CN101204573A - Medicine foe diabetes mellitus - Google Patents
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Abstract
The invention discloses a medicine for treating diabetes mellitus. The medicine is made by the following raw materials: balsam pear slices 30g, sophorae flavescentis radix 9g, coptis chinensis 30g, anemarrhenae rhizome 30g, spina date seed 15g, zingiberis rhizoma tostum 6g, monascus 3g, tangerine peel 9g, rhubarb 2g and persicae semen 6g. The invention achieves repeatable affirmation effect through the clinical test for treating 2-type diabetes mellitus by the heat clearing and cloud dropping method, which can improve a 2-type diabetes mellitus model rat abnormal glucose metabolism by means of the obvious improvement of insulin resistance and islet cell function. The invention has great significance on alleviating fatty liver and maintaining normal blood lipid.
Description
Technical field
The present invention relates to a kind of Chinese medicine medicine, particularly relate to a kind of Chinese medicine medicine for the treatment of diabetes.
Background technology
Diabetes are systemic diseases based on carbohydrate metabolism disturbance that insulin is absolute or relative deficiency causes in the body.Recent years, along with going deep into to the research of type 2 diabetes mellitus pathogenesis, particularly some new experimental techniques such as cell potential are measured, target gene suddenlys change, transgenic is synthetic, the application of fluorescin in the research mammalian gene expression, and the insulin resistant of islet cells self (comprising β, α and delta cell insulin resistant) problem is familiar with by people gradually.Current research is verified: have Insulin receptor INSR and downstream signal thereof transduction path on the islet cells equally; Islet cells is not only the secretory cell of insulin etc., also is the target cell of insulin action simultaneously.The same with peripheral tissues, any link obstacle in the insulin signaling approach of islet cells also can cause insulin resistant, i.e. insulin resistant on the islet cells level or be called the insulin resistant of islet cells self, it has directly caused the obstacle of islet cell function.Many scholars think that insulin resistant and the impaired this direct correlation in the islet cells level of beta Cell of islet function are the important steps in the type 2 diabetes mellitus pathogenesis.
The purpose for the treatment of diabetes is to correct carbohydrate metabolism disturbance, to eliminate symptom, promotes the recovery of islet function, improves insulin resistant.Doctor trained in Western medicine hypoglycemic drug diabetes curative effect is affirmed, but life-time service needs dosage gradually, has untoward reaction in various degree mostly, and can not control complication effectively.Previously summarizing about the method for Chinese traditional treatment type 2 diabetes mellitus has: benefiting qi and nourishing yin, invigorating the spleen to clear away damp pathogen, depressed liver-energy dispersing and QI regulating, nourishing kidney yin, blood circulation promoting and blood stasis dispelling, warming the kidney and strengthening the spleen.In the industry, generally approve determination of treatment based on pathogenesis obtained through differentiation of symptoms and signs in conjunction with using effectively blood sugar lowering of blood sugar lowering Western medicine, but the independent blood sugar reducing function of independent Chinese medicine of neither one or Chinese medicine compound obtains everybody generally approval.
Crowd's sickness rate of diabetes is 1%-5%, and the trend that day by day increases is arranged, and it and cancer, cardiovascular disease are called as worldwide three big diseases.To the study on prevention of diabetes, be related to population with healthy, important social benefit and practical significance are arranged.
Summary of the invention
The purpose of this invention is to provide a kind of medicine for the treatment of diabetes.
The medicine of treatment diabetes provided by the present invention is heat clearing away the turbid descending side, is mainly made by the raw material of following weight portion:
Fructus Momordicae charantiae slice 25-35 part Radix Sophorae Flavescentis 6-12 part Rhizoma Coptidis 25-35 part
Rhizoma Anemarrhenae 25-35 part Semen Ziziphi Spinosae 10-20 part Rhizoma Zingiberis Preparatum 4-8 part.
In the medicine of described treatment diabetes, also can comprise Radix Et Rhizoma Rhei and/or the weight portion that Pericarpium Citri Reticulatae that Monas cuspurpureus Went that weight portion is 2-4 part and/or weight portion be 6-12 part and/or weight portion be 1-3 part in the described raw material is the Semen Persicae of 4-8 part.
Generally make in the practical application by the raw material of following weight portion:
Fructus Momordicae charantiae slice 25-35 part Radix Sophorae Flavescentis 6-12 part Rhizoma Coptidis 25-35 part
Rhizoma Anemarrhenae 25-35 part Semen Ziziphi Spinosae 10-20 part Rhizoma Zingiberis Preparatum 4-8 part
Monas cuspurpureus Went 2-4 part Pericarpium Citri Reticulatae 6-12 part Radix Et Rhizoma Rhei 1-3 part
Semen Persicae 4-8 part.
Be preferably:
30 parts of 9 portions of Rhizoma Coptidis of 30 parts of Radix Sophorae Flavescentiss of Fructus Momordicae charantiae slice
6 parts of 15 portions of Rhizoma Zingiberis Preparatums of 30 parts of Semen Ziziphi Spinosaes of the Rhizoma Anemarrhenae
2 parts of 9 parts of Radix Et Rhizoma Rhei of 3 parts of Pericarpium Citri Reticulataes of Monas cuspurpureus Went
6 parts in Semen Persicae.
In order to make medicine have better mouthfeel, also can contain the correctives of 1-2 ratio of weight and number in the described medicine.
Medicine provided by the present invention can adopt the conventional method in this area to make, and for example decocting, water decoction-alcohol sedimentation or mill medicated powder are taken after mixing it with water and all can.For taking convenience, medicine provided by the present invention can adopt decoction electuary, powder or pill.For taking convenience, medicine provided by the present invention can be soft capsule, granule or oral liquid.
Drug usage consumption provided by the present invention is: be grown up 140g/ time, water 500ml decocts to 200ml, divide 2 times oral; The child is cut down according to the circumstance.
The purport of medicine provided by the present invention is the treatment type 2 diabetes mellitus, has following function and curative effect: blood sugar lowering, glycolated hemoglobin, blood fat, improve insulin sensitivity, and improve the secretory function of beta Cell of islet, alleviate fatty liver.
Medicine heat clearing away provided by the present invention the turbid descending side takes " fertile, sugared, network wholistic therapy pattern ", and in blood sugar lowering, active treatment is fat and intervene complication as early as possible, and energy is blood sugar lowering, glycolated hemoglobin obviously, but blood fat reducing.Select this side mainly to be based on following understanding: at first as the main formula of control type 2 diabetes mellitus early metaphase, the all nutritious surplus of most type 2 diabetes mellitus patients, the fat partially clinical characters of build, and the morbidity early metaphase many with dry mouth with bitter taste, the breast side of body or distension and fullness in the abdomen, be hard and dry, red tongue with yellow fur, wiry and frequent pulse or sliding performance such as really do one's bit inside, Chinese medical discrimination is a syndrome of liver-stomach heat.Secondly, surfeit and few moving are to cause the topmost environmental factors of type 2 diabetes mellitus.Surfeit is the heat Excessive Intake, eating and drinking without temperances, surfeit delicious food.Eating twice as usual, the intestines and stomach are to hinder, and middle Jiao stops up stagnant for it, and taste are for fear of lifting, and important department must not mediate, and finally causes transformation failure of spleen, temper stasis; The polyphagia delicious food, the overeating greasy food bringing about internal heat, the over-eating the food with sweet flavor bringing about abdominal distension, what hindered also is middle burnt mechanism of qi; Few moving is movable the minimizing, spleen governing limbs, muscle, and active minimizing must influence the strong fortune of spleen.Spleen can not be stomach Xingqi body fluid, and spleen does not loose smart, thing not reforming then be expectorant, for wet, for turbid, be fat, and then deteriorated case hundred is living.So middle Jiao's mechanism of qi obstacle is the basic of type 2 diabetes mellitus and insulin resistant generation.Liver controlling conveyance and dispersion helps fortuneization again, and middle burnt stasis with the passing of time, the liver catharsis is too late, and then form that liver temper stagnates, stagnation of liver-QI and stomach-QI, spleen liter, stomach fall, liver dredge can not, middle burnt atmosphere does not change, then can further stop up and be heat, stagnate and be the stasis of blood, " food gas is gone into stomach, the spleen transporting essential substances to the liver; excess of QI is in muscle; conveyance of turbid essence to the heart, transporting essential substances to vessel " is earlier by middle burnt gastric heat, then liver-heat, heat in the heart, heat in blood etc., heat consume again gas burn cloudy, deficiency of both QI and YIN, empty and do not change, increase the weight of turbid, the blood stasis of expectorant again, the differentiation of this pathogenesis has just formed the generation evolution of type 2 diabetes mellitus and insulin resistant.Fructus Momordicae charantiae slice is monarch in the heat clearing away the turbid descending side, and Radix Sophorae Flavescentis, Rhizoma Coptidis, the Rhizoma Anemarrhenae, Semen Ziziphi Spinosae and Rhizoma Zingiberis Preparatum are minister, and Monas cuspurpureus Went, Pericarpium Citri Reticulatae are assistant, and Radix Et Rhizoma Rhei, Semen Persicae are for making.
