CN104208210A - Traditional Chinese medicine composition for treating insulin resistance and related diseases, as well as preparation method and traditional Chinese medicine preparation thereof - Google Patents
Traditional Chinese medicine composition for treating insulin resistance and related diseases, as well as preparation method and traditional Chinese medicine preparation thereof Download PDFInfo
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Abstract
The invention discloses a traditional Chinese medicine composition for treating insulin resistance and related diseases. The traditional Chinese medicine composition is mainly prepared from the following traditional Chinese medicinal raw materials in parts by weight: 100-300 parts of hawthorn, 90-300 parts of radix polygonum multiflorum preparata, 120-360 parts of radix paeoniae alba and 100-500 parts of salvia miltiorrhiza. Correspondingly, the invention further provides a method for preparing the traditional Chinese medicine composition and a traditional Chinese medicine preparation of the traditional Chinese medicine composition. The traditional Chinese medicine composition is capable of simultaneously treating insulin resistance and related diseases to play a role in treating multiple diseases by one medicine, and has small toxic and side effect and low price.
Description
Technical field
The present invention relates to field of medicaments, particularly a kind ofly treat Chinese medicine composition of insulin resistant and relevant disease thereof and preparation method thereof, and the Chinese medicine preparation containing above-mentioned Chinese medicine composition.
Background technology
Insulin resistant (insulin resistance; IR) refer to that a variety of causes makes the target tissue of insulin action (being mainly liver, fat, skeletal muscle, vascular endothelial cell etc.) promote that its picked-up and the efficiency utilized and effect reduce to insulin, in early days, beta Cell of islet is the insulin (i.e. hyperinsulinemia) making blood glucose still maintain normal and compensatory supersecretion.
Insulin resistant is prevalent in multiple physiology and pathological state, in adolescence, geratic period, trimester of pregnancy, all can have insulin sensitivity index reduction in various degree and IR.Only have 15%-20% to there is IR in normal population, but body weight increase, blood glucose increases, blood pressure increases, blood fat disorder, hyperuricemia time, incidence rate and the degree of IR all significantly increase.
Insulin resistant is the basic feature of type 2 diabetes mellitus, is the key link of metabolism syndrome, is also the pathologic basis of the glutinous state of obesity, blood fat disorder, vascular endothelial dysfunction, cardiovascular disease and blood height.
The insulin of normal level has in vivo and promotes glycogen, fat and protein synthesis, promotes that glucose utilization and energy generate, suppresses steatolysis, strengthens cell proliferation and differentiation, maintenance inner skin cell function, suppression lipase thus many-sided effects such as lipotropism solution.
There is insulin resistant, the biological action of insulin weakens, make tissue to the biologically of a certain amount of insulin lower than expectation normal level, finally cause the sugar of muscle and fat to utilize to reduce, liver glyconeogenesis increases, adipose cell release free fatty increases, thus occurs blood glucose, dyslipidemia; The insulin long duration of action of high concentration is in vascular endothelial cell, vasodilatory material can be caused to reduce, pressor material such as the synthesis secretions such as Endothelin (ET) increase, thus there are dysarteriotony (Chen Linxi, Qin Xuping, Huang Qiulin etc., vascular endothelial cell pharmacology and clinical, People's Medical Officer Press, 2012, P164-166.); High-caliber insulin makes the inhibitor synthesis of plasma tissue type activator of plasminogen and release increase, suppress fibrin degradation, thus promote thrombosis (Yang Sheng, Xu Xiaoguang, Yue Guiying etc., propylene glycol alginate sodium sulfate on the impact of type 2 diabetes mellitus patients with lipid lectin from hemolymph and insulin resistant, Chinese practical internal medicine journal, 2004,24 (3): 183.); Long-term blood high insulin levels can cause blood viscosity to raise, and (dragon is for army building, Zhao Aiting, Luo Bingquan etc., the comprehensive study of Hypertensive Disease Insulin Resistance and microcirculation disturbance relation, China's hemorheology magazine, 2001,11 (2): 111-114.).
Occur that the abnormal expression of insulin resistant and related gene is closely related.After insulin and its receptors bind, signal is to intracellular transduction and amplify step by step, makes insulin play Metabolism regulation effect.IRS-1 (IRS-1) and Insulin receptor substrate-2 (IRS-2) play an important role in insulin signal transduction process, when the sudden change of IRS-1 and IRS-2 producer or abnormal expression, insulin signaling can be caused to be obstructed at endocellular transduction and to cause insulin resistant; GLUT4 (GLUT4) is present in muscle and adipose cell, IRS-1 phosphorylation under insulin action, makes GLUT4 insert to cell serous coat, accelerates the facilitory transport process of glucose, muscle is increased glucose uptake.GLUT4 abnormal expression or transposition are obstructed, and can cause insulin resistant after receptor; The quantity of glucose transporter 2 (GLUT2) reduces or active reduction, can reduce hepatic glucose picked-up, increase output (the insulin resistant) (Wang Jiyao of hepatic glycogen, internal medicine, People's Health Publisher, 2008, P974-975).Peroxisome proliferator-activated receptors γ (PPAR-γ) is mainly expressed in fatty tissue, regulates the Differentiation and proliferation of adipose cell.The activation energy of PPAR-γ suppresses fatty tissue secreting tumor necrosis factor α (TNF-α), blocks TNF-α inhibitory action to Insulin receptor INSR and IRS phosphorylation in signal transduction pathway, is conducive to improving insulin resistant.Beta 3 adrenoreceptor (β 3AR) mainly regulates steatolysis and the exothermal process of body, and the β 3AR of low expression can cause fatty tissue heat production to reduce, and causes lipopexia.Lipid is at Intracellular retention, interior fat is caused to increase, cause Abdominal obesity, cause insulin resistant (An Yali, Guangwei LI etc., beta 3 adrenoreceptor gene pleiomorphism and insulin resistant, obesity, China-Japan Friendship Hospital's journal, 2004,18 (6): 366-368.).
Current clinical treatment insulin resistant mainly applies euglycemic agent.Metformin recovers insulin to the suppression of adenyl cyclase by liver plasma membrane G-protein, reduce hepatic gluconeogenesis and output, promote anaerobic glycolysis, increase the peripheral tissues such as muscle to the picked-up of glucose and utilization, suppression or delay glucose improve carbohydrate metabolism in gastrointestinal absorption etc.But take metformin and may occur that many side effect are as gastrointestinal reaction, anaphylaxis etc., severe patient may bring out lactic acidosis; To serum insulin levels significantly rising person, have ketoacidosis tendency person, severe depletion of oxygen, heart failure, severe liver disease, kidney patient to be not suitable for using metformin.Thiazolidinediones can increase the sensitivity of insulin in peripheral tissues, alleviates insulin resistant, is a kind of euglycemic agent.Thiazolidinediones is not suitable for that insulin definitely lacks, ketoacidosis, serious and acute heart failure patient; Common adverse effect has headache, dizziness, weak, nauseating, diarrhoea, gently anemia, edema, body weight increase, hypercholesterolemia etc., and some patients may occur hepatic injury.
And, clinical in the lysis for the treatment of centered by insulin resistant at present, because the generation of insulin resistant is simultaneously with multiple relevant disease (as hyperinsulinemia, diabetes, hypertension, hyperlipidemia, metabolism syndrome, hyperuricemia, blood viscosity increase), need to take depressor, antidiabetic drug, lipid lowerers, antithrombotic simultaneously, even use insulin etc., take medicine of a great variety, dosage is large, and majority is Western medicine, toxic and side effects is more, especially gastrointestinal reaction and hepatic and renal function injure.In addition, existing medicine needs long-term taking, and medical expense is expensive.
Summary of the invention
Technical problem to be solved by this invention is, provides the Chinese medicine composition that a kind of toxic and side effects is little, price is low, can treat insulin resistant and relevant disease thereof simultaneously, plays the many sick effects of a medicine treatment.
Technical problem to be solved by this invention is also, provides the method preparing above-mentioned Chinese medicine composition.
Technical problem to be solved by this invention is also, provides the Chinese medicine preparation containing above-mentioned Chinese medicine composition.
