Application of forsythiaside A in preparation of medicine for treating type 2 diabetes mellitus and improving intestinal flora

Abstract

The invention belongs to the technical field of biological medicine, and particularly discloses application of forsythiaside A to preparation of medicine for treating type 2 diabetes mellitus and improving intestinal flora. Experiments prove that the forsythiaside A can mediate inflammation and insulin resistance at the same time, can improve structural damage and dysfunction of livers and pancreas, and can be used for preparing medicines for treating type 2 diabetes mellitus and improving intestinal flora. Glucose metabolism and lipid accumulation abnormity are improved; compared with the prior art, the compound has the advantages that the compound can be used for preparing medicines for treating type 2 diabetes mellitus, enhancing insulin sensitivity, obviously regulating blood sugar, improving insulin resistance and effectively improving intestinal flora, so that the compound has an important application value when being applied to preparation of medicines for treating type 2 diabetes mellitus and improving intestinal flora.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and in particular relates to a use of forsythiaside A (FTA) in preparing a medicine for treating type 2 diabetes (T2DM) and improving intestinal flora. Background Art

[0002] Diabetes is a chronic metabolic disease characterized by high blood sugar. It is estimated that by 2025, about 1.31 billion people will be affected by it, and this number is still rising. Long-term diabetes can cause complications such as cognitive dysfunction, affective disorders and obstruction, as well as kidney and liver diseases. Insulin resistance (IR) refers to the reduced sensitivity of insulin-targeted organs to insulin. It is a core risk factor for T2DM and a precursor to abnormal glucose and lipid metabolism. It is also an important cause of metabolic diseases such as obesity and hyperlipidemia. T2DM is also considered a systemic metabolic inflammatory disease. Chronic inflammation of the pancreatic islets and insulin target organs (such as the liver) can induce β-cell dysfunction and IR in T2DM. Impaired liver function and pancreatic β-cell dysfunction are closely related to systemic IR. Currently, drugs for the treatment of IR and T2DM include metformin, acarbose and liraglutide. However, side effects such as gastrointestinal reactions and increased cardiac load are inevitable, resulting in poor patient compliance during treatment. Therefore, safer and more effective innovative drugs are an important direction for research and development.

[0003] Forsythia leaf is the leaf of Forsythia supensa (Thunb.) Vahl, a traditional Chinese medicinal plant of the Oleaceae family. Studies have shown that Forsythia leaf is similar to Forsythia fruit in terms of chemical composition, but the content of active ingredients such as forsythia glycosides, FTA and rutin is significantly higher than that of fruit. As a traditional bulk medicinal material with the effects of clearing heat and detoxifying, Forsythia, Forsythia leaf and its FTA, forsythia glycosides, rutin, quercetin and other ingredients have shown good therapeutic effects on various inflammations such as tracheitis and neuroinflammation, and are also effective in immunomodulation. Forsythia leaf and its main ingredients such as forsythia glycosides and rutin also have a good lipid-regulating effect. Forsythia leaf water extract can significantly reduce the blood sugar level of diabetic mice. In addition, Forsythia leaf has a long history of application as a medicinal and edible resource, is rich in nutrients, and is both safe and health-promoting.

[0004] At present, there have been reports on the effects of Forsythia suspensa leaf extract on diabetes and improving intestinal flora, but the composition of Forsythia suspensa leaf extract is complex. According to literature reports, a total of 39 components have been identified in Forsythia suspensa leaf extract, including 8 phenylethanol glycosides, 5 lignans, 12 flavonoids, 3 organic acid compounds, 6 terpenes, 1 quinone, 2 phenolic compounds, 1 glycoside, and 1 other compound. However, there have been no reports on the use of FTA in the treatment of T2DM. Summary of the invention

[0005] The purpose of the present invention is to provide the use of FTA in the preparation of drugs for treating T2DM, and to provide a new treatment approach and method for improving insulin resistance and intestinal microecological environment.

[0006] In order to achieve the above purpose, the following technical solutions are adopted:

[0007] The present invention mainly protects the use of FTA in the preparation of drugs for treating T2DM and improving intestinal flora, wherein the source of the FTA is not limited, and the FTA obtained in any manner has the above-mentioned effects. The FTA is the main active ingredient in the drug, and it may also include pharmaceutically acceptable excipients, which are specifically determined according to the specific form of the drug, and all of which are pharmaceutical excipients that can be used in the prior art.

