CN102579585A - Blood sugar lowering medicinal composition and application thereof - Google Patents
Blood sugar lowering medicinal composition and application thereof Download PDFInfo
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- CN102579585A CN102579585A CN2011100025310A CN201110002531A CN102579585A CN 102579585 A CN102579585 A CN 102579585A CN 2011100025310 A CN2011100025310 A CN 2011100025310A CN 201110002531 A CN201110002531 A CN 201110002531A CN 102579585 A CN102579585 A CN 102579585A
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Abstract
The invention provides a blood sugar lowering medicinal composition and application thereof to preparing a blood sugar lowering medicament. The medicinal composition contains root bark of white mulberry and root of kudzu vine, and is characterized in that: the proportion of the root bark of white mulberry to the root of kudzu vine is 1:2-1:10 in parts by weight. The invention also relates to a preparation method of the medicinal composition and application of the preparation method to preparation of medicaments for treating hyperglycemia, diabetes or complications thereof. The invention also relates to application of the root of kudzu vine to delay intestinal absorption of an alpha-glucosidase inhibitor.
Description
Technical field
The present invention relates to a kind of pharmaceutical composition, particularly a kind of hypoglycemic pharmaceutical composition, and be used to prepare the purposes of hypoglycemic drug.
Background technology
Diabetes are a kind of by polygenes, the multifactor chronic metabolic disease that causes.Chinese medicine compound has many target spots, the synergistic advantage of multiple composition, compares with the medicine of single target spot and is more suitable for this type of treatment of diseases.The alpha-glucosidase inhibitor that with the acarbose is representative is through slowing down digesting and assimilating of carbohydrate; Can reduce type ii diabetes patient's post-prandial glycemia (PPHG); Increase insulin sensitivity and secretion capacity; Become the first-line treatment medicine of type ii diabetes, occupied the market share more than 30%, had vast market prospect.But still do not have at present and have the inhibiting compound Chinese medicinal preparation listing of alpha-glucosidase.
Previous research work and bibliographical information show that the 1-deoxynojirimycin (DNJ) in the Cortex Mori has stronger alpha-glucosaccharase enzyme inhibition, and Cortex Mori and Radix Puerariae compatibility have synergistic function.Experimental result shows that the blood sugar lowering effect of Mulberry pueraria root compound obviously is superior to single Cortex Mori extract and Radix Puerariae extract in the animal body, can be developed into to have the inhibiting Mulberry Pueraria lobota of alpha-glucosidase hypoglycemic compound.
Patent " a kind of hypoglycemic pharmaceutical composition and preparation method thereof " (application number CN200610099050.5) has been protected Cortex Mori: Radix Puerariae is 1～6: 1 compositions; Show in its patent specification; Certain when the Radix Puerariae consumption, and Cortex Mori: the Radix Puerariae ratio is 1～6: 1 o'clock, along with the increase of Cortex Mori consumption; Its alpha-glucosaccharase enzyme inhibition is enhanced gradually; Promptly when DNJ content in the Mulberry pueraria root compound increased, compound recipe also strengthened the alpha-glucosaccharase enzyme inhibition thereupon, shows the inhibitory action amount effect relationship of DNJ to alpha-glucosidase.
But still do not have at present research to Radix Puerariae the effect in Cortex Mori Radix Puerariae compound recipe (be called for short Mulberry pueraria root compound) set forth.
Summary of the invention
Inventor of the present invention has proved the effect of Radix Puerariae in the Mulberry pueraria root compound through experiment in large quantities, and promptly in the Mulberry pueraria root compound, when the Cortex Mori consumption was constant, along with the increase of Radix Puerariae consumption, hypoglycemic effect also strengthened thereupon, and new Mulberry pueraria root compound is provided.Particularly, the present invention relates to the following aspects:
One aspect of the present invention relates to a kind of pharmaceutical composition, and it contains Cortex Mori and Radix Puerariae, it is characterized in that the ratio of Cortex Mori and Radix Puerariae is counted 1: 2～1: 10 by weight, for example can be 1: 3～1: 10,1: 6～1: 10 or 1: 8～1: 10.
In embodiments of the invention, said pharmaceutical composition consists of Cortex Mori and Radix Puerariae.
In embodiments of the invention, the ratio of said Cortex Mori and Radix Puerariae count 1: 2 by weight, 1: 3,1: 4,1: 6,1: 8 or 1: 10.
In one embodiment of the invention, when the ratio of Cortex Mori and Radix Puerariae is 1: 8 and 1: 10, can obviously reduce fasting glucose and 30～120min blood glucose after the meal, and along with the increase of Radix Puerariae proportion, hypoglycemic effect is better.
In another embodiment of the invention, when the ratio of Cortex Mori and Radix Puerariae is 1: 3 and 1: 6, be with 6: 1 o'clock to compare at 3: 1 with the ratio of Cortex Mori and Radix Puerariae respectively, have stronger hypoglycemic activity, and blood sugar reducing function is more lasting.
Another aspect of the present invention relates to the purposes of pharmaceutical composition of the present invention in the preparation hypoglycemic drug.
Another aspect of the present invention relates to the purposes of pharmaceutical composition of the present invention in the medicine of preparation treatment hyperglycemia, diabetes or its complication.