By experimentation, think that heat clearing away the turbid descending side medicine is to alleviate insulin resistant to the dominant mechanism of the therapeutical effect of abnormal carbohydrate metabolism, and stimulate insulin secretion different diabetes rat models.Further this OLETF rat liver that can obviously raise, Skeletal Muscle Cell film Insulin receptor INSR adhesion are pointed out in mechanism research, reduce concentration of cortisol, and glicentin had certain reduction effect, can improve gathering and reducing triglyceride deposition in liver, the muscular tissue of high fat insulin resistance rat abdominal cavity fat simultaneously.
Good effect of the present invention is: preparation is that pure Chinese herbal medicine is made, and toxic and side effects is little, and treatment technology is simple, evident in efficacy, has wide application value.
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.
Description of drawings
Fig. 1 is the body weight change figure that high fat is fed stage two groups of rats
Fig. 2 is the absorption thermal change figure that high fat is fed stage two groups of rats
Fig. 3 respectively organizes the rat body weight variation diagram for irritating between gastric phase
Fig. 4 is for respectively organizing relatively block diagram of rat food ration for irritating between gastric phase
Fig. 5 is intravenous glucose tolerance test blood glucose dynamic change figure
Fig. 6 is intravenous glucose tolerance test insulin dynamic change figure
Fig. 7 is the Islet cells photo
The specific embodiment
Method therefor is conventional method if no special instructions among the following embodiment
One, active constituents of medicine and active constituents of medicine consumption
Fructus Momordicae charantiae slice 30g Radix Sophorae Flavescentis 9g Rhizoma Coptidis 30g
Rhizoma Anemarrhenae 30g Semen Ziziphi Spinosae 15g Rhizoma Zingiberis Preparatum 6g
Monas cuspurpureus Went 3g Pericarpium Citri Reticulatae 9g Radix Et Rhizoma Rhei 2g
Semen Persicae 6g
Two, one active constituents of medicine and consumption proportion prepare Chinese medicine set by step
1, the 140g medicine is put in the medicine pot;
2, add water 500ml (not being advisable), soaked 30-40 minute to have medicine;
3, the moderate heat decocting is 30 minutes, and extracting juice 200ml makes 30ml extractum.
40 OLETF rats (available from Japanese Otsuka Pharmaceutical Co., Ltd. Tokushima institute) conventional feed was raised to 32 weeks, body weight＞560g, be divided into 4 groups at random, 10 of diabetic model group, 10 of Western medicine groups, 10 of Chinese medicine small dose group, 10 of the heavy dose of groups of Chinese medicine.10 of Wistar rats (available from Chinese Academy of Medical Sciences's Experimental Animal Center) are normal control.
Medicine with embodiment 1 preparation experimentizes, and irritating stomach preceding is the solution of 4g crude drug/ml concentration with distilled water diluting.In 4 weeks of rat oral gavage administration, once a day, dosage is 1. normal control group: normal saline, 3ml/ are only; 2. model group: normal saline, 3ml/ are only; 3. Western medicine group: rosiglitazone (Smithkline Beecham Int'l, lot number: traditional Chinese medicines examination word X20000013), the 1.5mg/kg body weight; 4. Chinese medicine small dose group: heat clearing away of the present invention the turbid descending side solution, 9ml/kg body weight; 5. Chinese medicine is heavy dose of organizes: heat clearing away of the present invention the turbid descending side solution, 18ml/kg body weight.The following index of each group of 4 weeks back detection: 1) fasting glucose; 2) triglyceride and T-CHOL; 3) pathomorphology.Experimental data usefulness (
) expression, significant difference adopts the t check between group.
1) mensuration of fasting glucose
Rat tail vein is got blood, and One-touch II blood glucose meter (U.S. Johnson ﹠ Johnson) is measured the fasting glucose of rat.
As seen from Table 1, with normal group relatively, model group fasting glucose obviously raise (P＜0.001); After treating for 4 weeks, compare with model group, each treatment group fasting glucose has no significant change.
Table 1 is respectively organized fasting glucose relatively (x ± s mg/dl)
| Group | Mus number (only) | Dosage (g/kg) | Fasting glucose |
| The low dose of Chinese medicine heavy dose of normal group model group Western medicine group |
10 10 10 10 10 | - - 1.5mg/kg 36g/kg 72g/kg | 98.6±6.8 139.3±37.9 △△△ 126.9±9.3 116.8±12.6 132.1±21 |
Annotate: with the normal group ratio
△ △ △P＜0.001
2) serum triglycerides, T-CHOL are measured
Anaesthetize each treated animal with the pentobarbital sodium of 35mg/kg body weight, get blood 6ml from the carotid artery intubate, after leaving standstill 1 hour, centrifugal serum (3500rpm, 10min), T-CHOL adopts enzyme-linked method, triglyceride second phthalein acetone development process, measure at Hitachi-7150 full automatic biochemical apparatus (HITACHI7150, Automatic Analyzer).
As seen from Table 2, compare with normal group, model group triglyceride and T-CHOL be obviously rising (P＜0.01 and P＜0.001) all; Compare with model group, the content of Western medicine group and Chinese medicine heavy dose of group serum triglycerides and T-CHOL reduces (P＜0.05 and P＜0.01), and the Chinese medicine small dose group only content of T-CHOL reduces (P＜0.05), and content of triglyceride does not have notable difference.
Brief summary: the OLETF rat is during 32 ages in week, and triglyceride levels is 2 times of normal Wistar rats, and T-CHOL is its 5 times.Illustrate that this animal pattern has tangible hyperlipidemia, insulin resistant is often unusual with lipid metabolism.This Chinese medicine triglyceride reducing of the same energy with rosiglitazone and total cholesterol level illustrate that mechanism and its lipid metabolic disorder that can improve the type 2 diabetes mellitus patient that it improves insulin resistant (IR) are closely related.
Table 2 is respectively organized the comparison (x ± s) of triglyceride, T-CHOL
| Group | Mus number (only) | Dosage (g/kg) | Triglyceride (mg/dl) | T-CHOL (mg/dl) |
| The low dose of Chinese medicine heavy dose of normal group model group Western medicine group |
10 10 10 10 10 | - - 1.5mg/kg 36g/kg 72g/kg | 148.1±37.8 292.3±64.6 △△ 159.1±41.3 ** 256.3±66.5 176.4±63.7 ** | 18.1±9.3 86.3±18.1 △△△ 67.2±15.9 * 58.1±12.4 * 53.1±13.1 ** |
Annotate: with the normal group ratio
△ △P＜0.01,
△ △ △P＜0.001; With the model group ratio
*P＜0.05,
*P＜0.01
3) pathomorphology
After animal is got blood and puts to death, get in the pancreas, afterbody, the nearly about 1cm of the same position of hepatic portal liver organization
3Fully fix 24 hours in 10% formalin fixed liquid, paraffin embedding is used in ethanol dehydration step by step after the flowing water rinsing, the thick section of 5 μ m, and HE dyeing back neutral gum mounting, observed result is as follows under the light microscopic:
1. pancreas
Normal group pancreas lobule is intensive, and visible islets of langerhans boundary is clear.Islet cells is arranged in order, and endochylema is abundant, and karyon is circular placed in the middle, the blood capillary of visible more thin-walled between islet cells.
Model group pancreas lobule is separated by more adipose cell, and islets of langerhans obviously increases, and visible inflammatory cell soaks into therebetween.Visible circular cavity (fat change) in the islet cells endochylema has blood vessel, fibroplasia in the islets of langerhans.
Western medicine group pancreas lobule is separated by more adipose cell, and islets of langerhans obviously increases, and visible inflammatory cell soaks into therebetween.Visible circular cavity (fat change) in the islet cells endochylema has blood vessel, fibroplasia in the islets of langerhans, do not have remarkable morphology difference with model group.
Chinese medicine small dose group pancreas lobule major part is intensive, and the visible adipose cell of part is separated, and significantly alleviates than model group.Islets of langerhans obviously increases, and visible inflammatory cell infiltration therebetween, blood vessel and fibroplasia do not have remarkable morphology difference with model group.
The heavy dose of group of Chinese medicine pancreas lobule major part is intensive, and the visible adipose cell of part is separated, and significantly alleviates than model group.Islets of langerhans obviously increases, and visible inflammatory cell infiltration therebetween, blood vessel and fibroplasia do not have remarkable morphology difference with model group.
2. liver
Normal group lobules of liver structural integrity, central vein is placed in the middle, the liver rope to around radially distribute, the hepatocyte marshalling, karyon is clear, endochylema is abundant.
Model group lobules of liver structure is complete substantially, and central vein is placed in the middle, and hepatocyte is the change of vesicle fat around the central vein, and visible bulla fat change, and is in extensive range.
Western medicine group lobules of liver structure is complete substantially, and central vein is placed in the middle, and hepatocyte is the change of vesicle fat around the central vein, and visible bulla fat becomes the same model group of scope and degree.