[0008] As a preferred embodiment, the method for extracting and purifying FTA comprises the following steps:

[0009] (1) Accurately weigh 20 g of Forsythia suspensa leaf powder that has passed through a No. 3 sieve and put it into a 500 mL conical flask, add 400 mL of 60% ethanol, and extract in a water bath at 70° C. for 50 min to obtain an extract. Extract twice, combine the extracts, filter with a Buchner funnel to obtain a filtrate, and use a rotary evaporator to reduce pressure and concentrate to recover ethanol, then dry in an oven at 60° C. until hardened, and freeze-dry in a freeze dryer to obtain an extract;

[0010] (2) Dissolve the extract with 5% ethanol, set aside, and load it onto an AB-8 resin column. First, elute impurities with deionized water until the eluent is clear, then load the sample at a rate of 5 g per 100 g of resin. Then, elute with 7 BV of 30% ethanol solution at a flow rate of 3 BV / h, concentrate and recover ethanol, and the volume of the concentrate is 1 / 10 of the original eluent. Let stand at room temperature for 8 h; centrifuge, and take the supernatant;

[0011] (3) The supernatant was extracted twice at room temperature with water and saturated n-butanol in a volume ratio of 1:1, the water layer was discarded, and the n-butanol phases obtained from each extraction were combined, concentrated under reduced pressure at 50°C, and dried to obtain crude FTA;

[0012] (4) Octadecylsilane (ODS) was soaked in methanol for 24 h. The ODS slurry fully soaked in methanol was poured into a column with a diameter-to-height ratio of 1:30 along the inner wall of the column at room temperature using a glass rod. 2-3 BV of methanol was flushed with 1.0 mL / min of methanol, and then the column was equilibrated with 3 BV of 30% methanol solution. The prepared FTA crude product was dissolved in 30% methanol solution and loaded. 30% methanol-0.2% glacial acetic acid solution was used as the eluent to elute 10 BV, and 5-10 BV was collected. The organic solvent was recovered at low temperature, and FTA with a purity of more than 95% was obtained after freeze-drying.

[0013] Preferably, the temperature of the rotary evaporator in step (1) is set to 55° C. and the rotation speed is 40 r / min.

[0014] Preferably, the preparation method of the AB-8 resin column is as follows: soak the macroporous adsorption resin AB-8 in 95% ethanol solution for 24 hours to fully swell, elute with anhydrous ethanol until the eluent is free of white turbidity by adding an appropriate amount of distilled water, then wash with deionized water until there is no alcohol smell, then soak in 3% NaOH solution for 4 hours, wash with deionized water until neutral, then soak in 3% HCl solution for 4 hours, wash with deionized water until neutral, filter and set aside.

[0015] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0016] The present invention studies the effect of FTA on T2DM and intestinal flora for the first time. The results show that the FTA can mediate inflammation and insulin resistance at the same time, improve liver and pancreatic structural damage and dysfunction, improve glucose metabolism and lipid accumulation abnormalities; enhance insulin sensitivity, have a significant effect on regulating blood sugar, and improve insulin resistance. By acting on the intestinal microbial environment, beneficial bacteria are increased, harmful bacteria are inhibited, and intestinal flora imbalance is improved; the FTA comes from the new Chinese medicinal resource Forsythia suspensa leaves, which is both a medicine and food, and is a safe, effective, and multi-pathway drug. It can effectively overcome the problems of drug resistance, large side effects, and poor effectiveness caused by a single drug target.

[0017] The present invention will be further described in detail below with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The effect of forsythiaside A (hereinafter referred to as FTA) on the body weight of T2DM mice.

[0019] Figure 2 It is the effect of FTA on glucose metabolism; in the figure, A: fasting blood glucose value after four weeks of administration; B: fasting blood glucose value after the fourth week; C: oral glucose tolerance curve; D: area under the curve; E: liver glycogen content.

[0020] Figure 3This is the effect of FTA on the insulin resistance level; in the figure, A: insulin tolerance curve; B: area under the curve; C: serum insulin level; D: insulin resistance index.

[0021] Figure 4 This is the effect of FTA on inflammatory factors; in the figure, A: IL-1β level; B: IL-6 level; C: TNF-α level.

[0022] Figure 5 This is the effect of FTA on blood lipids; in the figure, A: serum TCHO content; B: serum TG content; C: serum LDL-C content; D: serum HDL-C content.

