CN110664834A

CN110664834A - Application of tartary buckwheat polysaccharide in preparation of medicine for treating non-alcoholic fatty liver disease

Info

Publication number: CN110664834A
Application number: CN201911111361.2A
Authority: CN
Inventors: 任强; 张立涛; 王建安; 王慧云
Original assignee: JINING MEDICAL COLLEGE
Current assignee: JINING MEDICAL COLLEGE
Priority date: 2019-11-14
Filing date: 2019-11-14
Publication date: 2020-01-10

Abstract

The invention relates to an application of tartary buckwheat polysaccharide in preparing a medicine for treating non-alcoholic fatty liver disease, which is characterized in that: the Fagopyrum tataricum polysaccharide is extract obtained from root of Fagopyrum tataricum of Fagopyrum of Polygonaceae by conventional water extraction method, and at least comprises the Fagopyrum tataricum polysaccharide in various dosage forms for preparing medicine for treating non-alcoholic fatty liver disease. The tartary buckwheat polysaccharide is an active ingredient which is purely natural, plant-derived, non-toxic and non-irritant and can obviously inhibit the non-alcoholic fatty liver.

Description

Application of tartary buckwheat polysaccharide in preparation of medicine for treating non-alcoholic fatty liver disease

Technical Field

The invention relates to the technical field of biology, in particular to application of tartary buckwheat polysaccharide in preparation of a medicine for treating non-alcoholic fatty liver disease.

Background

NAFLD (non alcoholic fatty liver disease) is a clinical pathological syndrome with liver histological changes similar to alcoholic liver disease but without excessive drinking history, with the improvement of living level and dietary structure of Chinese people, the increase of fatty substances taken by people from food and the movement mode of hypo-motility and multi-sitting lead to the increasing of the incidence rate of non-alcoholic fatty liver disease, the non-alcoholic fatty liver disease is a disease spectrum, the pathological changes are from light to heavy, namely 4 different pathological stages of hepatic steatosis, steatohepatitis and hepatic cirrhosis, the pathological features of the pathological stages are the same as that of alcoholic fatty liver disease, but without long-term excessive drinking history, the individual metabolic syndrome is the largest risk factor of the non-alcoholic fatty liver disease, the incidence rate of the non-alcoholic fatty liver disease and arterial sclerosis is increased, obesity, hyperglycemia, dyslipidemia and hypertension, the incidence rate of the non-alcoholic fatty liver disease and the arterial sclerosis, heart failure and nephropathy are closely related to 356326%, the incidence rate of the non-alcoholic liver disease is nearly 3560%, and the incidence rate of 3670% of the non-alcoholic liver disease is nearly 35% of 3670% of women in the age of ~ year-old people.

Although many drugs for the prevention and treatment of non-alcoholic fatty liver disease are being developed, no specific drug is currently available for the clinical treatment of non-alcoholic fatty liver disease. Because fatty liver does not exist as an independent disease, the treatment of fatty liver usually aims at the primary causes of the fatty liver according to the causes of the fatty liver, such as excessive alcohol intake, obesity, diabetes and the like, and the drugs only play a role in adjuvant therapy by keeping physical exercise and reasonable diet. Lipid lowering drugs are usually used to control obesity and improve dyslipidemia to alleviate non-alcoholic fatty liver disease, but these drugs have significant side effects. While fatty liver is treated, toxicity of heart and kidney may be increased, and some of them may have the effect of aggravating metabolic burden of liver and increasing deposition of lipid in liver. Therefore, the search for safe and effective therapeutic drugs has certain social value.

Radix Et rhizoma Fagopyri Tatarici (radix Et rhizoma Fagopyri Tatarici)Fagopyrum tataricum(L.) Gaertn) is an annual herb plant of the genus Fagopyrum of the family Polygonaceae, and is described in compendium of materia Medica: tartary buckwheat is bitter, mild and cold in nature and taste. Has effects of invigorating qi, refreshing mind, benefiting ears and eyes, lowering qi, relieving intestinal obstruction, and invigorating stomach. Meanwhile, the tartary buckwheat is also a small coarse cereal crop and a medicinal and edible homologous plant with rich nutritive value, and has unique dietary therapy and health care functions. The tartary buckwheat is sourced from China, the resources are rich, and the China has a habit of eating the tartary buckwheat.

The polysaccharide is a natural macromolecular active substance formed by connecting a large number of monosaccharides through glycosidic bonds, and becomes a new direction for the development of current food, medicine and functional health care products due to the wide source, diversity of biological functions and no toxic or side effect. The polysaccharide has good effect in reducing blood sugar, and mainly can promote insulin secretion, improve insulin sensitivity and sugar, protein and lipid metabolism, promote glycogen synthesis, and increase organism immunity and oxidation resistance and clearance in organism. So far, no report about the application of the tartary buckwheat polysaccharide in treating the non-alcoholic fatty liver disease exists.

Disclosure of Invention

The invention aims to solve the technical problem of providing the application of the tartary buckwheat polysaccharide in preparing the medicine for treating the non-alcoholic fatty liver disease.

In order to solve the problems, the application of the tartary buckwheat polysaccharide in preparing the medicine for treating the non-alcoholic fatty liver disease is characterized in that: the Fagopyrum tataricum polysaccharide is extract obtained from root of Fagopyrum tataricum of Fagopyrum of Polygonaceae by conventional water extraction method, and at least comprises the Fagopyrum tataricum polysaccharide in various dosage forms for preparing medicine for treating non-alcoholic fatty liver disease.

The tartary buckwheat polysaccharide at least comprises the tartary buckwheat polysaccharide in the preparation of the medicine for reducing the body weight of the body with the non-alcoholic fatty liver disease.

The tartary buckwheat polysaccharide at least comprises the tartary buckwheat polysaccharide in the preparation of the medicine for reducing the lipid deposition in the liver of the body with the non-alcoholic fatty liver disease.

