CN109180754B - Application of the combination of sweet tea and chitosan oligosaccharide in the preparation of anti-diabetic drugs - Google Patents

Abstract

The invention belongs to the field of antidiabetic drugs, and particularly relates to application of rubusoside and chitosan oligosaccharide in preparation of an antidiabetic drug. The invention firstly provides a method for extracting rubusoside by microwave extraction, which is characterized in that sweet tea powder after degreasing and impurity removal of petroleum ether is added into an acidic extraction solution for extraction, the extraction rate is up to 92.43%, and the method has the effects of high extraction efficiency, extraction time saving and preparation process simplification. The invention also provides the application of the combination of rubusoside and chitosan oligosaccharide in the preparation of antidiabetic drugs, and the research of the inventor finds that the combination of rubusoside and chitosan oligosaccharide can realize the comprehensive treatment effects of regulating blood sugar index, reducing blood fat and improving the pathological states of liver, kidney and pancreas, and the sum of the two effects is larger than the sum of single-component treatment, thereby providing support for the development of antidiabetic drugs.

Description

Application of the combination of sweet tea and chitosan oligosaccharide in the preparation of anti-diabetic drugs

technical field

The invention belongs to the field of anti-diabetic drugs, in particular to the application of sweet tea and chitosan oligosaccharides in the preparation of anti-diabetic drugs.

Background technique

An aging population, changes in lifestyle and eating habits, and rapid urbanization have led to a high risk of metabolic syndrome and diabetes. Diabetes mellitus is a serious metabolic disease characterized by elevated fasting blood sugar or postprandial blood sugar, impaired insulin secretion or sensitivity, and accumulated multiple organ damage. Hyperglycemia is the main feature of diabetes. Chronic hyperglycemia can lead to increased oxidative stress. Long-term high blood sugar levels can lead to a vicious circle of reactive oxygen species generation, damage to vascular endothelial cells, and various complications.

The treatment of diabetes and the prevention of its complications have always been regarded as a hot research topic in the medical community. In order to better control blood sugar levels, patients often need lifelong medication. At present, the most widely used oral drugs for clinical treatment are biguanide and sulfonic acid drugs, mainly by reducing the output of liver glucose, promoting the uptake and utilization of glucose by adipocytes and tissue cells, improving insulin resistance, or stimulating pancreatic islet β. Cells release insulin, which lowers blood sugar. However, long-term medication will bring gastrointestinal adverse reactions, hypoglycemia, etc., and bring adverse reactions to patients with liver and renal insufficiency, as well as pregnant and breastfeeding women, which limits the number of drug users.

As a deciduous shrub of the Rosaceae Rubus genus, sweet tea mainly grows in Guangxi Province, my country. Sweet tea contained in sweet tea leaves is a non-toxic, low-calorie, high-sweet non-sugar sweetener. In the process of food industry production and processing, sweet tea can be used as a substitute for sugar sweeteners, which is beneficial to reduce energy intake and reduce the occurrence of diseases such as obesity and metabolic syndrome.

In recent years, studies have shown that sweet tea has the potential effect of regulating blood sugar and blood lipids. Tian Cuiping and others found that administering steatine to Streptozotocin (STZ)-induced diabetic model mice can promote insulin secretion and reduce the blood sugar level of rats in the experimental group; Inhibits gluconeogenesis induced by L-a-alanine. Liang Xiaoqing et al. found that steatine not only reduced the damage of pancreatic islet cells, promoted insulin release, but also inhibited the secretion of glucagon to maintain normal lipid metabolism.