[0023] Figure 6 This is the effect of FTA on liver function; in the figure, A: serum alanine aminotransferase level; B: serum aspartate aminotransferase level.

[0024] Figure 7 The histopathological morphological changes of liver and pancreas were observed under an optical microscope after H&E staining.

[0025] Figure 8 This is the effect of FTA on pancreatic islet structure; in the figure, A: mouse pancreas immunofluorescence staining 400×; B: pancreatic β-cell area; C: pancreatic α-cell area; Note: Insulin (Insulin, green); Glucagon (Glucagon, red); DAPI (blue).

[0026] Fig. 9 The effect of FTA on apoptosis of pancreatic islet cells in T2DM mice; Note: The figure shows immunofluorescence TUNEL staining of mouse pancreas at 400×, in which TUNEL staining positive apoptotic cell nuclei (green) and DAPI staining cell nuclei (blue).

[0027] Fig.10 It is the OUT distribution and Pan / Core analysis between groups; in the figure, A. Pan species curve; B. Core species curve; C. Rank-abundance curve (phylum level); D. Rank-abundance curve (genus level).

[0028] Fig.11 It is the change of α diversity index of intestinal flora in each group of mice; In the figure, A. Aces index; B. Chao index; C. Shannon index; D. Ace index dilution curve; E. Chao index dilution curve; F. Shannon index dilution curve.

[0029] Fig.12 It is the β diversity of intestinal flora in each group of mice; In the figure, A. PCoA analysis; B. NMDS2 analysis.

[0030] Fig.13 It is the composition of mouse intestinal flora; A. Venn diagram of intestinal flora OUT; B. Community distribution diagram at the phylum level; C. Community distribution diagram at the genus level. DETAILED DESCRIPTION

[0031] The technical solutions in the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Although the representative embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and changes can be made to the present invention without departing from the scope of the present invention.

[0032] The preparation method of the present invention and the effects of its application are studied below through specific examples.

[0033] Embodiment 1:

[0034] This embodiment provides a method for extracting and purifying FTA, which is specifically obtained through the following steps.

[0035] The forsythia leaves used in this embodiment were collected from Anze County, Shanxi Province, and were identified as leaves of the forsythia plant of the genus Forsythia of the family Oleaceae.

[0036] (1) Accurately weigh 20 g of Forsythia suspensa leaf powder that has passed through a No. 3 sieve and put it into a 500 mL conical flask, add 400 mL of 60% ethanol, and extract in a water bath at 70° C. for 50 min to obtain an extract. Extract twice, combine the extracts, filter with a Buchner funnel to obtain a filtrate, and use a rotary evaporator (temperature 55° C., speed 40 r / min) to reduce pressure and concentrate to recover ethanol, then dry in a 60° C. oven until hardened, and freeze-dry in a freeze dryer to obtain an extract;

[0037] Preparation of AB-8 resin column: soak the macroporous adsorption resin AB-8 in 95% ethanol solution for 24 hours to fully swell, then elute with anhydrous ethanol until the eluent is free of white turbidity after adding an appropriate amount of distilled water, then wash with deionized water until there is no alcohol smell, then soak in 3% NaOH solution for 4 hours, wash with deionized water until neutral, then soak in 3% HCl solution for 4 hours, wash with deionized water until neutral, filter and set aside;

[0038] (2) Dissolve the extract with 5% ethanol, set aside, and load it onto an AB-8 resin column. First, elute impurities with deionized water until the eluent is clear, then load the sample at a rate of 5 g per 100 g of resin. Then, elute with 7 BV of 30% ethanol solution at a flow rate of 3 BV / h, concentrate and recover ethanol, and the volume of the concentrate is 1 / 10 of the original eluent. Let stand at room temperature for 8 h; centrifuge, and take the supernatant;

[0039] (3) The supernatant was extracted twice at room temperature with water and saturated n-butanol in a volume ratio of 1:1, the water layer was discarded, and the n-butanol phases obtained from each extraction were combined, concentrated under reduced pressure at 50°C, and dried to obtain crude FTA;

[0040] (4) Soak ODS in methanol for 24 hours, and pour the ODS slurry fully soaked in methanol into a column with a diameter-to-height ratio of 1:30 along the inner wall of the column at room temperature using a glass rod. Rinse 2 to 3 BV of methanol at 1.0 mL / min, and then balance the column with 3 BV of 30% methanol solution; dissolve the prepared FTA crude product in 30% methanol solution and load it, use 30% methanol-0.2% glacial acetic acid solution as eluent, elute 10 BV, collect 5-10 BV, recover the organic solvent at low temperature, and obtain FTA with a purity of more than 95% after freeze-drying.

