CN102233076B

CN102233076B - A kind of pharmaceutical composition to treat the application in the type 2 diabetes mellitus medicine that Fatty toxicity causes in preparation by reducing SREBP1-C

Info

Publication number: CN102233076B
Application number: CN201010156324.6A
Authority: CN
Inventors: 仝小林; 甄仲; 常柏; 周水平
Original assignee: Tasly Pharmaceutical Group Co Ltd
Current assignee: Beijing Zhongyitang Technology Co ltd
Priority date: 2010-04-27
Filing date: 2010-04-27
Publication date: 2015-08-19
Anticipated expiration: 2030-04-27
Also published as: CN102233076A

Abstract

The invention belongs to pharmaceutical field, relate to the application of a kind of pharmaceutical composition in the medicine of preparation attenuating SREBP1-c, wherein said pharmaceutical composition is that prescription is formulated by the raw material of Chinese medicine medicine of following weight portion: Radix Trichosanthis 540 parts, Radix Bupleuri 10-30 part, Fructus Aurantii Immaturus 3-15 part, Radix Et Rhizoma Rhei 1-6 part, Rhizoma Pinelliae 1-12 part, Radix Scutellariae 3-15 part, Rhizoma Coptidis 1-12 part, Radix Paeoniae Alba 3-15 part, Fructus Mume 5-20 part.

Description

A kind of pharmaceutical composition to treat the application in the type 2 diabetes mellitus medicine that Fatty toxicity causes in preparation by reducing SREBP1-C

Technical field

The invention belongs to pharmaceutical field, relate to the application of a kind of pharmaceutical composition at treatment type 2 diabetes mellitus.

Background technology

Type 2 diabetes mellitus (T2DM) is the worldwide public health problem of serious threat human health.The statistical data display that International Diabetes Federation (IDF) 2006 delivers, 20 years in the past, global diabetics number increased severely to 2.3 hundred million from 3,000 ten thousand.World Health Organization's expert expects, will reach 3.5 hundred million to global diabetics in 2025.India, China, the U.S. are followed successively by three maximum countries of global diabetics quantity, estimate diabetes mellitus in China subject population nearly 4,000 ten thousand, are only second to India, occupy second place of the world, account for 29.63% of the world.The control of T2DM foreseeable quite over a long time in will be all the severe challenge of facing mankind.

Type 2 diabetes mellitus is also Adult Onset's patients with type Ⅰ DM, how the sequela of 35 ~ 40 years old, accounts for diabetics more than 90%.The ability producing insulin in type 2 diabetes mellitus ward mate body not completely loses, and in some patient bodies, insulin even produces too much, but the action effect of insulin is had a greatly reduced quality, and the insulin therefore in patient body may be in a kind of state of relative shortage.The secretion of insulin in body can be stimulated by some oral drugs.But still have some patients to need to carry out insulinize as type 1 diabetes to the later stage.Type 2 diabetes mellitus roughly has following 3 features: first feature, and type 2 diabetes mellitus is apt to occur in adult, is especially many with middle-aged and elderly people.Repeatedly diabetes epidemiological study result all shows, type 2 diabetes mellitus morbidity age many 40-60 year, from 40 years old, prevalence increased gradually, peaked to the geratic period.The maturity-onset diabetes of Juvenile-onset is comparatively speaking, still very rare.Second feature is that the state of an illness generally compares mitigation, hidden, that is compared with type 1 diabetes, does not so bear down menacingly.Type 2 diabetes mellitus patient symptom is not obvious, may not be certain each patient have drink many, urinate many, the performance of outeat, majority also do not become thin significantly, and muscle power and body weight decline still more common to some extent certainly.3rd feature is exactly that patient does not often need to sustain life by insulinize.That is, patient does not beat insulin and is unlikely to ketoacidosis to occur soon and threat to life yet.So type 2 diabetes mellitus was non-insulin-dependent diabetes mellitus again originally.Type 2 diabetes mellitus patient also needs use of exogenous insulin sometimes, but majority is because glycemic control is undesirable, or because the chronic complicating diseases that there occurs acute complications or diabetes is heavier, and be to sustain life unlike type 1 diabetes patient.

Type 2 diabetes mellitus is the disease more more complicated than type 1 diabetes, and particularly insulin still can oneself but more easily be treated when producing in body in early days.Distribute due to early symptom slight (as without ketoacidosis and stupor etc.), therefore type 2 diabetes mellitus is many is diagnosed at rear for many years at disease progression more.But the complication that the improper meeting of control of type 2 diabetes mellitus causes such as renal failure, blind, wound healing slow, arteriopathy (comprising coronary artery disease) etc. is serious.

Its principal character of type 2 diabetes mellitus patient is insulin resistant, and relative insulin lacks and hyperglycemia; The increase (as deriving from the degraded of hepatic glycogen) of hepatic glucose production, particularly in the increase (typical reason is that insulin level is disorderly, and one of effect of insulin regulates and controls this function of liver just) on improper opportunity; The minimizing (receptor and post-receptor pathway defect) of the glucose transport of muscle and the middle insulin-mediated of fatty tissue (mainly); Islet beta cell function is impaired---lack the function of early stage insulin releasing when elevated blood glucose levels stimulates.

Chinese medicine can play more positive effect in treating diabetes." the strange sick opinion of Plain Questions ": " ... overflowing also of this five gas, name says the spleen-warm syndrome.Husband's five tastes entrance, is hidden in stomach, and spleen is its vital essence of trip, and body fluid at spleen, therefore makes us sweet taste in the mouth also.This fertile institute is sent out also, and this person must count the sweet and refreshing and many fertilizer of food also, overeating greasy food bringing about internal heat, and over-eating the food with sweet flavor bringing about abdominal distension, therefore its gas overflow, transfer to and quenching one's thirst ".Professor Tong little Lin is in conjunction with clinical epidemiological study, proposing to stop up the strongly fragrant caused interior fullness and heat of wood by soil is (obesity) T2DM basic pathogenesis, dissipating depression of QI heat clearing away is its basic therapy, establish dissipating depression of QI heat clearing away side medicine through Long-term clinical and confirm the clinical efficacy of dissipating depression of QI clearing away heat method, its mechanism of action has tentatively been set forth at integral level, histiocyte level and molecular level, comprise the dystopy deposition alleviating triglyceride, improve receptor number and adhesion, affect insulin signal transduction system etc.

The quick spirit of sugar of dissipating depression of QI heat clearing away side medicine is a kind of traditional Chinese compound medicine for the treatment of diabetes, is made up of Chinese medicines such as Radix Trichosanthis, Radix Bupleuri, Fructus Aurantii Immaturus, Radix Et Rhizoma Rhei.Through finding with placebo, the quick clever glycolated hemoglobin that reduces of sugar is obviously better than placebo, and sugared quick spirit significantly can not only improve insulin resistant, also has the effect significantly repairing and protect islet cell function.In addition, sugared quick spirit has good hypoglycemic effect to diabetics, has good regulating action for lipid metabolic disorder.

Sterol regulatory element binding protein (SREBPs) is that the film that a class is positioned in endoplasmic reticulum connects albumen, and SREBPs is a kind of nuclear factor, up to now, finds 3 kinds altogether: SREBP-1a, SREBP-1c and SREBP-2.SREBP-1c is one of them hypotype, and SREBP-1c is directed and differentiation 1 (ADD1) also known as adipose cell, mainly in liver and the adipose cell expression of rodent and the mankind.

First SREBPs synthesizes the precursor forms of the non-activity being incorporated into ER film, and it activates the adjustment being subject to sterin content in cell.SREBPs, after ER synthesis, is combined on ER film, combines with SREBP cracking activator protein (SCAP), forms SREBP-SCAP complex.When in cell, sterin content is higher, SREBPs-SCAP complex is retained in endoplasmic reticulum, but when sterin in cell exhaust lack time, SCAP-SREBP complex is transported to Golgi body, activates transcribing of cholesterol and fatty acid metabolism related gene.

