CN104758316A - Pharmaceutical composition for preventing and curing diabetes itches - Google Patents

Abstract

The invention discloses a pharmaceutical composition for preventing and treating diabetic itch, which contains tortoise shell and raw oyster as active ingredients. The raw oysters accounted for 5% to 12%. Animal experiments show that the medicinal composition for intervening and treating diabetes can effectively reduce the itching index of diabetic rats, lower blood sugar levels, reduce the area under the curve of glucose tolerance test and reduce the itching index of diabetic rats.

Description

A pharmaceutical composition for preventing and treating diabetic itch

technical field

The invention relates to a pharmaceutical composition for preventing and treating diabetic itch, in particular to a pharmaceutical composition for intervening and treating diabetic itch containing tortoise shell and raw oyster 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 onset of diabetes is slow, and what seriously endangers the health and life of diabetic patients is the various chronic complications of the disease, including diabetic peripheral neuropathy. Diabetes can damage all peripheral nerves, including the auditory nerve, leading to diabetic neuropathic hearing impairment. Although the clinical manifestations of peripheral nerve damage are different due to different parts, the mechanism of nerve damage is generally the same. Diabetes is a chronic inflammatory state, and the imbalance of inflammatory cytokines in the internal environment plays a key role in the occurrence and development of diabetes and its chronic complications. The plasma reticulum stress eventually leads to functional changes and even apoptosis of peripheral neurons, and then diabetic neuropathy occurs.

The treatment of diabetes and its complications 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.

Studies have shown that active secondary prevention of diabetes can effectively prevent or delay the occurrence of chronic complications, but the problem is that due to limitations in medical ethics and drug side effects, some chemical drugs cannot be used for preventive interventions . 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 at all levels.

The tortoise shell is the carapace and plastron of the tortoise of the turtle family. The tortoise shell has a strong quality and suppresses the potential; Hyperactivity of asthenic fire, bone steaming and hot flashes, night sweats and nocturnal emission; yin deficiency and wind movement, wriggling of hands and feet; deficiency of kidney yin, soreness of waist and knees, incompatibility of fontanel in children; mental disorder, palpitations and palpitations, insomnia and forgetfulness; menstrual blood disorder, vomiting blood and epistaxis, menstruation Too much, abdominal pain, uterine bleeding leukorrhea.

The main active ingredient of turtle shell is animal glue, cutin and various amino acids have anti-oxidation activity, so it can protect the degeneration of islet β cells and reduce lipid peroxidation. Tortoise shell extract caffeic acid and p-coumaric acid can inhibit the content of PGE2 and the expression of COX-2mRNA, indicating that turtle shell has anti-inflammatory effect. In addition, tortoise shell can significantly alleviate peripheral neuropathy in early diabetic rats.

Raw oysters, also known as oysters and clams. Raw oysters are suitable for people with deficiency and fever, children with weak constitution, hilar lymphatic tuberculosis, cervical lymphatic tuberculosis, scrofula, yin deficiency, dysphoria, insomnia, restlessness, cancer and radiotherapy (after chemotherapy). In addition, a team of American and Italian scientists found that eating raw oysters can improve libido, especially in spring. Raw oysters are rich in amino acids, vitamins, phospholipids and minerals, and are a high-quality source of potassium. Raw oysters have hypoglycemic, hypolipidemic, and antioxidant effects; 25 polyphenolic compounds isolated from raw oyster extracts all have significant activities of inhibiting α-glucosidase and scavenging free radicals, showing that raw oysters have antioxidative properties. effect.

Although it has been shown in the above prior art that tortoise shell and raw oyster may respectively have anti-oxidation, anti-inflammatory and hypoglycemic effects, the prior art does not provide the method of using tortoise shell and raw oyster. Especially because the intermediary metabolism of raw oysters in the body can be converted into fructose, without clarifying its mechanism and specific active ingredients, taking it without authorization will cause a sharp increase in blood sugar in diabetic patients, which is not conducive to the health of patients. Furthermore, the intermediate metabolites of tortoise shell can be converted into 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 secondary prevention of diabetes, how to effectively control the patient's blood sugar without producing side effects is one of the subjects proposed by the present invention. One of the purposes of the present invention.

Based on the existing problems in the prior art, the medicine applied for in this patent is a kind of medicine that has the function of preventing and improving diabetic peripheral neuropathy, which is extracted from pure natural tortoise shell and raw oyster. The occurrence of complications has a special effect. At the same time, the tortoise shell and raw oyster composition eliminates the effect of preventing diabetic neuropathy from being fully manifested when the two Chinese medicinal materials are taken alone through an appropriate ratio. As a secondary preventive intervention to treat diabetes, it has achieved unexpected therapeutic At the same time, it has great practicability in terms of cost and price convenience.

Contents of the invention

The technical problem to be solved by the present invention is to provide a pharmaceutical composition with intervention and therapeutic effect before the occurrence of diabetic peripheral neuropathy. In order to solve the above problems, the inventors have found a pharmaceutical composition for the intervention and treatment of diabetic itch after painstaking research, which is characterized in that it contains turtle shell and raw oyster as active ingredients.

