CN104825632A - Drug for intervention treatment of diabetic peripheral neuropathy (DPN) - Google Patents
Drug for intervention treatment of diabetic peripheral neuropathy (DPN) Download PDFInfo
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- CN104825632A CN104825632A CN201510208679.8A CN201510208679A CN104825632A CN 104825632 A CN104825632 A CN 104825632A CN 201510208679 A CN201510208679 A CN 201510208679A CN 104825632 A CN104825632 A CN 104825632A
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- egg yolk
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Landscapes
- Medicines Containing Plant Substances (AREA)
Abstract
The invention discloses a drug for the intervention treatment of diabetic peripheral neuropathy (DPN). The drug comprises the following effective components in percentage by weight: 45 to 80% of chicken egg yolk, and 5 to 15% of dried rehmannia root. The results of animal experiments show that the provided drug can effectively reduce the blood sugar level of diabetic rats, moreover, the area under the curve (AUC) in the glucose tolerance tests is reduced, and the DPN pain index of diabetic rates is also reduced.
Description
Technical Field
The invention relates to a medicine for intervening and treating diabetic peripheral neuropathy pain, in particular to a medicine for intervening and treating diabetic peripheral neuropathy, which contains egg yolk and dried rehmannia as effective components.
Background
Diabetes (diabetes) is a series of metabolic disorder syndromes of sugar, protein, fat, water, electrolyte and the like caused by hypofunction of pancreatic islets, insulin resistance and the like due to the action of various pathogenic factors on organisms, and is clinically characterized by hyperglycemia. The total prevalence rate of diabetes in China currently reaches 9.7 percent. The prevalence rate of the early stage of the concurrent diabetes is as high as 15.5 percent. According to the calculation made by the survey result, the total diabetes number in China reaches over 9 thousand and 2 million, and the diabetes number in the early stage reaches over 1 hundred million and 4 thousand and 8 million. Diabetes is an absolute or relative deficiency of insulin secretion by the beta cells of the islets of langerhans, with or without insulin resistance. The onset of diabetes is slow and the mechanism is not fully elucidated.
The treatment of diabetes almost needs to be throughout life, mainly including insulin, chemical drugs, traditional Chinese medicines and the like, and other methods which are not completely mature include islet transplantation and stem cell treatment. Although the methods are not few, the diabetes cannot be cured, and the incidence rate of the diabetes is in a rapidly rising state. The main reason for this is that during the development of diabetes, impaired islet beta cell function already exists before the onset of diabetes, and this impairment becomes irreversible with increasing degree. Unfortunately, the functional impairment of islet beta cells is essentially irreversible at the onset of diabetes, and the treatment of diabetes often begins after diabetes has occurred. This is the major reason why diabetes cannot be cured at present. Diabetes mellitus is a slow-onset disease that presents a serious health and life risk to diabetic patients as a variety of chronic complications of diabetes mellitus, including diabetic peripheral neuropathy, which can damage all peripheral nerves, including the auditory nerve, to diabetic neuropsychiatric hearing impairment. Although the clinical manifestations of peripheral nerve damage vary from site to site, the mechanisms involved in nerve damage are generally the same.
There are studies that have shown that active treatment of diabetes can effectively prevent or delay the onset of chronic complications. In this regard, the traditional Chinese medicine has proved effective and few side effects after thousands of years of practical application, and can be used for health preserving functions such as body function conditioning, health care and the like without medical ethical problems. Therefore, the traditional Chinese medicine can play an irreplaceable important role in the preventive intervention treatment of the diabetes. The smoked plum as a traditional Chinese medicine has sour and slightly astringent taste, has the functions of astringing lung, promoting the production of body fluid, astringing intestine and relieving ascaris, is commonly used for treating thirst and polydipsia (such as diabetes) due to the stimulation of salivary secretion, the production of body fluid and the thirst quenching, and the treatment of the thirst and the polydipsia by the smoked plum pill is recorded in 326 article in the treatise of the treatise on the typhoid fever, and the symptoms of easy hunger, thirst, fatigue and the like of patients are improved. The dark plum contains rich organic acids, such as citric acid, malic acid, oxalic acid, glycolic acid, fumaric acid and the like, wherein the high content of the organic acids is mainly citric acid and malic acid; in addition, mume fructus also contains chemical components such as flavonoids, terpenoids, saccharides, etc., and microelements such as Fe, Mg, Cu, Zn, etc. In recent experimental studies, 2 alkaloid compounds (2, 2, 6, 6-tetramethylpiperidone and tert-butyl urea) and superoxide dismutase (SOD) are also separated and identified from dark plum; the vitamin content of the dark plum is quite rich, and the dark plum contains vitamin C and vitamin B1 similar to other fruits, but the content of the vitamin B2 is hundreds of times higher than that of other fruits, and the content of the vitamin B reaches 5.6mg/100 g. These abundant chemical components make dark plum have a great many beneficial effects on the human body.