Chitosan oligosaccharide is a kind of oligosaccharide that can be directly dissolved in water and can be absorbed by the body, which is formed by physical, chemical and biological enzymatic degradation of chitosan. It is a kind of N-acetyl-D-amino. Glucose is a linear polysaccharide linked by β-1,4-glycosidic bonds. It has the characteristics of good biocompatibility, easy degradation, non-toxicity and antibacterial, and is widely used in the fields of biomedicine and pharmacy. Studies have shown that after the intervention of chitosan oligosaccharide in diabetic rats, the blood sugar level was significantly decreased, the serum insulin level was increased, and the TC and TG levels were decreased. Liu Bing et al. found that after administration of chitosan oligosaccharide in primary cultured islet cells and islet β cell line NIT-1 cells, chitosan oligosaccharide can promote islet cell proliferation and insulin secretion; at the same time, in STZ-induced experimental diabetes In rats, chitosan oligosaccharide can reduce hepatocyte steatosis, repair damaged hepatocytes, and enhance liver sensitivity to insulin. Zhu Junmei et al. made a model by injecting alloxan into the tail vein, and intervening with medium and high doses (300mg/kg, 500mg/kg) of chitosan oligosaccharide for a period of time could significantly reduce the blood sugar level of diabetic rats.

At home and abroad, there are many studies on the effect and mechanism of steatine and chitosan oligosaccharide alone on the treatment and improvement of diabetes, but there is no exploration on the effect of the combination of the two. In addition, most of them focus on the detection of blood sugar and the determination of various biochemical indicators, while the research on complications and multiple organ lesions caused by diabetes is relatively blank.

SUMMARY OF THE INVENTION

The present invention measures the blood sugar, blood lipid and other blood biochemical indexes of the mice by using different doses of sweet tea and chitosan oligosaccharide alone or in combination to intervene the experimental diabetic mice of the STZ model, and prepare the mice The pathological sections of the liver, kidney and pancreas were observed under the microscope, and the comparison between each group was made to evaluate the effect of steatine, chitosan oligosaccharide and the combined dose groups on diabetic mice. The results showed that steatine Combining medication with chitosan oligosaccharide can effectively reduce blood glucose biochemical indicators, insulin and blood lipid levels, restore liver and kidney functions, and provide a theoretical basis for clinical application of assisting in the treatment of diabetes.

The first aspect of the present invention provides a method for extracting sweet tea. The sweet tea powder after impurity removal is weighed, and an extraction solvent is added to extract in a microwave digestion apparatus. After the extraction is completed, the supernatant is separated by centrifugation, and the extraction solvent is added to continue. extract. Among them, the pH value of the extraction solvent is 2 to 4.

Preferably, the pH of the extraction solvent is 3.

Preferably, the preparation method of the above-mentioned sweet tea powder after impurity removal is as follows: take an appropriate amount of sweet tea leaves, pulverize them with a food processor and sieve them, place the sweet tea powder in a Soxhlet extractor, add petroleum ether to boil and extract, remove fat-soluble impurities and pigments . The solid samples were taken out and air-dried naturally.

Further preferably, in the above preparation method, the sweet tea leaves are pulverized with a food processor and passed through a 50-70 mesh sieve, more preferably, a 60 mesh sieve. Among them, 10g of sweet tea powder needs to be extracted by adding 150-200mL of petroleum ether, and the boiling time is 25-35min. More preferably, the extraction times are 3 times.

Preferably, the working power of the microwave digestion apparatus is 1000-1400W; more preferably, it is 1200W.

Preferably, the above extraction time is 25-35 min; more preferably, it is 30 min, and the extraction is performed three times.

Preferably, the ratio of the above sweet tea powder to the extraction solvent is 1g:30-50mL; more preferably, it is 1g:40mL.

In the second aspect of the present invention, there is provided steatine obtained by the above-mentioned extraction method.

In a third aspect of the present invention, a composition is provided, which is composed of sweet tea and chitosan oligosaccharide.

Preferably, the sweet tea in the composition is the sweet tea prepared by the above-mentioned extraction method.

Preferably, in the above composition, the dosage of steatin is 4.12-16.48 mg/kg, and the dosage of chitosan oligosaccharide is 6.87-27.47 mg/kg.

Further preferably, in the above composition, the mass ratio of steatin to chitosan oligosaccharide is 3:5.

In a fourth aspect of the present invention, there is provided the application of staphylulin and chitosan oligosaccharide in the preparation of antidiabetic drugs.

In a fifth aspect of the present invention, there is provided an application of staphylulin and chitosan oligosaccharide in the preparation of insulin-raising medicines.