The tartary buckwheat polysaccharide at least comprises the tartary buckwheat polysaccharide in the preparation of the medicine for reducing the contents of TG and TC in liver and plasma of a body with the non-alcoholic fatty liver disease.

The tartary buckwheat polysaccharide at least comprises the tartary buckwheat polysaccharide in the preparation of the medicine for reducing AST and ALT of the organism plasma of the non-alcoholic fatty liver disease.

The tartary buckwheat polysaccharide at least comprises the tartary buckwheat polysaccharide in the preparation of the medicine for reducing the LDL-C level of the plasma of the body with the non-alcoholic fatty liver disease.

The tartary buckwheat polysaccharide at least comprises the tartary buckwheat polysaccharide in the preparation of the medicine for improving the level of the liver and plasma HDL-C of the body with the non-alcoholic fatty liver disease.

The tartary buckwheat polysaccharide at least comprises the tartary buckwheat polysaccharide in the preparation of the medicine for improving the lipid metabolism disorder.

The dosage of the tartary buckwheat polysaccharide is 150 ~ 200 mg/kg/day.

Compared with the prior art, the invention has the following advantages:

1. the tartary buckwheat polysaccharide is an active ingredient which is purely natural, plant-derived, non-toxic and non-irritant and can obviously inhibit the non-alcoholic fatty liver.

2. The experiment of the invention proves that the tartary buckwheat polysaccharide can obviously reduce the weight of a non-alcoholic fatty liver model mouse, can obviously improve the hepatocyte damage caused by the non-alcoholic fatty liver and reduce the lipid accumulation in the liver. The lipid metabolism physiological and biochemical indexes such as Triglyceride (TG), cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), alanine Aminotransferase (ALT), glutamic-oxalacetic transaminase (AST) and the like are obviously improved, and the tartary buckwheat polysaccharide has an important effect on treating the non-alcoholic fatty liver.

The experimental procedures used in ⑴ ~ ⑷ below are all conventional unless otherwise specified.

Materials, reagents and the like used in ⑴ ~ ⑷ described below are commercially available unless otherwise specified.

⑴ ~ ⑷, the extraction and purification of the tartary buckwheat polysaccharide comprises the steps of crushing and soaking 5.0kg of tartary buckwheat, extracting with hot water, wherein the material-liquid ratio is 1: 10, the extraction time is 1h, filtering to obtain filtrate, adding hot water into residues for re-extraction, extracting for multiple times until the reaction of sugar in the extracting solution is not obvious through a sulfuric acid-phenol method, combining the filtrates, concentrating to 10 ~ 20% of the original volume, adding absolute ethyl alcohol until the concentration of the ethyl alcohol is 80%, standing overnight in a cold place, centrifuging, collecting precipitates, passing the precipitates through a 10 x 100cm macroporous resin chromatographic column, enabling the height of a D101 macroporous resin filler to be 65cm, enabling the sample loading amount to be 30g, enabling 500mL to be one collecting unit, enabling the flow rate to be 25mL/min, carrying out chromatography, and carrying out freeze drying to obtain the tartary buckwheat polysaccharide, wherein the tartary buckwheat polysaccharide consists of 9 monosaccharides, and the molecular weight is 1293 ~ 36973 Da.

[ Infrared Spectrum Scan ] an infrared spectrogram of the tartary buckwheat polysaccharide is shown in figure 1, and functional groups corresponding to each peak are detailed in Table 1.

TABLE 1 summary of the functional groups corresponding to the peaks of the Fagopyrum tataricum polysaccharide

[ monosaccharide analysis ] monosaccharide components were determined by HPLC, weighing 5mg polysaccharide samples in 5mL tubes with plug scales, adding 1mL of 2mol/L TFA, oven hydrolyzing at 121 ℃ for 2h, diluting to 50mL with water, filtering with 0.45 μm microporous membrane, and analyzing by injection. Using an ICS-5000 ion chromatograph (dean, usa), column: CarboPac PA20, flow rate: 0.5mL/min, detector: a pulsed amperometric detector. Chromatograms of monosaccharide standard (A) and hydrolyzed sample of Fagopyrum tataricum polysaccharide (B) are shown in FIG. 2.

The monosaccharide components in the tartary buckwheat polysaccharide are analyzed by high performance liquid chromatography and comprise: fucose of 0.77%, arabinose of 4.80%, rhamnose of 13.13%, galactose of 21.35%, glucose of 31.41%, xylose of 2.22%, mannose of 3.8%, galacturonic acid of 21.04%, glucuronic acid of 1.49%.

[ polysaccharide molecular weight determination ] using a Waters1525 high performance liquid chromatograph (with 2414 refractive index detector and Empower 3 workstation); a chromatographic column: an Ultra hydrogel ™ Linear (300 mm. times.7.8 mm); mobile phase: 0.1MNaNO 3; flow rate: 0.9 mL/min; column temperature: 45 ℃; sample preparation: the sample is dissolved in the mobile phase, filtered by a microporous filtering membrane and then fed.

Standards for molecular weight calibration curves: MW135350, MW36800, MW9750, MW2700, MW180, as shown in fig. 3, molecular weight results shown in table 2.

TABLE 2 molecular weight analysis of the total polysaccharides from Fagopyrum tataricum

The invention adopts a cell model and an animal model to investigate the improvement effect of the tartary buckwheat polysaccharide on the metabolic syndromes such as non-alcoholic fatty liver, obesity, lipid metabolism disorder and the like.

⑴ in vitro HepG2 cell model experiment:

1) preparing a reagent:

molding liquid: accurately weighing 12.8mg of palmitic acid and 14.1mg of oleic acid respectively, dissolving in 5mL of Phosphate Buffer Solution (PBS) in a water bath until the final concentration is 10 mM; then, a molding solution was prepared at a ratio of palmitic acid to oleic acid =2:1, and then filtered through a 0.45 μm filter to sterilize the molding solution.

