Application of sophora flower extract in preparation of preparations for intervening metabolic diseases

Abstract

The invention discloses an application of a sophora flower extract in preparation of a preparation for intervening metabolic diseases. According to the application, the preparation for intervening metabolic diseases is prepared from the sophora flowers with homology of medicine and food, it is proved that TMAO in plasma can be effectively removed, the preparation has nutrition, health care and treatment effects on patients, and the preparation is low in side effect after use, high in safety and good in compliance during use.

Description

Technical Field

[0001] The present application relates to the field of pharmaceutical technology, and in particular, to the use of a Sophora japonica flower extract in the preparation of a preparation for intervening in metabolic diseases. Background Art

[0002] The intestinal flora in the human body can decompose dietary choline or betaine and L-carnitine to form trimethylamine (TMA), which further enters the liver through the portal vein circulation in the intestine and generates trimethylamine N-Oxide (TMAO) under the action of flavin-dependent monooxygenase (FMOs) in the liver. The human kidney can eliminate TMAO in the blood. Generally speaking, the concentration of TMAO in the serum of healthy people remains at a normal level, but in many metabolic disease states, TMAO accumulates in the human body and its serum concentration increases significantly. Many literatures have confirmed that TMAO is a diagnostic marker for a variety of metabolic diseases. Furthermore, high levels of TMAO also promote the occurrence and development of cardiovascular events. Therefore, how to reduce the level of TMAO in the circulation and develop related drugs is one of the intervention methods for metabolic diseases. At present, the main methods to reduce the level of TMAO in the body include dietary adjustment, drug treatment and regulation of the intestinal microbiome. However, each of these methods has certain limitations. For example, dietary adjustments require a high degree of personal self-discipline, and the effects vary from person to person; drug treatments may be accompanied by side effects and microbial imbalances; and the intestinal microbiome is the only source of circulating TMAO, so targeting the intestinal flora to downregulate host circulating TMAO levels has huge translational potential. Summary of the invention

[0003] In view of the shortcomings of the existing methods, the present application proposes the use of a Sophora japonica flower extract in the preparation of a preparation for intervention in metabolic diseases, so as to solve the technical problems that the related technologies have significant side effects and unclear intervention effects.

[0004] The present application provides an application of a Sophora japonica flower extract in the preparation of a preparation for intervening in metabolic diseases.

[0005] Furthermore, the Sophora japonica flower extract comprises genistein.

[0006] Optionally, the metabolic disease comprises a metabolic disease of dyslipidemia.

[0007] Further optionally, the metabolic disease of abnormal blood lipid metabolism includes hyperlipidemia.

[0008] Furthermore, the Sophora japonica flower extract intervenes in the development of metabolic diseases by reducing the level of trimethylamine-N-oxide in serum.

[0009] Furthermore, the Sophora japonica flower extract intervenes in the development of metabolic diseases by reducing the abundance of intestinal pathogenic bacteria.

[0010] Optionally, the enteric pathogenic bacteria include Escherichia coli.

[0011] Alternatively, the preparation is administered via the gastrointestinal tract.

[0012] The beneficial technical effects brought about by the technical solution provided by the embodiment of the present application include: (1) The application of the present application uses Sophora japonica flowers, which are both medicinal and edible, to prepare a preparation for intervention in metabolic diseases. It has not only been proven to be able to effectively remove TMAO from plasma, but the preparation also has nutritional, health and therapeutic effects on patients, has low side effects after use, is highly safe, and has good compliance during use.

[0013] (2) The application of this application can further affect the abundance of intestinal pathogenic bacteria, providing practical evidence for the treatment of metabolic diseases related to TMAO levels through intervention of the intestinal microenvironment.

