Application of folium cortex eucommiae aqueous extract in preparation of product for reducing TMAO and / or TMA content

Abstract

The invention discloses an application of a folium cortex eucommiae aqueous extract in preparation of a product for reducing the content of TMAO and / or TMA. Experiments prove that the folium cortex eucommiae aqueous extract can regulate and control the composition of intestinal flora, so that generation of TMAO and TMA is reduced, and a new method is provided for follow-up treatment of TMAO-related diseases. Meanwhile, the anti-inflammatory, anti-oxidation and lipid-lowering characteristics of the folium cortex eucommiae possibly play a positive role in lowering the TMAO level, and due to the high safety of the folium cortex eucommiae, the folium cortex eucommiae is formally listed in a medicinal and edible substance catalog, and the problems that most of existing treatment methods are low in efficiency and large in side effect can be solved when the folium cortex eucommiae aqueous extract is used for intervening the TMAO related diseases.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and particularly relates to application of an aqueous extract of eucommia leaves in preparing a product with reduced TMAO and / or TMA content. Background Art

[0002] In recent years, a number of independent studies have shown that trimethylamine N-oxide (TMAO), a metabolite of intestinal flora, is closely related to the occurrence and development of a variety of major chronic diseases, including cardiovascular disease, chronic kidney disease, metabolic syndrome, type 2 diabetes, etc. Patients with the above diseases have significantly higher TMAO levels than normal people. TMAO is a metabolite of choline, betaine, and carnitine. Choline is first metabolized by intestinal flora to trimethylamine (TMA), and then oxidized to TMAO by flavin monooxygenase (FMO3) in the liver. As a risk factor that promotes disease development and increases all-cause mortality, TMAO may be a potential therapeutic target for the above diseases. Finding methods or intervention drugs that can effectively reduce TMAO levels is expected to provide new paths for clinical treatment.

[0003] Eucommia leaves are a substance that can be used as both medicine and food. They are widely used in traditional Chinese medicine to nourish the liver and kidneys, strengthen the tendons and bones, and other treatments. Modern science has revealed that Eucommia leaves are rich in a variety of bioactive ingredients, such as flavonoids, phenolic acids, lignans, and cyclopentane ether terpenes, which have been shown to have multiple pharmacological effects such as antioxidant, antihypertensive, and hypolipidemic effects. In recent years, researchers have found that the active ingredients in Eucommia leaves may exert their health benefits by regulating the composition or abundance of intestinal flora.

[0004] Although studies have been conducted to explore methods for reducing the content of TMAO and TMA, there are few studies on products for reducing the content of TMAO and TMA in the prior art, and most methods have problems such as low efficiency and significant side effects. Summary of the invention

[0005] 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 an aqueous extract of Eucommia ulmoides leaves in the preparation of a product that reduces the content of TMAO and / or TMA, and by using an aqueous extract of Eucommia ulmoides leaves to regulate intestinal flora, thereby reducing the production of TMAO and / or TMA, providing a new method for the subsequent treatment of TMAO-related diseases.

[0006] The invention also proposes the use of the eucommia leaf water extract in preparing a product for regulating the composition of intestinal flora.

[0007] The invention also provides a method for preparing the eucommia leaf water extract.

[0008] The present invention also provides a product with reduced TMAO and / or TMA content.

[0009] The invention also provides a product for regulating the composition of intestinal flora.

[0010] According to a first aspect of the present invention, a use of an aqueous extract of eucommia leaves in preparing a product with reduced TMAO and / or TMA content is proposed.

[0011] In some embodiments of the present invention, the TMAO level comprises serum TMAO level.

[0012] In some embodiments of the present invention, the TMA level comprises serum TMA level.

[0013] According to a second aspect of the present invention, a use of an aqueous extract of eucommia leaves in preparing a product for regulating the composition of intestinal flora is proposed.

[0014] In some embodiments of the present invention, regulating the intestinal flora composition includes increasing the abundance of Akkermansia, Oscillospira and Ruminococcus.

[0015] In some embodiments of the present invention, regulating the intestinal flora composition includes reducing the abundance of Actinobacteria and Proteobacteria.

