Application of cordate houttuynia dietary fiber in preparation of medicine for reducing blood sugar

Abstract

The invention discloses application of cordate houttuynia dietary fiber in preparation of a medicine for reducing blood sugar, and belongs to the technical field of biological medicine. The heartleaf houttuynia herb dietary fibers comprise heartleaf houttuynia herb soluble dietary fibers, and the extraction method of the heartleaf houttuynia herb soluble dietary fibers comprises the following steps: sieving heartleaf houttuynia herb rhizomes with a 65-mesh sieve, degreasing, mixing with distilled water, hydrolyzing with cellulase at 50 DEG C, adjusting the pH value to 4.9, carrying out 150rpm for 2 hours, boiling for 15 minutes to inactivate the enzyme, carrying out soluble dietary fiber separation and ethanol precipitation, and drying to obtain the heartleaf houttuynia herb soluble dietary fibers. And drying to obtain the cordate houttuynia soluble dietary fiber. Research results show that intake of the cordate houttuynia soluble dietary fiber can well regulate blood sugar and blood fat of mice and improve intestinal health of the mice, has a good effect on treatment of T2DM, and has potential in treatment of diabetic mice.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to the application of houttuynia cordata dietary fiber in the preparation of medicines for treating hypoglycemia. Background Art

[0002] Houttuynia cordata contains a variety of different bioactive compounds and has received widespread attention for its good medicinal value. Diabetes is a chronic disease that is related to the body's inability to metabolize glucose normally, resulting in atypical high blood sugar levels. The incidence of diabetes has increased dramatically worldwide. The conventional treatment for diabetes is drug therapy, which uses different drug treatment mechanisms to control blood sugar within a certain range and reduce damage to body tissues and organs.

[0003] At present, there are 12 types of traditional glucose-lowering drugs approved for the treatment of type 2 diabetes (T2DM), including biguanides, sulfonylureas, and thiazolidinediones. However, long-term use of such drugs is prone to drug tolerance and resistance, and can also lead to some side effects, making diabetic patients suffer unbearably, which has great limitations. Therefore, finding natural nutrients that can prevent and treat diabetes symptoms without side effects is of great significance for the prevention and treatment of diabetes. Summary of the invention

[0004] The purpose of the present invention is to provide an application of Houttuynia cordata dietary fiber in the preparation of a drug for treating hypoglycemia, so as to solve the problems existing in the above-mentioned prior art. The research results of the present invention show that the intake of SDF can better regulate the blood sugar and blood lipids of mice, improve the intestinal health of mice, and has a good effect on the treatment of T2DM.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] Technical solution 1: Application of Houttuynia cordata dietary fiber in the preparation of drugs for treating hypoglycemia.

[0007] The medicine includes a compound preparation of houttuynia cordata dietary fiber and other blood sugar lowering medicines, or a medicine obtained by mixing houttuynia cordata dietary fiber with a medical carrier or excipient.

[0008] Furthermore, the Houttuynia cordata dietary fiber includes Houttuynia cordata soluble dietary fiber.

[0009] Furthermore, the extraction method of the Houttuynia cordata soluble dietary fiber includes: defatting the Houttuynia cordata rhizome after passing it through a 65-mesh sieve, mixing it with distilled water, hydrolyzing it with cellulase at 50°C, adjusting the pH to 4.9, 150rpm for 2h, boiling it for 15min, separating the soluble dietary fiber and ethanol precipitation, and obtaining the Houttuynia cordata soluble dietary fiber after drying.

[0010] Furthermore, the degreasing treatment is to first soak the Houttuynia cordata powder obtained after passing through a 65-mesh sieve in ethyl acetate at 25°C for 3 hours, filter, centrifuge, then rinse with distilled water to remove the residual organic solvent, and finally dry it at 50°C.

[0011] Technical solution 2: A blood sugar lowering drug, the active ingredient of which includes Houttuynia cordata dietary fiber.

[0012] Furthermore, the Houttuynia cordata dietary fiber includes Houttuynia cordata soluble dietary fiber.

[0013] Technical solution three: A method for preparing a hypoglycemic drug using dietary fiber from Houttuynia cordata, comprising the step of using the dietary fiber in Houttuynia cordata as the active ingredient of the hypoglycemic drug.

[0014] Furthermore, the Houttuynia cordata dietary fiber includes Houttuynia cordata soluble dietary fiber.

[0015] The present invention discloses the following technical effects:

[0016] The present invention extracts total dietary fiber (TDF), soluble dietary fiber (SDF) and insoluble dietary fiber (IDF) from the rhizomes of Houttuynia cordata, and characterizes the physicochemical properties in vitro, and further studies the mechanism of action of soluble dietary fiber (SDF) in treating type 2 diabetes, and compares it with the metformin group and the combined treatment group. The functional groups and microstructure of DF were analyzed by infrared spectroscopy (FT-IR) and scanning electron microscopy (SEM), and it was found that SDF has a loose, porous structure, strong water holding capacity (WHC), oil holding capacity (OHC) and swelling capacity (SC), can improve the cholesterol adsorption and glucose adsorption capabilities, and the effect is better than IDF and TDF; high performance liquid chromatography analysis found that the main monosaccharide in DF is glucose; in diabetic mice, SDF can reduce serum total cholesterol, triglycerides and low-density lipoprotein cholesterol, regulate glucose tolerance and control blood sugar levels, and the effect is better than the metformin group; and And by 16SrRNA analysis, SDF changed the composition of the intestinal flora of diabetic mice, reduced the abundance of Lactobacillus, increased the relative abundance of Intestinimonas, Lachnoclostridium, and Blautia, reduced the ratio of Firmicutes to Bacteroidetes, and the phenotype of intestinal flora changed accordingly, and the effect was consistent with the metformin group and the combined group. The research results of the present invention show that the intake of SDF can better regulate the blood sugar and blood lipids of mice, improve the intestinal health of mice, and have a good effect on the treatment of T2DM. Houttuynia cordata dietary fiber has the potential for the treatment of diabetic mice. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0018] Figure 1 SEM analysis of SDF (a), IDF (b) and TDF (c) at 1000 times and SDF (d), IDF (e) and TDF (f) at 10000 times magnification;

[0019] Figure 2 It is the infrared spectra of SDF, IDF and TDF;

[0020] Figure 3 Effects of three dietary fibers, SDF, IDF and TDF, on glucose (a), cholesterol adsorption (b) and glucose diffusion (c);

[0021] Figure 4 is the effect of each treatment group on the body weight of diabetic mice;

[0022] Figure 5 The effects of each treatment group on blood glucose in diabetic mice;

[0023] Figure 6 The effect of each treatment group on glucose tolerance of diabetic mice;

[0024] Figure 7 The effect of each treatment group on the insulin level of diabetic mice;

[0025] Figure 8 The effects of each treatment group on glycosylated serum protein in diabetic mice;

[0026] Fig. 9 The effects of each treatment group on TC (a), TG (b), LDL-C (c), HDL-C (d) and HDL (e) blood lipids in diabetic mice;

[0027] Fig.10 This is the Ace Index analysis chart;

[0028] Fig.11 For chao1 index analysis;

[0029] Fig.12 This is the Shannon index analysis chart;

[0030] Fig.13 This is the Simpson index analysis chart;

[0031] Fig.14 is the principal coordinate analysis (PCoA) plot;

[0032] Fig.15 is a bar graph of species distribution at the genus level;

[0033] Fig.16 is the species distribution bar graph at the phylum level;

[0034] Fig.17 This is the inter-group analysis chart of LEfSe. DETAILED DESCRIPTION

[0035] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0036] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0037] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0038] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0039] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0040] Example 1

[0041] 1 Materials and methods

[0042] 1.1 Materials

[0043] Thermostable α-amylase, saccharifying enzyme, neutral protease, streptozotocin (STZ), metformin hydrochloride, Beijing Solebow; cellulase, Shanghai Baoman Biotechnology Co., Ltd.; total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C) detection kits, Beijing Solebow; Sinocare blood glucose meter, Hunan Sino Biosensor Co., Ltd.; mouse high-density lipoprotein (HDL) ELISA kit, mouse glycated protein (GSP) ELISA kit, mouse insulin (INS) enzyme-linked immunosorbent assay kit (ELISA), Wuhan Elaruite Biotechnology Co., Ltd.; chloroform, Sinopharm Chemical Reagent Co., Ltd.; Fourier transform infrared spectrometer, Nicoletis5; centrifuge, Eppendorf.

[0044] 1.2 Extraction methods of total dietary fiber, soluble dietary fiber and insoluble dietary fiber from Houttuynia cordata

[0045] Pick fresh rhizomes of Houttuynia cordata, wash with tap water to remove sand and dust, dry in the shade, and wait for use. Cut the roots of Houttuynia cordata into small pieces, grind them in a Chinese herbal medicine grinder 4 times, 5 seconds each time, pass through a 65-mesh sieve, collect the sieved Houttuynia cordata powder, put it in a sealed bag and store it in a cool and dry place for later use. Soak the Houttuynia cordata powder in ethyl acetate (1:4, v / v) at 25°C for 3h, filter, centrifuge, rinse with distilled water to remove residual organic solvents, filter, and dry at 50°C to obtain a defatted sample.

[0046] 1.2.1 Add distilled water to the defatted sample (10.0 g) at a solid-liquid ratio of 1:20 (g / mL). First, place the sample in a boiling water bath for gelatinization for 15 minutes and then shake it in a 50°C water bath for 30 minutes. Adjust the pH of the gelatinized sample to 5.5, add 0.5 g of thermostable α-amylase, and shake it in a 95°C water bath for 1 hour. Then adjust its pH value to 4.2, add 0.03 g of glucoamylase, and incubate it at 60°C for 1 hour. After hydrolysis, adjust the pH to 7.0, add 0.36 g of neutral protease, and enzymolyze it in a 55°C water bath for 2 hours. Finally, place the reaction mixture in a boiling water bath for inactivation for 15 minutes and cool it to room temperature. Add 4 times the volume of 95% ethanol, precipitate at 4°C overnight, filter and rinse, and fully dry the residue to obtain total dietary fiber (TDF).