The sixth aspect of the present invention provides the application of staphylulin combined with chitosan oligosaccharide in preparing a drug for restoring renal function, preferably, the drug for restoring renal function is a drug for restoring renal function for diabetic patients.

In a seventh aspect of the present invention, there is provided the application of staphylulin and chitosan oligosaccharide in the preparation of an anti-fatty liver drug.

The beneficial effects of the present invention

1. In the prior art, the method of macroporous resin adsorption is often used to separate and extract sweet tea, which can receive good extraction effect, but it is well known in the art that macroporous resin separation takes a long time and the process is complicated. The invention provides a method for extracting steatin from sweet tea powder by microwave extraction, which greatly saves the extraction time, the extraction process is simple, and the extraction rate can reach 92.43%. And in the research process of the present invention, it is found that: during microwave extraction, the pH value of the extraction solvent has a great influence on the extraction rate.

2. In the prior art, the improvement effect of chitosan oligosaccharide and steatin on diabetes has been disclosed in the literature, but the use of chitosan oligosaccharide and steatin in combination for the treatment of diabetes is not disclosed in the prior art. In the research process of the present invention, it is found that the combined use of steatin and chitosan oligosaccharide can realize the comprehensive therapeutic effect of regulating blood sugar index, reducing blood lipid, and improving the pathological state of liver, kidney and pancreas, which is greater than the sum of single component treatment. It is difficult to obtain a good and comprehensive hypoglycemic effect when the diabetic mice are treated with low doses of sweet tea and chitosan oligosaccharide alone.

Description of drawings

The accompanying drawings that form a part of the present application are used to provide further understanding of the present application, and the schematic embodiments and descriptions of the present application are used to explain the present application and do not constitute improper limitations on the present application.

Fig. 1 is the standard curve diagram of sweet tea;

Fig. 2 is the staining diagram of liver pathological tissue sections;

Among them, C in A-2 in Figure 2 is the model control group, the standard control group, and the blank control group, respectively. The F in D-2 in Figure 2 are the high, medium and low dose groups of the combined drug, respectively.

Figure 3 is a staining diagram of kidney histopathological sections;

Among them, C in A-3 in Figure 3 is the model control group, the standard control group, and the blank control group, respectively. The F in D-3 in Figure 3 are the high-dose, medium-dose and low-dose groups of the combination drug, respectively.

Figure 4 is a staining diagram of pancreatic histopathological sections;

Among them, C in A-4 in Figure 4 is the model control group, the standard control group, and the blank control group, respectively. The F in D-4 in Figure 4 are the high, medium and low dose groups of the combination drug, respectively.

Detailed ways

It should be noted that the following detailed description is exemplary and intended to provide further explanation of the application. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

It should be noted that the terminology used herein is for the purpose of describing specific embodiments only, and is not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly dictates otherwise, the singular is intended to include the plural as well, furthermore, it is to be understood that when the terms "comprising" and/or "including" are used in this specification, it indicates that There are features, steps, operations, devices, components and/or combinations thereof.

Terminology Explanation Section:

FBG (Fasting Blood-glucose): fasting blood sugar;

OGTT (Oral Glucose Tolerance Test): oral glucose tolerance test;

HG (Hepatic Glycogen): liver glycogen;

BUN (Urea Nitrogen): blood urea nitrogen;

Cre (Creatinine): creatinine;

INS (Insulin): insulin;

HbA1c (Hemoglobin A1c): glycosylated hemoglobin;

TC (Total Cholesterol): total cholesterol;

TG (Triglyceride): triglyceride;

HDL (High Density Lipoprotein): high density lipoprotein;

LDL (Low Density Lipoprotein): low density lipoprotein;

AST (Aspartate aminotransferase): aspartate aminotransferase;

ALT (Alanine aminotransferase): alanine aminotransferase;

As described in the background art, there is no research on the use of chitosan oligosaccharide and sweet tea in combination for diabetes in the prior art. effect was studied.

In order to enable those skilled in the art to understand the technical solutions of the present application more clearly, the technical solutions of the present application will be described in detail below with reference to specific embodiments and comparative examples.