[0016] According to a third aspect of the present invention, a method for preparing an aqueous extract of Eucommia ulmoides leaves is provided, the method comprising the following steps:

[0017] The dried Eucommia ulmoides leaves are mixed with water for the first water extraction, and a filtrate and a filter residue are obtained after filtering; the filter residue is mixed with water for the second water extraction, and the filtrate is collected and combined with the filtrate obtained from the first water extraction, and finally concentrated and dried.

[0018] In some embodiments of the present invention, the mixing mass ratio of the dried Eucommia ulmoides leaves and water is 1:(8-12).

[0019] In some embodiments of the present invention, the mixing mass ratio of the filter residue to water is 1:(8-12).

[0020] In some embodiments of the present invention, the extraction temperature of the first water extraction and the second water extraction is 80°C to 100°C.

[0021] In some embodiments of the present invention, the extraction time of the first water extraction and the second water extraction is 1 to 2 hours.

[0022] In some embodiments of the present invention, the concentration is carried out using vacuum concentration technology.

[0023] In some embodiments of the present invention, the drying is carried out using spray drying technology.

[0024] According to a fourth aspect of the present invention, a product with reduced TMAO and / or TMA content is provided, the product comprising an aqueous extract of eucommia leaves prepared by the preparation method described in the third aspect of the present invention.

[0025] In some embodiments of the present invention, the product further comprises pharmaceutically acceptable excipients.

[0026] In some embodiments of the present invention, the pharmaceutically acceptable excipients include at least one of a binder, a disintegrant, a lubricant, a coating agent, a suspending agent, a thickening agent and a surfactant.

[0027] In some embodiments of the present invention, the binder is selected from at least one of gum arabic, gelatin, dextrin, hydroxypropyl cellulose, methyl cellulose or polyvinyl pyrrolidone.

[0028] In some embodiments of the present invention, the disintegrant is selected from at least one of corn starch, potato starch, cross-linked polyvinyl pyrrolidone, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, cross-linked sodium carboxymethyl cellulose, carboxymethyl cellulose, carboxymethyl cellulose calcium or alginic acid.

[0029] In some embodiments of the present invention, the lubricant is selected from at least one of micro-powder silica gel, magnesium stearate, calcium stearate, stearic acid, talc or anhydrous silica gel.

[0030] In some embodiments of the present invention, the coating agent includes at least one of hydroxypropyl methylcellulose, hydroxypropyl cellulose, polyvinyl pyrrolidone, cellulose acetate phthalate, polyvinyl phthalate, ethyl cellulose, and cellulose acetate.

[0031] In some embodiments of the present invention, the suspending agent includes at least one of gum arabic, gelatin, methyl cellulose, sodium carboxymethyl cellulose, hydroxymethyl cellulose or aluminum stearate gel.

[0032] In some embodiments of the present invention, the surfactant is selected from at least one of lecithin, sorbitan monooleate or glyceryl monostearate.

[0033] According to a fifth aspect of the present invention, a product for regulating the composition of intestinal flora is provided, wherein the product comprises an aqueous extract of eucommia leaves prepared by the preparation method described in the third aspect of the present invention.

[0034] In some embodiments of the present invention, the product further comprises pharmaceutically acceptable excipients.

[0035] In some embodiments of the present invention, the pharmaceutically acceptable excipients include at least one of a binder, a disintegrant, a lubricant, a coating agent, a suspending agent, a thickening agent and a surfactant.

[0036] In some embodiments of the present invention, the binder is selected from at least one of gum arabic, gelatin, dextrin, hydroxypropyl cellulose, methyl cellulose or polyvinyl pyrrolidone.

[0037] In some embodiments of the present invention, the disintegrant is selected from at least one of corn starch, potato starch, cross-linked polyvinyl pyrrolidone, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, cross-linked sodium carboxymethyl cellulose, carboxymethyl cellulose, carboxymethyl cellulose calcium or alginic acid.

[0038] In some embodiments of the present invention, the lubricant is selected from at least one of micro-powder silica gel, magnesium stearate, calcium stearate, stearic acid, talc or anhydrous silica gel.

[0039] In some embodiments of the present invention, the coating agent includes at least one of hydroxypropyl methylcellulose, hydroxypropyl cellulose, polyvinyl pyrrolidone, cellulose acetate phthalate, polyvinyl phthalate, ethyl cellulose, and cellulose acetate.

