Sugarcane leaf polysaccharide composition for regulating intestinal flora function and application thereof

Abstract

The present application belongs to the technical field of sugarcane leaf polysaccharide regulating intestinal flora. The present application provides a sugarcane leaf polysaccharide composition, which comprises mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, xylose, arabinose and fucose in a mass ratio of 6.8:1.5:5.2:40.7:13.1:18.9:1.6:6.1:1.9. The composition of the present application can improve the imbalance of intestinal flora caused by obesity and hyperglycemia by reducing the ratio of Firmicutes / Bacteroidetes and increasing the abundance of probiotics.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of regulating intestinal flora by using polysaccharides in sugarcane leaves. BACKGROUND

[0002] There are about 100 million intestinal microorganisms, which encode more than 3 million genes and produce thousands of metabolites (short-chain fatty acids, bile acids, amino acids, etc.), thereby regulating the physiological functions of human hosts, such as nutrition, immunity, and energy metabolism, by affecting glycolipid metabolism. Metabolic syndrome is characterized by the coexistence of various metabolic disorders such as central obesity, hyperglycemia, hyperlipidemia, and hypertension, which can induce various clinical diseases such as diabetes and cardiovascular disease. With the development of China's social economy and the continuous improvement of the living standards of residents, the proportion of refined carbohydrates and oil foods in people's diet structure is increasing, and the restriction of working conditions has led people to form a sedentary lifestyle. The combination of various factors has promoted the occurrence and development of metabolic syndrome characterized by obesity.

[0003] Intestinal flora is closely related to human health, and research has shown that the ratio of Firmicutes to Bacteroidetes and the abundance of other metabolism-related bacteria are significantly imbalanced in patients with metabolic syndrome. Therefore, maintaining the homeostasis of intestinal flora is of great importance to the prevention and treatment of human health. Currently, there are two types of substances that can regulate intestinal flora: one is live probiotic preparations, which are commonly found in lactic acid bacteria and Bifidobacterium; the other is prebiotics, which are organic substances that are not digested and absorbed by the host but can selectively promote the metabolism and proliferation of beneficial bacteria in the body, thereby improving the health of the host. Common prebiotics include polysaccharides, oligosaccharides, microalgae, protein hydrolysates, and natural plants such as vegetables, Chinese herbal medicines, and wild plants. Among them, polysaccharides are a high-quality prebiotic that can improve metabolic disorders by regulating intestinal flora.

[0004] Sugarcane leaf polysaccharide is one of the effective components of sugarcane leaves. Studies have shown that it has a good improvement effect on type 1 diabetes, and also has anti-tumor, antioxidant, and cardiomyocyte protection effects. However, there have been no reports on the use of sugarcane leaf polysaccharide in regulating intestinal flora. SUMMARY

[0005] Therefore, the present application provides a sugarcane leaf polysaccharide composition, which comprises mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, xylose, arabinose, and fucoidan in a quality ratio of 6.8:1.5:5.2:40.7:13.1:18.9:1.6:6.1:1.9.

[0006] In specific embodiments of the present application, the sugarcane leaf polysaccharide composition is used in the preparation of a medicament or health food for regulating the function of intestinal flora.

[0007] In specific embodiments of the present application, the sugarcane leaf polysaccharide composition is used in the preparation of a medicament or health food for reducing the absorption of host energy in the intestine.

[0008] In specific embodiments of the present application, the sugarcane leaf polysaccharide composition is used in the preparation of a medicament or health food for reducing the ratio of Ascomycota / Bacteroidetes.

[0009] In specific embodiments of the present application, the sugarcane leaf polysaccharide composition is used in the preparation of a medicament or health food for increasing the abundance of Deferribacteres, Verrucomicrobia, Cyanobacteria and / or Tenericutes.

[0010] In specific embodiments of the present application, the sugarcane leaf polysaccharide composition is used in the preparation of a medicament or health food for increasing the abundance of beneficial bacteria in the intestine.

