CN105497205A - Medicine for interventionally treating diabetes - Google Patents

Abstract

The invention discloses medicine for interventionally treating diabetes. The medicine comprises, by weight percentage, 50-79% of prepared fructus mume and 10-16% of mulberry, wherein the prepared fructus mume and the mulberry are effective constituents. Animal experiments show that the medicine for interventionally treating the diabetes can effectively lower the blood sugar level of rats with the diabetes and reduce area under a glucose tolerance experimental curve.

Description

A drug for intervening in the treatment of diabetes

technical field

The invention relates to a medicine for intervention and treatment of diabetes, in particular to a pharmaceutical composition for intervention and treatment of diabetes containing mulberry and black plum as active ingredients.

Background technique

Diabetes (diabetes) is a series of metabolic disorder syndromes of sugar, protein, fat, water and electrolytes caused by a variety of pathogenic factors acting on the body leading to hypofunction of islets and insulin resistance. Clinically, it is characterized by hyperglycemia. The overall prevalence of diabetes in my country has reached 9.7%. The prevalence of prediabetes during the same period was as high as 15.5%. According to the calculations made based on the survey results, the total number of people with diabetes in my country is more than 92 million, and the number of people with pre-diabetes is more than 148 million. Diabetes is the absolute or relative deficiency of insulin secretion by pancreatic β cells, with or without insulin resistance. The pathogenesis of diabetes is slow, and the mechanism has not been fully elucidated.

The treatment of diabetes needs to last almost throughout life. The main treatments include insulin, chemical drugs and traditional Chinese medicine, etc. Other methods that are not yet fully mature include islet transplantation and stem cell therapy. Although there are many methods, none of them can cure diabetes, and the incidence of diabetes is rising rapidly. The main reason is that during the development of diabetes, the functional damage of islet β cells already exists before the onset of diabetes, and this damage becomes irreversible with the degree of aggravation. Unfortunately, at the onset of diabetes, the functional damage of islet β cells is basically irreversible, and the treatment of diabetes often starts after the onset of diabetes. This is the main reason why diabetes cannot be cured at present.

Some studies have shown that intervention treatment before the onset of diabetes can effectively prevent or delay the occurrence of the disease, but the problem is that due to the limitations of medical ethics and drug side effects, the current diabetes treatment drugs cannot be used for preventive intervention before the onset of diabetes. treat. This also provides a new entry point for the prevention and treatment of diabetes, that is, to find new and safe drugs, and to perform early preventive intervention treatment of islet β-cell function damage before the onset of diabetes.

In this regard, after thousands of years of practical application, traditional Chinese medicine has been proven to be effective with few side effects, and can be used for body function conditioning, health care and other health care functions without medical ethics issues. Therefore, traditional Chinese medicine can play an irreplaceable important role in the preventive intervention and treatment of diabetes. As a traditional Chinese medicine, ebony is sour and slightly astringent. It has the functions of astringing the lungs and promoting body fluid, and astringent intestines to relieve roundworm. Because it can stimulate the secretion of saliva and produce body fluid to quench thirst, it is often used to treat thirst and thirst (such as diabetes) In Article 326 of "Treatise on Febrile Diseases", it is recorded that Wumei Pills treat "diabetes" and improve symptoms such as hunger, thirst, fatigue and weakness in patients. Black plums are rich in organic acids, such as citric acid, malic acid, oxalic acid, glycolic acid, fumaric acid, etc., of which the higher content is mainly citric acid and malic acid; in addition, black plums also contain flavonoids and terpenes , carbohydrates and other chemical components as well as Fe, Mg, Cu, Zn and other trace elements. In recent years' experimental research, two alkaloid compounds (2,2,6,6-tetramethylpiperidone, tert-butylurea) and superoxide dismutase were also isolated and identified from Ume. (SOD); The vitamin content of ebony is also quite rich, it contains vitamin C and vitamin B1 similar to other fruits, but the content of vitamin B2 is hundreds of times that of other fruits, reaching 5.6mg/100g. These rich chemical components make ebony have many beneficial effects on the human body.

Studies have shown that in the control experiment between ebony and metformin, the blood sugar control of the ebony and metformin groups is good, with fasting blood glucose <7.0mmol/L and postprandial blood glucose <10.0mmol/L; the glycosylated hemoglobin (HbAlc) level of the ebony group From 7.66% to 6.78%, the level of glycosylated hemoglobin in the metformin group dropped from 8.23% to 6.76%, there was no statistical difference, indicating that ebony can improve the level of glycosylated hemoglobin in T2DM rats, and has obvious hypoglycemic effect. At the same time, ebony can repair and regenerate damaged pancreatic beta cells, restore the function of pancreatic beta cells and stimulate insulin secretion.