SREBP-1 is the major transcription regulatory factor of lipid homeostasis, the all genes needed for fatty acid synthesis can be activated, the increase of its protein level, particularly its N-terminal increases accordingly containing whorl spiral one leucine zipper structure fragment, directly can activate the enzyme of multiple participation fatty acid synthesis, comprise FAS, acetyl-CoA carboxylase (ACC) etc.Research shows, the increase of SREBP-1c can make liver be restricted the play a role synthesis of albumen of insulin, thus insulin action is declined, close with Relationship between insulin resistance.Also confirm that SREBP-1c and fatty acid metabolism and carbohydrate metabolism are closely related by the research such as transgenic mice and gene knockout mice, be the major transcription regulatory factor participating in lipogenesis gene, its expression regulates and controls by many factors such as AMPK.

Inventor passes through the deep research of the quick spirit of sugar, and the sugared quick spirit of unexpected discovery can lower SREBP1-c albumen and developed by molecule, for the therapeutic use of sugared Min Lingxin provides foundation.

Summary of the invention

The object of the invention is to provide a kind of pharmaceutical composition preparing the application in the medicine lowering SREBP1-c albumen and developed by molecule.

Pharmaceutical composition of the present invention (is also called the quick clever preparation of sugar, dissipating depression of QI fever-clearing preparation), it is that prescription is formulated by the raw material of Chinese medicine medicine of following weight portion: Radix Trichosanthis 5-40 part, Radix Bupleuri 10-30 part, Fructus Aurantii Immaturus 3-15 part, Radix Et Rhizoma Rhei 1-6 part, Rhizoma Pinelliae 1-12 part, Radix Scutellariae 3-15 part, Rhizoma Coptidis 1-12 part, Radix Paeoniae Alba 3-15 part, Fructus Mume 5-20 part.

Middle Fructus Crataegi can also be added in above-mentioned prescription, the formula obtained is: Radix Trichosanthis 10-30 part, Radix Bupleuri 1030 parts, Fructus Aurantii Immaturus 3-15 part, Radix Et Rhizoma Rhei 1-6 part, Rhizoma Pinelliae 1-12 part, Radix Scutellariae 3-15 part, Rhizoma Coptidis 1-12 part, Radix Paeoniae Alba 3-15 part, Fructus Mume 5-20 part, Fructus Crataegi 3-15 part.

Middle Fructus Crataegi can also be added in above-mentioned prescription, Rhizoma Polygonati, Radix Ginseng and Fructus Momordicae charantiae, the formula obtained is: Radix Trichosanthis 10-30 part, Radix Bupleuri 10-30 part, Fructus Aurantii Immaturus 3-15 part, Radix Et Rhizoma Rhei, 1-6 part, Rhizoma Pinelliae 1-12 part, Radix Scutellariae 3-15 part, Rhizoma Coptidis 1-12 part, Radix Paeoniae Alba 3-15 part, Fructus Mume 5-20 part, Fructus Crataegi 3-15 part, Rhizoma Polygonati 3-15 part, Radix Ginseng 3-15 part, Fructus Momordicae charantiae 3-15 part.

Preferred formula of the present invention is: Radix Trichosanthis 9 parts, Radix Bupleuri 12 parts, Fructus Aurantii Immaturus 9 parts, Radix Et Rhizoma Rhei 3 parts, the Rhizoma Pinelliae 6 parts, Radix Scutellariae 9 parts, Rhizoma Coptidis 6 parts, the Radix Paeoniae Alba 9 parts, Fructus Mume 9 parts.

Or:

Radix Trichosanthis 30 parts, Radix Bupleuri 12 parts, Fructus Aurantii Immaturus 9 parts, Radix Et Rhizoma Rhei 3 parts, the Rhizoma Pinelliae 6 parts, Radix Scutellariae 9 parts, Rhizoma Coptidis 6 parts, the Radix Paeoniae Alba 9 parts, Fructus Mume 15 parts, Fructus Crataegi 9 parts.

Or:

Radix Trichosanthis 30 parts, Radix Bupleuri 12 parts, Fructus Aurantii Immaturus 9 parts, Radix Et Rhizoma Rhei 3 parts, the Rhizoma Pinelliae 6 parts, Radix Scutellariae 9 parts, Rhizoma Coptidis 6 parts, the Radix Paeoniae Alba 9 parts, Fructus Mume 15 parts, Fructus Crataegi 9 parts, Rhizoma Polygonati 9 parts, Radix Ginseng 9 parts, 9 parts, Fructus Momordicae charantiae.

More than in composition, weight calculates with crude drug, and part is weight portion, if in grams, above composition can be made into a pharmaceutical preparation 5-50 preparation unit, and described preparation unit refers to, the final drug preparation made, as made solid preparation 5-50 unit, oral liquid 5-50ml etc.

More than composition can be made into the preparation of 1-6 taking dose, and as tablet, make 18, each taking dose can be 3-18 sheet, can take 1-6 time altogether.As granule, making 6 bags, each serving using 1-2 bag, 36 times can be taken altogether.

More than composition is by weight as proportioning, can increase according to corresponding proportion when producing or reduce, as large-scale production can by kilogram in units of, or in units of ton, small-scale production also can in units of milligram, weight can increase or reduce, but the constant rate of raw medicinal herbs weight proportion between each composition.

The ratio of above weight proportion is through that science screening obtains, and for especial patient, as serious symptom or mild, fat or modest patient, can the proportioning of amount of corresponding adjustment composition, and increase or reduce being no more than 300%, drug effect is constant.

Single medicinal material more than in composition, especially ministerial drug and adjuvant drug, also can be replaced by the suitable Chinese medicine with the identical property of medicine, its drug effect of the Chinese medicine preparation after replacement is constant.

Chinese medicine composition of the present invention, being that the raw material of Chinese medicine by being formed by above-mentioned formula is processed through extraction or other modes, making pharmaceutically active substance, subsequently, with this material for raw material, when needing, add medicine acceptable carrier, make according to the routine techniques of galenic pharmacy.Described active substance can obtain by extracting raw material of Chinese medicine respectively, also can be obtained by the common raw material of Chinese medicine that extracts, also can obtain by other means, as: by pulverizing, squeezing, calcine, grind, sieve, percolation, extraction, water extraction, alcohol extraction, ester carry, the material that method obtains, these active substances can be extractum form such as ketone is carried, chromatography, can be dry extract also can be fluid extract, need to determine to make different concentration according to the difference of preparation.

Pharmaceutically active substance in Chinese medicine composition of the present invention, its in the formulation shared percentage by weight can be 0.1-99.9%, all the other are medicine acceptable carrier.Pharmaceutical composition of the present invention, exists in a unit, and described unit dosage form refers to the unit of preparation, as every sheet of tablet, and every capsules of capsule, every bottle of oral liquid, granule every bag etc.

Chinese medicine composition of the present invention can be any pharmaceutically useful dosage form, and these dosage forms comprise: tablet, sugar coated tablet, film coated tablet, enteric coated tablet, capsule, hard capsule, soft capsule, oral liquid, suck agent, granule, electuary, pill, powder, unguentum, sublimed preparation, suspensoid, powder, solution, injection, suppository, ointment, plaster, cream, spray, drop, patch.Preparation of the present invention, preferably peroral dosage form, as: capsule, tablet, oral liquid, granule, pill, powder, sublimed preparation, unguentum etc.

Chinese medicine composition of the present invention, the preparation of its oral administration can containing conventional excipient, and such as binding agent, filler, diluent, tablet agent, lubricant, disintegrating agent, coloring agent, flavoring agent and wetting agent, can carry out coating to tablet if desired.

