CN107468681B - The drug and pharmaceutical composition of prevention or treatment prediabetes - Google Patents
The drug and pharmaceutical composition of prevention or treatment prediabetes Download PDFInfo
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Abstract
It is a kind of prevention or treatment prediabetes drug and its application, can effectively reduce prediabetes conversion diabetes relative risk.The drug includes a effective amount of flavone compound such as following formula I, it optionally include pharmaceutically acceptable carrier and/or excipient, the dosage form of the drug is tablet, oral disnitegration tablet, sustained release tablets, granule, capsule, freeze drying powder injection, pill, suspending agent, syrup, emulsion or injection.
Description
Technical field
The present invention relates to the drugs and pharmaceutical composition that prevent or treat prediabetes, can be effectively reduced forerunner's sugar
Urine disease is converted into the relative risk of diabetes B；Belong to field of pharmaceutical biology.
Background technique
Prediabetes refers to that blood glucose is higher than euglycemia range, and is lower than the state of diabetes glucose range.Forerunner
Diabetes are the preliminary stages of diabetes B, if prediabetic does not change its animation, the patient more than 70%
It will develop as diabetes B.Prediabetes mainly includes stand alone impaired glucose tolerance (impaired glucose
Tolerance, IGT), stand alone impaired fasting glucose (impaired fasting glucose, IFG) and IGT and IFG merge
Type.Wherein IGT accounts for 80% of prediabetes or more, prediabetes and diabetes B be all as caused by insulin resistance,
But the diagnosis of prediabetes and the diagnosis of diabetes are essentially different, and are primarily characterized in that fasting blood for IGT
Sugar is between 3.2mM and 5.5mM, and postprandial 2 hours blood glucose is between 7.8 and 11.1mM.
It is an incremental process that prediabetes, which is converted into diabetes B,.First with the Zeng Jia ﹑ society at age
With the variation of individual factor, script healthy population is caused to be in movement Bu Zu ﹑ diet and irregular state of resting.This state
It will lead to free fatty acid in blood to increase, interior fat is constantly accumulated, and is chronically at high pressure conditions so as to cause insulin.This
It is to cause the initial inducement of diabetes and a kind of sub-health state.With continuing for sub-health state, the insulin sensitivity of body
Property reduce, insulin secretion compensatory increases, and the high pressure conditions of insulin have been further aggravated.This stage blood glucose is still in just
Normal state, but insulin content significantly increases in blood, Atherosclerosis Risk increases.This stage has hyperinsulinism
The risk of mass formed by blood stasis.With the lasting aggravation of insulin resistance, insulin secretion then will appear slight defect, although still can compensatory one
Part, but blood glucose has had already appeared exception, but its degree for being still not up to diabetes, referred to as prediabetes stage.
The stage complication risk of various diabetes is larger, but with biggish reverse possibility and relative to insulin resistance rank
Duan Rongyi is detected.But the stage such as intervenes without reasonable, with being further aggravated for insulin resistance, blood glucose into one
Step increases, and is finally then converted into diabetes B.Diabetic phase can not reverse, and can only lean on insulin therapy or orally-taken blood sugar reducing
Drug maintains.
The prevention diabetes B best evidence is from the research to impaired glucose tolerance crowd at present.2015, whole world sugar was resistance to
The abnormal illness rate of amount is 6.7%, and there are about 3.18 hundred million people, the illness rate to the year two thousand forty, diabetes exception is up to 7.8%, about
It is 4.8 hundred million, most of impaired glucose tolerance patient lives in low middle income country's (69.2%), Middle and North America and Caribbean Haiti
Area's illness rate is 15.0%, and European illness rate is only 4.8%.Wherein, it is print that impaired glucose tolerance patient populations, which come first three,
It spends (36,500,000), the U.S. (35,800,000) and Indonesia (29,000,000).There are about 26,700,000 impaired glucose tolerance patients, illness for China
Number is the 4th, the whole world, it is contemplated that undiagnosed patient, the number can be bigger.The impaired glucose tolerance patient age of about half
Less than 50 years old, if this some patients cannot suitably intervene, there will be very high risk to develop into diabetes B.And it is worth
It obtains it is noted that there are about the impaired glucose tolerance patient ages of 1/3 (29.8%) between 20-39 years old, they are following to occur 2 types sugar
Urinate the dangerous (International Diabetes Federation.IDF Diabetes Atlas 2016) of disease.
The complication of most diabetes is in the prediabetes stage with regard to disease incidence with higher and onset risk.It is cardiovascular
The research at disease research center, which demonstrates prediabetes, can significantly improve the incidence of cardiovascular disease.American National nutrition
Health survey shows that 18% prediabetic has kidney trouble.It is found in the works in diabetes mellitus prevention, forerunner's glycosuria
Disease can equally lead to retinopathy, wherein 7.9% patient's retinopathy does not make further progress, and 12.6% trouble
Person can then be further developed into cataract.