Chinese medicine small dose group lobules of liver structural integrity, central vein is placed in the middle, and vesicle that as seen hepatocyte is dispersed in around the central vein and bulla fat become, and fat range degree and model group significantly alleviate.
The heavy dose of group of Chinese medicine lobules of liver structural integrity, central vein is placed in the middle, and vesicle that as seen hepatocyte is dispersed in around the central vein and bulla fat become, and fatty range degree and model group significantly alleviate, and be not remarkable with Chinese medicine small dose group difference.
Brief summary: OLETF rat model group islet tissue fatty infiltration is serious, points out this rat model owing to weight increase, and cholesterolemia and triglyceride increase, and lipidosis can take place pancreatic tissue, and this is the major reason that finally influences islet function.Chinese medicine can significantly alleviate the fatty infiltration of pancreatic tissue, and this effect of rosiglitazone is not obvious, points out this Chinese medicine closely related therewith to the protective effect mechanism of pancreatic function.The extensive fatty infiltration of OLETF rat model group hepatic tissue, the rounded cavity shape of hepatocyte steatosis forms signet ring cell.And see that the liver rope twists, sinus hepaticus is squeezed phenomenon.Two Chinese drug-treated group hepatic tissue fatty infiltrations significantly alleviate, and are identical with the rosiglitazone group, and the effect of pointing out Chinese medicine to alleviate the hepatic tissue fatty infiltration is similar to rosiglitazone, all has the sedimentary effect of the interior fat of alleviating.The more fat-free liver person of type 2 diabetes mellitus and Patients with Fatty Liver exists tangible disorders of lipid metabolism, insulin resistant and overweight, and the complication of trunk takes place more.Therefore the effect that alleviates fatty liver and keep normal lipid of heat clearing away the turbid descending side takes place significant to prevention and the complication that reduces type 2 diabetes mellitus insulin resistant and trunk.
1) preparation of diabetes model and administration
50 of male Wistar rats (available from Chinese Academy of Medical Sciences's Experimental Animal Center), body weight 180-220g, age in 6-8 week.Streptozotocin (STZ) (Sigma, lot number: 202-263-7) in ice bath, be mixed with 1% solution with the 0.1mol/L citrate buffer solution, pH value 4.4.After the rat fasting 12 hours, get 40 at random, left lower quadrant intraperitoneal injection STZ, 65mg/kg body weight.After three days, survey rat blood sugar, more than 16.7mmol/L, be defined as diabetes, select 30 of diabetes rats, be divided into diabetic model group (10), Chinese drug-treated group (10) and Western medicine group (10).Getting 10 rats is the normal control group, intraperitoneal injection equal-volume citrate buffer solution.0.1mol/L pH4.4 citrate buffer solution compound method is as follows:
A liquid: citric acid 21.01g distilled water 1000ml;
B liquid: sodium citrate 29.4lg distilled water 1000ml;
A liquid 44ml+B liquid 56ml.
Medicine with embodiment 1 preparation experimentizes, and irritating stomach preceding is the solution of 4g crude drug/ml concentration with distilled water diluting.Metformin (Beijing Double-Crane Pharmaceutical Co., Ltd, lot number: 990825) be made into 1% concentration with normal saline.Each organizes equal 4 weeks of gastric infusion of rat, and once a day, dosage is 1. normal control group: normal saline 3ml/ only; 2. model group: normal saline 3ml/ only; 3. Western medicine group: 1% metformin 10ml/kg body weight; 4. the heat clearing away the turbid descending side solution 9ml/kg body weight of Chinese drug-treated group: 4g/ml.
2) blood sugar detection
Under the non-fasting state of animal, 9 o'clock mornings, the tail vein was got blood, before the administration and administration second, each is surveyed once all around the time.The second, all around detecting the blood glucose time was administration (water) after 1 hour.Rat tail vein is got blood, measures the fasting glucose of rat with One-touch II blood glucose meter (U.S. Johnson ﹠ Johnson).
Experimental data represents that with mean ± standard deviation significant difference adopts the t check between group.
As seen from Table 3, model group blood glucose is than normal group obviously raise (P＜0.001); After treating for 2 weeks, Chinese medicine and metformin all can make blood glucose decline (P＜0.05); Blood glucose decline more obvious (P＜0.01) during the 4th week.
Table 3 respectively organize non-fasting glucose relatively (
Mmol/L, n=10)
| Group | Dosage (g/kg) | Before the | Treat | 2 weeks of | Treat | 4 weeks of back |
| Normal model metformin Chinese medicine | - - 0.1 36 | 4.17±0.28 19.93±1.98 △△△ 20.41±1.71 △△△ 19.64±1.77 △△△ | 3.87±0.21 20.07±5.18 △△△ 15.06±1.58 * 15.52±1.50 * | 4.37±0.68 18.83±1.63 △△△ 14.68±2.2 ** 15.05±1.23 ** |
Annotate: with the normal group ratio
△ △ △P＜0.001 is with the model group ratio
*P＜0.05;
*P＜0.01
3) mensuration of serum insulin, glucagon, hydrocortisone, insulin-like growth factor-i (IGF-1) and motilin (MTL)
35mg/kg body weight pentobarbital sodium anesthetized animal is used in administration after 4 weeks, the carotid artery intubate is got blood, 2 kinds of modes are handled: after 1. blood leaves standstill 1 hour, centrifugal serum (3500rpm, 10min), detect serum insulin, glucagon, hydrocortisone, insulin-like growth factor-i with insulin radioimmunity test kit, glucagon radioimmunity test kit, hydrocortisone radioimmunity test kit, insulin-like growth factor-i radioimmunity test kit (the sharp biotechnology center of Beijing sea section, lot number is respectively defends the accurate word R-11 of medicine, R-64, R-69, R-70); 2. blood is put in the anticoagulant tube (aprotinin 30 μ l/ prop up, and 10%EDTA 40 μ l/ prop up), and (3500rpm 10min), detects motilin with motilin radioimmunity test kit (the sharp biotechnology center of Beijing sea section, lot number is for defending the accurate word R-67 of medicine) to centrifugal blood plasma.Experimental data represents that with mean ± standard deviation significant difference adopts the t check between group.
As seen from Table 4, model group glucagon and hydrocortisone serum content all raise, and insulin content reduces (P＜0.001); Treatment back each group and model group ratio, Chinese medicine can reduce hydrocortisone content (P＜0.05), but insulin and glucagon content are not had obvious influence, and metformin does not all have influence to the content of insulin, glucagon and hydrocortisone.
As seen from Table 5, compare with normal group, model group insulin-like growth factor-i (IGF-1) and motilin content all reduce (P＜0.001 and P＜0.05); Treatment back each group and model group ratio, Chinese medicine can raise insulin-like growth factor-i and motilin content (P＜0.001 and P＜0.05), metformin does not have obvious influence to insulin-like growth factor-i and motilin content.
The comparison that insulin, glucagon and hydrocortisone change after 4 weeks of table 4 treatment (
, n=10)
| Group | Dosage (g/kg) | Insulin (μ U/ml) | Glucagon (pg/ml) | Hydrocortisone (ng/ml) |
| Normal model metformin Chinese medicine | - - 0.1 36 | 21.52±4.24 10.70±2.08 △△△ 9.41±3.35 △△△ 9.73±2.24 △△△ | 175.91±53.61 362.88±64.37 △△△ 281.84±94.91 △△ 269.23±48.06 △△ | 16.45±3.40 23.56±6.37 △ 19.19±6.08 16.27±4.17 * |
Annotate: with the normal group ratio
△ △ △P＜0.001,
△ △P＜0.01,
△P＜0.05 is with the model group ratio
*P＜0.05
The comparison that insulin-like growth factor-i and motilin change after 4 weeks of table 5 treatment (
, n=10)
| Group | Dosage (g/kg) | IGF-1(ng/ml) | MTL(pg/ml) |
| Normal model metformin Chinese medicine | - - 0.1 36 | 1456.85±183.64 387.28±90.60 △△△ 348.99±102.32 582.14±78.93 *** | 233.46±25.99 202.65±20.60 △ 209.01±7.13 237.27±25.61 * |
Annotate: with the normal group ratio
△ △ △P＜0.001,
△P＜0.05; With the model group ratio
* *P＜0.001,
*P＜0.05
4) the small intestinal absorption function is measured
Adopting dextrorotation xylose absorption experiment method to carry out the small intestinal absorption function measures.After 1 hour, first tail vein is got blood and is surveyed blood glucose with batch animal last administration, and each group gavages 4%D-xylose solution 0.3g/kg then, and blood is got in anesthesia after 1 hour, preparation serum.Experimentize according to the following steps then: 1. amplify 1 part of Mus serum in the 50ml conical flask, 8 parts of adding distil waters add 0.5 part in 0.3mol/L sulphuric acid again, add 0.5 part of 10% sodium tungstate again, and all with adding with shaking, centrifugation removes deproteinize, promptly gets nonprotein filtrate.2. get 4 in test tube, the 1st pipe is put distilled water 2ml for blank pipe; The 2nd pipe concentration (0.05mg/ml) the xylose titer 2ml that lowers; The 3rd pipe is put high concentration (0.1mg/ml) xylose titer 2ml; The 4th pipe is put nonprotein filtrate 2ml.3. in above each pipe, all respectively add 1% orcin reagent 2ml, mixing.4. put in the boiling water 1 hour, and respectively managed adding distil water after the cooling and be diluted to 25ml, mixing.5. use 721 spectrophotometer 630nm photometry density, proofread and correct blank pipe optical density, read and respectively manage optical density value to zero point.6. calculate the K value earlier: K=low concentration standard pipe optical density/0.025+ high concentration standard pipe optical density/0.05.7. xylose content (mg/L) in the serum=(measure pipe optical density/K) * 500.Experimental data represents that with mean ± standard deviation significant difference adopts the t check between group.