[0014] Additional aspects and advantages of the present application will be partially given in the following description, which will become apparent from the following description, or will be understood through the practice of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The above and / or additional aspects and advantages of the present application will become apparent and easily understood from the following description of the embodiments in conjunction with the accompanying drawings, in which: Figure 1 A technical roadmap for application verification experiments for this application; Figure 2 Based on Figure 1 The results of serum TMAO level test of each group of mice before and after intervention with Sophora japonica extract were obtained; Figure 3 Based on Figure 1 The PcoA graphs of the changes in intestinal flora of each group of mice before (left) and after (right) intervention with Sophora japonica extracts were obtained; Figure 4 Based on Figure 1 The obtained TMAO level and RDA graph of intestinal flora in the high-fat model group (HFD) and intervention group (GEN); Figure 5 Based on Figure 1 The feces of mice in the normal control group (NFD), high-fat model group (HFD) and intervention group (GEN) were obtained. CutC and CnA Gene abundance quantification results; Figure 6 Based on Figure 1 The feces of mice in the normal control group (NFD), high-fat model group (HFD) and intervention group (GEN) were obtained. CnA Results of Spearman correlation analysis between gene abundance and serum TMAO levels; Figure 7 Based on Figure 1 The obtained results of incubation of mouse feces with d9-choline and d3-L-carnitine in the normal control group (NFD), high-fat model group (HFD) and intervention group (GEN); Figure 8 Based on the whole genome analysis of 1520 bacterial strains CnA List of genes specifically distributed in three bacterial genera within the phylum Proteobacteria; Fig. 9 Based on Figure 1 The obtained LEfSe graphs of intestinal flora in the normal control group (NFD), high-fat model group (HFD), and intervention group (GEN); Fig.10 Based on Figure 1 The abundance of Escherichia coli in the intestines of each group of mice was quantitatively determined using the qPCR method. DETAILED DESCRIPTION

[0016] The embodiments of the present application are described below in conjunction with the drawings in the present application. It should be understood that the implementation methods described below in conjunction with the drawings are exemplary descriptions for explaining the technical solutions of the embodiments of the present application and do not constitute a limitation on the technical solutions of the embodiments of the present application.

[0017] Those skilled in the art will appreciate that, unless expressly stated, the "said" and "the" used herein may also include plural forms. It should be further understood that the term "including" used in the specification of the present application refers to the presence of the features, integers, steps, operations, elements and / or components, but does not exclude the implementation of other features, information, data, steps, operations, elements, components and / or combinations thereof supported by the technical field. The term "and / or" used herein refers to at least one of the items defined by the term, for example, "A and / or B" may be implemented as "A", or as "B", or as "A and B".

[0018] In order to make the objectives, technical solutions and advantages of the present application clearer, the implementation methods of the present application will be further described in detail below with reference to the accompanying drawings.

[0019] First, the basic terms involved in this application are explained: Sophora japonica extract includes genistein. Genistein, also known as genistein, genistein or genistein isoflavone, is a natural flavonoid compound extracted from Pterocarpus or Leguminosae plants (such as genistein, soybean, clover, Sophora japonica, Sophora japonica, broad bean root, etc.), which has a variety of biological activities and pharmacological effects, such as antioxidant effect: Genistein is a powerful antioxidant that can scavenge free radicals, reduce oxidative stress damage to cells, and delay the aging process. It can also effectively lower cholesterol, inhibit the oxidation of low-density lipoprotein cholesterol (LDL), and keep LDL in the blood at a low level, improve the abnormal peroxidation state in the body caused by high fat, and alleviate peroxidation damage to the body; cardiovascular protection effect: Genistein can protect the cardiovascular system by improving endothelial function, lowering blood pressure, reducing platelet aggregation and other mechanisms. It can also upregulate LDL receptors and increase LDL receptor activity, thereby promoting cholesterol clearance; antibacterial effect: Genistein has an inhibitory effect on a variety of pathogens and can enhance the body's ability to resist infection.

[0020] Metabolic disease refers to a disease caused by a disorder in the body's metabolic process, which leads to the accumulation or deficiency of certain metabolic substances such as amino acids, organic acids, fatty acids, sugars and hormones.

[0021] In this example, an animal model of hyperlipidemia was constructed using mice, and the intervention group was fed with genistein, and the positive control of the intervention group was fed with normal saline.

[0022] refer to Figure 1 In the examples of this application, a high-fat and high-cholesterol (18% fat, 1.2% cholesterol) hyperlipidemia mouse model was constructed to observe the effect of genistein on plasma TMAO in the hyperlipidemia mouse model. All HFD mice were randomly divided into four groups, with eight mice in each group. Experimental groups: normal control group (ordinary feed, NFD group), model group (high-fat and high-cholesterol feed, HFD group), model intervention group (100 mg / kg genistein was gavaged on the basis of high-fat and high-cholesterol feed, GEN group).

[0023] After 12 weeks of intervention according to the above embodiment, serum was extracted from the blood of mice, and 50 μL of each sample was taken to detect the content of TMAO.