The prediabetes stage is best period and the critical period of diabetes B prevention, is reinforced to prediabetes
Intervening has important meaning to prevention diabetes B.With had tens of kinds of alternative diabetes B therapeutic agent phases
Than the research of prediabetes therapeutic agent is still at the initial stage, and prediabetes and diabetes have many common ground, still
Also there is apparent difference, it is evaluate prediabetes therapeutic agent one that prediabetes could be effectively prevent, which to be converted into diabetes,
A important indicator is generally converted into relative risk reduced rate (the The relative risk of diabetes B with prediabetes
Reduction of progression from prediabetes to diabetes, RRRD) it indicates.Many have compared with
The drug of good blood sugar reducing function can not be effectively reduced prediabetes and be converted into diabetes B risk, such as carry out clinic
The chemical drug of evaluation, such as melbine, acarbose, voglibose clinically have preferable hypoglycemic effect, but grind
Study carefully and show that these drugs reduce diabetes B relative risk reduced rate and are not better than life style adjustment (weight-reducing, movement etc.),
Middle acarbose is earliest and uniquely to carry out the prediabetes drug and China's sales volume of clinical test in multiple countries
Maximum oral hypoglycemic drug, reduce diabetes B risk be only 25-31% (Deloreme, S., Curr.Opin,
Pharmacol, 2005,5,184-189), which is alpha-glucosidase inhibitor, significant can must alleviate carbohydrate in diet
Hydrolysis, effectively control postprandial blood sugar increase.But acarbose mechanism of action is relatively single, to the improvement results such as complication compared with
It is weak, cause the probability for reducing diabetes B relative risk lower.For prediabetes drug, because application is
Sub-healty adults, and non-patient, safety are also highly important index.Thiazolidine dione compounds, such as Rosiglitazone and pyrrole
Lattice column ketone is the strong agonist of PPAR γ, can improve insulin sensitivity, and then play drop by the expression of up-regulation PPAR γ
Sugar, the effect for improving complication, in clinical test, the reduction diabetes B relative risk of Rosiglitazone and Pioglitazone drops
Low rate is adjusted higher than life style, but because such drug is too strong to PPAR γ agonist activity, is put on weight, hepatotoxicity,
And the risk of cardiovascular disease and bladder cancer is increased, as the therapeutic agent of diabetes, by the country such as European Union disabling or limit
With can not also be used in the prevention and treatment of prediabetes (Tabake AG.Lancet, 2012,379,2279-2290).It is so far
Only, clinically lack prediabetes therapeutic agent.
The interference method of 1. prediabetes of table
Flavone compound is widely present in the plant of nature, platymiscium secondary metabolite.Flavone compound
Very wide in plant kingdom's distribution, most of form for being combined into glycoside or carbon glycosyl with sugar exists in plant, and also some is with trip
Exist from form.Flavonoids composition generally has preferable biological safety, even if largely taking, human body will not be caused obvious
Side effect, such as the safety of baicalein is confirmed in the clinical I phase of human body, daily intakes 100-2800mg
Baicalein to people safety, adverse reaction is low, has no stomach and intestine side reaction and the damage to liver, kidney.Flavone compound
In have the compound of medical value very much, such as luteolin, Quercetin, oroxin A and baicalein, these compounds are used
In prevention and treatment cardiovascular and cerebrovascular disease, senile hypertension, cerebral hemorrhage, coronary heart diseases and angina pectoris are prevented and treated；With cough-relieving, eliminating the phlegm, relievings asthma and resist
The effect of bacterium；With protect liver, solution liver poison, treatment acute hepatitis, chronic hepatitis, cirrhosis and free radical resisting and oxidation resistant effect；Have
Effect identical with phytoestrogen.In treating diabetes field, flavonoids chemical analysis, which is also reported to have, inhibits glycosidase, drop
Effect (Sarinya, A., the et al., Molecules, 2011,16,2075- of the blood glucose of low diabetes B mouse, rat
2083；Satoh, T., et al., Apoptosis, 2015,14,1459-1471), but it treats prediabetes, and it is especially high
Effect reduces the effect of diabetes B relative risk, and there is not been reported.
Summary of the invention
The purpose of the present invention is to provide drugs and pharmaceutical composition that diabetes are gone before prevention and treatment, can be effectively reduced forerunner
Diabetes are converted into the relative risk of diabetes B, to solve the defects of efficient low, side effect is big in the prior art.
In order to achieve the above object, the invention adopts the following technical scheme:
It is prepared by the pharmaceutical composition the present invention relates to the flavone compound of such as Formulas I and comprising the flavone compound
Application in the drug of prevention or treatment prediabetes,
Wherein: R1、R2、R3、R4、R5It can be identical or different；R1 represents hydrogen, hydroxyl, the 1 molecule Portugal linked in the form of oxygen glycosides
Grape glycosyl；R2、R3、R4、R5Represent hydrogen or hydroxyl.
