Application of Jerusalem artichoke polysaccharide in the preparation of drugs for regulating intestinal flora

Abstract

This invention discloses the application of Jerusalem artichoke polysaccharide in the preparation of drugs for regulating intestinal flora, belonging to the field of intestinal flora drugs. The drugs for regulating intestinal flora use Jerusalem artichoke polysaccharide as the active ingredient. These drugs improve intestinal disorders by increasing the proliferation of beneficial intestinal bacteria and the content of short-chain fatty acids, metabolites of intestinal flora. The Jerusalem artichoke polysaccharide prepared by this invention contains uronic acid and pyranose, and is mainly composed of fructose and glucose, with an average molecular weight of 2228 Da. The preparation method of Jerusalem artichoke polysaccharide in this invention has high yield, low cost, and is suitable for industrial production.

Description

Technical Field

[0001] This invention belongs to the field of intestinal flora drugs, specifically the application of Jerusalem artichoke polysaccharide in the preparation of drugs that regulate intestinal flora. Background Technology

[0002] Numerous studies have shown that the pathogenesis of type 2 diabetes mellitus (T2DM) is closely related to gut microbiota dysbiosis caused by nutritional excess in daily diets. Dietary patterns involving excessive intake of nutrients such as salt, sugar, and fat are key contributing factors to this dysbiosis. Nutritional excess significantly disrupts the diversity and stability of the gut microbiota, with a core characteristic being a decrease in the relative abundance of beneficial bacteria and an increase in the relative abundance of opportunistic pathogens. This imbalance further induces intestinal inflammatory responses, ultimately mediating insulin resistance through multiple pathways, thereby promoting the development of T2DM. The gut microbiota plays an irreplaceable role in regulating human physiological functions, immune responses, and maintaining metabolic and nutritional balance. Its core functions are mainly reflected in the following aspects: First, the gut microbiota can participate in the metabolism of non-digestible dietary residues. Its metabolites can not only provide energy for intestinal peristalsis, but also provide an important energy source for the host by generating short-chain fatty acids (SCFAs). Second, the gut microbiota maintains the integrity of the intestinal barrier function by regulating the growth and differentiation of intestinal epithelial cells, thereby effectively resisting the invasion of pathogens and harmful substances and reducing the disruption of intestinal homeostasis.

[0003] In recent years, polysaccharides, as natural biological macromolecules, possess multiple biological activities such as antioxidation, anti-inflammation, and immunomodulation, and have gradually become potential candidates for the treatment of type 2 diabetes mellitus (T2DM). Polysaccharides can improve T2DM-related symptoms through various mechanisms, including regulating glucose metabolism, improving insulin sensitivity, and promoting the recovery of β-cell function. Jerusalem artichoke, a perennial herb, is cultivated to improve saline-alkali environments, hence its large-scale planting in saline-alkali areas near the Yellow River Delta. Jerusalem artichoke also contains various active substances; inulin, a well-known polysaccharide, can be added to the diet to reduce blood lipid production and plasma triglyceride concentration, thereby lowering the risk of atherosclerosis. In addition, it contains coumarin, sesquiterpene chlorogenic acid, caffeic acid, and proteins, making it a beneficial vegetable for controlling various diseases. However, there are few reports on how Jerusalem artichoke polysaccharides improve T2DM by regulating gut microbiota structure. Summary of the Invention

[0004] Purpose of the invention: This invention provides an application of Jerusalem artichoke polysaccharide in the preparation of drugs that regulate intestinal flora. The preparation method of this invention has high yield and low cost, which is conducive to industrial production. The Jerusalem artichoke polysaccharide obtained has a stable structure and high activity, and can change the structure of intestinal flora and regulate intestinal function. It can be applied to the preparation of functional products that improve intestinal function.

[0005] This invention discloses the application of Jerusalem artichoke polysaccharide in the preparation of drugs for regulating intestinal flora. The drugs for regulating intestinal flora are preparations made with Jerusalem artichoke polysaccharide as the active ingredient, plus pharmaceutically acceptable excipients or replicable components. Excipients refer to those that shape the formulation and regulate the properties of the drug, such as microcrystalline cellulose. In compound plant-derived preparations, replicable components refer to solubilizing excipients and physical stabilizers.