Oil red stock solution, weighing 500mg of oil red, transferring 80mL of isopropanol, putting a beaker into a water bath kettle at 37 ℃ for dissolving, carrying out constant volume and refrigerating for storage, mixing the oil red stock solution and water = 3: 2 before use, standing for 5 ~ 10min, and filtering with a 0.45 mu m filter membrane when in use.

And (3) a tartary buckwheat polysaccharide solution: 0.1g of tartary buckwheat polysaccharide is weighed and dissolved in 10mL of PBS, and the final concentration after dissolution is 1000 mg/mL.

2) Cell culture:

HepG2 cells were cultured in RPMI medium supplemented with 10% fetal bovine serum at 37 ℃ with 5% CO₂Culturing is carried out under the environment.

3) Influence of tartary buckwheat polysaccharide on cell proliferation activity of HepG 2:

adding the cells into a 96-well cell culture plate, culturing for 24h, adding tartary buckwheat polysaccharide solutions with different concentrations, and culturing for 24h, and detecting the cell activity by an MTT method.

4) Establishing a non-alcoholic fatty liver model and carrying out administration treatment:

after the cells adhere to the wall, HepG2 cells are treated by a molding solution with the final concentration of 1mM for 24 hours, and oil red staining is carried out to determine whether molding is successful. Then, the cell model for constructing the non-alcoholic fatty liver is treated by adopting low concentration (15 mug/mL) and high concentration (1000 mug/mL) of tartary buckwheat polysaccharide, and after 24 hours, the collected cells are frozen at-80 ℃ for standby.

5) Detection of physiological and biochemical indexes:

after the cells are collected and ultrasonically broken, physiological and biochemical indexes such as TG, TC, LDL-C, HDL-C, ALT, AST and the like are measured according to the operation steps in the kit instruction.

6) Analysis of key gene expression:

after collecting cells, total RNA is extracted by using a Trizol method, then cDNA is synthesized by using a Takara reverse transcription kit, and the expression of genes related to fat de novo synthesis is detected by adopting a fluorescence quantitative method.

7) The statistical method comprises the following steps:

all data are presented as mean ± sem, plotted using GraphPad Prism 5 software, and variance determined by one-way anova at p <0.05 levels.

Analysis of experimental results of in vitro HepG2 cell model:

1) HepG2 cell culture status:

HepG2 cells were normal in morphology, intact in structure, almost free of any cell debris, epithelial-like in morphology, grown in monolayer and small aggregated form on the body wall, and the growth state of the cells was good as shown in FIG. 4.

2) Effect of fagopyrum tataricum polysaccharide on cell proliferation viability of HepG 2:

the MTT method is used for detecting the influence of the tartary buckwheat polysaccharide on the cell proliferation of HepG2, and the results are shown in figure 5, wherein the influence of the tartary buckwheat polysaccharide with different concentrations on the cell proliferation capacity is different and is more than 90% of the proliferation activity of a control group. When the concentration is higher than 62.5 mu g/mL, the cell proliferation activity is obviously improved, and no significant difference exists compared with a control group (p is larger than 0.05), and the result shows that the tartary buckwheat polysaccharide has no toxic effect on HepG2 cells, but can enhance the cell proliferation activity.

3) Non-alcoholic fatty liver HepG2 cell model staining analysis:

after oil red dyeing is adopted, compared with a control group, more red fat drops appear in the model group, the range is wider, and the success of model construction is shown; the number of red lipid droplets in the cells treated by the tartary buckwheat polysaccharide is obviously reduced, and the result shows that the tartary buckwheat polysaccharide can obviously reduce the accumulation of fat as shown in figure 6.

4) Effect of fagopyrum tataricum polysaccharide on fat content in HepG2 cells:

fat in the human body exists mainly in the forms of TG and TC. The content in the cells is measured by using the kit, the change of fat in the cells can be shown, and the result is shown in figure 7, the TG and TC contents in the model group are respectively 656.4nmol/mg total protein and 978.8nmol/mg total protein, and compared with the control group (235.6 nmol/mg total protein and 166.8nmol/mg total protein), the content is respectively and significantly increased by 178.6% and 486.8% (p is less than 0.05), which indicates that fat accumulation occurs in the non-alcoholic fatty liver cell model, and indicates that the model is successfully constructed. After the tartary buckwheat polysaccharide is adopted for treatment, the content of TG and TC is obviously reduced, the content of TG in low-concentration cells is 356.6nmol/mg total protein, and the content of TC is 372.6nmol/mg total protein; and compared with a control group, the content of TG in a high-concentration group is 334.2 nmol/mg total protein, the content of TC in the high-concentration group is 541.4nmol/mg total protein, and no significant difference exists, so that the tartary buckwheat polysaccharide can effectively relieve fat accumulation in cells.

5) Effect of fagopyrum tataricum polysaccharide on lipoprotein content in HepG2 cells:

the LDL-C content in the control group is 226.6 nmol/mg total protein, while the LDL-C content in the cells of the model group is 785.5nmol/mg total protein, which reaches 3.5 times of that of the control group, and significant difference (p < 0.05) appears, the LDL-C content in the tartary buckwheat polysaccharide group is reduced compared with that of the model group, wherein the LDL-C content in the low-concentration polysaccharide group is 362.4 nmol/mg total protein, and the LDL-C content in the high-concentration polysaccharide group is 260 nmol/mg total protein (figure 8A). However, for HDL-C, the HDL-C content was significantly lower in the model group than in the control group, but was significantly higher in the Fagopyrum tataricum polysaccharide group (FIG. 8B). The results show that the tartary buckwheat polysaccharide can effectively reduce the content of LDL-C in cells, prevent cholesterol from being accumulated in blood vessels in the transportation process, reduce atherosclerotic lesion and liver fat degeneration, and simultaneously can improve the content of HDL-C in the cells and promote the metabolism of the cholesterol in the liver.