Another aspect of the present invention relates to preparation of drug combination method of the present invention, and it may further comprise the steps:
(1) get raw material Cortex Mori and Radix Puerariae, its ratio is counted 1: 2～1: 10 by weight;
(2) prepare Cortex Mori extract and Radix Puerariae extract respectively, its preparation method is for to pass through water or organic solvent such as ethanol extraction respectively with Cortex Mori and Radix Puerariae, or extracts the laggard single step purification of advancing, and obtains extract;
(3) with obtaining pharmaceutical composition of the present invention behind the Cortex Mori of step (2) and the Radix Puerariae extract mix homogeneously, i.e. Mulberry pueraria root extract.
Wherein Cortex Mori and the Radix Puerariae extract in the step (2) preferably adopts following method for preparing:
The method for preparing of Cortex Mori extract is one of them in the following method:
A. the Cortex Mori medical material is used water extraction through cutting or pulverizing, and extraction time is 1～3 time; The preferred extraction 3 times, extraction temperature are room temperature～boil, and preferably boil extraction; Quantity of solvent is 10～30 times of weight portions of medical material weight, and preferred 20-30 doubly obtains Cortex Mori medical material aqueous extract; Extracting solution is centrifugal after concentrating, and supernatant concentration becomes thick paste to obtain Cortex Mori extract A, or gets Cortex Mori extract A through spray drying, decompression or constant pressure and dry;
B. Cortex Mori medical material aqueous extract concentrates back adding ethanol precipitation; Concentration of alcohol scope 50%～90%; The preferred alcohol concentration range is 70～80%, and deposition is abandoned it, supernatant concentration; Become thick paste to obtain Cortex Mori extract B after reclaiming ethanol, or get Cortex Mori extract B through spray drying, decompression or constant pressure and dry;
C. it is centrifugal that Cortex Mori medical material aqueous extract concentrates the back, and supernatant is crossed strongly acidic cation-exchange, and resin can be selected models such as 001*4,001*7, D61, D001cc; Preferred 001*7 (732 type); The upper prop effluent is abandoned it, collects the ammonia eluent of 0.1～1N, preferably uses the ammonia eluting of 0.5N; Eluent obtains Cortex Mori extract C after concentrating, or gets Cortex Mori extract C through spray drying, decompression or constant pressure and dry;
D. it is centrifugal that Cortex Mori medical material aqueous extract concentrates the back, and supernatant is crossed strongly acidic cation-exchange, and resin can be selected models such as 001*4,001*7, D61, D001cc; Preferred 001*7 (732 type), the upper prop effluent is abandoned it, collects the ammonia eluent of 0.1～1N; Eluent concentrates and flings to ammonia, and pH value 8～9, concentrated solution continue to go up strong basic type anion-exchange resin; Resin can be selected models such as 201*4, D201; Preferred 201*4 type, weak-type cation exchange resin column in upper prop effluent and water lotion (washing is to the neutral) series connection, resin can be selected models such as WK40,110, D113, D151; Preferred D151 type; The PH that collection is got off from the weak-type cation exchange resin column is 8～9 effluent and water lotion, and merging obtains Cortex Mori extract D after concentrating, or gets Cortex Mori extract D through normal pressure, drying under reduced pressure, lyophilization or spray drying;
E, Cortex Mori medical material aqueous extract concentrate back centrifugal; Last powder or granular pattern carbon column; Extracting solution also can pass through the precipitate with ethanol supernatant and fling to behind the ethanol at last activated-charcoal column, and the upper prop water that finishes is washed till ninhydrin reaction and uses 10～50% ethanol elution, preferred 15～25% ethanol elution when negative instead; With 1-naphthols reagent qualitative detection total sugar; When the molish loss for reaction, finish eluting, eluent obtains Cortex Mori extract E after concentrating, or gets Cortex Mori extract E through normal pressure, drying under reduced pressure, lyophilization or spray drying;
The method for preparing of Radix Puerariae extract is one of them in the following method:
F. the Radix Puerariae medical material is used water extraction through cutting or pulverizing, and extraction time is 1～3 time; The extraction temperature is a room temperature～boil, and preferably boils extraction, and quantity of solvent is 10～30 times of weight portions of medical material weight; Preferred 20～30 times, the aqueous extract of acquisition Radix Puerariae medical material, it is centrifugal that extracting solution concentrates the back; Supernatant continues to be condensed into thick paste and obtains Radix Puerariae extract F, or gets Radix Puerariae extract F through spray drying, decompression or constant pressure and dry;
G. the Radix Puerariae medical material is through cutting or pulverizing, and using concentration is 30～95% alcohol reflux, preferred 70～80% ethanol extraction; Extraction time is 1～3 time, preferably extracts 3 times, and quantity of solvent is 10～30 times of weight portions of medical material weight; Preferred 20～30 times, the alcohol extract of acquisition Radix Puerariae medical material, it is centrifugal that extracting solution concentrates the back; Supernatant continues to be condensed into thick paste and obtains Radix Puerariae extract G, or gets Radix Puerariae extract G through spray drying, decompression or constant pressure and dry;
The aqueous extract of H, Radix Puerariae medical material concentrates the back and adds the ethanol precipitate with ethanol; Concentration of alcohol scope 50%～90%; The preferred alcohol concentration range is 70～80%; Deposition is abandoned it, and supernatant continues to be condensed into thick paste and obtains Radix Puerariae extract H, or gets Radix Puerariae extract H through spray drying, decompression or constant pressure and dry;
The alcohol extract of I, Radix Puerariae medical material is concentrated into does not have alcohol back adding water precipitation, and deposition is abandoned it, obtains Radix Puerariae extract I after the supernatant concentration, or gets Radix Puerariae extract I through spray drying, decompression or constant pressure and dry;
After the water of J, Radix Puerariae medical material or alcohol extract concentrate, last non-polar macroporous resin post, the optional models such as AB-8, DS401, D101, D3520, NKA-9, D4006 of selecting of resin model; Preferred AB-8, DS401; Upper prop effluent and water lotion are abandoned it, collect 50～95% ethanol elution, preferred 70% ethanol elution; Eluent obtains Radix Puerariae extract J after concentrating, or gets Radix Puerariae extract J through spray drying, decompression or constant pressure and dry.