Embodiment 1 Study on the extraction method of sweet tea

1 material

1.1 Main reagents

Sweet tea standard product (purity 98%, purchased from Meikang Biotechnology Co., Ltd.), sweet tea leaves (produced in Jinxiu County, Guangxi), cellulase purchased from Beijing Soleibo Technology Co., Ltd., acetonitrile (chromatographically pure), Petroleum ether (analytical grade), hydrochloric acid (36.0-38.0%), nitric acid (65.0-68.0%), and sulfuric acid (95.0-98.0%) were purchased from Sinopharm Chemical Reagent Co., Ltd.

1.2 Main instruments

High performance liquid chromatograph (L-2130, Japan HITACHI company), food processor (JYL-C012, Joyoung Co., Ltd.), high-speed centrifuge (5424 type, Germany Eppendorf company), ultrasonic (KQ-500DE, Kunshan ultrasonic Instrument Co., Ltd.), microwave digestion apparatus (Milestone, Italy).

2 methods

2.1 Sample pretreatment

Take an appropriate amount of sweet tea leaves, crush them with a food processor and pass through a 60-mesh sieve. Weigh 10g of sweet tea powder, put it in a Soxhlet extractor twice, add 150-200 mL of petroleum ether, boil and extract for 30 minutes, extract 3 times each time, and remove the fat. Soluble impurities and pigments. The samples were taken out and air-dried in a fume hood.

2.2 Microwave extraction method

Accurately weigh 0.50 g of the sweet tea powder after removal of impurities, add water as an extraction solvent at a solid-to-liquid ratio of 1:40, and extract in a microwave digestion apparatus. Under the condition of constant raw material quality, three factors, pH value of extracting solution, microwave power and extracting time, were screened by L ₉ (3 ⁴ ) orthogonal design experiment to explore the optimum conditions for microwave extraction.

Table 1-1 Factor levels of microwave extraction method

After the extraction was completed, after cooling, the supernatant after centrifugation was taken, and the residue was added to the solvent to continue the extraction, and the extraction was carried out 3 times in total. The content of sweet tea in the extract was determined and the extraction percentage was calculated.

2.3 Determination of sweet tea content

The content of sweet tea was determined by high performance liquid chromatography, and the specific method was referred to GB8027-2014. Chromatographic conditions: C18 reverse chromatographic column, 250mm×4.6mm, particle size 5μm. Mobile phase: acetonitrile: sodium phosphate buffer = 32:68. Mobile phase flow rate: 1ml/min. Detection wavelength: 210nm. Injection volume: 10 μL. Column temperature: 40°C.

2.3.1 Standard curve drawing

Precisely weigh 0.0200g of steatine standard product in a 10ml volumetric flask, dissolve it in 30% acetonitrile aqueous solution to obtain a solution of 2.0g/L, and then dilute it respectively to prepare 1.6g/L, 1.2g/L , 0.8g/L, 0.4g/L standard solutions, the solvent was used as a blank, and the peak area was measured at 210nm.

2.4 Statistical analysis

The content of sweet tea in the sample was calculated by the standard curve, the extraction rate was calculated, and SPSS20.0 was used for statistical analysis.

3 results

3.1 Standard curve of sweet tea

The content of steatin was determined by high performance liquid chromatography, the abscissa was the concentration of steatin (g/L), and the ordinate was the measured peak area. The regression equation was obtained according to the standard curve: y=2E+06x+6982.6, and the correlation coefficient was: R=0.99988. The results are shown in Figure 1.

3.2 Microwave extraction method

The present invention adopts the orthogonal experimental design to study the experimental parameters of the microwave extraction method, and the results involved in the orthogonal experiment are shown in the following table.

Table 1-2 Orthogonal test and results of microwave extraction method

Taking the measured sweet tea extraction as the reference index, the results in the table show that the pH value of the extract and the power of the microwave digester have a significant effect on the extraction rate (P<0.1), and the effect of pH value is more significant (P<0.1). P<0.05), the effect of leaching time on the extraction rate was not significant (P>0.05). The range analysis results showed that the influence of each factor on the extraction rate was pH value>power>extraction time. The results show that the optimum extraction condition of microwave extraction method is A ₃ B ₃ C ₂ , that is, when the pH value of the extract is equal to 3, and the power of the microwave digester is 1200W, the extraction rate can reach 92.43% for 30 minutes.