[0041] Embodiment 2:

[0042] The pharmacodynamic evaluation of FTA in improving insulin resistance in T2DM is as follows: the FTA used can be prepared by the method in Example 1, or a commercially available product can be used.

[0043] 1. Experimental Methods

[0044] 1. Model establishment, grouping and drug administration of T2DM mice

[0045] SPF male C57BL / 6J mice were randomly divided into a high-fat and high-sugar diet group and a normal diet group after adaptive feeding for 1 week. The normal diet group was fed with a maintenance diet, and the high-fat and high-sugar diet group (HFD) was fed with a high-fat diet (66.5% basal feed + 10% lard + 20% sucrose + 2.5% cholesterol + 1% sodium cholate). After 4 weeks of feeding, the mice were fasted for 12 hours and had free access to water. The mice in the HFD group were intraperitoneally injected with STZ 45mg / kg (0.1mmol / L, pH 4.0 citric acid buffer, prepared and used immediately), and fed normally 1 hour after the injection. After 72 hours, the tail vein blood was collected to test the fasting blood glucose (FBG). If FBG ≥ 11.1mmol / L, the T2DM model was considered to be successfully established. For mice whose blood sugar did not reach the standard, STZ injection was performed again one week later until the model was successfully established.

[0046] The successfully modeled T2DM mice were randomly divided into 4 groups, with 10 mice in each group. They were model group (Model), metformin group (Met, 200 mg / kg metformin), low-dose FTA group (FTA-L, 40 mg / kg), and high-dose FTA group (FTA-H, 80 mg / kg). The control group (Contro1) was fed with mouse maintenance feed, and the Model group, Met group, and FTA group were fed with high-fat and high-sugar feed. The drugs in the Met group and the FTA administration group were dissolved in normal saline and used immediately. The Control group and the Model group were gavaged with the same dose of normal saline as the administration group, and the administration was continued for 4 weeks.

[0047] 2. General index determination

[0048] Body weight: The body weight of mice in each group was recorded at a fixed time every week during the experiment.

[0049] Fasting blood sugar: After fasting for 6 hours, the fasting blood sugar value was measured. The tail tip vein blood sugar of each group of mice was measured in turn with a blood glucose meter once a week.

[0050] 3. Oral glucose tolerance test (OGTT)

[0051] After 3 weeks of administration, the mice were fasted for 15 hours and their fasting blood glucose was measured. After the end of the administration, an oral glucose tolerance test was performed. Before the experiment, the mice were fasted overnight and had free access to water during the period. Each mouse was gavaged with glucose (2g / kg body weight). At 0min, 30min, 60min, 90min, and 120min after gavage, blood was collected from the tail vein to measure the blood glucose concentration of each mouse and draw an oral glucose tolerance test (OGTT) curve.

[0052] AUC = (0min blood glucose + 2×30min blood glucose + 3×60min blood glucose + 2×120min blood glucose) / 4.

[0053] 4. Insulin tolerance test

[0054] After 4 weeks of administration, insulin was diluted with normal saline and prepared into the corresponding concentration. The insulin dosage for the mouse insulin tolerance test (ITT) was 0.75 IU / kg, and the dosage for intraperitoneal injection was 0.1 mL / 10 g. Fast for 4 hours in the morning and drink water normally. Tests were performed in the afternoon, and the blood sugar test before injection was recorded as 0 min. The injection amount of insulin was calculated according to body weight. Blood was collected from the tail vein at 30 min, 60 min, 90 min, and 120 min, and the blood sugar value was measured with a blood glucose meter.

[0055] AUC = (0min blood glucose + 2×30min blood glucose + 3×60min blood glucose + 2×120min blood glucose) / 4.

[0056] Fasting insulin (Fins) detection and HOMA-IR calculation: Take out the frozen serum and thaw it in a 4°C refrigerator. Use the mouse insulin ELISA kit according to the instructions of the kit to detect the serum insulin (Fins) content; FBG is the fasting blood glucose value of the mouse. The insulin resistance index is calculated according to the following formula: HOMA-IR = FBG (mmol / L) × Fins (mIU / L) / 22.5;

[0057] 5. Determination of liver glycogen content

[0058] The glycogen content in liver tissue homogenate was detected according to the instructions of mouse glycogen ELISA kit.

