CN117959305A - Application of xanthine in preparation of medicines for preventing obesity or regulating intestinal tract bacteria activity - Google Patents

Abstract

The invention discloses an application of xanthine in preparing medicines for preventing obesity or regulating intestinal tract bacteria activity, wherein the xanthine can prevent the rapid increase of the weight of mice and epididymal fat caused by high-fat diet, has the potential of being an ideal candidate compound for preventing obesity, and provides a new solving way for controlling overweight and obesity prevalence.

Description

Application of xanthine in preparation of medicines for preventing obesity or regulating intestinal tract bacteria activity

Field of the art

The invention belongs to the technical field of medicines, and particularly relates to a novel application of xanthine in preparation of a medicament for preventing obesity or regulating intestinal tract bacteria activity.

(II) background art

Obesity refers to a state in which body fat excessively accumulates due to an imbalance between energy intake and expenditure, and is a major risk factor for non-infectious diseases such as type 2 diabetes, hypertension, dyslipidemia, cardiovascular disease, and some cancers. The World Health Organization (WHO) defined obesity as a major global public health problem in 1997. The diseases, psychological and social problems caused by obesity are increasingly serious, and the problems of obesity are urgently solved, which have certain influence on the economy and development of a plurality of countries.

Enteric bacteria play an important role in the development and progression of obesity, and they can affect weight gain by increasing dietary energy harvesting, promoting fat deposition, possibly modifying locomotor activity, having a central effect on satiety, and inducing systemic inflammation.

The intestinal microbiota of the human body consists of more than 100 trillion individuals of bacteria whose composition varies significantly from individual to individual, where the dietary structure has a large impact on the structure of the intestinal microbiota. In recent years, intestinal microbiota has been considered an important factor in the development and progression of metabolic diseases, including obesity, regulation of energy metabolism, inflammation and the intestinal brain axis. It plays a vital role in the utilization of dietary energy and aids in the synthesis of intestinal peptides, such as GLP-1 and PYY, which are vital to the maintenance of energy balance in the host. Dietary nutrients, such as carbohydrates, fibers, proteins and fats, significantly shape the role of the intestinal microbiota in the development of obesity. For example, high fat intake promotes the absorption of lipopolysaccharide from intestinal microorganisms by the intestinal tract, thereby increasing circulating endotoxin levels, triggering the release of various inflammatory cytokines. However, the interactions between diet, intestinal flora and obesity are complex, and the mechanism of action of intestinal flora in obesity is largely underutilized.

Intestinal flora-mediated endogenous and exogenous metabolism also play a key role in disease progression and drug treatment/toxicity. The bioconversion of primary bile acids to secondary bile acids is mediated primarily by the intestinal flora, which regulates the signal pathway in the host, altering the progression of cancer and liver fibrosis. Intestinal microbial dipeptidyl peptidase 4 (DPP 4) can degrade active glucagon-like peptide-1 (GLP-1) and is therefore identified as a potential hypoglycemic target. Other intestinal microbial metabolic enzymes identified as promising new drug targets include beta-glucuronidase, choline trimethylamine lyase and 3-beta-hydroxysteroid dehydrogenase.

Prevention of obesity mainly includes various methods of controlling diet, exercise, and medicines. While diet and exercise control is important to improve obesity, its effect is not ideal because of its difficulty in sustaining. Drugs remain the primary means of preventing obesity. In recent years, development of a drug for preventing obesity has been a great challenge for technical and social reasons. From the past experience, obese patients are often at high risk of vascular disease and are accompanied by co-morbidity, while many classical drugs targeting the central nervous system cause certain adverse effects of cardiovascular, drug dependence and abuse.

(III) summary of the invention

The invention aims to provide an application of xanthine in preparing medicines for preventing obesity or regulating intestinal bacteria activity, wherein xanthine takes host intestinal bacteria and metabolites thereof as targets, regulates metabolism abnormality, improves glucose tolerance and insulin sensitivity, can prevent rapid increase of weight of mice and epididymal fat caused by high-fat diet, has potential as ideal candidate compounds for preventing obesity, lays solid scientific basis and theoretical basis for developing special medical foods and health care products for preventing obesity or regulating intestinal bacteria, and brings more choices for obese patients in China, thereby reducing harm of obesity-related diseases and improving health level of residents.

The technical scheme adopted by the invention is as follows:

the invention provides an application of xanthine in preparing a medicament for preventing obesity or regulating intestinal tract bacteria activity.

Further, the medicine is a medicine for regulating the ratio of the phylum firmicutes to the phylum bacteroides in the intestinal bacteria.