[0047] 1.2.2 The defatted sample (10.0 g) was added to distilled water at a solid-liquid ratio of 1:20 (g / mL), and hydrolyzed with 0.2% (w / w) cellulase at 50°C, and continuously shaken (150 rpm) for 2 hours at pH 4.9. The enzyme was then inactivated by boiling for 15 min, followed by SDF separation and ethanol precipitation, and dried to obtain soluble dietary fiber (SDF).

[0048] 1.2.3 Weigh 10 g of defatted sample, add distilled water at a solid-liquid ratio of 1:20 (g / mL), adjust the pH value to 1.0 with 0.1 mol / L HCl solution, hydrolyze in a constant temperature water bath at 80°C for 90 min, filter, add 1.4% NaOH solution at a solid-liquid ratio of 1:20 (g / mL) to the residue, alkaline hydrolyze in a constant temperature water bath at 75°C for 80 min, filter, and dry the residue at 70°C to constant weight to obtain insoluble dietary fiber (IDF).

[0049] 1.3 Scanning electron microscopy (SEM) analysis of Houttuynia cordata dietary fiber

[0050] The surfaces of TDF, SDF and IDF were sprayed with metal, and the spatial structure of dietary fiber was analyzed using a scanning electron microscope (USA - Thermo Fisher Scientific - NOVANANOSEM230, EDS X-MAX50) at a magnification of 10000× and a voltage of 5 kV.

[0051] 1.4 FT-IR analysis of Houttuynia cordata dietary fiber

[0052] Infrared spectroscopy analysis was performed on TDF, SDF and IDF samples. The potassium bromide method was used to analyze TDF, SDF and IDF samples in the FT-IR spectrophotometer with a wave number range of 4000-500 cm -1 .

[0053] 1.5 Determination of water holding capacity, oil holding capacity and swelling capacity of dietary fiber

[0054] 1.5.1 Water holding capacity determination

[0055] Using the direct drying method, dietary fiber and distilled water were weighed in a conical flask at a mass ratio of 1:50, shaken at room temperature, and then allowed to stand for 2 h. The mixture was filtered with filter paper of known mass, and the filter paper and dietary fiber residue were weighed and placed in a constant temperature drying oven at 105°C for constant weight. Finally, the mixture was weighed and the measurement was repeated 3 times.

[0056] Water holding capacity (g / g) = (mass of sample after holding water / g - mass of sample before holding water / g) / mass of sample / g.

[0057] 1.5.2 Determination of oil holding capacity

[0058] Weigh dietary fiber and soybean oil in a centrifuge tube at a mass ratio of 1:20, shake well and place in a 37°C water bath for 1 hour, then centrifuge at 4000r / min for 10 minutes, discard the upper oil layer, weigh the residue, and repeat the measurement 3 times.

[0059] Oil holding capacity (g / g) = (mass of sample after oil holding / g - mass of sample before oil holding / g) / mass of sample / g.

[0060] 1.5.3 Determination of expansion force

[0061] Weigh dietary fiber and deionized water in a test tube at a mass ratio of 1:20, record the dry weight volume of the dietary fiber, mix well and let stand at room temperature for 24 hours, and record the wet weight volume of the dietary fiber.

[0062] Swelling force (mL / g) = (volume of sample after water retention / mL - sample volume / mL) / sample mass / g.

[0063] 1.6 Determination of Glucose Adsorption Capacity (GAC) of Dietary Fiber

[0064] Accurately weigh 0.5 g of sample, add 100 mL of glucose solution of different concentrations (10, 50, 100 and 200 mmol / L) respectively, stir evenly and place in a shaker at 37 ° C for 6 hours, take out and centrifuge at 4000 r / min for 20 minutes, then take 2 mL of supernatant, add 3 mL of 3,5-dinitrosalicylic acid (DNS), and determine the glucose content colorimetrically at a wavelength of 540 nm.

[0065] Glucose adsorption capacity (mmol / L) = (glucose concentration before adsorption / glucose concentration after adsorption) × volume of supernatant / sample mass (g).

[0066] 1.7 Determination of cholesterol adsorption capacity (CAC) of dietary fiber

[0067] The o-phthalaldehyde method was used. Egg yolk and distilled water were completely mixed at a solid-liquid ratio of 1:9 (g / mL) to form a uniformly diluted egg yolk solution. 50 g of the diluted egg yolk solution was added to SDF (150 mg), IDF (150 mg) and TDF (150 mg) samples (W). The pH of the mixture was adjusted to 7.0 or 2.0, and then incubated in a shaking water bath at 37°C for 2 hours. The blank was a diluted egg yolk solution without SDF, IDF and TDF. Centrifuge at 1000×g for 10 minutes, and the absorbance of the supernatant was measured at 535 nm. In the diluted egg yolk solution containing SDF, IDF and TDF, the cholesterol level was measured as Cd, while in the diluted egg yolk solution without SDF, IDF and TDF, the cholesterol level was measured as Cb.

[0068] The CAC calculation formula is: CAC (mg / g) = (Cb-Cd) / W.