[0059] 6. Determination of physiological and biochemical indicators

[0060] Four blood lipid items: Detect the levels of total cholesterol (TC), triglycerides (TG), high-density lipoprotein (HDL-C) and low-density lipoprotein (LDL-C) in serum according to the kit instructions.

[0061] Determination of inflammatory factor content: The expression levels of IL-1β, IL-6, and TNF-α in serum were detected according to the instructions of the ELISA kit.

[0062] 7. Histopathological analysis

[0063] H&E staining: The liver and pancreatic tissues were fixed with 4% paraformaldehyde, dehydrated with ethanol and toluene, embedded in paraffin, and stained with H&E. The pathological tissue changes were observed under a microscope.

[0064] 8. Pancreatic Immunofluorescence

[0065] The sections were dewaxed and rehydrated with a series of dewaxing solutions and ethanol. Then, the sections were antigen retrieved using citric acid antigen retrieval solution (pH 6.0). The sections were washed three times with phosphate-buffered saline (PBS, pH 7.4) for 5 min each time and blocked with 3% BSA for 30 min. The sections were incubated with primary antibodies insulin (1:800) and glucagon (1:500) at 4°C overnight. Then, the sections were incubated with secondary antibodies Alexa Fluor (1:400). 488-conjugated goat anti-mouse IgG (H+L) (1:400) and Cy3-labeled goat anti-rabbit IgG (H+L) (1:300) were incubated for 50 min at room temperature in the dark. DAPI dye was used to label the cell nuclei. Images were scanned and analyzed by Image J for subsequent analysis.

[0066] The sections were dewaxed and rehydrated with a series of dewaxing solutions and ethanol, and proteinase K working solution was added for repair, and PBS (pH7.4) was washed three times, 5 minutes each time. Then, the membrane breaking working solution was added to cover the tissue, incubated at room temperature for 20 minutes, and washed three times with PBS (pH7.4), 5 minutes each time. After equilibration at room temperature, the reaction solution in the TUNEL kit was added, and after washing with PBS (pH7.4), DAPI staining solution was added to counterstain the cell nucleus, and the sections were sealed. The sections were observed under a fluorescence microscope and images were collected.

[0067] 9. Data Analysis

[0068] GraphPad Prism statistical software (version 9.0; GraphPad Software, Inc., San Diego, CA, USA) was used. All data are presented as mean ± SD, and statistical significance was determined by one-way analysis of variance and Tukey test, with the confidence level of statistical significance set at P < 0.05.

[0069] 2. Results Analysis

[0070] 1. Effect of forsythiaside A (hereinafter referred to as FTA) on body weight of T2DM mice

[0071] After 4 weeks of drug intervention, the body weight of the model group (Model) was significantly higher than that of the normal group (Contro1), and the positive control metformin (Met) group was able to significantly reduce the body weight of T2DM mice (P < 0.01). There was no significant difference in body weight between the FTA low-dose group (FTA-L), FTA high-dose group (FTA-H) and the Model group (P>0.05) ( Figure 1 ).

[0072] 2. Effects of FTA on glucose metabolism in T2DM mice

[0073] FTA can reduce the blood glucose level in T2DM mice ( Figure 2 A in the figure). At the 4th week of administration, the blood glucose levels of FTA-L and FTA-H were significantly lower than those of the Model group (P<0.01), and there was no significant difference between the blood glucose levels of the FTA-H group and the Met group (P>0.05) ( Figure 2 B).

[0074] FTA-L and FTA-H can significantly reduce the glucose tolerance impairment caused by diabetes ( Figure 2C), the AUC values ​​showed that there were extremely significant differences between FTA-L and FTA-H and the Model group (P<0.01). Among the different doses of FTA, the AUC value of the FTA-H group was lower than that of the FTA-L group, and reached an extremely significant level (P<0.01). Compared with the positive drug metformin group (Met), there was no significant difference between the FTA-H group and the positive drug group (P>0.05), indicating that it is comparable to the positive drug in improving the body's ability to absorb and utilize glucose ( Figure 2 D).

[0075] Compared with the Control group, the liver glycogen content of the Model group mice was significantly reduced, and the difference reached a very significant level (P<0.0 1)( Figure 2 E). The liver glycogen content in FTA-L and FTA-H groups gradually increased compared with that in Model group. The difference between FTA-H group and Model group was extremely significant (P<0.01), but there was no significant difference between FTA-H group and positive drug Met group (P>0.05).