In embodiments of the invention, adopt method for preparing A-E to prepare Cortex Mori extract; In embodiments of the invention, adopt method for preparing F-J to prepare Radix Puerariae extract.
In one embodiment of the invention, adopt method for preparing C or D to prepare Cortex Mori extract, in another embodiment of the invention, adopt method for preparing F or J to prepare Radix Puerariae extract.
In embodiments of the invention, the content of DNJ is 1.45%-60% in the Cortex Mori extract, and content of puerarin is 10%-35.4% in the Radix Puerariae extract.
Mix to be selected from Cortex Mori extract that A-E makes and to be selected from the Radix Puerariae extract that F-J makes, obtain pharmaceutical composition of the present invention, i.e. the Mulberry pueraria root extract.In one embodiment of the invention, the extract that respectively C and J, D and J is made mixes, and obtains the Mulberry pueraria root extract.
Another aspect of the present invention relates to the purposes of Radix Puerariae in postponing the alpha-glucosidase inhibitor intestinal absorption.In said purposes, Radix Puerariae can be that the Radix Puerariae crude drug makes through pulverizing, and also can be the extract for preparing according to above-mentioned method for preparing, and promptly Radix Puerariae carries out the product that purification obtains through water or ethanol extraction or after extracting.In one embodiment of the invention, Radix Puerariae is the product that obtains through behind ethanol extraction and the macroporous resin column purification.
Said alpha-glucosidase inhibitor is DNJ, N-Me-DNJ, GAL-DNJ or fagomine, and in one embodiment of the invention, said alpha-glucosidase inhibitor is DNJ.
The beneficial effect of the invention
1. the present invention has further proved the effect of Radix Puerariae in the Mulberry pueraria root compound; Cortex Mori and the ratio range of Radix Puerariae in compound recipe are further expanded as Cortex Mori: the Radix Puerariae ratio is 1: 2～1: 10; Promptly when the Cortex Mori consumption is constant; Along with the increase of Radix Puerariae consumption, the hypoglycemic effect of compound recipe strengthens thereupon.
2. the present invention has tentatively proved the partial action mechanism of compound recipe of the present invention through the pharmacokinetics test; Be that composition in the Radix Puerariae to processes such as in animal body absorption of DNJ, metabolism, distribution, drainages influence has taken place; Retardation the fast Absorption of intestinal DNJ; The DNJ of bigger concentration is fully contacted with a-glucosidase on the intestinal brush border; The inhibitory action of enzyme is strengthened, causes part disaccharidase and can not be hydrolyzed into glucose absorption and go into blood, make post-prandial glycemia become lower, steadily and blood sugar reducing function more lasting.
3. the present invention has proved that Radix Puerariae is to the synergism of Cortex Mori in the Mulberry pueraria root compound; Overcome the prejudice that in this compound recipe Cortex Mori that former studies thinks played a major role, only needed a small amount of Radix Puerariae; And new mechanism of action has been proposed, for new direction has been opened up in the development of Remedies for diabetes.
Description of drawings
Fig. 1. the curve during medicine of DNJ (40mg/kg) behind rat orally give Cortex Mori and the Mulberry pueraria root compound.Wherein abscissa is time after the administration (h of unit), and vertical coordinate is DNJ concentration (ng/mL of unit).
The specific embodiment
To combine embodiment that embodiment of the present invention are described in detail below, but it will be understood to those of skill in the art that the following example only is used to explain the present invention, and should not be regarded as limiting scope of the present invention.Unreceipted actual conditions person among the embodiment carries out according to the condition of normal condition or manufacturer's suggestion.The unreceipted person of production firm of agents useful for same or instrument, being can be through the conventional products of commercial acquisition.
Experimental example:
The Cortex Mori extract of experimental example 1,2 is according to embodiment 3 preparations, and Radix Puerariae extract is according to embodiment 10 preparations, and the Mulberry pueraria root extract is according to embodiment 11 preparations.