As seen from Table 6, model group is compared serum dextrorotation xylose content with normal group obviously increase (P＜0.001), and promptly the small intestinal absorption function strengthens; Treatment back each group and model group ratio, metformin can obviously reduce serum dextrorotation xylose content (P＜0.001), promptly suppressed the absorption function of small intestinal, but Chinese medicine does not have tangible influence to the small intestinal absorption function.
The comparison of small intestinal absorption function after 4 weeks of table 6 treatment (
, n=10)
| Group | Dosage (g/kg) | Serum dextrorotation xylose content (mg/L) |
| Normal model metformin Chinese medicine | - - 0.1 36 | 22.34±9.78 164.45±38.75 △△△ 68.36±31.09 *** 166.80±147.19 |
Annotate: with the normal group ratio
△ △ △P＜0.001 is with the model group ratio
* *P＜0.001
5) observation of liver cell and muscle cell Insulin receptor INSR adhesion
The Kilp method of employing improvement (Liu Hongfang, Tong Xiaolin, Wang Qingguo etc. dissipating depression of QI clearing stomach side is to the influence of diabetes rat liver and Skeletal Muscle Cell Insulin receptor INSR. Beijing University of Chinese Medicine's journal, 2002,25 (2): 35-37) carry out the cell membrane preparation.Adopt the coomassie brilliant blue staining method to measure memebrane protein concentration (list of references: Duan Jun, Tong Xiaolin, Wang Xia, etc. dissipating depression of QI clearing stomach side induces the influence of early stage insulin resistance rat insulin sensitivity to high fat diet. Chinese practical Chinese and western medicine magazine, 2006,19 (3): 327-330).The Nishimura method of employing improvement (list of references: Zhao Yu, Tong Xiaolin, Liu Subin. dissipating depression of QI clearing stomach side is to the influence of type 2 diabetes mellitus β cell function. Chinese practical Chinese and western medicine magazine, 2005,18 (16): 712-715) measure the insulin adhesion.Experimental data represents that with mean ± standard deviation significant difference adopts the t check between group.
As seen from Table 7, the variation of liver, Skeletal Muscle Cell film Insulin receptor INSR combination rate is after 4 weeks of treatment: model group obviously raises (P＜0.05) than normal group, and Chinese drug-treated group and metformin group all reduce (P＜0.05) than model group.
4 week of table 7 treatment back livers, Skeletal Muscle Cell film Insulin receptor INSR combination rate (
, n=10)
| Group | The ratio (%) of single-minded binding capacity and total binding | |
| Liver | Muscle | |
| Normal model metformin Chinese medicine | 34.38±12.06 55.51±9.53 △ 33.70±11.01 * 42.96±11.03 * | 12.37±6.46 30.72±11.71 △ 15.10±9.28 * 14.69±6.55 * |
Annotate: with the normal group ratio
△P＜0.05 is with the model group ratio
*P＜0.05
6) immunohistochemical observation of pancreatic tissue Fas receptor and Fas-L part
Each treated animal sacrificed by exsanguination, it is conventional fixing to get pancreas, paraffin embedding, the thick section of 5 μ m.Show the expression of islets of langerhans Fas and Fas-L with the immunohistochemical ABC Faxian, the immunohistochemical staining process is as follows: paraffin section de-waxing is to water → 3%H
2O
2Incubated at room 10 minutes, to eliminate the activity → distilled water flushing of endogenous peroxydase, PBS liquid soaks 5 minutes → 10% normal goats serum sealing (PBS dilution), incubated at room 10 minutes → serum deprivation inclines, drip anti-(anti-Fas of rabbit and Fas-L polyclonal antibody) (Beijing Zhong Shan company) of I working solution respectively, put 4 ℃ of refrigerator overnight → PBS flushings in the wet box, anti-(the anti-rabbit igg of biotinylated goat) (Beijing Zhong Shan company) of 5 minutes * 3 times → dropping II, 37 ℃ of temperature were incubated 30 minutes, the PBS flushing, 5 minutes * 3 times, drip 1: 200 III anti-(Radix Cochleariae officinalis enzyme labelling chain enzyme avidin) (Beijing Zhong Shan company), 30 minutes → PBS of 37 ℃ of incubations flushing, 5 minutes * 3 times → DAB colour developing → tap water fully washes → haematoxylin redyeing, tap water washes more than 15 minutes → step by step dehydration of alcohol → dimethylbenzene transparent → gummy mounting.Light microscopic is observed down, and the result is as follows:
1. Fas antigen
Normal group: the visible light brown granule of minute quantity cell in the islets of langerhans, become faint expression, most cells are negative.
Model group: the islets of langerhans inner cell is the product of brownish red positive reaction widely as seen, and the strong positive that slabbing distributes is expressed.
Western medicine group: the as seen brown widely positive reaction product of islets of langerhans inner cell, the medium positive expression that slabbing distributes.
Chinese drug-treated group: the cell of expressing that is positive in the islets of langerhans is dispersed in distribution, and product is light brown.
2. Fas part
Normal group: the Fas part expression that is negative in the islets of langerhans.
Model group: the visible light brown granule of minute quantity cell in the islets of langerhans becomes faint expression.
The Western medicine group: the as seen brown widely positive reaction product of islets of langerhans inner cell, medium-strong positive that slabbing distributes is expressed.
Chinese drug-treated group: the as seen brown widely positive reaction product of islets of langerhans inner cell, medium-strong positive that slabbing distributes is expressed.
In sum, find the muroid empty stomach in the experiment after 12 hours, each organizes blood glucose and normal group does not have tangible difference, so this research is observed the influence of this medicine to blood glucose by non-fasting glucose.Chinese medicine began just to have significantly to fall non-fasting glucose effect in 2 weeks of administration, and each organizes the effect of insulin concentration not being had raising, illustrated that its blood sugar reducing function by insulin-mediated, is different from sulphanylureas, and this is consistent with OLETF rat model result.Chinese medicine reduces the concentration of hydrocortisone in the serum, but glucagon is not had obvious increase effect, and the result is consistent during with OLETF model high insulin levels, and no matter the insulin concentration height is described, Chinese medicine all can improve liver and transfer the function that reaches feelings will by reducing concentration of cortisol in the serum.This medicine can improve the motilin level, but small intestinal absorption function and model group are than no obvious change, show that the heat clearing away the turbid descending can increase the secretion of motilin, this experimental result with the OLETF rat is consistent, affirmed the turbid descending function of recovering stomach more, promote the mechanism of gastric emptying promptly to be this, Chinese medicine can not suppress the absorption function of small intestinal, illustrates that its mechanism of action is different with biguanides.Diabetes rat liver, muscle cell membrane acceptor binding force increase in this experiment, may increase relevant with the Insulin receptor INSR quantity that insulin concentration decline in the blood causes.Though cell membrane Insulin receptor INSR quantity increases during diabetes, acceptor binding force increases, and blood glucose does not still fall, and shows that the insulin effect is incomplete, has rate-limiting reaction in the cell, i.e. opposing behind the Insulin receptor INSR, and this respect awaits further to study.Chinese medicine can make the unusual Insulin receptor INSR adhesion that raises of STZ diabetes model reduce, but the Insulin receptor INSR adhesion of OLETF diabetes rat model reduction is raise, and points out the dual regulation of this medicine.Normal group Fas antigen is negative expression in most cells, and small amounts of cells is faint expression, and model group Fas antigen is strong positive expresses, illustrate diabetes take place after the antigenic expression of Fas strengthen, show the reinforcement of islet cells apoptosis.Chinese medicine and Western medicine all can make the antigenic expression of Fas weaken, and the degree that weakens of Chinese medicine is better than Western medicine, have suppressed immunoreactive process in the islets of langerhans after oral Chinese medicine is described.Fas part expressions that be negative in the normal group islets of langerhans, model is formed faint expression, and the Western medicine group all becomes medium-strong positive expression with Chinese drug-treated group.Confirm that further this Chinese medicine can play a protective role to pancreatic tissue, thereby delay the depletion of islet cells.
