Application of houttuynia cordata polysaccharide in preparation of medicine for treating type 2 diabetes mellitus

Abstract

The invention discloses application of houttuynia cordata polysaccharide in preparation of a medicine for treating type 2 diabetes, and belongs to the technical field of polysaccharide. The preparation method of the herba houttuyniae polysaccharide specifically comprises the following steps: by taking herba houttuyniae as a raw material, drying, crushing, degreasing, treating by utilizing ultrasonic waves and a compound enzyme, taking supernate, carrying out alcohol precipitation, removing protein by virtue of a Sevag method, putting into a rotary evaporator to remove a residual solvent, purifying by virtue of a DEAE Sepharose Fast Flow chromatographic column and a Sephacryl S-300 gel chromatographic column, and finally freeze-drying, so as to obtain the herba houttuyniae polysaccharide. The medicine for treating type 2 diabetes mellitus is a preparation prepared by taking houttuynia cordata polysaccharide as an active component and adding pharmaceutically acceptable auxiliary materials or auxiliary components. The medicine for treating type 2 diabetes mellitus can regulate the intestinal flora structure, which is expressed by increasing the relative abundance of beneficial bacteria to restore the balance of the intestinal flora and increasing the content of short-chain fatty acid (SCFAs) in the intestinal tract at the same time.

Description

Technical Field

[0001] The present invention relates to the technical field of polysaccharides, and in particular to use of Houttuynia cordata polysaccharide in preparing a medicine for treating type 2 diabetes mellitus (T2DM). Background Art

[0002] Diabetes mellitus is a chronic disease characterized by abnormally elevated blood sugar levels. Type 2 diabetes (T2DM), characterized by hyperglycemia, impaired glucose tolerance, and low-grade inflammation, is associated with insulin resistance or insufficient insulin secretion and accounts for 90% to 95% of all diabetes cases. Currently, most treatments for T2DM act on specific tissues. For example, metformin hydrochloride inhibits hepatic glycogen output, reducing intestinal glucose absorption; sulfonylureas and glinides stimulate insulin secretion from beta cells; and α-glucosidase inhibitors inhibit α-glucosidase activity in the body, lowering blood sugar levels. However, long-term use of these drugs can cause a variety of adverse reactions, such as hypoglycemia, rash, and anemia. Therefore, there is a need to identify novel, safe, effective, and low-side-effect active ingredients for the treatment of T2DM.

[0003] The safety of plants with both medicinal and edible properties is a topic of widespread concern, with polysaccharides (PGs) becoming a research hotspot in the food, medical, and chemical sectors. As a key dietary component and an essential bioactive component in human life, PGs hold broad promise in biochemistry and medicine. Studies have shown that PGs exhibit low toxicity and high efficacy against metabolic diseases.

[0004] Houttuynia cordata (Houttuynia cordata Thunb.) is a plant belonging to the family Saururaceae. Its bioactive components primarily include polysaccharides, flavonoids, and phenolic acids. Polysaccharides extracted from Houttuynia cordata exhibit immunomodulatory, antiviral, and anti-inflammatory activities. Furthermore, studies have shown that Houttuynia cordata polysaccharides can inhibit the activity of α-amylase and α-glucosidase. However, there are no reports on Houttuynia cordata polysaccharides in the treatment of type 2 diabetes by regulating the intestinal microbiota. Summary of the Invention

[0005] Based on the problems existing in the background technology, the present invention provides a new use of Houttuynia cordata polysaccharide, specifically the use of Houttuynia cordata polysaccharide in the preparation of drugs for treating T2DM.

[0006] The present invention is implemented through the following technical solutions:

[0007] The invention discloses use of houttuynia cordata polysaccharide in preparing medicine for treating type 2 diabetes.

[0008] Preferably, the drug for treating type 2 diabetes is a preparation prepared with Houttuynia cordata polysaccharide as the active ingredient and pharmaceutically acceptable excipients or auxiliary ingredients.

[0009] Preferably, the drug for treating type 2 diabetes improves intestinal disorders caused by type 2 diabetes by regulating intestinal flora.

[0010] The present invention also discloses a method for preparing the Houttuynia cordata polysaccharide, which comprises the following steps:

[0011] S1. Dry and crush the Houttuynia cordata raw material, and then degrease it by reflux ethanol;

[0012] S2. Add distilled water to the defatted raw material, adjust the pH, perform ultrasonic extraction, and obtain the supernatant A after centrifugation and filtration;

[0013] S3. Add complex enzyme to supernatant A for enzymatic hydrolysis. After enzymatic hydrolysis, inactivate the enzyme in boiling water bath and centrifuge to obtain supernatant B.

