CN101874832B - Wine-steamed Chinese goldthread processed product as well as preparation method and application thereof - Google Patents
Wine-steamed Chinese goldthread processed product as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention provides wine-steamed Chinese goldthread as well as a preparation method and application thereof. The wine-steamed Chinese goldthread as an insulin sensitizer and a PPAR gamma activator has the functions of inhibiting the differentiation of 3T3-L1 pre-adipocytes, improving the utilization rate of glucose of the 3T3-L1 pre-adipocytes, reducing blood fat, improving insulin resistance and treating diabetes mellitus. The wine-steamed Chinese goldthread and extracts thereof have the functions of inhibiting the differentiation of the 3T3-L1 pre-adipocytes, improving the utilization rate of the glucose of the 3T3-L1 pre-adipocytes and reducing the blood fat and the application in improving insulin resistance or treating diabetes mellitus, have a better specific effect than that of berberine hydrochloride and crude Chinese goldthread as well as extracts thereof, and the safety obviously higher than that of the crude Chinese goldthread.
Description
Technical field
The present invention relates to a kind of wine and steam Rhizoma Coptidis, the present invention also provides this wine to steam method for preparing and the purposes of Rhizoma Coptidis.
Background technology
According to World Health Organization (WHO) " World Health Report in 2008 ": urbanization, aging and global lifestyle change triplicity make Chronic Non-Communicable Diseasess such as diabetes become the more and more main disease and the cause of death.According to diabetology branch of Chinese Medical Association " Chinese type 2 diabetes mellitus guideline of prevention and treatment (2007 editions) ": in the Chinese diabetes afflicted, be main (accounting for 93.7%) with the type 2 diabetes mellitus, expectation in 2025 will reach the fifty-five million people.Diabetes spp is in the traditional Chinese medical science sick category of " quenching one's thirst "; Chinese medical theory is thought the pathogenesis of " quenching one's thirst ": because of addiction to greasy and sweet foods; Or after the emotional stress, intemperate sexual intercourse, calentura etc., accumulate in hot and suffocating, it is not normal to gasify; Body fluid is precise and tiny can not normally fail cloth and under let out the deficiency of YIN scorching (GB/T 16751.1-1997 tcm clinical practice diagnosis and treatment term disease part)." all accumulating in the course of time drunk, and can not have and quenches one's thirst." (the Tang Dynasty " prescriptions worth thousand gold ")
Rhizoma Coptidis is a conventional Chinese medicine, and the traditional Chinese medical science is recognized the purposes of " control and quench one's thirst " very early, and it is how on the books that successive dynasties book on Chinese herbal medicine and well-known doctor test case, beam generation " Mingyi Bielu " record " only quenching one's thirst "; The Tang Dynasty " Newly Revised Canon of Materia Medica " record " Rhizoma Coptidis, treat yearningly for ".Ming Dynasty's " little justice of beautiful machine " record " wine steams coptis teeta pill, controls and quenches one's thirst, and it is immoderate to two or three liters to drink water, and urinates five or seven ten times, and it is thin and weak to generate heat, xerostomia, and food is like famine, this diseases due to endogenous heat of ZANG FU organs.Modern nontoxic with bitter in the mouth, heat extraction healthy energy, the thick intestinal of quenching one's thirst.The people who quenches one's thirst, taste are disliked wet, and Rhizoma Coptidis is right.Rhizoma Coptidis is clean, and half jin, one liter of wine, Tang Chongzheng.Dry usefulness during volt, right end, the ball that drips, WUZI is big, under the preceding warm water of per 50 balls food "; Ming Dynasty's Compendium of Material Medica record " control and quench one's thirst, steam Rhizoma Coptidis " with wine; Korea's doctor's nationality " Dong-eui-bo-gam " draws Ming Dynasty's " Compendium of medicine " " control the key medicine of quenching one's thirst, steeping in wine steams, and dries to be the end, honeyed pill, five or seven ten balls under the plain soup "; Qing Dynasty's essentials of Matea Medica record " list is quenched one's thirst with controlling ".
The Chinese medicine Rhizoma Coptidis also is flavour of a drug commonly used in the Chinese medicine prescription of treatment diabetes (diabetes), and the domestic diabetes analysis of drug use result of hospital showed in 2006, in the product of rank top ten; TANGMAIKANG KELI (containing Rhizoma Coptidis, market share 22.1%, about 100,000,000 yuan), JINQI JIANGTANG PIAN (contain Rhizoma Coptidis; Market share 11.6%; About 5,000 ten thousand yuan), the 2nd, 4 of apportions, extensive use in tcm clinical practice.
But this herbalist nationality is to controlling the Rhizoma Coptidis decoction pieces (processed product) of the suitable usefulness of quenching one's thirst; Or do not elaborate; Or the system and generally it; Also lack in the modern study the experimental evidence of the different processed products of Rhizoma Coptidis on " control and quench one's thirst " purposes, cause in the tcm clinical practice practice even proposition " Rhizoma Coptidis is not the principal agent of treatment diabetes (diabetes) ".
The research of Rhizoma Coptidis treatment diabetes (diabetes) at present mainly concentrates on gives birth on the Rhizoma Coptidis total alkaloids that extracts in article Rhizoma Coptidis and single active ingredient (berberine) and the article of the giving birth to Rhizoma Coptidis; [Huang Xiaoxia; Ou Yangqin. Rhizoma Coptidis exempts to fry in shallow oil the clinical observation of granule therapy hepatogenous diabetes. Tianjin pharmacy .2008; Report such as 20 (3): 41-42]; Exempt to fry in shallow oil granule and insulin combination is used all effectively blood sugar lowering at clinical independent use insulin and berberine, but berberine exempts to fry in shallow oil granule and the insulin combination effect is better, and berberine exempts to fry in shallow oil granule and insulin combination set of applications blood glucose value is controlled more stablely.[Li-Qin Tang; Wei Wei; Li-MingChen; Et al.Effects of berberine on diabetes induced by alloxan anda high-fat/high-cholesterol diet in rats.Journal ofEthnopharmacology.2006,108 (1): 109～115] report, to the diabetic hyperlipidemia rat model (high fat/hypercholesterolemia/alloxan) of diet induced; Berberine has suppressed the diabetes process, and mechanism of action maybe be relevant with effects such as blood sugar lowering, blood lipid regulation metabolism.[bright red is defended, and Tang Li can. and Rhizoma Coptidis total alkaloids is studied the mice blood sugar reducing function. Yunnan Chinese medicine magazine .2008,29 (7): 59～60] report, Rhizoma Coptidis total alkaloids has remarkable hypoglycemic activity, to the not influence of blood sugar level of normal mouse.
Berberine has clearly effect to improving type 2 diabetes mellitus patients with insulin resistance aspect, and the various decoction pieces specifications of Rhizoma Coptidis all contain the berberine composition, and the effect of different Chinese goldthread processed products also has difference; And the Chinese medicine clinical efficacy such as Cortex Phellodendri, Radix Berberidis that contains the berberine composition also has difference; Medication puzzle by tcm clinical practice prescription, as far back as the Qing Dynasty " repairing the thing guide " just point out " process of preparing Chinese medicine fails to understand that the property of medicine is not true; then soup side do not have standard and disease do not test yet "; Therefore the present invention excavates herbal experience, systematic study wine steam Rhizoma Coptidis and " control and quench one's thirst " purposes and safety thereof, control metabolism syndrome, diabetes or the complication relevant with insulin resistant had certain meaning.