1) preparation of diabetes model
105 of Sprague-Dawley (SD) male rats (purchasing Experimental Animal Center) in dimension tonneau China, the normal diet adaptability was fed after 2 days, and 85 of modeling groups give high lipid food (Chinese Academy of Medical Sciences's Experimental Animal Center; Heat consists of: carbohydrate 20%, protein 21%, fat 59% (fat is mainly from safflower oil)) to feed, 20 of matched groups continue to give normal diet, and (Jiujiang, Beijing pellet factory produces; Carbohydrate 64%, protein 23%, fat 13%) feed.
Regularly detect the body weight and absorption heat of rat, record data, experimental data represents that with mean ± standard deviation significant difference adopts the t check between group.
Urea chain assistant rhzomorph (STZ) is with reference to the method for embodiment 3, and facing with before being mixed with concentration is 1% solution, 4 ℃ of preservations.After rat fed for 9 weeks, fasting overnight.The modeling group is through abdominal cavity shot STZ 15mg/kg body weight, and the tail vein is got blood and surveyed non-fasting glucose after 1 week, is the modeling success greater than 16.7mmol/L, become mould 60 rats, be divided into model group, treatment by Chinese herbs group and western medicine group at random, each 20,25 of Cheng Mo rats are not picked out.Matched group is injected isodose citrate buffer solution.
The body weight change that high fat is fed stage two groups of rats as shown in Figure 1, the variation of taking in heat as shown in Figure 2.The result shows, the body weight no significant difference (was respectively 260 ± 8.4 when high lipid food group rat and normal diet group rat were finished in the 1st, 2 weeks of feeding, 263 ± 33.4 and 301 ± 22.2,302 ± 32.2), from the 3rd week, two groups of body weight begin to occur significant difference (417 ± 25,382 ± 14.6, P＜0.01), to the 9th weekend, high lipid food rat feeding body weight is 632 ± 56.7g, and normal diet rat feeding body weight is 521 ± 40.5g, statistics has notable difference, and high fat group rat occurs significantly fat.The heat of high lipid food group is taken in early days the stage apparently higher than the normal diet group, but the 3rd week is later on along with the Mus growth in age, matched group is taken in gradually to be increased, on average taking in the heat matched group during the 8th week is 521 kilojoules, high fat group is 452 kilojoules, may be different with the feedstuff put-on method relevant, and the former consumes the more intake of having exaggerated, but the high fat diet nursing also has been described, simultaneously even under the not high situation of absorption heat, also can cause tangible obesity.
2) administration of rat
Medicine with embodiment 1 preparation experimentizes, and irritating stomach preceding is the solution of 3.7g crude drug/ml concentration with distilled water diluting.Elegant enlightening sheet (available from U.S. GlaxoSmithKline PLC China Investment Ltd.), every contains rosiglitazone maleate 4mg, irritates the preceding solution that is made into 0.75mg/ml concentration with distilled water of stomach.
Normal diet is fed rat, irritates stomach every day once, 4 weeks of continuous irrigation stomach.It is as follows to irritate stomach dosage: 1. matched group: distilled water is irritated stomach, 4ml/kg body weight; 2. model group: irritate stomach, 4ml/kg body weight to distilled water; 3. Chinese drug-treated group: heat clearing away the turbid descending side solution is irritated stomach, 4.2ml/kg body weight; 4. Western medicine group: Avandia solution is irritated stomach, 4ml/kg body weight.Observe the general state of rat, active situation writes down rat body weight and food ration weekly and changes.
During each organizes rat oral gavage body weight change as shown in Figure 3, food ration is more as shown in Figure 4.The result shows, after the Cheng Mo, tangible weight loss does not appear in the model group rat, ingest and also not significantly increase of amount of drinking water, the characteristics of incidence that relatively meets human type 2 diabetes mellitus, irritate stomach after 4 weeks, the model group rat body weight with irritate stomach before do not compare and increase, the body weight of matched group meets the normal growth rule of rat, and Avandia is irritated stomach group 4 weeks of rat oral gavage, and body weight increases about 30 grams steadily, may be the result that blood glucose improves on the one hand, also relevant with the effect of Western medicine weight increase, about 14 grams of Chinese drug-treated group weight increase may improve relevant with blood glucose.Four treated animals are irritating between gastric phase food ration no significant difference every day, all between the 30-32 gram.
3) oral glucose tolerance test (OGTT)
Irritate before the stomach and irritate stomach after 4 weeks, rat fasting overnight 12 hours, the tail vein was got blood and was surveyed fasting glucose (FBG) and glucose fully and irritate 2 hours blood glucose (P2hBG) behind the stomach morning next day, and it is 2g/kg body weight (concentration 40%) that glucose is irritated the stomach amount.(card BIOSEN5030 in the Germany) goes up and detects blood glucose value at quick glucose analyser.Handle experimental data with the SPSS13.0 statistical analysis software, the gained data with mean ± standard deviation (
) expression, mean is relatively added up with the t check between two groups of Normal Distribution, relatively adopts variance analysis between many groups.
As seen from Table 8, blood sugar level will be apparently higher than normal group (P＜0.01) after 2 hours for model group fasting blood glucose level and filling stomach glucose, no change before and after the blank treatment; Suitable before Chinese drug-treated group and the treatment of Western medicine group with the model group blood sugar level, no significant difference, the treatment back all obviously descends with 2 hours after the meal blood sugar levels on an empty stomach than model group, Western medicine group fasting glucose descends more obvious, and 2 hours after the meal blood glucose of Chinese drug-treated group are better than the Western medicine group, but this species diversity does not have statistical significance.
OGTT fasting glucose and 2 hours blood sugar levels are relatively before and after each group treatment of table 8
| Grouping | Example number (only) | FBG(mmol/L) | P2hBG(mmol/L) | ||
| Before the treatment | After the treatment | Before the treatment | After the treatment | ||
| Normal group model group Chinese drug-treated group |
8 8 9 9 | 4.46±0.15 8.99±3.78 ## 7.79±3.48 8.05±3.81 | 4.42±0.17 12.5±6.47 ## 6.19±3.51 * 5.69±1.70 ** | 6.09±0.32 15.08±8.68 ## 16.03±4.86 15.1±5.67 | 6.08±0.32 18.22±6.11 ## 11.01±6.26 * 12.72±4.5 * |
Annotate: compare with normal group
##P＜0.01; Compare with model group
*P＜0.05,
*P＜0.01
4) detection of blood fat, basal insulin and saccharifying serum albumin
Irritated in 4 weeks after stomaches finish, every group of picked at random 8-9 rat, fasting overnight 12 hours, inferior morning, the rat jugular vein was got blood 2.5ml, centrifuging and taking serum ,-70 ℃ of preservations.T-CHOL (TC), HDL-C (HDL-C), low-density lipoprotein cholesterol (LDL-C) and C-VLDL (VLDL-C) pass through enzyme-linked method, triglyceride (TG) second phthalein acetone development process, measure at Hitachi-7150 full automatic biochemical apparatus (HITACHI7150, Automatic Analyzer).Measure radioimmunological kit (Linco) by rat insulin and measure basal insulin (FINS).On Hitachi-7600 instrument, detect the saccharifying serum albumin by microtrabeculae layer method.Handle experimental data with the SPSS13.0 statistical analysis software, the gained data with mean ± standard deviation (
) expression, mean is relatively added up with the t check between two groups of Normal Distribution, relatively adopts variance analysis between many groups.
As seen from Table 9, the triglyceride levels of model group and very low density lipoprotein (VLDL) level raise than matched group, meet the blood fat performance of diabetes, but have only the latter that statistical significance is arranged, and Chinese drug-treated group has the decline of statistical significance.
Table 9 is respectively organized the blood fat situation relatively
| Grouping | The example number | TC(mmol/L) | TG(mmol/L) | HDL-C (mmol/L) | LDL-C (mmol/L) | VLDL-C (mmol/L) |
| Normal group model group Chinese drug-treated group |
8 9 9 9 | 1.86±0.3 1.92±0.65 1.77±0.36 2.13±0.31 | 1.08±0.33 1.61±1.05 1.17±0.57 1.25±0.42 | 0.55±0.05 0.63±0.16 0.52±0.1 0.64±0.07 | 0.26±0.05 0.31±0.12 0.35±0.13 0.36±0.1 | 0.49±0.15 0.8±0.47 # 0.48±0.21 * 0.57±0.19 |
Annotate: compare with normal group
#P＜0.05; Compare with model group
*P＜0.05
As seen from Table 10, model group saccharifying serum albumin level is significantly higher than matched group, treatment by Chinese herbs 4 week back saccharifying serum albumin levels obviously descend (P＜0.01), near matched group, Avandia also makes saccharifying serum albumin level obviously descend (P＜0.05) after treating for 4 weeks, shows that two kinds of medicines all have reliable hypoglycemic effect; The insulin result is nonnormal distribution; so carry out statistical analysis after getting its natural logrithm; the result shows; the model group basal insulin level is starkly lower than matched group (P＜0.01), and after the Chinese and western drugs treatment, basal insulin level all has certain recovery; compare with model group; two treatment groups all have significant significant difference (Chinese drug-treated group P＜0.01, Western medicine group P＜0.05), illustrate that two kinds of medicines all have the certain protection effect to the islet function of this rat model.