[0014] S4. Add ethanol to the supernatant B, let it stand, and centrifuge to obtain the precipitate;

[0015] S5. The precipitate was redissolved in water and the protein was removed by the Sevag method;

[0016] S6. Collect the solution after protein removal, remove the residual solvent, and dry it to obtain crude Houttuynia cordata polysaccharide.

[0017] S7. Dissolve the crude Houttuynia cordata polysaccharide in distilled water, pass it through a DEAE Sepharose Fast Flow column, collect the distilled water eluate and concentrate it;

[0018] S8. The eluate was purified again by a Sephacryl S-300 gel chromatography column using a 0.1 mol / L NaCl solution for elution. The eluate was collected, dialyzed, and freeze-dried to obtain Houttuynia cordata polysaccharide.

[0019] Preferably, in step S2, the material-liquid ratio of the defatted raw material to distilled water is 1:(35-40) (W / V), the pH is adjusted to 5, and the ultrasonic time is 40-50 min.

[0020] Preferably, in step S3, the complex enzyme is cellulase and pectinase in a mass ratio of 2:1; and the added amount of the complex enzyme is 1.5-2.0% (w / w).

[0021] Preferably, in step S3, the enzymatic hydrolysis temperature is 50-55° C., and the enzymatic hydrolysis time is 40-50 min.

[0022] Beneficial effects of the present invention:

[0023] The present invention first discovered that Houttuynia cordata polysaccharide prepared by the present invention has a significant therapeutic effect on T2DM. Animal experiments investigating the effect of Houttuynia cordata polysaccharide on T2DM mice revealed that the polysaccharide can regulate the structure of the intestinal flora, increasing the relative abundance of beneficial bacteria, restoring the balance of the intestinal flora, and increasing the content of short-chain fatty acids (SCFAs) in the intestine. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The accompanying drawings are used to further explain the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings:

[0025] Figure 1 This is the infrared spectrum of Houttuynia cordata polysaccharide;

[0026] Figure 2 This is the monosaccharide composition diagram of Houttuynia cordata polysaccharide;

[0027] Figure 3 The abundance of the intestinal flora at the phylum level of mice;

[0028] Figure 4 The difference in intestinal flora between the low-dose Houttuynia cordata polysaccharide group and the model group at the genus level;

[0029] Figure 5 The difference in intestinal flora between the Houttuynia cordata polysaccharide high-dose group and the model group at the genus level;

[0030] Figure 6 is the content of acetic acid, propionic acid and butyric acid in the mouse intestine;

[0031] Figure 7 This is the Spearman correlation heat map between mouse intestinal flora and short-chain fatty acids. DETAILED DESCRIPTION

[0032] The technical solution of the present invention is further described in detail below with reference to specific embodiments, but the protection scope of the present invention is not limited to the following embodiments.

[0033] Example 1

[0034] 1. Preparation of Polysaccharides

[0035] A method for preparing Houttuynia cordata polysaccharide comprises the following steps:

[0036] (1) The raw material of Houttuynia cordata is dried and crushed, and then defatted by refluxing with 80% ethanol;

[0037] (2) Take the defatted powder, add distilled water at a solid-liquid ratio of 1:40 (W / V), adjust the pH to 5, sonicate for 50 minutes, and centrifuge to obtain the supernatant;

[0038] (3) Add 1.5% (w / w) of complex enzyme (cellulase:pectinase = 2:1) to the supernatant, and enzymolysis temperature is 55°C for 50 min;

[0039] (4) Inactivate the enzyme in a boiling water bath for 10 min and centrifuge to obtain the supernatant;

[0040] (5) Add 4 times the volume of ethanol to the supernatant and let it stand for 24 hours. Centrifuge at 5000 rpm for 5 minutes to obtain the precipitate.

[0041] (6) The precipitate was redissolved in water and the protein was removed by the Sevag method;

[0042] (7) collecting the solution after protein removal and placing it in a rotary evaporator to remove the residual solvent, and then freeze-drying it in vacuum to obtain crude Houttuynia cordata polysaccharide;

[0043] (8) Dissolve the crude Houttuynia cordata polysaccharide in distilled water, pass it through a DEAE Sepharose Fast Flow chromatography column, collect the distilled water eluate and concentrate it;

[0044] (9) The eluate was purified again by Sephacryl S-300 gel chromatography column, eluted with 0.1 mol / L NaCl solution, and the eluate was collected, dialyzed, and freeze-dried to obtain Houttuynia cordata polysaccharide.