For reaching above-mentioned technique effect, the invention provides a kind of Chinese medicine composition for the treatment of insulin resistant and relevant disease thereof, described Chinese medicine composition is made primarily of the raw material of Chinese medicine of following weight proportion:
Fructus Crataegi 100-300,
Radix Polygoni Multiflori Preparata 90-300,
Radix Paeoniae Alba 120-360,
Radix Salviae Miltiorrhizae 100-500.
As the improvement of such scheme, described Chinese medicine composition is made primarily of the raw material of Chinese medicine of following weight proportion:
Fructus Crataegi 180-240,
Radix Polygoni Multiflori Preparata 180-240,
Radix Paeoniae Alba 120-300,
Radix Salviae Miltiorrhizae 120-360.
Correspondingly, present invention also offers a kind of preparation method for the treatment of the Chinese medicine composition of insulin resistant and relevant disease thereof, comprising:
A, get Fructus Crataegi, Radix Polygoni Multiflori Preparata, the Radix Paeoniae Alba, Radix Salviae Miltiorrhizae by formula, pulverized;
B, carry out extracting and concentrating with methanol, ethanol or acetone, obtain the first extracting solution;
C, the medicinal residues water extraction after extracting is concentrated, obtain the second extracting solution;
D, by the first extracting solution, second extracting solution merge.
As the improvement of such scheme, described step B comprises:
Add 6-10 times amount, methanol, ethanol or acetone that concentration is 60-80% soaks 20-40min;
Reflux, extract, 1-3 time, each 1-3h;
Merge extractive liquid, concentrating under reduced pressure recycling design, obtain the first extracting solution.
As the improvement of such scheme, described step B comprises:
Add 8 times amount, concentration be 70% methanol, ethanol or acetone soak 30min;
Reflux, extract, 2 times, each 2h;
Merge extractive liquid, concentrating under reduced pressure recycling design, obtain the first extracting solution.
As the improvement of such scheme, described step C comprises:
By the medicinal residues 6-10 times amount water soaking 20-40min after extraction;
Water extraction 1-3 time, first time 45-75min, second time 30-60min;
Merge extractive liquid, concentrating under reduced pressure, obtain the second extracting solution.
As the improvement of such scheme, described step C comprises:
By the medicinal residues 8 times amount water soaking 30min after extraction;
Water extraction 2 times, first time 60min, second time 45min;
Merge extractive liquid, concentrating under reduced pressure, obtain the second extracting solution.
Correspondingly, present invention also offers the preparation method of the Chinese medicine composition of another kind for the treatment of insulin resistant and relevant disease thereof, comprising:
A, take Fructus Crataegi, Radix Polygoni Multiflori Preparata, the Radix Paeoniae Alba, Radix Salviae Miltiorrhizae by formula;
B, adding water did not have medical material, soaked 20-40min;
C, be heated to water and open, little fire continues to decoct 10-30min, filters, obtains the first medicinal liquid;
D, continuing to add water did not have medical material, and water is opened rear little fire and continued to decoct 10-30min, filters, obtains the second medicinal liquid;
E, merge the first medicinal liquid, the second medicinal liquid.
Correspondingly, the present invention also provides a kind of Chinese medicine preparation for the treatment of insulin resistant and relevant disease thereof, and described Chinese medicine preparation contains above-mentioned any one Chinese medicine composition.
As the improvement of such scheme, described Chinese medicine preparation is decoction, powder, capsule, tablet, granule or pill.
Implement the present invention and there is following beneficial effect:
The composition of Chinese medicine composition of the present invention is the active component of the medicines such as Fructus Crataegi, Radix Polygoni Multiflori Preparata, the Radix Paeoniae Alba, Radix Salviae Miltiorrhizae, by finding the drug effect of said composition and study on mechanism, it can treat insulin resistant and relevant disease thereof simultaneously, play the many sick effects of a medicine treatment, and toxic and side effects is little, price is low, specific as follows:
One, compositions drug action of the present invention is remarkable, has good therapeutical effect to insulin resistant and relevant disease thereof.Mechanism of action of the present invention is clear and definite, and research finds that said composition targeting is in the related pathways of glucose-lipid metabolism, by the expression of regulation and control IRS IRS-1, IRS-2 gene, improves body to the sensitivity of insulin; By the expression of upregulation of glucose transport Protein G LUT2, GLUT4mRNA, promote that body is to the picked-up of glucose, increase body to the consumption of glucose; By raising the expression of PPAR-γ, β 3AR mRNA, promote the catabolism of adipose cell; The effect of control insulin resistant is played from gene level.Meanwhile, said composition also has improves hemorheology, reduces endothelin-1 (ET-1) level, plays the effect preventing and treating thrombosis and protection vascular endothelial function.Said composition plays the effect for the treatment of insulin resistant and relevant disease thereof by many aspects.
Two, the present invention is to improve centered by insulin resistant, by regulating the signal path relevant to insulin resistant, to treat insulin resistant for starting point, and then play the treatment hyperinsulinemia relevant to insulin resistant, the various diseases such as diabetes, hypertension, hyperlipidemia, metabolism syndrome, hyperuricemia, blood viscosity increase, reach the effect of " the many diseases of a medicine ", improve patient compliance, alleviate economy and the physiological load of patient.
Three, the present invention is Chinese medicine preparation, and medicine composition is simple, and dose is little, and has obvious therapeutical effect through effect experiment checking to insulin resistant and relevant disease thereof, and toxic and side effects is little.
Four, the present invention is the extract of natural plant crude drugs, and crude drug is cheap, wide material sources, and extraction process is simple, can realize large-scale industrialization application.
Accompanying drawing explanation
Fig. 1 is the flow chart of preparation method one embodiment of Chinese medicine composition of the present invention;
Fig. 2 is the flow chart of another embodiment of preparation method of Chinese medicine composition of the present invention.
Detailed description of the invention
For making the object, technical solutions and advantages of the present invention clearly, below in conjunction with accompanying drawing, the present invention is described in further detail.
The invention provides a kind of Chinese medicine composition for the treatment of insulin resistant and relevant disease thereof, described Chinese medicine composition is made primarily of the raw material of Chinese medicine of following weight proportion:
Fructus Crataegi 100-300,
Radix Polygoni Multiflori Preparata 90-300,
Radix Paeoniae Alba 120-360,
Radix Salviae Miltiorrhizae 100-500.
Wherein, insulin resistant relevant disease of the present invention, refers to the relevant disease caused by insulin resistant, as hyperinsulinemia, diabetes, hypertension, hyperlipidemia, metabolism syndrome, hyperuricemia, blood viscosity increase.
Preferably, described Chinese medicine composition is made primarily of the raw material of Chinese medicine of following weight proportion:
Fructus Crataegi 180-240,
Radix Polygoni Multiflori Preparata 180-240,
Radix Paeoniae Alba 120-300,
Radix Salviae Miltiorrhizae 120-360.
Fructus Crataegi, warm in nature, sour in the mouth, sweet, return spleen, stomach, Liver Channel, function invigorating the stomach and promoting digestionization is stagnant, promoting flow of QI and blood dissipating blood stasis.Modern pharmacological research finds, Fructus Crataegi mainly containing flavones ingredient, comprises hyperin, Quercetin, flavone polymer; Organic acid, comprises citric acid, Crataegolic acid etc., also containing lactone, saccharide and glycoside etc.Modern pharmacological research finds that Fructus Crataegi has and promotes that fat digestion, cardioprotection, blood pressure lowering, blood fat reducing, antioxidation improve the effects such as immunity, antibacterial, antitumor.
Radix Polygoni Multiflori Preparata, bitter in the mouth, sweet, puckery, warm in nature, return liver, the heart, kidney channel.Function invigorating the liver and kidney, benefiting essence-blood, black beard and hair, bone and muscle strengthening.Modern pharmacological research finds that the main component of Radix Polygoni Multiflori has phospholipid, Anthraquinones, glucose glycoside, gallic acid and various trace elements.Effects such as there is endocrine regulation function, improve immunity of organisms, blood fat reducing, atherosclerosis, antioxidation protect the liver.