Example 2 Therapeutic effect of sweet tea combined with chitosan oligosaccharide on diabetic mouse model

1 material

1.1 Experimental animals and feed

310 SPF grade Kunming male mice, weighing 26±2g, were provided by the Experimental Animal Center of Shandong University. Rats and mice maintenance feed: provided by Beijing Keao Xieli Feed Co., Ltd.

1.2 Main reagents

Glycated hemoglobin kit, insulin kit, and liver glycogen kit were purchased from Nanjing Jiancheng Bioengineering Institute; streptozotocin was purchased from Beijing Soleibao Technology Co., Ltd.; sweet tea was purchased from Xi'an Tianfeng Biotechnology Co., Ltd. ; Chitooligosaccharide was purchased from Shandong Weikang Biomedical Technology Co., Ltd.; Metformin was purchased from Beijing Jingfeng Pharmaceutical Co., Ltd.; Physiological saline was purchased from Shandong Huaxin Pharmaceutical Group Co., Ltd.; concentrated sulfuric acid and glucose were purchased from Sinopharm Chemical Reagent Co., Ltd. .

1.3 Main instruments

Multifunctional microplate reader (Tecan Infinite200pro sequence, Tecan, Switzerland), digital display electric heating constant temperature water tank (Model 8302, Beijing Changfeng Instrument Co., Ltd.), low temperature centrifuge (5430R, Eppendorf, Germany), vortex mixer ( G560E, Scientific Industries, USA), blood glucose meter (Sinonuo Biosensing Co., Ltd.), rotary microtome (RM2235, LEICA), microscope (DX45, OLYMPUS).

2 methods

2.1 Preparation of diabetic model mice

After 5 days of adaptive feeding, 13 mice were randomly selected as blank control group. The remaining 297 mice were used to prepare the diabetes model. After fasting for 24 hours, the animals were given a one-time intraperitoneal injection of STZ solution (prepared for immediate use) at 65 mg/kg. After 7 days of normal food and water intake, the animals were fasted for 5 hours, and blood was collected from the tail vein to measure fasting blood glucose (FBG). Mice with 14.0-25.0mmol/L were regarded as hyperglycemic mice.

2.2 Handling of experimental animals

According to the random number table method, the mice were divided into 11 groups according to their body weight and FBG, namely: (1) high-dose steatin group (300 mg/kg/d.bw), (2) steatine medium-dose group (150 mg/kg/d.bw) d.bw), (3) low-dose steatin group (75mg/kg/d.bw), (4) high-dose group of chitosan oligosaccharide (500mg/kg/d.bw), (5) medium-dose of chitosan oligosaccharide Group (250mg/kg/d.bw), (6) low-dose group of chitosan oligosaccharide (125mg/kg/d.bw), (7) combined with high-dose group ((150mg of sweet tea + 250mg of chitosan oligosaccharide)/kg/ d.bw), (8) combined with the middle-dose group ((75 mg of staphylococcus + 125 mg of chitosan oligosaccharide)/kg.bw), (9) combined with the low-dose group ((37.5 mg of staphylococcus + 62.5 mg of chitosan oligosaccharide)/kg. bw), (10) standard control group (metformin 150 mg/kg.bw), (11) model control group (normal saline). The reagents in each group were prepared with 10 mL of normal saline according to the above dose, and all of them were prepared as they were. The daily survival status of the mice was recorded, and their water intake was recorded. The mice were weighed by gavage at 4w, 8w, and 12w.

Oral glucose tolerance (OGTT) tests were performed after the experiment. Take blood from the eyeball first, take 0.3-0.5 mL of blood into a disodium ethylenediaminetetraacetic acid (EDTA) anticoagulant centrifuge tube, quickly and fully shake it, and store it in a -70°C refrigerator; then take 0.8-1 mL of blood from an ordinary centrifuge tube, 4 After being stored at ℃ for 2 hours, centrifuged, and the supernatant was taken and stored in a -70 ℃ refrigerator. After that, the mice were sacrificed, and the liver, pancreas and kidney were taken for pathological sections and observed under a microscope.