[0011] In specific embodiments of the present application, the beneficial bacteria in the intestine include: Bacteroides acidifaciens, Mucispirillum sp., Faecalibacterium prausnizii, Lactobacillus reuteri, Akkermansia muciniphila, Alistipes timonensis, Bacteroides sartorii, Parabaceroides distasonis, Alistipes indistinctus, Alistipes shahii, Bacteroides uniformis, Parabacteroides goldsteinii and Bacteroides thetaiotaomicron.

[0012] The composition of the present application can regulate intestinal flora, enhance intestinal and body immunity, and help intestinal nutrition and absorption. BRIEF DESCRIPTION OF DRAWINGS

[0013] Figure 1 PMP pre-column derivatization liquid chromatogram of monosaccharide standard and polysaccharide hydrolysate.

[0014] Wherein 1 is mannose, 2 is rhamnose, 3 is glucuronic acid, 4 is galacturonic acid, 5 is glucose, 6 is galactose, 7 is xylose, 8 is arabinose, 9 is fucose.

[0015] Figure 2 Figure 1 is the Alpha diversity index chart of the intestinal flora changes of each group of mice after intervention of sugarcane leaf polysaccharide.

[0016] Figure 3 Figure 2 is the PcoA chart of the intestinal flora changes of each group of mice after intervention of sugarcane leaf polysaccharide.

[0017] Figure 4 Figure 3 is the NMDS chart of the intestinal flora changes of each group of mice after intervention of sugarcane leaf polysaccharide. DETAILED DESCRIPTION

[0018] The present application will be further described below in conjunction with the accompanying drawings and examples, which are merely illustrative and the present application is not limited by these examples.

[0019] Example 1

[0020] 1.1. Dialysis of sugarcane leaf polysaccharide

[0021] Activation of dialysis bag: The dialysis bag was cut into small pieces, washed with pure water, boiled in hot water for 30 min, and then washed with pure water to complete the activation.

[0022] Dialysis of sugarcane leaf polysaccharide: 50 mg of sugarcane leaf polysaccharide was dissolved in water, dialyzed in a dialysis bag using pure water for 3 days, with water change every 12 h during the period, and after dialysis, concentrated under reduced pressure on a 38 rotary evaporator, freeze-dried to obtain a dialyzed sugarcane leaf polysaccharide sample.

[0023] 1.2. Determination of monosaccharide composition of sugarcane leaf polysaccharide

[0024] Hydrolysis of polysaccharide: 5 mg of polysaccharide was weighed, 2 mL of 2M trifluoroacetic acid solution was added, sealed, and placed in a constant temperature oven at 120°C for 2 h, then taken out and cooled, concentrated under reduced pressure on a rotary evaporator at 38°C, and repeatedly added with 3 times of methanol solution, evaporated to dryness, and finally, added with ultrapure water to completely dissolve, thus obtaining a hydrolyzed sample of sugarcane leaf polysaccharide.

[0025] Derivatization of polysaccharide: 600 μL of the hydrolyzed sample and standard solution (standard solution was prepared at 2.5 mg / mL) was added with 300 μL of 0.3 M sodium hydroxide solution and 300 μL of 1-phenyl-3-methyl-5-pyridinium ketone (PMP) methanol solution, vortexed, and reacted in water bath at 70 °C for 60 min, then cooled and placed, added with 300 μL of 0.3 M hydrochloric acid solution for neutralization, and membrane was passed to obtain the derivatized sample of sugarcane leaf polysaccharide.

[0026] Monosaccharide composition analysis of polysaccharide: Waters 2695 high performance liquid chromatography analysis, mobile phase: 0.1 M phosphate buffer-acetonitrile = 83:17, flow rate: 1 mL / min; column temperature: 30 °C; wavelength: 254 nm.

[0027] Monosaccharide composition of sugarcane leaf polysaccharide: as shown in Figure 1 Table 1, the sugarcane leaf polysaccharide mainly contains galacturonic acid, galactose, glucose, mannose and arabinose, and a small amount of glucuronic acid, fucose, xylose and rhamnose.