The pharmacodynamic experiment (1) of experimental example 1 different proportioning Mulberry pueraria root compounds
Adopt male ICR mouse (available from Beijing Vital River Experimental Animals Technology Co., Ltd.) 64; Every group 8, body weight is 24-28g, and overnight fasting (since first day 5:00pm on an empty stomach; The next morning begins modeling); Through the modeling of the freshly prepared streptozotocin of quick tail vein injection (STZ) solution, the latter's dosage is 140mg/kg, and modeling makes the free diet of mice after finishing.(daytime is 3-4h on an empty stomach) measures at modeling 2 all laggard line space abdomen blood glucose, and the model that can be used as success of blood glucose more than 11.1mM selected for use.Concrete modeling method sees also pharmacological experiment guide-new drug and finds and pharmacological evaluation that H.G. Wo Geer writes, Science Press's calendar year 2001 front page.
Receive reagent thing (specimen in use is respectively according to embodiment 3,10 and 11 preparations) with what each group mice continuous irrigation stomach gave each group then; Extract dosage when wherein Cortex Mori and Radix Puerariae use separately is respectively 0.04g/kg and 0.21g/kg, and the dosage of Cortex Mori extract is 0.04g/kg when using the Mulberry pueraria root compound, and the consumption of Radix Puerariae is referring to proportioning in the table 1; Irritate stomach every day 2 times; Continuous irrigation stomach 2 months carried out the starch tolerance test of model mice after 2 months, concrete grammar is following: water 4h is can't help in the fasting on daytime of STZ diabetic mice; Irritate respectively then stomach give with the 0.15g/ml gelatinizing after starch mixing each group together receive the reagent thing; Dosage is identical with long term administration dosage, and wherein the dosage of starch is 3g/kg, and blank (for normal mouse) and model control group only give starch; Giving the mensuration that the 0min of starch, 30min, 60min and 120min carry out blood glucose then, result of the test is seen table 1.
The Mulberry pueraria root compound of the different proportionings of table 1 gives starch tolerance test result (1) after 2 months continuously
*, p＜0.05, * *, n=8 is compared with model control group in p＜0.01; Each ratio is the weight ratio of raw medicinal material in the table.
The The above results explanation, the independent use of Radix Puerariae medical material does not have hypoglycemic activity in animal body, and 30-120min blood glucose is compared with model control group does not after the meal have obviously difference.At Cortex Mori consumption one regularly; Radix Puerariae consumption proportion bigger (1: 2～1: 10), the potentiation of Radix Puerariae is strong more, and fasting glucose of mice and post-prandial glycemia are controlled well more; When Cortex Mori: the ratio of Radix Puerariae reaches 1: 8～and 1: 10 the time, fasting glucose and post-prandial glycemia have obtained remarkable reduction.But when the ratio of Radix Puerariae during greater than 1: 10, the hypoglycemic effect of Mulberry pueraria root compound has the trend of reduction on the contrary, explains that the amount of Radix Puerariae neither be the bigger the better.
The pharmacodynamic experiment (2) of experimental example 2 different proportioning Mulberry pueraria root compounds
Adopt 24 of male ICR mouses, 8 every group, body weight is 24-28g, and all the other experimental techniques are with experimental example 1.
The dosage of Cortex Mori extract is 0.12g/kg when wherein using 3: 1 compound recipes of Mulberry Pueraria lobota; The dosage of Radix Puerariae extract is 0.21g/kg; When using 6: 1 compound recipes; The dosage of Cortex Mori extract is 0.12g/kg, and the dosage of Radix Puerariae extract is 0.105g/kg, and the dosage of 1: 3 and 1: 6 compound recipe by that analogy.Experimental result is seen table 2, table 3.
The Mulberry pueraria root compound of the different proportionings of table 2 gives starch tolerance test result (2) after 2 months continuously
*, p＜0.05, * *, compare with model control group p＜0.01; #, p＜0.05 is with 3: 1 groups of comparisons; N=8; Each ratio is the weight ratio of raw medicinal material in the table.
Table 2 is the result show; Improving Radix Puerariae consumption hypoglycemic effect does not separately obviously improve; 3: 1 Mulberry pueraria root compound can obviously reduce the blood glucose of model mice 30min and 60min, explains that this compound recipe can reduce the effect of blood glucose in diabetic mice really through alpha-glucosaccharase enzyme inhibition performance; And the effect that ratio is 1: 3 a Mulberry pueraria root compound not only reduces 30min and 60min blood glucose obviously is better than 3: 1 compound recipes; Can also obviously reduce the blood glucose of model mice 120min; Show that its blood sugar reducing function is more lasting; Therefore as a whole, proportioning is that the hypoglycemic effect of 1: 3 Mulberry pueraria root compound will obviously be superior to 3: 1 Mulberry pueraria root compounds.
The Mulberry pueraria root compound of the different proportionings of table 3 gives starch tolerance test result (3) after 2 months continuously
*, p＜0.05, * *, compare with model control group p＜0.01; #, p＜0.05, ##, p＜0.01 is with 6: 1 groups of comparisons; N=8
Table 3 is the result show; Improving Radix Puerariae consumption hypoglycemic effect does not separately obviously improve; 6: 1 Mulberry pueraria root compound can obviously reduce the blood glucose of model mice 30min and 60min, explains that this compound recipe can reduce the effect of blood glucose in diabetic mice really through alpha-glucosaccharase enzyme inhibition performance; And the effect that ratio is 1: 6 a Mulberry pueraria root compound not only reduces 30min and 60min blood glucose obviously is better than 6: 1 Mulberry pueraria root compounds; Can also obviously reduce the blood glucose of model mice fasting glucose and 120min; Show that it can reduce fasting glucose and blood sugar reducing function is more lasting; Therefore as a whole, proportioning is that the hypoglycemic effect of 1: 6 Mulberry pueraria root compound will obviously be superior to 6: 1 Mulberry pueraria root compounds.