Research shows that in a control experiment of dark plum and metformin, the blood sugar control condition of dark plum and metformin groups is good, the fasting blood sugar is less than 7.0mmol/L, and the postprandial blood sugar is less than 10.0 mmol/L; the glycosylated hemoglobin (HbAlc) level of the dark plum group is reduced from 7.66% to 6.78%, the glycosylated hemoglobin level of the metformin group is reduced from 8.23% to 6.76%, and no statistical difference exists, so that the dark plum can improve the glycosylated hemoglobin level of a T2DM rat and has an obvious blood sugar reducing effect. Meanwhile, the dark plum has the effects of repairing and regenerating damaged islet beta cells, so that the function of the islet beta cells is recovered, and insulin secretion is stimulated.
Egg yolk, name of traditional Chinese medicine. Is egg yolk of pheasant family chicken. The fresh egg is shelled, the egg white is removed, and the egg yolk is remained for use. The "Renzai of materia Medica": tonify middle-jiao and Qi, nourish kidney and nourish yin, moisten lung and relieve cough, and treat hematemesis due to consumptive disease. Modern research shows that lecithin, triglyceride, cholesterol and ovoflavin in egg yolk have great effect on development of nervous system and body. After being digested by human body, the lecithin can release choline, and the choline can improve the memory of various age groups. The egg yolk powder has the advantages that the egg yolk powder is strong in strength and capable of keeping all nutrients of egg yolk, the concept of homology of medicine and food is inherited, natural energy of the egg yolk is released by science and technology, the egg yolk powder is healthy and strong, and the developed egg yolk powder is also suitable for infants. In addition, researches show that the egg yolk has the effects of reducing blood sugar, reducing blood fat and resisting oxidation; the 25 polyphenol compounds separated from the substance extracts have obvious activities of inhibiting alpha-glucosidase and removing free radicals, and show that the egg yolk has antioxidation.
Rehmannia root, radix rehmanniae, as a nutrient supplement, can nourish the middle-jiao and earth. For traumatic injuries and exhaustion, it is also known as tonifying blood and tonifying injury from the book Jing. It is indicated for middle energizer injury, namely, it can tonify yin and nourish blood. The smell is mild, and the nourishing effect cannot be obtained when the viscera are deficient. It is indicated for five internal injuries of five organs due to overstrain and seven injuries of men and female injuries due to cyst leakage and bleeding. For arthralgia syndrome due to blood flow, the blood is deficient and the arthralgia is obstructed, so the nourishing is sufficient, the natural flow and the ocean overflow, and the arthralgia syndrome is treated with . Filling bone marrow and muscle with long muscle can be regarded as the most important meaning for tonifying. The dried rehmannia has the effects of reducing blood sugar and blood fat and resisting oxidation; the 35 polyphenol compounds separated from the dry rehmannia root extract have obvious activities of inhibiting alpha-glucosidase and eliminating free radicals, and show that the dry rehmannia root has an anti-oxidation effect.