[0028] Table 1 Mass ratio of monosaccharide components of sugarcane leaf polysaccharide

[0029]

[0030] Example 2

[0031] 2.1. Animal experiment of sugarcane leaf polysaccharide (PSL)

[0032] Experimental animals: ob / ob male mice were purchased from the Experimental Animal Center of the Chinese Academy of Medical Sciences, and were allowed to freely eat and drink under the conditions of 25 °C, humidity of 40%-60%, and 12 / 12 h alternating day and night environment, and were adaptively fed for one week.

[0033] Mouse breeding feed: conventional feed for mice (including feeding of ob / ob mice) was purchased from Beijing Huafukang Biotechnology Co., Ltd., and the item number was 1032.

[0034] The ob / ob group mice were randomly divided into 2 groups, 8 mice in each group, and the experimental group was PSL-H 1.0 g / kg, and the model ob / ob group was fed with water.

[0035] After 8 weeks of treatment, the mouse feces were frozen at -80 °C. At the end of the experiment, the mice were anesthetized with 10% chloral hydrate and sacrificed after fasting for 12 h, and the intestinal tissues were taken on ice. The cecal contents were sent for 16s rDNA amplicon sequencing analysis to reveal the diversity and richness changes of the mouse intestinal flora. The experimental grouping is shown in Table 1.

[0036] Table 1 Experimental grouping and treatment

[0037]

[0038] Example 3

[0039] 3.1. Sugarcane leaf polysaccharides regulate the structure of intestinal flora in mice

[0040] 3.1.1 Experimental method:

[0041] 1) Extraction of genomic DNA and PCR amplification After extracting the genomic DNA of the samples by CTAB method, the sample purity and concentration were detected by agarose gel electrophoresis, and the qualified samples were uniformly diluted to 20 ng / ul for PCR amplification. In the amplicon sequencing analysis based on 16s rRNA gene, the V3-V4 region amplification primer was F: CCTAYGGGRBGCASCAG; R: GGACTACNNGGGTATCTAAT. The amplification system (25 μL) was 5x reaction buffer 5 μL, 5x GC buffer 5 μL, dNTP (2.5 mM) 2 μL, Forward primer (10 uM) 1 μL, Reverse primer (10 uM) 1 μL, DNA Template 2 μL, ddH2O 8.75 μL, Q5 DNA Polymerase 0.25 μL.

[0042] 2) Mixing and purification of PCR products

[0043] 2) Mixing and purification of PCR products

[0044] PCR products were detected by electrophoresis using 2% agarose gel; according to the concentration of PCR products, equal amount of mixing was carried out, and after mixing thoroughly, 2% agarose gel electrophoresis was used to detect PCR products, and the product was recovered using the gel recovery kit provided by qiagen company.

[0045] 3) Library construction and machine

[0046] Library construction was performed using TruSea@DNA PCR-Free Sample Preparation Kit library kit, and the constructed library was quantified by Qubit and Q-PCR. After the library was qualified, Illumina platform was used for paired-end sequencing of community DNA fragments.

[0047] 4) Bioinformatics analysis

[0048] Firstly, the primer fragments of the sequences were cut off using cutadapt with the setting of -O 10 and the sequences with unmatched primers were discarded; the sequences were spliced using the fastq_mergepairs module of Vsearch; the spliced sequences were quality controlled using the fastq_filter module; the duplicate sequences were removed using the derep_fulllength module; the sequences after removing duplicates were clustered at the level of 98% similarity using the cluster_size module, and chimeras were removed using the uchime_denovo module; then, the chimeras in the sequence set after quality control were filtered using a perl script (https: / / github.com / torognes / vsearch / wiki / VSEARCH-pipeline), so as to obtain high-quality sequences; the high-quality sequences were clustered at the level of 97% similarity using the cluster_size module, and the representative sequences and OTU table were output respectively. Finally, the singletons OTUs (i.e., the OTUs with an abundance of 1 in all samples by default) in the OTU table and their representative sequences were removed. The insertion and deletion errors in the nucleic acid sequences were corrected using the RDP FrameBot software (https: / / github.com / rdpstaff / Framebot) based on the seed protein sequences of the corresponding functional genes downloaded from the RDP website. The amino acid length filtering threshold was set to 50, and the de novo mode was turned on, and the protein sequences that met the specific requirements after passing the verification were added to the reference sequence, and the other parameters used the default values. After FrameBot analysis, the corrected nucleic acid sequences and protein sequences can be obtained, and the non-target fragments will be removed, and the corrected nucleic acid sequences will be used for subsequent analysis.