Further, the drug is a drug enriched in intestinal species including actinomycetes (such as NM07-P-09sp 004793665) and Thick-walled bacteria (Merdicola).

Further, the medicament is a medicament for preventing a rapid increase in body weight and/or epididymal fat weight caused by High Fat Diet (HFD).

Further, the drug is a drug that improves glucose tolerance and insulin sensitivity.

Further, the drug is a drug for regulating host-intestinal bacteria xanthine and bile acid metabolism abnormality.

The xanthine is a host-microorganism symbiotic metabolite, widely exists in tissues, body fluids and intestinal flora of animals and human bodies, can be converted into uric acid through xanthine oxidase, has an alkaloid structure which is a bicyclic compound which is formed by combining imidazole pyrimidine, contains four nitrogen atoms in a molecular structure, and has the following structural formula:

compared with the prior art, the invention has the beneficial effects that:

(1) When the high-fat diet and xanthine are simultaneously given to the mice, the weight and epididymal fat of the mice are significantly reduced compared with the mice fed only the high-fat diet, which indicates that the xanthine has a significant preventive effect on the weight gain induced by the high-fat diet.

(2) The xanthine-fed high-fat diet mice of the invention obviously improve the relative abundance of NM07-P-09sp004793665 actinomycetes strains in intestinal tracts, and simultaneously have enrichment effect on Merdicola bacteria in intestinal microbiota of obese mice, which indicates that the xanthine can regulate the activity of intestinal bacteria and has the effect of enriching intestinal bacteria.

(3) The xanthine-treated high fat diet mice of the invention have significantly reduced levels of xanthine, 6-oxopurine, and adenine in the cecum, and xanthine treatment significantly altered the abundance of a variety of primary and secondary bile acids. Xanthine can influence the intestinal metabolic phenotype of high-fat fed mice, and can regulate host-intestinal bacteria bile acid metabolism abnormality, which is probably a key for regulating body weight, and high-concentration xanthine is beneficial to the inhibition of obesity.

The invention lays a solid scientific basis and theoretical foundation for developing special medical food and health care product medicines for preventing obesity or regulating intestinal flora by taking the intestinal bacteria and metabolites thereof as targets.

(IV) description of the drawings

FIG. 1, effect of xanthine on index of high fat diet mice; (A) High Fat Diet (HFD), bifidobacterium longum, xanthine and xanthine oxidase inhibitors intervened in mice (n=8) for 6 weeks. (B) body weight change curve. (C) Weight gain values between the end of diet (8 weeks) and the beginning. (D) liver weight. (E) epididymal White Adipose Tissue (WAT) weight. (F) Plasma glucose concentration after Oral Glucose Tolerance Test (OGTT). (G) area under OGTT curve. (H) serum TC. (I) serum TG. (J) serum LDL-C. (K) serum HDL-C.

FIG. 2, effect of xanthine on mice with a weight gain amplitude of 40% later; (A) High Fat Diet (HFD), bifidobacterium longum, xanthine and xanthine oxidase inhibitors intervened in HFD-induced obese resistant mice (n=8) for 6 weeks. (B) body weight curve. (C) body weight at the end of the intervention. (D) liver weight. (E) epididymal White Adipose Tissue (WAT) weight. (F) fasting blood glucose. (G) Plasma glucose concentration after Oral Glucose Tolerance Test (OGTT). (H) area under OGTT curve.

FIG. 3, effect of xanthine on mice with a weight gain of the first 40%; (A) High Fat Diet (HFD), bifidobacterium longum, xanthine and xanthine oxidase inhibitors intervened in HFD-induced obese mice (n=8) for 6 weeks. (B) body weight curve. (C) body weight at the end of the intervention. (D) liver weight. (E) epididymal White Adipose Tissue (WAT) weight. (F) fasting blood glucose. (G) Plasma glucose concentration after Oral Glucose Tolerance Test (OGTT). (H) area under OGTT curve.

Figure 4, modulation of intestinal microbiota in HFD fed mice by xanthine metabolic pathways (bifidobacterium longum, xanthine and xanthine oxidase inhibitors) (n=8). Phylum (a) and level of intestinal microbiota. The intestinal microbiota at the level of (B). (C) principal component analysis of intestinal strain level in HFD group mice. (D) principal component analysis of intestinal strain level in DIR group mice. (E) principal component analysis of intestinal strain level in DIO group mice.