[0040] In some embodiments of the present invention, the suspending agent includes at least one of gum arabic, gelatin, methyl cellulose, sodium carboxymethyl cellulose, hydroxymethyl cellulose or aluminum stearate gel.

[0041] In some embodiments of the present invention, the surfactant is selected from at least one of lecithin, sorbitan monooleate or glyceryl monostearate.

[0042] The present invention has at least the following beneficial effects:

[0043] The present invention provides an application of an aqueous extract of eucommia leaves in the preparation of a product that reduces the content of TMAO and / or TMA. Experiments have confirmed that the aqueous extract of eucommia leaves can regulate the composition of intestinal flora, thereby reducing the production of TMAO and TMA, and providing a new method for the subsequent treatment of TMAO-related diseases. At the same time, the anti-inflammatory, antioxidant and lipid-lowering properties of eucommia leaves may also play a positive role in reducing TMAO levels. In view of the high safety of eucommia leaves, it has been officially included in the catalog of medicinal and edible homologous substances. The use of eucommia leaf aqueous extracts to intervene in TMAO-related diseases can overcome the problems of low efficiency and large side effects of most existing treatment methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0045] Figure 1The figure is a graph showing the weight changes of mice in each group in Example 2 of the present invention;

[0046] Figure 2 The result graph of TMAO and TMA levels in mouse serum after treatment with the water extract of Eucommia ulmoides leaves in Example 2 of the present invention; wherein, compared with the control group, #### represents P<0.0001; compared with the model group, * represents P<0.05, ** represents P<0.01, and **** represents P<0.0001;

[0047] Figure 3 This is a graph showing the analysis results of the effect of the eucommia leaf water extract on the alpha diversity index of intestinal flora in Example 3 of the present invention;

[0048] Figure 4 This is the NMDS analysis diagram of the Eucommia ulmoides leaf water extract group in Example 3 of the present invention;

[0049] Figure 5 The figure is a bar graph showing the distribution of LDA values ​​of each group of samples in Example 3 of the present invention;

[0050] Figure 6 is a species taxonomy cladogram of each group of samples in Example 3 of the present invention;

[0051] Figure 7 This is a diagram showing the analysis results of the effect of the water extract of Eucommia ulmoides leaves on the abundance of bacterial flora at the mouse phylum level in Example 3 of the present invention;

[0052] Figure 8 This is a graph showing the analysis results of the effect of the water extract of Eucommia ulmoides leaves on the abundance of TMAO metabolism-associated bacterial flora at the phylum level in Example 3 of the present invention; wherein, compared with the control group, # represents P<0.05; compared with the model group, * represents P<0.05;

[0053] Fig. 9 This is a diagram showing the analysis results of the effect of the aqueous extract of Eucommia ulmoides leaves on the abundance of the bacterial flora at the level of the genus Mouse in Example 3 of the present invention;

[0054] Fig.10 This is a result diagram of the effect of the aqueous extract of Eucommia ulmoides leaves on the abundance of TMAO metabolism-associated bacteria at the genus level in Example 3 of the present invention; wherein, compared with the control group, # represents P<0.05; compared with the model group, * represents P<0.05. DETAILED DESCRIPTION

[0055] 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.

[0056] Example 1 Preparation of Eucommia Leaf Water Extract

[0057] The present embodiment prepares a kind of Eucommia leaf water extract, and its preparation method comprises the following steps:

[0058] 1) Put the dried Eucommia leaves into an extraction tank, mix the Eucommia leaves with purified water at a mass ratio of 1:10, and perform the first water extraction; during the extraction process, control the temperature at 90°C (80-100°C), and the extraction time is 1.5h (1-2h);

[0059] 2) After the first water extraction is completed, the extract is separated from the Eucommia leaf residue using a filtration device, and the filtrate is collected; the residue is mixed with purified water at a mass ratio of 1:10, and a second water extraction is performed under the same extraction conditions as the first water extraction, and the filtrate is collected after the water extraction is completed;

[0060] 3) The filtrates obtained from the two water extractions are combined and concentrated by vacuum concentration technology, and the concentrated extract is dried by a spray dryer to obtain a dry powdery substance, i.e., the Eucommia ulmoides leaf water extract.