Preferably, the flavone compound of Formulas I is selected from luteolin, Quercetin, oroxin A and baicalein, structure
It is as follows:
Preferably, pharmaceutical composition of the drug of the present invention with above-mentioned flavone compound or comprising the compound
For effective ingredient, the dosage of above-mentioned flavone compound or the composition comprising any of the above-described compound in drug is referred to
The relative risk that prediabetes is converted into diabetes B can be reduced.
The dosage of drug of the invention can change with the mode and the severity of disease to be treated of administration.So
And when drug administration amount of the invention reaches 10-800mg/kg, preferably 20-700mg/kg, most preferably 30-650mg/kg mouse
When the dosage of weight is given daily, the relative risk that prediabetes is converted into diabetes B can effectively reduce, preferably daily
It is given with 1-3 separated dosage, or is administered with sustained release forms.For most of large mammal, according to mouse and
People's dose lonvestion value is 9.1:1, is 60kg calculating with the weight of people, and daily accumulated dose is 66-5274mg, preferably 132-
4615mg, more preferably 198-4286mg.This dosage is adjusted to provide optimal treatment response.For example, by treatment situation
Needs, dosage separated several times can be given once daily, or dosage is reduced pari passu.It is calculated according to being administered daily 3 times, In
Unit dose in drug is 22-500mg, preferably 44-500mg, more preferably 66-500mg.
Another object of the present invention is to provide a kind of prevention or the drugs for the treatment of prediabetes, include a effective amount of formula
I flavone compound and pharmaceutical composition comprising Formulas I flavone compound.
Drug of the present invention further includes pharmaceutically acceptable carrier and/or excipient.It is pharmaceutically subjected to
Carrier and/or excipient be one or more common fillers, adhesive, wetting agent, disintegrating agent, sorbefacient, table
Face activating agent, absorption carrier, lubricant or corrigent carrier.Filler can be selected from starch, sucrose, lactose or microcrystalline cellulose
Element；Adhesive is selected from cellulose derivative, alginates, gelatin or polyvinylpyrrolidone；Disintegrating agent be selected from sodium carboxymethyl starch,
Hydroxypropylcellulose, cross-linked carboxymethyl cellulose, agar, calcium carbonate or sodium bicarbonate；Surfactant can be hexadecanol or
Lauryl sodium sulfate；Lubricant is selected from talcum powder, calcium stearate and magnesium, superfine silica gel powder or polyethylene glycol etc..
The dosage form of drug of the present invention can be tablet, oral disnitegration tablet, sustained release tablets, granule, capsule, freeze-dried powder
Injection, pill, suspending agent, syrup, emulsion or injection.
Required for the various pharmaceutical dosage forms of drug of the invention can be prepared according to the conventional production process of pharmaceutical field
Preparation.For example, tablet can be ordinary tablet, diaphragm, enteric coatel tablets etc., luteolin, Quercetin, oroxin A, radix scutellariae can be used
Aglycon and comprising any of the above-described pharmaceutical composition dry powder is added appropriate diluent and is selected from starch, dextrin, mannitol, micro-
Crystalline cellulose, suitable adhesive are selected from water, ethyl alcohol, cellulose, starch, gelatin, and suitable disintegrating agent is selected from carboxymethyl starch
Sodium, low-substituted hydroxypropyl cellulose, sodium alginate and lubricant appropriate are selected from magnesium stearate, talcum powder, polyethylene glycol,
Sweetener is added, D- xylose, xylitol, maltitol, STEVIA REBAUDIANA element, aspartame are selected from, routinely wet granulation, after dry
Tabletting after whole grain or dry granulation, for example film coating piece are selected from cellulose family, polyethylene glycols with filmogen, by normal
Rule coating, is distributed into airtight bottle or aluminium-plastic panel.Capsule can be for conventional capsule agent, enteric capsule etc., can be by reseda
Element, Quercetin, oroxin A, baicalein and include that appropriate auxiliary material choosing is added in any of the above-described pharmaceutical composition dry powder
From calcium carbonate, mannitol, magnesia, superfine silica gel powder etc., proper lubrication agent is selected from talcum powder, magnesium stearate, glycol ester, poly- silicon
Ketone and adhesive appropriate are selected from mineral oil, edible oil, and appropriate sweetener is added, and are selected from D- xylose, xylitol, malt
Sugar alcohol, STEVIA REBAUDIANA element, aspartame, are mixed into dry powder or particle are made, be packed into capsule, be divided in airtight bottle or plastic-aluminum
In plate.
The present invention has the advantages that prediabetes can be prevented and treated, prediabetes is effectively reduced and is converted into 2 types
The relative risk of diabetes, Small side effects.
Specific embodiment
Following non-limiting embodiments can with a person of ordinary skill in the art will more fully understand the present invention, but not with
Any mode limits the present invention.
Detailed description of the invention:
Fig. 1 is the PPAR γ Transcription Activity In Nuclei of flavones composition, Rosiglitazone and acarbose in embodiment 3, A: sweet-scented osmanthus
Careless element, B: Hu Pi Su ﹑ C: oroxin A ﹑ D: baicalein, E: oroxin B, F: mangiferin, G: chrysin, H: white poplar is yellow
- two Pu grape Tang Gan ﹑ I of element: Rosiglitazone, J: acarbose and K:DMSO.