[0069] 1.8 Determination of glucose diffusion in the glucose-dietary fiber system

[0070] The glucose-dietary fiber system consisted of glucose and 2.5 mg / mL or 10 mg / mL DF. -1 or 10 mg mL -1 TDF, SDF and IDF and blank; 5mL) were dialyzed in 200mL deionized water, pH 7.0, 37°C. The glucose content in 1.0mL solution was measured after 60, 120, 180, 240, 300 and 360 minutes. 200μL of glucose was quickly mixed with 200μL of 5% phenol and 1mL of concentrated sulfuric acid, and the mixture was incubated in the dark for 30 minutes. The absorbance was measured at 490nm, and the glucose concentration was interpolated from the glucose calibration curve. The experimental data were fitted to a parabolic equation to calculate the maximum diffusion velocity (Vmax) of glucose, as follows:

[0071] y=ax 2 +bx+c

[0072] Where y is the glucose content (mg / mL); x is the time (h); a, b, and c are coefficients. The following formula is used to calculate the instantaneous diffusion rate (dy / dx):

[0073] dy=2ax+b

[0074] When x approaches 0, dy=Vmax=b.

[0075] 1.9 Analysis of Monosaccharide Composition of Dietary Fiber

[0076] 10 mg of TDF, SDF and IDF powder were added to 4 mL of 4 mol / L trifluoroacetic acid (TFA) respectively, and hydrolyzed at 110 ° C for 3 h. After the temperature dropped to 25 ° C, 200 μL of methanol was added, and the reaction was repeated three times to remove excess TFA. Subsequently, 1.0 mL of distilled water was added to re-dissolve the dry sample and stored at 4 ° C. Then, a monosaccharide stock solution (1.0 mg / mL) containing mannose, ribose, rhamnose, glucuronic acid, galacturonic acid, arabinose, caramel, galactose and xylose was prepared.

[0077] Then, the sample solution was mixed with 0.3 mol / L NaOH and 0.5 mol / L 1-phenyl-3-methyl-5-pyrazolone (PMP) methanol solution. The mixture was incubated at 70°C for 1 h, and then neutralized with 0.3 mol / L HCl solution. The mixture was extracted with chloroform (1 mL) and centrifuged at 3000×g for 10 min three times to obtain the supernatant. The supernatant was subjected to high performance liquid chromatography, C 18 Column (4.6×200 mm, 5 μm) analysis. Chromatographic detection conditions are 83% dipotassium phosphate (0.05 M) and 17% acetonitrile, temperature 30° C., flow rate 1.0 mL / min, detection wavelength 250 nm.

[0078] Example 2 The hypoglycemic effect of dietary fiber in type 2 diabetes

[0079] 1 Induced type 2 diabetes model mice

[0080] Animal experiment: 4-week-old male C57BL / 6 mice were divided into a healthy group and a diabetic model group after acclimation for 1 week. The healthy group was given a standard diet throughout the experiment. After 1 week of adaptation, they were fed with normal mouse chow (control group, n=8) and high-fat diet (HFD) (model group, n=32).

[0081] After 3 weeks of dietary intervention, mice in the model group were intraperitoneally injected with streptozotocin (STZ) (50 mg / kg, pH 4.5, dissolved in 0.1 M cold citric acid buffer) twice, 72 h apart. Animals in the blank group were injected with an equal volume of citric acid buffer (pH 4.5). Mice were fasted for 12 h before injection. Fasting blood glucose (FBG) of mice in the model group was measured 3 and 10 days after the last STZ injection. If the fasting blood glucose concentration was greater than 11.1 mmol / L after 12 h, the mice were selected as T2DM mice.

[0082] The mice were randomly divided into 5 groups (4 mice per cage, n = 8 mice per group) for analysis: (1) blank group, non-diabetic (NC); (2) diabetic group (DMC); (3) metformin (300 mg / kg BW / d) group (Met); (4) soluble dietary fiber (800 mg / kg BW / d) group (SDF); (5) SDF + metformin (800 mg / kg + 300 mg / kg BW / d) group (SMet). 4-week-old male C57BL / 6J mice [qualification certificate number SCXK (E) 2022-0030, Hubei, China] were purchased from Beijing Weitong Lihua Co., Ltd. All animal experiments were in accordance with the guidelines for animal experiments issued by the National Institutes of Health (NIH, Publication No. 85-23, 1996) and were approved by the ethics committee.

[0083] 2. Sample collection and preparation

[0084] After the 8-week treatment period, all mice were fasted for 12 h, and blood was collected from the eyeballs. The blood samples were placed at room temperature for 2 h and then centrifuged at 4°C, 3500 rpm for 10 min. Serum was collected and stored at -80°C for later use.

[0085] 2.1 Oral glucose tolerance test (OGTT)

[0086] One day before the end of the experiment, mice were fasted for 14 h and then orally administered 20% (w / v) glucose solution at a dose of 2 g / kg. Blood was collected from the tail vein at 0, 30, 60, 90 and 120 minutes after administration. Blood glucose levels were measured using a Sinocare blood glucose meter and recorded as BG0, BG30, BG60, B90 and B120.

[0087] 2.2 Determination of fasting blood glucose and body weight

[0088] During the animal breeding period, the mice were weighed at a fixed time each week and tail blood was collected using a blood glucose meter for fasting blood glucose testing. The mice were fasted overnight the day before the test.