Mulberry is the ripe fruit of the Moraceae plant mulberry. Traditional Chinese medicine believes that "Mulberry is sweet and cold, which can nourish the liver and kidney, nourish blood and dispel wind, soothe the nerves and nourish the heart, and delay aging." Mulberry is rich in fructose, amino acid, vitamins, phospholipids and minerals, etc. Excellent source of potassium. Like mulberry leaves, mulberry has hypoglycemic, hypolipidemic and antioxidant effects; 25 polyphenolic compounds isolated from mulberry ethyl acetate extract have significant activity of inhibiting α-glucosidase and scavenging free radicals, showing Mulberry has antioxidant effect.

Although the above prior art shows that ebony and mulberry may have hypoglycemic effects respectively, the prior art does not provide the method of using ebony and mulberry. Especially because mulberry is rich in fructose, if the mechanism and specific active ingredients are not clear, the rich fructose will cause a sharp increase in the blood sugar of diabetic patients, which is not conducive to the health of patients. Furthermore, ebony is rich in citric acid, malic acid, and succinic acid. Eating too much will not only damage the teeth, but also damage the spleen and stomach.

In the pre-diabetes intervention treatment stage, how to effectively control the patient's blood sugar without side effects is one of the subjects proposed by the present invention. One of the purposes of the present invention.

Based on the problems existing in the prior art, the medicine applied for in this patent is a kind of medicine with hypoglycemic function, which is extracted from pure natural plant ebony and mulberry, which can prevent the damage of pancreatic beta cells and protect the function of pancreatic beta cells. Has special curative effect. At the same time, the combination of ebony and mulberry eliminates the side effects of the two Chinese herbal medicines when they are taken alone through an appropriate proportion. Also has great practicality.

Contents of the invention

The technical problem to be solved by the present invention is to provide a pharmaceutical composition for intervening and treating diabetes before the onset of diabetes. In order to solve the above problems, the inventors have found a pharmaceutical composition for intervention and treatment of diabetes after painstaking research, which is characterized in that it contains ebony and mulberry as active ingredients.

A pharmaceutical composition for intervention and treatment of diabetes according to the present invention, the preferred active ingredient accounts for the percentage of the total weight of the pharmaceutical composition: the black plum accounts for 50% to 79%, and the mulberry fruit accounts for 10% to 16%. .

In the pharmaceutical composition for intervention and treatment of diabetes described in the present invention, more preferably, the black plum accounts for 79%, and the mulberry fruit accounts for 16%.

The pharmaceutical composition for intervention and treatment of diabetes in the present invention further includes pharmaceutically acceptable carriers and/or excipients.

The present invention also provides an application of a pharmaceutical composition in the intervention and treatment of diabetes, which is characterized in that the pharmaceutical composition contains ebony and mulberry as active ingredients.

The present invention also provides an application of a pharmaceutical composition in the intervention and treatment of diabetes, preferably, the percentage of the active ingredient in the total weight of the pharmaceutical composition is: the black plum accounts for 50% to 79%, and the mulberry fruit accounts for 10% ~16%.

The present invention also provides an application of a pharmaceutical composition in the intervention and treatment of diabetes, more preferably, the black plum accounts for 79%, and the mulberry fruit accounts for 16%.

Description of drawings

Fig. 1: The distillate extraction figure of an example of the embodiment of the present invention.

Fig. 2: A comparison chart of the effect of the composition of the present invention on lowering blood sugar in diabetic rats.

Figure 3: A comparative graph of the effect of the composition of the present invention on reducing the area under the curve of the glucose tolerance test in diabetic rats.

detailed description

Embodiments of the present invention will be described in detail below, but the embodiments of the present invention are not limited by the following specific embodiments.

1. Separation and collection of mulberry and ebony volatile oil

Volatile oil, also known as essential oil, is a general term for a class of oily liquids immiscible with water that can be obtained by steam distillation. Common methods for extracting volatile oil from the fresh fruits of mulberry and ebony or their quick-frozen and refrigerated fresh fruits include distillation, solvent extraction, pressing, and supercritical liquid extraction. In this embodiment, the steam distillation method adopted by the volatile oil of general traditional Chinese medicine is adopted. The specific method is a conventional method, and the extraction methods described in patent documents such as CN201940071U, CN2928228Y, and CN2686690Y can also be used.