The filler be suitable for comprises cellulose, mannitol, lactose and other similar filler.Suitable disintegrating agent comprises starch, polyvinylpyrrolidone and starch derivatives, such as sodium starch glycollate.Suitable lubricant comprises, such as magnesium stearate.The suitable acceptable wetting agent of medicine comprises sodium lauryl sulphate.

By mixing, fill, the method that tabletting etc. are conventional prepares solid oral composition.Repeatedly mix and active substance can be made to be distributed in those compositionss of a large amount of filler of whole use.

The form of oral liquid can be such as aqueous or oily suspensions, solution, Emulsion, syrup or elixir, or can be the composite dry products of a kind of available water before use or other suitable carrier.This liquid preparation can containing conventional additive, such as suspending agent, such as sorbitol, syrup, methylcellulose, gelatin, hydroxyethyl-cellulose, carboxymethyl cellulose, aluminium stearate gel or hydrogenated edible fats, emulsifying agent, such as lecithin, anhydro sorbitol monooleate or arabic gum; Non-aqueous carrier (they can comprise edible oil), the oily ester of the such as ester of almond oil, fractionated coconut oil, such as glycerol, propylene glycol or ethanol; Antiseptic, such as para hydroxybenzene methyl ester or propyl p-hydroxybenzoate or sorbic acid, and if need, can containing conventional flavouring agent or coloring agent.

For injection, the fluid unit dosage form of preparation contains active substance of the present invention and sterile carrier.According to carrier and concentration, this compound can be suspended or dissolve.The preparation of solution is normally by being dissolved in a kind of carrier by active substance, filter-sterilized before being loaded a kind of suitable bottle or ampoule, then seals.Adjuvant such as a kind of local anesthetic, antiseptic and buffer agent also can be dissolved in this carrier.In order to improve its stability, by freezing for this compositions after loading bottle, and under vacuo water can be removed.

Chinese medicine composition of the present invention, applicable medicine acceptable carrier is optionally added when being prepared into medicament, described medicine acceptable carrier is selected from: mannitol, sorbitol, sodium pyrosulfite, sodium sulfite, sodium thiosulfate, cysteine hydrochloride, TGA, methionine, vitamin C, EDETATE SODIUM, Ethylenediaminetetraacetic Acid Calcium Salt, the alkali-metal carbonate of monovalence, acetate, phosphate or its aqueous solution, hydrochloric acid, acetic acid, sulphuric acid, phosphoric acid, aminoacid, sodium chloride, potassium chloride, sodium lactate, xylitol, maltose, glucose, fructose, dextran, glycine, starch, sucrose, lactose, mannitol, silicon derivative, cellulose and its derivates, alginate, gelatin, polyvinylpyrrolidone, glycerol, POLYSORBATE 80, agar, calcium carbonate, calcium bicarbonate, surfactant, Polyethylene Glycol, cyclodextrin, beta-schardinger dextrin-, phospholipid material, Kaolin, Pulvis Talci, calcium stearate, magnesium stearate etc.

Compositions of the present invention according to the situation determination usage and dosage of patient, can take three every day in use, each 1-20 agent, as: 1-20 bag or grain or sheet.

Compositions of the present invention is through following methods preparation, and concrete steps are as follows:

The preparation method of the quick clever preparation of sugar of the present invention is: (1) gets above-mentioned raw materials in proportion, (2) ethanol of 4 times amount 70-90% is added, 50-80 DEG C of decompression recycling ethanol, 70-90 DEG C of vacuum drying, pulverize 80 mesh sieves, obtain drug extract; Add stone and make acceptable dosage form on any number of pharmaceutics.

Pharmaceutical composition of the present invention and preparation method thereof can with reference to Chinese patent CN1726929.

The therapeutic effect of pharmaceutical composition of the present invention is further illustrated by following experiment.

Test the impact of a sugared quick spirit on spontaneous type 2 diabetes mellitus rat glucose-lipid metabolism

1. materials and methods

1.1 laboratory animal

With spontaneous type 2 diabetes mellitus rat model OLETF Mus and its homology non diabetic controls Mus LETO Mus for experimental subject.Rat is all male, OLETF 40, LETO rat 10, surrounding age, is introduced by Japanese Otsuka Pharmaceutical Co., Ltd. Tokushima institute.

Rat no-special pathogen level (SPF level, specific pathogen-free) condition place an order cage raise, raise with standard feed.Ambient temperature controls at 22-25 DEG C, and humidity is 55 ± 5%, 12/12 h light dark cycle (light application time 7:00-19:00), freely obtains food and drinking-water.

Raise place: Institute of Radiation Medicine, Chinese Academy of Medical Sciences Experimental Animal Center SPF level animal feeding room.

1.2 grouping and administrations

1.2.1OLETF diabetes diagnosis grouping standard

The regular row oral glucose tolerance test (OGTT) of rat.Test front fasting overnight 15 hours, can't help water, give 30% glucose solution gavage (get glucose powder 82.5g, adding distil water is settled to 250ml, and heating for dissolving is clear liquid, and obtaining concentration is 30% glucose solution) by 2g/kg.

In on an empty stomach and after glucose load 30,60,90 and 120min get blood, measure blood glucose value with card BIOSEN5030 rapid glucose analyser in Germany.After glycemic peaks > 16.7mmol/L and load, 120min blood glucose > 11.1mmol/L examines as diabetes, possesses an above-mentioned person for impaired glucose tolerance.

1.2.2 laboratory animal grouping and administration

During to 30 weeks, have into mould OLETF Mus 27, be divided into model group, rosiglitazone group, quick clever group of sugar at random, LETO is blank group.

Rosiglitazone group: rosiglitazone (Avandia): the every sheet of Tianjin company limited of Smithkline Beecham product is containing rosiglitazone maleate 4mg, purchased from by U.S.'s GlaxoSmithKline PLC (China) Investment Co., Ltd, be stirred to before use and dissolve completely, with distilled water diluting, dosage is 3mg/kg/d ^-1.

Sugar quick clever group (that is: dissipating depression of QI heat clearing away group), adopt the formulated of the embodiment of the present invention 1 to become extractum, 4 DEG C of Refrigerator stores, with distilled water diluting, given low is 9mg/kg/d ^-1(calculating by contained crude drug gauge).

Model group and blank group with equivalent distilled water gavage, the equal gastric infusion of each group 12 weeks, every day 1 time.

1.3 experimental technique

1.3.1 laboratory animal food ration

Weigh rat food ration 1 time weekly.Give the enough feedstuffs of rat pre-cast weekly, after one week, take surplus (it is 8 a.m. Monday that feedstuff surplus weighs the time).

1.3.2 laboratory animal weight

Weight: claim Mus weight and record with electronics weighing machine weekly, the time of taking is all a whole morning 9:00.

1.3.3 oral glucose tolerance test

Within every 4 weeks, oral glucose tolerance test (OGTT) is carried out before rat administration and after administration.Fasting overnight 15 hours before test, can't help water, give 30% glucose solution gavage by 2g/kg, on an empty stomach and after glucose load 30,60,90 and 120min cut tail and get blood, measure blood glucose.During oral carbohydrate tolerance experiment in 32 weeks, synchronous inner canthus gets blood, measures insulin.

Blood glucose is measured by card BIOSEN5030 rapid glucose analyser in Germany.

Insulin assay is with putting the method for exempting from (with Linco company rat insulin medicinal box special, special messenger is with batch mensuration).

Insulin, Area under the curve of blood glucose calculate (AUC): the computing formula adopting approximate trapezoid, and 0-120min blood glucose AUC=1/2 (0min value+120min is worth)+30min is worth+60min and is worth+90min value.

1.3.4 four items of blood lipid tests, serum free fatty acid, glycated serum protein detect

After gavage terminates, Rat Septal curfew eats 12 hours, secondary morning rat taking blood from jugular vein 2.5ml, centrifuging and taking serum, preserves standby survey for-70 DEG C.