[0061] Example 2 Investigating the effect of Eucommia ulmoides leaf aqueous extract on serum TMA / TMAO levels based on animal models

[0062] This example is based on a mouse model to test the effect of the Eucommia leaf water extract prepared in Example 1 on serum TMA / TMAO levels. The specific experimental methods and results are as follows:

[0063] 1. Model construction:

[0064] 1) Experimental animals:

[0065] Healthy SPF-grade 8-week-old C57BL / 6J male mice weighing approximately 20 g were selected. The feeding conditions included: room temperature, relative humidity of approximately 55%, and 12 h of light and darkness per day. All mice were adaptively fed for 1 week before the formal start of the experiment. During the feeding period, stable feeding conditions and sufficient feed and drinking water were ensured.

[0066] 2) Grouping and processing:

[0067] All 8-week-old C57BL / 6J male mice were randomly divided into 5 groups after being fed for 1 week: control group, model group, low-dose group of Eucommia leaf aqueous extract and high-dose group of Eucommia leaf aqueous extract; each group had 10 to 15 mice. Except for the control group, all groups were fed with feed containing 1.0wt% choline, and the Eucommia leaf aqueous extract was dissolved in normal saline and administered by gavage for 8 weeks. The control group and model group were gavaged with an equal amount of normal saline; the grouping and treatment of mice in each group are shown in Table 1.

[0068] Table 1 Drug administration of mice in each group

[0069]

[0070] 2. Weight recording and sample collection:

[0071] 1) Weight record:

[0072] During the modeling and drug administration period, the mice were weighed and recorded using an electronic balance. Figure 1 shown.

[0073] Depend on Figure 1 It can be seen that there is no statistically significant difference in body weight between the control group and the high-choline feed after 8 weeks of feeding, indicating that the high-choline feed has no significant effect on the body weight of mice, and there is no statistically significant difference between the Eucommia leaf water extract intervention group and the control group, indicating that the dosage setting and intervention period of the Eucommia leaf water extract have no obvious toxic and side effects on mice.

[0074] 2) Serum sample collection:

[0075] Place the mouse in a side-lying position on a flat surface, press the skin around the eye with the index finger and thumb to make the eyeball protrude and become congested; use ophthalmic forceps to quickly pinch off the eyeball, turn the mouse upside down with its head downward, and massage the mouse body from the back of the body toward the eye to allow the blood in the eye socket to flow into the prepared centrifuge tube; separate the upper serum after centrifugation at 4°C and store in a -80°C refrigerator.

[0076] 3) Collection of cecal contents:

[0077] The mice were dissected to expose the cecum. The cecum was carefully separated using sterile forceps and scissors, and its contents were collected into sterile cryotubes. The cryotubes were immediately placed in a liquid nitrogen tank. After collection, the cryotubes were removed from the liquid nitrogen and transferred to a -80°C refrigerator.

[0078] 3. Detection of serum TMA and TMAO levels by liquid chromatography-mass spectrometry (HPLC-MS):

[0079] 1) Chromatographic conditions:

[0080] Liquid chromatograph: Waters UPLC I-Class;

[0081] Chromatographic column: Waters ACQUITY UPLC BEH HILIK 1.7 μm (2.1 × 50 mm);

[0082] Online filter: Waters CRITICAL CLEANTM;

[0083] Autosampler temperature: 10°C;

[0084] Column temperature: 40°C;

[0085] Mobile phase: Phase A is 10 mM ammonium acetate aqueous solution, phase B is acetonitrile;

[0086] Flow rate: 0.3 mL / min;

[0087] Injection volume: 5 μL.

[0088] The gradient elution program for the analytical samples was as follows: 0-0.5 min, 70% B phase; 0.5-2.0 min, 10% B phase; 2.4-3.0 min, 70% B phase.

[0089] 2) Mass spectrometry conditions:

[0090] The ion source selected was an electrospray ionization source, and 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 / h, and the desolvation gas temperature was 500°C.