Summary of the invention
Technical scheme of the present invention has provided a kind of Wine-steamed Chinese goldthread processed product.Another technical scheme of the present invention has provided the method for preparing and the purposes of this processed product.
The invention provides a kind of Wine-steamed Chinese goldthread processed product; It is to form steaming in yellow wine or the Chinese liquor adding SHENGHUANG company; The finger printing of the Chinese goldthread processed product of preparation is as shown in Figure 1; The leading indicator composition comprises the hydrochlorate of jateorhizine, Columbamine, epiberberine, coptisine, palmatine, berberine and above alkaloid component thereof; Wherein, the weight proportion of jateorhizine, Columbamine, epiberberine, coptisine, palmatine, berberine is: 4-7: 5-9: 11-17: 23-35: 23-27: 100.
Further preferably, the weight proportion of jateorhizine, Columbamine, epiberberine, coptisine, palmatine, berberine is: 4.7-6.8: 5.7-8.3: 11.5-16.7: 23.9-34.5: 23.4-26.5: 100.
Further preferably, the weight proportion of described jateorhizine, Columbamine, epiberberine, coptisine, palmatine, berberine is: 5.6: 7.1: 14.8: 31.0: 24.5: 100.
Wine-steamed Chinese goldthread processed product of the present invention is to be prepared from following method: get clean Rhizoma Coptidis, add yellow wine or Chinese liquor moistening 1-6 hour, heating steams 3～8 hours, takes out, and drying promptly gets, and wherein the consumption proportion of Rhizoma Coptidis and yellow wine or Chinese liquor is:
20～120 parts of 100 parts of Rhizoma Coptidis, yellow wine or Chinese liquor.
Further preferably, described wine steams in the processing procedure of Rhizoma Coptidis, and per 100 portions of Rhizoma Coptidis are mixed thoroughly for 40 parts with wine, moistening 6 hours, and heating steams 8 hours, takes out drying.
The present invention also provides a kind of method for preparing described Chinese goldthread processed product, and it is to be prepared from following method: get clean Rhizoma Coptidis, added the yellow wine moistening 1-6 hour, heating steams 3～8 hours, takes out, and drying promptly gets, and wherein the consumption proportion of Rhizoma Coptidis and yellow wine is:
100 parts of Rhizoma Coptidis, 20～120 parts of yellow wine.
Further preferably, described wine steams in the processing procedure of Rhizoma Coptidis, and per 100 portions of Rhizoma Coptidis are mixed thoroughly for 40 parts with wine, moistening 6 hours, and heating steams 8 hours, takes out drying.
The present invention also provides described Chinese goldthread processed product to have the purposes in the medicine that suppresses adipose cell differentiation before the 3T3-L1, improves glucose utilization rate or blood fat reducing function in preparation.
Wherein, described medicine is to improve insulin resistant, reduces the medicine of glyconeogenesis.
Wherein, described medicine is the medicine of treatment diabetes and complication thereof.
The present invention also provides the purposes of this Chinese goldthread processed product in the medicine of preparation euglycemic agent and PPAR gamma agonist.Comparing with the article of giving birth to Rhizoma Coptidis, berberine, significantly increase PPAR γ expression, can significantly improve insulin resistant, is the euglycemic agent with DEVELOPMENT PROSPECT.
The present invention also provides described Chinese goldthread processed product purposes in the medicine of preparation alpha-glucosidase inhibitor.
The present invention also provides a kind of pharmaceutical composition that suppresses the preceding adipose cell differentiation of 3T3-L1, improves preceding adipose cell glucose utilization rate of 3T3-L1 or blood fat reducing function that has, and it is to be prepared from following method: the weighting profit requires the described wine of 1-4 to steam Rhizoma Coptidis, decocte with water 1～3 time; Each 0.5～3 hour, each amount of water was 6～10 times of amounts, filters; Merge extractive liquid; Concentrate, subsequent use, add acceptable accessories or complementary composition and be prepared into preparation pharmaceutically commonly used.
Further preferably, it is to be prepared from following method: get wine and steam Rhizoma Coptidis, and decocte with water 3 times, each 3 hours, each amount of water was 10 times of amounts, filtered, merge extractive liquid, concentrates, and is subsequent use.
Wherein, described preparation is an oral formulations.
Wine of the present invention steams Rhizoma Coptidis to be compared with giving birth to article Rhizoma Coptidis and berberine hydrochloride, has tangible effect, and drug effect is clear and definite, for clinical a kind of new selection is provided.
Below, foregoing of the present invention is remake further detailed description through the specific embodiment of embodiment form.But should this be interpreted as that the scope of the above-mentioned theme of the present invention only limits to following instance.All technology that realizes based on foregoing of the present invention all belong to scope of the present invention.
Description of drawings
Fig. 1 wine steams Rhizoma Coptidis sample HPLC collection of illustrative plates (wherein, 1-Jatrorrhizine chloride 2-Columbamine 3-epiberberine 4-coptisine 5-palmatine 6-berberine hydrochloride)
Rat blood serum HPLC finger printing after the administration of Fig. 2 wine steaming Rhizoma Coptidis (wherein, a: the blank serum chromatogram of reference substance chromatogram: b c: wine steams chromatogram in the Rhizoma Coptidis body, 1-Columbamine 2-epiberberine 3-coptisine 4-berberine)
Fig. 3 wine steam Rhizoma Coptidis extract extractum to 3T3-L1 before the influence of adipose cell model propagation
Adipose cell before adipose cell and 3T3-L1 adipose cell (* 200) A:3T3-L1 before the 3T3-L1 of Fig. 4 microscopically; The B:3T3-L1 adipose cell; C:3T3-L1 adipose cell (oil red O stain)
Fig. 5 wine steam Rhizoma Coptidis extract extractum to 3T3-L1 before the influence of adipose cell strain differentiation
Fig. 6 wine steams Rhizoma Coptidis and extracts the influence of extractum to 3T3-L1 adipose cell IR model glucose utilization
Fig. 7 wine steams the influence (Western blot method) that Rhizoma Coptidis is expressed PPAR γ
The specific embodiment
The preparation of embodiment 1 Wine-steamed Chinese goldthread processed product of the present invention
Get clean Rhizoma Coptidis, mix (the clean medical material of every 1kg is with yellow wine or Chinese liquor 0.2kg) thoroughly, put in the pot with wine, moistening 2 hours, heating steams 5 hours, takes out drying.
The preparation of embodiment 2 Wine-steamed Chinese goldthread processed products of the present invention
Get clean Rhizoma Coptidis, mix (the clean medical material of every 1kg is with yellow wine or Chinese liquor 0.4kg) thoroughly, put in the pot with wine, moistening 4 hours, heating steams 8 hours, takes out drying.
The preparation of embodiment 3 Wine-steamed Chinese goldthread processed products of the present invention
Get clean Rhizoma Coptidis, mix (the clean medical material of every 1kg is with yellow wine or Chinese liquor 0.6kg) thoroughly, put in the pot with wine, moistening 6 hours, heating steams 4 hours, takes out drying.
The preparation of embodiment 4 Wine-steamed Chinese goldthread processed products of the present invention
Get clean Rhizoma Coptidis, mix (the clean medical material of every 1kg is with yellow wine or Chinese liquor 0.8kg) thoroughly, put in the pot with wine, moistening 1 hour, heating steams 7 hours, takes out drying.
The preparation of embodiment 5 Wine-steamed Chinese goldthread processed products of the present invention
Get clean Rhizoma Coptidis, mix (the clean medical material of every 1kg is with yellow wine or Chinese liquor 1kg) thoroughly, put in the pot with wine, moistening 3 hours, heating steams 3 hours, takes out drying.