Triglyceride (TG), T-CHOL (TC), low-density lipoprotein cholesterol (LDL-C), HDL-C (HDL-C).TC, LDL-C, HDL-C adopt enzyme process, and TG adopts acetylacetone,2,4-pentanedione development process, and transaminase, Hitachi-7150 automatic clinical chemistry analyzer carries out, is detected by Gate of Pervasive Peace hospital laboratory; Glycated serum protein BecKmanCX-5 automatic clinical chemistry analyzer measures, and sample is with a collection of mensuration.

1.3.5 Hyperinsulin euglycemic clamp

Rat Septal curfew eats, can't help water, intraperitoneal injection of anesthesia is carried out with the pentobarbital sodium of 2% after weighing, dorsal position is fixed, anaesthetize effective after, cut skin of neck, blunt separation subcutaneous tissue and muscle, be separated and expose in right carotid and left side neck or external jugular vein, carries out in right carotid and left side neck or external jugular vein intubate, threading ligation, fixing (U.S. produces epidural anesthesia silica gel catheter, internal diameter 0.6mm, external diameter 1mm), and be full of conduit with the normal saline containing heparin 50U/ml, keep it unobstructed.Arterial cannulation surveys blood glucose to get blood, and venous cannulation connects mini three links pipe, and the port of export is connected with vein, and two entrance points respectively micro-injection pump digital with micro computer are connected with infusion of insulin and glucose.After intubate, after rat is left standstill 30 minutes, measure blood glucose.And it is incubated simultaneously.

Insulin, 20% glucose are pumped into by jugular vein with 2 digital micro-injection pumps of micro computer respectively, and blood preparation is obtained by carotid duct.The syringe that the intubate rat carotid artery conduit leaving standstill 30 minutes connects is taken off, gets blood one, in 30s, measure basal plasma glucose value by miniature blood glucose meter.

During beginning, (infusion rates is 8mU/kgmin to the fugitive Iletin II (Lilly) of first constant infusion ^-1, face used time insulin 0.5% bovine serum albumin and dilute).Within every 10 minutes, measure blood glucose once, when blood glucose value exceeds the scope of basic value ± 0.5mmol/L, namely starting infusion concentration is 20% glucose, adjustment glucose infusion rate (i.e. glucose infusion rate, GIR), make blood glucose value control about the scope of basic value ± 0.5mmol/L, if blood glucose value exceeds the scope of basic value ± 0.5mmol/L, can glucose infusion be continued.Still within every 10 minutes, survey a blood glucose after glucose infusion, and within the shortest time, adjust glucose infusion rate according to blood glucose value, blood sugar recovery is arrived in the scope of basic value ± 0.5mmol/L.So repeatedly carry out, when continuous three blood glucose values are all stabilized in above-mentioned scope, namely reach steady statue.By three the GIR values being in stable state of clamp, (unit is mgkg ^-1min ^-1) average.This index is for judging whether the degree that there is IR and IR.

1.3.6 the acquisition and processing of rat pancreas

After Hyperinsulin euglycemic clamp terminates, namely put to death rat, open abdominal cavity, between spleen, stomach and duodenum crook to dissociate pancreas, weigh after filter paper wipe dry, cut along the pancreas longitudinal axis, get tail of pancreas portion of tissue and be placed on ice pan, be cut into 1mm rapidly ³fritter, 4 DEG C of glutaraldehydes are fixed, for Electron microscopy.

It is liquid-solid fixed that the fresh pancreatic tissue of part puts into Bouins, for morphologic detection; The fresh pancreatic tissue of part is placed in rapidly in cryopreservation tube, and Liquid nitrogen storage, for molecular Biological Detection.

1.3.7 the acquisition and processing of rat liver tissue

Part fresh liver tissue is put into 10% neutral formalin solution and is fixed, for morphologic detection; Part fresh liver is organized and is placed in rapidly in cryopreservation tube, and Liquid nitrogen storage, for molecular Biological Detection.

1.3.8 fatty in rat abdomen acquisition and processing

Put to death rat, get epididymis, mesentery, fold back properitoneal fat weigh, count visceral fat weight, and calculate visceral fat weight percentage of liveweight percentage ratio; The all fat of the fresh testis of part is put into 10% neutral formalin solution and is fixed, for morphologic detection.

1.3.9 the acquisition and processing of rat skeletal muscle

The fresh Gastrocnemius Muscle of Cancer of part is put into 10% neutral formalin solution and is fixed, for morphologic detection; The fresh Gastrocnemius Muscle of Cancer of part is placed in rapidly in cryopreservation tube, and Liquid nitrogen storage, for molecular Biological Detection.

1.3.10 rat skeletal muscle and liver AMPK detect process in early stage

Take out liver of laboratory animal, muscular tissue respectively, and 500 milligrams of tissue weights of weighing

Put 15 milliliters of conical centrifuge tube of pre-cooling into

Add GENMED and clear up liquid (Reagent A) cleaning

Pump cleaning liquid

Be moved into a liquid nitrogen cryopreservation pipe

At once put liquid nitrogen container into spend the night

Next day takes out from liquid nitrogen container, at once with grinding rod pulverize be organized into Powdered

Put 15 milliliters of conical centrifuge tube into

Add the GENMED lysate (Reagent B) be placed in ice groove

Powerful vortex shakes 30 seconds, fully mixes

Put ice groove into and hatch 30 minutes, every 10 minutes of period, powerful vortex shook 30 seconds

At once centrifugal 10 minutes of 4 DEG C of desk centrifuges are put into

Carefully pipette supernatant to new 15 milliliters of aseptic centrifuge tubes ,-80 DEG C of preservations

1.3.11 pancreas and liver HE staining procedure:

Paraffin cuts dewaxing to water.

Distilled water flushing 5 minutes.

Cut into slices into Harris haematoxylin 15 minutes.

Running water 5 minutes.

0.5% hydrochloride alcohol breaks up 30 seconds

Running water 10 minutes.

Eosin stains 10 minutes.Change twice tap water.

Gradient alcohol dehydration, the transparent mounting of dimethylbenzene I, II

1.3.12 statistical method

Adopt SPSS16.0 software to take statistics analysis, experimental data measurement data is with mean ± standard deviation represent, between two groups of Normal Distribution, mean compares and adds up with t inspection, with batch more than compare between group and adopt one factor analysis of variance (One-wayANOVA), compare between two and adopt least significant difference (LSD).

2. result

2.1 laboratory animal ordinary circumstances

OLETF rat comparatively LETO is fat, and movable slow, lazyness is moved, hair color is withered matt.

Experimental session gets blood unexpected death animal 5, wherein OLETF 3, LETO 2.

2.2 rat food rations compare

In experimental period, model group, dissipating depression of QI heat clearing away group, rosiglitazone group food ration compare no significant difference, and three groups of food rations are all higher than LETO group (P < 0.05).

Table 1 experimental rat food ration compares

Note: compare with age in week, model group compares with normal group ^##p < 0.01, ^#p < 0.05; Treatment group compares with model group ^*p < 0.01, ^*p < 0.05

The comparison of 2.3 rat weights

As shown in table 2, in experimental period, model group, dissipating depression of QI heat clearing away group and rosiglitazone group weight are all significantly higher than LETO group (p < 0.01), and dissipating depression of QI heat clearing away group weight is starkly lower than model group and rosiglitazone group (p < 0.05)

The change of table 2 experimental rat weight

The comparison of 2.4 rat abdominal cavity fat contents

Within 42 weeks, cut open and kill result display, fat content comparatively LETO group obviously increase (P < 0.05) in model group abdomen, dissipating depression of QI heat clearing away group comparatively model group reduces (P < 0.05), rosiglitazone group comparatively model group increases (P < 0.05), in table 3, the change of this body weight and abdomen fat content may recover with islet function, and blood sugar level declines relevant.