In addition to extracting the volatile oil from the fresh fruit mentioned above, the raw materials can also be dried by various drying methods, and then the traditional Chinese medicine extract can be prepared by the general method adopted in this field. As long as the main components of the raw materials are not destroyed, the present invention can choose any The main components of mulberry and ebony are extracted by means, and the volatile oil is preferably separated and collected, because the sugar and other side effects can be reduced to a reasonable range in the volatile oil.

2. Preparation of Compositions

The volatile oils separated and collected from the mulberry and ebony are mixed according to a specific ratio to prepare the composition stock solution. According to different clinical needs, it can be made into pills, powders, tablets, suppositories, granules, films, capsules, microcapsules, dropping pills, aerosols, injections, ointments, liquors, syrups, oral Solution, gel or liposome. The specific implementation method can be carried out according to the standard of Chinese Pharmacopoeia 2000 edition.

3. The effect detection of composition of the present invention

3.1 Animal source and model preparation

Healthy male SD rats, aged 2 months and weighing 150-200 g, were provided by the Experimental Animal Center of Zhejiang Province. Before the experiment, the animals were placed in the laboratory to adapt to the environment for a week, free to drink water and fed with standard rat food, keep the cage clean, the room temperature was controlled at 23±2°C, natural light (raised in the Experimental Animal Center of Ningbo University Medical College), relative humidity 60% to 70%. First, 6 rats were randomly selected as the normal control group and fed with common feed for four weeks. All the other rats were fed with high-sugar and high-fat feed (15% lard, 20% sucrose, 13 egg yolk powder, 2% sodium cholate, 50% common feed) for four weeks and then with a dose of 30 mg/kg (once a week, continuously 2 weeks) One-time intraperitoneal injection of 1% streptozotocin (STZ, American Sigma Company, batch number: SO130-1g) citric acid buffer solution (prepared with 0.1mol/L sodium citrate buffer solution with a pH of 4.2, now prepared The normal control group was injected with the same amount of normal saline synchronously in the tail vein.

Two weeks later, the tail of the model-making rats was cut and blood was taken to measure fasting blood glucose and random blood glucose. Twelve rats with fasting blood glucose (FBG) ≥ 7.8mol/L and random blood glucose ≥ 11.1mol/L were selected and randomly divided into 6 rats in the diabetes model group were fed with 0.9% normal saline every day; 6 rats in the positive drug control group were fed with mulberry preparation every day.

3.2 Interventions

After the successful establishment of the diabetes model, the intervention treatment began. The ebony mulberry preparation was administered by intragastric administration at a dose of 5g/kg, once a day, for 4 consecutive weeks. The normal control group was given the same amount of normal saline at the same time, and the rats were gavaged continuously for 4 weeks.

The experimental group of the composition of the present invention was gavaged according to the dosage of 200ml/kg, and the normal control group and the negative control group were gavaged with normal saline of solvents such as gavage. Wherein when configuring the composition of the present invention, the remainder except the mulberry and ebony distillate is distilled water. The time interval of gavage was 12 hours.

3.3 Collection and processing of specimens

After the treatment, the rats in each group were measured for fasting blood glucose, weighed, and related records were made, and then the rats were seized and directly cut off one side of the femoral artery to cause their death. Then the rats were dissected and the pancreas was quickly taken out, and 4°C normal saline After rinsing, fix with 4% formaldehyde for 24 hours, soak in distilled water for 4 hours. Take out conventional graded alcohol dehydration, make xylene transparent, embedding in paraffin, slice continuously with LEICA2135 paraffin microtome, thickness 5 μm, take 1 slice every 20 slices, and take 6 slices for each specimen, and mount them on polylysine-coated carriers respectively. Store on glass slides at room temperature.

3.4 Observation indicators

3.4.1 General situation

Changes in food intake, drinking water, urine output, and mental status of the rats were observed daily. Body weight was measured before modeling, after modeling, and after treatment.

3.4.2 Blood glucose measurement

3.4.2.1 Fasting blood glucose and random blood glucose

The fasting blood glucose (FBG) (mmol/L) of animals in each group was measured before modeling, after modeling, and after treatment, and random blood glucose was measured every 3 days. Blood was collected from the tail vein of the rats, fasting blood glucose was measured with an automatic blood glucose meter, and relevant records were made.