Although the above prior art shows that the egg yolk and the dry rehmannia root respectively have the effect of reducing blood sugar, the prior art does not provide a method for using the egg yolk and the dry rehmannia root. Especially, since egg yolk is rich in fructose, the fructose, which is rich after being taken without any intention, causes a rapid increase in blood sugar level of a diabetic patient, and is not good for the health of the patient, without understanding the mechanism and specific active ingredients thereof. Furthermore, dry rehmannia is rich in citric acid, malic acid and succinic acid, and when eaten more, the dry rehmannia can not only damage teeth, but also damage spleen and stomach.
The invention aims to provide an effective and low-cost pharmaceutical composition for effectively controlling the blood sugar of a patient without generating side effects in the prevention and treatment stage of diabetes and meet the requirements of different people taking the composition.
Based on the problems in the prior art, the medicine applied by the patent is a medicine with the function of reducing blood sugar, which is prepared by mixing, processing and refining pure natural medicines of egg yolk and dried rehmannia, and has special curative effects on preventing the damage of islet beta cells and protecting the function of the islet beta cells. Meanwhile, the composition of the egg yolk and the dry rehmannia root eliminates the side effect of the two traditional Chinese medicinal materials when being taken independently through proper proportion, and has great practicability when being used as a medicament for early intervention treatment of diabetes, and the composition has unexpected treatment effect and is convenient in cost and price.
Disclosure of Invention
The invention aims to solve the technical problem of providing a medicine for intervening and treating diabetic peripheral neuropathy pain before the onset of diabetes. In order to solve the problems, the inventor of the invention finds a medicine for intervening and treating diabetic peripheral neuropathy pain through intensive research, and is characterized by containing egg yolk and dry rehmannia as effective components.
The invention relates to a medicament for intervening and treating diabetic peripheral neuropathy pain, which preferably comprises the following active ingredients in percentage by weight: the egg yolk accounts for 45-80 percent, and the dried rehmannia accounts for 5-15 percent.
The medicine for intervening and treating diabetic peripheral neuropathy pain is characterized in that more preferably, the egg yolk accounts for 80% and the dried rehmannia accounts for 15%.
The medicine for intervening and treating the diabetic peripheral neuropathy pain further comprises a pharmaceutically acceptable carrier and/or excipient.
The invention also provides an application of the medicine composition in intervening and treating diabetic peripheral neuropathy pain, which is characterized in that the medicine composition contains egg yolk and dry rehmannia as effective components.
The invention also provides an application of the pharmaceutical composition in interventional therapy of diabetic peripheral neuropathy pain, preferably, the active ingredients account for the following percentage by weight of the total weight of the pharmaceutical composition: the egg yolk accounts for 45-80 percent, and the dried rehmannia accounts for 5-15 percent.
The invention also provides application of the pharmaceutical composition in intervention treatment of diabetic peripheral neuropathy pain, and more preferably, the egg yolk accounts for 80% and the dried rehmannia accounts for 15%.
Drawings
FIG. 1: an example of the embodiment of the present invention is a distillate extraction diagram.
FIG. 2: the composition of the invention has a comparative graph of the effect of reducing the blood sugar of diabetic rats.
FIG. 3: the composition of the present invention has a comparative graph of the effect of decreasing the area under the glucose tolerance test curve of diabetic rats.
FIG. 4: the composition has the function of reducing the pain index of diabetic rat peripheral neuropathy
Detailed Description
The embodiments of the present invention will be specifically described below, but the embodiments of the present invention are not limited by the following specific embodiments.
1. Separation and collection of volatile oil of egg yolk and dried rehmannia root
Essential oils, also known as essential oils, are a generic name for water-immiscible oily liquids that can be obtained with steam distillation. The common methods for extracting volatile oil from fresh medicinal materials of ovum gallus Domesticus flavus and rehmanniae radix or quick-frozen and refrigerated medicinal materials thereof include distillation, solvent extraction, squeezing, and supercritical fluid extraction. In the embodiment, a steam distillation method adopted by common traditional Chinese medicine volatile oil is adopted, and a specific method is a conventional method, and extraction methods described in patent documents such as CN201940071U, CN2928228Y, CN2686690Y and the like can also be adopted.