The pharmacokinetic of experimental example 3 Mulberry pueraria root compounds of the present invention
1, medicine and reagent: 1-deoxynojirimycin (DNJ) is available from last Hiroad standing grain medical sci-tech Development Co., Ltd, purity 98.91%; (Miglitol MIG) is provided by Zhejiang Medicine Co miglitol; Cortex Mori extract and Cortex Mori-Radix Puerariae extract provide (Cortex Mori extract is pressed the method preparation of embodiment 4, and the Mulberry pueraria root extract is according to the method preparation of embodiment 12) by the Beijing WBL Peking University Biotech Co., Ltd; Trifluoroacetic acid aqueous solution is a U.S. Fisher product; The analytical pure ammonium formate is Beijing Chemical Plant's product; All waters are ultra-pure water.
2, instrument: 1200 type liquid chromatographic systems by U.S. Agilent company (Agilent) (contain the quaternary gradient pump; Automatic sampler, column oven) and Q-Trap 3200 types of u.s.a. applied biosystem company (ABI) series connection level Four bar-ion trap mass spectrometers form.
3, liquid phase separation: adopt Japanese TOSOH company TSK Gel amide 80 (4.5mm * 150mm, 5um) chromatographic column is separated, column temperature is 50 ℃.Mobile phase is acetonitrile-biphase isocratic elution of 6.5mM ammonium formate aqueous solution (pH 5.5) (75: 25), flow velocity 0.3ml/min.Effluent all gets into mass spectrograph analysis.
4, Mass Spectrometer Method: adopt the electron spray ionisation mode to carry out ionizing, scan with many reaction detection (MRM) pattern of cation, the ion pair of DNJ and MIG is respectively m/z164 → 146 and m/z 208 → 146.Ion source and other relevant parameters are optimized for: spray voltage 5500v; Remove a bunch voltage (DP) 36v; Outlet voltage (EP) 9v; Gas curtain gas (Curtain gas 10psi); 375 ℃ of heating-up temperatures; Gas 160psi; Gas 260psi; The collision energy of DNJ and MIG (CE) is respectively 16v and 20v; Collision pond inlet voltage (CEP) is respectively 10v and 16v; Collision pond outlet voltage (CXP) is respectively 4v and 10v.
5, the preparation of standard solution: precision takes by weighing DNJ standard substance 1mg, and water is mixed with the standard reserving solution of 1mg/ml.This standard reserving solution reuse water diluted concentration is 125.0,100.0,50.0,20.0,10.0,5.0,2.0,1.0,0.5, and the series of standards DNJ solution of 0.2ug/mL is deposited in-20 ℃ of refrigerators subsequent use.
The preparation of inner mark solution: precision takes by weighing MIG 1mg, and water is mixed with the interior mark storing solution of 1mg/ml.This solution again dilute with water to process concentration be mark liquid in the 10ug/mL MIG, deposit in-20 ℃ of refrigerators subsequent use.
The preparation of standard plasma sample: the SD rat blank plasma 90uL behind the accurate absorption fasting 12h, add the above-mentioned variable concentrations DNJ of 10uL standard solution, promptly get concentration behind the vortex mixing and be respectively 12.5,10.0; 5.0,2.0,1.0,0.5; 0.2,0.1,0.05, the DNJ standard plasma sample of 0.02ug/mL.
6, standard curve: get above-mentioned DNJ standard plasma sample; According to plasma sample processing method (Kiyotaka Nakagawa; Occurrence of Orally Administered Mulberry 1-Deoxynojirimycin in Rat plasma J.Agric Food.2007; 558928-8933) handle sample, and carry out LC-MS/MS and analyze, the peak area of record DNJ and MIG.PC with DNJ is an abscissa, and the peak area ratio of DNJ and MIG is that vertical coordinate carries out linear regression, gets the blood plasma standard curve of DNJ.
7, zoopery: healthy male Sprague-Dawley rat (200-220g), 5 every group, purchase animal institute in the Chinese Academy of Medical Sciences.Test and under narcotism, carry out the operation of jugular vein intubate the previous day.Postoperative overnight fasting (more than 12 hours), oral administration gavage gives Cortex Mori extract or Mulberry pueraria root extract, and the dosage of 1-deoxynojirimycin in two preparations equates, is 40mg/kg.After (0min) and the administration 5,15,30,60,90,120,180,240,360,480min gets blood 0.2mL from the jugular vein intubate before administration.The blood that takes out is put into the 1.5mL centrifuge tube that contains heparin, centrifugal 3min separated plasma in the 8000rpm/min centrifuge immediately.Replenish the normal saline that 0.2mL contains heparin (40IU/mL) immediately after getting blood at every turn.Blood plasma is deposited in-20 ℃ and is preserved to be measured down.