Radix Paeoniae Alba bitter in the mouth, acid, cold nature.Return liver, spleen channel.The pain relieving of function suppressing the hyperactive liver, nourishing blood for regulating menstruation, astringing YIN to stop sweating.Main component has peoniflorin, paeonol, flavone, monoterpene, triterpene and glycosides compound thereof.The effects such as modern pharmacological research finds to have protecting the liver, ease pain, antiinflammatory, immunity moderation power, calmness, anti-bacteria and anti-virus.
Radix Salviae Miltiorrhizae, bitter in the mouth, cold nature, GUIXIN, Liver Channel.Function stasis-dispelling and pain-killing, promoting blood circulation to restore menstrual flow, clear away heart-fire relieving restlessness.Main pharmacological components has Radix Salviae Miltiorrhizae acid, salvianolic acid, Tanshinone I, tanshinone ⅡA, cryptotanshinone I, iso tanshinone I etc. more than 40 to plant modern pharmacology research to find the effects such as Radix Salviae Miltiorrhizae has antithrombotic, changes hemorheological property, resists myocardial ischemia, antibacterial, antiinflammatory antitumor.
In clinical practice, the dosage of Chinese medicine composition of the present invention is 0.4-3gkg
-1d
-1.Preferably, the dosage of Chinese medicine composition is 0.6-2gkg
-1d
-1.
It should be noted that, raw material of Chinese medicine of the present invention can be had the Chinese medicine of same or similar function substitute, and these medical materials all can be concocted according to method in " Chinese Pharmacopoeia " or " Chinese medicine voluminous dictionary ".
Correspondingly, see Fig. 1, present invention also offers a kind of preparation method for the treatment of the Chinese medicine composition of insulin resistant and relevant disease thereof, comprising:
S101, get Fructus Crataegi, Radix Polygoni Multiflori Preparata, the Radix Paeoniae Alba, Radix Salviae Miltiorrhizae by formula, pulverized;
The Chinese medicine composition of Fructus Crataegi, Radix Polygoni Multiflori Preparata, the Radix Paeoniae Alba, Radix Salviae Miltiorrhizae composition is as follows with the formula of parts by weight:
Fructus Crataegi 100-300 part,
Radix Polygoni Multiflori Preparata 90-300 part,
Radix Paeoniae Alba 120-360 part,
Radix Salviae Miltiorrhizae 100-500 part.
Preferably, the Chinese medicine composition of Fructus Crataegi, Radix Polygoni Multiflori Preparata, the Radix Paeoniae Alba, Radix Salviae Miltiorrhizae composition is as follows with the formula of parts by weight:
Fructus Crataegi 180-240 part,
Radix Polygoni Multiflori Preparata 180-240 part,
Radix Paeoniae Alba 120-300 part,
Radix Salviae Miltiorrhizae 120-360 part.
S102, carry out extracting and concentrating with methanol, ethanol or acetone, obtain the first extracting solution;
As step S102 mono-preferred embodiment, comprising:
Add 6-10 times amount, methanol, ethanol or acetone that concentration is 60-80% soaks 20-40min;
Reflux, extract, 1-3 time, each 1-3h;
Merge extractive liquid, concentrating under reduced pressure recycling design, obtain the first extracting solution.
As the embodiment that step S102 is better, comprising:
Add 8 times amount, concentration be 70% methanol, ethanol or acetone soak 30min;
Reflux, extract, 2 times, each 2h;
Merge extractive liquid, concentrating under reduced pressure recycling design, obtain the first extracting solution.
As the embodiment of step S102 the best, comprising:
Add 8 times amount, concentration is the soak with ethanol 30min of 70%;
Reflux, extract, 2 times, each 2h;
Merge extractive liquid, concentrating under reduced pressure recycling design, obtain the first extracting solution.
S103, the medicinal residues water extraction after extracting is concentrated, obtain the second extracting solution;
As step S103 mono-preferred embodiment, comprising:
By the medicinal residues 6-10 times amount water soaking 20-40min after extraction;
Water extraction 1-3 time, first time 45-75min, second time 30-60min;
Merge extractive liquid, concentrating under reduced pressure, obtain the second extracting solution.
As the embodiment that step S103 is better, comprising:
By the medicinal residues 8 times amount water soaking 30min after extraction;
Water extraction 2 times, first time 60min, second time 45min;
Merge extractive liquid, concentrating under reduced pressure, obtain the second extracting solution.
S104, by the first extracting solution, second extracting solution merge.
Preferably, the first extracting solution, the second extracting solution merging are concentrated into extractum.
The present invention is the extract of natural plant crude drugs, and crude drug is cheap, wide material sources, and extraction process is simple, can realize large-scale industrialization application.
It should be noted that, the methanol described in the preparation method shown in Fig. 1, ethanol or acetone, refer to methanol, ethanol, acetone or their aqueous solution.
Correspondingly, see Fig. 2, present invention also offers the preparation method of the Chinese medicine composition of another kind for the treatment of insulin resistant and relevant disease thereof, comprising:
S201, take Fructus Crataegi, Radix Polygoni Multiflori Preparata, the Radix Paeoniae Alba, Radix Salviae Miltiorrhizae by formula.
The Chinese medicine composition of Fructus Crataegi, Radix Polygoni Multiflori Preparata, the Radix Paeoniae Alba, Radix Salviae Miltiorrhizae composition is as follows with the formula of parts by weight:
Fructus Crataegi 10-30 part,
Radix Polygoni Multiflori Preparata 9-30 part,
Radix Paeoniae Alba 12-36 part,
Radix Salviae Miltiorrhizae 10-50 part.
Preferably, the Chinese medicine composition of Fructus Crataegi, Radix Polygoni Multiflori Preparata, the Radix Paeoniae Alba, Radix Salviae Miltiorrhizae composition is as follows with the formula of parts by weight:
Fructus Crataegi 12-24 part,
Radix Polygoni Multiflori Preparata 12-24 part,
Radix Paeoniae Alba 18-30 part,
Radix Salviae Miltiorrhizae 15-36 part.
S202, adding water did not have medical material, soaked 20-40min;
Preferably, add water and do not have medical material, soak 30min.
S203, be heated to water and open, little fire continues to decoct 10-30min, filters, obtains the first medicinal liquid;
Preferably, be heated to water and open, little fire continues to decoct 15min, filters, obtains the first medicinal liquid.
S204, continuing to add water did not have medical material, and water is opened rear little fire and continued to decoct 10-30min, filters, obtains the second medicinal liquid;
Preferably, continuing to add water did not have medical material, and water is opened rear little fire and continued to decoct 15min, filters, obtains the second medicinal liquid.
S205, merge the first medicinal liquid, the second medicinal liquid.
Correspondingly, the present invention also provides a kind of Chinese medicine preparation for the treatment of insulin resistant and relevant disease thereof, and described Chinese medicine preparation contains the Chinese medicine composition of above-mentioned any embodiment.
Described Chinese medicine preparation is decoction, powder, capsule, tablet, granule or pill.
For making above-mentioned dosage form be achieved, operable adjuvant in pharmaceutical preparation need be added in preparation process: as filler, disintegrating agent, lubricant, suspending agent, binding agent, sweeting agent, correctives, antiseptic etc.Filler comprises: starch, lactose, mannitol, microcrystalline Cellulose, sucrose, xylitol etc.; Disintegrating agent comprises: starch, microcrystalline Cellulose, carboxymethyl starch sodium, low-substituted hydroxypropyl cellulose sodium etc.; Lubricant comprises: Pulvis Talci, silicon dioxide, sodium lauryl sulphate, magnesium stearate etc.; Suspending agent comprises: polyvinylpyrrolidone, microcrystalline Cellulose, agar, hydroxy methocel etc.; Binding agent comprises: starch slurry, polyvinylpyrrolidone, hydroxy methocel etc.; Sweeting agent comprises: saccharin sodium, aspartame, sucrose, cyclamate, enoxolone etc.; Correctives comprises: sweeting agent and various essence; Antiseptic comprises: parabens, benzoic acid, sodium benzoate, benzalkonium bromide, chlorhexidine acetate, eucalyptus oil, sorbic acid and its esters etc.