In addition to the above-mentioned extraction of volatile oil from fresh fruit, the raw material can be dried by various drying methods, and then the Chinese medicinal extract can be prepared by the general method adopted in the field, so long as the main components of the raw material are not damaged, the main components of the egg yolk and the dried rehmannia root can be extracted by any method, wherein the volatile oil is preferably separated and collected, because the sugar content and other side effect components in the volatile oil can be reduced to a reasonable range.
2. Preparation of the composition
Mixing the volatile oils respectively separated and collected from ovum gallus Domesticus flavus and rehmanniae radix at a certain ratio to obtain a composition stock solution. And making into pill, powder, tablet, suppository, granule, membrane, capsule, microcapsule, dripping pill, aerosol, injection, unguent, medicated liquor, syrup, oral solution, gel or liposome according to different clinical requirements. The specific implementation can be implemented according to the standard of the Chinese pharmacopoeia 2000 edition.
3. Effect testing of the compositions of the invention
3.1 animal sources and model preparation
Healthy male SD rats aged 2 months and weighing 150-200 g are provided by the centers of Experimental animals in Zhejiang province. The animals are placed in a laboratory for one week before the experiment, are fed with free drinking water and standard rat feed, are kept clean in a cage, are controlled at the room temperature of 23 +/-2 ℃, are naturally illuminated (raised in the center of laboratory animals of Ningbo university medical college), and have the relative humidity of 60-70%. First, 6 rats were randomly selected as normal control groups and fed with normal feed for four weeks. The remaining rats were fed with high-sugar high-fat diet (15% lard, 20% sucrose, 13 egg yolk powder, 2% sodium cholate, 50% normal diet) for four weeks, and were then intraperitoneally injected with 1% streptozotocin (STZ, Sigma, usa, lot No.: S0130-1g) in a single dose of 30mg/kg (1 week 1 time, 2 consecutive weeks) in a buffer solution of citric acid (prepared in buffer solution of 0.1mol/L sodium citrate at pH 4.2, ready to use), and the normal control group was injected with the same amount of physiological saline simultaneously into the tail vein.
After two weeks, the tail of the rat is cut, blood is taken for measuring fasting blood glucose and random blood glucose, 12 rats with fasting blood glucose value (FBG) being more than or equal to 7.8mol/L and random blood glucose value being more than or equal to 11.1mol/L are selected and randomly divided into 6 rats in the diabetes model group, and the rat is gavaged with 0.9 percent of physiological saline every day; the positive drug control group contains 6 positive drugs, and the dark plum mulberry preparation is administrated by intragastric administration every day.
3.2 intervention measures
And starting intervention treatment after the diabetes model is successfully made. The preparation is administered by gavage at a dose of 5g/kg for 1 time per day for 4 weeks. The normal control group was administered with an equal amount of physiological saline simultaneously and the gavage was continued for 4 weeks.
The composition experimental group is perfused with physiological saline solution of solvents such as 200ml/kg, normal control group and negative control group. Wherein the rest of the distillate of the egg yolk and the dried rehmannia root is distilled water when the composition is prepared. The gavage time interval was 12 hours.
3.3 Collection and handling of specimens
After treatment is finished, all groups of rats measure fasting blood glucose, weigh, make related records, then grasp the rats and directly cut off one side femoral artery to cause death, then dissect the rats and rapidly take pancreas, wash the pancreas with 4 ℃ physiological saline, fix the pancreas with 4% formaldehyde for 24 hours, and soak the rat in distilled water for 4 hours. Taking out conventional gradient alcohol for dehydration, making xylene transparent, embedding paraffin, continuously slicing by LEICA2135 paraffin slicer to obtain slices with thickness of 5 μm, taking 1 slice every 20 slices, taking 6 slices of each sample, respectively mounting on glass slides coated with polylysine, and storing at room temperature for later use.
3.4 Observation index
3.4.1 general case
The rats were observed daily for changes in food intake, water intake, urine volume, mental status, etc. The body weight was measured before and after molding, respectively.
3.4.2 blood glucose determination
3.4.2.1 fasting and random blood glucose
Fasting Blood Glucose (FBG) (mmol/L) of each group of animals is measured before modeling, after modeling and after treatment respectively, random blood glucose is measured once every 3 days, the rats are fasted for 12h before the fasting blood glucose is measured, blood is taken from the tail vein of the rat on the next day, the fasting blood glucose is measured by an automatic glucometer, and relevant records are made.