FIG. 5, intestinal flora microbiota interaction network of HFD fed mice. (A) The fill color of the node represents a classification at the gate level, while the shape of the node represents whether the species appears to vary significantly between different groups in HFD fed mice. (B) The fill color of the nodes reflects the relative abundance ratio of each species in the different groups, and the border color represents the classification at the gate level. The connection indicates that the correlation value between two nodes is greater than 0.6.

Figure 6, intestinal metabolite interaction network of HFD fed mice. The filled color of the nodes reflects the relative abundance ratio of each molecule in the different groups, and the border color indicates whether the compound appears to vary significantly between the different groups in HFD fed mice. The connection indicates that the correlation value between two nodes is greater than 0.6.

(Fifth) detailed description of the invention

The invention will be further described with reference to the following specific examples, but the scope of the invention is not limited thereto:

The C57BL/6J mice of the invention are from Jackson Laboratory, bar Harbor, ME, USA; high fat diet refers to 60% kcal fat, D12492, RESEARCH DIETS inc. Xanthine oxidase inhibitor (topiroxostat) was weighed 3mg before use, dissolved in physiological saline, and fixed to a volume of 10mL, and administered by gavage. Before using, 20.0mg of xanthine is weighed, dissolved in normal saline and fixed to 10mL of volume, and then the xanthine is administrated by stomach irrigation.

Bifidobacterium longum (Bifidobacterium longum, accession number GDMCC No.1.520, available from the microorganism strain collection of Guangdong province) was inoculated into ATYP M2246B medium (available from Shandong Tuo Pu Biotechnology Co., ltd.) and cultured at 37℃and 200rpm for 18-20 hours, and the wet cells were collected and adjusted to a concentration of 5X 10 ⁸ cfu/mL with sterile physiological saline, and administered by gavage.

Example 1 the effect of three interventions on intestinal xanthine metabolism on high fat diet fed mice, obese mice and obese resistant mice was evaluated.

1. High fat diet fed mice

100 Male C57BL/6J mice of 6 weeks of age were fed normal diet for one week for adaptation. The mice were then randomly divided into 4 groups: (a) high fat diet control group (HFD); (B) High fat diet + bifidobacterium longum treatment group (HFD-B), bifidobacterium longum at 0.1mL/10g, once daily; (C) High fat diet + xanthine treatment group (HFD-X), xanthine dose 0.1mL/10g, once daily; (D) High fat diet + xanthine oxidase inhibitor treated group (HFD-XI) with xanthine oxidase inhibitor dosage of 0.1mL/10g once daily. Body weight was recorded once a week, feed consumption was recorded every two days, the body weight change curve is shown in figure 1B, and the body weight gain values before and after 8 weeks of high fat feed feeding are shown in figure 1C. Feces were collected once before and after 8 weeks of feeding with high fat diet.

At the end of week 8, 8 mice were taken from each group and serum was assayed for Total Cholesterol (TC), triglyceride (TG), low density cholesterol (LDL-C), high density cholesterol (HDL-C), respectively, as shown in FIG. 1 for results at H, I, J, K.

At the end of week 8, 8 mice per group were tested for oral glucose tolerance (OGTT), as follows: the feed was removed from each group of mice the evening prior to the experiment, keeping the cage litter clean and free of food residues, ensuring that the mice were fasted for 12 hours without water. Blood glucose was measured in tail vein for 0min, and then 2g/kg of glucose was infused in stomach, and blood glucose was measured with a glucometer at 15, 30, 60, 90, 120min time points after the stomach infusion, and data were recorded, and the results are shown in FIG. 1 at F, G.

At the end of week 8, 8 mice per group were dissected, the liver, epididymal fat, plasma and cecal contents were collected, and the liver and epididymal white fat weights were weighed, respectively, and the results are shown in figure 1 at D, E.

As a result, as shown in fig. 1, xanthine significantly alleviated weight gain caused by the high fat diet of mice during the course of the 8 weeks of the experiment, while bifidobacterium longum and xanthine oxidase inhibitors showed only slight reduction in the latter stages. Meanwhile, after 8 weeks, the body weight of the mice in the xanthine treated group (mice fed HFD with xanthine, HFD-X) was reduced by 22.6% (p < 0.001) compared to the HFD group, while the other two groups were reduced by only 14% (p < 0.05). The research results show that three key factors in the xanthine metabolic pathway have remarkable preventive effects on obesity caused by high fat diet, wherein the xanthine effect is most remarkable, and bifidobacterium longum with xanthine metabolic capability and xanthine oxidase inhibitor only show a certain preventive effect in the later stage.