Fig. 2 is influence of the baicalein to SOCS3 in the HepG2 cell of insulin resistance in embodiment 5, and data indicate
Method is average ± standard deviation, same letter (it a, b, c, d and e) is displayed without significant difference (p > 0.05), different letters
Between have significant difference (p < 0.05).
Fig. 3 is baicalein in embodiment 5 to the downstream SOCS3 pIRS1/IRS1 (Fig. 3 A and 3B), pAkt1/Akt1 (Fig. 3 A
And 3C) and pGSK3 β/GSK3 β (Fig. 3 A and 3D) influence.Data representation is average ± standard deviation, same letter (a, b, c
And it d) is displayed without significant difference (p>0.05), there is significant difference (p<0.05) between different letters.
Fig. 4 is influence of the baicalein to glucose consumption (A) and glycogen levels (B) in embodiment 5.Data representation is
Average ± standard deviation, same letter (a, b and c) are displayed without significant difference (p > 0.05), have between different letters significant
Sex differernce (p < 0.05).
Fig. 5 is oroxin A in embodiment 7 and Rosiglitazone to prediabetes liver protection.Scheme A: Oroxylum indicum
The influence of glycosides A and Rosiglitazone to liver structure；Scheme B: the shadow of oroxin A and Rosiglitazone to liver organization liquid ALT level
It rings；Scheme C: the influence of oroxin A and Rosiglitazone to liver organization liquid AST level.Wherein figure B and figure C, data explicit representation are
Mean+SD does not have significant difference (p>0.05) between same letter, have between different letters significant difference (p<
0.05)。
Specific embodiment
Following non-limiting embodiments can with a person of ordinary skill in the art will more fully understand the present invention, but not with
Any mode limits the present invention.
The material and method that the present invention uses
1. material
Luteolin used herein, Quercetin, oroxin A, oroxin B, baicalein, mangiferin, quercitrin
Element, chrysin and chrysin-diglucoside, purchased from Nantong Fei Yu company or by this laboratory according to literature method from plant
It is prepared in object, purity is all larger than 95%, and confirmed the correctness of structure by means such as mass spectrum and nuclear magnetic resonance.In the present invention
The kunming mice and basal feed used is purchased from SPF Experimental Animal Center, streptozotocin (STZ) purchase purchased from Dalian Medical Univ
From Beijing Suo Laibao Biotechnology Co., Ltd.Anti-p-GSK3 β antibody and anti-GSK3 β antibody is purchased from the U.S.
CST Co., Ltd.Anti-SOCS3body is purchased from Britain Abcam Co., Ltd.Anti-p-Akt1antibody and anti-
Akt1antibody is purchased from Shenyang all creation Co., Ltd.HepG2 cell and H293T cell are purchased from U.S. ATCC cell bank.
2. method:
2.1 prediabetes modeling methods:
16-20g kunming mice is classified as 2 groups after adapting to environment 3 days, wherein one group, feeding high confectionery object high in fat
The water of (36.4% starch, 25.6% butter, 20% albumen, 1% cholesterol and 0.1% bile acid) and 3% sucrose, by 5 weeks
After diet induced insulin resistance, tail vein injection 30mg/kg STZ.After injection the 7th day and 21 days progress oral glucoses it is resistance to
By experiment (OGTT).2 blood glucose values meet is less than 7.8mM and postprandial 2 hours blood glucose in 7.8mM between 11.1mM on an empty stomach
Mouse is prediabetes mouse.Another group of kunming mice, feeding standard feed is infused simultaneously when model group injects STZ always
Penetrate sodium citrate solution (STZ is dissolved in this solution).7th day and 21 days progress Oral glucose tolerance tests after injection.2 blood glucose
Value meets fasting blood-glucose less than 5.5mM and postprandial 2 hours blood glucose is less than 7.8mM mouse for normal mouse.
2.2 oral glucose tolerance test methods:
Mouse fasting 16 hours, 2g/kg glucose solution is taken orally, tail portion takes blood, while using Glucose estimation kit
It is measured in 0 minute, 30 minutes and 120 minutes blood glucose.
2.3 pharmaceutical intervention prediabetes mouse experiments
Compound used in zoopery is dissolved in 0.5% carboxymethylcellulose sodium solution in embodiment, fills daily
Stomach is administered once, the carboxymethylcellulose sodium solution of model group stomach-filling 0.5%, and in experimental period, model group and administration group are fed simultaneously
The high-sugar-fat-diet of same recipe in food and modeling experiment is normal to organize standard feed of the feeding without high sugared composition high in fat.It gives
After medicine terminates experiment, oral glucose tolerance test, measurement weight (Body Wight, B.W.) are carried out.