6) Effect of fagopyrum tataricum polysaccharide on ALT and AST activity in HepG2 cells:

in the ALT assay, the control cells contained 132U/g total protein, whereas the model cells contained a reduction in ALT content of 29U/g total protein, which was 25% of the control (FIG. 9A); in the AST detection, the AST content in the control group is 223.4U/g total protein, and the AST content in the model group is 4U/g total protein, so that the AST content is reduced by 55 times (FIG. 9B); the results indicated that the liver was damaged in the model group. The concentrations of the two enzymes are improved in the tartary buckwheat polysaccharide group, the ALT content in the low-concentration tartary buckwheat polysaccharide group cell is 80.9U/g total protein, the AST content is 92U/g total protein, the ALT content in the high-concentration tartary buckwheat polysaccharide group cell is 59.3U/g total protein, and the AST content is 28.1U/g total protein, and the result shows that the tartary buckwheat polysaccharide has a good protection effect on the liver cell, and can effectively relieve the inflammation degree of the liver cell and the formation of fatty liver.

7) Effect of tartary buckwheat polysaccharide on fat synthesis genes:

in the non-alcoholic fatty liver disease model group, the gene expression levels of closely related genes SREBP-1, ACLY, ACC and FASN of fat de novo synthesis are significantly increased compared with the control group, and the polysaccharide group of tartary buckwheat is reduced to different degrees, and is even lower than the control group, so that the de novo synthesis of fat is greatly inhibited, and the effect of treating the non-alcoholic fatty liver disease is achieved (figure 10).

⑵ in vitro BEL7404 cell model experiment:

1) preparing a reagent: the same experiment as HepG2 cells.

2) Cell culture: the same experiment as HepG2 cells.

3) Establishing a non-alcoholic fatty liver model and carrying out administration treatment: the same experiment as HepG2 cells.

4) Detection of physiological and biochemical indexes: the same experiment as HepG2 cells.

5) The statistical method comprises the following steps: the same experiment as HepG2 cells.

In vitro BEL7404 cell model experiment result analysis:

1) BEL7404 cell culture state:

BEL7404 cells have normal shape, complete structure, almost no cell fragments, epithelial cell-like shape, and adherent growth in small aggregates in the form of blocks, and the growth state of the cells is good and has no pollution, and can be used for further experiments as shown in FIG. 11.

2) Effect of fagopyrum tataricum polysaccharides on cell viability of BEL 7404:

the MTT method is used for detecting the influence of the tartary buckwheat polysaccharide on the BEL7404 cell proliferation, the results are shown in figure 12, the influence of different concentrations of the tartary buckwheat polysaccharide on the cell proliferation capacity is different, except for low concentrations (15.625 mu g/mL and 31.25 mu g/mL), the high concentration group can increase the cell viability, statistical analysis shows that the cell proliferation viability can be obviously enhanced (p is less than 0.05) when the concentration of the tartary buckwheat polysaccharide is 250 mu g/mL and 500 mu g/mL, and the results show that the tartary buckwheat polysaccharide hardly has any toxic effect on the BEL7404 cell, but can enhance the cell proliferation viability.

3) Analysis of hyperlipidemic cell model staining:

for the constructed BEL7404 cell high fat model, more red fat drops appear in the model group and the range is wider than that in the control group after oil red staining is adopted, so that the model construction is proved to be successful; the number of red lipid droplets was significantly reduced after treatment with the tartary buckwheat polysaccharide, indicating that the tartary buckwheat polysaccharide can significantly reduce the accumulation of fat as shown in fig. 13.

4) Influence of tartary buckwheat polysaccharides on fat content in cells:

the results are shown in fig. 14, where TC and TG contents were 2163.86nmol/mg total protein and 487.3nmol/mg total protein in the model group, respectively, and 671.5nmol/mg total protein and 177.5nmol/mg total protein were significantly increased by 3.22-fold and 2.75-fold, respectively, compared to the control group (p < 0.05), indicating that fat accumulation occurred in the hyperlipidemic cell model. In the tartary buckwheat polysaccharide group, the TC low-concentration group has the content of 802.4nmol/mg total protein, the TC high-concentration group has the content of 845.3nmol/mg total protein, and the TC low-concentration group and the TC high-concentration group are reduced by 2.7 times and 2.6 times relative to the model group; the content of the TG low-concentration group is 179.9nmol/mg total protein, the content of the TG high-concentration group is 267.8nmol/mg total protein, and the content is reduced by 2.7 times and 1.8 times respectively relative to the model group, and experimental results show that the tartary buckwheat polysaccharide can effectively relieve fat accumulation in cells.

5) Influence of tartary buckwheat polysaccharides on lipoprotein content in cells:

the HDL-C content in the model group is greatly reduced compared with that in the control group, the HDL-C content in the low-concentration group is remarkably increased (p is less than 0.05) compared with that in the control group, and the HDL-C content in the high-concentration group is also remarkably increased (figure 15A). While the control group contained 204.2nmol/mg total protein of LDL-C, the model group contained 486.1nmol/mg total protein of LDL-C, and showed a significant increase (p < 0.05), while the Fagopyrum tataricum polysaccharide group contained decreased LDL-C, the low concentration group contained 318.6nmol/mg total protein, and the high concentration group contained 298.9nmol/mg total protein, as compared to the model group (FIG. 15B). The results show that the tartary buckwheat polysaccharide can improve the content of HDL-C in cells and promote the metabolism of cholesterol in the liver. Meanwhile, the content of LDL-C in cells can be effectively reduced, cholesterol is prevented from being accumulated in blood vessels in the transportation process, and atherosclerotic lesions and liver fat degeneration are reduced.