2.3 Determination of indicators

2.3.1 General Status Observation

Observe the general living conditions of the mice, including food intake, water intake, body shape, hair color, mental state, skin condition, etc., and record any abnormalities in time.

2.3.2 Fasting blood glucose determination

The experimental mice were fasted for 5 hours and the tail was sterilized with an iodine cotton ball. The tail vein was punctured with a blood collection needle. After the first drop of blood was discarded, the blood glucose value was measured with a blood glucose meter and the data was recorded. After the measurement was completed, a cotton ball was used to stop the bleeding.

2.3.3 Oral glucose tolerance

After 12 weeks, the mice in each group were subjected to OGTT test, fasting for 5 hours, and the mice in each group were given corresponding doses of the test substance. After 15-20 minutes, 2.0g/kg.bw glucose solution was orally administered, and the blood glucose of tail vein was measured at 0h, 0.5h, 1h and 2h, and the area under the curve was calculated according to the following formula:

Area under the blood sugar curve = 1/4×0h blood sugar value + 1/2×0.5h blood sugar value + 3/4×1h blood sugar value + 1/2×2h blood sugar value

2.3.4 INS determination method

Mouse serum insulin was determined by biotin double antibody sandwich enzyme-linked immunosorbent assay, and the specific steps were operated according to the instructions.

2.3.5 HG determination method

Take 75 mg of liver tissue in a test tube, boil it in boiling water for 20 minutes, cool it with running water, and then add double distilled water to prepare a glycogen detection solution. The absorbance was measured using a microplate reader according to the kit instructions.

2.3.6 Determination method of AST

Use an automatic biochemical analyzer to measure the above indicators in serum, and operate according to the kit instructions.

2.4 Pathological sections of mouse liver, kidney, pancreas and eyeball

After the animals were sacrificed at low temperature, their livers, kidneys, and pancreas were immersed in 10% formaldehyde for fixation, dehydrated, dipped in wax, embedded, sliced, and stained with HE (dewaxing, rehydration, staining, dehydration, transparency, solidification). seal) observation under the microscope.

2.5 Statistical analysis

表示，组间比较方差齐时用方差分析，方差不齐时用Kruskal-Wallis检验，组间多重比较用SNK法。Statistical analysis was performed with SPSS 20.0, and continuous variables obeying normal distribution were used as mean ± standard deviation

When the variance is homogeneous, the analysis of variance is used for the comparison between groups, the Kruskal-Wallis test is used when the variance is unequal, and the SNK method is used for multiple comparisons between groups.

3. Results

3.1 Observation of the mouse model

Table 2-1-1 Basic condition of mice

Compared with the normal group, the diabetic model mice showed symptoms of three more and one less. The mice had sparse hair, decreased gloss, damp and peculiar litter, and individual mice had slight bleeding from the nose and nose, ascites, and extreme weight loss. Compared with the model control group, the survival status of the diabetic mice in the standard control group, each dose group of sweet tea, each dose group of chitosan oligosaccharide, and each dose group of combined action was improved, especially the diseased mice in the middle and high dose groups. Skin color, mental state and litter condition improved significantly.

3.2 Blood sugar levels

Table 2-2-1 Fasting blood glucose levels of mice in high-dose group

aP<0.05 compared with blank control group; bP<0.05 compared with model control group; cP<0.05 compared with standard control.

Table 2-2-2 Fasting blood glucose level of mice in middle dose group

aP<0.05 compared with blank control group; bP<0.05 compared with model control group; cP<0.05 compared with standard control.

Table 2-2-3 Fasting blood glucose levels of mice in low-dose group before and after intervention

aP<0.05 compared with the blank control group; bP<0.05 compared with the model control group; cP<0.05 compared with the standard control.

3.2 Oral glucose tolerance level

Table 2-3-1 Oral glucose tolerance level of mice in high-dose group

aP<0.05 compared with blank control group; bP<0.05 compared with model control group; cP<0.05 compared with standard control.