2.4 prediabetes are converted into the relative risk reduced rate of diabetes B:
The blood glucose value of oral glucose sugar tolerance measuring is classified, statistics each group euglycemia (NM) meets
Prediabetes standard blood glucose (Pre-DM) and meet diabetes B standard blood glucose (DM) mouse quantity, calculate each group account for
The ratio of total experimental animal, according to method (Steurer J et al.Praxis, 1997,86 (15): before 614) calculating of document
Drive the relative risk reduced rate (RRRD) that diabetes are converted into diabetes B:
RRRD=(IDC-IDT)/IDC (1)
Wherein: IDT (the incidence of diabetes in the treatment group): drug treatment group
The incidence of diabetes B；IDC (the incidence of diabetes in the control group): model group 2
The incidence of patients with type Ⅰ DM.
The building and determination of activity of 2.5 PPAR γ nuclear transcriptional activation models:
Carrier cell is H293T cell, and H293T cell uses the fetal calf serum DMEM culture medium containing 10% to be trained
It supports, culture contains 5% CO in 37 DEG C ﹑2Insulating box in.Contain PPAR γ's by 2000 cotransfection of liposome, 1 μ g first
The plasmid of gene, plasmid and 1 μ g that 1 μ g contains fluorescein expressing gene contain the plasmid of PPAE gene.Steps are as follows: logarithmic phase
H293T cell inoculation in 24 orifice plates, cell inoculation amount be 2.5 × 104A/hole, final volume are 500 holes μ L/, are connect
Take it is suitable mixing plasmid and 50 μ L the DMEM without blood plasma, gently concussion mix, be configured to mixed liquor 1.Take 1 μ L's simultaneously
The DMEM without blood plasma of the μ of liposome 2000 and 50 L, gently concussion mixes, and is incubated for 5 minutes, is configured to mixed liquor 2.1 He of mixed liquor
Mixed liquor 2 merges, and mixes gently, and is incubated for 20 minutes, final mixed liquor is added in 24 well culture plates at room temperature then.In
The CO of 37 DEG C ﹑ 5%2It is cultivated under complete wet environment.The cell of 3 plasmids is filtered out while is transferred to using antibiotic method.It is transfecting
After 48 hours, a certain amount of luteolin (A) ﹑ Quercetin (B) ﹑ oroxin A (C) ﹑ baicalein (D) ﹑ oroxin B is added
(E) ﹑ mangiferin (F) ﹑ chrysin (G) ﹑ chrysin-diglucoside (H) ﹑ Rosiglitazone (I) ﹑ acarbose (J) or
DMSO (K), all untested compound concentration are that 30 μ g/L are incubated for 24 hours jointly, then detect it with fluorescence detection reagent kit
Fluorescence intensity.Its detection method is as follows: 24 orifice plates taken out from incubator, discard culture medium, after washing 3 times with PBS solution, and every hole
The lysate of 200 μ L is added, cracks 15 minutes on ice, prepares lysate.The luciferase inspection of 50 μ L is added in every measurement pipe
The lysate or 50 μ L Gol reagents of test agent and 50 μ L, measure its luminous quantity, wherein the activity of each group luciferase respectively
For experimental group uciferase activity divided by control group uciferase activity.
2.6 flavones compositions influence experimental method to SOCS3 and downstream passages:
Containing 100nM pancreas with (0.0,0.6,1.2,2.4 and 4.8 μM) the processing culture of the flavones composition of various concentration respectively
HepG2 cell 8 days in the element culture solution of island changes the liquid once for cell culture fluid every 2 days, and dosing again, after 8 days, is cleaned carefully with PBS
Born of the same parents extract total protein of cell.Use SOCS3/ β-actin, pGSK3 β/GSK3 β, pAkt1/ in western blotting method measurement cell
Akt1 and pIRS1/IRS1 ratio.Wherein Akt1 is 1:1000 dilution, and pAkt1 is 1:1000 dilution, and GSK3 β is that 1:1000 is dilute
It releases, p-GSK3 β is 1:1000 dilution.
Cell is cultivated 36 hours in the culture medium containing 1 μM of insulin, it, will after cell length to the 80% of culture medium
HepG2 cell is uniformly laid on 24 orifice plates, replace the culture medium of serum-free, and starved cells 12 hours, what is more renewed contained 1 μM
Insulin serum free medium, and the flavones composition (0.05 ﹑ 0.5 and 5 μM) of various concentration is added and DMSO is handled 24 hours, it surveys
Determine the glucose content of culture medium and calculates glucose utilization.The culture medium on plate is sucked, is cleaned cell 3 times, is added with PBS
Enter suitable protein lysate, lytic cell.It takes out part lysate and measures protein concentration using BCA method.Remaining lysate, root
It is diluted according to protein concentration, is prepared into 0.1% glycogen detection liquid, anthrone developing solution is then added with 1:2, is developed the color.With
The glucose of 0.01mg/mL is titer, using the glycogen content in State Standard Colorimetry computation organization, glucose and glycogen
Conversion coefficient is 1.11, i.e., the absorbance that 100g glycogen anthrone reagent develops the color is equal to what 111g glucose anthrone reagent developed the color
Absorbance.