[0089] 2.3 Glycated serum protein (GSP) determination

[0090] Before GSP determination, mice were fasted for 24 h, and orbital venous blood was collected. GSP levels were determined after centrifugation at 1000×g for 15 min. Mouse glycated serum enzyme-linked immunosorbent assay (ELISA) was used to determine GSP levels.

[0091] 2.4 Determination of insulin (INS) levels

[0092] The mouse orbital blood was collected in a centrifuge tube and centrifuged at 4000 g and 4°C for 10 min. The upper serum sample was collected and stored in a refrigerator at -80°C. Mouse insulin (INS) enzyme-linked immunosorbent assay kit (ELISA) was used to measure insulin levels.

[0093] 2.5 Determination of lipid-lowering function indicators

[0094] Kits were used to detect the levels of total blood cholesterol (TC), total serum cholesterol (TG), high-density lipoprotein (HDL), low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) in serum.

[0095] 2.6 Intestinal microbial analysis

[0096] After the experiment, fresh stool samples were collected, colon contents were collected, and 16SrDNA high-throughput sequencing was used to determine the richness of intestinal flora. After sequencing was completed on the machine, the raw data was obtained. Sequence overlap paired-end sequencing data were assembled, and quality control and chimera filtering were performed to obtain high-quality, clean data.

[0097] 2.7 Statistical analysis

[0098] Data are presented as mean ± standard deviation (SD) of three replicates. All graphical representations were generated using GraphPad Prism software (version 7.0, GraphPad software Inc., San Diego, CA, USA). Two groups of data were compared using Student's t test, multiple groups were compared using Tukey's multiple comparison test, one-way ANOVA; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; NS, not significant.

[0099] 3 Experimental results

[0100] 3.1 Preparation of Houttuynia cordata dietary fiber

[0101] In the early stage, the extraction rate of dietary fiber from Houttuynia cordata was analyzed by single factor experiment. The single factor control of the experiment based on temperature, time, material-liquid ratio and pH improved the extraction rate of dietary fiber. The extraction rates of three dietary fibers (TDF, SDF and IDF) extracted from Houttuynia cordata powder by optimized enzymatic hydrolysis and alkaline method were 36.52%, 3.27% and 31.38% respectively.

[0102] 3.2 SEM images of fiber samples

[0103] Scanning electron microscopy observation of dietary fiber can provide a morphological and structural basis for the comparison of its physical and chemical properties such as water holding capacity and swelling capacity. Figure 1 shown. Figure 1 a shows that SDF has a loose, porous, coral-like structure and an uneven surface. Figure 1 b shows that IDF particles exhibit a rod-like structure and more regular morphology with more voids compared to SDF particles. Figure 1 c shows that TDF has an arc-shaped porous structure. This porous structural feature of DF is conducive to the fixation of molecules in the network structure or pores, enhancing the binding ability of DF; and the loose structure of DF increases the adsorption capacity, water holding capacity, oil holding capacity and swelling capacity of cholesterol and glucose. In summary, the results show that SDF after enzymatic hydrolysis has better physiological functions than IDF and TDF.

[0104] 3.3 FT-IR analysis of Houttuynia cordata dietary fiber

[0105] The FTIR spectra of SDF, IDF and TDF are as follows Figure 2 As shown, the wave numbers of the three DFs are almost the same. The recorded wave number is 3261.32 cm -1 、3380.20cm -1 and 3337.40cm -1 The signal reflects the vibration of polysaccharide hydroxyl groups in water, while 1598.50 cm -1 、1647.64cm -1 and 1614.35cm -1 The peak at 1409.87 cm is related to CO- vibration. -1 、1411.45cm -1 and 1417.79cm -1 The peak at 1016.75 cm-1 is probably related to the stretching vibration of C=O. -1 、1004.07cm -1 and 1016.75cm -1 The signal at , which may be the absorbance peak assigned to the COC stretching vibration, indicates that SDF has a typical structure of polysaccharide compounds.

[0106] 3.4 Determination of water holding capacity, oil holding capacity and swelling capacity of dietary fiber

[0107] The degradation degree and physiological function of dietary fiber are closely related to water retention and swelling power. The results of WHC, OHC and SC are shown in Table 1. The WHC and SC of TDF (3.42 g / g, 6.25 mL / g) are significantly higher than those of IDF (1.38 g / g, 3.14 mL / g) (p<0.05). SDF is easily soluble in water and has good WHC and SC. The results showed that the differences between TDF and IDF in WHC and SC may be related to the content of SDF; WHC is beneficial to increase the amount and speed of human defecation, reduce rectal pressure, and is very effective in preventing intestinal diseases; OHC can help control body weight and abnormal blood lipids by interfering with the intestinal absorption of fat; and higher SC is conducive to causing satiety, which increases the volume effect of the intestine and affects the absorption of other components in food.