[0091] 3) Solution preparation and sample processing

[0092] ① Preparation of reference substance stock solution: Accurately weigh 1 mg of TMA-HCl (the following reference substances were purchased from Beijing Manhag Biotechnology Co., Ltd., CAS No. 593-81-7) and TMAO (CAS No. 1184-78-7) reference substances, transfer them to volumetric flasks, dissolve them with 50% methanol aqueous solution, make up to volume and shake well to obtain 1 mg / mL reference substance stock solutions, and store them in a -20°C refrigerator.

[0093] ② Preparation of internal standard reference substance stock solution: Accurately weigh 1 mg of D9-TMA-HCl (CAS No. 18856-86-5) and D9-TMAO (CAS No. 1161070-49-0) reference substances, transfer them to volumetric flasks, dissolve them with 50% methanol aqueous solution, make up to volume and shake well to obtain 1 mg / mL internal standard reference substance stock solutions, and store them in a -20°C refrigerator.

[0094] ③ Preparation of standard curve working solution: Take the TMA-HCl and TMAO reference substance stock solutions (concentration of 1 mg / mL) prepared in step ①, dilute them with 50% methanol aqueous solution, and dilute the TMA-HCl and TMAO reference substances to final concentrations of 2.000 μg / mL and 100.000 μg / mL, respectively. After stepwise dilution, use them as standard curve working solutions.

[0095] ④ Preparation of internal standard working solution: Take the D9-TMA-HCl and D9-TMAO internal standard reference substance stock solutions (concentration of 1 mg / mL) prepared in step ②, and dilute them with acetonitrile to internal standard working solutions with final concentrations of D9-TMA-HCl and D9-TMAO of 50.000 ng / mL and 800.000 ng / mL, respectively.

[0096] ⑤ Take 50 μL of the serum sample to be tested, add 150 μL of the 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 mix, and inject 5 μL; the results are shown in Table 2 and Figure 2 shown.

[0097] Table 2 Detection results of TMAO and TMA content in serum of mice in each group

[0098]

[0099]

[0100] Note: Compared with the control group, #### Represents P < 0.0001; compared with the model group, * represents P < 0.05, ** represents P < 0.01, **** Represents P < 0.0001.

[0101] From Table 2 and Figure 2 It can be seen that compared with the control group, the serum TMAO level of the model group mice given choline feed was significantly increased; compared with the model group, the TMAO level of the low and high dose groups of Eucommia leaf water extract was significantly reduced (P < 0.0001); compared with the model group, the serum TMA level of the low and high dose groups of Eucommia leaf water extract was also significantly reduced (P < 0.05), indicating that Eucommia leaf water extract can reduce the TMAO and TMA levels of choline-treated mice.

[0102] Example 3: Investigating the effect of Eucommia ulmoides leaf aqueous extract on intestinal flora composition based on animal models

[0103] This example detects and analyzes the effect of the Eucommia leaf water extract prepared in Example 1 on the composition of intestinal flora based on a mouse model. The test sample used is the cecal content sample collected in Example 2. The specific experimental method and results are as follows:

[0104] 1. Sequencing:

[0105] 1) Extraction of total DNA from microbiome:

[0106] The total DNA of the test samples was extracted using the DNeasy PowerSoil Kit of Mo Bio / QIAGEN, and the extracted DNA was detected; the absorbance of DNA was measured at 260 nm and 280 nm using a fluorescence spectrophotometer (Quantifluor-ST Huorometer, Promega, E6090) and a DNA quantification kit Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen, P7589), the concentration of DNA was detected, and the quality of DNA was detected by 1% agarose gel electrophoresis; the concentration of DNA solution was adjusted, the DNA working solution was stored at 4°C, and the storage solution was stored at -20°C.

[0107] 2) PCR amplification of target fragment:

[0108] First, the variable region of the 16S rRNA gene was amplified, and a PCR pre-experiment was performed on the specific V region of the sample DNA; large-scale PCR amplification (Pyrobest DNA Polymerase, TaKaRa, DR500A);

[0109] The primer sequences used to amplify 16S rRNA are:

[0110] 338F:ACTCCTACGGGAGGCAGCA(SEQ ID NO:1);

[0111] 806R: GGACTACHVGGGTWTCTAAT (SEQ ID NO: 2);

[0112] Among them, H represents A, C or T, V represents A, C or G, and W represents A or T.

