Control group, after Choline feeding, the abundance of Dubosiella, Clostridium and Klebsiella increased significantly (accounting for 22.67%, 6.66% and 4.69%, respectively), the abundance of Faecalibaculum, Lactobacillus and Escherichia-Shigella increased (accounting for 14.60%, 5.76% and 3.22%, respectively), the abundance of Akkermansia and Bacteroides did not change significantly, and the abundance of Parabacteroides decreased significantly. Compared with the Choline group, the abundance of Dubosiella, Clostridium, Faecalibaculum and Escherichia-Shigella in the Choline+GCK(L) group and the Choline+GCK(H) group was significantly reduced; the abundance of Klebsiella decreased after GCK administration, and the decrease was significant in the Choline+GCK(H) group; the abundance of Lactobacillus decreased significantly in the Choline+GCK(H) group; the abundance of Akkermansia increased in a dose-dependent manner; the abundance of Bacteroides did not change significantly; the abundance of Parabacteroides increased significantly. It can be seen that GCK administration regulates the abundance of intestinal flora, reduces the abundance of Dubosiella, Faecalibaculum, Escherichia-Shigella, Lactobacillus, Clostridium and Klebsiella, and inhibits the production of TMA and TMAO.

[0110] The embodiments of the present invention are described in detail above in conjunction with the accompanying drawings, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. Application of ginsenoside CK in the preparation of products with reduced TMAO and / or TMA content.

2. The use according to claim 1, characterized in that: TMAO and TMA are TMAO and TMA produced by choline metabolism.

3. A product, characterized in that: The product contains ginsenoside CK.

4. The product according to claim 3, characterized in that The product described is a pharmaceutical product.

5. The product according to claim 3, characterized in that The medicine also includes a pharmaceutical carrier and / or a pharmaceutical excipient; preferably, the pharmaceutical carrier includes at least one of a diluent, an excipient, a filler, a binder, a disintegrant, an absorption promoter, a surfactant, an adsorption carrier, a lubricant, a sweetener and a flavoring agent; and / or the dosage form of the medicine is one of a powder, a tablet and a granule.

6. The product according to any one of claims 3 to 5 is used in any of the following: (1) Application in the preparation of products with reduced TMAO and / or TMA content; (2) Application in the preparation of products for regulating intestinal flora; (3) Application in the preparation of antibacterial agents; (4) Application in the preparation of drugs for treating cardiovascular diseases and chronic kidney disease.

7. The use according to claim 6, characterized in that: The regulating of the intestinal flora includes regulating the abundance of the intestinal flora to reduce the content of TMAO and / or TMA.

8. The use according to claim 7, characterized in that: The regulation of intestinal flora abundance includes reducing the abundance of Firmicutes; and / or increasing the abundance of Bacteroidota or Verrucomicrobiota.

9. The use according to claim 7, characterized in that: The regulation of intestinal flora abundance includes reducing the abundance of at least one of Dubosiella, Clostridium, Faecalibaculum, Lactobacillus, Klebsiella and Escherichia-Shigella; and / or increasing the abundance of at least one of Akkermansia and Parabacteroides.

10. A method for inhibiting the production of TMAO and / or TMA, characterized in that: The method comprises the following steps: introducing ginsenoside CK into an animal body.

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