The comparison of fat content, fat body ratio in table 342W experimental rat abdomen

The change of 2.5 Oral Administration in Rats carbohydrate tolerance test glucose levels

As shown in table 4, model group and LETO group before the treatment after AUC obvious difference (P < 0.01) all the time.With course advancement, model group AUC rises gradually, two treatment groups compare AUC and decline with model group, at the end of experiment, dissipating depression of QI heat clearing away group and rosiglitazone group AUC are all starkly lower than model group (P < 0.01), rosiglitazone group is slightly lower than dissipating depression of QI heat clearing away group, but this species diversity does not have statistical significance.

Table 442W respectively organizes each time point blood glucose (mmol/L) of OGTT and AUC (mmolmin/L) change

Note: AUC, compares with age in week, compares ##P < 0.01, #P < 0.05 with normal group; * * P < 0.01, * P < 0.05 is compared with model group; Dissipating depression of QI heat clearing away group compares with rosiglitazone group ^{△ △}p < 0.01, ^△p < 0.05

The change of 2.6 experimental rat insulin secretion functions

Oral Administration in Rats carbohydrate tolerance test is with the change of pacing insulin secretion and area under curve, under the synchronous insulin curve of model group OGTT, area is greater than LETO group, in conjunction with positive sugared clamp experiment result, hints model group also exists obvious insulin resistant, after dissipating depression of QI heat clearing Chinese medicine and rosiglitazone in treating, under two groups of insulin curves, area significantly increases, and points out two groups to have a better role to B cell insulin secretion function.As shown in table 5

Table 542W respectively organizes each time point insulin (ng/ml) of OGTT and AUC (ngmin/ml) change

Note: AUC, compares with age in week, compares ##P < 0.01, #P < 0.05 with normal group; * * P < 0.01, * P < 0.05 is compared with model group

OGTT synchronous insulin release test is passed through during 42W, we calculate insulin secretion index (Δ I30/ Δ G30), result display model group comparatively LETO obviously declines, and dissipating depression of QI heat clearing away and rosiglitazone group comparatively model group obviously raise, dissipating depression of QI heat clearing away group is better than rosiglitazone.As shown in table 6

Table 642W tests each group of insulin secretion index and compares (ng/mmol)

Note: compare ##P < 0.01, #P < 0.05 with normal group; * * P < 0.01, * P < 0.05 is compared with model group;

2.7 experiments each group of Hyperinsulin euglycemic clamp results contrast

As shown in table 7, model group rats Steady state glucose infusion rate comparatively LETO group significantly declines, (P < 0.01), show that rat model has obvious insulin resistant, after Chinese drug-treated group and Western medicine group therapeutic intervention, glucose infusion rate is significantly increased (P < 0.01), and both displays all have the effect of good improvement insulin resistant.

Table 742W tests each group of Hyperinsulin euglycemic clamp result

Note: compare ##P < 0.01, #P < 0.05 with normal group; * * P < 0.01, * P < 0.05 is compared with model group

2.8 experiments each group of Blood Lipid situation

The triglyceride levels of model group extremely significantly increases, cholesterol, free fatty acid levels obviously increase, record similar to document, meet OLETF Blood Lipid feature, dissipating depression of QI heat clearing Chinese medicine has the effect significantly reducing triglyceride, cholesterol, free fatty are also had to the decline of statistical significance, display has good Regulation serum lipids.As shown in table 8

Table 8 42W tests each group of blood fat situation and compares

3 brief summaries

3.1OLETF-meets doctor trained in Western medicine diabetes diagnosis, embodies the animal model of pathogenesis feature

Raised through about 30 weeks time, part OLETF rat blood sugar obviously raises, oral administration carbohydrate tolerance test, 120min blood glucose > 11.1mmol/L after partial rat glycemic peaks > 16.7mmo1/L and load, reach diabetes diagnostic criterion, diabetes model success.

3.2 sugared quick spirits lose weight, and reduce fat content in abdomen

In this laboratory observation to OLETF comparatively LETO group body weight, abdomen, athero all obviously increases.Through treatment in 12 weeks, sugared quick spirit alleviated OLETF weight, and reduces fat content in abdomen, compares, embody greater advantage with rosiglitazone.

3.3 sugared quick spirits effectively improve glucose-lipid metabolism level

The result of study of this experiment shows, the quick spirit of sugar can improve the abnormal carbohydrate metabolism of type 2 diabetes mellitus rat model, its hypoglycemic mechanism preliminary analysis is two aspects: one is the improvement of insulin resistant, we judge with the goldstandard of insulin sensitivity evaluation, there is obvious insulin resistant in model group rats, Chinese drug-treated group and Western medicine group insulin resistant all obviously improve, its glucose infusion rate is even greater than Normal group, Chinese drug-treated group has significant difference compared with model group, and this is consistent with research team previous investigation; Two is improvement of islet cells secrete function, and result shows, and under the insulin curve of rat model, area obviously declines, and Chinese drug-treated group and Western medicine group significantly improve after treatment.Meanwhile, Chinese drug-treated group significantly improves insulin secretion index (Δ I30/ Δ G30), improves the pattern of insulin secretion.

The quick spirit of sugar also demonstrates good Regulation serum lipids, while fat-reducing, improve lipid metabolism.The disorder of type 2 diabetes mellitus glucose-lipid metabolism is an entirety, the two connects each other, influence each other, therefore from the organic conception of disease, to consider in treatment comprehensively, avoid independent blood sugar lowering or lipid-lowering therapy, affect the treatment, blood sugar lowering, tune fat must be placed on status of equal importance, this is for the process controlling diabetes, and the generation development preventing and treating complication has great importance.

The quick spirit of experiment disaccharide is on the impact of spontaneous type 2 diabetes mellitus rat liver AMPK

1. materials and methods

1.1 laboratory animals (with experiment one)

1.2 groupings and administration (with experiment one)

1.3. the collection of experimental rat liver specimens and HE dyeing (with experiment one)

1.4. index and detection

1.4.1 liver AMPK active level measures

Key instrument: Herolab mini desktop centrifuge, V-1800PC spectrophotometer and cuvette

Main agents: GENMED company organization AMPK kinase activity colorimetric assay detection kit

1.4.1.1 process in early stage

Take out laboratory animal fresh liver tissue respectively, and weighing 500 milligrams of tissue weights

Put 15 milliliters of conical centrifuge tube of pre-cooling into

Add GENMED and clear up liquid (Reagent A) cleaning

Pump cleaning liquid

Be moved into a liquid nitrogen cryopreservation pipe

At once put liquid nitrogen container into spend the night

Put 15 milliliters of conical centrifuge tube into

Add the GENMED lysate (Reagent B) be placed in ice groove

Powerful vortex shakes 30 seconds, fully mixes

At once put centrifugal 10 minutes of 4 DEG C of desk centrifuges into, speed is 10000g

Carefully pipette supernatant to new 1.5 milliliters of aseptic centrifuge tubes ,-80 DEG C of preservations

Preparation of determine

Get out testing sample (such as lysis suspension etc.), be placed in ice groove

Set spectrophotometer (temperature is 30 DEG C): wavelength is 340nm, 1 minute, interval, reading 6 times (totally 5 minutes), and zero setting

1.4.1.2 ground control measures

Pipette GENMED buffer (Reagent C) to new cuvette

Add GENMED enzymatic liquid (Reagent D)

Add GENMED negative fluid (Reagent G)

Put 30 DEG C of incubators into and leave standstill 2 minutes

Spectrophotometer, zero setting

Take out cuvette

1.4.1.3 sample determination

Pipette GENMED buffer (Reagent C) to new cuvette

Add GENMED enzymatic liquid (Reagent D)

Add 5 microlitre testing samples, put 30 DEG C of incubators into and leave standstill 2 minutes

Add GENMED reactant liquor (Reagent E)

Add GENMED substrate solution (Reagent F)

Topple over for several times up and down, mixing (being limited within 3 seconds)

At once put spectrophotometer into detect, this is sample readout:

340 wavelength readings 0 minute-340 wavelength readings 5 minutes

According to formula, calculate AMPK enzymatic activity a quantitative index

1.4.2 real time fluorescent quantitative PGR method detects the mrna expression of liver SREBP-1

Key instrument

Applied Biosystems company of the GeneAmp 5700TaqMAN PCR instrument U.S.