3.4.2.2 Oral Glucose Tolerance Test (OGTT)
Oral Glucose Tolerance Test (OGTT) is carried out before, after and after molding. After the rats are fasted for 12h, the rats are gazed with 50% glucose solution according to the dose of 2g/kg, and the tail vein blood is taken for measuring the blood sugar at 0h, 0.5h, 1h and 2 h. After the experiment is finished, glucose tolerance curves of rats in each group are drawn.
3.4.3 pathomorphology
After the treatment is finished, the animals are sacrificed and the pancreas is rapidly taken out, washed clean by 4 ℃ physiological saline, fixed by 4 percent formaldehyde for 24 hours and soaked in distilled water for 4 hours. Dehydrated by conventional gradient alcohol, cleared by xylene, embedded by paraffin, serially sectioned by IEICA2135 paraffin microtome, 5 μm thick, mounted on a glass slide coated with polylysine, stained by hematoxylin-eosin staining and observed under an optical microscope.
3.4.4 immunohistochemistry
HIF-1 alpha affinity purification antibody, instant SABC kit, DAB color kit were prepared. Placing the slices in an oven at 60 ℃ for 1h, strictly operating according to the kit operating instructions, performing conventional dewaxing on the slices, and soaking in distilled water for 2 minutes; adding 3% hydrogen peroxide at room temperature for 10 minutes; washing with distilled water for 1min × 3 times; placing the slices in 0.01mol/L citric acid buffer solution, heating by microwave until boiling, cutting off the power for 15 minutes, heating again until boiling, and naturally cooling; adding 0.02mol/LPBS for washing for 1 minute multiplied by 3 times; adding 5% BSA antigen for blocking, and throwing off excessive liquid at room temperature for 20 minutes; adding primary antibody (1: 100) for dilution, and incubating for 1 hour at 37 ℃; washing with 0.02mol/LPBS for 2 minutes multiplied by 3 times; adding biotinylated secondary antibody dropwise, and incubating for 20 minutes at 37 ℃; washing with 0.02mol/LPBS for 2 minutes multiplied by 3 times; dripping horseradish peroxidase-labeled streptoenzyme ovalbumin, and incubating for 20 minutes at 37 ℃; 0.02mol/LPBS washing for 5 minutes multiplied by 4 times; DAB color development, and reaction time is controlled under a mirror; counterstaining with hematoxylin and violet for 10 seconds, and washing with tap water; dehydrating, transparent, neutral resin sealing sheet. Observed under a light mirror, the specific stain is sheet-shaped brown yellow particles. A negative blank with 0.1mol/LPBS solution instead of primary antibody was set up simultaneously for each batch of staining. Under an optical microscope (400 times), 4 fields are randomly selected from each section, the number of positive cells in each field is counted, and finally the average number of each group is compared.
3.4.5RT-PCR
Measuring the concentration of RNA after extracting RNA from tissues, wherein A1/A2 is 1.8-2.0, centrifuging the RNA for a short time, adding 1ul of M-MLV reverse transcriptase, 5ul of 5 XTT buffer solution, 2ul of Oligo (dT)18(20mol/L), 2ul of dNTP (20umol/L), RNA2ug and supplemented deionized water to 20ul of volume, carrying out reverse transcription reaction at 42 ℃ for 1h, inactivating M-MIV at 95 ℃ for 5min, centrifuging again, heating at 70 ℃ for 5min, stopping the reaction, placing on ice for carrying out the reverse transcription reaction, sequentially adding reaction reagents into a PCR reaction tube according to the operation instructions of a kit strictly, inserting the PCR tube into a PCR instrument, and carrying out denaturation at 95 ℃ for 5 min; performing 35 times of cyclic amplification reaction (94 deg.C, 1 min; annealing temperature is determined by primer (50-60 deg.C), 1 min; 72 deg.C, 1 min); and keeping the temperature of 72 ℃ for 7min, taking out the PCR reaction tube, carrying out electrophoresis detection on the reaction product, taking out the agarose gel after the electrophoresis is finished, and gently placing the agarose gel on a gel imager or an ultraviolet transmission instrument for imaging. Estimating the size of the amplified band according to the DNA molecular weight standard, and forming an electronic file by the electrophoresis result or taking a picture by a camera system.