[0076] 3. Effect of FTA on insulin resistance in T2DM mice

[0077] like Figure 3 A in Figure 2 shows that the Met group and the FTA-L and FTA-H groups can significantly improve the insulin resistance of T2DM mice. Compared with the Model group, the AUC values ​​of the FTA-L and FTA-H groups decreased significantly, and the difference with the AUC values ​​of the Model group reached an extremely significant level (P<0.01) ( Figure 3 At the same time, the AUC values ​​of mice in the FTA group after intervention were not significantly different from those in the positive drug group metformin and the Control group (P>0.05) ( Figure 3 C). Compared with the Model group, the serum insulin content of T2DM mice treated with FTA was significantly decreased (P<0.01), and there was no significant difference between the two groups (P>0.05) ( Figure 3 D). The insulin resistance index of the FTA-L group and the FTA-H group decreased sharply compared with the Model group, indicating that FTA can significantly improve the insulin sensitivity of diabetic mice and effectively alleviate the insulin resistance of T2DM mice.

[0078] 4. Effects of FTA on inflammatory factors in T2DM mice

[0079] Compared with the Control group, the levels of IL-1β, IL-6, and TNF-α in the Model group were significantly increased (P<0.01) ( Figure 4 ). FTA-L and FTA-H groups could significantly reduce the elevated IL-1β in T2DM mice (P<0.05) ( Figure 4 A in the figure). As the dosage concentration increases, FTA has a stronger effect on reducing the levels of IL-6 and TNF-α in T2DM mice. There is no significant difference between the FTA-L and FTA-H groups and the positive drug Met group, indicating that it is comparable to metformin in improving the inflammatory factors IL-6 and TNF-α in T2DM ( Figure 4 B and C in ).

[0080] 5. Effect of FTA on blood lipid levels in T2DM mice

[0081] FTA can reduce the TCHO and TG content of T2DM mice, and the difference between FTA-L and Model group reached a very significant level (P<0.01). There was no significant difference in TCHO and TG content between FTA-L and FTA-H and Control group (P>0.05) ( Figure 5 A and B in ).

[0082] The LDL-C content of FTA-L and FTA-H was significantly different from that of the Model group (P < 0.01), but there was no significant difference between the Control group and the metformin group (P > 0.05). Both doses can effectively inhibit the excessive accumulation of LDL-C in serum, reverse the dyslipidemia caused by T2DM, and restore it to normal levels ( Figure 5 FTA had no significant effect on HDL-C level (P>0.05) ( Figure 5 D).

[0083] Effects of 6FTA on liver function in T2DM mice

[0084] The Met group, FTA-L group and FTA-H group could significantly reduce the serum alanine aminotransferase (ALT) level in T2DM mice (P<0.01)( Figure 6 A). After intervention in the Met group, the serum aspartate aminotransferase (AST) level in T2DM mice decreased significantly and returned to normal levels, and there was no statistical difference between the two groups (P>0.05). Compared with the Model group, both FTA-L and FTA-H could significantly reduce the AST level, and the FTA-H group was significantly better than the FTA-L group (P<0.01)( Figure 6 B).

[0085] 7. Effects of FTA on the morphology of liver and pancreas in diabetic mice

[0086] After H&E staining, the pathological morphological changes of liver tissue were observed under an optical microscope ( Figure 7). The liver tissue structure of the Control group was normal, the liver lobule structure was clear, the liver cells had no degeneration and necrosis, the liver cords were radially distributed from the central vein to the surrounding, there was a clear nucleus in the center, and there was no obvious stenosis and dilation of the liver sinusoids. The liver cells in the Model group were irregularly arranged, with blurred boundaries, uneven cytoplasmic staining, and lipid droplets of different sizes. Compared with the Model group, the liver lesions in the FTA administration group and the metformin group were significantly alleviated.

[0087] The pancreatic islets of T2DM mice were atrophied compared with those of the control group. FTA-L and FTA-H administration at different doses helped maintain the integrity of the pancreatic islet structure in diabetic mice.