6) Effect of fagopyrum tataricum polysaccharide on AST and ALT activity in cells:

in the AST detection (FIG. 16A), the AST content in the cells of the control group is 205.8U/g total protein, the AST content in the cells of the model group is 63.2U/g total protein, the AST content is reduced by 3.2 times, and the significant reduction (p < 0.05) is generated; in the ALT assay (FIG. 16B), the amount of total protein in the control cells was 43.5U/g, while the ALT content in the model cells decreased to 6.4U/g total protein, which was 14.7% of the control cells, with a significant decrease (p < 0.05) indicating liver damage in the model. The concentration of the two enzymes in the tartary buckwheat polysaccharide group is increased, the AST content in the cells of the low-concentration group is 117.2U/g total protein, the ALT content is 19.8U/g total protein, the AST content in the cells of the high-concentration group is 77.1U/g total protein, and the ALT content is 11.8U/g total protein, and the result shows that the tartary buckwheat polysaccharide has a better protective effect on the liver cells.

⑶ in vivo animal model experiment:

1) preparation of high-fat diet-induced non-alcoholic fatty liver model mice:

the invention adopts a method that C57BL/6 mice are fed with high-fat feed for 12 weeks to establish a non-alcoholic fatty liver animal model, and the curative effect of the non-alcoholic fatty liver animal model is verified by a gavage tartary buckwheat polysaccharide mode.

The specific process comprises the following steps: in the experiment, 42 healthy C57BL/6 male mice are randomly divided into a control group, a high-fat feed model group and a tartary buckwheat polysaccharide group, and the high-fat feed model group and the high-fat feed of the tartary buckwheat polysaccharide group are fed for 12 weeks to construct a high-fat model mouse. The Fagopyrum tataricum extract is administered by gavage for 1 time/day, and each mouse is 250 mg/kg. The model group and the blank control group are given with physiological saline with corresponding volume, and blood is taken by cutting head after 1 month of gastric lavage, and blood plasma is taken by centrifugation and is reserved at minus 80 ℃. Picking up the whole liver, washing blood stain with normal saline for 2 times, placing on filter paper, completely absorbing water, and keeping at-80 ℃ for later use.

2) Change in body weight of mice:

c57BL/6 male mice were fed with high-fat diet for 12 weeks, and a non-alcoholic fatty liver disease model was successfully constructed. The mass of the model group, the control group (given distilled water with the same volume) and the tartary buckwheat polysaccharide group is measured after 45 days of gastric lavage, the result is shown in figure 17, the weight of the non-alcoholic fatty liver model group mice is increased by nearly 1 time compared with the control group, the significant difference occurs, the weight of the tartary buckwheat polysaccharide group mice does not obviously increase, and the experimental result shows that the tartary buckwheat polysaccharide can reduce the weight of the non-alcoholic fatty liver mice.

3) Results of pathological tissue section of non-alcoholic fatty liver:

liver tissue sections of the model group and the tartary buckwheat polysaccharide group are subjected to oil red staining, and the result shows that the red staining of the liver of the tartary buckwheat polysaccharide group is obviously lower than that of the model group (figure 18), which indicates that the tartary buckwheat polysaccharide has the function of treating the non-alcoholic fatty liver.

4) Detection of physiological and biochemical indexes in mouse liver:

0.05 g of mouse liver was taken, 0.5mL of lysate was added thereto and homogenized, and then centrifuged at 8000 rpm for 10min to obtain the supernatant. The physiological and biochemical indexes such as TG, TC, LDL-C, HDL-C, ALT, AST and the like are detected according to the steps of a kit instruction.

Analyzing the experimental result of the liver of the animal model mouse:

1) influence of tartary buckwheat polysaccharide on fat content in liver:

the TG content in the liver of the control group was 600.4nmol/mg of total protein, and the TG content in the model group was 1794.9nmol/mg, which was 3 times that of the control group (FIG. 19A); the TC content was 467.8nmol/mg, and 1390nmol/mg in the model group was 2.9 times that in the control group (FIG. 19B); a significant increase (p < 0.05) was observed (fig. 19B), indicating successful construction of non-alcoholic fatty liver animal model. In the tartary buckwheat polysaccharide group, compared with the model group, the contents of TG and TC are obviously reduced, the content of TG is 1264nmol/mg total protein (figure 19A), and the content of TC is 1134nmol/mg total protein (figure 19B), which shows that the tartary buckwheat polysaccharide can effectively relieve the deposition of lipid in the liver.

2) Influence of tartary buckwheat polysaccharide on lipoprotein content in mouse liver:

the LDL-C content in the liver of the model group is obviously reduced (p is less than 0.05), while the LDL-C content in the tartary buckwheat polysaccharide group is increased, which shows that the tartary buckwheat polysaccharide can promote the transportation of cholesterol and is beneficial to the reduction of the cholesterol content in the liver (figure 20). The content of the HDL-C control group in the liver is 41.7nmol/mg total protein, the content of the model group in the liver is 9.7nmol/mg total protein (figure 21), and the content is remarkably reduced (p is less than 0.05); the content of HDL-C in the tartary buckwheat polysaccharide group is increased, which shows that the tartary buckwheat polysaccharide can effectively relieve the deposition of cholesterol and the like in the liver, so that the high-density lipoprotein level in liver cells is basically recovered to be normal, the metabolic transport of the cholesterol is facilitated, and the liver can better play a role.

3) Effect of tartary buckwheat polysaccharides on ALT and AST activity in mouse liver:

as shown in FIG. 22, the ALT content in the liver of the control group was 108.7U/g total protein, the ALT content in the liver of the model group was 97U/g total protein, and no significant decrease was observed, but the AST content in the control group was 113.3U/g total protein, and the AST content in the liver of the model group was 65.9U/g total protein, indicating that the liver of the mouse with successful modeling had relatively severe damage. In the tartary buckwheat polysaccharide group, the contents of the two enzymes are increased, the ALT is 130.6U/g total protein, and the AST is 95.9U/g total protein, and the result shows that the tartary buckwheat polysaccharide has stronger capability of restoring the liver function.