[0113] PCR amplification uses Pfu high-fidelity DNA polymerase from Quanshijin Company, and the amplification conditions are:

[0114] ① Initial denaturation: 98°C, duration 3 minutes, this step is usually used to separate the DNA double strands;

[0115] ② Cycle steps:

[0116] Denaturation: 98°C, 30 seconds;

[0117] Annealing: 52°C, 30 seconds (at this temperature, the primer binds to the template);

[0118] Extension: 72°C, 45 seconds (DNA polymerase synthesizes new DNA chains);

[0119] This cycle will be repeated 25 times, that is, 25 cycles will be performed;

[0120] ③Final extension: After completing 25 cycles, a final extension at 72°C for 5 minutes was performed to ensure that all DNA chains were fully extended.

[0121] At the same time, a negative control (ultrapure water) is set up. The negative control can detect microbial contamination such as the environment and reagents. Any sample group with bands amplified by the negative control cannot be used for subsequent experiments.

[0122] 3) Purification and recovery of amplified products by magnetic beads:

[0123] Add 0.8 times the volume of magnetic beads (Vazyme VAHTSTM DNAClean Beads) to 25 μL of the PCR product obtained by amplification in step 2), shake and fully suspend, adsorb on a magnetic rack for 5 minutes, and carefully aspirate the supernatant with a pipette; then add 20 μL of 0.8 times the volume of magnetic bead washing solution, shake and fully suspend, place on a magnetic rack for adsorption for 5 minutes, and carefully aspirate the supernatant; add 200 μL of 80% ethanol, place on the magnetic rack in reverse so that the magnetic beads are adsorbed to the other side of the PCR tube, and aspirate the supernatant after sufficient adsorption; let it stand at room temperature for 5 minutes until the alcohol evaporates completely and cracks appear on the magnetic beads; add 25 μL of ElutionBuffer for elution; place the PCR tube on the adsorption rack for 5 minutes, fully adsorb, and remove the supernatant to a clean 1.5 mL centrifuge tube for storage.

[0124] 4) Fluorescence quantification of amplified products:

[0125] The PCR amplification products were quantified by fluorescence, the fluorescence reagent was Quant-iT PicoGreen dsDNAAssay Kit, and the quantitative instrument was Microplate reader (BioTek, FLx800). 5) Sequencing library preparation:

[0126] Illumina's TruSeq Nano DNA LT Library Prep Kit was used to prepare the sequencing library. First, the amplified products were repaired at the end of the sequence. The protruding bases at the 5' end of the DNA sequence were removed by End Repair Mix 2 in the kit, and a phosphate group was added to fill the missing bases at the 3' end. A base was added to the 3' end of the DNA sequence to prevent the DNA fragment from self-ligating, and to ensure that the target sequence could be connected to the sequencing adapter (there is a protruding T base at the 3' end of the sequencing adapter). A sequencing adapter containing a library-specific tag (i.e., Index sequence) was added to the 5' end of the sequence so that the DNA molecule could be fixed on the Flow Cell. BECKMAN AMPure XP Beads was used to remove the self-ligated fragments of the adapter through magnetic bead screening, and the library system after adding the adapter was purified. The DNA fragments connected to the adapter were PCR amplified to enrich the sequencing library template, and the library enrichment product was purified again using BECKMAN AMPure XP Beads. The library was finally selected and purified by 2% agarose gel electrophoresis.

[0127] 6) Perform high-throughput sequencing on the machine:

[0128] Before sequencing, the library needs to be quality checked on the Agilent Bioanalyzer using the Agilent High Sensitivity DNA Kit. A qualified library has only a single peak and no connectors. Afterwards, the library is quantified on the Promega QuantiFluor fluorescence quantification system using the Quant-iT PicoGreen dsDNA Assay Kit, and the concentration of a qualified library should be above 2 nM. After gradient dilution of each qualified sequencing library (Index sequence cannot be repeated), mix them in the corresponding proportion according to the required sequencing amount, and denature them into single strands with NaOH for sequencing.

[0129] 2. Data Analysis:

[0130] 1) First, conduct a preliminary screening of the original data from high-throughput sequencing based on sequence quality; retest and supplement the problematic samples.