Pharmacia Biotech company of the gel image analysis instrument ImageMaster VDS U.S.

High speed refrigerated centrifuge Germany BACKMAN company Allagra.21R Centrifcige

Ultraviolet spectrophotometer Germany BACKMAN company DU640 type

Electrophresis apparatus Liuyi Instruments Plant, Beijing OYY-III-5 type

Main agents:

Trizol(Invitrogen)

RevertAidTM First Strand cDNA Synthesis Kit(Fermentas)

SYBR Green PCR Master Mix(Applied Biosystems)

Primer adopts primer premier5.0 design, and by match, Parkson company synthesizes.

1.4.2.1 extract total serum IgE from tissue

● get after about 100mg tissue adds 1ml Trizol, be ground to tissue completely fragmentation, liquid thickness, room temperature places 5 minutes, makes its abundant cracking.

● add chloroform by 200ul chloroform/mlTrizol, shake up 15 seconds, room temperature places 15 minutes.

● centrifugal 15 minutes of 4 DEG C of 12000g.

● draw upper strata aqueous phase, in the centrifuge tube of another pre-cooling.

● add isopropyl alcohol (pre-cooling) mixing by 0.5ml isopropyl alcohol/ml Trizol, room temperature places 5-10min.

● 4 DEG C of centrifugal 10min of 12000g, abandon supernatant, RNA is sunken at the bottom of pipe.

● add 75% ethanol (pre-cooling) by 1ml 75% ethanol/ml Trizol, gentle concussion centrifuge tube, suspend precipitation.

● 4 DEG C of centrifugal 5min of 7500g, abandon supernatant as far as possible.

● room temperature dries 5-10min.

● with 50 μ l RNase-free H ₂o, dissolves RNA sample, 55-60 DEG C of 10min.

● survey the quantitative RNA concentration of OD value.

1.4.2.2 the concentration of spectrophotometry nucleic acid

With the spectrophotometer of argon lamp, before using thermally-stabilised 15 minutes in advance.

● draw 6 μ l RNA sample, after adding water to 1.5ml mixing, proceed in spectrophotometric quartz cuvette.

● 1ml water suppressed zero first used by spectrophotometer.

● read optical density respectively at 260nm and 280nm, the concentration of RNA sample is: OD260* nucleic acid extension rate * 40/1000; Concentration unit is μ g/ μ l.

1.4.2.3RNA sample reverse transcription

● take out RNA template, reagent, after room temperature is melted, be placed on immediately in ice bath, DL device mixes, of short duration centrifugal.

● template denaturation:

Template RAN x ul(5.0ug)

Oligo(dT) ₁₈primer 0.5ug/ul 1.0ul

DEPC treated water is to 12.0ul

Mixing, of short duration centrifugal, 70 DEG C of temperature baths 10 minutes, to be placed in ice bath at least 1 minute immediately.

● preparation reaction mixture (often pipe is by following amounts preparation)

5×reaction buffer 4.0ul

10mM dNTP Mix 2.0ul

RNase Inhibitor (20U/ul) 1.0ul

Compound mixed solution amount is sample number N+2.

● often pipe adds the above-mentioned mixed liquor mixing of 7.0uL.37 DEG C of temperature are bathed 5 minutes.

● often pipe adds 1.0uL M-MuLV Reverse Transciptase (200u/uL), and 42 DEG C of temperature are bathed 60 minutes, and 70 DEG C of temperature are bathed 10 minutes.-20 DEG C save backup.

1.4.2.4 fluorescence quantitative PCR detection

● primer and reagent

SYBR Green PCR Master Mix：ABI(Applied Biosystems)，Catalog No.43049155

Primer sequence:

β-actin-FP：5’-GAG ACC TTC AAC ACC CCA GCC-3’

β-actin-RP:5 '-AAT GTC ACG CAC GAT TTC CC-3 ' amplified fragments is 263bp.

SREBP: amplified fragments is 168bp

Upstream: 5 '-CGC TAC CGT TCA TCT ATC AAT G-3 '

Downstream: 5 '-ACT TCG CAG GGT CAG GTT CT-3`

● primer dilutes: diluted by final concentration 10pmol/ul by primer, and concrete grammar is V (ul)=O.D. value × 33ug/10M (DEPC process water volume needed for V=, M=primer molecule amount)

● the preparation of reaction mixture:

Take out 2 × Buffer, RNase-free water, cDNA is put at room temperature, melts, is placed on immediately in ice bath, DL device mixes, of short duration centrifugal.

The contained reagent of every person-portion mixed liquor (reaction system V=25ul) sees the following form

Component Volnme

2×SYBR Green PCR Master Mix， 12.5ul

xxx-F primer(10pmol/ul) 1ul

xxx-R primer(10pmol/ul) 1ul

Nuclease-free H ₂O 8.5ul

Total Volume 23ul

After preparing mixed liquor, in each reaction tube, add 23ul mixed liquor.

● application of sample: each reaction tube adds 2.0ul cDNA template, and DL device mixes, of short duration centrifugal (being less than 5sec).

● carry out at GeneAmp 5700 quantitative real time PCR Instrument, reaction condition is as follows:

95.0℃：10min

72.0℃：50sec

72.0℃：5min

More than reaction setting is all carried out on the computer be connected with PCR instrument, and each circulation computer records the fluorescence signal value in reaction tube automatically, and describes curve.Reaction terminates by PE 5700 software analysis result, automatic calculation in quantity numerical value.

Experimental result is using the Ct value ratio of reference gene and genes of interest as the mRNA relative expression quantity of genes of interest, (because Ct value and actual expression are inversely, in order to intuitively, the Ct value ratio of genes of interest and Beta-actin is got inverse when analytical data by us, and namely the Ct value ratio of Beta-actin and target gene is as the relative expression quantity of its mRNA)

1.4.3 Immunohistochemical Method detects liver SREBP-1 protein expression

Instrument: various conventional reagent and equipment are provided by central laboratory of clinical research institute of China-Japan Friendship Hospital.Ultra cold storage freezer ULTRA LOW-70 DEG C, Japanese SANYO company; Oriental cherry flat push type microtome, Japanese SAKURA PTERATOMECRM-440; Exhibition sheet device, Japanese SAKURA Slide warner PS-53; Rotary automatic dehydrator, Japanese SAKURATissue-Tek Tissue processor; Tissue embedding machine, Japanese SAKURA Tissue-Tek TECTM5 SMC1-042-SO

Reagent: SREBP-1 rabbit against murine one anti antibody, Santa Cruz Biotechnology Products; Two resist for EnVisionTM system; DAB nitrite ion (all conventional reagent are Denmark DAKO Products).

1.4.3.1 fixing, dehydration, transparent, waxdip, embedding

After flesh tissue is fixing, gradient alcohol dehydration (75% ethanol 1 hour → 80% ethanol 1 hour → 90% ethanol 1 hour → 95% ethanol 1 hour → 100% ethanol 1 hour → 100% ethanol 1 hour → dimethylbenzene, 45 minutes → dimethylbenzene 45 minutes), last waxdip embeds into wax stone.

1.4.3.2 slicing treatment

Microscope slide process: newly purchase microscope slide and soak 24-48 hour, running water 2-3 hour, distilled water immersion 1 hour through washing, 60 DEG C of baking boxs are dried, and be soaked in the chromic acid gelatin solution of new preparation, pull out after soaking, natural drying is for subsequent use.