Analysis of animal model mouse plasma experiment results:

1) effect of tartary buckwheat polysaccharides on lipoprotein content in mouse plasma:

the content of plasma HDL-C in the control group is 0.13 mmol/mg total protein, the content in the model group is 0.04 mmol/mg total protein, and the content is remarkably reduced (p is less than 0.05); the HDL-C content was increased in the Fagopyrum tataricum polysaccharide group (FIG. 23A). The LDL-C content in the model group was significantly increased (p < 0.05), while the LDL-C content in the tartary buckwheat polysaccharide group was decreased (FIG. 23B).

2) Effect of tartary buckwheat polysaccharides on fat content in mouse plasma:

the TC content in the plasma of the control group is 1.03 mmol/mg, and the TC content in the model group is 3.2 mmol/mg, which is 3 times of that of the control group (FIG. 24A); the TG content in the plasma of the control group is 1.8 mmol/mg total protein, the TG content in the model group is 2.4 mmol/mg, and the TG content reaches 1.3 times of that of the control group (figure 24B); significant increase (p < 0.05) was observed (fig. 23), indicating successful construction of non-alcoholic fatty liver animal model. In the tartary buckwheat polysaccharide group, the content of TC is 1.8 mmol/mg total protein (figure 24A), and the cholesterol content is obviously reduced compared with that of the model group; TG content was 0.3 mmol/mg total protein (fig. 24B), significantly reduced compared to control (p < 0.05). The tartary buckwheat polysaccharide can effectively relieve the deposition of lipid in blood plasma and effectively reduce the content of blood fat.

3) Effect of fagopyrum tataricum polysaccharide on AST and ALT activity in mouse plasma:

the content of AST in the plasma of the control group is 22.5U/L, the content of AST in the model group is 69U/L, and the model group has a significant increase (p < 0.05) compared with the control group (FIG. 25A); the ALT content in the plasma of the control group is 25.5U/L, the ALT content in the model group is 40U/L, and the ALT content in the model group is obviously increased (figure 25B), which indicates that the liver of the mice successfully molded is seriously damaged, and the AST and ALT content in the plasma is increased. In the tartary buckwheat polysaccharide group, the contents of the two enzymes are reduced, AST is reduced to 27.9U/L, and ALT is reduced to 18.2U/L.

⑷ evaluation of toxicity of tartary buckwheat polysaccharide:

1) feeding zebra fish and collecting embryos:

the method comprises the steps of breeding 4-month-old wild AB zebra fishes in an environment at 28.0 +/-1 ℃, ensuring 14 hours of light and 10 hours of darkness and pH =6.8 ~.5, feeding the wild AB zebra fishes twice a day, feeding solid feed and hatched shrimp larvae at 8 am and 17 pm respectively every time, feeding the bred zebra fishes of the proper age after 15 minutes, separating and breeding the bred zebra fishes in advance for one week, feeding the bred zebra fishes and the hatched shrimps according to the ratio of 1:1 of male ratio after feeding the bred zebra fishes and 14 pm of the day before spawning, separating the bred zebra fishes and the hatched shrimps by a transparent plastic partition plate, drawing out the middle partition plate at eight am next day to enable the bred zebra fishes and the bred zebra fishes to freely mate and spawn, collecting healthy eggs produced in 30min, cleaning the eggs at the moment by using an E3 culture medium, removing impurities such as excrement, feed and the like, removing unfertilized fish eggs with poor quality, uniformly cleaning the selected fish eggs, placing the cleaned eggs in a constant-temperature culture box with the diameter of 10.0 +/-2 cm, and the culture medium of 10.5 ℃ and removing the eggs.

2) In vitro toxicity screening of zebra fish:

healthy 5hpf roe was evenly distributed into 24-well plates, 20 roe per well. Different concentrations of tartary buckwheat polysaccharide (0. mu.g/mL, 50. mu.g/mL, 100. mu.g/mL, 250. mu.g/mL, 500. mu.g/mL, 1000. mu.g/mL) prepared by using the E3 solution are respectively added into each well for 3mL, and two wells are arranged for each concentration. The development of the embryos was observed at 24, 48 and 72hpf, respectively, and the cumulative mortality and teratogenicity of the embryos at 24, 48, 72 and 96hpf, as well as the hatchability and body length of 72 and 96hpf were counted. The mortality rate, the teratogenicity rate and the hatching rate are calculated in a mode that the accumulated number of the dead fish eggs, the malformation fish eggs and the hatching fish eggs in each hole at the time point is divided by the total number of the fish eggs (20) and multiplied by one hundred percent. The heart rate was monitored by the number of beats completed at 1min under a Leica DMi8 microscope 4X field of view. The body length was calculated by measuring the straight line distance from the center of the eye to the tail tip under the 4X field of a Leica DMi8 microscope, and the detailed results are shown in FIGS. 26 and 27.

3) In vitro toxicity screening results of zebra fish:

when the concentration of the tartary buckwheat polysaccharide is less than 250 mu g/mL, the growth and development of the zebra fish embryo/larva are not influenced. When the concentration exceeds 250 mu g/mL, growth and development of the zebra fish are retarded.

The results show that the tartary buckwheat polysaccharide has no obvious influence on the food intake of normal feed-fed mice and high-fat feed diet-induced non-alcoholic fatty liver model mice, but can obviously reduce the weight, the lipid deposition of the liver, the TG and TC contents of the liver and the liver function damage indexes-AST and ALT of the model mice. Reduce TG, TC and LDL-C level in the plasma of mice, increase HDL-C level, and improve lipid metabolism disorder. Besides in vivo animal models, 2 non-alcoholic fatty liver in vitro cell models are established, and the tartary buckwheat polysaccharide is found to reduce the lipid deposition of liver cells, reduce the levels of TG, TC and LDL-C, increase the level of HDL-C and regulate abnormal fat metabolism so as to improve the non-alcoholic fatty liver in a dose-dependent manner. Meanwhile, qRT-PCR detection finds that the tartary buckwheat polysaccharide can inhibit the expression of a fat synthesis gene. The safety evaluation result of the zebra fish shows that the toxicity of the tartary buckwheat polysaccharide is very low, and the safety is high.