The processing procedure of embodiment 6 Wine-steamed Chinese goldthread processed products of the present invention is preferred
Get clean Rhizoma Coptidis, mix (the clean medical material of every 1kg is with yellow wine or Chinese liquor 1.2kg) thoroughly, put in the pot with wine, moistening 5 hours, heating steams 6 hours, takes out drying.
More than the Wine-steamed Chinese goldthread processed products of 6 Test Example preparation, measure total alkaloid content, carry out thin-layer chromatographic analysis simultaneously.
The total alkaloids assay method: get the about 0.8g of Golden Thread, the accurate title, decide. puts in the apparatus,Soxhlet's, and an amount of with hydrochloric acid-methanol (1: 100).Heating and refluxing extraction till extracting liquid colourless, is transferred to extracting solution in the 25mL measuring bottle, with a small amount of washing container of hydrochloric acid-methanol (1: 100), in washing liquid and the people's extracting solution, and adds to scale.According to column chromatography (appendix VI C) test, precision is measured 5mL, is added on the alumina column (the about 9mm of internal diameter, the neutral alumina 5g that have handled well; Wet method dress post is with the about 30mL prewashing of ethanol) on, with ethanol 35mL gradation eluting, put in the 50mL measuring bottle; Add ethanol dilution to scale, shake up, the accurate 2mL that draws; Put in the 50mL measuring bottle, (0.05mol/L) is diluted to scale with sulfuric acid solution, shakes up; Is blank according to spectrophotography with sulfuric acid solution (0.05mol/L), measures trap in the 345nm wavelength, presses berberine hydrochloride (C
20H
18ClNO
4) absorptance
Be 728 its content of calculating.
Table 1 Wine-steamed Chinese goldthread processed technology Uniform Design table and result
Content comprehensive grading=∑ (in the table 2 in each alkaloid/table 3 each alkaloid peak * 100)
Effect comprehensive grading=∑ (in the table 3 in each dose groups glucose utilization rate/table 3 each dose groups glucose utilization rate peak * 100)
The Wine-steamed Chinese goldthread processed technology assay of table 2 result
The Wine-steamed Chinese goldthread processed technology sample of table 3 is to the influence of 3T3-L1 cell model glucose utilization rate
(1) adopt statistical analysis software that content comprehensive grading result is carried out stepwise regression analysis showed, obtain regression equation:
Y＝570.02-0.41*X1+4.18*X2
P=0.019, regression equation have the significance meaning
(2) adopt statistical analysis software that content comprehensive grading result is carried out polynary secondary stepwise regression analysis, obtain regression equation 2:
Y＝574.45-0.17*X1-0.08*X3*X3-0.04*X1*X3+0.71*X2*X3
P=0.0021, regression equation have the significance meaning
Factor level combination during high target: yellow wine consumption 20%, 6 hours moistening time, steaming time 8 hours.
(3) adopt statistical analysis software that characteristic chemical constituent (Columbamine) content is carried out stepwise regression analysis showed, obtain regression equation 3:
Y＝0.4864-0.0109*X3
P=0.055, regression equation do not have the significance meaning
(4) adopt statistical analysis software pairing effect comprehensive grading result to carry out stepwise regression analysis showed, obtain regression equation:
Y＝211.69-0.63*X1
P=0.040, regression equation have the significance meaning
(5) adopt statistical analysis software pairing effect comprehensive grading result to carry out polynary secondary stepwise regression analysis, obtain regression equation:
Y＝179.68+0.41*X1-0.01*X1*X1+0.09*X1*X3-0.57*X2*X3
P=0.049, regression equation have the significance meaning
Factor level combination during high target: yellow wine consumption 60%, 1 hour moistening time, steaming time 8 hours.
By equation 1,2,3,4,5, in conjunction with intuitive analysis and cost effectiveness analysis, confirm that the technological parameter of wine steaming Rhizoma Coptidis is: get clean Rhizoma Coptidis, every 100kg is with yellow wine or Chinese liquor 40kg, and moistening 6 hours steams 8 hours, promptly gets.
Predictive value according to the content comprehensive grading of equation 2 is 584
Predictive value according to the effect comprehensive grading of equation 5 is 182
More than predicting the outcome shows, steams at the wine of confirming under the technological parameter of Rhizoma Coptidis, and the result of content and effect is more satisfactory, and also the factor level with Uniform Design numbers 2 is provided with close.
Therefore the technological parameter of confirming wine steaming Rhizoma Coptidis is: get clean Rhizoma Coptidis, every 100kg is with yellow wine or Chinese liquor 40kg, and moistening 6 hours steams 8 hours, promptly gets.
Embodiment 7 wine of the present invention steam the method for quality control of Rhizoma Coptidis
Wine steams the method for quality control of Rhizoma Coptidis
(1) thin layer chromatography is differentiated
These article of getting powder 0.1g adds methanol 50ml, supersound process 30min, and filtrating is as need testing solution.Get Jatrorrhizine chloride and add methanol according to article, Columbamine, hydrochloric acid epiberberine, hydrochloric acid coptisine, palmatine hydrochloride and berberine hydrochloride and process the solution that every 1ml contains 0.1mg, as reference substance solution.Precision is measured each reference substance solution 1.5ml and is added in the same 10ml measuring bottle, is diluted to scale with methanol, as mixing reference substance solution.Test according to thin layer chromatography (an appendix VI of Chinese Pharmacopoeia version in 2005 B); Draw each 10 μ l of above-mentioned three kinds of solution, put respectively on same silica gel g thin-layer plate, with cyclohexane extraction-ethyl acetate-isopropyl alcohol-methanol-ammonia-triethylamine (3: 3.5: 1: 1.5: 0.5: 1) and with behind the ammonia presaturation 20min; Launch; Take out, dry, put under the ultra-violet lamp (365nm) and inspect.In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the speckle of same color.
(2) assay
Method: adopt XtimateTM C18 (4.6mm * 250mm, 5 μ m) chromatographic column, be mobile phase A with 0.1% triethylamine (ammonium bicarbonate, it is 10 that ammonia is transferred PH), acetonitrile is a Mobile phase B, carries out gradient elution; Flow velocity: 1mL.min-1, column temperature: 30 ℃, detect wavelength 270nm.The result: Jatrorrhizine chloride is at 0.848 μ g.mL
-1～16.96 μ g.mL
-1(r=0.9997), Columbamine is at 1.248 μ g.mL
-1～24.96 μ g.mL
-1(r=0.9999), epiberberine is at 2.048 μ g.mL
-1～40.96 μ g.mL
-1(r=0.9999), coptisine is at 3.648 μ g.mL
-1～72.96 μ g.mL
-1(r=0.9999), palmatine is at 2.88 μ g.mL
-1～57.6 μ g.mL
-1(r=0.9998), berberine hydrochloride is at 13.248 μ g.mL
-1～264.96 μ g.mL
-1, (r=0.9996) present good linear relationship.Average recovery and RSD thereof are respectively: 102.4% (RSD is 1.17%), 101.8% (RSD is 1.30%), 100.3% (RSD is 1.76%), 100.7% (RSD is 1.75%), 101.2% (RSD is 1.53%) and 97.9% (RSD is 1.97%).Sample repeatability is good, and is stable in 12h.Conclusion: method is easy and simple to handle, accuracy is high, good stability, can be used for wine and steams 6 kinds of alkaloids content mensuration in the Rhizoma Coptidis.