[0131] 2) The original sequences that have passed the initial quality screening are divided into libraries and samples according to the index and barcode information, and the barcode sequences are removed.

[0132] 3) Perform sequence denoising or OTU clustering according to the QIIME2 dada2 analysis process or the Vsearch software analysis process.

[0133] 4) Display the specific composition of each sample (group) at different species taxonomic levels to understand the overall overview.

[0134] 5) According to the distribution of ASV / OTU in different samples, the Alpha diversity level of each sample is evaluated, and the sparseness curve is used to reflect whether the sequencing depth is appropriate.

[0135] 6) At the ASV / OTU level, the distance matrix of each sample was calculated, and the beta diversity differences and difference significance between different samples (groups) were measured through a variety of unsupervised sorting and clustering methods combined with corresponding statistical test methods.

[0136] 7) At the level of species taxonomic composition, various unsupervised and supervised sorting, clustering and modeling methods are used, combined with corresponding statistical test methods, to further measure the differences in species abundance composition between different samples (groups) and try to find marker species.

[0137] 8) According to the composition distribution of species in each sample, construct an association network, calculate the topological index, and try to find the key species.

[0138] 9) Based on the sequencing results, the bacterial metabolic function of the sample can be predicted, the differential pathways can be found, and the species composition of specific pathways can be obtained; the results obtained are as follows: Figures 3 to 10 shown.

[0139] Depend on Figure 3 and Figure 4 It can be seen that α diversity analysis showed that compared with the model group, the α diversity of the intestinal flora of mice increased after intervention with the aqueous extract of Eucommia leaves, and the Good's Coverage index and Pielou_e index were significantly increased (* represents P<0.05, ** represents P<0.01, compared with the model group), indicating that the aqueous extract of Eucommia leaves increased the richness and diversity of the intestinal flora community; β polymorphism compared the composition of different intestinal microbial communities, and the NMDS analysis based on the Bray-Curtis distance matrix showed that the flora structure of mice fed with high choline changed significantly after intervention with the aqueous extract of Eucommia leaves.

[0140] Depend on Figure 5 and Figure 6It can be seen that the present embodiment uses LEfSe to analyze the flora with significant differences between different groups, and by setting the linear discriminant analysis (LDA) numerical value to evaluate the influence size of the significantly different flora, the LDA threshold is set to 3, and the LDA value distribution bar graph and cluster tree diagram show that Gemmatimonadetes (Gemmamonadetes) and Sphingomonadales (Sphingomonadales) are the main flora of the model group, and the levels from order to genus are significantly increased. Compared with the model group, the LDA diagram shows that Staphylococcaceae (Staphylococcus family) is the dominant flora of the low-dose Eucommia water extract group, and the levels from family to genus are significantly increased. The cluster tree diagrams of Actinomycetales (Actinomycetales) and Corynebacterium (Corynebacterium) in the high-dose Eucommia water extract group reflect that they have increased significantly from class to genus levels.

[0141] Depend on Figure 7 and 8It can be seen that species abundance clustering showed that at the phylum level, the main dominant bacteria in the intestinal flora of mice in the control group were Firmicutes and Actinobacteria, accounting for 69.67% and 22.17% of the total intestinal bacteria, respectively, followed by Verrucomicrobia, Bacteroidetes and Proteobacteria, accounting for 5.10%, 2.17% and 0.74%, respectively. Among them, related studies have found a causal relationship between Actinobacteria and Proteobacteria and increased TMAO (references Huang Y, Zhang J, Zhang Y, et al. Red meat intake, faecal microbiome, serum trimethylamine N-oxide and hepatic steatosis among Chinese adults. Liver Int. 2024; 44(5): 1142-1153. and Fu BC, Hullar MAJ, Randolph TW, et al. Associations of plasma trimethylamine N-oxide, choline, carnitine, and betaine with inflammatory and cardiometabolic risk biomarkers and the fecal microbiome in the Multiethnic Cohort Adiposity Phenotype Study. Am J Clin Nutr. 2020; 111(6): 1226-1234.). After feeding with high choline diet, the abundance of Actinobacteria and Proteobacteria in the intestinal flora of mice increased significantly (accounting for 32.13% and 1.86%). After administration of Eucommia leaf extract, compared with the model group, the abundance of Proteobacteria in the low-dose Eucommia water extract group and the Actinobacteria in the high-dose Eucommia water extract group decreased (accounting for 0.59% and 21.60%, respectively). Therefore, Eucommia leaf water extract may reduce the production of TMAO by reducing the abundance of Actinobacteria and Proteobacteria.