It should be noted that, the Chinese medicine composition contained by described Chinese medicine preparation, and the preparation method of this Chinese medicine composition is consistent with aforementioned, does not repeat them here.
The present invention is set forth further below with specific embodiment
Embodiment 1
The preparation of medicine capsule of the present invention
Formula: Fructus Crataegi 200g, Radix Polygoni Multiflori Preparata 180g, Radix Paeoniae Alba 240g, Radix Salviae Miltiorrhizae 300g
Preparation method: take Fructus Crataegi, Radix Polygoni Multiflori Preparata, the Radix Paeoniae Alba, Radix Salviae Miltiorrhizae, adds 8 times amount 70% soak with ethanol 30min, reflux, extract, 2 times, 2h/ time, and merge extractive liquid, is also concentrated; By medicinal residues 8 times amount water soaking 30min, water extraction twice, first time 1h, second time 45min, merge extractive liquid, merged by extracting solution, spraying dry, adds 1% magnesium stearate, incapsulates and makes 1000 capsules.
Embodiment 2
The preparation of medicinal tablet of the present invention
Formula: Fructus Crataegi 180g, Radix Polygoni Multiflori Preparata 240g, Radix Paeoniae Alba 300g, Radix Salviae Miltiorrhizae 240
Preparation method: take Fructus Crataegi, Radix Polygoni Multiflori Preparata, the Radix Paeoniae Alba, Radix Salviae Miltiorrhizae, adds 8 times amount 70% soak with ethanol 30min, reflux, extract, 2 times, 2h/ time, and merge extractive liquid, is also concentrated; By medicinal residues 8 times amount water soaking 30min, water extraction twice, first time 1h, second time 45min, merge extractive liquid, merged by extracting solution, spraying dry, adds 1% magnesium stearate, and 1000 tablets of tablets made by tabletting.
Embodiment 3
The preparation of medicinal granule of the present invention
Formula: Fructus Crataegi 180g, Radix Polygoni Multiflori Preparata 300g, Radix Paeoniae Alba 360g, Radix Salviae Miltiorrhizae 120
Preparation method: take Fructus Crataegi, Radix Polygoni Multiflori Preparata, the Radix Paeoniae Alba, Radix Salviae Miltiorrhizae, adds 8 times amount 70% soak with ethanol 30min, reflux, extract, 2 times, 2h/ time, and merge extractive liquid, is also concentrated; By medicinal residues 8 times amount water soaking 30min, water extraction twice, first time 1h, second time 45min, merge extractive liquid, merged by extracting solution, spraying dry, adds soluble starch, makes granule.
Embodiment 4
The preparation of medicine concentrated pill of the present invention
Formula: Fructus Crataegi 240g, Radix Polygoni Multiflori Preparata 240g, Radix Paeoniae Alba 180g, Radix Salviae Miltiorrhizae 360g
Preparation method: take Fructus Crataegi, Radix Polygoni Multiflori Preparata, the Radix Paeoniae Alba, Radix Salviae Miltiorrhizae, adds 8 times amount 70% soak with ethanol 30min, reflux, extract, 2 times, 2h/ time, and merge extractive liquid, is also concentrated; By medicinal residues 8 times amount water soaking 30min, water extraction twice, first time 1h, second time 45min, merge extractive liquid, merges extracting solution, is concentrated into extractum, adds dextrin, make concentrated pill.
Embodiment 5
The preparation of decoction of the present invention
Formula: Fructus Crataegi 12g, Radix Polygoni Multiflori Preparata 12g, Radix Paeoniae Alba 18g, Radix Salviae Miltiorrhizae 15g
Preparation method: take above-mentioned medical material, add water and there be not medical material, soaks 30min, is heated to water and opens, and little fire continues to decoct 15min, and filter, continuing to add water did not have medical material, and water is opened rear little fire and continued to decoct 15min, merges medicinal liquid and get final product.
Embodiment 6
The preparation of medicine capsule of the present invention
Formula: Fructus Crataegi 100g, Radix Polygoni Multiflori Preparata 90g, Radix Paeoniae Alba 120g, Radix Salviae Miltiorrhizae 100g
Preparation method: take Fructus Crataegi, Radix Polygoni Multiflori Preparata, the Radix Paeoniae Alba, Radix Salviae Miltiorrhizae, adds 7 times amount 60% methanol and soaks 35min, reflux, extract, 2 times, 2h/ time, and merge extractive liquid, is also concentrated; By medicinal residues 7 times amount water soaking 25min, water extraction twice, first time 1h, second time 45min, merge extractive liquid, merged by extracting solution, spraying dry, adds 1% magnesium stearate, incapsulates and makes 1000 capsules.
Embodiment 7
The preparation of medicinal tablet of the present invention
Formula: Fructus Crataegi 260g, Radix Polygoni Multiflori Preparata 200g, Radix Paeoniae Alba 200g, Radix Salviae Miltiorrhizae 280
Preparation method: take Fructus Crataegi, Radix Polygoni Multiflori Preparata, the Radix Paeoniae Alba, Radix Salviae Miltiorrhizae, adds 6 times amount 80% soak with ethanol 20min, reflux, extract, 2 times, 3h/ time, and merge extractive liquid, is also concentrated; By medicinal residues 6 times amount water soaking 20min, water extraction twice, first time 45min, second time 30min, merge extractive liquid, merged by extracting solution, spraying dry, adds 1% magnesium stearate, and 1000 tablets of tablets made by tabletting.
Embodiment 8
The preparation of medicinal granule of the present invention
Formula: Fructus Crataegi 300g, Radix Polygoni Multiflori Preparata 300g, Radix Paeoniae Alba 360g, Radix Salviae Miltiorrhizae 500
Preparation method: take Fructus Crataegi, Radix Polygoni Multiflori Preparata, the Radix Paeoniae Alba, Radix Salviae Miltiorrhizae, adds 10 times amount 60% acetone and soaks 10min, reflux, extract, 3 times, 3h/ time, and merge extractive liquid, is also concentrated; By medicinal residues 10 times amount water soaking 40min, water extraction 3 times, first time 75min, second time 60min, merge extractive liquid, merged by extracting solution, spraying dry, adds soluble starch, makes granule.
Embodiment 9
The preparation of medicine concentrated pill of the present invention
Formula: Fructus Crataegi 250g, Radix Polygoni Multiflori Preparata 220g, Radix Paeoniae Alba 200g, Radix Salviae Miltiorrhizae 320g
Preparation method: take Fructus Crataegi, Radix Polygoni Multiflori Preparata, the Radix Paeoniae Alba, Radix Salviae Miltiorrhizae, adds 8 times amount 70% soak with ethanol 30min, reflux, extract, 2 times, 2h/ time, and merge extractive liquid, is also concentrated; By medicinal residues 8 times amount water soaking 30min, water extraction twice, first time 1h, second time 45min, merge extractive liquid, merges extracting solution, is concentrated into extractum, adds dextrin, make concentrated pill.
Embodiment 10
The preparation of decoction of the present invention
Formula: Fructus Crataegi 13g, Radix Polygoni Multiflori Preparata 13g, Radix Paeoniae Alba 20g, Radix Salviae Miltiorrhizae 16g
Preparation method: take above-mentioned medical material, add water and there be not medical material, soaks 20min, is heated to water and opens, and little fire continues to decoct 10min, and filter, continuing to add water did not have medical material, and water is opened rear little fire and continued to decoct 10min, merges medicinal liquid and get final product.
Embodiment 11
The preparation of decoction of the present invention
Formula: Fructus Crataegi 15g, Radix Polygoni Multiflori Preparata 15g, Radix Paeoniae Alba 25g, Radix Salviae Miltiorrhizae 20g
Preparation method: take above-mentioned medical material, add water and there be not medical material, soaks 40min, is heated to water and opens, and little fire continues to decoct 20min, and filter, continuing to add water did not have medical material, and water is opened rear little fire and continued to decoct 20min, merges medicinal liquid and get final product.