[0024] Take 50ul serum, add 150ul d9-TMAO internal standard (50% methanol), mix, centrifuge for 10 minutes (13.3×1000 rpm, 4℃); take 50ul supernatant and add 100ul mobile phase A to mix, then transfer the mixture to the injection bottle for determination; mobile phase preparation: phase A: 0.1% ammonium fluoride + 0.1% formic acid + water, phase B: 0.1% ammonium fluoride + 0.1% formic acid + methanol. Remove bubbles before mobile phase is put on the machine: after mobile phase AB is prepared, place it in an ultrasonic machine for 5min to help remove bubbles. Mass spectrometer detection. Chromatographic column: Phenomenex Luna Omega 3um Polar C18, 100 x 3.0 mm. Mobile phase: phase A: 2 mM ammonium fluoride + 0.1% formic acid aqueous solution; phase B: 2 mM ammonium fluoride + 0.1% formic acid methanol solution. Mass spectrometry parameters: APCI source, spray voltage: 5500 V; temperature: 400°C; nebulizer gas: 60 psi; auxiliary heating gas: 55 psi; curtain gas: 25 psi; CAD: 10.

[0025] After 12 weeks (3 months) of the above intervention, the plasma of mice in each group was collected to detect the TMAO content. Figure 2 The figure shows the effect of genistein on the TMAO content in mouse serum. It can be seen from the figure that the TMAO content of mice in the model group is the highest. After using genistein, it has a significant effect on the TMAO content, and the content decreases significantly, so it has a significant effect on the removal of TMAO.

[0026] Further analyze the test data.

[0027] refer to Figure 3 The PcoA graph was created using the data on changes in intestinal flora in each group of mice before (left) and after (right) intervention with Sophora japonica extract.

[0028] PCoA, or principal coordinate analysis, is a dimensionality reduction technique for multidimensional data that aims to explore the relationship between samples in high-dimensional space. PCoA has a wide range of applications in many fields, including but not limited to: Ecology: used to explore how environmental factors or experimental conditions affect the composition of samples, and whether the microbial community structure of samples under different environments or conditions is different; Genetics: In genetic studies, PCoA can be used to reveal similarities or differences between samples or individuals; Metagenomics: Displays similarities and differences between samples, helping to identify changes in microbial community structure at different time points or between different treatment groups.

[0029] The functions and features of PCoA graphs include: Data dimensionality reduction: PCoA graphs can reduce high-dimensional data to low-dimensional space, which is convenient for data visualization and interpretation. Sample relationship display: In the PCoA graph, each point represents a sample, and the distance between points reflects the similarity or difference between samples. Points with closer distances indicate higher similarity, while points with farther distances indicate lower similarity. Group differentiation: If the data contains sample grouping information, the PCoA graph can help identify whether different groups are clearly separated in multidimensional space, thereby understanding the differences between different groups.

[0030] Figure 3 The results showed that before the addition of genistein for intervention, the intestinal flora of mice in each group had a high overlap, suggesting that there was no significant difference in the intestinal flora of each group of mice before feeding with a normal diet (NFD) and feeding with high fat and high cholesterol (NFD and GEN); after 12 weeks of genistein intervention, the clustering of the intestinal flora of mice showed significant differences from the high-fat model group of intervention (between HFD and GEN), but also showed significant differences from the normal control group (NFD).

[0031] refer to Figure 4 , combined with the data obtained in the above process, we further prepared the RDA graphs of TMAO levels and intestinal flora in the high-fat model group (HFD) and the intervention group (GEN).

[0032] RDA diagram, or Redundancy Analysis diagram, is a ranking method based on the combination of Correspondence Analysis and multiple regression analysis. RDA analysis is a direct extension of multiple regression, which simulates the impact of the explanatory matrix X on the response matrix Y, rather than a single response variable. In RDA analysis, the contribution of the explanatory variables to the response variables is revealed by calculating the regression relationship between the explanatory variables (usually environmental factors) and the response variables (usually species data). RDA diagrams visualize the results of RDA analysis, usually using two-dimensional or three-dimensional coordinates to represent the relationship between samples, explanatory variables, and response variables. In ecology, RDA diagrams can be used to analyze the impact of environmental variables (such as climate and soil) on the distribution of species communities.