[0108] Table 1

[0109]

[0110] 3.5 In vitro intestinal function studies of dietary fiber

[0111] 3.5.1 Glucose adsorption capacity (GAC)

[0112] Glucose can attach to the DF network structure, limiting the interaction with the human digestive tract, thereby creating a physical barrier and delaying the diffusion of glucose. Studies have found that Houttuynia cordata DF can effectively adsorb glucose at different glucose concentrations (10-200mM), such as Figure 3 As shown in a, the adsorption capacity of SDF for glucose was significantly higher than that of IDF (p<0.05). Figure 1 The SEM images in the Figure 3 show that enzymatic hydrolysis resulted in smaller particle size of SDF and a larger specific surface area than IDF, which can absorb more glucose. This may be another factor for the better glucose adsorption capacity of SDF. In general, due to the high content of IDF in TDF, no significant difference was found between the glucose adsorption capacity of IDF and TDF (p>0.05). Therefore, SDF has the strongest adsorption capacity for glucose.

[0113] 3.5.2 Cholesterol adsorption (CAC)

[0114] The CAC of DF is classified as physical adsorption or chemical adsorption. Physical adsorption is mainly affected by DF particle size, porosity and temperature, while chemical adsorption is affected by DF charge and hydrophobic groups. CAC was evaluated at two different pH values, 2.0 and 7.0, to simulate cholesterol adsorption in the gastrointestinal system and small intestine environment, respectively. By studying the effect of DF at pH 2.0 and 7.0 on the amount of cholesterol adsorbed ( Figure 3 b). The results showed that at the same pH level, the adsorption capacity of SDF for cholesterol (0.32 and 0.43 mg / g) was significantly higher than that of IDF (0.28 and 0.26 mg / g) and TDF (0.21 and 0.30 mg / g) (p<0.05). SEM analysis showed that SDF had a honeycomb network structure, and the surface and structural properties of SDF enabled it to bind cholesterol molecules at the physical level through adsorption and chemical chelation. Therefore, SDF could balance cholesterol levels and show better CAC.

[0115] 3.5.3 Determination of glucose diffusion in glucose dietary fiber system

[0116] Dietary fiber can not only competitively inhibit the active transport of glucose to reduce postprandial blood sugar, but also increase the viscosity of intestinal contents, slow down the diffusion of glucose, and delay the release of glucose from starch to reduce blood sugar levels. Figure 3c), the results showed that the three types of DF significantly inhibited glucose diffusion at low concentration (2.5 mg / mL) and high concentration (10 mg / mL) (P<0.05). SDF had the lowest glucose diffusion rate, and the SEM image showed that SDF had a rough, porous surface, which may affect the diffusion of glucose. Therefore, SDF and IDF with higher ability to inhibit glucose diffusion may be beneficial for the treatment of diabetes.

[0117] 3.6 Analysis of Monosaccharide Composition of Dietary Fiber

[0118] The monosaccharide composition of TDF, SDF and IDF is shown in Table 2. Glucose is the most abundant monosaccharide in the three DFs (36.62%, 74.53% and 38.32%, respectively), galactose is the second most abundant monosaccharide in SDF, and arabinose is the most abundant monosaccharide in SDF and TDF (10.41% and 11.17%, respectively). Studies have shown that arabinose can treat diabetes by affecting intestinal microbiota and related metabolites. Studies have also shown that a polysaccharide composed of glucose, galactose, arabinose and rhamnose can reduce the blood glucose level of diabetic mice. Therefore, the three types of DF extracted from Houttuynia cordata, especially SDF, are rich in monosaccharides that have therapeutic effects on diabetes and may have a positive effect on the treatment of diabetes.

[0119] Table 2

[0120]

[0121]

[0122] 3.7 The hypoglycemic effect of dietary fiber in type 2 diabetes

[0123] 3.7.1 Effects of drug administration on body weight and blood glucose in diabetic mice

[0124] During the entire experimental period, the body weight and blood glucose levels of diabetic mice were recorded at a fixed time every week to study the effects of Houttuynia cordata dietary fiber on their body weight and blood glucose. Figure 4 As shown in the figure, after 8 weeks of administration, the body weight of mice in the SDF group was significantly lower than that in the DMC group (p<0.05), and its effect on the body weight of diabetic mice reached the same therapeutic effect as that of the Met group, indicating that drug intervention has a significant effect on the decrease in mouse body weight. Figure 5. After the model was established, the blood glucose level of mice was 11.11-16.40mmoL / L, which was 2 times higher than that of healthy mice, indicating that the mouse hyperglycemia model was successfully established. After 8 weeks of oral administration, the administration of SDF, Met and SMet had a significant effect on the reduction of blood glucose in diabetic mice. The diabetic mice in the DMC group still maintained a high blood glucose level of 12.07mmoL / L at the end of the experiment. Compared with the DMC group, the blood glucose levels of the SDF, Met and SMet groups decreased significantly after 8 weeks of administration (p<0.05), and there was no significant difference between the blood glucose of the SDF group and the Met group and the SMet group (p>0.05). Specifically, the blood glucose values ​​of the SDF, Met and SMet groups decreased by 29.08%, 22.78% and 23.18%, respectively. , among which SDF had the best hypoglycemic effect. The results showed that the hypoglycemic effects of SDF and Met were similar. More importantly, SDF is prepared from natural plant resources, and no side effects on diabetic mice were observed in the experiment.