[0088] 8. Effects of FTA on pancreatic islet structure in diabetic mice

[0089] The number of pancreatic β cells in the model group mice was significantly reduced, and the number of α cells was significantly increased. The FTA-H group can significantly reverse the abnormal number of β cells in diabetic mice, improve the abnormal structure of the pancreatic islets, and maintain the balance of pancreatic β cells and α cells ( Figure 8 A, B, C in the figure).

[0090] 9. Apoptosis of pancreatic tissue in T2DM mice after FTA intervention

[0091] Compared with the pancreatic tissue of the Control group, a large number of positive apoptotic cells appeared in the Model. After intervention in the FTA-H group, the number of abnormal apoptotic cells in the pancreas of T2DM mice was significantly reduced, indicating that FTA can effectively alleviate the abnormal large-scale apoptosis of pancreatic cells in T2DM mice ( Fig. 9 ).

[0092] From the above, it can be seen that the FTA provided by the present invention can effectively reduce the hyperglycemia of T2DM mice, restore the damaged liver and pancreatic structure and function, alleviate the inflammatory environment, restore the ability to absorb and utilize glucose, enhance lipid metabolism function, and comprehensively improve insulin resistance, indicating that the substance has a significant anti-insulin resistance effect. Therefore, it can be used as the main ingredient to prepare drugs for the treatment of T2DM.

[0093] Example 3 Effect of FTA on intestinal flora of T2DM mice

[0094] 1. Experimental Methods

[0095] After the entire experimental period, the colon contents of mice in the Control group, Model group and FTA-H group were collected in sterile centrifuge tubes, and the samples were quickly frozen in liquid nitrogen and transferred to a -80°C refrigerator for storage, and then 16S rRNA microbial diversity analysis was carried out.

[0096] Alpha diversity and Pan / Core species analysis were used to carry out annotation and evaluation analysis of species; Venn analysis was used to analyze community composition; Linear discriminant analysis effectsize (LEfSe) difference analysis was used to identify differentially dominant bacterial communities; Principal co-ordinates analysis (PCoA) and non-metric multidimensional scaling (NMDS) were used to study the similarities or differences in sample community composition.

[0097] The Spearman correlation coefficient analysis was performed between the relevant indicators used to evaluate FTA in improving T2DM in pharmacodynamic studies, including blood lipid levels (TCHO, TG, LDL-C, HDL-C), glucose metabolism ability (fasting blood glucose level and liver glycogen content), insulin resistance (serum insulin level and insulin resistance index), liver function (ALT, AST), inflammatory factor levels (TNF-α, IL-1β, IL-6) and bacterial genus abundance.

[0098] 2. Experimental results

[0099] 2.1 Analysis of intestinal microbial diversity sequencing results

[0100] ( Fig.10 In the figure (A), as the number of samples increases, the Pan analysis results show an increase in the total number of OUTs; the Core analysis results show a decrease in the number of shared OTUs ( Fig.10 B). The Rank-abundance curve evaluation sample results show ( Fig.10 In C and D), the abundance of species in each group of samples at the phylum and genus levels is high and the distribution is uniform. The overall analysis results show that the sequencing samples are sufficient and the data are reasonable, which can be used for subsequent analysis.

[0101] 2.2α Diversity

[0102] Chao, ACE, and Sobs index analysis can be used to analyze the alpha diversity of intestinal flora, thereby evaluating the richness and diversity of microbial communities. Fig.11 AC showed that the ACE, Chao, and Sobs indexes of the Model group were significantly lower than those of the Control group (P < 0.05). Compared with the Model group, the ACE, Chao, and Shannon indexes of the intestinal flora of mice increased after FTA intervention, but did not reach a significant level (P > 0.05). Fig.11The DF in the middle shows that the dilution curves of ACE, Chao, and Shannon indexes have all tended to be flat, which indicates that the amount of sequence data is sufficient and reasonable, which can fully represent the sample information and ensure that the analysis results are reasonable and reliable.

[0103] 2.3 β diversity

[0104] Beta diversity can be used to measure the similarities or differences in the overall community structure of microorganisms in different groups. The trends between groups can be analyzed by principal coordinate analysis (PCoA) and non-metric multidimensional scaling (NMDS). PCoA results show ( Fig.12 Middle A), the Control group, Model group and FTA group were obviously separated in spatial distance, and the NMDS results were consistent with the PCoA results ( Fig.12 (B) This indicates that FTA treatment can significantly change the community structure and aggregation of intestinal flora in T2DM mice.