Table 4 wine steams the assay result of Rhizoma Coptidis
Above assay The data SPSS statistical software is carried out the comparative analysis (Independent-Samples T Test) of two sample means; Statistic analysis result shows that the content of Columbamine in the wine steaming Rhizoma Coptidis and the article of giving birth to decoction pieces relatively have significance to change (P=0.017) (finger printing is seen Fig. 1); Simultaneously the serum chemistry finger printing find to go in the blood component chromatographic peak of Columbamine obvious; Columbamine possibly be that wine steams one of important component relevant with the drug effect biological effect in the Rhizoma Coptidis, see Fig. 2.
Steam each experiment of Rhizoma Coptidis technology number mensuration result's (table 2) by wine among the embodiment 6, steam Rhizoma Coptidis in conjunction with each batch wine among the embodiment 7 and measure result's (table 4), merge each formation table 5 as a result
Table 5 wine steams Rhizoma Coptidis assay result (mg/g)
Table 6 wine steams Rhizoma Coptidis assay result (ratio content is 100 calculating with content of berberine hydrochloride)
Press 99% confidence interval (lower limit, higher limit) of statistical significance, U
α/2=2.56 (α=0.01): lower limit=meansigma methods-U
α/2* standard deviation, higher limit=meansigma methods+U
α/2* standard deviation
In sum; Wine steams in the Rhizoma Coptidis, and the weight proportion of jateorhizine, Columbamine, epiberberine, coptisine, palmatine, berberine is: 4.74-6.84: 5.67-8.32: 11.54-16.66: 23.89-34.49: 23.38-26.46: 100 or: 4-7: 5-9: 11-17: 23-35: 23-27: 100
(3) chemical fingerprint
The principal character peak that wine steams the Rhizoma Coptidis chemical fingerprint comprises jateorhizine, Columbamine, epiberberine, coptisine, palmatine, berberine, also comprises the hydrochlorate (Fig. 1) of above alkaloid component.
The optimum chromatogram condition that wine steams Rhizoma Coptidis HPLC finger printing is: Xtimate
TMC
18(4.6 * 250mm, 5 μ m); 30 ℃ of column temperatures; Flow velocity 1.0ml/min; Detect wavelength: 270nm; Mobile phase: the aqueous solution of 0.1% triethylamine (ammonium bicarbonate, ammonia is regulated PH to 10)-acetonitrile (binary gradient elution); Sample size: 10ul; Sampling time: 45min, elution program is following:
Mobile phase: A pump: the aqueous solution of 0.1% triethylamine (ammonium bicarbonate, ammonia is regulated PH to 10); B pump: acetonitrile.
Temporal sequence: 0～15min, A pump: 90% → 75%; B pump: 10% → 25%
15～25min, A pump: 75% → 70%; B pump: 25% → 30%
25～40min, A pump: 70% → 55%; B pump: 30% → 45%
40～50min, A pump: 55% → 90%; B pump: 45% → 10%
Wine steams Rhizoma Coptidis need testing solution method for preparing: get wine and steam Golden Thread 0.1g, the accurate title, decide, and puts in the conical flask, adds hydrochloric acid-methanol (1: 100) 50ml, and supersound process 30min filters, and crosses the 0.45um microporous filter membrane, gets subsequent filtrate, promptly gets.
Wine steams Rhizoma Coptidis water extraction extractum need testing solution method for preparing: get wine and steam Rhizoma Coptidis 25g, the accurate title, decide, and puts in the beaker, adds water 150ml, decocts 3h, decocts 3 times, merges decocting liquid, is concentrated into the 1g/g concentrated solution.Get the 0.1g concentrated solution, the accurate title, decide, and puts in the conical flask, adds hydrochloric acid-methanol (1: 100) 50ml, and supersound process 30min filters, and crosses the 0.45um microporous filter membrane, gets subsequent filtrate, promptly gets.
(4) serum chemistry finger printing
The principal character peak that wine steams in the Rhizoma Coptidis serum chemistry finger printing comprises Columbamine, epiberberine, coptisine, berberine, also comprises the hydrochlorate (Fig. 2) of above alkaloid component.
The whole animal model is irritated the preparation of stomach sample: get wine and steam Rhizoma Coptidis 100g, add water 600ml, decoct 2h, decoct 3 times, merge decocting liquid, be concentrated into 1g/ml, promptly get and irritate the stomach sample.
Test method: get 5 of SD rats.Fasting 12h (freely drinking water) weighs, and irritate stomach according to 5g/kg and give wine steaming Rhizoma Coptidis concentrated solution (1g/ml), 180min after the administration, femoral artery is got blood, 37 ℃ of water-bath 15min, the centrifugal 15min of 3600rpm, separation of serum is pastille serum.Get each time point pastille serum 1ml and place 5ml centrifuge tube with cover respectively, add acetonitrile 3ml vortex 3min respectively, the centrifugal 10min of 3600r/min; Get supernatant, dry up, add 50% methanol, 75 μ l in 37 ℃ of Nitrogen evaporators; Vortex 3min, the centrifugal 10min of 120000r/min promptly gets.
Chromatographiccondition with " chemical fingerprint " item down.
(5) its controlling index
| Controlling index | Moisture | Total ash | Acid-insoluble ash | Ethanol-soluble extractives |
| Meansigma methods (n=10) | 9.10％ | 3.13％ | 0.81％ | 29.63％ |
The preparation technology of experimental example 7 Wine-steamed Chinese goldthread processed products extraction extractum of the present invention is preferred
According to Uniform Design and decocting extraction process characteristics, investigate three factors such as amount of water, extraction time, extraction time, select the uniform designs table U of mixed-level
6(6 * 3
2).
Take by weighing Rhizoma Coptidis 10g, add water to ormal weight, decocting extracts the stipulated time, extracts stipulated number, and extracting solution is concentrated into 10mL.
Table 7 wine steams the influence factor of Rhizoma Coptidis decocting extraction process and is horizontally disposed with
Table 8 wine steams the Uniform Design harmony in the exterior result of Rhizoma Coptidis decocting extraction process
(1) adopt the DPS statistical software that the total alkaloid content result is carried out stepwise regression analysis showed, obtain regression equation 1:
Y＝1.6680+0.3209*X1+0.5200*X3
P=0.0121; Regression equation has the significance meaning
(2) adopt the DPS statistical software that the total alkaloid content result is carried out polynary secondary stepwise regression analysis, obtain regression equation 2:
Y＝2.1836+0.1235*X1*X3+0.0044*X2*X3
P=0.0164; Regression equation has the significance meaning
Factor level combination during high target: amount of water is 100ml, and extraction time is that 3 hours, extraction time are 3 times
Can know that by equation 1,2 the remarkable factor that Rhizoma Coptidis decocting extraction process is influenced is extraction time, extraction time, secondly is amount of water; From the parameters optimization of having confirmed wine steaming Rhizoma Coptidis decocting extraction process be: get clean Rhizoma Coptidis; Decocte with water 3 times, each 3 hours, each amount of water was 10 times of amounts; Filter merge extractive liquid.
2.3.3 verification method and result
Press the optimization decocting extraction process parameter of confirming under the 2.3.2 item, carry out demonstration test, can be known by table 9, wine steams the decocting extraction process of Rhizoma Coptidis and stablizes feasible.
Table 9 wine steams the demonstration test harmony in the exterior result of Rhizoma Coptidis decocting extraction process
Experimental example 8 wine of the present invention steam the preparation that Rhizoma Coptidis is extracted extractum
According to the optimization technology that experimental example 6,7 provides, extraction has prepared wine and has steamed Rhizoma Coptidis extraction extractum confession pharmacological evaluation, on the basis that total alkaloids is measured, has also measured the content of total polysaccharides.According to the optimization technology that experimental example 7 provides, also extract the extraction extractum that has prepared living article Rhizoma Coptidis and supply pharmacological evaluation, on the basis that total alkaloids is measured, also measured the content of total polysaccharides.