Embodiment 12
The preparation of decoction of the present invention
Formula: Fructus Crataegi 20g, Radix Polygoni Multiflori Preparata 20g, Radix Paeoniae Alba 30g, Radix Salviae Miltiorrhizae 25g
Preparation method: take above-mentioned medical material, add water and there be not medical material, soaks 40min, is heated to water and opens, and little fire continues to decoct 20min, and filter, continuing to add water did not have medical material, and water is opened rear little fire and continued to decoct 20min, merges medicinal liquid and get final product.
Be further elaborated the present invention below in conjunction with experimental data, the present invention, and to be obtained by above-mentioned preparation method for crude drug with Fructus Crataegi, Radix Polygoni Multiflori Preparata, the Radix Paeoniae Alba, Radix Salviae Miltiorrhizae, hereinafter referred to as SHBD, its drug action and case study as follows:
I, SHBD Contained Serum is on the impact of HepG2 cell insulin resistant model
One, experiment material
Cell: HepG2 cell strain, Tumor Hospital Attached to Zhongshan Univ. grants.
Animal: SD male rat, SPF level, body weight 180 ~ 220g, purchased from Guangdong Medical Lab Animal Center, production licence number: SCXK (Guangdong) 0105817.
Medicine: Chinese medicine composition SHBD: Fructus Crataegi, Kangmei Pharmaceutical Co., Ltd's (product batch number: 120304861); Radix Polygoni Multiflori Preparata, Kangmei Pharmaceutical Co., Ltd's (product batch number: 111203551); The Radix Paeoniae Alba, Kangmei Pharmaceutical Co., Ltd (product batch number: 120205421), Radix Salviae Miltiorrhizae, Guangzhou Lingnan prepared slices of Chinese crude drugs company limited (product batch number: 091008)
Metformin hydrochloride tablet, Shanghai Shi Guibao pharmaceutical Co. Ltd of Sino-U.S. (product batch number: 1204101)
Simvastatin blade, Hangzhou Mo Shadong pharmaceutical Co. Ltd (product batch number: 110593).
Reagent: RPMI 1640 culture medium, Hyclone; Hyclone, GIBCO; The pancreatin of 0.25%, Hyclone; Insulin, Sigma; Glucose determination reagent box, Meikang Biotech Co., Ltd., Ningbo; G6Pase ELISA kit.
Instrument: carbon dioxide cell incubator, Thermo; Micro imaging system is inverted by IX71+DP72 type, Olympus; ST16R frozen type common bench centrifuge, Thermo; Electric-heated thermostatic water bath, Shanghai hundred allusion quotation instrument and equipment company limited; Microplate reader, Bio-Rad; The multi-functional automatic biochemistry analyzer of ultramicron, Trilogy; Intelligent constant-temperature digital display electric jacket, Henan Ai Bote development in science and technology company limited; Rotary Evaporators, IKA;
Two, experimental technique
(1) medicinal liquid extracts and concentrates
Medicinal liquid: obtain by the above-mentioned detailed description of the invention of the present invention, be settled to 0.864gml
-1.
Diformin tablet medicinal liquid: distilled water dissolves diformin tablet, and concentration is 20mgml
-1.
Simvastatin Tablets medicinal liquid: distilled water dissolves Simvastatin Tablets, and concentration is 1mgml
-1.
(2) preparation of Contained Serum
Male SD rat, 180-220g, Normal group is divided at random by body weight, simvastatin group, metformin group, SHBD group, give normal saline respectively, simvastatin, metformin and SHBD medicinal liquid, by every 100g body weight 1ml gavage, 2 administrations every day, successive administration 3 days, after last 1 administration 1.5h, anesthetized rat, abdominal aortic blood, leave standstill several minutes, centrifugal, get serum, each group of serum mixes respectively, 56 DEG C of deactivation 30min, through the filtration sterilization of 0.22 μm of syringe needle micropore filter, thus prepare each group of Contained Serum,-20 DEG C of Refrigerator stores, for subsequent use.
(3) HepG2 insulin resistant model is set up and pharmaceutical intervention process
By be in exponential phase HepG2 cell dissociation after, RPMI 1640 culture medium suspendible cell.After cultured cell to cell attachment, with containing 1 × 10
-8molL
-1the culture medium of insulin acts on HepG2 cell 36h and sets up HepG2 cell insulin resistant model.
Experiment is grouped into the large, medium and small dosage group of SHBD (Contained Serum dilution is 15%, 10%, 5%), metformin group (Contained Serum dilution is 15%), simvastatin group (Contained Serum dilution is 15%), normal group and model group.After HepG2 insulin resistant model modeling success, give different Contained Serums respectively and intervene 24h.(4)
3h-D-glucose participates in the impact of testing inspection SHBD Contained Serum on HepG2 cell tryptase insulin resistance Modeling glucose uptake ratio
After HepG2 insulin resistant model modeling success, give different Contained Serums respectively and intervene 24h.Add
3h-D-glucose (1.3 μ Ciml
-1in culture fluid) hatch 2h, add 4 DEG C of phosphate buffer quick wash cells with cessation reaction.Cell, with after 20%KOH 0.5ml cracking, is transferred in test tube.Get the cell pyrolysis liquid of 100 μ l, adopt Coomassie Brilliant Blue to measure protein concentration.4 DEG C of alcoholic solution wash 2 times.Room temperature 3000rmin
-1abandoning supernatant after centrifugal 5min, is inverted precipitate (glycogen) and is deposited on the filter paper of diameter 2cm (repeatedly rinsing), filter paper cold preservation in the ethanol agitator treating 4 times of 4 DEG C, 3h altogether.Filter paper is dried to be placed in 10ml scintillation solution and measures radiocounting.According to
3the specific activity of H-D-glucose, the protein concentration of cell pyrolysis liquid and the radioactivity of glycogen, calculate
3h-D-glucose participate in rate, unit is mmolgProh
-1.Formula is: the radiocounting (cpm) of the uptake ratio=glycogen of glucose/[
3protein concentration (the gl of specific activity (the Bq/mmol) × cell pyrolysis liquid of H-D-glucose
-1) × cell pyrolysis liquid volume (ml) × incubative time (h)]
(5) SHBD Contained Serum is on the impact of HepG2 cell tryptase insulin resistance Modeling glucose consumption
With containing 1 × 10
-8molL
-1the culture medium of insulin acts on HepG2 cell 36h and sets up HepG2 cell insulin resistant model, after model is set up, after Contained Serum process 24h, collect each group of cell culture medium supernatant, the multi-functional automatic biochemistry analyzer of ultramicron detects the glucose content of culture medium supernatant, and calculates the glucose utilization of each group of cell.
(6) SHBD Contained Serum is to the expression regulation of HepG2 cell insulin resistant model carbohydrate metabolism correlation factor IRS-1, IRS-2, GLUT2, GLUT4
After insulin resistant cell model modeling success, each Contained Serum effect 24h, discards culture medium.
(1) cell total RNA extracts
PBS cleans 2 times, every 10cm
2add the TRIZOL reagent of 1ml in order to cell lysis in the cultured cell of growth, be transferred in centrifuge tube by interior celliferous lysate, liquid-transfering gun is pressure-vaccum repeatedly, and room temperature leaves standstill 5min.Add chloroform, thermal agitation 15sec, after solution is fully emulsified, room temperature leaves standstill 5min, and rear 12,000g, 4 DEG C of centrifugal 15min, Aspirate supernatant is transferred in another new centrifuge tube.Isopyknic isopropyl alcohol is added in supernatant, after mixing of turning upside down, 15 ~ 30 DEG C of standing 10min.Rear 12,000g, 4 DEG C of centrifugal 10min.Careful supernatant discarded, adds the ethanol 1ml of 75% lentamente, turns upside down gently, carefully discard ethanol after 12,000g, 4 DEG C of centrifugal 5min along centrifugal tube wall.Drying at room temperature precipitation 2 ~ 5min, adds appropriate RNase-free water dissolution precipitation.The absorbance of UV spectrophotometer measuring RNA, calculates content and the purity of total serum IgE.
(2) reverse transcription reaction:
1. genomic DNA except dereaction
Room temperature 5min, 4 DEG C.
