Application of Momordica charantia exosomes in the preparation of hypoglycemic drugs

Abstract

The present invention belongs to the field of pharmaceutical technology and discloses the application of bitter gourd exosomes in the preparation of hypoglycemic drugs. The bitter gourd exosomes are extracted by the following steps: cleaning fresh bitter gourd, cutting it open to remove seeds, and squeezing out bitter gourd juice; continuously centrifuging the bitter gourd juice, centrifuging at 1000 g for 10 minutes, 3000 g for 20 minutes, and 10000 g for 40 minutes at 4°C, taking the supernatant after centrifugation and discarding the precipitate; ultracentrifuging the obtained supernatant in a cryo-ultracentrifuge at 150000 g for 90 min at 4°C, discarding the supernatant after centrifugation and taking the precipitate, and suspending the precipitate in 1-2 ml of phosphate buffer; filtering the suspension with a 0.22 μm filter membrane, and then ultracentrifuging the filtrate again in a cryo-ultracentrifuge at 150000 g for 90 min at 4°C, discarding the supernatant after centrifugation and taking the precipitate, and suspending the precipitate in 1-2 ml of phosphate buffer; filtering the suspension again with a 0.22 μm filter membrane to obtain sterile bitter gourd exosomes. The present invention shows that bitter gourd exosomes can significantly reduce the fasting blood glucose, postprandial blood glucose and blood lipids of mice.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and particularly relates to the application of bitter melon exosomes in the preparation of hypoglycemic drugs. Background Art

[0002] Diabetes mellitus (DM) is a prevalent metabolic disease characterized by hyperglycemia, which seriously affects people's quality of life and health status. Currently, the main treatment methods for diabetes include oral hypoglycemic drugs or insulin injection, etc., which can temporarily maintain blood sugar at a relatively normal level. However, adverse reactions such as hypoglycemia caused by excessive insulin injection dosage cannot be well avoided.

[0003] T1DM is an autoimmune disease characterized by β - islet cell dysfunction and β - islet cell death caused by autoreactive T lymphocytes, resulting in absolute insulin deficiency and elevated blood sugar levels. T1DM mainly occurs in children and adolescents. Due to the unclear pathogenic mechanism, many patients rely on exogenous insulin administration for life. In recent years, studies have shown that exosomes are involved in the autoimmune response of islet cells and have the potential to be used as new therapeutic drugs and drug delivery carriers in the treatment of T1DM. Currently, there is no specific report on the hypoglycemic effect of plant exosomes. Summary of the Invention

[0004] In view of the deficiencies in the prior art, the present invention provides the application of bitter melon exosomes in the preparation of hypoglycemic drugs.

[0005] To achieve the above object, the present invention adopts the following technical scheme:

[0006] The application of bitter melon exosomes in the preparation of hypoglycemic drugs, wherein the bitter melon exosomes are extracted by the following steps:

[0007] 1.1. Wash fresh bitter melons, cut them open to remove seeds, and extract bitter melon juice;

[0008] 1.2. Centrifuge the bitter melon juice continuously. At 4°C, centrifuge at 1000g for 10 minutes, 3000g for 20 minutes, and 10000g for 40 minutes. After centrifugation, take the supernatant and discard the precipitate;

[0009] 1.3. Ultra - centrifuge the obtained supernatant in a cryo - ultracentrifuge at 4°C at 150000g for 90 min. After centrifugation, discard the supernatant and take the precipitate. Suspend the precipitate in 1 - 2 ml of phosphate buffer;

[0010] 1.4. Filter the suspension with a 0.22 μm filter membrane. The filtrate is centrifuged again in a refrigerated ultracentrifuge at 4 °C and 150,000 g for 90 min. After centrifugation, discard the supernatant and collect the precipitate. Suspend the precipitate in 1 - 2 ml of phosphate buffer solution;

[0011] 1.5. Filter the suspension again with a 0.22 μm filter membrane to obtain sterile bitter gourd exosomes.

[0012] Use a mouse model of type 1 diabetes mellitus (TIDM) induced by streptozotocin (STZ) injection, and treat with bitter gourd exosomes at different concentrations by intraperitoneal injection. Administer the drug continuously for two weeks, and record data such as the fasting blood glucose, glucose tolerance, daily water intake, and food intake of the mice for two weeks.

[0013] The results show that 400 μg / kg of bitter gourd exosomes can significantly reduce the fasting blood glucose, postprandial blood glucose, and blood lipids of mice.