8, sample treatment: the accurate absorption prepares blood plasma 100 μ L in the step 7; Be transferred in the 1.5mL centrifuge tube, add 10 μ L inner mark solutions, add the 0.4mL trifluoroacetic acid aqueous solution behind the vortex mixing again; Ultrasonic 1min behind the mixing; And in 4 ℃ of placement 30min, centrifugal 10min gets supernatant 20 μ L injection LC-MS/MS appearance then and carries out the analysis of DNJ.
9, date processing
Blood drug level-time graph according to every rat; With pharmacokinetics software WinNonlin (Pharsight Corporation; The U.S.); Adopt non-compartment model to calculate the pharmacokinetic parameter of DNJ, comprise peak time (Tmax), peak concentration (Cmax), lower area of blood concentration-time curve AUC each administration rat
0-tAnd AUC
0-∝Data relatively adopt the t check analysis between group, are judged as less than 0.05 with the p value to have significant difference.
10, result of study
Behind rat orally give Cortex Mori and the Mulberry pueraria root compound, the plasma drug level-time data of DNJ (40mg/kg) such as table 4 are with shown in Figure 1, and pharmacokinetic parameter is as shown in table 5.
The plasma drug level of DNJ (40mg/kg) behind table 4 rat orally give Cortex Mori and the Mulberry pueraria root compound
The P value is Cortex Mori group and the statistical result relatively of Mulberry pueraria root compound group corresponding index.
The pharmacokinetic parameter of DNJ (40mg/kg) behind table 5. rat orally give Cortex Mori and the Mulberry pueraria root compound
The P value is Cortex Mori group and the statistical result relatively of Mulberry pueraria root compound group corresponding index in the table.
11, result and discussion
Above-mentioned experimental result shows, behind the orally give rat Cortex Mori extract, DNJ can fast Absorption, and (peak time) reaches maximum plasma concentration 10.66 ± 2.66ug/mL when average 0.35h (21min).When Cortex Mori and Radix Puerariae composition compound recipe, the absorption of DNJ is slightly slow, and the average out to peak time is 0.45h (27min), does not have significant difference between two groups.Although peak time does not significantly change, peak concentration obviously is reduced to 5.27 ± 2.75ug/mL, has statistical significant difference (p＜0.05).Relatively find that 5,15 during with 30min after administration during two groups blood drug level, the blood drug level of DNJ is obviously different, and Mulberry pueraria root compound group is starkly lower than Cortex Mori group (p＜0.05); Two groups TG-AUC (AUC) is also obviously different, (6.76 ± 3.01h*ug/mL) AUC (9.94 ± 1.47h*ug/mL) the significances minimizings (p＜0.05) of DNJ when giving Cortex Mori of the AUC of DNJ when giving the Mulberry pueraria root compound.This result shows, the absorption total amount that rat gives DNJ behind the Mulberry pueraria root compound is lower than giving behind the Cortex Mori absorption total amount of DNJ.Relatively two groups elimination half-life, find difference, explain that compound recipe only influences the absorption of DNJ, and do not change the elimination of DNJ.
This experimental result shows, the chemical constituent in the Radix Puerariae extract suppresses the absorption of DNJ in the Cortex Mori, makes that giving the plasma drug level of DNJ behind the Mulberry pueraria root compound is lower than and gives the Cortex Mori folk prescription, absorbs the elimination that gets into intravital DNJ but do not influence.
The pharmacokinetic result of Mulberry pueraria root compound has the support effect to pharmacodynamics experimental result in the mice body.Chemical constituent retardation in the Radix Puerariae extract fast Absorption of intestinal DNJ; The DNJ of bigger concentration is fully contacted with a-glucosidase on the intestinal brush border; Inhibitory action to enzyme strengthens, and makes part disaccharidase can not be hydrolyzed into glucose absorption and goes into blood, and it is lower and steady that post-prandial glycemia becomes; And the consumption of Radix Puerariae extract is big more, and is also big more to the DNJ inhalation effects in the Cortex Mori extract, and the animal blood glucose of 30-120min after the meal reduces, and fluctuation diminishes.
Embodiment:
In following examples, DNJ and content of puerarin are used high effective liquid chromatography for measuring.
Cortex Mori medical material (DNJ content 0.27%) 10kg is through after the cutting; Extract with water boil, extraction time is 3 times, and the amount of water of 3 extractions is respectively 7,5,5 times of weight portions of medical material weight; Extraction time was respectively 2,1,1 hours; It is centrifugal that extracting solution concentrates the back, and the supernatant spray drying gets Cortex Mori extract 1300g, DNJ content 1.45%.
Embodiment 2
Cortex Mori medical material (DNJ content 0.27%) 10kg is through after the cutting, and 50 ℃ of water temperature lixiviates are got, and extraction time is 3 times; The amount of water that extracts for 3 times is respectively 7,5,5 times of weight portions of medical material weight, and extraction time was respectively 2,1,1 hours, and extracting solution adds 95% ethanol 80L when being concentrated into volume 20L; The limit edged stirs, and room temperature is placed and spent the night, and deposition is abandoned it; Get supernatant concentration, adopt spray drying to get Cortex Mori extract 890g, DNJ content 1.97% behind the recovery ethanol.