2. Obese mice, obese resistant mice

150 Male C57BL/6J mice of 6 weeks of age were fed normal diet for one week for adaptation. The mice were then fed a high fat diet. After 8 weeks of high fat diet feeding, 80 of the mice were divided into DIO groups (n=40, the weight gain was 40% at the top, i.e. obese mice) and DIR groups (n=40, the weight gain was 40% at the bottom, i.e. obese resistant mice) according to the degree of weight gain. The two groups are subdivided into four groups (n=10), and DIO groups continue to feed with high fat; DIO-B group, high fat diet + bifidobacterium longum treatment group, bifidobacterium longum dose 0.1mL/10g, once daily; DIO-X group, high fat diet + xanthine treated group, xanthine dose 0.1mL/10g, once daily; DIO-XI group, high fat diet + xanthine oxidase inhibitor treated group, xanthine oxidase inhibitor dosage was 0.1mL/10g, once daily. The DIR group is subdivided into the four groups described above (DIR-XI).

All mice were treated for 6 weeks and body weights were individually weighed weekly and at the end of the intervention.

Blood was taken through the tail vein one week before the end of the experiment for oral glucose tolerance determination.

After the mice were sacrificed by cervical dislocation after the end of the experiment, the liver and epididymal fat of the mice were taken and used to characterize the development of obesity and changes in the functional gene composition of the intestinal bacteria during this period.

As shown in FIGS. 2 and 3, the effect of xanthine on DIR and DIO mice was studied. During the whole 6 weeks of treatment, there was no significant effect on weight gain, liver weight, epididymal white adipose tissue weight, etc., due to the relatively large individual differences exhibited. However, in the OGTT test, the glucose solution was infused at a dose of 0.1mL/10g for DIO mice in each group, and the blood glucose of each group rose rapidly within 0-15min, began to fall after 15min and tended to stabilize after 60min, with oral xanthine providing a significant improvement in glucose tolerance for DIO mice.

EXAMPLE 2 high throughput sequencing of 16S rRNA for analysis of intestinal bacteria composition

(1) Microbial genomic DNA was extracted from fecal samples of mice (4 groups of mice after 8 weeks of high fat diet feeding in example 1 and 4 groups of DIO mice and DIR mice after 6 weeks of treatment) using TIANamp Stool DNA kit (DP 328, tiangen). The V3-V4 region of the 16S rRNA gene was amplified using universal primers 338F (5-ACTCCTACGGGAGGCAGCA-3) and 806R (5-GGACTACHVGGGTWTCTAAT-3). The amplification conditions were as follows: predenaturation at 95 ℃ for 3 min, 30 cycles were started: denaturation at 95℃for 30 seconds, annealing at 55℃for 30 seconds, and primer extension at 72℃for 45 seconds. Amplicons were purified using Ampure XP beads (A63881, beckman) and quantified using a Qubit 3.0 fluorometer using a Qubit dsDNA HS assay kit (Q32854, invitrogen) and then sequenced on a Illumina Miseq PE platform by Kaitai Biotechnology Co., hangzhou.

(2) The original sequence was analyzed using a QIIME2 2018.4 (Quantitative Insights Into Microbial Ecology, USA) insert-based microbiome analysis platform. Sequences were denoised and mass filtered with DADA2 using the q2-DADA insert, which can remove low mass sequences (average mass score <25 per 50bp window), primer sequences and chimeras, and retain specific post-splice sequences. The resulting specific sequences were subjected to taxonomic analysis using classify-sklearn and Greengenes 2.2022.10% OTU reference sequences, and finally relative abundance tables were derived.

The intestinal flora of the mice of the experiment C57BL/6J mainly comprises actinomycota (Actinobacteria), bacteroides (Bacteroidetes), proteus (Proteus), thick-walled mycota (Firmicutes _A) and thick-walled mycota (Firmicutes _D). The firmicutes and bacteroides are main doors, and the relative abundance is high. As shown in fig. 4a, at the portal level, the intestinal microbiota composition of the three mouse models showed Firmicutes _a (15.7% -64.7%), bacteroidetes (0.6% -59.5%) and actinomycetes (0.6% -53.0%) to be the main intestinal bacterial portal. Compared with DIR and DIO mice, the abundance of the firmicutes and actinomycetes in the intestinal tracts of mice fed with HFD is significantly increased, the abundance of the bacteroides is significantly reduced, and the xanthine treatment significantly increases the relative abundance of actinomycetes in the intestinal tracts of mice fed with HFD, with some mice having an abundance as high as 53.0%.