2. reverse transcription reaction (reactant liquor is formulated in and carries out on ice)
37℃15min，85℃5sec，4℃。
(3) Real Time PCR reacts
1. primer sequence
2. PCR reaction system
Reaction condition: Stage 1: denaturation, 95 DEG C, 30sec, 1Reps; Stage 2:PCR reacts, 95 DEG C, 5sec, 60 DEG C, 34sec, 40Reps.
After PCR reaction terminates, verified amplification efficiency and the primer specificity of primer by primer amplification curve and solubility curve, according to 2
-△ △ Ctcalculate the relative expression quantity of each gene, wherein, the Ct that △ Ct equals genes of interest group deducts the Ct of reference gene group, wherein, β-actin is reference gene, and the △ Ct that-△ △ Ct equals Contained Serum group deducts the △ Ct of matched group, with model group be contrast, then model group 2
-△ △ Ctvalue is 1.
(7) Contained Serum is to HepG2 cell insulin resistant model lipid metabolism correlation factor PPAR-γ and β
3the expression regulation of AR
Experimental procedure is with (six), and primer sequence is:
(9) statistical analysis
Experiment different batches cell repeats more than 3 times, carries out one factor analysis of variance, experimental data mean ± standard deviation
represent, with P < 0.05 for there being statistical significance.
Three, experimental result
1.SHBD Contained Serum is on the impact of HepG2 cell tryptase insulin resistance Modeling glucose uptake ratio
Insulin resistant cell model glucose uptake rate after drug effect
Compared with normal group, △ P<0.05; Compared with model group, * P<0.05.
Compared with normal group, model group glucose uptake rate significantly reduces (P<0.05); After pharmaceutical intervention, in the heavy dose of group of metformin group, SHBD and SHBD, dosage group glucose uptake rate significantly raises, and has significant difference (P<0.05) compared with model group.
2.SHBD Contained Serum is on the impact of HepG2 cell tryptase insulin resistance Modeling glucose consumption
Insulin resistant cell model glucose utilization after drug effect
Compared with normal group, △ P<0.05; Compared with model group, * P<0.05.
Compare with normal group, model group glucose utilization significantly reduces (P<0.05); After pharmaceutical intervention, metformin group, SHBD big or middle dosage group glucose utilization comparatively model group significantly increases (P<0.05).
The impact that 3.SHBD Contained Serum is expressed HepG2 cell tryptase insulin resistance fasting insulin receptor substrate IRS-1, IRS-2mRNA
Insulin resistant cell model IRS-1, IRS-2mRNA relative expression quantity after drug effect
Compared with normal group, △ P<0.05; Compared with model group, * P<0.05.
Compared with normal group, model group IRS-1, IRS-2mRNA relative expression quantity significantly reduce (P<0.05); After pharmaceutical intervention, compared with model group, in the heavy dose of group of metformin group, SHBD and SHBD, dosage group IRS-1, IRS-2mRNA relative expression quantity significantly raise (P<0.05); Simvastatin group IRS-2mRNA relative expression quantity also has remarkable rising (P<0.05) compared with model group, but comparatively SHBD heavy dose group significantly reduces (P<0.05).
The impact that 4.SHBD Contained Serum is expressed the transport protein of HepG2 cell tryptase insulin resistance Modeling glucose GLUT2, GLUT4mRNA
Insulin resistant cell model GLUT2, GLUT4mRNA relative expression quantity after drug effect
Compared with normal group, △ P<0.05; Compared with model group, * P<0.05.
Compared with normal group, model group GLUT2, GLUT4mRNA relative expression quantity significantly reduce (P<0.05); After pharmaceutical intervention, compared with model group, the big or middle dosage group GLUT2 of metformin group, SHBD, GLUT4mRNA relative expression quantity significantly raise (P<0.05).
Four, experiment conclusion
Experimental result shows, and SHBD Contained Serum can improve HepG2 cell tryptase insulin resistance Modeling glucose uptake ratio, increases insulin resistant model cell to the consumption of glucose; Meanwhile, SHBD Contained Serum can raise IRS IRS-1, IRS-2 and glucose transporter GLUT
2, GLUT
4the expression of mRNA.Result shows, SHBD side's Contained Serum is expressed by regulating IRS, increases cell to the sensitivity of insulin; Simultaneously by the expression of upregulation of glucose transport albumen, increase cell to the picked-up of glucose, thus play increase insulin resistant cell to the sensitivity of insulin, improve insulin resistant cell to the effect of glucose consumption.
II, SHBD side is on the impact of insulin resistant model rat
One, experiment material
Laboratory animal: SD male rat, SPF level, body weight 180 ~ 220g, 100, purchased from Guangdong Medical Lab Animal Center, production licence number: SCXK (Guangdong) 0105817.
The high sugared high salt emulsion formulations raw material of high fat: yolk powder, cholesterol, bovine bile, propylthiouracil, white sugar, Sal, Adeps Sus domestica.
Medicine: Chinese medicine composition SHBD: Fructus Crataegi, Kangmei Pharmaceutical Co., Ltd's (product batch number: 120304861); Radix Polygoni Multiflori Preparata, Kangmei Pharmaceutical Co., Ltd's (product batch number: 111203551); The Radix Paeoniae Alba, Kangmei Pharmaceutical Co., Ltd's (product batch number: 120205421); Radix Salviae Miltiorrhizae, Guangzhou Lingnan prepared slices of Chinese crude drugs company limited (product batch number: 091008)
Metformin hydrochloride tablet, Shanghai Shi Guibao pharmaceutical Co. Ltd of Sino-U.S. (product batch number: 1204101)
Simvastatin Tablets, Hangzhou Mo Shadong pharmaceutical Co. Ltd (product batch number: 110593).
Reagent: pyrophosphoric acid diethylester (DEPC), Sigma; Chloroform (analytical pure), Guangzhou Chemical Reagent Factory; Isopropyl alcohol (analytical pure), Guangzhou Chemical Reagent Factory; Ethanol (analytical pure), Guangzhou Chemical Reagent Factory; PCR primer, TAKARA; Trizol Plus, TAKARA, Lot:A8601-1; Prime Script RT reagent Kit with gDNA, TAKARA, Lot:BK1301; SYBR Premix Ex Taq II, TAKARA, Lot:BK1303.
Instrument: intelligent constant-temperature digital display electric jacket, Henan Ai Bote development in science and technology company limited; Rotary Evaporators, SENCO company; Electronic balance, Chinese mugwort Kohler Co; Biological functional system, Chengdu TME Technology Co., Ltd.; The multi-functional automatic biochemistry analyzer of ultramicron animal specific, Trilogy; Automatic blood rheological instrument, Pulisheng Instruments Co., Ltd., Beijing; Microscope, OLYMPUS.
Two, experimental technique
(1) foundation of Rat model of insulin-resistant and veterinary method
Healthy male SD rat 100, SPF level, body weight 180 ~ 220g, adaptability is raised 1 week, is only set to normal group by body weight random selecting 10, and remainder 90 is for modeling.Before modeling group rat, 4 week every day gave not containing the high sugared high salt Emulsion gavage 1ml/100g/d of high fat of propylthiouracil.From the 5th week, give the high fat containing propylthiouracil high sugared high salt Emulsion gavage 1ml/100g/d, continuous 2 weeks.Normal group Isodose distilled water gavage, normal water.All feeding rats normal diets, feedstuff and water are changed once every day.
The high sugared high salt Emulsion preparation of high fat: Adeps Sus domestica 20%, cholesterol 5%, yolk powder 6%, bovine bile 1%, white sugar 20%, salt 10%.Get 20g Adeps Sus domestica, 5g cholesterol, 6g yolk powder, 1g bovine bile, 20g white sugar, 10g salt puts into beaker, add 100ml distilled water, put into the heating of constant temperature electric heating cover and fully stir until dissolve completely, the high sugared high salt Emulsion of high fat, continuous gavage is after 4 weeks, 0.1% propylthiouracil is added in Emulsion, more continuous gavage 2 weeks.Emulsion 4 DEG C of stored refrigerated, use front 37 DEG C of heating in water bath to melt.