In conclusion, the tartary buckwheat polysaccharide is safe and has good effects of protecting the liver and treating the non-alcoholic fatty liver.

Drawings

The following describes embodiments of the present invention in further detail with reference to the accompanying drawings.

FIG. 1 is an infrared spectrum of the tartary buckwheat polysaccharide of the invention.

FIG. 2 is the chromatogram of the monosaccharide control (A) and the chromatogram of the acid hydrolysis of the tartary buckwheat polysaccharide (B). The numbers of the chromatographic peaks are respectively 1-fucose, 2-arabinose, 3-rhamnose, 4-galactose, 5-glucose, 6-xylose, 7-mannose, 8-fructose, 9-galacturonic acid and 10-glucuronic acid.

FIG. 3 is a gel column chromatogram of the tartary buckwheat polysaccharide of the present invention.

FIG. 4 shows morphological features of HepG2 cells (scale bar represents 100 μm).

FIG. 5 shows the effect of the Fagopyrum tataricum polysaccharides of this invention on the proliferation activity of HepG2 cells.

FIG. 6 shows the results of oil red staining of HepG2 cells (scale bar represents 100 μm).

FIG. 7 shows the effect of the Fagopyrum tataricum polysaccharides on the levels of triglyceride (A) and cholesterol (B) in a non-alcoholic fatty liver cell model (p < 0.05).

FIG. 8 shows the effect of the Fagopyrum tataricum polysaccharides on the levels of LDL-C (A) and HDL-C (B) (p < 0.05) in the non-alcoholic steatohepatocyte model.

FIG. 9 shows the effect of Fagopyrum tataricum polysaccharides on activity of glutamic pyruvic transaminase (A) and glutamic oxaloacetic transaminase (B) in non-alcoholic fatty liver cell model (p < 0.05).

FIG. 10 shows the effect of the Fagopyrum tataricum polysaccharides of the present invention on the expression of genes involved in de novo synthesis of fat (. p < 0.05).

FIG. 11 shows the morphological characteristics of BEL7404 cells (scale bar represents 100 μm).

FIG. 12 shows the effect of the Fagopyrum tataricum polysaccharides of this invention on BEL7404 cell viability (. p. < 0.05).

FIG. 13 shows the results of oil red staining of BEL7404 cells; the scale represents 100 μm.

FIG. 14 shows the effect of the Fagopyrum tataricum polysaccharides of this invention on the cholesterol (A) and triglyceride (B) levels in the model of hyperlipidemic cells (p < 0.05).

FIG. 15 shows the effect of the Fagopyrum tataricum polysaccharides of this invention on the content of HDL cholesterol (A) and LDL cholesterol (B) in a model of high fat cells (. p < 0.05).

FIG. 16 shows the effect of Fagopyrum tataricum polysaccharides on the activity of glutamic oxaloacetic transaminase (A) and glutamic pyruvic transaminase (B) in the model of high fat cells (. about.p < 0.05).

FIG. 17 shows the body weight changes of mice in the control group, model group and Fagopyrum tataricum polysaccharide group of the present invention.

FIG. 18 is an oil red staining chart of mouse liver tissue sections.

FIG. 19 shows the effect of Fagopyrum tataricum polysaccharides on the levels of triglyceride (A) and cholesterol (B) in liver of non-alcoholic fatty liver animal model (. about.p < 0.05).

FIG. 20 shows the effect of the Fagopyrum tataricum polysaccharides on low density lipoprotein cholesterol content in liver of non-alcoholic fatty liver animal model.

FIG. 21 shows the effect of the Fagopyrum tataricum polysaccharides on the high density lipoprotein cholesterol content in the liver of non-alcoholic fatty liver animal model.

FIG. 22 shows the effect of Fagopyrum tataricum polysaccharides on the activity of glutamic-pyruvic transaminase (A) and glutamic-oxalacetic transaminase (B) in non-alcoholic fatty liver animal model (p < 0.05).

FIG. 23 shows the effect of Fagopyrum tataricum polysaccharides on the plasma levels of HDL cholesterol (A) and LDL cholesterol (B) in non-alcoholic fatty liver animal models (p < 0.05).

FIG. 24 shows the effect of Fagopyrum tataricum polysaccharides on the levels of cholesterol (A) and triglyceride (B) in plasma of non-alcoholic fatty liver animal model (p < 0.05).

FIG. 25 shows the effect of Fagopyrum tataricum polysaccharides on the activity of glutamic-oxaloacetic transaminase (A) and glutamic-pyruvic transaminase (B) in non-alcoholic fatty liver animal models (p < 0.05).

FIG. 26 shows the state of the Fagopyrum tataricum polysaccharides of this invention incubated with the zebrafish embryo/larvae at 24/48/72hpf (scale bar 250 μm).

FIG. 27 shows the physiological indexes of the zebra fish larvae incubated with different concentrations of the tartary buckwheat polysaccharides. Cumulative teratogenicity of zebrafish larvae at 72hpf n =20 (a); body length of zebrafish larvae at 72hpf n =8 (B); cumulative mortality of zebrafish larvae at 72hpf n =20 (C); heart rate n =20 (D) for 72hpf (p <0.05 and p <0.01 compared to control).

Detailed Description

The tartary buckwheat polysaccharide is an extract obtained by adopting a conventional water extraction method from the roots of tartary buckwheat of Fagopyrum of Polygonaceae, and at least comprises the tartary buckwheat polysaccharide in various dosage forms for preparing the medicine for treating the non-alcoholic fatty liver disease.

The tartary buckwheat polysaccharide can also at least comprise the tartary buckwheat polysaccharide in the preparation of medicaments for reducing the body weight of the body with the non-alcoholic fatty liver disease.