[0006] Jerusalem artichoke polysaccharides (JIH) regulate gut microbiota by increasing the proliferation of beneficial bacteria and the levels of short-chain fatty acids (SCFAs), a metabolic product of gut microbiota. Furthermore, JIH can alleviate hyperglycemia symptoms by increasing the levels of SCFAs (acetic acid, propionic acid, and butyric acid). JIH can also improve hyperglycemia symptoms by regulating the relative abundance of beneficial bacteria such as *Dubosiella*, *Oscillibacter*, and *Colidextribacter* at the genus level, thereby altering the gut microbiota structure.

[0007] Furthermore, the preparation method of the Jerusalem artichoke polysaccharide includes the following steps:

[0008] Step 1: Dry the fresh Jerusalem artichoke tubers in an oven and then crush them.

[0009] Step 2: Weigh out Jerusalem artichoke tubers, add distilled water and compound enzyme, and first enzymatically hydrolyze the resulting mixed solution for a period of time, and then perform enzyme inactivation treatment;

[0010] Step 3: The mixed solution after enzymatic hydrolysis and enzyme inactivation is subjected to ultrasonic-assisted extraction, followed by centrifugation to collect the supernatant;

[0011] Step 4: Concentrate the supernatant, add ethanol, let stand, and centrifuge to collect the precipitate;

[0012] Step 5: Redissolve the precipitate in distilled water to remove protein;

[0013] Step 6: Collect and concentrate the solution after removing the protein, remove the solvent, and freeze-dry to obtain Jerusalem artichoke crude polysaccharide;

[0014] Step 7: Dissolve the crude Jerusalem artichoke polysaccharide in distilled water, pass it through a DEAE Sepharose FastFlow chromatography column, and elute with distilled water.

[0015] Step 8: Pass the eluent through a Sephacry S-300 gel chromatography column and elute with 0.1 mol / L NaCl solution. Dialyze the eluent and freeze-dry it to obtain Jerusalem artichoke polysaccharide.

[0016] Preferably, in step two, the ratio of Jerusalem artichoke tuber, compound enzyme, and distilled water is 2g:(1.5-2)g:100ml; the compound enzyme is composed of cellulase and pectinase, and the mass ratio of cellulase to pectinase is 1:1.

[0017] Preferably, in step two, the mixed solution obtained in step two is placed in a constant temperature water bath, and enzymatically hydrolyzed by magnetic stirring at 50°C for 2 hours. Then, the water bath is heated to 100°C and heated for 10-20 minutes to inactivate the enzyme.

[0018] Preferably, in step three, the conditions for ultrasonic-assisted extraction are: ultrasonic power 450 W, 60 ℃ for 60-70 minutes; centrifugation conditions are: 3000 r / min, centrifugation for 10 minutes.

[0019] Preferably, in step four, anhydrous ethanol is added to the concentrated supernatant, the volume ratio of the concentrated supernatant to anhydrous ethanol is 1:3, the temperature is 4°C, and the precipitate is collected after centrifugation.

[0020] The Jerusalem artichoke polysaccharide prepared by the above method contains uronic acid and pyranose. The Jerusalem artichoke polysaccharide is mainly composed of fructose (92.30%) and glucose (7.71%), and its average molecular weight is 2228 Da.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] 1. The Jerusalem artichoke polysaccharide prepared by this invention, as shown in animal experiments, can regulate the intestinal flora structure, specifically by increasing the relative abundance of beneficial bacteria and the content of SCFAs (short-chain fatty acids).