2.7 alpha-glucosaccharase enzyme inhibition activity measuring methods:
SD rat (200~220g) fasting 16h, etherization take small intestine after putting to death, split, with the PBS buffer solution of pre-cooling
Rinse, after according to 1:10 (W/V) be added PBS buffer solution.It is homogenized, 4 DEG C, 10000r/min, is centrifuged after small intestine is cut into broken section
15min takes supernatant as test alpha-glucosidase mother liquor.
Enzyme reaction system total volume is 500 μ L, including enzyme solution, test compound, acarbose, maltose, buffer are each
100μL.Enzyme and test compound, acarbose mix, 37 DEG C of incubation 30min, maltose solution starting reaction.37 DEG C of incubations,
React 30min.Glucose Oxidase kit measurement generates concentration of glucose, measures sample light absorption value under microplate reader 505nm.
The measuring method of inhibiting rate
Inhibitor is calculated to the inhibiting rate of enzyme according to formula (2),
Inhibiting rate (%)=(1- sample sets (A)/control group (A)) × 100 (2)
Wherein: sample sets (A): the light absorption value after inhibitor is added；Control group (A): control buffering is added in without inhibitor
The light absorption value of liquid and enzyme.
Embodiment 1: flavones composition is converted into the influence of diabetes B relative risk reduced rate to mouse prediabetes
Prediabetes model is made according to experimental method 2.1, flavones composition reduction prediabetes is carried out and is converted into 2 types sugar
Sick relative risk rate experiment is urinated, experimental period is 8 weeks, and flavones composition is luteolin, Quercetin, oroxin A, scutelloside
Member, oroxin B, mangiferin, chrysin, chrysin-diglucoside.Dosage is 200mg/kg.While with
The acarbose of 20mg/kg and the Rosiglitazone of 4mg/kg are positive control, and prediabetes is converted into the phase of diabetes B
To risk reduced rate and weight statistics in table 2.
2. flavones composition of table is converted into the relative risk reduced rate of diabetes B and the shadow of weight to mouse prediabetes
It rings
It learns as can be seen from Table 2, different flavones compositions, reduces prediabetes and be converted into the several of diabetes B
Rate has biggish difference, and wherein luteolin, Quercetin, oroxin A and baicalein have better drug activity.Example
Oroxin A is such as administered, mouse transforming only 25% prediabetes is diabetes B, and model group, then before having 80%
It drives diabetic mice and is converted into diabetes B, the RRRD value for the oroxin A being computed is 68.75%.It is administered simultaneously Roger's column
Ketone dramatically increases mouse weight, and influence of each flavones to mouse weight is then smaller.Show that flavones ingredient side effect is lower.
Embodiment 2: the relative risk that the flavones composition of various dose is converted into diabetes B to mouse prediabetes drops
The influence of low rate
Prediabetes model is made according to experimental method 2.1, carries out various dose Mu rhinoceros Cao Su ﹑ Hu Pi Su ﹑ oroxin A
Prediabetes is reduced with baicalein and is converted into the experiment of diabetes B relative risk rate, and experimental period is 8 weeks, various flavones
The dosage of composition is 40,400 and 600mg/kg.Its prediabetes is converted into the relative risk reduced rate system of diabetes B
It counts in table 3.
3. various dose flavones of table is converted into the influence of the relative risk reduced rate of diabetes B to mouse prediabetes
From table 3 it can be seen that the Mu rhinoceros Cao Su ﹑ Hu Pi Su ﹑ oroxin A and baicalein of various dose can be effective
The probability of mouse prediabetes conversion diabetes B is reduced, wider range of effective dose is according to mouse and people's dose ratio
9.1:1 is calculated, and people's dosage is that 4.3-65.9mg/kg is effective, is calculated according to adult weight 60kg, is administered daily dosage at least
It is effective in 258-3956mg.Mu rhinoceros Cao Su ﹑ Quercetin, oroxin A and baicalein all have higher forerunner
Diabetes are converted into diabetes B relative risk reduced rate, and drug effect is better than known generally acknowledged prediabetes therapeutic agent Ah
Card wave sugar and Rosiglitazone, and Rosiglitazone once reports that there is highest prediabetes to be converted into diabetes B relative risk
Reduced rate, but because toxic side effect is by limitation use.So in terms of reducing the relative risk that prediabetes is converted into diabetes, wood
Rhinoceros Cao Su ﹑ Hu Pi Su ﹑ oroxin A and baicalein have better effect, are better than existing pre-diabetes therapeutic agent.
Embodiment 3: the composition of different flavones compositions is converted into diabetes B relative risk drop to mouse prediabetes
The influence of low rate
Prediabetes model is made according to experimental method 2.1, carrying out flavones composition composition reduces mouse prediabetes
The experiment of diabetes B relative risk rate is converted, experimental period is 8 weeks, and composition includes: oroxin A and baicalein twenty percent
Three composition compositions of part composition and oroxin A, baicalein and luteolin, various flavones compositions are in the composition
Dosage be 200mg/kg.The relative risk reduced rate that composition is converted into diabetes B to prediabetes influences statistics
In table 4.