The assay method of total alkaloids: precision takes by weighing extractum 0.4g, to the 25mL measuring bottle, adds ethanol dilution to scale, shakes up, according to the column chromatography test.Precision is measured 5mL, puts on the alumina column of having handled well (wet method dress post is with ethanol 30mL prewashing for the about 0.9cm of internal diameter, neutral alumina 5g), with ethanol 35mL eluting, collects eluent and puts in the 50mL measuring bottle,, shakes up to scale with ethanol dilution.Precision is measured in the 2mL transposition 50mL measuring bottle, with 0.05mol/l H2SO4 solution, is diluted to scale, shakes up.According to ultraviolet spectrophotometry, in the 345nm wavelength, measure trap, be 728 to calculate by the absorptance of berberine hydrochloride, promptly get.
The assay method of total polysaccharides: precision takes by weighing the yellow processed product article that extracted and extracts extractum, puts in the centrifuge tube, adds dehydrated alcohol to 80%, mixing; Centrifugal 10min (3000r/min), abandoning supernatant, with the repeatedly washing precipitation of 80% ethanol, colourless to centrifuged supernatant; Deposition makes dissolving with the boiling water gradation, and is centrifugal, and supernatant shifts and puts in the 100mL measuring bottle; The accurate 5mL of absorption shifts and puts in the 25mL measuring bottle, and thin up is to scale, as test sample.Draw need testing solution, glucose reference substance solution (0.06mg/mL) 2mL respectively to tool plug test tube, add anthrone reagent 8mL, vortex 30s; Boiling water 10min; Be cooled to room temperature, measure its absorbance at the 620nm place, calculate the content of the Rhizoma Coptidis total polysaccharides of calculating with glucose.
Table 10 wine steams Rhizoma Coptidis and extracts extractum total alkaloids/total polysaccharides mensuration result
Can know that by last data total alkaloid content is starkly lower than the article of giving birth to Rhizoma Coptidis in the wine steaming Rhizoma Coptidis extraction extractum.
Below prove beneficial effect of the present invention through pharmacodynamics test.
Test Example 1 wine steam Rhizoma Coptidis extract extractum to 3T3-L1 before the value-added influence of adipose cell
The adipose cell strain is the cell strain that mice embryonic is originated, had the fibroblast form before the 3T3-L1; Hormone cocktail at classics; Be under insulin, dexamethasone and 1-methyl-xanthic stimulation of 3-isobutyl group; Having the ability that is divided into mature fat cell, is the cell model of studying fat and external lipocyte proliferation and differentiation at present in the world.Observe its influence through adipose cell before intervening mice, for the treatment of the type 2 diabetes mellitus of obesity provides possible approach to this cell proliferation and differentiation.
Method: adipose cell suspension before the 3T3-L1 is pressed 5 * 10
5The density of/ml is inoculated into 48 well culture plates, in the DMEM high glucose medium that contains 10% calf serum, at 37 ℃, 5%CO
2, cultivate under the saturated humidity condition, 2d changes liquid 1 time.After treating that the preceding adipose cell of 3T3-L1 is adherent, the reagent thing that receives that adds variable concentrations by design dosage is respectively intervened, and adds equal-volume drug solvent PBS in the blank group culture fluid, and every concentration group is all established 6 multiple holes.At 37 ℃, volume fraction 5%CO
2The middle 2d that cultivates.Behind the 48h, the PBS rinsing adds high sugared culture fluid 400 μ l of DMEM and 5mg/ml MTT 40 μ l, hatches 4h.Culture fluid is removed in suction, and adds solvent DMSO 300 μ l, and 490nm measures the OD value of each group.Calculate the preceding lipocyte proliferation rate of change of 3T3-L1.
Relative change rate (%)=(the average OD value of the experimental group/average OD value-1 of blank group) * 100%
Table 11 wine steam Rhizoma Coptidis extract extractum to 3T3-L1 before the influence of lipocyte proliferation
The rate of increase 1: the propagation rate of change of the relative blank control group of each dose groups;
The rate of increase 2: wine steaming group is with respect to the propagation rate of change of giving birth to article with dose groups.
Annotate: compare with the blank group,
*P＜0.05,
*P＜0.01;
Can know that by table 11, Fig. 3 wine steams Rhizoma Coptidis and promotes the preceding lipocyte proliferation effect of 3T3-L1 to be superior to coptis root.
Test Example 2 wine steam Rhizoma Coptidis and extract extractum to the influence to adipose cell differentiation before the 3T3-L1
Method: adipose cell suspension before the 3T3-L1 is pressed 5 * 10
5The density of/ml is inoculated into 48 well culture plates, in the DMEM high glucose medium that contains 10% calf serum, at 37 ℃, 5%CO
2, cultivate under the saturated humidity condition, 2d changes liquid 1 time.After treating that the preceding adipose cell of 3T3-L1 reaches contact inhibition; DMEM complete culture solution with the Dex, 0.5mmol/L IBMX and the 5mg/L insulin that contain 1 μ mol/L is induced differentiation; Remove Dex and IBMX behind the 48h,, change normal complete culture solution and cultivated 8～12 days with 10mg/L insulin continuation effect 48h again; Change liquid every other day, when 3T3-L1 cell more than 90% is the adipose cell phenotype.When inducing differentiation, the reagent thing that receives that adds variable concentrations by design dosage is respectively intervened, and adds equal-volume drug solvent PBS in the blank group culture fluid, and every concentration group is all established 6 multiple holes.Medicine and differentiation culture liquid are changed to the differentiation end synchronously.After inducing differentiation to finish, inhale and remove culture fluid, cell with 4% paraformaldehyde solution fixed cell 10min, is inhaled and is removed fixative after PBS cleans 3 times, and the PBS rinsing adds oil red O dye liquor room temperature dyeing 15min.Discard oil red O dye liquor, the PBS rinsing is removed unnecessary dye liquor 3 times; Put microscopically and observe, film making; Add isopropyl alcohol, oil red O in the extracting adipose cell, 510nm measures the OD value, calculates the cell differentiation relative change rate.
Differentiation relative change rate (%)=(the average OD value of the experimental group/average OD value-1 of blank group) * 100%
Adipose cell is fusiformis before the undifferentiated 3T3-L1, and no fat drips existence in the endochylema, the similar (Fig. 4-A) with fibroblast of form.Adipose cell induces differentiation to become sophisticated adipose cell under the combined effect of dexamethasone, 3-isobutyl-1-methylxanthine and insulin gradually before the 3T3-L1, and occur fat in the endochylema and drip, and gather along with the intensification of differentiation degree and to increase (Fig. 4-B).Oil red O is fatty characteristic stain, and the fat in the endochylema of dyeing back drips and take on a red color (Fig. 4-C).
Table 12 wine steam Rhizoma Coptidis extract extractum to 3T3-L1 before the influence
of adipose cell strain differentiation
Annotate: compare * P＜0.05, * * P＜0.01 with the blank group;
By table 12, Fig. 5 can know, wine steams Rhizoma Coptidis has 2 dose groups obviously to suppress the differentiation (P＜0.01) of adipose cell before the 3T3-L1, and this and the effect of positive drug rosiglitazone maleate are just the opposite; Simultaneously, compare with coptis root, wine steams Rhizoma Coptidis and suppresses differentiation rate all apparently higher than coptis root.