In modeling the 6th weekend evenings fasting 12 hours, chloral hydrate intraperitoneal anesthesia next day 10%, eye socket got blood 1ml/ only, 3000rpm, and centrifugal 15min gets serum, and multi-functional automatic biochemistry analyzer detects rat blood sugar, and adopts and put method of exempting from and detect insulin content.Calculate insulin sensitivity index (insulin sensitivity index, ISI).ISI=Ln (FBG × FINS)
-1(FBG: fasting glucose, FINS: Diagnostic Value of Fasting Serum insulin).
(2) animal grouping and administration
Choosing the successful rat of modeling and be divided into 6 groups at random, is model group, simvastatin group, metformin group, the large, medium and small dosage group of SHBD, together with normal group totally 7 groups respectively.
Model group and the high sugared high salt Emulsion 1ml/100g/d of each medication group gavage in morning height fat, each medication in afternoon group is gavage simvastatin, metformin and the large, medium and small dosage of SHBD medicinal liquid respectively, model group and normal group gavage normal saline, normal water.All feeding rats normal diets, feedstuff and water are changed once every day, continuous 6 weeks.
Prepared by medicinal liquid: obtain by the above-mentioned detailed description of the invention of the present invention, be settled to 0.864gml
-1, be the heavy dose of group of SHBD; Middle dosage group thin up is 0.432gml
-1; Small dose group thin up is 0.216gml
-1; By 100g body weight 1ml gastric infusion.
Simvastatin medicinal liquid: distilled water dissolves Simvastatin Tablets, and liquor strength is 0.5mgml
-1, by 5mgkg
-1d
-1dosed administration.
Metformin medicinal liquid: distilled water dissolves diformin tablet, and liquor strength is 10mgml
-1, by 100mgkg
-1d
-1dosed administration.
Animals administer dosage is according to " the pharmacological experimental methodology " (third edition, Xu Shuyun, Bian Rulian, Chen Xiu edits, People's Health Publisher, 2002) " between subordinate list 11-8 humans and animals, pressing the dose,equivalent ratio of body surface area conversion " (P1861), be converted into rat dose,equivalent according to the dosage of people:
Rat dose,equivalent=(every consumption per day × 0.018 of people)/0.2
(3) collection of specimens
Medication is after 6 weeks, and process the upper fasting evening before yesterday, the intraperitoneal anesthesia of m seq 10% chloral hydrate, abdominal aortic blood, separation of serum, detects serum blood glucose and insulin level; Get whole blood and blood plasma and detect hemorheology of rat and plasma ET.
Get rat liver, hind leg skeletal muscle and omental adipose tissue, put into centrifuge tube ,-80 DEG C of Refrigerator stores are for subsequent use.For detecting the expression of IRS-1, IRS-2, GLUT2, GLUT4mRNA in tissue, and and PPAR-γ, β
3the expression of AR mRNA.
(4) detection method
1. IRS-1, IRS-2, GLUT in liver organization
2, β
3gLUT in AR mrna expression, skeletal muscle
4the expression of PPAR-γ mRNA in mrna expression and omental adipose tissue
(1) extraction of liver total RNA:
Each group of rat liver, skeletal muscle and omental adipose tissue is taken out in-80 DEG C of refrigerators, PBS cleaning once, every 100mg liver adds the TRIZOL reagent of 1ml, and the centrifuge tube including rat tissue and lysate is put into the homogenate of Multifunctional biological sample homogenizer, room temperature leaves standstill 5min.4 DEG C, 12,000g, centrifugal 5min, gets supernatant, adds the chloroform of supernatant 1/5 volume, thermal agitation 15sec, and after solution is fully emulsified, room temperature leaves standstill 5min, and rear 12,000g, 4 DEG C of centrifugal 15min, Aspirate supernatant is transferred in another new centrifuge tube.Isopyknic isopropyl alcohol is added in supernatant, after mixing of turning upside down, 15 ~ 30 DEG C of standing 10min.Rear 12,000g, 4 DEG C of centrifugal 10min.Careful supernatant discarded, adds the ethanol 1ml of 75% lentamente, turns upside down gently, carefully discard ethanol after 12,000g4 DEG C of centrifugal 5min along centrifugal tube wall.Drying at room temperature precipitation 2 ~ 5min, adds appropriate RNase-free water dissolution precipitation.
(2) reverse transcription reaction:
1. genomic DNA except dereaction
Room temperature 5min, 4 DEG C.
2. reverse transcription reaction (reactant liquor is formulated in and carries out on ice)
37℃15min，85℃5sec，4℃。
(3) Real Time PCR reacts
1. primer sequence
2. PCR reaction system
Reaction condition: Stage 1: denaturation, 95 DEG C, 30sec, 1Reps; Stage 2:PCR reacts, 95 DEG C, 5sec, 60 DEG C, 34sec, 40Reps.
After PCR reaction terminates, verified amplification efficiency and the primer specificity of primer by primer amplification curve and solubility curve, according to 2
-△ △ Ctcalculate the relative expression quantity of each gene, wherein, the Ct that △ Ct equals genes of interest group deducts the Ct of reference gene group, and wherein, GAPDH is reference gene, and the △ Ct that-△ △ Ct equals medication group deducts the △ Ct of matched group, take model group as contrast, then model group 2
-△ △ Ctvalue is l.
Three, experimental result
1.SHBD side is on the impact of insulin resistant model rat blood sugar and serum insulin
Each experimental group rat fasting blood-glucose, serum insulin and insulin sensitivity index level
Compared with normal group, △ P<0.05; Compared with model group, * P<0.05.
Compared with normal group, model group rats fasting glucose (FBS) and serum insulin levels (Ins) significantly raise (P<0.05), insulin sensitivity index (ISI) reduces ((P<0.05)), and Rat model of insulin-resistant modeling success is described; After pharmaceutical intervention, compared with model group, metformin group and SHBD big or middle dosage group rat fasting blood-glucose (FBG) and serum insulin levels (Ins) significantly reduce (P<0.05), and insulin sensitivity index (ISI) raises; Compared with model group, simvastatin group, SHBD small dose group FBS, Ins, ISI there are no significant difference (P>0.05).
5.SHBD side is on the impact of insulin resistant model hemorheology of rat
Each experimental group hemorheology of rat level
Compared with normal group, △ P<0.05; Compared with model group, * P<0.05.
Compared with normal group, model group rats whole blood viscosity and plasma viscosity all significantly raise (P<0.05), illustrate that hemorheology of rat changes, and blood viscosity increases after the high sugared high salt Emulsion of high fat is fed; After pharmaceutical intervention, compared with model group, simvastatin group and SHBD big or middle dosage group whole blood viscosity and plasma viscosity all significantly reduce (P<0.05).
6.SHBD side is on the impact of insulin resistant model rat plasma Endothelin (ET-1)
Each experimental group rat plasma level of ET
Compared with normal group, △ P<0.05; Compared with model group, * P<0.05.
Compared with normal group, model group plasma ET significantly raises (P<0.05), and illustrate that insulin resistant causes vascular endothelial function impaired, endothelin level synthesis secretion increases; After pharmaceutical intervention, compared with model group, in the heavy dose of group of metformin group, simvastatin group, SHBD and SHBD, dosage group plasma ET significantly reduces (P<0.05).
4.SHBD side is on the impact of insulin resistant model rat liver IRS-1, IRS-2, GLUT2, β 3AR mrna expression level
Each experimental group rat liver IRS-1, IRS-2, GLUT2, β 3AR mRNA relative expression quantity
Compared with normal group, △ P<0.05; Compared with model group, * P<0.05.
Compare with normal group, model group rats liver IRS-1, IRS-2, GLUT2, β
3aR mRNA relative expression quantity all significantly reduces (P<0.05); After pharmaceutical intervention, compared with model group, metformin group and the big or middle dosage group IRS-1 of SHBD, IRS-2, GLUT2mRNA relative expression quantity significantly raise (P<0.05), and SHBD heavy dose group β 3AR mRNA relative expression quantity significantly raises (P<0.05).
5.SHBD side is to insulin resistant model rat skeletal muscle GLUT
4the impact of mrna expression level
Each experimental group rat skeletal muscle GLUT4mRNA relative expression quantity
Compared with normal group, △ P<0.05; Compared with model group, * P<0.05.