[0045] 2. Polysaccharide Infrared Spectroscopy (FT-IR) Analysis

[0046] 2 mg of Houttuynia cordata polysaccharide (HCP) was ground and mixed with 200 mg of anhydrous potassium bromide powder, and pressed into transparent sheets. -1 -400cm -1 The Fourier transform infrared spectroscopy (FT-IR) was used to scan the wave number range. Figure 1 The infrared spectrum of Houttuynia cordata polysaccharide is shown. -1 The stretching vibration absorption peak of OH is at 2922.72cm -1 The CH stretching vibration absorption peak is at 1637.91cm -1 The asymmetric stretching vibration of C=O in the carboxyl group is the characteristic absorption peak of uronic acid. -1 The strong absorption at may be due to the stretching vibration of CC and CO in the pyran ring, and the stretching vibration of COC in the glycosidic bond.

[0047] 3. Determination of Monosaccharide Composition and Molecular Weight of Houttuynia Cordata Polysaccharide

[0048] Take 5mg HCP, add 1mL 2mol / L trichloroacetic acid, and hydrolyze at 121℃ for 2h. After drying with nitrogen, wash with 3mL methanol and repeat three times. After dissolving the dried sample in 5mL sterile water, take 0.2mL monosaccharide standard solution and polysaccharide sample hydrolyzate for 1-phenyl-3-methyl-5-pyrazolone (PMP) derivatization. Use high performance liquid chromatography system to analyze the monosaccharide composition. Take 5mg HCP, add 1mL 0.05mol / L NaCl solution, centrifuge at 8000rpm for 10min, take the supernatant and filter through 0.22μm filter membrane, then place the sample in a 2mL injection bottle for use. Molecular weight is determined by high performance gel permeation chromatography. Figure 2 The monosaccharide composition of Houttuynia cordata polysaccharide is shown in FIG. HCP is mainly composed of mannose (32.46%), galactose (20.97%) and glucose (13.47%), with an average molecular weight of 15-18 kDa.

[0049] Test Example 1

[0050] 1. T2DM mouse model establishment and grouping

[0051] 60 healthy male Kunming mice (SPF grade, 4 weeks old, 20±4g) were randomly selected as the normal group after one week of adaptive feeding and continued to be fed with basic maintenance feed. Except for the mice in the normal group, the remaining 48 mice were fed with high sugar and high fat (HSHF) feed for 4 weeks, and then intraperitoneally injected with streptozotocin (STZ, 45mg / kg BW) dissolved in 0.1mol / L citric acid buffer solution every other day for three consecutive days. The mice in the normal group were intraperitoneally injected with the same dose of 0.1M citric acid buffer during the same period. One week later, the fasting blood glucose (FBG) of the mice was measured by tail tip blood sampling. If FBG ≥11.1mmol / L, it indicated that the T2DM mouse model was successfully established. T2DM mice were randomly divided into four groups: model group (Model, n=12), low-dose HCP group (HCP-L, 100 mg / kg / d, n=12), high-dose HCP group (HCP-H, 300 mg / kg / d, n=12), and metformin hydrochloride group (MET, 100 mg / kg / d, n=12). The mice were gavaged daily for four weeks according to the dose. After the experiment, the mice were dissected and the cecal contents were stored at -80°C for further study.

[0052] 2. Short-chain fatty acid analysis

[0053] The SCFAs content in cecal contents was determined by gas chromatography (GC). First, 10 μL of acetic acid, propionic acid, and butyric acid as standards were placed in a 1.5 mL centrifuge tube, and anhydrous ether was added to the centrifuge tube to 1 mL. Next, the diluted standards were further diluted with anhydrous ether to produce samples of different concentrations for gas chromatography analysis. The standard curve corresponding to each SCFA was calculated based on the concentration and peak area of ​​the standards. 0.5 mg of cecal contents were weighed into a 5 mL centrifuge tube, 0.5 mL of deionized water was added and mixed, followed by 0.125 mL of phosphoric acid and mixed, and then 0.25 mL of anhydrous ether was added twice to extract SCFAs. The supernatant was further filtered through a 0.22 μm nylon 6 needle filter and 0.4 mL of anhydrous ether was added before gas chromatography analysis.

[0054] 3. Intestinal flora analysis

[0055] Total DNA was extracted from the cecal contents using the CTAB method, and the DNA sample was diluted to 1 ng μL -1 PCR amplification was performed using primers 341F (5′-CCTAYGGGRBGCASCAG-3′) and 806R (5′-GGACTACNNGGGTATCTAAT-3′) to amplify the V3-V4 region of 16S rDNA. The PCR products were then subjected to high-throughput sequencing. The PCR products were combined with 1×TAE buffer and detected by electrophoresis on a 2% agarose gel. The sequencing library was prepared by NEB The DNA was constructed using the Ultra DNA LibraryPrep Kit (Illumina, USA) and sequenced and analyzed on the Illumina NovaSeq PE 250 platform.