A pharmaceutical composition for preventing and treating diabetic itch according to the present invention, preferably, the active ingredients account for the percentage of the total weight of the pharmaceutical composition: the turtle shell accounts for 62% to 83%, and the raw oyster accounts for 5% to 12%. %.

In the pharmaceutical composition for preventing and treating diabetic itch according to the present invention, more preferably, the turtle shell accounts for 83%, and the raw oyster accounts for 12%.

The pharmaceutical composition for preventing and treating diabetic itch in the present invention also includes pharmaceutically acceptable carriers and/or excipients.

The present invention also provides an application of a pharmaceutical composition in the intervention and treatment of diabetic itch, which is characterized in that the pharmaceutical composition contains tortoise shell and raw oyster as active ingredients.

The present invention also provides an application of a pharmaceutical composition in the intervention and treatment of diabetic itch. Preferably, the active ingredients account for 62% to 83% of the total weight of the pharmaceutical composition, and the raw oyster accounts for 62% to 83%. 5% to 12%.

The present invention also provides an application of a pharmaceutical composition in the intervention and treatment of diabetic itch, more preferably, the turtle shell accounts for 83%, and the raw oyster accounts for 12%.

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.

Figure 4: The composition of the present invention has the effect of reducing the itching index of diabetic rats

Detailed ways

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 volatile oil from turtle shell and raw oyster

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 Morus alba 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 tortoiseshell and raw oysters are extracted by means of methods, and the volatile oil is preferably separated and collected, because the volatile oil can reduce sugar and other side effects to a reasonable range.

2. Preparation of Compositions

The above volatile oils separated and collected from turtle shells and raw oysters are mixed according to a specific ratio to prepare the stock solution of the composition. 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: S0130-1g) citric acid buffer (prepared with 0.1mol/L sodium citrate buffer with pH 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. 16 rats with fasting blood glucose (FBG) ≥ 7.8mol/L and random blood glucose ≥ 11.1mol/L were selected and randomly divided into Eight rats in the diabetes model group were fed with 0.9% normal saline every day; eight rats in the positive drug control group were fed with turtle shell and raw oyster preparations every day.

3.2 Interventions

After the successful establishment of the diabetes model, the intervention treatment began. Tortoise shell and raw oyster were 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 Morus alba 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 observe.

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 section, and the number of positive cells in each field of view was counted respectively, and finally compared with the mean number of each group.

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 white 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.

3.4.7Western blot

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

1000g of tortoiseshell and raw oysters (or frozen fresh) were mashed and homogenized with a mixer, placed in a distillation flask as shown in Figure 1, and the distillation products were collected after distillation. Wherein the distillation product of tortoise shell is 452g, and the yield is 45.2%, and the distillation product of raw oyster is 337g, and the yield is 33.7%.

The above-mentioned distillation products were formulated in different proportions, and fed to model rats and normal rats as controls. The specific experimental examples and comparative 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 diabetic model rats taking tortoise shell alone

Group e is diabetic model rats taking raw oysters 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 diabetic model rats taking tortoise shell alone

Group e is diabetic model rats taking raw oysters alone

Fig. 4 shows that the medicine applied for by this patent has the effect of reducing the itching index 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 medication groups, taking The normal control group is 1, and the rest are 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 diabetic model rats taking tortoise shell alone

Group e is diabetic model rats taking raw oysters 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 control group, feeding the distillate of tortoise shell or raw oyster alone, although the hyperglycemia in the diabetic model rats has a certain inhibitory effect, but the composition of the present invention can reduce the blood sugar of the rats to a lower level s level. ,

2. Especially according to the ratio of the present invention, tortoise shell and raw oysters are mixed and used, and the results show that the blood sugar concentration of rats is controlled at a normal value between 7-10 mg/dl, and an unexpected effect has been achieved.

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

Claims (7)

1. A pharmaceutical composition for preventing and treating diabetes itch is characterized by containing tortoise plastron and raw oyster as effective components.

2. The pharmaceutical composition of claim 1, wherein the effective components comprise, by weight: the tortoise plastron accounts for 62-83 percent, and the raw oyster accounts for 5-12 percent.

3. The pharmaceutical composition of claim 2, wherein said tortoise plastron is 83% and said raw oyster is 12%.

4. The pharmaceutical composition of any one of claims 1 to 4, further comprising a pharmaceutically acceptable carrier and/or excipient.

5. The application of a pharmaceutical composition in intervening treatment of diabetic itching is characterized in that the pharmaceutical composition contains tortoise plastron and raw oyster as effective components.

6. The use according to claim 5, wherein the active ingredients comprise, in percentages by weight of the total weight of the pharmaceutical composition: the tortoise plastron accounts for 62-83 percent, and the raw oyster accounts for 5-12 percent.

7. The use of claim 6, wherein the tortoise plastron is 83% and the raw oyster is 12%.