[0014] Beneficial effects: The present invention shows that bitter gourd exosomes can significantly reduce the fasting blood glucose, postprandial blood glucose, and blood lipids of mice. Description of the Drawings

[0015] Figure 1 It is a graph showing the changes in the fasting blood glucose of mice after two weeks of intraperitoneal injection of bitter gourd exosomes at different concentrations in the examples of the present invention;

[0016] Figure 2 It is a graph showing the changes in the glucose tolerance level of mice after two weeks of intraperitoneal injection of bitter gourd exosomes at different concentrations in the examples of the present invention ( Figure 2 a is the glucose tolerance level of the mice in the first week; Figure 2 b is the glucose tolerance level of the mice in the second week);

[0017] Figure 3 It is a graph showing the changes in the water intake of mice after two weeks of intraperitoneal injection of bitter gourd exosomes at different concentrations in the examples of the present invention;

[0018] Figure 4 It is a graph showing the changes in the food intake of mice after two weeks of intraperitoneal injection of bitter gourd exosomes at different concentrations in the examples of the present invention;

[0019] Figure 5 It is a graph showing the changes in the body weight of mice after two weeks of intraperitoneal injection of bitter gourd exosomes at different concentrations in the examples of the present invention;

[0020] Figure 6 It is a graph showing the changes in the total serum cholesterol of mice two weeks after intraperitoneal injection of bitter gourd exosomes at different concentrations in the examples of the present invention. Detailed Embodiments

[0021] The present invention will be described below with reference to specific embodiments. Those skilled in the art can understand that these embodiments are only used to illustrate the present invention and do not limit the scope of the present invention in any way.

[0022] Example

[0023] 1. Experimental materials and instruments

[0024] 1.1 Experimental materials: Fresh bitter gourd, BCA kit, mouse cholesterol ELISA kit, streptozotocin (STZ), citric acid, sodium citrate, 0.9% normal saline, glucose, blood glucose test strips.

[0025] 1.2 Experimental instruments: 37°C incubator, laminar flow hood, microplate reader, electronic balance, blood glucose meter, pipette, insulin syringe.

[0026] 2. Experimental procedures

[0027] 2.1 Extraction of bitter gourd exosomes

[0028] Exosomes of fresh bitter gourd samples were obtained by continuous differential centrifugation and ultracentrifugation;

[0029] 2.1.1. Wash the fresh bitter gourd, cut it open to remove the seeds, and squeeze out the bitter gourd juice;

[0030] 2.1.2. Centrifuge the bitter gourd juice continuously. At 4°C, centrifuge at 1000g for 10 minutes, 3000g for 20 minutes, and 10000g for 40 minutes. After centrifugation, take the supernatant and discard the precipitate;

[0031] 2.1.3. Ultracentrifuge the obtained supernatant. In a refrigerated ultracentrifuge, at 4°C, centrifuge at 150000g for 90 min. After centrifugation, discard the supernatant and take the precipitate. Suspend the precipitate in 1-2 ml of phosphate buffer;

[0032] 2.1.4. Filter the suspension with a 0.22 μm filter membrane. The filtrate is again ultracentrifuged in a refrigerated ultracentrifuge at 4°C at 150000g for 90 min. After centrifugation, discard the supernatant and take the precipitate. Suspend the precipitate in 1-2 ml of phosphate buffer;

[0033] 2.1.5. Filter the suspension again with a 0.22 μm filter membrane to obtain sterile bitter gourd exosomes.

[0034] Determine the protein concentration of bitter gourd exosomes using a BCA kit according to the manufacturer's instructions.

[0035] 2.2 Establishment of an experimental type 1 diabetes mouse model

[0036] After the purchased mice were adaptively fed for 2 weeks, the modeling was started. They were randomly divided into a model group (50 mice) and a normal control group (5 mice). The mice were fasted for 12 - 14 hours, and a certain amount of STZ solution (100 mg / kg) was intraperitoneally injected into the mice in the model group. When injecting, STZ was dissolved in 0.1 mol / L sodium citrate buffer (pH 4.5), prepared on ice in the dark, and injected as soon as possible within 30 min after dissolution. The normal control group was not treated. Five days after injection, the mice were fasted for 8 h, and 20 μl of blood was taken from the tail vein of the modeled mice to measure the content of fasting serum glucose. A fasting blood glucose value of ≥ 11.1 mmol / L in the mice was used as the standard for successful establishment of the experimental type 1 diabetes mouse model.