3.4.2.2 Oral glucose tolerance test (OGTT)

Oral glucose tolerance test (OGTT) was performed before modeling, after modeling and after treatment. After fasting for 12 hours, the rats were fed with 50% glucose solution by gavage at a dose of 2 g/kg, and blood glucose was measured by taking tail vein blood at 0 h, 0.5 h, 1 h, and 2 h. After the experiment, the glucose tolerance curves of rats in each group were drawn.

3.4.3 Pathomorphology

After the treatment, the animals were sacrificed and the pancreas was removed quickly, rinsed with normal saline at 4°C, fixed with 4% formaldehyde for 24 hours, and soaked in distilled water for 4 hours. Routine gradient alcohol dehydration, xylene transparency, paraffin embedding, LEICA2135 paraffin microtome serial section, thickness 5μm, mounted on polylysine-coated glass slides, stained with hematoxylin-eosin staining under an optical microscope Observed.

3.4.4 Immunohistochemistry

Prepare HIF-1α affinity pure antibody, ready-to-use SABC kit, and DAB chromogenic kit. Take the above-mentioned slices and place them in a 60°C oven for 1 hour, and operate in strict accordance with the operation instructions of the kit. Perform routine dewaxing of the slices, soak in distilled water for 2 minutes; add 3% hydrogen peroxide at room temperature for 10 minutes; wash with distilled water for 1 minute x 3 times ; Place slices in 0.01mol/L citrate buffer, heat to boiling with microwave, power off for 15 minutes, heat again to boil and cool naturally; add 0.02mol/LPBS to wash for 1 minute x 3 times; add 5% BSA antigen to block , at room temperature for 20 minutes, shake off excess liquid; add dropwise primary antibody (1:100) to dilute, and incubate at 37°C for 1 hour; wash with 0.02mol/LPBS for 2 minutes×3 times; dropwise add biotinylated secondary antibody, and incubate at 37°C 20 minutes; wash with 0.02mol/LPBS for 2 minutes×3 times; add horseradish peroxidase-labeled streptavidin dropwise, incubate at 37°C for 20 minutes; wash with 0.02mol/LPBS for 5 minutes×4 times; develop color with DAB, The reaction time was controlled under the microscope; hematoxylin counterstained for 10 seconds, rinsed with tap water; dehydrated, transparent, and mounted with neutral resin. Observed under a light microscope, the specific staining was flaky brown-yellow granules. For each batch of staining, a negative blank control with 0.1mol/LPBS solution instead of the primary antibody was established simultaneously. Under an optical microscope (400 times), 4 fields of view were randomly selected for each slice, and the number of positive cells in each field of view was counted respectively, and finally the average number of each group was used for comparison.

3.4.5 Immunofluorescence staining

Tissue frozen sections were fixed with acetone (cold acetone) at room temperature for 10 minutes. After fixation, use a capillary pipette to draw appropriately diluted immune serum and drop it on top of it. Place it in a staining box to maintain a certain humidity, act at 37°C for 30 minutes, and then use 0.01mol /1pH7.2PBS, wash twice for 10min, absorb or dry the remaining liquid with water; then add indirect fluorescent antibody (such as rabbit anti-human γ-globulin 1 fluorescent antibody, etc.), and then wash twice with PBS , 10min, staining for 30min, 37°C, washed twice for 10min with buffered saline, stirred, mounted with buffered glycerol, and observed under a confocal laser microscope. Control staining: replace the immune serum with normal rabbit serum or human serum, and then stain with the above method, the result should be negative.

3.4.6 RT-PCR

After extracting RNA from the tissue, measure the concentration, A1/A2 should be between 1.8-2.0, centrifuge the RNA briefly, add M-MLV reverse transcriptase 1ul, 5×RT buffer 5ul, Oligo(dT)18 (20mol/L ) 2ul, dNTP (20umol/L) 2ul, RNA 2ug, add deionized water to 20ul volume, reverse transcription reaction at 42°C for 1h, inactivate M-MLV at 95°C for 5min, then centrifuge, heat at 70°C for 5min, stop the above reaction, and place Perform reverse transcription reaction on ice, add reaction reagents to the PCR reaction tube in strict accordance with the kit operation instructions, then insert the PCR tube into the PCR instrument, denature at 95°C for 5 min; perform 35 cycles of amplification reaction (94°C, 1min; the annealing temperature depends on the primer (50-60°C), 1min; 72°C, 1min); then keep the temperature at 72°C for 7min, take out the PCR reaction tube, and perform electrophoresis detection on the reaction product. After the electrophoresis, take out the agarose gel, Gently place on a gel imager or image on a UV transilluminator. Estimate the size of the amplified band according to the DNA molecular weight standard, and archive the electrophoresis results in electronic files or take pictures with a camera system.