[0125] 3.7.2 Effects of drug administration on glucose tolerance in diabetic mice

[0126] The oral glucose tolerance test is an effective and simple method to detect impaired glucose metabolism. Its results are more accurate than fasting blood glucose alone and can also be used to assess pancreatic β-cell function and glucose metabolism. OGTT is performed on the last day of the experiment. Figure 6 As shown in the figure, after feeding glucose, the blood glucose of the NC group returned to normal 2 hours later, while the blood glucose of the other groups increased sharply 0.5 hours after feeding (p<0.05). Except for the NC group, the mice in the other groups showed obvious glucose intolerance (p<0.05). The glucose tolerance of the SDF group was significantly higher than that of the DMC group (p<0.05), and the glucose tolerance of the SDF group was stronger than that of the Met group and the SMet group. The results showed that the SDF treatment group significantly improved the glucose tolerance of diabetic mice.

[0127] 3.7.3 Effects of drug administration on insulin content in diabetic mice

[0128] STZ selectively destroys pancreatic islet cells, leading to dysfunction of β-cell insulin secretion and inducing hyperglycemia in mice. Soluble dietary fiber slows down the absorption of postprandial glucose by prolonging digestion time, thereby balancing glucose and insulin levels. Figure 7As shown. The study showed that the serum insulin level in the DMC group was significantly higher than that in the NC group, and the difference was statistically significant (p<0.05), indicating that the model was successfully established. After treatment, the serum insulin level in the SDF group was significantly lower than that in the DMC group (p<0.05). And there was no significant difference in the serum insulin level between the SDF group and the Met group and the SMet group (p>0.05). These results show that the adjustment of serum insulin levels in the SDF group is similar to that in the Met group and the SMet group. The intake of SDF can enhance the blood sugar regulation ability of diabetic mice and enhance the sensitivity of target organs to insulin.

[0129] 3.7.4 Effects of drug administration on the content of glycosylated serum protein in diabetic mice

[0130] Glycated serum protein (GSP) value reflects the degree of blood sugar control within 2-3 weeks and is a sensitive indicator of diabetes control. The GSP content in the DMC group was higher than that in the NC group, with a significant difference (p<0.05), indicating that the model was successfully established. After drug treatment, the GSP content in the SDF group was significantly lower than that in the DMC group (p<0.05), and there was no significant difference in GSP content between the SMet group, SDF group and Met group (p>0.05) ( Figure 8 ). These results indicate that SDF has a good ability to reduce the GSP level in diabetic mice, and the effect is similar to that of the Met group and SMet group.

[0131] 3.7.5 Effects of drug administration on blood lipid levels in diabetic mice

[0132] The abnormal secretion of insulin caused by long-term hyperglycemia will further induce metabolic disorders of blood lipids. The synthesis of HDL-C requires insulin to activate lipoprotein lipase, and the decomposition of LDL-C also requires insulin to promote receptor apolipoprotein mediation. The body produces glucose through the decomposition of liver glycogen and converts free fatty acids produced by the deterioration of adipose tissue to obtain metabolic energy. When the consumption rate of sugar slows down, fatty acids become the main source of energy, reducing intracellular glucose utilization and aggravating diabetic symptoms. When the consumption rate of sugar slows down, fatty acids become the main source of energy, reducing intracellular glucose utilization and aggravating diabetic symptoms. Glucose and lipid metabolism are related in many ways, and dyslipidemia in diabetic patients is the most prominent clinical manifestation. The effects of SDF, Met and SMet on TC, TG, LDL-C, HDL and HDL-C levels in diabetic mice are as follows: Fig. 9As shown. Compared with the DMC group, the TC, TG, and LDL-C levels of mice treated with drugs were significantly reduced (p<0.05); the HDL and HDL-C levels were significantly increased (p<0.05). SMet had the best effect in the determination of TC and HDL-C content, and there was no significant difference compared with the SDF group (p>0.05). Met had the best effect in the determination of TG and LDL-C, which was better than the SDF treatment group. SDF had the best effect in the determination of HDL, which was similar to the Met group and SMet group. The results showed that SDF has a good effect in alleviating glucose and lipid metabolism.

[0133] 3.7.6 Intestinal microbial analysis

[0134] 3.7.6.1 Effect of SDF on intestinal flora of hyperglycemic mice

[0135] The results are as follows Figure 10-Figure 14 As shown, the alpha diversity index of intestinal flora diversity showed a significant difference between the NC group and the DMC group (p<0.05). Compared with the DMC group, the SDF-treated group significantly increased the alpha diversity. Comparison between the SDF group and the Met and SMet-treated groups showed that the three drugs had similar improvement effects on the intestinal flora, as evidenced by the Ace, Simpson, Shannon, and Chao 1 indices. This increase indicates that the SDF-treated group restored or even exceeded the intestinal microbial species diversity of diabetic mice. In addition, beta diversity analysis was performed to evaluate the differences between sample groups using Anonim analysis. Principle coordinate analysis (PCoA) based on Bray-Curtis distance revealed a clear separation between the NC group and the diabetic group ( Fig.14 ). Notably, the SDF group showed less variability and higher consistency compared with the DMC group, as evidenced by the smaller range of its 95% confidence ellipse. The first two principal coordinates accounted for 40.65% of the between-group variance. These results suggest that diabetes significantly alters the gut microbiota, which can be effectively offset by dietary fiber. Improved gut microbiota diversity, especially in the SDF group, may contribute to the hypoglycemic effects of these fibers by cultivating beneficial bacteria and producing metabolites that promote glucose transport and metabolism.