[0037] 2.3 Grouping of experimental animals and administration of drugs

[0038] In this experiment, a total of 40 mice were successfully modeled. The successfully modeled mice were randomly divided into: a model group, a 200 μg / kg drug administration group, a 400 μg / kg drug administration group, and an 800 μg / kg drug administration group. After successful modeling, the experimental mice in each drug administration group began to be intraperitoneally injected with drugs. The drugs were administered continuously for two weeks, and the body weight, water intake, and food intake of each group were recorded every day. The blood glucose and glucose tolerance indexes of the mice were measured every week, as shown in Figures 1-5 .

[0039] 2.4 Glucose tolerance test

[0040] 2.4.1 At 5 pm the day before the experiment, the mice were transferred to clean cages and fasted for 16 hours until 9 am the next day;

[0041] 2.4.2 At 9 am the next day, the glucose tolerance test was started. The weight of each mouse was weighed, and the fasting blood glucose was first measured and recorded;

[0042] 2.4.3 The normal group and the diabetic model group were given the same volume of normal saline. After the mice were allowed to adapt for 30 min, preparations were made to intraperitoneally inject glucose IPGTT (20% glucose solution, 0.01 ml / g), and the timing was started;

[0043] 2.4.4 At 15 min, 30 min, 60 min, 90 min, and 120 min, the blood glucose values of each mouse at each time point were measured;

[0044] 2.4.5 After the experiment was completed, the feed was replenished for each cage of mice.

[0045] 2.5 Serum cholesterol determination

[0046] 2.5.1 After the collected whole blood specimens were placed at room temperature for 2 hours, they were centrifuged at 3000 g for 10 minutes to obtain the supernatant;

[0047] 2.5.2 Take out the required strips from the aluminum foil bag after equilibrating at room temperature for 20 min. Seal the remaining strips with a self-sealing bag and return them to 4°C.

[0048] 2.5.3 Set up standard wells and sample wells. Add 50 μL of standards with different concentrations to each standard well; add 50 μL of the sample to be tested to the sample wells; do not add anything to the blank wells.

[0049] 2.5.4 Except for the blank wells, add 100 μL of the detection antibody labeled with horseradish peroxidase (HRP) to each of the standard wells and sample wells. Seal the reaction wells with a sealing film and incubate in a 37°C water bath or incubator for 60 min.

[0050] 2.5.5 Discard the liquid, pat dry on absorbent paper. Fill each well with washing solution (350 μL), let stand for 1 min, discard the washing solution, and pat dry on absorbent paper. Repeat the washing process 5 times (or use a plate washer).

[0051] 2.5.6 Add 50 μL of Substrate A and B to each well and incubate at 37°C in the dark for 15 min.

[0052] 2.5.7 Add 50 μL of stop solution to each well and measure the OD value of each well at a wavelength of 450 nm within 15 min.

[0053] 2.5.8 Use the measured OD values of the standards as the abscissa and the concentration values of the standards as the ordinate to plot a standard curve on graph paper or using relevant software, and obtain the linear regression equation. Substitute the OD value of the sample into the equation to calculate the concentration of the sample, see Figure 6 .

[0054] Refer to Figure 1 , Figure 2 and Figure 6 , the experimental results show that: Momordica charantia exosomes can reduce the fasting blood glucose, postprandial blood glucose and blood lipids of mice. Among them, 400 μg / kg Momordica charantia exosomes can significantly reduce the fasting blood glucose, postprandial blood glucose and blood lipids of mice.

Claims

1. Use of Momordica charantia exosomes in the preparation of antidiabetic drugs, wherein the Momordica charantia exosomes are extracted by the following steps: 1.

1. Clean the fresh bitter gourd, cut it open to remove the seeds, and extract bitter gourd juice; 1.

2. Centrifuge the bitter gourd juice continuously. At 4°C, centrifuge at 1000g for 10 minutes, 3000g for 20 minutes, and 10000g for 40 minutes. After centrifugation, take the supernatant and discard the precipitate; 1.

3. Ultracentrifuge the obtained supernatant in a refrigerated ultracentrifuge at 4°C at 150000g for 90 min. After centrifugation, discard the supernatant and take the precipitate. Suspend the precipitate in 1 - 2 ml of phosphate buffer; 1.

4. Filter the suspension through a 0.22 - μm filter membrane. The filtrate is again ultracentrifuged in a refrigerated ultracentrifuge at 4°C at 150000g for 90 min. After centrifugation, discard the supernatant and take the precipitate. Suspend the precipitate in 1 - 2 ml of phosphate buffer; 1.

5. Filter the suspension through a 0.22 - μm filter membrane again to obtain sterile bitter gourd exosomes.

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