3.4.7 Westemblot

The protein was extracted from the tissue and quantified. After the protein was separated by 10% SDS polyacrylamide gel electrophoresis, it was transferred to the nitrocellulose membrane by electrophoresis. After washing the membrane, add the primary antibody (the primary antibody was diluted to an appropriate concentration with TBST) and incubate for 1 h. After washing the membrane again, add the horseradish peroxidase (HRP)-labeled secondary antibody diluted with Western secondary antibody diluent and incubate for 1 h. The chromogenic solution was developed for 1 hour, and the molecular weight and optical density of the target band were analyzed with a gel image analysis system.

Example

The fresh fruit (or frozen fresh fruit) of 1000g mulberry and ebony is pulverized and homogenized with a blender respectively, placed in a distillation flask as shown in Figure 1, and the distillation product is collected after distillation. Wherein the distillation product of mulberry is 452g, and the yield is 45.2%, and the distillation product of ebony is 337g, and the yield is 33.7%.

The above-mentioned distilled products were formulated in different proportions, and fed to model rats and normal rats as controls. The specific experimental examples and their examples are shown in Table 1. After the four-week feeding experiment, the blood index detection values of the rats in each group were counted, and the concentration of serum glucose (in mg/dl) was measured by the glucose oxidase method.

What Fig. 2 shows is the result of a typical experimental example of the composition of the present invention, and each experimental group is the average value of three repeated experiments.

in,

Group a is the blank control of normal rats

Group b is the blank control of diabetic model rats

Group c is the blood glucose concentration after the intervention treatment of the composition of the present invention in diabetic model rats

Group d is the diabetic model rats that take black plum alone

Group e is diabetic model rats taking mulberries alone

Fig. 3 shows that the medicine applied for by this patent has the effect of reducing the area under the curve of the glucose tolerance test of diabetic rats. Among the figures, a is the normal group, group b, group c, group d and group e are respectively the diabetes blank control group and different For the medication group, the normal control group was taken as 1, and the rest were compared with it. in,

Group a is the blank control of normal rats

Group b is the blank control of diabetic model rats

Group c is the blood sugar concentration of diabetic model rats after the intervention treatment of the composition of the present invention.

Group d is the diabetic model rats that take black plum alone

Group e is diabetic model rats taking mulberries alone

The results of other embodiments of the present invention and comparative examples are as shown in Table 1, according to the results shown in Table 1, after the pharmaceutical composition of the invention is used, the technical effects obtained are as follows:

1. Compared with the matched group, the distillate of ebony plum or mulberry is fed alone, although the hyperglycemia in the diabetic model rats has a certain inhibitory effect, the composition of the present invention can reduce the blood sugar of the rats to a lower level s level.

2. Especially according to this example of the present invention, the system ebony plum and mulberry are used in combination, and the results show that the blood sugar concentration of rats is controlled at a normal value between 7-10mg/dl, and unexpected effects have been achieved.

Table 1. Blood glucose concentration of rats after 4 weeks in each experimental example and control example

Claims (7)

1. A medicine for intervening and treating diabetes, characterized in that it contains black plum and mulberry as active ingredients. 2. The pharmaceutical composition according to claim 1, characterized in that, the percentages of the active ingredients in the total weight of the pharmaceutical composition are: the black plum accounts for 50% to 79%, and the mulberry fruit accounts for 10% ~16%. 3. The pharmaceutical composition according to claim 2, characterized in that, the black plum accounts for 79%, and the mulberry fruit accounts for 16%. 4. The pharmaceutical composition according to any one of claims 1-4, further comprising pharmaceutically acceptable carriers and/or excipients. 5. An application of a pharmaceutical composition in the intervention and treatment of diabetes, characterized in that the pharmaceutical composition contains black plum and mulberry as active ingredients. 6. The application according to claim 5, characterized in that, the percentages of the active ingredients in the total weight of the pharmaceutical composition are: the ebony is 50% to 79%, and the mulberry is 10% to 16%. %. 7. The application according to claim 6, characterized in that 79% of the black plum is made, and 16% of the mulberry.