[0105] 2.4 Analysis of bacterial flora composition at the phylum and genus levels

[0106] The total number of OUTs in the three groups was 417 (13 in A). After classification based on abundance ratio, the intestinal flora of mice in different groups was divided into 11 bacterial phyla, and those with combined abundance less than 0.01 were classified as others. The top three dominant bacterial phyla in the microbiota of mice in each group were Bacteroidetes, Firmicutes, and Verrucomicrobiota ( Fig.13 (B). The results of this study showed that there was no significant change in the abundance of Firmicutes and Bacteroidetes between the normal group and the model group. The abundance of Firmicutes decreased in the FTA group, while the abundance of Bacteroidetes increased. The abundance of Actinobacteriota and Proteobacteria in the Model group increased significantly compared with the Control group. The abundance of Actinobacteriota and Proteobacteria in the FTA group decreased significantly compared with the Model group, while the abundance of Verrucomicrobiota increased sharply.

[0107] Depend on Fig.13As shown in Figure C, compared with the Control group, the abundance of Akkermansia, Alloprevotella, Lachnospiraceae_NK4A136_group, and unclassified_f_Lachnospiraceae in the Model group decreased; the abundance of Dubosiella decreased. Compared with the Model group, the abundance of Akkermansia, Alloprevotella, Lachnospiraceae_NK4A136_group, and unclassified_f_Lachnospiraceae increased after FLEE intervention.

[0108] In summary, FTA can effectively improve the intestinal flora of T2DM mice.

[0109] The above examples are merely illustrative of the present invention and do not constitute a limitation on the protection scope of the present invention. All designs that are the same or similar to the present invention fall within the protection scope of the present invention.

Claims

1. Use of forsythiaside A in the preparation of drugs for treating type 2 diabetes and improving intestinal flora.

2. The use according to claim 1, characterized in that The extraction and purification method of forsythiaside A comprises the following steps: (1) Accurately weigh 20 g of Forsythia suspensa leaf powder that has passed through a No. 3 sieve and put it into a 500 mL conical flask, add 400 mL of 60% ethanol, and extract in a water bath at 70° C. for 50 min to obtain an extract. Extract twice, combine the extracts, filter with a Buchner funnel to obtain a filtrate, and use a rotary evaporator to reduce pressure and concentrate to recover ethanol, then dry in an oven at 60° C. until hardened, and freeze-dry in a freeze dryer to obtain an extract; (2) Dissolve the extract with 5% ethanol, set aside, and load it onto an AB-8 resin column. First, elute impurities with deionized water until the eluent is clear, then load the sample at a rate of 5 g per 100 g of resin. Then, elute with 7 BV of 30% ethanol solution at a flow rate of 3 BV / h, concentrate and recover ethanol, and the volume of the concentrate is 1 / 10 of the original eluent. Let stand at room temperature for 8 h; centrifuge, and take the supernatant; (3) The supernatant was extracted twice at room temperature with water and saturated n-butanol in a volume ratio of 1:1, the water layer was discarded, and the n-butanol phases obtained from each extraction were combined, concentrated under reduced pressure at 50°C, and dried to obtain the crude product of forsythiaside A; (4) Soak ODS in methanol for 24 h, and pour the ODS slurry fully soaked in methanol into a column with a diameter-to-height ratio of 1:30 along the inner wall of the column at room temperature using a glass rod. Rinse 2-3 BV of methanol at 1.0 mL / min, and then balance the column with 3 BV of 30% methanol solution; dissolve the prepared crude product of Forsythiaside A in 30% methanol solution and load it, use 30% methanol-0.2% glacial acetic acid solution as eluent, elute 10 BV, collect 5-10 BV, recover the organic solvent at low temperature, and obtain Forsythiaside A with a purity of more than 95% after freeze-drying.

3. The use according to claim 2, characterized in that: The temperature of the rotary evaporator in step (1) is set to 55° C. and the rotation speed is 40 r / min.

4. The use according to claim 2, characterized in that: The preparation method of the AB-8 resin column is as follows: soaking the macroporous adsorption resin AB-8 in a 95% ethanol solution for 24 hours to fully swell, eluting with anhydrous ethanol, washing until the eluent is free of white turbidity by adding an appropriate amount of distilled water, then washing with deionized water until there is no alcohol smell, then soaking with a 3% NaOH solution for 4 hours, washing with deionized water until neutral, then soaking with a 3% HCl solution for 4 hours, washing with deionized water until neutral, filtering and setting aside.

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