[0142] Depend on Fig. 9 and Fig.10It can be seen that at the genus level, the main intestinal bacterial genera in the control group are: Allobaculum, Corynebacterium, Adlercreutzia, Ruminococcus, Desulfovibrio, Akkermansia, Prevotella, Oscillospira, Lactobacillus, and Bacteroides. Among them, related studies have found a causal relationship between Akkermansia, Oscillospira and Ruminococcus, which are negatively correlated with increased TMAO (references Wang X, Li X, Dong Y. Vitamin D Decreases Plasma Trimethylamine-N-oxide Level in Mice by Regulating Gut Microbiota. Biomed Res Int. 2020; 2020: 9896743. and Gao X, Zhang H, Li K, et al. Sandalwood seed oil improves insulin sensitivity in high-fat / high-sucrose diet-fed rats associated with altered intestinal microbiota and its metabolites. Food Funct. 2021; 12(20): 9739-9749.). Compared with the control group, the abundance of Akkermansia, Oscillospira and Ruminococcus decreased after feeding with a high choline diet (accounting for 0.10%, 0.14% and 0.57%, respectively). Compared with the model group, the abundance of Akkermansia, Oscillospira and Ruminococcus in the low-dose and high-dose Eucommia water extract groups increased. This shows that the administration of Eucommia leaf water extract can regulate the abundance of intestinal flora, increase the abundance of Akkermansia, Oscillospira and Ruminococcus, and inhibit the production of TMAO.

[0143] In summary, the present invention experimentally confirms the effect of Eucommia leaf water extract in reducing TMAO / TMA levels, and regulates intestinal flora through Eucommia leaf water extract, thereby reducing the production of TMAO and / or TMA, providing a new method and idea for the subsequent treatment of TMAO-related diseases.

[0144] 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 an aqueous extract of eucommia leaves in preparing products with reduced TMAO and / or TMA content.

2. Application of Eucommia ulmoides leaf water extract in the preparation of products for regulating intestinal flora composition.

3. The use according to claim 2, characterized in that: The regulating the intestinal flora composition includes reducing the abundance of Actinobacteria and Proteobacteria; And / or, regulating the intestinal flora composition includes increasing the abundance of Akkermansia, Oscillospira and Ruminococcus.

4. A method for preparing an aqueous extract of Eucommia ulmoides leaves, characterized in that: The preparation method comprises the following steps: The dried Eucommia ulmoides leaves are mixed with water for the first water extraction, and a filtrate and a filter residue are obtained after filtering; the filter residue is mixed with water for the second water extraction, and the filtrate is collected and combined with the filtrate obtained from the first water extraction, and finally concentrated and dried.

5. The preparation method according to claim 4, characterized in that: The mixing mass ratio of the dried Eucommia ulmoides leaves and water is 1:(8-12); Preferably, the mixing mass ratio of the filter residue to water is 1:(8-12).

6. The preparation method according to claim 4, characterized in that: The extraction temperature of the first water extraction and the second water extraction is 80°C to 100°C; Preferably, the extraction time of the first water extraction and the second water extraction is 1 to 2 hours.

7. A product with reduced TMAO and / or TMA content, characterized in that The product comprises the eucommia leaf water extract prepared by the preparation method as described in any one of claims 4 to 6.

8. A product for regulating the composition of intestinal flora, characterized in that: The product comprises the eucommia leaf water extract prepared by the preparation method as described in any one of claims 4 to 6.

9. The product according to claim 7 or 8, characterized in that The product also includes pharmaceutically acceptable excipients.

10. The product according to claim 9, characterized in that The pharmaceutically acceptable auxiliary materials include at least one of a binder, a disintegrant, a lubricant, a coating agent, a suspending agent, a thickening agent and a surfactant.

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