Wax stone is cut into 4 microns of slabs by paraffin slicing machine, tissue is moved in the constant-temperature hot water container of 40 DEG C, after tissue is launched completely, invest on microscope slide, lie on exhibition sheet device, the section of mounting 56 DEG C of calorstats spend the night, and take out for subsequent use.

1.4.3.3 immunohistochemical staining step:

Dewaxing, aquation tissue slice.

Distilled water rinsing,

Drip 0.3%H ₂o ₂block endogenous peroxydase, hatch 15 minutes.

Distilled water rinsing, is placed in TBS 5 minutes totally 3 times.

Primary antibodie hatches 2 hours

Distilled water rinsing, is placed in TBS 5 minutes totally 3 times

Drip EnvisionTM and hatch 60 minutes.

TBS rinsing 5 minutes totally 3 times.

Color source DAB solution develops the color 3 minutes, distilled water color development stopping.

Gradient alcohol dehydration, mounting rubber seal sheet.

Basis of microscopic observation result.

Establish negative control during experiment simultaneously, adopt TBS to replace primary antibodie as blank negative control.Occur in hepatocyte endochylema that brown yellow granule is positive.

1.4.3.4 graphical analysis

The dyeing of the SREBP of liver and quantitatively: the medical image analysis system analysis adopting China Academy of TCM's molecular immune room, often open section and select 5 visuals field, often organize and at least select 8 sections, the gross area measuring stained positive region in each visual field represents the quantity of this Materials Cell, measure the expression that average optical (MOD) value represents this material of unit are cell, finally with the Average value compare often organized.

1.4.4 statistical procedures

Completed by SPSS16.0 statistical package, measurement data mean ± standard deviation represent, compare between group more than same batch and adopt one factor analysis of variance (One-wayANOVA), compare between two and adopt least significant difference (LSD).

2 results

The lipometabolic comparison of 2.1 experimental rat (see experiment one)

2.2 experiments each group of liver lipids dystopy deposition conditions

Normal group (LETO): portal area small artery, venule and gallbladder tube structure are normal, and hepatocyte is monokaryon or double-core, endochylema powder contaminates, and even particle size is consistent.

Model group: liver rope arrangement disorder, without radial arrangement, the fat differed in size in a large number in endochylema drips; Some liver cell nuclears are then squeezed to obtain obvious off normal by fat drop.

Rosiglitazone group: under light microscopic, fat becomes, and swelling of liver cell is not obvious, has tiny fat to drip in part cell.

Dissipating depression of QI heat clearing away group of the present invention: hepatocyte fat becomes not obvious, and lobules of liver and hepatic sinusoid clear in structure, cellularity is more complete.

The protein expression of 2.3 experimental rat liver SREBP-1C

SABC shows, and the immunoreation positive products of SREBP-1C is brown yellow granule, is expressed in hepatocyte.In the hepatocyte of LETO rat, the expression region of SREBP-1C is comparatively limited to, and intensity is more weak.The expression of the SREBP-1C of model group rats significantly strengthens, and rosiglitazone group and dissipating depression of QI heat clearing away group are expressed weak compared with model group.

Quantitative analysis results shows: compared with normal group, and in model group hepatocyte, the cell area of SREBP-1C stained positive and average optical (MOD) value are significantly higher than normal group (P < 0.01); Compared with model group, in dissipating depression of QI heat clearing away group hepatocyte, the cell area of SREBP-1C stained positive and MOD are significantly lower than model group (P < 0.01) (as shown in table 9)

Table 9 respectively group rat liver SREBP-1C positive expression region area and MOD value compares

The mrna expression of 2.4 experiments each group of rat liver SREBP-1C

2.4.1 the mensuration of spectrophotography nucleic acid concentration

Ultraviolet spectrophotometer measures RNA, λ 260nm/ λ 280nm between 1.8-2.0.

2.4.2 amplified production melting point curve

The melting point curve of SREBP-1C and β-actin gene is single peak, and does not widen distortion, illustrates that this experiment can ensure the specificity of amplified production.

2.4.3 the gene expression of SREBP-1c in hepatic tissue

Compared with normal group, in model group hepatocyte, SREBP-1C mrna expression is significantly higher than normal group (P < 0.01); Compared with model group, in sugar quick spirit group hepatic tissue, SREBP-1CmRNA is significantly lower than model group (P < 0.01).As shown in table 10

Table 10 is group liver β-actin and SREBP-1 respectively _cluciferase expression amount odds ratio comparatively (%)

By above-mentioned experiment, the present invention proves that the quick spirit of medicine sugar has good therapeutic effect for type 2 diabetes mellitus and complication thereof further.Quick attenuating SREBP1-c albumen and the developed by molecule of having quick access to information of sugar, thus improve type 2 diabetes mellitus people lipid metabolism level, reduce the deposition of lipid in peripheral tissues, finally reach the effect for the treatment of and prevention type 2 diabetes mellitus.

Type 2 diabetes mellitus of the present invention is the type 2 diabetes mellitus that Fatty toxicity causes, or the type 2 diabetes mellitus that sugared toxicity causes.

Detailed description of the invention

By following specific embodiment, the invention will be further described, but not as limitation of the present invention.

The preparation of embodiment 1, the quick curing capsule of sugar of the present invention

Get Radix Trichosanthis 30g, Radix Bupleuri 12g, Fructus Aurantii Immaturus 9g, Radix Et Rhizoma Rhei 3g, Rhizoma Pinelliae 6g, Radix Scutellariae 9g, Rhizoma Coptidis 6g, Radix Paeoniae Alba 9g, Fructus Mume 15g, Fructus Crataegi 9g, Rhizoma Polygonati 9g, Radix Ginseng 9g, Fructus Momordicae charantiae 9g;

Add the ethanol of 4 times amount 80%, 60 DEG C of reflux, extract, 3 times, each 1.5 hours, merge extractive liquid, ethanol are reclaimed in 70 DEG C of pressurizations, and 80 DEG C of vacuum dryings, pulverized 80 mesh sieves, and obtained drug extract;

Add lactose and make capsule.

The preparation of the quick clever tablet of embodiment 2, sugar of the present invention

Add dextrin and make tablet.

The preparation of the quick clever granule of embodiment 3, sugar of the present invention

60 DEG C of forced air dryings, granulate, granulate, obtains granule.

The preparation of embodiment 4, the quick drop pills of sugar of the present invention

The Polyethylene Glycol added, mix homogeneously, melting, upper pill dripping machine, makes drop pill.

The preparation of the quick clever oral cavity disintegration tablet of embodiment 5, sugar of the present invention

Add 5% polyvinylpolypyrrolidone, the magnesium stearate of 0.1%, the microcrystalline Cellulose of 50%, make soft material with ethanol in proper amount solution, granulate, 60 DEG C of forced air dryings, granulate, granulate, tabletted, obtains oral cavity disintegration tablet.

The preparation of the quick curing capsule agent of embodiment 6, sugar of the present invention

Add appropriate amount of starch, sucrose and magnesium stearate, granulate, incapsulate, obtain capsule.

The preparation of the quick clever tablet of embodiment 7, sugar of the present invention

With starch, sodium carboxymethyl cellulose, Pulvis Talci mix homogeneously, granulate, namely tabletting obtains tablet.

The preparation of embodiment 8, the quick Ling oral liquid of sugar of the present invention

With syrup 4g, be dissolved in the pure water of 100ml, homogenizing, filter, through high-temperature short-time sterilization (135 DEG C, 4s).Sterile filling, subpackage, obtained oral liquid.

The preparation of embodiment 9, the quick Ling oral liquid of sugar of the present invention

With syrup 5g, be dissolved in the pure water of 100ml, homogenizing, filter, through high-temperature short-time sterilization (135 DEG C, 4s).Sterile filling, subpackage, obtained oral liquid.