Each group treatment back saccharifying serum albumin of table 10 and basal insulin level are relatively
| Grouping | Example number (only) | Saccharifying serum albumin (mmol/L) | Basal insulin (ng/ml) |
| Normal group model group Chinese drug-treated group |
8 9 9 9 | 3.29±0.27 3.8±0.37 ## 3.2±0.49 ** 3.31±0.28 ** | 2.76±1.21 0.77±0.32 ## 1.48±0.63 ** 1.22±0.5 * |
Annotate: compare with normal group
##P＜0.01; Compare with model group
*P＜0.05,
*P＜0.01
5) the positive glucose clamp test of hyperinsulinism
The rat jugular vein was got behind the blood 2-3 days, every group of every day got a rat and began fasting overnight in batches 12 hours, weighed, and the tail vein is got blood and surveyed basic blood glucose value, carries out intraperitoneal anesthesia with 2% pentobarbital sodium (2.5ml/kg body weight) then.Method according to Zhou Shuiping, separate in rat right carotid and the left side neck or external jugular vein, the 75IU/ml anticoagulant heparin, (U.S. produces the epidural anesthesia silica gel catheter to carry out intubate, internal diameter 0.6mm, external diameter 1mm), arterial cannulation is surveyed blood glucose and stable state insulin to get blood, and venous cannulation is to connect micro-injection pump with infusion of insulin and glucose.After intubate finishes, rat left standstill measure blood glucose after 30 minutes, begin test, elder generation's fugitive Iletin II (Lilly) of constant infusion (infusion rates is 8mu/kgmin-1, faces the time spent insulin and dilutes with 0.5% bovine serum albumin) during beginning was got blood from left neck artery and is surveyed blood glucose 1 time in per 10 minutes, when blood glucose drops to basic blood glucose+1.0mmol/L, beginning is infusion 20% glucose solution in a small amount, and constantly adjusts glucose infusion rate according to blood sugar level, makes blood glucose reach stable state gradually.All clamp is in basic blood glucose ± 0.2mmol/L level with all rat blood sugars, and the glucose infusion rate under the record stable state in continuous 30 minutes is got average.The stable state insulin is surveyed in right common carotid artery blood-letting during off-test.Leave and take the tissue specimen of pancreas, liver, get whole abdominal cavity fats and weigh.Handle experimental data with the SPSS13.0 statistical analysis software, the gained data with mean ± standard deviation (
) expression, mean is relatively added up with the t check between two groups of Normal Distribution, relatively adopts variance analysis between many groups.
As seen from Table 11, under the stable state blood glucose situation close with insulin, model group rat stable state glucose infusion rate has had insulin resistant than matched group 20.4% (P＜0.01) that descended; Glucose infusion rate is significantly increased (P＜0.01) behind Chinese drug-treated group and the Western medicine group therapeutic intervention, compares with model group, and the glucose infusion rate of Chinese drug-treated group has improved 47%, and the Western medicine group has improved 56%.
Table 11 is respectively organized the positive glucose clamp of hyperinsulinism result of the test relatively
| Grouping | Example number (only) | Stable state glucose (mmol/L) | Stable state insulin (ng/ml) | Glucose infusion rate (mg/kgmin) |
| Normal group model group Chinese drug-treated group |
6 6 6 6 | 4.8±0.3 4.6±0.2 4.6±0.2 4.8±0.3 | 10.75±1.27 12.43±0.93 12.03±1.03 11.87±1.09 | 8.61±1.02 6.85±0.76 ## 10.07±1.4 ** 10.67±1.24 ** |
Annotate: compare with normal group
##P＜0.01; Compare with model group
*P＜0.01.
As seen from Table 12, under every experiment empty stomach state, the model group rat body weight is a little less than matched group, but no difference of science of statistics, anatomical results shows that fat content in abdominal cavity also reduces, also no difference of science of statistics; Body weight and the fat content in abdominal cavity of Western medicine group and Chinese drug-treated group treatment back rat all are significantly higher than model group, and the variation of this body weight and abdomen fat content may recover with islet function, and blood sugar level descends relevant.
Rat body weight respectively organized by table 12 and abdomen fat content compares
| Grouping | Example number (only) | Body weight (g) | Abdomen fat (g) | Abdomen fat percentage of liveweight percentage ratio (%) |
| Normal group model group Chinese drug-treated group Western medicine group | 12 10 11 13 | 552.3±29.9 510±95.13 # 564±64.9 583±77.03 * | 29.9±5.23 20.34±15.9 34.1±18.12 * 35.9±16.4 * | 4.71±0.81 3.73±2.54 ## 5.83±2.53 * 5.97±2.1 * |
Annotate: compare with model group
*P＜0.05,
*P＜0.01
6) intravenous glucose tolerance test (IVGTT)
After each organizes 4 weeks of rat administration, put to death previous crops intravenous injection glucose-insulin release test.Step is as follows: rat is from test proxima luce (prox. luc) 20:00 fasting, test day 8:00 intraperitoneal injection 3% pentobarbital sodium 80mg/kg anesthetized animal, after jugular vein, carotid artery intubate are kept somewhere about 30min, give glucose load 0.5g/kg (concentration 50%) from vein, before load (0), load back 5,10,30,60, the 120min 1ml that takes a blood sample respectively surveys blood glucose, insulin.At once survey blood glucose, surplus blood specimen is centrifugal, and it is to be checked that blood plasma is stored in-20 ℃ of refrigerators.It is 1ml that each blood sampling time point institute blood sampling erythrocyte is added 0.9% normal saline to cumulative volume, feeds back rat to guarantee that blood volume is constant from vein.Regularly push heparin sodium (100IU/ml) with anticoagulant from vein in the process of the test.Handle experimental data with the SPSS13.0 statistical analysis software, the gained data with mean ± standard deviation (
) expression, mean is relatively added up with the t check between two groups of Normal Distribution, relatively adopts variance analysis between many groups.
The result is shown in Fig. 5 and table 13 for the experiment change of blood sugar, and experiment insulin result of variations is shown in Fig. 6 and table 14.Each time point model group rat blood sugar all is significantly higher than matched group among the IVGTT, and treatment group each point blood glucose all obviously descends than model group, and area Chinese drug-treated group and Western medicine group all significantly are lower than model group under 0-30 minute while and the 0-120 minute blood glucose curve.Corresponding secretion of insulin curve display, the whole secretion of model group is very low, and Chinese drug-treated group and Western medicine group secretion of insulin amount are then obviously greater than model group, but the secretion peak early stage than matched group delayed.
Table 13 is respectively organized area comparison under the blood glucose curve
| Grouping | Example number (only) | Area under the 0-30min blood glucose curve | Area under the 0-120min blood glucose curve |
| Normal group model group Chinese drug-treated group |
5 5 6 6 | 41.98±8.07 87.47±19.08 ## 51.55±15.59 * 57.88±23.72 * | 54.85±12.35 125.97±23.36 ## 72.34±22.50 ** 80.62±36.82 * |
Annotate: compare with normal group
##P＜0.01; Compare with model group
*P＜0.05,
*P＜0.01
Table 14 is respectively organized vein carbohydrate tolerance insulin secretion area under curve
| Grouping | Example number (only) | 0-10min | 0-30min | 0-60min | 0-120min |
| Normal group model group Chinese drug-treated group |
5 5 6 6 | 3.07±1.68 0.48±0.6 ## 2.3±1.99 ** 2.64±1.57 ** | 3.75±2.03 0.62±0.79 2.93±2.69 3.25±1.93 | 4.27±2.41 0.68±0.82 3.69±3.31 3.9±2.33 | 5.04±2.93 0.79±20.03 ## 4.6±3.87 ** 4.69±2.73 ** |
Annotate: compare with normal group
##P＜0.01; Compare with model group
*P＜0.05,
*P＜0.01
7) pancreatic tissue paraffin section insulin (Insulin), glucagon (Glucagon), substrate 1 (IRS-1) and Insulin receptor INSR (IRc) immunohistochemical analysis
1. pancreatic tissue paraffin section Insulin, Glucagon and IRS-1 immunohistochemical staining
A) paraffin section dimethylbenzene I, II dewaxing is each 15 minutes, and gradient ethanol promptly goes out promptly (concentration is respectively 100%, 100%, 95%, 95%, 90%, 80%, 70%, 50%) to water;
B) flowing water embathed 5 minutes;
C) 0.3% H
2O
2Solution oxide 15 minutes;
D) flowing water embathed 5 minutes, and PBS washes 5 minutes * 3 times;
E) drip 1: 100 Insulin, the one anti-(A056429 that suitably dilutes respectively, U.S. DAKO), Glucagon one anti-(A056529, U.S. DAKO) and IRS-1 one anti-(2382, U.S. Cell Signaling), establish negative control simultaneously and replace I anti-, 4 ℃ of refrigerator overnight incubation with PBS;
F) PBS washes 5 minutes * 3 times;
G) drip 1: 400 biotin labeling two anti-(EnVision
TMSystem, DAK company), keep somewhere 1.5 hours (room temperature) in the wet box;
H) PBS washes 5 clocks * 3 time;
I) colour developing of DAB solution is 2 minutes;
J) flowing water embathed 5 minutes;
K) gradient alcohol dehydration (concentration is respectively 50%, 70%, 80%, 90%, 95%, 100%), transparent each 15 minutes of dimethylbenzene I, II;
L) DPX mounting;
2. pancreatic tissue paraffin section IRc immunohistochemical staining
A), b) two steps are with preceding identical
C) 0.3% H
2O
2 Methanol solution oxidation 15 minutes;
D) flowing water embathes and carries out antigen retrieval (putting in 5 minutes the 0.1mmol/LPH6.0 citrate buffer solution of high fiery preheating 5 minutes → fluid infusion of middle high fire heating → middle high fiery 5 minutes) after 5 minutes;
E) after room temperature natural cooling 30-40 minute, PBS washes 5 minutes * 3 times;
F) drip one of 1 of suitably dilution: 50IRc and resist (BA0951, Wuhan doctor's moral company), establish negative control simultaneously and replace I anti-, 4 ℃ of refrigerator overnight incubation with PBS;
Following steps are identical with pancreatic tissue paraffin section Insulin, Glucagon and IRS-1 immunohistochemical staining.