[0023] 2. The method for preparing Jerusalem artichoke polysaccharide of the present invention has high yield, low cost, and is conducive to industrial production. Attached Figure Description

[0024] Figure 1 Infrared spectrum of Jerusalem artichoke polysaccharide;

[0025] Figure 2 Diagram showing the monosaccharide composition of Jerusalem artichoke polysaccharides;

[0026] Figure 3 A schematic diagram showing the content of short-chain fatty acids in the intestine of T2DM mice;

[0027] Figure 4AA schematic diagram of the differential bacterial communities between the high-dose Jerusalem artichoke polysaccharide group and the model group at the genus level in T2DM mice;

[0028] Figure 4B This is a schematic diagram of the differential bacterial communities between the low-dose Jerusalem artichoke polysaccharide group and the model group in T2DM mice at the genus level. Detailed Implementation

[0029] The technical solution of the present invention will be described in detail below through embodiments, but the scope of protection of the present invention is not limited to the embodiments described.

[0030] Example 1: Preparation of Jerusalem artichoke polysaccharides

[0031] 1. The specific preparation steps of Jerusalem artichoke polysaccharide are as follows:

[0032] (1) Soak and wash the fresh Jerusalem artichoke tubers with water, filter out the mud, put them in an oven at 60°C for 5 hours, and crush them into pieces about 1cm in size.

[0033] (2) Enzymatic hydrolysis: Weigh the crushed Jerusalem artichoke tubers, add distilled water and compound enzyme to obtain a mixed solution, place the mixed solution in a 50 ℃ constant temperature water bath and stir magnetically for 2 hours, then heat to 100 ℃ and heat for 10-20 minutes to inactivate the enzyme.

[0034] In the mixed solution, the ratio of Jerusalem artichoke tuber, compound enzyme, and distilled water is 2g: 2g: 100ml; the compound enzyme consists of cellulase and pectinase, with a mass ratio of cellulase to pectinase of 1:1.

[0035] (3) After enzymatic hydrolysis, the mixed solution was extracted for 60 minutes at 450 W ultrasonic power and 60 ℃. After cooling, it was centrifuged at 3000 r / min for 10 minutes to obtain the supernatant.

[0036] (4) Concentrate the supernatant to one-quarter of the original supernatant, add anhydrous ethanol, control the ethanol volume content in the alcohol precipitation system (concentrated supernatant: anhydrous ethanol = 1:3, V / V) to be 75%, the temperature to be 4℃, let stand for 24 hours, centrifuge at 4000r / min for 10 minutes and take the precipitate.

[0037] (5) After redissolving the precipitate in a small amount of distilled water, remove the protein using the Sevag method (chloroform: n-butanol = 4:1, v / v);

[0038] (6) Collect and concentrate the solution after removing the protein, remove the solvent, and freeze dry under vacuum at -55~-50℃ to obtain Jerusalem artichoke crude polysaccharide;

[0039] (7) Dissolve Jerusalem artichoke crude polysaccharide in distilled water to prepare a 10 mg / ml solution, and elute with distilled water using a DEAE Sepharose Fast Flow chromatography column;

[0040] (8) The eluent was passed through a Sephacry S-300 gel chromatography column and eluted with 0.1 mol / L NaCl solution. The eluent was dialyzed with pure water and freeze-dried at -55~-50℃ to obtain Jerusalem artichoke polysaccharide.

[0041] 2. Infrared spectral analysis of Jerusalem artichoke polysaccharides

[0042] Weigh 2 mg of Jerusalem artichoke polysaccharide, mix it with 200 mg of anhydrous potassium bromide powder, grind it in a mortar and press it into transparent sheets, and then heat it at 4000 cm⁻¹. -1 -400cm -1 The wavelength range was scanned using a Fourier transform infrared spectrometer. The infrared spectrum of Jerusalem artichoke polysaccharide is shown below. Figure 1 As shown, by Figure 1 It can be seen that HTP is at 3428cm -1 and 2925cm -1 The two absorption peaks at 1630 cm⁻¹ and 1740 cm⁻¹ are characteristic of carbohydrates, representing the OH stretching vibration and CH stretching vibration, respectively. Furthermore, no band was observed near 1740 cm⁻¹, indicating the absence of a carbonyl functional group in the HTP molecule; while the band at 1630 cm⁻¹... -1 The signal at this location is related to the bending vibration of OH. 1428cm -1 The absorption peak at 1029 cm⁻¹ is a characteristic peak of -COOH, indicating that HTP contains a certain amount of uronic acid. -1 The absorption band at this location indicates that pyranose may be present in Jerusalem artichoke polysaccharides.