Above-mentioned experimental result shows; Wine steams Rhizoma Coptidis suppresses adipose cell before the 3T3-L1 under doses differentiation; The prompting Wine-steamed Chinese goldthread processed product maybe not can cause the gathering of fat and causes weight increase; This has certain meaning to control metabolism syndrome or the complication relevant with insulin resistant, and its effect is superior to coptis root.
Test Example 3 wine steam Rhizoma Coptidis extract extractum to 3T3-L1 before the influence of adipose cell insulin resistant (IR) model glucose utilization
Method: after cell differentiation is complete, removes blank control group and add the normal cultured base, all the other each groups all add the dexamethasone of 1 μ mol/L, the insulin of 1 μ mol/L carries out the insulin resistant of adipose cell, acts on 3d.After treating that the adipose cell insulin resistant is set up; Use no phenol red DMEM high glucose medium instead; The reagent thing that receives that adds variable concentrations by design dosage is respectively intervened 3d; Add equal-volume drug solvent PBS in the blank group culture fluid, every concentration group is all established 6 multiple holes, observes the improvement effect of medicine to insulin resistant.Get the content of cell culture supernatant 505nm colorimetric determination glucose behind the 72h, calculate grape cell sugar and utilize the relative change rate.
Table 13 wine steams Rhizoma Coptidis and extracts the influence of extractum to 3T3-L1 adipose cell IR model glucose utilization
Annotate: compare with model control group,
*P＜0.05,
*P＜0.01;
By table 13, Fig. 6 can know, compares with blank control group; The 3T3-L1 adipose cell is under dexamethasone and insulin action; Model group obviously reduces the sensitivity of insulin, and the picked-up of glucose obviously reduces in the culture fluid, shows adipose cell insulin resistant model modeling success (P＜0.01).Compare with model group; Wine steam Rhizoma Coptidis can be obviously or part reduce the content (P＜0.05～0.01) of glucose in the culture fluid; Improve the utilization rate of adipose cell glucose, improve insulin resistant, act on similar with the positive drug rosiglitazone maleate; Simultaneously, wine steams Rhizoma Coptidis has the glucose utilization relative change rate of 3 dose groups to be higher than coptis root.
Test Example 4 wine steam Rhizoma Coptidis extract extractum to 3T3-L1 before the influence of adipose cell PPAR γ
(Peroxisome Proliferator ActivatedReceptors, PPARs), PPARs comprises three kinds of hypotypes such as PAR α, PPAR γ and PPAR δ to the peroxisome proliferator activated receptor.PPAR γ then mainly expresses in liver and fat, muscular tissue, and promote that differentiation, the fatty acid of fatty tissue is synthetic and store, be adipogenic key substance.PPAR γ can further activate the adipose cell atomization with after part or activator combine, and participates in regulating expression, the lipid of adipose cell specific gene and stores and metabolism.Typical medicaments in the PPAR gamma agonist is a rosiglitazone at present; It is the PPAR part of synthetic; It has insulin sensitivity enhancing and hypoglycemic activity, propagation and migration that can be through activating PPAR γ performance improvement insulin resistant (IR), blood sugar lowering, inhibition vascular smooth muscle in the target tissue, improves various biological effect such as endothelial function.
To adipose cell model before the 3T3-L1, the reagent thing that receives that adds variable concentrations by design dosage is respectively intervened, and Western blot detects PPAR γ expression, and the result shows: compare with the article of giving birth to, wine steams Rhizoma Coptidis significantly rises the expressing quantity of PPAR γ, sees Fig. 7.
Adipose cell is through the berberine intervention before the In vitro culture 3T3-L1; In 3T3-L1 adipose cell atomization, the PPAR γ mRNA of berberine group adipose cell is low by 48% than matched group, and the protein immunoblotting result shows that also berberine suppresses the expression [Zhou Libin of PPAR γ; Yang Ying; Tang Jinfeng, etc. berberine is to the glycometabolic influence of adipose cell. the journal .2002 of Shanghai Second Emdical University, 22 (5): 412-414].And the increase that PPAR γ expresses can promote the utilization of glucose and the enhanced sensitivity of insulin [Cheng Feng, Jiang Hualiang; Chen Kaixian waits the progress of .PPAR gamma agonist. Chinese pharmaceutical chemistry magazine 2003,13 (2): 110-118; 124] Liu Yi; Lou Shaoying, He Yanming, etc. berberine to 3T3-L1 before the influence of lipocyte proliferation and differentiation associated gene PPAR γ, what protein expression of C/EBP α mRNA. Chinese combination of Chinese and Western medicine magazine .2008; 28 (11): 10051009, berberine makes PPAR γ expression reduce 82%.
Press BandScan5.0, default value:
| Numbering | Wine steams Rhizoma Coptidis | The article of giving birth to Rhizoma Coptidis |
| PPAR γ expresses | 35284 | 3343 |
| Confidential reference items are expressed | 8355 | 13473 |
| Proofreading and correct PPAR γ expresses | 4.22 | 0.25 |
| Barren relatively correction PPAR γ expresses | 0.68 | 0.04 |
| The correction PPAR γ that gives birth to article relatively expresses | 17.02 | 1.00 |
The result shows after confidential reference items are proofreaied and correct, and compares with the article of giving birth to Rhizoma Coptidis, and wine steams Rhizoma Coptidis makes PPAR γ expression increase by 17.02 times.
Above document shows with the result of test: with give birth to article Rhizoma Coptidis, berberine is different, wine steaming Rhizoma Coptidis obviously increases the expression of PPAR γ, can promote the utilization of glucose and the enhanced sensitivity of insulin, promises to be one type of type 2 diabetes mellitus medicine.
This tests explanation, and cornel of the present invention is processed Rhizoma Coptidis and can be used as the euglycemic agent use, and euglycemic agent is meant thiazolidinedione (TZDs) derivant, claims rosiglitazone again.Its effect for reducing fat of nineteen eighty-two research finds to also have the losing weight and reducing blood sugar effect simultaneously.It mainly works through combination and activation peroxisome proliferator-activated receptor PPAR γ; The PPAR γ receptor back that is activated generates enzyme and regulates the expression of GAP-associated protein GAP with carbohydrate metabolism through induced lipolysis; Promote the differentiation of adipose cell and other cells; And improve the sensitivity of cell to insulin action, alleviate insulin resistant, so be called as euglycemic agent.(Zhang Wen, the developing history of treatment diabetes medicament, " Chinese licensed pharmacist "; 2008 2 phases) euglycemic agent also comprises the retinoid receptor agonist except comprising the euglycemic agent agonist, (Cai Xiaohua such as beta 3 receptor agonist; Deng, insulin sensitizer diabetes medicament progress, Huaihua are cured special journal; 2002,1 (2).
Above-mentioned result of the test shows that wine steams Rhizoma Coptidis and has the 3T3-L1 of improvement adipose cell insulin resistant, strengthens the ability of adipose cell to glucose uptake and utilization, has the effect that improves insulin resistant from the cell in vitro level; Simultaneously; Wine steams Rhizoma Coptidis suppresses adipose cell before the 3T3-L1 under doses differentiation; Prompting wine steams Rhizoma Coptidis and when the increase cell is to glucose uptake, can not cause the gathering of fat and cause weight increase that this has certain meaning to control metabolism syndrome or the complication relevant with insulin resistant.