3.4.6Western blot
Extracting protein from tissues and quantifying, separating the protein by 10% SDS polyacrylamide gel electrophoresis, transferring the protein onto a nitrocellulose membrane by using electricity, weighing skimmed milk powder after membrane transfer, preparing the skimmed milk powder into a TBST buffer solution to prepare 5% concentration for sealing, adding a primary antibody (the primary antibody is diluted to a proper concentration by TBST) for incubation for 1h after membrane washing, adding a secondary antibody labeled by horseradish peroxidase (HRP) and diluted by a Western secondary antibody diluent after membrane washing again for incubation for 1h, developing the color for 1h by using DAB color developing solution, and analyzing the molecular weight of a target zone and the net light density value by using a gel image analysis system.
Examples
1000g of fresh (or frozen fresh) fruits of egg yolk and dried rehmannia respectively are homogenized by a blender, placed in a distillation flask as shown in FIG. 1, and distilled products are collected after distillation. Wherein the distilled product of egg yolk was 643g, the yield was 64.3%, and the distilled product of dry rehmannia was 427g, the yield was 42.7%.
The above distilled products were formulated in various proportions and fed to model rats and normal rats as controls, and specific experimental examples and comparisons are shown in table 1. After the feeding experiment for four weeks is finished, blood index detection values of rats in each group are counted, and the concentration of serum glucose (unit is mg/dl) is measured by a glucose oxidase method.
FIG. 2 shows the results of a typical experimental example of the composition of the present invention, each experimental group being the average of three replicates. Wherein,
group a is a blank control of normal rats
Group b is blank control of diabetic model rats
Group c is the blood sugar concentration of the diabetes model rat after the intervention treatment of the composition
Group d diabetic model rats administered with egg yolk alone
Group e diabetic model rats taking dried rehmannia alone
Fig. 3 shows that the medicine applied by the patent has the effect of reducing the area under the glucose tolerance test curve of diabetic rats, wherein a is a normal group, b, c, d and e are respectively a diabetic blank control group and different medicine groups, the normal control group is taken as 1, and the rest are compared with the normal control group. Wherein,
group a is a blank control of normal rats
Group b is blank control of diabetic model rats
Group c is the blood sugar concentration diabetes of the diabetes model rats after the intervention treatment of the composition
Group d diabetic model rats taking dried rehmannia alone
Group e diabetic model rats administered with egg yolk alone
The results of other examples and comparative examples of the present invention are shown in table 1, and it is understood from the results shown in table 1 that the technical effects obtained by using the pharmaceutical composition of the present invention are:
1. compared with a control group, the distilled liquid of smoked plum or mulberry which is fed alone has a certain inhibition effect on hyperglycemia of rats in a diabetes model, but the composition can reduce the blood sugar of the rats to a lower level. ,
2. particularly, the smoked plum and the mulberry are mixed according to the proportion of the invention, and the result shows that the blood sugar concentration of the rat is controlled to be a normal value between 7 and 10mg/dl, and the unexpected effect is 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 diabetic peripheral neuropathy pain is characterized by comprising egg yolk and dry rehmannia as effective components.
2. The pharmaceutical composition of claim 1, wherein the effective components comprise, by weight: the egg yolk accounts for 45-80 percent, and the dried rehmannia accounts for 5-15 percent.
3. The pharmaceutical composition of claim 2, wherein said egg yolk is 80% and said dried rehmannia is 15%.
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 peripheral neuropathy pain is characterized in that the pharmaceutical composition contains egg yolk and dry rehmannia 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 egg yolk accounts for 45-80 percent, and the dried rehmannia accounts for 5-15 percent.
7. The use of claim 6, wherein said egg yolk is 80% and said dried rehmannia is 15%.
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