[0043] 3. Determination of monosaccharide composition and molecular weight of Jerusalem artichoke polysaccharide

[0044] Weigh 5 mg of Jerusalem artichoke polysaccharide and add 1 mL of 2 M trifluoroacetic acid (TFA) solution. Heat at 60 °C for 30 minutes. Purge with nitrogen and dry. Wash with 3 mL of 99.99% methanol, then dry again. Repeat the methanol washing three times. Dissolve in 5 mL of sterile water and transfer to a chromatographic vial for analysis. Analyze the monosaccharide composition using an ICS 5000+ ion chromatography system (Thermo Fisher Scientific, USA) equipped with a Dionex CarboPac PA20 (150 × 3.0 mm, 10 μm) liquid chromatography column. Weigh another 5 mg of Jerusalem artichoke polysaccharide and add 1 mL of 0.05 M NaCl solution to prepare a 5 mg / mL sample solution. Filter the solution through a 0.22 μm microporous membrane and transfer the sample to a 2 mL vial. Determine the molecular weight using high-performance gel permeation chromatography (HPGPC) with a polymer-based water-soluble SEC (GFC) column (8 × 300 mm). Figure 2 The diagram shown is a representation of the monosaccharide composition of Jerusalem artichoke polysaccharides. Figure 2 As shown, Jerusalem artichoke polysaccharide is mainly composed of fructose (92.30%) and glucose (7.71%), with an average molecular weight of 2228 Da.

[0045] Example 2

[0046] 1. Construction of T2DM mouse model.

[0047] Sixty healthy SPF-grade male ICR mice (4 weeks old, 20±3g) were purchased from Yangzhou University Medical Center and acclimatized for one week in a constant-temperature animal room (23±2℃). The mice were kept under 12-hour light per day to simulate day and night. During this acclimatization period, they were fed a normal diet and had free access to water. After one week of acclimatization, 12 mice were randomly selected as the normal group and continued to be fed a basal maintenance diet. The remaining 48 mice were fed a high-sugar, high-fat diet for four weeks. Then, streptozotocin (STZ, 45mg / kg BW) dissolved in 0.1M sodium citrate buffer was injected intraperitoneally for four consecutive days. The normal group mice received the same dose of 0.1M sodium citrate buffer intraperitoneally during the same period. One week later, all mice were fasted for 12 hours, and fasting blood glucose was measured by blood collection from the tail tip. A fasting blood glucose level >11.1mM indicated successful establishment of the T2DM mouse model.

[0048] T2DM mice were randomly divided into four groups: the model group, the high-dose Jerusalem artichoke polysaccharide group (HTP-H, 300 mg / kg / d, n=12), the low-dose Jerusalem artichoke polysaccharide group (HTP-L, 100 mg / kg / d, n=12), and the metformin hydrochloride group (MET, 100 mg / kg / d, n=12). Each group was administered the prescribed dose by gavage once daily for four weeks, with free access to water.

[0049] 2. Methods for determining the content of short-chain fatty acids.

[0050] The content of short-chain fatty acids was determined by gas chromatography. Acetic acid, propionic acid, and butyric acid were used as standards for quantitative detection. 10 μL of each standard was placed in a 1.5 mL centrifuge tube and slowly diluted to 1 mL with anhydrous diethyl ether. The three standards were further diluted with anhydrous diethyl ether to seven different concentrations for gas chromatographic analysis. Standard curves for each short-chain fatty acid were obtained based on the concentration and peak area of ​​the standards.

[0051] 50 mg of mouse cecal contents were mixed with 0.5 mL of deionized water, and 0.125 mL of phosphoric acid was added. The mixture was vortexed. Then, 0.25 mL of anhydrous diethyl ether was added, and vortexing was continued. The mixture was centrifuged to extract short-chain fatty acids. The extract was filtered through a 0.22 μm microporous membrane before analysis. Chromatographic conditions were as follows: HPINNOWAX column (30 m × 0.25 mm × 0.25 μm), nitrogen as carrier gas, injection port temperature 260 ℃, split ratio 10:1, injection volume 1 μL, column flow rate 1 mL / min. The initial column temperature was 100 ℃ and held for 1 minute, then increased to 200 ℃ at 5 ℃ / min and held for 2 minutes.