Test Example 5 wine steam Rhizoma Coptidis and extract the influence of extractum to diabetes whole animal model
The design dosage of table 14 whole animal test
The influence of 1 pair of mice euglycemia
(1) test method
Get 70 of ICR mices, male and female half and half, body constitution amount 18～22g.Be divided into 7 groups by body constitution amount stratified random, 12 every group, promptly normal control group, metformin matched group, berberine hydrochloride matched group, coptis root group, wine steam the high, medium and low dose groups of Rhizoma Coptidis.Each organizes mice by table 14 design dosage gastric infusion (every quantitative 0.3ml of mice), every day 1 time, continuous 7 days.45min after the last administration (water 8h is can't help in fasting), mouse orbit is got blood, the centrifugal 10min of 3000r/min, separation of serum is pressed the kit measurement description and is measured mice fasting glucose (FBG).
(2) result of the test
Can know that by table 15 result wine steams each dose groups of Rhizoma Coptidis does not all have obviously influence (P＞0.05) to normal mouse fasting glucose (FBG).
The influence of table 15 couple normal mouse FBG
2 pairs of high concentration glucoses cause the influence of chmice acute blood sugar increasing
(1) test method
Get 80 of ICR mices, male and female half and half, body constitution amount 18～22g.Be divided into 8 groups by body constitution amount stratified random, 12 every group, promptly normal control group, model control group, metformin matched group, berberine hydrochloride matched group, coptis root group, wine steam the high, medium and low dose groups of Rhizoma Coptidis.Each organizes mice by table 14 design dosage gastric infusion (every quantitative 0.3ml of mice), every day 1 time, continuous 7 days.10min (water 8h is can't help in fasting) after the last administration in the 7th day, mouse peritoneal injection high concentration glucose injection 2g/kg (0.1ml/ is only) duplicating model, blank control group gives isometric normal saline.The blood glucose (Glu) of 120min after the mensuration modeling.
(2) result of the test
Can know by table 16, compare, cause the acute rising of fasting glucose immediately behind the model group mouse peritoneal injection high concentration glucose with blank control group.
Compare with model group, wine steams the Rhizoma Coptidis height, middle dose groups all can obviously reduce the mouse blood sugar rising (P＜0.01) that high dense glucose causes; Simultaneously, compare with coptis root, the blood sugar reducing function that wine steams Rhizoma Coptidis obviously is superior to coptis root, and effect is superior to berberine hydrochloride.
Table 16 pair high concentration glucose causes the influence of chmice acute blood sugar increasing
The inhibitory action of 3 pairs of normal mouse alpha-glucosidases
(1) test method
Get 80 of ICR mices, male and female half and half, body constitution amount 18～22g.Be divided into 8 groups by body constitution amount stratified random, 12 every group, promptly normal control group, model control group, metformin matched group, berberine hydrochloride matched group, coptis root group, wine steam the high, medium and low dose groups of Rhizoma Coptidis.Each organizes mice by table 14 design dosage gastric infusion (every quantitative 0.3ml of mice), every day 1 time, continuous 7 days.The 7th day, animal fasting 8h measured mice fasting glucose (0min), respectively organizes mice administration 1 time after the mensuration, and irritated clothes sucrose solution 5g/kg (50g-100ml, 0.3ml/ are only) duplicating model immediately, and blank control group gives isometric normal saline.After measure irritating before the stomach (0min) and irritating stomach 30,60 and 120min blood glucose, calculate each time point changes of blood glucose rate, adopt area (AUC) under the computing formula calculated curve of approximate trapezoid.
(2) result of the test
The influence of table 17 pair normal mouse alpha-glucosidase
Can know by table 17 result; Wine steams Rhizoma Coptidis high dose group and berberine hydrochloride group can obviously suppress to irritate the blood sugar increasing of obeying sucrose and causing (P＜0.05-0.01); Act on similarly, show that medicine has certain inhibitory action to alpha-glucosidase with acarbose, maybe with the absorption that suppresses the starch based carbohydrate; It is relevant to reduce post-prandial glycemia, and its effect possibly be that wine steaming Rhizoma Coptidis is controlled one of approach of quenching one's thirst.
4 pairs of alloxan cause the influence of diabetic mice glyconeogenesis
(1) test method
Get 80 of ICR mices, male and female half and half, body constitution amount 18～22g.Be divided into 8 groups by body constitution amount stratified random, 10 every group, promptly normal control group, model control group, metformin matched group, berberine hydrochloride matched group, coptis root group, wine steam the high, medium and low dose groups of Rhizoma Coptidis.Mice fasting 18h (advising 3 fasting in preceding 1 day afternoon, 9 modelings morning on the 2nd) before the test.Morning on the 2nd mouse tail vein injection alloxan solution 60mg/kg modeling, injection back 4h model mice is irritated stomach 50% glucose, 0.4ml/ only.Modeling next day, except that blank control group and model group, all the other each groups are pressed table 14 dosage gastric infusion (every quantitative 0.3ml of mice), every day 1 time, continuous 7 days.45min (water 8h is can't help in fasting) after the last administration in the 7th day, lumbar injection L-alanine (2g/kg) is measured injection preceding (0min) and injection back 60 and 120min blood glucose, adopts the computing formula of approximate trapezoid to calculate AUC.
(2) result of the test
Can know by table 18, compare with blank control group, behind the model group mouse tail vein injection alloxan 72h; Symptoms such as polyphagia, polydipsia, polyuria all appear; Model group fasting glucose content obviously raises, and shows that alloxan causes mice diabetes model modeling success, behind the model group lumbar injection L-alanine; Blood glucose raises immediately, model group glyconeogenesis unusual obviously (P＜0.01).
Compare with model group, Wine-steamed Chinese goldthread processed product can obviously reduce the blood sugar increasing (P＜0.01) that mouse peritoneal injection L-alanine causes, shows that medicine can alleviate the unusual of glyconeogenesis, possibly be that wine steaming Rhizoma Coptidis is controlled one of approach of quenching one's thirst.
5 pairs of alloxan cause the influence of diabetic mice
(1) test method
Get 80 of ICR mices, male and female half and half, body constitution amount 18～22g.Be divided into 8 groups by body constitution amount stratified random, 10 every group, promptly normal control group, model control group, metformin matched group, berberine hydrochloride matched group, coptis root group, wine steam the high, medium and low dose groups of Rhizoma Coptidis.Mice fasting 18h (advising 3 fasting in preceding 1 day afternoon, 9 modelings morning on the 2nd) before the test.Morning on the 2nd mouse tail vein injection alloxan solution 60mg/kg modeling, injection back 4h model mice is irritated stomach 50% glucose, 0.4ml/ only.Modeling next day, except that blank control group and model group, all the other each groups are pressed table 14 dosage gastric infusion (every quantitative 0.3ml of mice), every day 1 time, continuous 9 days.
(2) observation index
1) to the influence of mice glucose carbohydrate tolerance (OGTT)
The 7th day, animal fasting 8h, after the last administration 10 minutes; Irritate the stomach glucose (3g/kg, 10ml/kg), measure irritate before the stomach (0min) and irritate stomach after 30; 60 and 120min blood glucose, calculate each time point changes of blood glucose rate, adopt area (AUC) under the computing formula calculated curve of approximate trapezoid.
2) to the biochemical influence of mouse blood
120min (water 8h is can't help in fasting) after the last administration in the 9th day; Mouse orbit is got blood; The centrifugal 10min of 3000r/min; Separation of serum is measured the content of mice glycolated hemoglobin (GHb), saccharifying serum albumin (GSP), fasting glucose (FBG), blood lactic acid (LD), serum cholesterol (TC) and triglyceride (TG) respectively by the kit measurement description.