4. flavone composition of table is converted into the influence of the relative risk reduced rate of diabetes B to mouse prediabetes
From table 4, it can be seen that various flavones compositions are combined equally, and there is reduction mouse prediabetes to be converted into 2
The effect of patients with type Ⅰ DM relative risk.
Embodiment 4: effect of the flavones composition to PPAR γ nuclear transcriptional activation
The effect of PPAR γ nuclear transcriptional activation is an important target spot of insulin sensitivity enhancing, and prediabetes is along with insulin
It resists, flavones composition is of great significance to the effect of PPAR γ nuclear transcriptional activation to the prevention and treatment of prediabetes.
As shown in Figure 1, Rosiglitazone to PPAR γ Transcription Activity In Nuclei Qiang ﹑ Mu rhinoceros Cao Su ﹑ Hu Pi Su ﹑ oroxin A and
Chrysin also has stronger PPAR γ Transcription Activity In Nuclei, shows that these flavones can increase pancreas islet by activation PPAR γ
Plain sensibility, and A 's Bo Tang ﹑ radix scutellariae Gan Yuan ﹑ oroxin B ﹑ mangiferin and chrysin-diglucoside do not have substantially
PPAR γ Transcription Activity In Nuclei.The above result shows that Mu rhinoceros Cao Su ﹑ Hu Pi Su ﹑ oroxin A is by improving the record of PPAR γ consideration convey
Activity reduces mouse prediabetes and is converted into diabetes B relative risk.
Embodiment 5: influence of the flavones composition to SOCS3 and downstream passages
SOCS3 is also the key protein of insulin sensitivity enhancing signal path, and the downward of SOCS3 expression quantity can activate IRS/
Akt signal path, and then improve insulin sensitivity.
In the flavones of measurement, after only baicalein and HepG2 cell are incubated for jointly, the variation (figure of SOCS3 expression quantity
2), the influence of other flavones compositions is weaker or without influencing (result is not shown).Compared with 100nM insulin, 0.6 μM, 1.2 μ
M, 2.4 μM and 4.8 μM of baicalein makes the expression of SOCS3 have dropped 17.7%, 21.1%, 37.1% and 47.8% respectively,
These results prove that baicalein can be such that SOCS3 expression quantity lowers, and have dose dependent.
Further study influence of the baicalein to the downstream SOCS3 insulin signaling pathway.Use various concentration radix scutellariae
It is found behind HepG2 cell 8 days of aglycon processing 100nM insulin stimulating, baicalein can be improved in a manner of dose-dependent
The phosphorylation level of the IRS1 of insulin stimulating and the phosphorylation level of Akt1 reduce the phosphorylation level of GSK3 β.When with 4.8 μ
When the baicalein of M handles HepG2 cell, baicalein can make the phosphorylation level of IRS1 increase 1.97 times, make Akt1's
Phosphorylation level increases 2.89 times, and the phosphorylation level of GSK3 β is made to decline 68% (Fig. 3).
The phosphorylation degree of GSK3 β and the activity of glycogen synthetase are closely related, and the decline of GSK β phosphorylation level can be led
The raising for causing glycogen synthase activity, and then leads to the rising of intracellular glycogen content, thus measure cell glucose consumption and
Influence of the intracellular glycogen levels to determining baicalein to insulin downstream signaling pathway is very necessary.Baicalein energy
The enough consumption and glycogen levels that glucose inside cells are dramatically increased in a manner of dose-dependent.It is handled when with 5 μM of baicalein
When HepG2 cell, baicalein can make glucose consumption level increase 2.58 times, and glycogen content is made to increase 1.89 times (Fig. 4).
Baicalein can make the Portugal of cell by reducing the expression quantity of SOCS3 and activating the downstream insulin signaling pathway of SOCS3
Grape sugar consumption and glycogen levels increase, and then increase insulin sensitivity.
Embodiment 6: inhibiting effect of the flavones composition to alpha-glucosidase
Carbohydrate composition in food can be effectively prevented to the inhibiting effect of alpha-glucosidase to hydrolyze, and then reduce postprandial blood
Sugar, various flavones compositions are as shown in table 5 to the inhibitory activity of alpha-glucosidase.Acarbose shows strong alpha-glucosidase
Inhibitory activity.Mu rhinoceros Cao Su ﹑ Hu Pi Su ﹑ Mang Guo Gan ﹑ baicalein and oroxin A to the inhibitory activity of alpha-glucosidase also compared with
By force, in addition to positive control acarbose, activity is preferably baicalein.Bai Yanghuang element ﹑ chrysin-diglucoside, the wooden butterfly
Butterfly glycosides B and Rosiglitazone are to alpha-glucosidase unrestraint activity.