As redying, can develop the color at DAB, after flowing water embathes, to contaminate 1 minute with the Mayer hematoxylin, back flowing water embathes, dehydration, transparent, mounting.
Mayer hematoxylin compound method is as follows:
Hematoxylin 1g, first vitriol 50g, sodium iodate 0.2g, distilled water 1000ml, chloral hydrate 50g, citric acid 1g, with hematoxylin, first vitriol and sodium iodate add successively in the distilled water, heated and stirred slightly, treat complete to spend the night after molten, add chloral hydrate and citric acid and be heated to and boil 5 minutes, cooled and filtered is standby.
3. computer picture quantitative analysis
SABC section: adopt the quantitative analysis of LEICA-Q550IW computer multi-functional true color pathological image analytical system, measure IRc respectively, each islets of langerhans area and the corresponding average optical density value of full islets of langerhans (MOD) in IRS-I, Insulin and the Glucagon stained.
As seen from Table 15, the result is as follows:
The IRS-1 SABC is expressed: matched group dyeing is dark-brown, and strong positive is expressed, and fills the air the endochylema that is distributed in whole islet cells; The positive expression that the main islets of langerhans middle section of model group rat disperses or is dispersed in is pale brown color; The positive expression of Chinese drug-treated group rat obviously strengthens and increases than model group, is dark brown yellow, in the cell cytosol of main islets of langerhans middle section; The Western medicine group is similar to the Chinese drug-treated group expression.Average optical (MOD) value relatively, model group is minimum, with matched group, Chinese drug-treated group and Western medicine group the difference of remarkable statistical significance is arranged.
The IRc SABC is expressed: bulk dyeing is lighter, and matched group dyeing is the light brown positive expression, fills the air the middle section that is distributed in islet cells; The weak positive expression that the model group rat mainly is dispersed at the islets of langerhans middle section is light brown yellow; The positive expression of Chinese drug-treated group rat obviously strengthens and increases than model group, is light brown, and the islets of langerhans middle section is more; The Western medicine group is similar to the Chinese drug-treated group expression.Average optical (MOD) value relatively, model group is minimum, with matched group, Chinese drug-treated group and Western medicine group the difference of remarkable statistical significance is arranged.
The IRS SABC is expressed: matched group dyeing is dark brown yellow, and strong positive is expressed, and fills the air the middle section that is distributed in islet cells; The model group rat is brown mainly at the positive expression of islets of langerhans middle section disperse; The positive expression of Chinese drug-treated group rat limits to than the expression zone of matched group and is discontinuous, is pale brown color positive expression; The Western medicine group is to fill the air and is distributed in islets of langerhans and gets middle section, pale brown color positive expression.Average optical (MOD) value relatively, model group is minimum, with matched group, Chinese drug-treated group and Western medicine group the difference of remarkable statistical significance is arranged.
The Glc SABC is expressed: matched group dyeing is pale brown color, is distributed in the neighboring area of islets of langerhans, and strong positive is expressed; Increase in the positive expression zone of model group rat, and periphery is main, and middle section also has expression, is dark brown yellow; The positive expression of Chinese drug-treated group rat increases than the expression zone of matched group, but the expression of central area is few than model group, is pale brown color positive expression; The Western medicine group is similar to the Chinese drug-treated group expression.Average optical (MOD) value relatively, model group is than the matched group height, but no difference of science of statistics is compared the difference that does not also have remarkable statistical significance with Chinese drug-treated group with the Western medicine group, also be that model group is more to expressing the statistics of carrying out area in the zone simultaneously, but no difference of science of statistics.From each group rat Langerhans islet specimen, choose the close individuality of insulin expression situation, carry out the glicentin average optical density value and express the comparison of area, the area that the glicentin of Chinese drug-treated group and Western medicine group is expressed still obviously reduces than model group, and the enhancing of insulin to the effect of glicentin expression inhibiting is described to a certain extent.
Table 15 immunohistochemical staining average optical (MOD) value relatively
| Grouping | Example number (only) | IRc | IRS-1 | INS | Glc |
| Normal group model group Chinese drug-treated group |
5 5 5 5 | 0.231±0.006 0.218±0.007 ## 0.229±0.009 ** 0.226±0.004 ** | 0.268±0.009 0.244±0.008 ## 0.277±0.01 ** 0.262±0.04 ** | 0.247±0.01 0.221±0.01 ## 0.249±0.03 ** 0.264±0.03 ** | 0.305±0.02 0.316±0.02 # 0.312±0.01 0.323±0.02 |
Annotate: compare with normal group
##P＜0.01; Compare with model group
*P＜0.01
8) rat Langerhans islet separates and functional examination
1. experimental rat fasting overnight, 2% pentobarbital sodium intraperitoneal anesthesia, wipes of alcohol wash disinfection, fixedly rat.
2. with the operating theater instruments that disinfects in advance, open the thoracic cavity, abdominal cavity, break heart, inhale the liquid of dehematizing with sterile gauze rapidly, liver is upwards dug, found stomach and small intestinal junction, small intestinal is opened to the right, can expose pancreas, clamp pancreas to the duodenum inlet with mosquito forceps, the collagenase (Sigma) of avoiding being injected into pancreas enters small intestinal, the collagenase of the 0.5mg/ml that extraction 13ml filters, thrust common bile duct with the trocar, after pulling out nook closing member, fix, inject about collagenase 10ml with the ophthalmology tweezer, can see clearly that pancreas expands, careful complete the peeling of pancreas.
3. pancreas is put into the centrifuge tube of 50ml, more remaining collagenase is added, 37 ℃ of water-baths 20 minutes, concussion in 10 minutes therebetween is once, allow its abundant cracking, add 4 ℃ Hanks liquid, stop digestion, piping and druming with strength, 4 ℃ of centrifugal 2000rpm, 5 minutes, remove supernatant, Hanks washes, drainage screen, centrifugal again, 4 ℃ of centrifugal 2000rpm, 10 minutes, remove clean supernatant, every pipe adds 5ml 25%-4 ℃-Ficoll, and (Ficoll 400,50g as far as possible; Hepes, 0.475g; Hanks 150ml), adds the back centrifuge tube is tilted, slowly add 23%Ficoll 5ml successively, 21%Ficoll 5ml, 11%Ficoll 5ml, 4 ℃ are centrifugal, 400rpm, 5 minutes, reuse 2500rpm, 20 minutes, islets of langerhans can be suspended between 11% and 21%, also has between 21% and 23%.
The Hanks liquid making method is as follows:
Anhydrous CaCl
2: 0.14g; KCl:0.4g; Na
2HPO
47H
2O:0.09g; MgCl
26H
2O:0.1g; MgSO
47H
2O:0.1g; NaCl:8.0g; NaHCO
3: 0.35g; KH
2PO
4: 0.6g; Anhydrous glucose: 1.0g; Phenol red: 0.01g.Add ddH2O to 1000ml, transfer pH to 7.2 ~ 7.4,0.22 μ m filtering with microporous membrane packing.
4. carefully the islets of langerhans sucking-off, add Hanks and wash, centrifugal, 2000rpm 10 minutes, 1600rpm is 10 minutes again, 1200rpm 10 minutes, abandon supernatant, add 1640 (RPMI-1640 culture medium fully) (10% serum) about about 1ml, piping and druming, sucking-off 1-2 drips, put into plate, select under anatomical lens, islet cells can the group of being gathered into, and resembles the Fructus Ananadis comosi shape.Adding 1640 (not exclusively RPMI-1640 culture medium) cultivates.