The fasting blood glucose value and the area under the glucose tolerance curve of the combined high-dose group were higher than those of the blank control group and lower than the model control group (P<0.05). The area under the tolerance curve increased; compared with the high-dose steatin group, the hypoglycemic ratio was higher; compared with the high-dose chitosan oligosaccharide group, the hypoglycemic effect was significantly better, and the hemoglobin level and the area under the glucose tolerance curve were significantly reduced (P <0.05).

Table 2-3-2 Oral glucose tolerance level of mice in middle dose group

aP<0.05 compared with blank control group; bP<0.05 compared with model control group; cP<0.05 compared with standard control.

The fasting blood glucose level and the area under the glucose tolerance curve of the combined middle-dose group were higher than those of the blank control group, but lower than those of the model control group (P<0.05). The area under the tolerance curve was significantly increased (P<0.05); compared with the middle-dose group of chitosan oligosaccharide, the hypoglycemic effect was significantly better, and the hemoglobin level and the area under the glucose tolerance curve were significantly decreased (P<0.05).

Table 2-3-3 Oral glucose tolerance level of mice in low-dose group

aP<0.05 compared with blank control group; bP<0.05 compared with model control group; cP<0.05 compared with standard control.

The fasting blood glucose level and the area under the glucose tolerance curve of the combined low-dose group were higher than those of the blank control group, and the area under the glucose tolerance curve was lower than that of the model control group (P<0.05). The level and the area under the oral glucose tolerance curve were significantly increased (P<0.05); compared with the low-dose steatin group, the level of glycosylated hemoglobin was significantly increased (P<0.05); compared with the low-dose chitosan oligosaccharide group, the control of blood sugar The effect was significantly better, and the hemoglobin level and the area under the glucose tolerance curve were significantly reduced (P<0.05).

3.3 Serum insulin

Table 2-4-1 Serum insulin levels of mice in high-dose group

aP<0.05 compared with blank control group; bP<0.05 compared with model control group; cP<0.05 compared with standard control.

The serum insulin level in the combined high-dose group was significantly lower than that in the blank control group and the standard control group (P<0.05), but higher than that in the model control group (P<0.05).

Table 2-4-2 Serum insulin levels of mice at medium doses

aP<0.05 compared with blank control group; bP<0.05 compared with model control group; cP<0.05 compared with standard control.

The serum insulin level of the middle-dose group in the combined effect was significantly lower than that of the blank control group and the standard control group (P<0.05), and higher than that of the model control group (P<0.05).

Table 2-4-3 Insulin levels of mice in low-dose group

aP<0.05 compared with blank control group; bP<0.05 compared with model control group; cP<0.05 compared with standard control.

The serum insulin level in the combined low-dose group was significantly lower than that in the blank control group and the standard control group (P<0.05), but higher than that in the model control group (P<0.05).

3.4 Determination of liver function level

Table 2-5-1 Levels of liver glycogen and transaminase in mice in high-dose group

aP<0.05 compared with blank control group; bP<0.05 compared with model control group; cP<0.05 compared with standard control.

The level of HG in the combined high-dose group was significantly higher than that in the model control group, but lower than that in the standard control group and the blank control group (P<0.05). The level of AST in the combined high-dose group was higher than that in the model control group (P<0.05).

Table 2-5-2 Levels of liver glycogen and transaminase in middle dose mice

aP<0.05 compared with blank control group; bP<0.05 compared with model control group; cP<0.05 compared with standard control.

Table 2-5-3 The liver glycogen and transaminase levels of mice in the low-dose group

aP<0.05 compared with blank control group; bP<0.05 compared with model control group; cP<0.05 compared with standard control.

The level of HG in the combined low-dose group was lower than that in the standard control group and the blank control group (P<0.05). Compared with the model control group, the AST level in the combined low-dose group was significantly higher than that in the standard control group and blank control group (P<0.05).

The level of HG in the middle-dose group of combined effect was significantly higher than that in the model control group, but lower than that in the standard control group and the blank control group (P<0.05). The level of AST in the middle-dose group of combined effect was higher than that in the blank control group and the standard control group (P<0.05).