Compare with normal group, model group rats skeletal muscle GLUT4mRNA relative expression quantity significantly reduces (P<0.05); After pharmaceutical intervention, compared with model group, metformin group and SHBD big or middle dosage group GLUT4mRNA relative expression quantity significantly raise (P<0.05).
6.SHBD side is on the impact of insulin resistant model omental adipose tissue PPAR-γ mrna expression level
Each experimental group rat omental adipose tissue PPAR-γ mRNA relative expression quantity
Compared with normal group, △ P<0.05; Compared with model group, * P<0.05.
Compare with normal group, model group rats omental adipose tissue PPAR-γ mRNA relative expression quantity significantly reduces (P<0.05); After pharmaceutical intervention, compared with model group, simvastatin group and SHBD big or middle dosage group PPAR-γ mRNA relative expression quantity significantly raise (P<0.05).
Four, experiment conclusion
Experimental result shows, SHBD can significantly reduce insulin resistant model rat fasting blood-glucose and serum insulin levels, improves the insulin sensitivity index of rat model; Can the blood high viscosity state caused by insulin resistant be reduced, reduce rat model whole blood viscosity and plasma viscosity; Can the vascular endothelial injury caused by insulin resistant be reduced, reduce Plasma endothelin-1 levels.After being intervened by SHBD side, rat model regulates glycometabolic gene IRS-1, IRS-2, GLUT
2, GLUT
4mrna expression raises, and regulates lipometabolic β 3AR and PPAR-γ mrna expression to raise, the effectiveness of relevant disease demonstrating SHBD side's treatment insulin resistant further and cause.
In sum, the present invention is made up of Fructus Crataegi, Radix Polygoni Multiflori Preparata, the Radix Paeoniae Alba, Radix Salviae Miltiorrhizae, has following characteristics:
One, compositions drug action of the present invention is remarkable, has good therapeutical effect to insulin resistant and relevant disease thereof.Mechanism of action of the present invention is clear and definite, and research finds that said composition targeting is in the related pathways of glucose-lipid metabolism, by the expression of regulation and control IRS IRS-1, IRS-2 gene, improves body to the sensitivity of insulin; By the expression of upregulation of glucose transport Protein G LUT2, GLUT4mRNA, promote that body is to the picked-up of glucose, increase body to the consumption of glucose; By raising the expression of PPAR-γ, β 3AR mRNA, promote the catabolism of adipose cell; The effect of control insulin resistant is played from gene level.Meanwhile, said composition also has improves hemorheology, reduces endothelin-1 (ET-1) level, plays the effect preventing and treating thrombosis and protection vascular endothelial function.Said composition plays the effect for the treatment of insulin resistant and relevant disease thereof by many aspects.
Two, the present invention is to improve centered by insulin resistant, by regulating the signal path relevant to insulin resistant, to treat insulin resistant for starting point, and then play the treatment hyperinsulinemia relevant to insulin resistant, the various diseases such as diabetes, hypertension, hyperlipidemia, metabolism syndrome, hyperuricemia, blood viscosity increase, reach the effect of " the many diseases of a medicine ", improve patient compliance, alleviate economy and the physiological load of patient.
Three, the present invention is Chinese medicine preparation, and medicine composition is simple, and dose is little, and has obvious therapeutical effect through effect experiment checking to insulin resistant and relevant disease thereof, and toxic and side effects is little.
Four, the present invention is the extract of natural plant crude drugs, and crude drug is cheap, wide material sources, and extraction process is simple.
The above is the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention; can also make some improvements and modifications, these improvements and modifications are also considered as protection scope of the present invention.
Claims (10)
1. treat a Chinese medicine composition for insulin resistant and relevant disease thereof, it is characterized in that, described Chinese medicine composition is made primarily of the raw material of Chinese medicine of following weight proportion:
Fructus Crataegi 100-300,
Radix Polygoni Multiflori Preparata 90-300,
Radix Paeoniae Alba 120-360,
Radix Salviae Miltiorrhizae 100-500.
2. Chinese medicine composition as claimed in claim 1, it is characterized in that, described Chinese medicine composition is made primarily of the raw material of Chinese medicine of following weight proportion:
Fructus Crataegi 180-240,
Radix Polygoni Multiflori Preparata 180-240,
Radix Paeoniae Alba 120-300,
Radix Salviae Miltiorrhizae 120-360.
3. prepare a method for the Chinese medicine composition as described in any one of claim 1-2, it is characterized in that, comprising:
A, get Fructus Crataegi, Radix Polygoni Multiflori Preparata, the Radix Paeoniae Alba, Radix Salviae Miltiorrhizae by formula, pulverized;
B, carry out extracting and concentrating with methanol, ethanol or acetone, obtain the first extracting solution;
C, the medicinal residues water extraction after extracting is concentrated, obtain the second extracting solution;
D, by the first extracting solution, second extracting solution merge.
4. preparation method as claimed in claim 3, it is characterized in that, described step B comprises:
Add 6-10 times amount, methanol, ethanol or acetone that concentration is 60-80% soaks 20-40min;
Reflux, extract, 1-3 time, each 1-3h;
Merge extractive liquid, concentrating under reduced pressure recycling design, obtain the first extracting solution.
5. preparation method as claimed in claim 4, it is characterized in that, described step B comprises:
Add 8 times amount, concentration be 70% methanol, ethanol or acetone soak 30min;
Reflux, extract, 2 times, each 2h;
Merge extractive liquid, concentrating under reduced pressure recycling design, obtain the first extracting solution.
6. preparation method as claimed in claim 3, it is characterized in that, described step C comprises:
By the medicinal residues 6-10 times amount water soaking 20-40min after extraction;
Water extraction 1-3 time, first time 45-75min, second time 30-60min;
Merge extractive liquid, concentrating under reduced pressure, obtain the second extracting solution.
7. preparation method as claimed in claim 6, it is characterized in that, described step C comprises:
By the medicinal residues 8 times amount water soaking 30min after extraction;
Water extraction 2 times, first time 60min, second time 45min;
Merge extractive liquid, concentrating under reduced pressure, obtain the second extracting solution.
8. prepare a method for the Chinese medicine composition as described in any one of claim 1-2, it is characterized in that, comprising:
A, take Fructus Crataegi, Radix Polygoni Multiflori Preparata, the Radix Paeoniae Alba, Radix Salviae Miltiorrhizae by formula;
B, adding water did not have medical material, soaked 20-40min;
C, be heated to water and open, little fire continues to decoct 10-30min, filters, obtains the first medicinal liquid;
D, continuing to add water did not have medical material, and water is opened rear little fire and continued to decoct 10-30min, filters, obtains the second medicinal liquid;
E, merge the first medicinal liquid, the second medicinal liquid.
9. treat a Chinese medicine preparation for insulin resistant and relevant disease thereof, it is characterized in that, described Chinese medicine preparation is containing, for example the Chinese medicine composition described in any one of claim 1-2.
10. Chinese medicine preparation as claimed in claim 9, it is characterized in that, described Chinese medicine preparation is decoction, powder, capsule, tablet, granule or pill.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101574423A (en) * | 2008-05-05 | 2009-11-11 | 天津天士力制药股份有限公司 | Application of medicine combination in preparing medicament for reducing insulin resistance |
| CN101618176A (en) * | 2008-07-04 | 2010-01-06 | 陈民 | Chinese medicament for treating dyslipidemia and atherosclerosis |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101574423A (en) * | 2008-05-05 | 2009-11-11 | 天津天士力制药股份有限公司 | Application of medicine combination in preparing medicament for reducing insulin resistance |
| CN101618176A (en) * | 2008-07-04 | 2010-01-06 | 陈民 | Chinese medicament for treating dyslipidemia and atherosclerosis |
Non-Patent Citations (1)
| Title |
|---|
| 刘江等: "山楂叶总黄酮防治大鼠胰岛素抵抗及脂肪肝的实验研究", 《华东师范大学学报(自然科学版)》, no. 06, 30 November 2008 (2008-11-30), pages 127 - 132 * |
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