[0052] 3. Methods for analyzing gut microbiota.

[0053] Gut microbiota sequencing was performed using CTAB to extract genomic DNA from mouse cecal contents. PCR amplification of the V3+V4 variable regions was conducted using primers 341F (5'-CCTAYGGGRBGCASCAG-3') and 806R (5'-GGACTACNNGGGTATCTAAT-3'). PCR products were purified by 1xTAE 2% agarose gel electrophoresis. The target bands were recovered using a Universal DNA (TianGen, China) purification and recovery kit, and finally sequenced using an Illumina sequencing platform.

[0054] 4. Statistical data analysis methods.

[0055] Short-chain fatty acid data are expressed as mean ± standard deviation (SD). One-way ANOVA, least significant difference (LSD), and Duncan's multiple range test were used to assess statistical significance between groups. Kruskal-Wallis test was used to determine statistical significance, followed by Dunn's post-hoc test and Bonferroni correction. All statistical analyses were performed using SPSS 16.0 (IBM, New York, USA) and Graphpad Prism 7.0 (Graphpad Software, Inc. USA). p < 0.05 was considered statistically significant.

[0056] 5. Results of the effect of Jerusalem artichoke polysaccharide on short-chain fatty acids in T2DM mice.

[0057] Short-chain fatty acids, primarily including acetic acid, propionic acid, and butyric acid, are produced by gut microbiota through the metabolism of complex carbohydrates. Acetic acid regulates host energy metabolism by promoting the secretion of intestinal hormones, reducing systemic inflammatory cytokines, influencing appetite, and increasing energy expenditure and lipid oxidation. Furthermore, butyric acid can improve insulin sensitivity and reduce inflammatory responses in adipocytes by stimulating the production of glucagon-like peptide-1 (GLP-1). Propionic acid helps improve insulin sensitivity and inhibits hepatic lipid accumulation. Butyric acid has been shown to increase energy expenditure in obese mice and can improve symptoms of type 2 diabetes mellitus (T2DM) glucose metabolism disorder by promoting pancreatic β-cell differentiation.

[0058] Figure 3 The content of short-chain fatty acids in the intestine of T2DM mice, by Figure 3 It can be seen that, compared with the model group, the content of acetic acid, propionic acid and butyric acid in the Jerusalem artichoke polysaccharide group (HTP) was significantly increased (p < 0.05).

[0059] 6. Effects of Jerusalem artichoke polysaccharides on gut microbiota in T2DM mice

[0060] The gut microbiota is a complex community of various microorganisms that establishes a close symbiotic relationship with the host. It plays a vital role in maintaining health, metabolizing indigestible dietary components, synthesizing certain vitamins, preventing the invasion of pathogens, and contributing to the maturation and regulation of the immune system.

[0061] Figure 4A and 4B This section showcases some bacterial communities exhibiting significant differences at the genus level among the HTP-L group, HTP-H group, and model group. Figure 4AIt was found that, compared with the model group, the relative abundance of *Dubosiella* was significantly increased in the HTP-L group, while the relative abundance of *Limosilactobacillus* and other lipid metabolites was significantly decreased. Studies have shown that *Limosilactobacillus* can prevent obesity by inhibiting lipid accumulation by reducing the expression of key lipid metabolism regulators. Simultaneously, *Limosilactobacillus* can regulate glucose metabolism by downregulating hepatic glucose synthesis genes, ultimately reducing hepatic glucose production and improving fasting blood glucose levels. Figure 4B It was found that four weeks after HTP-H gavage administration, compared with the model group, the relative abundance of Oscillibacter and Coridextribacter was significantly increased in the HTP-H group, while the relative abundance of Lachnospiraceae was significantly decreased. Lachnospiraceae is a Gram-positive anaerobic bacterium, and an increase in its relative abundance can cause symptoms such as insulin dysfunction and inflammation. Furthermore, a decrease in the relative abundance of Lachnospiraceae increases the content of unsaturated fatty acids and bile acids, and also increases the activity of key lipid synthases, thereby inhibiting intestinal lipid absorption and ultimately improving the symptoms of dyslipidemia.