[0033] Figure 4 In the experiment, the environmental vectors were TMAO and TMA levels. Although there was some overlap in the intestinal flora composition between the HED group and the GEN group, the similarity was generally low. TMAO and TMA showed a certain correlation with the composition of the intestinal flora, and the correlation trend between TMAO and TMA and the intestinal flora was opposite, which was consistent with the Figure 2The results shown correspond to those in the previous study, suggesting that the intervention measures of the GEN group can reduce the serum TMAO level of mice in the HFD group, among which the intestinal flora may be the key factor.

[0034] refer to Figures 5 to 8 TMA is a precursor of TMAO, which is mainly encoded by the intestinal flora and breaks down the enzyme complex of choline or carnitine in the diet ( CutC / CutD and CntA / CntB ) is generated. Among them, the enzyme gene mainly involved in the cleavage of choline is CutC / CutD , the enzyme gene mainly involved in the cleavage of carnitine is the enzyme gene for metabolizing choline CntA / CntB Here, we adopt CutC and AHr The primers were used to quantify the CutC and CnA Gene abundance. Figure 5 Compared with NFD, the feces of HFD mice showed CutC Gene or CnA The abundance of genes increased significantly, but the feces of mice in the GEN group CutC There was no significant difference in gene abundance between the HFD group and the control group. CnA The gene abundance was significantly lower than that of the HFD group. AHr Whether the abundance is correlated with serum TMAO levels, Spearman correlation analysis (such as Figure 6 As shown) found AHr The abundance of choline was significantly positively correlated with the serum TMAO level of mice. The feces of mice in the NFD, HFD, and GEN groups were collected after nearly 90 days of intervention, and in vitro incubation experiments were performed with d9-choline and d3-L-carnitine, respectively. Figure 7 The results showed that after 16 hours of in vitro incubation with d9-choline chloride, the d9-TMA content in the microbiota of the HFD group mice was significantly increased compared with the NFD group, while there was no significant difference between the GEN group and the HFD group. This result is also consistent with the previous results that the HFD group mice CutC The gene abundance was significantly higher than that in the NFD group. In the in vitro incubation experiment of d3-L-carnitine in the microbiota, the d3-TMA content produced by the microbiota of the HFD group mice was significantly higher than that of the NFD group; the d3-TMA content produced by the microbiota of the GEN group mice was significantly lower than that of the HFD group, which was consistent with the AHr The gene abundance is significantly reduced. CutC and AHr In which bacteria are the genes distributed? Reference was made to the genome sequence of 1520 bacterial strains reported by Zou et al., 2019. CutC and CnA Gene quantitative analysis revealed CutC The gene is widely distributed in 90% of bacteria, and AHr The genes are specifically distributed in three genera in the Proteobacteria: Escherichia coli ( Escherichia coli ), Klebsiella pneumoniae ( Klebsiella pneumoniae ) and Citrobacter lucidum ( Citrobacter portucalensis ) (As shown in Figure 8) 。

[0035] Furthermore, the data obtained from the above process were used to generate LEfSe diagrams of the intestinal flora in the normal control group (NFD), high-fat model group (HFD), and intervention group (GEN).

[0036] The LEfSe (Linear Discriminant Analysis Effect Size) graph is an effective tool for displaying the difference in flora between different groups in a microbial community. It combines linear discriminant analysis (LDA) and statistical tests to graphically display species with significant differences between groups (i.e., Biomarkers). The LEfSe graph includes an LDA value distribution bar plot. The LDA value distribution bar plot displays significantly different species whose LDA score is greater than the preset threshold. The color and length of the bar represent the size of the group and LDA score, i.e., the degree of influence of the different species. The horizontal axis represents the absolute value of the LDA score, and only species greater than the preset threshold will be displayed in the graph.

[0037] Fig. 9 The enriched intestinal flora in the HFD and GEN groups included Escherichia coli ( Escherichia coli ), further confirmed in the LDA value distribution histogram that Escherichia coli ( Escherichia coli ) is one of the species with significant differences.

[0038] In order to further confirm the abundance of E. coli in each group, the abundance of E. coli in the intestine of each group of mice was quantitatively measured by qPCR. The results are shown in Fig.10 As shown in the figure, the abundance of E. coli in the HFD group increased significantly, and after intervention with genistein, the abundance value of E. coli in the GEN group dropped to near normal levels.