5. inhale and remove old culture fluid in the culture bottle, use instead and cultivate dyeing in the rearmounted 5%CO2 of dithizone dyeing liquor of equivalent, the 37 ℃ of steam-laden CO2 gas incubator and observe under inverted microscope after 15-30 minute and take pictures, photo is seen Fig. 7.
6. the culture fluid in sucking-off 24 well culture plates, cell uses Kreb-Ringers liquid preincubate after 30 minutes, add final concentration and be the glucose incubation 1 hour of 5.6mM, wash with Kreb-Ringers liquid after the sucking-off culture supernatant and add the Kreb-Ringers liquid that contains the 16.7mM glucose behind the cell 2 times and continue to hatch sucking-off culture supernatant after 1 hour, put-20 ℃ of insulin contents to be measured.
The Kreb-Ringers liquid making method is as follows:
NaCl, 119mmol/L; KCl, 4.74mmol/L; CaCl
2, 2.54mmol/L; MgSO
4, 1.19mmol/L; KH
2PO
4, 1.19mmol/L; NaHCO
3, 25mmol/L; Hepes, 10mmol/L; BSA, 0.1%; Transfer pH to 7.2-7.4,0.22 μ m filtering with microporous membrane packing.
7. handle experimental data with the SPSS13.0 statistical analysis software, the gained data with mean ± standard deviation (
) expression, mean is relatively added up with the t check between two groups of Normal Distribution, relatively adopts variance analysis between many groups.
As seen from Table 16, secretion of insulin situation model group obviously reduces behind demonstration 5.6mM and the 16.7mM glucose incubation, and Chinese drug-treated group and Western medicine group all have the raising of statistical significance.But still differ greatly with matched group, may with experimental session once CO2 gas incubator die an evening, influenced the functional status relevant (normal group does not involve) of islet cells.
Table 16 variable concentrations glucose incubation is to the influence of islet cells insulin secretion
| Grouping | Islets of langerhans number (individual) | 5.6mM glucose insulin ng/ml | 16.7mM glucose islets of langerhans ng/ml |
| Normal group model group Chinese drug-treated group |
50 50 50 50 | 21.72±1.03 1.072±0.38 1.91±1.2 * 1.34±0.22 * | 21.93±1.87 1.47±0.91 2.676±1.13 * 1.70±1.03 * |
In sum, the result of study of this experiment shows that the heat clearing away the turbid descending can obviously improve the abnormal carbohydrate metabolism of type 2 diabetes mellitus rat model, no matter from fasting glucose, still oral glucose back 2 hours blood glucose of loading, and saccharifying serum albumin level can be affirmed its blood sugar lowering curative effect.Its hypoglycemic mechanism is two aspects: the one, and the improvement of insulin resistant, goldstandard with the insulin sensitivity evaluation is judged, there is obvious insulin resistance in the model group rat, Chinese drug-treated group and Western medicine group insulin resistant all obviously improve, its glucose infusion rate even greater than the normal control group, and Chinese drug-treated group is compared with the Western medicine group and is not had notable difference; The 2nd, the improvement of islet cell function, the result shows, the basal insulin level of rat model is starkly lower than matched group (P＜0.01), only is 28% of normal rat level, and Chinese drug-treated group and Western medicine group basal insulin level after treating has improved 92% and 58% (P＜0.01) respectively; The result of vein glucose tolerance shows that secretion of insulin that glucose stimulates almost disappears in model group and totally has islet cell function impaired serious, treatment by Chinese herbs group and western medicine group, no matter be the insulin secretion (0-10 minute, 0-30 minute insulin secretion area under curve) of phase morning, still the insulin secretion (0-120 minute insulin secretion area under curve) of whole process all improves significantly, so area just obviously reduces than model group under 0-30 minute and the 0-120 minute glucose curve.This experimental applications STZ people cause the destruction of islet function that blood glucose is increased; experimental result shows; the expression of the Insulin receptor INSR of Chinese drug-treated group rat Langerhans islet and receptor substrate-1 obviously strengthens than model group; with Western medicine group no significant difference; in conjunction with our research in the past; though heat clearing away the turbid descending side can not influence that STZ causes the destruction of islet cell function; but can improve because sugared toxicity; the effect of the inhibition of islet cells insulin signaling transduction path being played the protection islet cell function that fat toxicity causes just realizes improving the secretory function of islet cells by the insulin resistant that improves islet cells self.Rat model has still shown the plasma lipid profile of diabetes, be that triglyceride raises and very low density lipoprotein (VLDL) raises, and very low density lipoprotein (VLDL) of Chinese drug-treated group rat and model group relatively have the decline of statistical significance, pointed out Chinese medicine that dyslipidemia is had the positive therapeutic effect, and this effect can not all improve with insulin secretion, insulin sensitivity increases to be explained, because the Western medicine rosiglitazone has improved islet function and insulin resistant equally, but do not observe above-mentioned result, and cholesterol levels also slightly raises, and this may also be the advantage of Chinese medicine clinical practice.The expression of the experimental result prompting Chinese drug-treated group of SABC and the insulin of Western medicine group islet cells all obviously strengthens than model group, but do not demonstrate the inhibition that the islet cells glicentin is expressed, may be with the model group rat because beta Cell of islet destroys and the hypertrophy of the alpha Cell of islet that the nursing of high fat causes is more relevant, though the function by the treatment islet cells is recovered to some extent, the insulin that increases is not enough to the effect of α cell inhibiting by paracrine.
Claims (6)
1. medicine for the treatment of diabetes, make by the raw material of following parts by weight:
Fructus Momordicae charantiae slice 25-35 part Radix Sophorae Flavescentis 6-12 part Rhizoma Coptidis 25-35 part
Rhizoma Anemarrhenae 25-35 part Semen Ziziphi Spinosae 10-20 part Rhizoma Zingiberis Preparatum 4-8 part.
2. medicine according to claim 1 is characterized in that: also comprise in the raw material of described medicine:
Monas cuspurpureus Went 2-4 part Pericarpium Citri Reticulatae 6-12 part.
3. medicine according to claim 2 is characterized in that: also comprise in the raw material of described medicine:
Radix Et Rhizoma Rhei 1-3 part Semen Persicae 4-8 part.
4. medicine according to claim 3 is characterized in that: described medicine is made by the raw material of following parts by weight:
30 parts of 9 portions of Rhizoma Coptidis of 30 parts of Radix Sophorae Flavescentiss of Fructus Momordicae charantiae slice
6 parts of 15 portions of Rhizoma Zingiberis Preparatums of 30 parts of Semen Ziziphi Spinosaes of the Rhizoma Anemarrhenae
2 parts of 9 parts of Radix Et Rhizoma Rhei of 3 parts of Pericarpium Citri Reticulataes of Monas cuspurpureus Went
6 parts in Semen Persicae.
5. according to claim 1 or 2 or 3 or 4 described medicines, it is characterized in that: the dosage form of described medicine is decoction, electuary, powder or pill.
6. according to claim 1 or 2 or 3 or 4 described medicines, it is characterized in that: described medicine is soft capsule, granule or oral liquid.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102847095A (en) * | 2011-07-01 | 2013-01-02 | 广州中一药业有限公司 | Pharmaceutical composition for treating diabetes |
| CN103301433A (en) * | 2013-06-24 | 2013-09-18 | 芜湖乐锐思信息咨询有限公司 | Diabetes mellitus treatment medicine |
| WO2014040366A1 (en) * | 2012-09-13 | 2014-03-20 | Zhao Junhong | Toxin expelling powder comprising root of buckwheat and preparation process thereof |
| CN107149670A (en) * | 2017-05-23 | 2017-09-12 | 杨红 | Treat medicinal herb tea of diabetes and preparation method thereof |
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| CN1095952A (en) * | 1993-06-04 | 1994-12-07 | 陈顺志 | A kind of production method of medicated tea |
| CN1364468A (en) * | 2001-01-19 | 2002-08-21 | 赵国林 | Wall breaking and extracting method for Chinese herbal medicine and formulation |
| CN1636556A (en) * | 2003-11-18 | 2005-07-13 | 汪景山 | Compound coenzyme-Q10 prepn |
| CN1895291A (en) * | 2006-06-09 | 2007-01-17 | 陈永丽 | Nano-Chinese medicinal biological product and its preparation |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102847095A (en) * | 2011-07-01 | 2013-01-02 | 广州中一药业有限公司 | Pharmaceutical composition for treating diabetes |
| WO2014040366A1 (en) * | 2012-09-13 | 2014-03-20 | Zhao Junhong | Toxin expelling powder comprising root of buckwheat and preparation process thereof |
| CN103301433A (en) * | 2013-06-24 | 2013-09-18 | 芜湖乐锐思信息咨询有限公司 | Diabetes mellitus treatment medicine |
| CN107149670A (en) * | 2017-05-23 | 2017-09-12 | 杨红 | Treat medicinal herb tea of diabetes and preparation method thereof |
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