[0049] Alpha diversity analysis: using the unflattened ASV / OTU table, the "qiime diversity alpha-rarefaction" command was called, and the parameters "--p-steps 10 --p-min-depth 10 --p-iterations 10" were set, that is, the minimum rarefaction depth was 10, the parameter "--p-max-depth" was set to 95% of the sequence amount of the lowest sequencing depth sample in all samples, and then 10 depth values were uniformly selected between this depth and the minimum depth, and each depth value was rarefied for 10 times, and the selected alpha diversity index was calculated. The average value of the scores at the maximum rarefaction depth was taken as the alpha diversity index, and the Chao1 and Observed species indices were used to represent the richness, and the Shannon and Simpson indices were used to represent the diversity.

[0050] Species taxonomy annotation: Using QIIME2's classify-sklearn algorithm (Bokulich et al., 2018) (https: / / github.com / QIIME2 / q2-feature-classifier): For each ASVs' feature sequence or each OTU's representative sequence, use the pre-trained Naive Bayes classifier to do species annotation in QIIME2 software with default parameters.

[0051] Distance matrix and PCoA analysis: Using the rarefied ASV / OTU table, call the "qiime diversity core-metrics-phylogenetic" or "qiime diversity core-metrics" command according to the presence or absence of the tree file, calculate four kinds of distance matrixes such as Jaccard, Bray-Curtis, unweighted UniFrac and weighted UniFrac, or two kinds of distance matrixes such as Jaccard and Bray-Curtis, and do PCoA analysis on these distance matrixes, output QZV file. Drag the QZV file into the corresponding area of https: / / view.qiime2.org / to realize visualization. At the same time, PCoA analysis can be done in R using R script to output the PCoA coordinates of sample points, and draw them into a two-dimensional scatter plot.

[0052] Non-metric multidimensional scaling analysis (NMDS): By dimensionality reduction of sample distance matrix, simplify data structure, so as to describe the distribution characteristics of samples under a certain distance scale. Unlike PCoA analysis, NMDS analysis does not rely on the calculation of eigenvalues and eigenvectors, but through the rank ordering of sample distance, the ordering of samples in low-dimensional space is as consistent as possible with the proximity relationship between each other (rather than the exact distance value). Therefore, NMDS analysis is not affected by the numerical value of sample distance, only considers the size relationship between each other, and for complex structure data, the ordering result may be more stable. The smaller the stress value (Stress) of NMDS result is, the better, generally considered that when the value is less than 0.2, the result of NMDS analysis is more reliable.

[0053] 3.1.2 Experimental results:

[0054] 1) Sugarcane leaf polysaccharide regulates intestinal flora structure

[0055] As Figure 2, by Alpha diversity analysis, after PSL-H treatment, the Chao1 and Observed species index representing richness decreased, and the Shannon index also decreased significantly, indicating that the richness and diversity of intestinal flora decreased. Studies have shown that low diversity within a certain range represents a healthier state. Using the weighted unifrac algorithm, it can be seen from Figure 2 that after PSL-H treatment, the structure of the intestinal microbial community of mice changed significantly. In addition, the smaller the stress value (Stress) of the NMDS analysis result is, the better, as shown in Figure 2 , the stress value is 0.0904, indicating that the result of NMDS analysis is reliable, which indicates that the intervention of PSL-H will change the structure of the intestinal flora of mice.

[0056] 2) Regulation of sugarcane leaf polysaccharide on intestinal flora at the level of door

[0057] The species abundance at the level of door of the intestinal flora of mice was analyzed, and the top 10 species with relative abundance were compared. As shown in Table 2, the most abundant bacterial phylum in the two groups of mice was Bacteroidetes, Firmicutes, and Proteobacteria. Among them, most of the Bacteroidetes bacteria are polysaccharide-degrading bacteria, which generate short-chain fatty acids and maintain intestinal health, while the Firmicutes have genes that metabolize sugar and have the function of absorbing a large amount of energy from a small amount of food. Studies have found that in people with metabolic disorders such as obesity, the ratio of Firmicutes / Bacteroidetes increases, while in the present application, the ratio of Firmicutes / Bacteroidetes before intervention is 1.92, and the ratio of Firmicutes / Bacteroidetes after intervention is 0.94, which significantly decreases, to a certain extent, the energy absorption of the host in the intestine.