[0039] E. coli is widely present in the intestines of warm-blooded animals and is one of the normal flora of the intestine. In humans, E. coli is a conditional pathogen. Beneficial effects include: as part of the normal intestinal flora, E. coli helps maintain intestinal health and function; participates in the synthesis of vitamin K and certain B vitamins, helps digestion and absorption of nutrients; participates in the regulation of the immune system, helps prevent the invasion of pathogenic microorganisms; E. coli in the normal intestine can also inhibit the growth of harmful bacteria through competitive exclusion mechanisms and maintain the balance of intestinal flora. Pathogenic effects include: when E. coli enters the body through food, water or other routes and colonizes in places where it should not exist (such as the urethra, bladder or blood), or when pathogenic E. coli enters the intestine, it will cause various diseases. E. coli can cause a variety of diseases, including urinary tract infections, sepsis and other systemic infections.

[0040] E. coli can decompose choline, which is a way for it to obtain carbon sources. In the process of decomposing choline, E. coli will produce TMA, which will then be converted into TMAO in the liver. A high-fat diet will indirectly promote the decomposition of choline by E. coli, leading to an increase in the level of TMAO in the blood. At present, in order to control the level of TMAO in the blood, one way is to reduce the intake of high-fat foods by controlling the diet and control the excessive proliferation of E. coli. Another way is to use drugs to increase mitochondrial activity and increase the consumption of TMAO. However, there is no clear practice to control the level of TMAO by controlling the abundance of E. coli. The application of this application provides evidence for this intervention idea.

[0041] In summary, the Sophora japonica extract has the prospect of being used in the preparation of preparations for intervention in metabolic diseases. Furthermore, the Sophora japonica extract at least includes genistein. The metabolic diseases involved include metabolic diseases with abnormal blood lipid metabolism, such as hyperlipidemia. The Sophora japonica extract intervenes in the development of metabolic diseases by reducing the level of TMAO in serum, and this intervention may be achieved by reducing the abundance of intestinal pathogens, and the intestinal pathogens involved include Escherichia coli. The preparation involved can exert its effect through gastrointestinal administration, such as oral or intragastric administration. Furthermore, the Sophora japonica extract and a pharmaceutically acceptable carrier are made into a specific form of preparation, such as an excipient, a sweetener, etc. The application of the present application uses Sophora japonica, which is a medicine and food, to prepare a preparation for intervention in metabolic diseases. It has not only been proven that TMAO in plasma can be effectively removed, but the preparation has both nutritional, health care and therapeutic effects on patients, low side effects after use, high safety, and good compliance during use.

[0042] Those skilled in the art will appreciate that the various operations, methods, steps, measures, and schemes in the processes discussed in this application may be alternated, changed, combined, or deleted. Furthermore, other steps, measures, and schemes in the various operations, methods, and processes discussed in this application may also be alternated, changed, rearranged, decomposed, combined, or deleted. Furthermore, steps, measures, and schemes in the related art that are similar to those disclosed in this application may also be alternated, changed, rearranged, decomposed, combined, or deleted.

[0043] The terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of the features. In the description of this application, unless otherwise specified, "plurality" means two or more.

[0044] In the description of this specification, specific features, structures, materials or characteristics may be combined in an appropriate manner in any one or more embodiments or examples.

[0045] The above is only a partial implementation method of the present application. It should be pointed out that for ordinary technicians in this technical field, without departing from the technical concept of the scheme of the present application, other similar implementation methods based on the technical ideas of the present application are also within the protection scope of the embodiments of the present application.

Claims

1. Application of Sophora japonica flower extract in the preparation of preparations for intervention of metabolic diseases.

2. The use according to claim 1, characterized in that The Sophora japonica flower extract includes genistein.

3. The use according to claim 1, characterized in that The metabolic diseases include metabolic diseases of abnormal blood lipid metabolism.

4. The use according to claim 3, characterized in that The metabolic diseases of abnormal blood lipid metabolism include hyperlipidemia.

5. The use according to claim 1, characterized in that The Sophora japonica flower extract intervenes in the development of metabolic diseases by reducing the level of trimethylamine-N-oxide in serum.

6. The use according to claim 1, characterized in that The Sophora japonica flower extract intervenes in the development of metabolic diseases by reducing the abundance of intestinal pathogenic bacteria.

7. The use according to claim 6, characterized in that The intestinal pathogenic bacteria include Escherichia coli.

8. The use according to claim 1, characterized in that The preparation is effective by gastrointestinal administration.

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