Inhibitory activity of the 5. flavones composition of table to alpha-glucosidase
It can be seen that Mu rhinoceros Cao Su ﹑ Hu Pi Su ﹑ Mang Guo Gan ﹑ baicalein and Oroxylum indicum from the result of embodiment 4,5 and 6
Glycosides A has the inhibitory activity of insulin-sensitizing effect and alpha-glucosidase simultaneously, can be played a role by multiple target point, this
It is that these drugs and combinations thereof have the preferable master for reducing prediabetes and being converted into the ability of diabetes B relative risk
Want reason.And acarbose only has the inhibitory activity of alpha-glucosidase, Rosiglitazone only has the effect of insulin sensitivity enhancing, although
It is stronger to the effect of single target spot, but because activity is single, and blood glucose value is higher, insulin resistance is that prediabetes exists simultaneously
The problem of, existing drug can not play a role for multiple target spots and its prediabetes conversion diabetes B relative risk
The main reason for reduced rate is lower.Likewise, it has been found that chrysin only has insulin sensitivity enhancing work in flavones composition
Only there is alpha-glucosidase with, mangiferin, it is relatively low to reduce prediabetes conversion diabetes B relative risk rate,
The weaker reason of existing medicine effect can be confirmed.
The liver protection of 7. oroxin A of embodiment and Rosiglitazone to prediabetes mouse
Mu rhinoceros Cao Su ﹑ Hu Pi Su ﹑ baicalein and oroxin A administration group in Example 1, model group, normal group and
Rosiglitazone group the 8th week mouse liver sample terminated when experiment, make H-E coloring pathological section, and measure AST and ALT etc.
Main liver function indexes, the Antioxidant Indexes such as measurement T-SOD and MDA.Optical microscopy undertissue learn variation the results show that normal
Hepatic tissue liver cell size is suitable, exception and lesion does not occur, and serious lesion occurs in model group liver, and cell is swollen
Greatly, fatization is serious, or even situations such as meronecrosis of large area and fat vacuole occurs, Rosiglitazone group liver organization fat
Change without be improved significantly, and Mu rhinoceros Cao Su ﹑ Hu Pi Su ﹑ baicalein and oroxin A administration after liver organization cell
Oedema obviously weakens, and fatization phenomenon substantially reduces, wherein oroxin A, Rosiglitazone and normal mice and forerunner's glycosuria
The histotomy figure of sick mouse liver is as shown in Figure 5A.
Fig. 5 B shows the level of the ALT and AST of the liver organization liquid of oroxin A and Rosiglitazone administration group, this refers to
Mark has reacted the functional level of liver.ALT and AST level is substantially less than normal mice in prediabetes mouse liver tissue fluid,
The level of the ALT and AST of the group liver organization liquid of oroxin A administration group are significantly higher than model group, illustrate that flavones composition has
Protect liver, the ability that prevention liver function is lost.ALT the and AST level of Rosiglitazone administration group is then lower, illustrates that Roger arranges
Ketone maintains the ability of liver function weaker.
MDA and T-SOD has reacted liver organization oxidative stress.As shown in table 6, MDA in liver organization liquid is normally organized
Level is lower than model group, and T-SOD level is higher than model group, illustrates that prediabetes mouse receives stronger oxidation stress,
It is easy to appear oxidative damage.The MDA level of oroxin A group is lower than model group, and T-SOD level is higher than model group, illustrates the wooden butterfly
The oxidation stress that butterfly glycosides A effectively improves liver is horizontal, prevents oxidative damage.Though Rosiglitazone group MDA is horizontal to be lower than model
Group is higher than oroxin A group, is higher than model group though the T-SOD of D group is horizontal but is lower than oroxin A group, illustrates Rosiglitazone
The ability for preventing oxidative damage is weaker than oroxin A.
The influence of 6. oroxin A of table and Rosiglitazone to prediabetes mouse liver oxidative stress.
Data explicit representation is mean+SD, same index, do not have between same letter significant difference (p >
0.05), there is significant difference (p < 0.05) between different letters.
Claims (8)
1. the flavone compound of Formulas I reduces prediabetes in preparation as sole active agent and is converted into diabetes B
Application in the drug of relative risk,
Wherein: R1、R2、R3、R4、R5It can be identical or different；R1Represent hydrogen, hydroxyl, 1 molecule glucose connected in the form of oxygen glycosides
Base；R2、R3、R4、R5Represent hydrogen or hydroxyl；
Formulas I flavone compound is selected from luteolin, Quercetin, oroxin A and baicalein.
2. application according to claim 1, the prediabetes is selected from stand alone impaired glucose tolerance, stand alone fasting blood
Sugared impaired or stand alone impaired glucose tolerance and stand alone impaired fasting glucose combination type.
3. application according to claim 1 or 2, the dosage form of the drug is tablet, granule, capsule, pill, suspension
Agent, syrup, emulsion or injection.
4. application according to claim 3, the tablet is oral disnitegration tablet or sustained release tablets.
5. application according to claim 3, the injection is freeze drying powder injection.
6. application according to claim 1 or 2, unit dose of the flavone compound in drug is 22-
500mg。
7. application according to claim 6, unit dose of the flavone compound in drug is 44-500mg.
8. application according to claim 6, unit dose of the flavone compound in drug is 66-500mg.
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