Cortex Mori medical material (DNJ content 0.27%) 10kg boils extraction through after the cutting, and extraction time is 2 times; The amount of water that extracts for 2 times is 10 times of weight portions of medical material weight, and extraction time is 2 hours, and it is centrifugal that extracting solution concentrates the back; Supernatant is crossed the H+732 strongly acidic cation-exchange, and the resin volume is 6L, and the upper prop effluent is abandoned it; Collect the ammonia eluent 30L of 0.5N, eluent concentrates the back spray drying and gets Cortex Mori extract 150g, DNJ content 12.6%.
Embodiment 4
After the cutting of 10 kilograms of processes of Cortex Mori medical material (DNJ content 0.27%), 50 ℃ of water temperature lixiviates are got, and extraction time is 3 times; The amount of water that extracts for 3 times is respectively 7,5,5 times of weight portions of medical material weight, and extraction time was respectively 2,1,1 hours, and is centrifugal behind the extracting solution concentrating under reduced pressure; Get supernatant, last 001*7 gel type cation exchange resin column, column volume is 10L; The pH value of upper prop liquid is 4～5, and effluent PH drops to 1～2, and using deionized water to be eluted to pH value is 5～6; Reuse 35L concentration is the ammonia eluting of 0.5M, and ammonia eluent concentrating under reduced pressure is waved most ammonia, and PH is 8～9; 201*4 gel type strong base type anion-exchange resin column (column volume is 6L) on the concentrated solution, the upper prop effluent of PH to 13～14 and water lotion (washing is to neutral) series connection D151 macropore weak-type cation exchange resin column (column volume is 10L), the PH that collection is got off from the weak-type cation exchange column is 8～9 effluent and water lotion; Spray drying obtains Cortex Mori extract 35g, DNJ content 60%.
After the cutting of 10 kilograms of processes of Cortex Mori medical material (DNJ content 0.27%), 50 ℃ of water temperature lixiviates are got, and extraction time is 2 times; The amount of water that extracts for 2 times is respectively 5,5 times of weight portions of medical material weight, and extraction time was respectively 1,1 hour, and is centrifugal behind the extracting solution concentrating under reduced pressure; Get supernatant granular pattern carbon column, amount of activated is 8kg, and upper prop finishes and uses 15% ethanol elution when water is washed till the ninhydrin reaction feminine gender instead; With 1-naphthols reagent qualitative detection total sugar; When the molish loss for reaction, finish eluting, eluent concentrates the back drying under reduced pressure and gets Cortex Mori extract 320g, DNJ content 5.41%.
Embodiment 6
After 10 kilograms of processes of Radix Puerariae medical material (puerarin content 4%) cut into little fourth; Boil extraction, extraction time is 3 times, and the amount of water of 3 extractions is 10 times of weight portions of medical material weight; Extraction time is 1 hour; It is centrifugal that extracting solution concentrates the back, and the supernatant spray drying gets Radix Puerariae extract 2.6kg, puerarin content 10%.
Embodiment 7
After 10 kilograms of processes of Radix Puerariae medical material (puerarin content 4%) cut into little fourth; Use 70% alcohol reflux, extraction time is 2 times, and quantity of solvent is 10,7 times of weight portions of medical material weight; Extraction time is 2 hours, 1 hour; The extracting solution concentrating under reduced pressure, the concentrated solution spray drying gets Radix Puerariae extract 1.9kg, puerarin content 16.8%.
After 10 kilograms of processes of Radix Puerariae medical material (puerarin content 4%) cut into little fourth, boil extraction, extraction time is 3 times; The amount of water that extracts for 3 times is 10 times of weight portions of medical material weight, and extraction time is 1 hour, and extracting solution adds 95% ethanol 80L when being concentrated into volume 20L; The limit edged stirs, and room temperature is placed and spent the night, and deposition is abandoned it; Get supernatant concentration, adopt spray drying to get Radix Puerariae extract 2.0kg, puerarin content 12% behind the recovery ethanol.
After 10 kilograms of processes of Radix Puerariae medical material (puerarin content 4%) cut into little fourth, use 70% alcohol reflux, extraction time is 2 times; Quantity of solvent is 10,7 times of weight portions of medical material weight, and extraction time is 2 hours, 1 hour, adds the water of 40L when extracting solution is evaporated to 20L; The limit edged stirs, and room temperature is placed and spent the night, and deposition is abandoned it; Get supernatant concentration, spray drying gets Radix Puerariae extract 1.7kg, puerarin content 18.3%.
After 10 kilograms of processes of Radix Puerariae medical material (puerarin content 4%) cut into little fourth, water or 70% ethanol extraction, extraction time were 2 times; Quantity of solvent is 10,7 times of weight portions of medical material weight, and extraction time is 2 hours, 1 hour, when extracting solution is concentrated into 20L (alcohol extract need be waved most ethanol); Centrifugal, get AB-8 type macroporous resin column on the supernatant, the resin volume is 12L; Upper prop effluent and water lotion are abandoned it; Collect 70% ethanol elution, eluent concentrates the back spray drying and gets Radix Puerariae extract 800g, puerarin content 35.4%.