[0136] 3.7.6.2 Effects of colonic flora composition in diabetic mice

[0137] At the genus level ( Fig.15), the Lactobacillus in diabetic mice was significantly lower than that in normal mice (p<0.05), while the abundance of Intestinimonas, Lachnoclostridium, and Blautia was significantly increased (p<0.05). After drug feeding, there was a significant difference between the SDF group and the DMC group (p<0.05). The richness of Lactobacillus in the SDF group increased by 155.42%, which was similar to that of the Met group (155.42%) and better than that of the SMet group (121.67%); the richness of Intestinimonas in the SDF group decreased by 111.69%, which was lower than that of the Met group (138.15%) and the SMet group (128.65%); the richness of Lachnoclostridium in the SDF group decreased by 236.50%, which was weaker than that of the SMet group (320.79%) and better than that of the Met group (155.77%); the richness of Blautia in the SDF group decreased by 190.14%, which was weaker than that of the Met group (931.67%) and the Met group (231.95%). The above results show that SDF significantly improved the composition of the above intestinal flora and promoted the richness of the intestinal flora.

[0138] At door level ( Fig.16 ), of which Firmicutes and Bacteroidetes accounted for about 90% of the total community, dominating the intestinal microbiome. The ratio of Firmicutes to Bacteroidetes (F / B ratio) in the DMC group was significantly higher than that in the NC group (p<0.05), and a low F / B ratio is considered to be a possible sign of health. The relative abundance of Firmicutes in the DMC, NC, and SDF groups was 80.77%, 34.12%, and 70.33%, respectively, and the relative abundance of Bacteroidetes was 8.92%, 58.10%, and 16.42%, respectively. The abundance of Firmicutes in DMC was higher than that in the normal group, while that of Bacteroidetes was lower than that in the normal group. The ratio of Firmicutes to Bacteroidetes (F / B) increased from 0.68 (NC) to 13.83 (DMC). After treatment, the F / B value of the SDF group was 5.01, which significantly restored this ratio, and the effect was lower than that of the Met group (3.97) and the SMet group (3.51). The results showed that SDF may play a key role in diabetes-related intestinal diseases by regulating Firmicutes and Bacteroidetes, and has a positive effect on the recovery of damaged intestinal flora in diabetic mice.

[0139] 3.7.6.3 Analysis of LEfSe groups

[0140] Fig.17A comparative analysis of the abundance of gut microbiota in five groups was presented, and potential biomarkers were identified. Certain microbes such as Firmicutes, Clostridia, and Streptococcus were more abundant in T2DM mice. In contrast, potentially beneficial microbes such as uncultured_Erysipelotrichales_bacterium and Lachnospiraceae Faecalibaculum were more prevalent in the group treated with SDF compared to the Met and SMet groups. Studies have shown that people with higher insulin resistance have more bacteria from the Lachnospiraceae family in their intestines, and studies have also found that Erysipelotrichaceae can promote the production of SCFAs, and increased concentrations of SCFAs in the intestine stimulate intestinal L cells to secrete GLP-1, promote the proliferation of pancreatic β cells, and increase their ability to secrete insulin. These results suggest that the SDF group can beneficially improve the structure of the intestinal flora in diabetic mice and thus regulate T2DM.

[0141] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. Application of Houttuynia cordata dietary fiber in the preparation of drugs for treating hypoglycemia.

2. The use according to claim 1, characterized in that: The houttuynia cordata dietary fiber includes houttuynia cordata soluble dietary fiber.

3. The use according to claim 2, characterized in that: The extraction method of houttuynia cordata soluble dietary fiber comprises: houttuynia cordata rhizome is defatted after passing through a 65-mesh sieve, mixed with distilled water, hydrolyzed with cellulase at 50°C, pH adjusted to 4.9, 150rpm for 2h, boiled for 15min, soluble dietary fiber separated and ethanol precipitated, and dried to obtain the houttuynia cordata soluble dietary fiber.

4. The use according to claim 3, characterized in that: The degreasing treatment is to soak the Houttuynia cordata powder obtained after passing through a 65-mesh sieve in ethyl acetate at 25° C. for 3 hours, filter it with suction, centrifuge it, rinse it with distilled water to remove the residual organic solvent, and finally dry it at 50° C.

5. A drug for lowering blood sugar, characterized in that: The active ingredients include Houttuynia cordata dietary fiber.

6. The drug according to claim 5, characterized in that The houttuynia cordata dietary fiber includes houttuynia cordata soluble dietary fiber.

7. A method for preparing a hypoglycemic drug using Houttuynia cordata dietary fiber, characterized in that: The method comprises the step of using the dietary fiber in Houttuynia cordata as the active ingredient of the blood sugar lowering drug.

8. The method according to claim 7, characterized in that The houttuynia cordata dietary fiber includes houttuynia cordata soluble dietary fiber.

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