The preparation of embodiment 10, the quick curing capsule of sugar of the present invention

Get Radix Trichosanthis 9g, Radix Bupleuri 12g, Fructus Aurantii Immaturus 9g, Radix Et Rhizoma Rhei 3g, Rhizoma Pinelliae 6g, Radix Scutellariae 9g, Rhizoma Coptidis 6g, Radix Paeoniae Alba 9g, Fructus Mume 9g;

Add lactose and make capsule.

The preparation of embodiment 11, the quick curing capsule of sugar of the present invention

Get Radix Trichosanthis 30g, Radix Bupleuri 12g, Fructus Aurantii Immaturus 9g, Radix Et Rhizoma Rhei 3g, Rhizoma Pinelliae 6g, Radix Scutellariae 9g, Rhizoma Coptidis 6g, Radix Paeoniae Alba 9g, Fructus Mume 15g, Fructus Crataegi 9g;

Add lactose and make capsule.

The preparation of the quick clever tablet of embodiment 12, sugar of the present invention

Add dextrin and make tablet.

The preparation of the quick clever tablet of embodiment 13, sugar of the present invention

Add dextrin and make tablet.

The preparation of the quick clever granule of embodiment 14, sugar of the present invention

60 DEG C of forced air dryings, granulate, granulate, obtains granule.

The preparation of the quick clever granule of embodiment 15, sugar of the present invention

60 DEG C of forced air dryings, granulate, granulate, obtains granule.

The preparation of embodiment 16, the quick drop pills of sugar of the present invention

The preparation of embodiment 17, the quick drop pills of sugar of the present invention

The preparation of the quick clever oral cavity disintegration tablet of embodiment 18, sugar of the present invention

The preparation of the quick clever oral cavity disintegration tablet of embodiment 19, sugar of the present invention

The preparation of the quick curing capsule agent of embodiment 20, sugar of the present invention

The preparation of the quick curing capsule agent of embodiment 21, sugar of the present invention

The preparation of the quick curing capsule agent of embodiment 22, sugar of the present invention

The preparation of the quick curing capsule agent of embodiment 23, sugar of the present invention

The preparation of the quick curing capsule agent of embodiment 24, sugar of the present invention

The preparation of the quick curing capsule agent of embodiment 25, sugar of the present invention

The preparation of the quick clever injectable powder of embodiment 26, sugar of the present invention

Add glucose 4.5g, sodium thiosulfate 0.9g and distilled water 1ml again, after said components mix homogeneously, lyophilization, subpackage, obtains injectable powder.

The preparation of the quick clever injectable powder of embodiment 27, sugar of the present invention

The preparation of the quick clever injectable powder of embodiment 28, sugar of the present invention

Group component in above-described embodiment can expand or reduce in scale according to need of production simultaneously.

Claims

1. a pharmaceutical composition is preparing the application in the medicine lowering SREBP1-c albumen and developed by molecule, wherein said pharmaceutical composition is by lowering SREBP1-c albumen and developed by molecule, improve lipid metabolism level, thus reduce the hyperlipidemia of lipid caused by the deposition of peripheral tissues, described pharmaceutical composition is that prescription is formulated by the raw material of Chinese medicine medicine of following weight portion: Radix Trichosanthis 5-40 part, Radix Bupleuri 10-30 part, Fructus Aurantii Immaturus 3-15 part, Radix Et Rhizoma Rhei 1-6 part, Rhizoma Pinelliae 1-12 part, Radix Scutellariae 3-15 part, Rhizoma Coptidis 1-12 part, Radix Paeoniae Alba 3-15 part, Fructus Mume 5-20 part.

2. a pharmaceutical composition is preparing the application in the medicine lowering SREBP1-c albumen and developed by molecule, wherein said pharmaceutical composition is by lowering SREBP1-c albumen and developed by molecule, improve lipid metabolism level, thus reduce the spontaneous type 2 diabetes mellitus of lipid caused by the deposition of peripheral tissues, described pharmaceutical composition is that prescription is formulated by the raw material of Chinese medicine medicine of following weight portion: Radix Trichosanthis 5-40 part, Radix Bupleuri 10-30 part, Fructus Aurantii Immaturus 3-15 part, Radix Et Rhizoma Rhei 1-6 part, Rhizoma Pinelliae 1-12 part, Radix Scutellariae 3-15 part, Rhizoma Coptidis 1-12 part, Radix Paeoniae Alba 3-15 part, Fructus Mume 5-20 part.

3. application according to claim 1 and 2, it is characterized in that, middle Fructus Crataegi can also be added in pharmaceutical composition prescription, the formula obtained is: Radix Trichosanthis 10-30 part, Radix Bupleuri 10-30 part, Fructus Aurantii Immaturus 3-15 part, Radix Et Rhizoma Rhei 1-6 part, Rhizoma Pinelliae 1-12 part, Radix Scutellariae 3-15 part, Rhizoma Coptidis 1-12 part, Radix Paeoniae Alba 3-15 part, Fructus Mume 5-20 part, Fructus Crataegi 3-15 part.

4. application according to claim 1 and 2, it is characterized in that, middle Fructus Crataegi can also be added in pharmaceutical composition prescription, Rhizoma Polygonati, Radix Ginseng and Fructus Momordicae charantiae, the formula obtained is: Radix Trichosanthis 10-30 part, Radix Bupleuri 10-30 part, Fructus Aurantii Immaturus 3-15 part, Radix Et Rhizoma Rhei, 1-6 part, Rhizoma Pinelliae 1-12 part, Radix Scutellariae 3-15 part, Rhizoma Coptidis 1-12 part, Radix Paeoniae Alba 3-15 part, Fructus Mume 5-20 part, Fructus Crataegi 3-15 part, Rhizoma Polygonati 3-15 part, Radix Ginseng 3-15 part, Fructus Momordicae charantiae 3-15 part.

5. application according to claim 1 and 2, is characterized in that, pharmaceutical composition prescription is: Radix Trichosanthis 9 parts, Radix Bupleuri 12 parts, Fructus Aurantii Immaturus 9 parts, Radix Et Rhizoma Rhei 3 parts, the Rhizoma Pinelliae 6 parts, Radix Scutellariae 9 parts, Rhizoma Coptidis 6 parts, the Radix Paeoniae Alba 9 parts, Fructus Mume 9 parts.

6. application according to claim 3, is characterized in that, pharmaceutical composition prescription is: Radix Trichosanthis 30 parts, Radix Bupleuri 12 parts, Fructus Aurantii Immaturus 9 parts, Radix Et Rhizoma Rhei 3 parts, the Rhizoma Pinelliae 6 parts, Radix Scutellariae 9 parts, Rhizoma Coptidis 6 parts, the Radix Paeoniae Alba 9 parts, Fructus Mume 15 parts, Fructus Crataegi 9 parts.

7. application according to claim 4, it is characterized in that, pharmaceutical composition prescription is: Radix Trichosanthis 30 parts, Radix Bupleuri 12 parts, Fructus Aurantii Immaturus 9 parts, Radix Et Rhizoma Rhei 3 parts, the Rhizoma Pinelliae 6 parts, Radix Scutellariae 9 parts, Rhizoma Coptidis 6 parts, the Radix Paeoniae Alba 9 parts, Fructus Mume 15 parts, Fructus Crataegi 9 parts, Rhizoma Polygonati 9 parts, Radix Ginseng 9 parts, 9 parts, Fructus Momordicae charantiae.

8. application according to claim 1 and 2, is characterized in that, described pharmaceutical composition by lowering SREBP1-c albumen and developed by molecule, thus improves type 2 diabetes mellitus people lipid metabolism level.

9. the application according to claim 1 or 2 any one, is characterized in that, described pharmaceutical composition, by lowering SREBP1-c albumen and developed by molecule, is treated and prevents type 2 diabetes mellitus and complication thereof.

10. application according to claim 9, is characterized in that, described type 2 diabetes mellitus is the type 2 diabetes mellitus that the type 2 diabetes mellitus that causes of Fatty toxicity or sugared toxicity cause.