Embodiment 11
Cortex Mori medical material (DNJ content 0.27%) 5kg boils extraction through after the cutting, and extraction time is 2 times; The amount of water that extracts for 2 times is 10 times of weight portions of medical material weight, and extraction time is 2 hours, and it is centrifugal that extracting solution concentrates the back; Supernatant is crossed the H+732 strongly acidic cation-exchange, and the resin volume is 3L, and the upper prop effluent is abandoned it; Collect the ammonia eluent 15L of 0.5N, eluent concentrates the back spray drying and gets Cortex Mori extract.
After 10 kilograms of processes of Radix Puerariae medical material (puerarin content 4%) cut into little fourth, water or 70% ethanol extraction, extraction time were 2 times; Quantity of solvent is 10,7 times of weight portions of medical material weight, and extraction time is 2 hours, 1 hour, when extracting solution is concentrated into 20L (alcohol extract need be waved most ethanol); Centrifugal, get AB-8 type macroporous resin column on the supernatant, the resin volume is 12L; Upper prop effluent and water lotion are abandoned it, collect 70% ethanol elution, and eluent concentrates the back spray drying and gets Radix Puerariae extract.
Above-mentioned Cortex Mori and Radix Puerariae extract mix homogeneously in V-Mixer are obtained the Mulberry pueraria root extract, be respectively 1.09% and 32.2% with high effective liquid chromatography for measuring DNJ and content of puerarin.
Embodiment 12
After the cutting of 10 kilograms of processes of Cortex Mori medical material, 50 ℃ of water temperature lixiviates are got, and extraction time is 3 times; The amount of water that extracts for 3 times is respectively 7,5,5 times of weight portions of medical material weight, and extraction time was respectively 2,1,1 hours, and is centrifugal behind the extracting solution concentrating under reduced pressure; Get supernatant, last 001*7 gel type cation exchange resin column, column volume is 10L; The pH value of upper prop liquid is 4～5, and effluent PH drops to 1～2, and using deionized water to be eluted to pH value is 5～6; Reuse 35L concentration is the ammonia eluting of 0.5M, and ammonia eluent concentrating under reduced pressure is waved most ammonia, and PH is 8～9; 201*4 gel type strong base type anion-exchange resin column (column volume is 6L) on the concentrated solution; The upper prop effluent of PH to 13～14 and water lotion (washing is extremely neutral) series connection D151 macropore weak-type cation exchange resin column (column volume is 10L), the PH that collection is got off from the weak-type cation exchange column is 8～9 effluent and water lotion, and is subsequent use behind the concentrating under reduced pressure after effluent and water lotion merge.
After 20 kilograms of processes of Radix Puerariae medical material cut into little fourth, water or 70% ethanol extraction, extraction time were 2 times; Quantity of solvent is 10,7 times of weight portions of medical material weight, and extraction time is 2 hours, 1 hour, when extracting solution is concentrated into 20L (alcohol extract need be waved most ethanol); Centrifugal, get AB-8 type macroporous resin column on the supernatant, the resin volume is 12L; Upper prop effluent and water lotion are abandoned it, collect 70% ethanol elution, and the eluent concentrating under reduced pressure is subsequent use.
With spray drying behind the concentrated solution mix homogeneously of Cortex Mori and Radix Puerariae, obtain Mulberry pueraria root extract 1320g, be respectively 1.7% and 33.4% with high effective liquid chromatography for measuring DNJ and content of puerarin.
Although the specific embodiment of the present invention has obtained detailed description, it will be understood to those of skill in the art that.According to disclosed all instructions, can carry out various modifications and replacement to those details, these change all within protection scope of the present invention.Four corner of the present invention is provided by accompanying claims and any equivalent thereof.
Claims (9)
1. pharmaceutical composition, it contains Cortex Mori and Radix Puerariae, it is characterized in that the ratio of Cortex Mori and Radix Puerariae is counted 1: 2～1: 10 by weight.
2. the pharmaceutical composition of claim 1, the ratio of wherein said Cortex Mori and Radix Puerariae is counted 1: 3～1: 10 by weight.
3. the pharmaceutical composition of claim 2, the ratio of wherein said Cortex Mori and Radix Puerariae is counted 1: 6～1: 10 by weight.
4. the pharmaceutical composition of claim 3, the ratio of wherein said Cortex Mori and Radix Puerariae is counted 1: 8～1: 10 by weight.
5. each pharmaceutical composition of claim 1-4 is in the purposes of preparation in the hypoglycemic drug.
6. each the purposes of pharmaceutical composition in the medicine of preparation treatment hyperglycemia, diabetes or its complication of claim 1-4.
7. each preparation of drug combination method of claim 1-4.
8. the purposes of Radix Puerariae in postponing the alpha-glucosidase inhibitor intestinal absorption.
9. the purposes of claim 8, wherein said alpha-glucosidase inhibitor is the 1-deoxynojirimycin.
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| CN104474026A (en) * | 2014-11-21 | 2015-04-01 | 西南大学 | Mulberry resource long-acting hypoglycemic medicine composition as well as granules and preparation method thereof |
| CN107693577A (en) * | 2017-11-06 | 2018-02-16 | 威海松龄诺可佳中药饮片股份有限公司 | A kind of composition of plant extracts and its preparation method and application |
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