[0062] In summary, the enzymatic hydrolysis of Jerusalem artichoke polysaccharides significantly increased the sugar content and reduced the molecular weight, improving solubility and providing a more suitable molecular structure for subsequent processing and utilization. In T2DM mice, Jerusalem artichoke polysaccharide intervention significantly increased the levels of acetic acid, propionic acid, and butyric acid in the cecal contents, indicating that Jerusalem artichoke polysaccharides can alleviate hyperglycemia symptoms by regulating intestinal flora metabolites. Furthermore, it was found that Jerusalem artichoke polysaccharides improved hyperglycemia symptoms in T2DM mice by altering the intestinal flora structure at the genus level by regulating the relative abundance of beneficial bacteria such as *Dubosiella*, *Oscillibacter*, and *Colidextribacter*.

[0063] As described above, although the invention has been shown and described with reference to specific preferred embodiments, it should not be construed as limiting the invention itself. Various changes in form and detail may be made without departing from the spirit and scope of the invention.

Claims

1. Use of a Jerusalem artichoke polysaccharide in the preparation of a drug for regulating intestinal flora.

2. Use according to claim 1, characterized in that, The drug for regulating intestinal flora is a preparation prepared by taking Jerusalem artichoke polysaccharide as an active ingredient and adding pharmaceutically acceptable adjuvants or replication components.

3. Use according to claim 1, characterized in that, The preparation method of the Jerusalem artichoke polysaccharide comprises the following steps: Step one, put fresh Jerusalem artichoke tubers into an oven for drying and then crush them; Step two, weigh the Jerusalem artichoke tubers, add distilled water and a composite enzyme, and first enzymatically hydrolyze the obtained mixed solution for a period of time, and then perform enzyme inactivation treatment; Step three, perform ultrasonic-assisted extraction on the mixed solution after enzyme hydrolysis and enzyme inactivation treatment, and then centrifuge to collect the supernatant; Step four, concentrate the supernatant, add ethanol and stand still, centrifuge to obtain the precipitate; Step five, dissolve the precipitate in distilled water and remove the protein; Step six, collect and concentrate the solution after removing the protein, remove the solvent, and freeze-dry to obtain Jerusalem artichoke crude polysaccharide; Step seven, dissolve the Jerusalem artichoke crude polysaccharide in distilled water, pass it through a DEAE Sepharose FastFlow chromatography column, and elute it with distilled water; Step eight, pass the eluate through a Sephacry S-300 gel chromatography column, elute it with a 0.1 mol / L NaCl solution, dialyze the eluate, freeze-dry it, and obtain Jerusalem artichoke polysaccharide.

4. Use according to claim 3, characterized in that, In step two, the ratio of the use amount of Jerusalem artichoke tubers, composite enzyme, and distilled water is 2g: (1.5-2) g: 100ml; the composite enzyme is composed of cellulase and pectinase, and the mass ratio of cellulase to pectinase is 1:

1.

5. Use according to claim 3, characterized in that, In step two, place the mixed solution obtained in step two in a constant-temperature water bath kettle, first magnetically stir it at 50℃ for 2 hours for enzyme hydrolysis, and then heat the water bath kettle to a temperature of 100℃, and heat it for 10-20 minutes for enzyme inactivation treatment.

6. Use according to claim 3, characterized in that, In step three, the ultrasonic-assisted extraction conditions are: ultrasonic power 450 W, 60℃ for 60-70 minutes; and the centrifugation conditions are: 3000 r / min, centrifugation for 10 minutes.

7. The use according to claim 1, characterized in that, In step four, add anhydrous ethanol to the concentrated supernatant, and the volume ratio of the concentrated supernatant to anhydrous ethanol is 1:3, the temperature is 4℃, and the alcohol precipitation is centrifuged to obtain the alcohol precipitate.

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