[0058] In addition, after the intervention of sugarcane leaf polysaccharide, the abundance of Proteobacteria did not change, while the abundance of Deferribacteres, Verrucomicrobia, Cyanobacteria and Tenericutes at the level of door significantly increased.

[0059] Table 2 Effect of sugarcane leaf polysaccharide on the top 10 species with relative abundance at the level of door

[0060]

[0061] Table 3 Effect of sugarcane leaf polysaccharide on Firmicutes / Bacteroidetes (F / B)

[0062]

[0063] 3) Regulation of intestinal flora at the species level by sugarcane leaf polysaccharides

[0064] The relative abundance of the top 50 species was compared by analyzing the phylum level species abundance of the mouse intestinal flora. The top 50 species with significant changes in relative abundance are shown in Table 4. PSL-H significantly increased the abundance of beneficial bacteria in the intestines of ob / ob mice, including Bacteroides acidifaciens, Mucispirillum sp., Faecalibacterium prausnizii, Lactobacillus reuteri, Akkermansia muciniphila, Alistipes testimonensis, Bacteroides sartorii, Parabacteroides distasonis, Alistipes indistinctus, Alistipes shahii, Bacteroides uniformis, Parabacteroides goldsteinii, and Bacteroides thetaiotaomicron.

[0065] Among them, short-chain fatty acid-producing bacteria such as Bacteroides acidifaciens, Faecalibacterium prausnizii, Lactobacillus reuteri, Akkermansia muciniphila, Alistipe testimonensis, Bacteroides sartorii, Alistipes indistinctus, Alistipes shahii, Bacteroides thetaiotaomicron, etc. significantly increased, short-chain fatty acids have a positive effect on maintaining intestinal function, glucose homeostasis, appetite, energy metabolism regulation, inflammation and immune capacity, and tumors and colon cancer, etc., thereby improving the health of the body.

[0066] Akkermansia muciniphila, a mucin-decomposing bacterium, is abundant in the intestinal tract at 1%-3%, and studies have shown that oral administration of Akkermansia muciniphila can improve the symptoms of metabolic diseases in mice, and is a candidate drug for the treatment of type 2 diabetes and obesity. In the present application, after administration of sugarcane leaf polysaccharide, the abundance of Akkermansia muciniphila increased from 0.06% to 0.27%, an increase of 4.5 times.

[0067] Table 4 Influence of sugarcane leaf polysaccharide on species with significant changes at the species level

[0068]

[0069] Although the present application has been described in detail through the above examples, it should be recognized that the above description should not be considered as limiting the present application, and various modifications and alternatives of the present application will be apparent to those skilled in the art after reading the above. Therefore, the scope of protection of the present application should be defined by the appended claims.

Claims

1. Use of a Saccharum officinarum leaf polysaccharide composition comprising mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, xylose, arabinose and fucose in a mass ratio of 6.8:1.5:5.2:40.7:13.1:18.9:1.6:6.1:1.9 in the manufacture of a medicament or a health food for reducing host energy absorption in the gut, in the manufacture of a medicament or a health food for reducing the ratio of Firmicutes / Bacteroidetes, in the manufacture of a medicament or a health food for increasing Deferribacteres, Verrucomicrobia and / or Cyanobacteria, or in the manufacture of a medicament or a health food for increasing the abundance of beneficial bacteria in the gut. The intestinal beneficial bacteria include: Bacteroides acidifaciens , Mucispirillum sp., Faecalibacterium prausnizii , Lactobacillus reuteri , Akkermansia muciniphila , Alistipes timonensis , Bacteroides sartorii , Parabaceroides distasonis , Alistipes indistinctus , Alistipes shahii, Bacteroides uniformis , Parabacteroides goldsteinii and Bacteroides thetaiotaomicron .

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