3) to the influence of murine liver tissue hepatic glycogen
Mice is taken out hepatic tissue rapidly after putting to death, and weighs, and is frozen subsequent use in the Caused by Metastasis liquid nitrogen.Prepare liver tissue homogenate by hepatic glycogen test kit description, measure the Mouse Liver content of glycogen.
(3) result of the test
1) to the influence of mice glucose carbohydrate tolerance (OGTT)
Can know by table 19; Compare with blank control group; Behind the model group mouse tail vein injection alloxan 72h; Symptoms such as polyphagia, polydipsia, polyuria all occur, model group mice saccharification hemoglobin content and fasting glucose content obviously raise, and show that alloxan causes mice diabetes model modeling success (P＜0.01).
With model group relatively, Wine-steamed Chinese goldthread processed product can obviously reduce unusual (P＜0.01) of mice carbohydrate tolerance, and shows certain ageing with the prolongation of administration time.
2) to the biochemical influence of mouse blood
Can know by table 20, compare that the content of model group mice glycolated hemoglobin (GHb), saccharifying serum albumin (GSP), fasting glucose (FBG), blood lactic acid (LD), serum cholesterol (TC) and triglyceride (TG) obviously raises with blank control group.Compare with model group, wine steam Rhizoma Coptidis can be obviously or part reduce the content (P＜0.05-0.01), improve the unusual of diabetic mice blood biochemical of GHb, GSP, FBG, LD, TC and TG.Especially wine steaming Rhizoma Coptidis can obviously reduce the unusual of model mice serum lactic acid, and the effect that increases blood lactic acid with the positive drug metformin is opposite, and prompting wine steams Rhizoma Coptidis can not cause ketoacidosis in blood sugar lowering side effect.
In sum, wine steams Rhizoma Coptidis and can bring into play hypoglycemic activity to the damage of beta Cell of islet or the function of improving the β cell of damaged through weakening alloxan.
Brief summary: wine steams Rhizoma Coptidis and has the tangible effect that improves diabetes, but euglycemia is not had obvious influence, and it acts on and improves insulin resistant, reduces glyconeogenesis, and it is relevant to suppress the alpha-glucosidase effect.
Test Example 6 wine steam Rhizoma Coptidis and extract extractum to acute toxicity test
The grouping of table 21 medicine and dosage design
180 of ICR mices, male and female half and half are divided into 18 groups by body constitution amount stratified random; Divide into groups and dosage press table 21 design, every mice is all by 20ml/kg administration (water 8h is can't help in fasting), observes after the mice administration poisoning symptom of appearance in 14 days respectively; Death condition and death time, body weight change and feed situation; Dead mice is dissected immediately, and the perusal record is the organ and the pathological change situation of infringement mainly, and main organs is stored in 10% formalin.Check the death toll of each treated animal at last, calculate the LD of each medicine
50
(3) result of the test
The acute toxicity tests shows, gives birth to the LD of article Rhizoma Coptidis
50Be 4.4679g/kg, use according to " Chinese Pharmacopoeia 2005 editions " Rhizoma Coptidis human clinical and recommend maximum (body mass press 60kg) calculating in 5g/ days that i.e. 0.0833g/kgd is equivalent to 53.6 times of human body dosage; The LD50 that wine steams Rhizoma Coptidis is 7.5475g/kg, is equivalent to 90.6 times of human body dosage, approaches clinical safety dosage (be equivalent to human body 100 times), and Rhizoma Coptidis wine steams to be concocted back toxicity and obviously reduces.
Above result of study shows that total alkaloid content and total polysaccharides content are higher than the processed with vinegar Rhizoma Coptidis in the living article Rhizoma Coptidis.But in the test of pesticide effectiveness; Wine steams the result of study of Rhizoma Coptidis on isolated cells model, two levels of whole animal model and shows that its effect is superior to giving birth to the article Rhizoma Coptidis; Explain that should steam Rhizoma Coptidis with wine in the tcm clinical practice treats " diabetes " (diabetes); Embodied the tcm clinical practice administration features of " concocting ", for the suitable processed product of selection of clinical provides experimental basis with disease.
Claims (2)
1. Wine-steamed Chinese goldthread processed product is characterized in that: described wine steams in the processing procedure of Rhizoma Coptidis, and per 100 portions of Rhizoma Coptidis are mixed thoroughly for 40 parts with yellow wine or Chinese liquor, moistening 6 hours, and heating steams 8 hours, takes out drying.
2. method for preparing the described Chinese goldthread processed product of claim 1 is characterized in that: described wine steams in the processing procedure of Rhizoma Coptidis, and per 100 portions of Rhizoma Coptidis are mixed thoroughly for 40 parts with wine, moistening 6 hours, and heating steams 8 hours, taking-up, drying.
3. the purposes of the described Wine-steamed Chinese goldthread processed product of claim 1 in the medicine of preparation PPAR gamma agonist.
4. purposes according to claim 3 is characterized in that: described medicine is medicine or the euglycemic agent that suppresses the preceding adipose cell differentiation of 3T3-L1, improves the preceding adipose cell glucose utilization rate of 3T3-L1.
5. according to claim 3 or 4 described purposes, it is characterized in that: described medicine is the blood fat reducing function, improves insulin resistant, reduces the medicine of glyconeogenesis.
6. according to claim 3 or 4 described purposes, it is characterized in that: described medicine is the medicine of treatment diabetes and complication thereof.
The described Wine-steamed Chinese goldthread processed product of claim 1 the preparation alpha-glucosidase inhibitor medicine in purposes.
8. one kind has the pharmaceutical composition that suppresses the preceding adipose cell differentiation of 3T3-L1, improves the preceding adipose cell glucose utilization rate of 3T3-L1, and it is characterized in that: it is to be prepared from following method: the weighting profit requires 1 described wine to steam Rhizoma Coptidis, decocte with water 3 times; Each 3 hours, each amount of water was 10 times of amounts, filters; Merge extractive liquid; Concentrate, subsequent use, add acceptable accessories or complementary composition and be prepared into preparation pharmaceutically commonly used.
9. pharmaceutical composition according to claim 8 is characterized in that: described preparation is an oral formulations.
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| CN106236865B (en) * | 2016-08-30 | 2019-11-08 | 成都合迅医药技术有限公司 | A kind of concocting method of prepared RHIZOMA COPTIDIS with vino |
| CN106173110A (en) * | 2016-08-31 | 2016-12-07 | 洪雅县瓦屋山药业有限公司 | A kind of manufacture method of the refined even tea being of value to diabetes |
| CN110133158B (en) * | 2019-06-28 | 2022-05-20 | 雅安迅康药业有限公司 | HPLC fingerprint detection method of wine steamed coptis chinensis |
| CN113101318B (en) * | 2021-05-31 | 2022-08-30 | 山东农业大学 | A Coptidis rhizoma extractive solution and its application in treating animals infected with Staphylococcus pseudointermedius |
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2010
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1686228A (en) * | 2005-04-14 | 2005-10-26 | 中国科学院武汉植物园 | Coptic root baking processing method |
| CN1958000A (en) * | 2006-11-03 | 2007-05-09 | 中国科学院长春应用化学研究所 | Method for processing rhizoma coptidis |
| CN101053655A (en) * | 2007-05-23 | 2007-10-17 | 中国科学院长春应用化学研究所 | Preparing method for traditional Chinese medicine coptidis rhizome stir-fried with ginger juice |
Non-Patent Citations (1)
| Title |
|---|
| 杨金梅等.RP-HPLC法同时测定黄连及酒黄连中3种生物碱的含量.《中华中医药学会中药炮制分会2008年学术研讨会论文集》.2008, * |
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| CN101874832A (en) | 2010-11-03 |
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