The tartary buckwheat polysaccharide also can be used for preparing a medicament for reducing lipid deposition in the liver of a body with the non-alcoholic fatty liver disease.

The tartary buckwheat polysaccharide can also at least comprise the tartary buckwheat polysaccharide in the preparation of medicines for reducing the contents of TG and TC in liver and plasma of a body with the non-alcoholic fatty liver disease.

The tartary buckwheat polysaccharide also can be used for preparing a medicine for reducing AST and ALT in the plasma of a non-alcoholic fatty liver disease organism.

The tartary buckwheat polysaccharide can also at least comprise the tartary buckwheat polysaccharide in the preparation of medicaments for reducing the LDL-C level of plasma of organisms with non-alcoholic fatty liver diseases.

The tartary buckwheat polysaccharide can also at least comprise the tartary buckwheat polysaccharide in the preparation of medicaments for improving the level of liver and plasma HDL-C of a body with the non-alcoholic fatty liver disease.

The tartary buckwheat polysaccharide also can at least comprise the tartary buckwheat polysaccharide in the preparation of medicaments for improving the lipid metabolism disorder.

In the medicine, the dosage of radix Et rhizoma Fagopyri Tatarici polysaccharide is 150 ~ 200 mg/kg/day.

The embodiment provides an application of tartary buckwheat polysaccharide in preparing a medicament for treating non-alcoholic fatty liver disease, wherein the tartary buckwheat polysaccharide is prepared into a tablet of the medicament for treating the non-alcoholic fatty liver disease by using excipients (such as starch, dextrin, sucrose, mannitol, sorbitol and the like which are well known in the art) with the addition amount of 10 percent and the total addition amount of 90 percent.

The specific preparation method of the tablet comprises the following steps: the radix Et rhizoma Fagopyri Tatarici polysaccharide is lyophilized or vacuum dried into dry powder, or directly spray dried into dry powder from concentrated solution of radix Et rhizoma Fagopyri Tatarici polysaccharide, and the dry powder is mixed with excipient and compressed into tablet. Wherein: the excipient comprises diluent, adhesive, wetting agent, disintegrating agent, lubricant and glidant. The diluent can be starch, dextrin, sucrose, etc.; the humectant can be water, ethanol, isopropanol, etc.; the binder can be starch slurry, methyl cellulose, hydroxypropyl methyl cellulose, ethyl cellulose, etc.; the disintegrant can be dry starch, microcrystalline cellulose, low-substituted hydroxypropyl cellulose; the lubricant and glidant may be talc, silicon dioxide, stearate, tartaric acid, liquid paraffin, polyethylene glycol, and the like.

The above examples are further detailed illustrations of the present invention, but are not meant to be any limitation of the present invention. Various alternatives and modifications can be devised by those skilled in the art without departing from the spirit and scope of the invention as defined by the appended claims.

Claims

1. The application of the tartary buckwheat polysaccharide in preparing the medicine for treating the non-alcoholic fatty liver disease is characterized in that: the Fagopyrum tataricum polysaccharide is extract obtained from root of Fagopyrum tataricum of Fagopyrum of Polygonaceae by conventional water extraction method, and at least comprises the Fagopyrum tataricum polysaccharide in various dosage forms for preparing medicine for treating non-alcoholic fatty liver disease.

2. The use of the tartary buckwheat polysaccharide as defined in claim 1 for preparing a medicament for treating non-alcoholic fatty liver disease, wherein: the tartary buckwheat polysaccharide at least comprises the tartary buckwheat polysaccharide in the preparation of the medicine for reducing the body weight of the body with the non-alcoholic fatty liver disease.

3. The use of the tartary buckwheat polysaccharide as defined in claim 1 for preparing a medicament for treating non-alcoholic fatty liver disease, wherein: the tartary buckwheat polysaccharide at least comprises the tartary buckwheat polysaccharide in the preparation of the medicine for reducing the lipid deposition in the liver of the body with the non-alcoholic fatty liver disease.

4. The use of the tartary buckwheat polysaccharide as defined in claim 1 for preparing a medicament for treating non-alcoholic fatty liver disease, wherein: the tartary buckwheat polysaccharide at least comprises the tartary buckwheat polysaccharide in the preparation of the medicine for reducing the contents of TG and TC in liver and plasma of a body with the non-alcoholic fatty liver disease.

5. The use of the tartary buckwheat polysaccharide as defined in claim 1 for preparing a medicament for treating non-alcoholic fatty liver disease, wherein: the tartary buckwheat polysaccharide at least comprises the tartary buckwheat polysaccharide in the preparation of the medicine for reducing AST and ALT of the organism plasma of the non-alcoholic fatty liver disease.

6. The use of the tartary buckwheat polysaccharide as defined in claim 1 for preparing a medicament for treating non-alcoholic fatty liver disease, wherein: the tartary buckwheat polysaccharide at least comprises the tartary buckwheat polysaccharide in the preparation of the medicine for reducing the LDL-C level of the plasma of the body with the non-alcoholic fatty liver disease.

7. The use of the tartary buckwheat polysaccharide as defined in claim 1 for preparing a medicament for treating non-alcoholic fatty liver disease, wherein: the tartary buckwheat polysaccharide at least comprises the tartary buckwheat polysaccharide in the preparation of the medicine for improving the level of the liver and plasma HDL-C of the body with the non-alcoholic fatty liver disease.

8. The use of the tartary buckwheat polysaccharide as defined in claim 1 for preparing a medicament for treating non-alcoholic fatty liver disease, wherein: the tartary buckwheat polysaccharide at least comprises the tartary buckwheat polysaccharide in the preparation of the medicine for improving the lipid metabolism disorder.

9. The use of the Fagopyrum tataricum polysaccharide of claim 1 ~ 8 in the preparation of a medicament for the treatment of non-alcoholic fatty liver disease, wherein the Fagopyrum tataricum polysaccharide is used in an amount of 150 ~ 200 mg/kg/day.