4. Staining results of pathological sections

The results of liver slices are shown in Figure 2: Among them, the model control group (A in Figure 2): the arrangement of liver cells is extremely disordered, the liver cord is blurred, most liver cells in the field of view have fatty degeneration, a few liver cell nuclei are obviously squeezed, and some The cells showed vacuolar-like changes.

Standard control group (B in Figure 2): the arrangement of hepatocytes is relatively regular, the hepatic cord is relatively clear, and only individual cells have steatosis in the visual field, and no fat vacuoles are seen.

In the blank control group (C in Figure 2), the hepatic lobule structure was complete, the hepatic cord was clear, the hepatocytes were regularly arranged, the size of the nucleus was the same, and no cell degeneration was found.

Among them, in the low-dose combination group (F in Figure 2), the arrangement of hepatocytes was disordered, the hepatic cord was blurred, and about half of the hepatocytes in the visual field showed fatty degeneration, some cells showed vacuolar-like changes, and some hepatocyte nuclei were squeezed; Compared with the high-dose group, the damage was lighter, and no vacuolar degeneration was found. In the middle-dose group (E in Figure 2), fatty degeneration of a few cells was still seen. In the high-dose group (D in Figure 2), the hepatocytes were arranged more regularly. The hepatic cord was clear, and only individual cells showed steatosis.

The results of kidney slices are shown in Figure 3: Model control group (A in Figure 3): The glomerular volume was significantly increased, the mesangial matrix was significantly proliferated, the internal structure was not clear, and a large number of inflammatory cells were infiltrated in the renal interstitium; renal tubular epithelium The cells were edematous, the nucleus was squeezed to the edge, and individual cells appeared degeneration.

Standard control group (B in Figure 3): The glomerular structure is relatively clear, the degree of volume increase is low, a small amount of neutrophil infiltration can be seen in the renal interstitium, and the renal tubular epithelial cells can be seen edema without degeneration.

Blank control group (C in Figure 3): The glomerular structure is complete and clear, the size is normal, the tubular capillary basement membrane is evenly distributed, the renal tubular cells are not degenerated, the renal small organ cavity is not exuded, and there is no inflammatory cell. infiltration.

Combined action group: The structure of the glomerulus in the three groups was relatively clear, and there was no significant increase in volume, a small amount of neutrophil infiltration in the renal interstitium, edema of renal tubular epithelial cells, and no cell degeneration; the low-dose group (Figure 3). F) Mild hyperplasia of the mesangial matrix is seen.

The results of pancreas slices are shown in Figure 4: Model control group (A in Figure 4): Islets are irregular in shape, small in area, and small in number of cells. Very few cells have vacuolar degeneration, obvious pyknosis, and the size of blood vessel lumen. different.

Standard control group (B in Figure 4): The number of pancreatic islets in the visual field is large, the shape is irregular, the number of cells is large, the arrangement is relatively regular, a small amount of inflammatory cell infiltration can be seen in the islets, the size of the vascular lumen is basically the same, and there is no expansion.

Blank control group (C in Figure 4): In the normal group, the pancreatic islet structure of the pathological section of the pancreas was complete, the islet cells were more regularly arranged, and the blood vessel wall was uniform without expansion.

Combined action group: the islets in the middle and low dose groups were small in area, regular in shape, and less in number of cells, with very few cells showing vacuolar degeneration, and some nuclei of different sizes in the low dose group; in the high dose group (D in Figure 4) ) The islet cells were numerous and regularly arranged, with a small amount of inflammatory cell infiltration and no cellular degeneration.

The above descriptions are only preferred embodiments of the present application, and are not intended to limit the present application. For those skilled in the art, the present application may have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of this application shall be included within the protection scope of this application.

Claims (1)

1. the application of a composition in preparing insulin-raising medicine, repairing renal function medicine or anti-fatty liver medicine, is characterized in that, described composition is made up of sweet tea and chitosan oligosaccharide; The quality of sweet tea and chitosan oligosaccharide The ratio is 3:5.

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