[0056] 4. Data Analysis

[0057] All data are presented as mean ± standard deviation (SD). Statistical significance between groups was assessed using one-way analysis of variance (ANOVA), least significant difference (LSD), and Duncan's multiple range test. Statistical significance was set at p < 0.05. All statistical analyses were performed using SPSS 16.0 and Graphpad Prism 9.0. Differential microbiota were plotted using the Wekemo Bioincloud platform, and a heat map of the correlation between intestinal microbiota and short-chain fatty acids was created.

[0058] 5. Effects of Houttuynia cordata polysaccharide on the intestinal flora structure of T2DM mice

[0059] At the phylum level, the species abundance of the normal group was the highest, and the relative abundance of Bacillota increased in the Houttuynia cordata polysaccharide group ( Figure 3At the genus level, compared with the model group, the relative abundances of Allobaculum, Limosilactobacillus, and Massiliomicrobiota in the HCP-L group were significantly increased (p < 0.05), while the relative abundances of Prevotellaceae, Desulfovibrio, and Colidextribacter were significantly decreased (p < 0.05) ( Figure 4 Compared with the model group, the relative abundance of Faecalibaculum and Massiliomicrobiota in the HCP-H group increased significantly (p < 0.05), while the abundance of harmful bacteria such as Prevotellaceae and Colidextribacter decreased significantly (p < 0.05) ( Figure 5 The results showed that Houttuynia cordata polysaccharide could regulate the intestinal flora structure of T2DM mice, increase the relative abundance of beneficial bacteria in the intestines of T2DM mice, and restore the balance of intestinal flora.

[0060] Short-chain fatty acids (SCFAs) mainly include acetic acid, propionic acid and butyric acid. SCFAs can promote the secretion of glucagon-like peptide-1, thereby reducing fasting blood sugar levels and alleviating the symptoms of hyperglycemia in T2DM. Figure 6 It can be seen that compared with the model group, the content of acetic acid, propionic acid and butyric acid in the HCP group increased significantly (p < 0.05). Figure 7 It can be seen that bacterial genera such as Akkermansia, Christensenella, and Faecalibaculum are positively correlated with acetic acid, propionic acid, and butyric acid. Therefore, HCP can promote the increase of beneficial bacteria in the intestine, thereby promoting the production of acetic acid, propionic acid, and butyric acid.

[0061] In summary, Houttuynia cordata polysaccharide can regulate the intestinal flora structure to treat T2DM.

[0062] The above embodiments are merely examples of the present invention, and the scope of protection is subject to the claims. Any modification, replacement or improvement based on the core concept of the present invention shall fall within the scope of protection of this patent.

Claims

1. Use of Houttuynia cordata polysaccharide in the preparation of a drug for treating type 2 diabetes.

2. The use according to claim 1, characterized in that The medicine for treating type 2 diabetes is a preparation prepared by taking Houttuynia cordata polysaccharide as an active ingredient and adding pharmaceutically acceptable excipients or auxiliary ingredients.

3. The use according to claim 1, characterized in that The drug for treating type 2 diabetes improves intestinal disorders caused by type 2 diabetes by regulating intestinal flora.

4. The use according to claim 1, characterized in that The preparation method of Houttuynia cordata polysaccharide comprises the following steps: S1. Dry and crush the Houttuynia cordata raw material, and then degrease it by reflux ethanol; S2. Add distilled water to the defatted raw material, adjust the pH, perform ultrasonic extraction, and obtain the supernatant A after centrifugation and filtration; S3. Add complex enzyme to supernatant A for enzymatic hydrolysis. After enzymatic hydrolysis, inactivate the enzyme in boiling water bath and centrifuge to obtain supernatant B. S4. Add ethanol to the supernatant B, let it stand, and centrifuge to obtain the precipitate; S5. The precipitate was redissolved in water and the protein was removed by the Sevag method; S6. Collect the solution after deproteinization, remove the residual solvent, and dry to obtain crude Houttuynia cordata polysaccharide; S7. Dissolve the crude Houttuynia cordata polysaccharide in distilled water, pass it through a DEAE Sepharose FastFlow chromatography column, collect the distilled water eluate and concentrate it; S8. The eluate was purified again by a Sephacryl S-300 gel chromatography column using a 0.1 mol / L NaCl solution for elution. The eluate was collected, dialyzed, and freeze-dried to obtain Houttuynia cordata polysaccharide.

5. The use according to claim 4, characterized in that In step S2, the material-liquid ratio of the defatted raw material and distilled water is 1:(35-40) (W / V), the pH is adjusted to 5, and the ultrasonic time is 40-50 minutes.

6. The use according to claim 4, characterized in that In step S3, the complex enzyme is cellulase and pectinase, with a mass ratio of 2:1; the added amount of the complex enzyme is 1.5-2.0% (w / w).

7. The use according to claim 4, characterized in that In step S3, the enzymatic hydrolysis temperature is 50-55° C., and the enzymatic hydrolysis time is 40-50 min.

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