The proportion of the first 20 common bacteria in HFD fed, DIR and DIO mouse gut microbiota is shown in figure 4B. These species together account for 43.3-79.7% of the intestinal microbiota of these mice. The main 3 dominant species are one unclassified Lacrimispora, one unclassified Lachnospiraceae and Amulumruptor caecigallinarius. However, most HFD-B group mice contained less Amulumruptor caecigallinarius, except for a few mice of HFD-B group. But xanthine treatment significantly increased the relative abundance of the actinomycete strain labeled NM07-P-09sp004793665 in HFD fed mice. Xanthines also significantly enriched the unclassified Merdicola (genus thick-walled bacteria) of the DIR and DIO mouse intestinal microbiota.

As shown in fig. 4 by C, D and E, analysis of the principal components of intestinal microbiota composition at the species level demonstrated that oral xanthine significantly altered the intestinal microbiota composition of some mice fed HFD, the predominant bacterial species responsible for this alteration was NM07-P-09sp004793665 and Amulumruptor caecigallinarius, whereas the significant changes in these microbiota groups were sufficient by themselves to increase insulin sensitivity, increase energy expenditure and reduce fat content.

Example 3 interactions between different intestinal microbial strains were understood by microbial interaction network analysis.

The method comprises the steps of processing the relative abundance data of the intestinal strain level obtained in the example 2 by using a correlation analysis tool on a OmicStudio platform, selecting spearman correlation calculation method, screening the data according to the absolute value of the correlation coefficient being larger than 0.6, and leading the corresponding result into Cytoscape to form a corresponding microbial interaction network diagram so as to know the interaction among different intestinal microbial strains.

The results are shown in figure 5a, where there are a total of 5001 correlations (r is greater than 0.6 absolute) in a network containing 226 enterobacteria species, indicating the existence of considerable interconnectivity between nodes within the gut microbiota. The network consisted of five major classes, including actinomycota, bacteroidetes, proteus, firmicutes_a and firmicutes_d, whereas figure 5B shows the specific abundance of the four groups in HFD fed mice.

The critical flora in this network map generally exhibits the highest connectivity, whereas many species of bacteroides are critical flora, where positive correlation is present between the relative abundance of bacteroides species, and furthermore, positive correlation exists between Mucispirillum schaedleri, but significant negative correlation is present with bacteroides critical flora.

Example 4 non-target metabonomics methods were used to study the host-microorganism co-metabolism profile of high fat diet fed mice.

The method comprises the following steps of carrying out non-targeted metabonomics analysis on cecal content collected in a high-fat diet feeding mouse experiment in the embodiment 1 by adopting LC-MS, generating a metabolite network with a matching peak of more than 5 and a cosine similarity of more than 0.6 by utilizing a characteristic-based molecular network platform (FBMN) (http:// GNPS. Ucsd. Edu) in GNPS, visualizing the molecular network of FBMN by Cytoscape, and discussing the influence of xanthine metabolic pathway on the host-microbiota co-metabolism characteristic of HFD feeding mice.

The results are shown in figure 6, of the 1939 compounds detected, 464 showed significant correlation in relative abundance between groups HFD, HFD-B, HFD-X and HFD-XI (|r| > 0.6) and distributed among 70 molecular families. Of these, the relative abundance of 629 molecules between the intervention group and the HFD group showed a significant difference, with 173 features shown in fig. 6 and the remaining 456 features present as single points (not shown in fig. 6). Of the 464 features with significant abundance correlations, 215 features were initially identified as bile acids, fatty acids, steroids, amino acids, and peptides. Notably, the relative abundance of xanthines in the HFD-X group was significantly higher than that in the HFD group (p < 0.05). Furthermore, another product in the xanthine metabolic pathway, 6-oxopurine, shows a tendency to be regulated by these interventions. Xanthine treatment significantly alters the abundance of a variety of primary and secondary bile acids, such as cholate, cholic acid, deoxycholate, and 12-ketodeoxycholate.

The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.

Claims (6)

1. An application of xanthine in preparing medicine for preventing obesity or regulating intestinal tract bacteria activity is provided.

2. The use according to claim 1, wherein the medicament is a medicament for regulating the ratio of firmicutes/bacteroidetes in enterobacteria.

3. The use according to claim 1, wherein the medicament is enriched in intestinal species including actinomycetes and thick-walled bacteria.

4. The use according to claim 1, wherein the medicament is a medicament for preventing a rapid increase in body weight and/or epididymal fat weight caused by a high fat diet.

5. The use according to claim 1, wherein the medicament is a medicament for improving glucose tolerance and insulin sensitivity.

6. The use according to claim 1, wherein the medicament is a medicament for modulating host-enterobacteria xanthine and bile acid metabolism abnormalities.