CN101371869B - Inhibitor originated from alpha-glucosidase of natto and preparation method thereof - Google Patents
Inhibitor originated from alpha-glucosidase of natto and preparation method thereof Download PDFInfo
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Abstract
The invention discloses an alpha-glucosaccharase inhibitor which is extracted from natto and a preparation method thereof. The alpha-glucosaccharase inhibitor is an active ingredient which has the alpha-glucosaccharase inhibition function and extracted by aqueous or ethanol from natto. The preparation method comprises steps such as aqueous extraction and ethanol extraction that are carried on solid fermented natto, concentration, centrifugation and drying or the steps such as concentration, ethanol precipitation, centrifugation and drying that are carried on liquid fermented natto products; or the active ingredient is further extracted by macroporous resin and organic solvent or refined and purified by C18 column to obtain the alpha-glucosaccharase inhibitor.
Description
Technical field
The present invention relates to a kind of alpha-glucosidase inhibitor and preparation method thereof, particularly a kind of alpha-glucosidase inhibitor that derives from natto and preparation method thereof.
Background technology
Natto is a kind of nutritious high-quality leavened food, and it is to utilize bacillus natto (Bacillus natto) to be seeded in the soybean of boiling to form at suitable temperature bottom fermentation, is a kind of traditional fermented food of Japan.Be rich in several physiological active substances such as Nattokinase, soybean isoflavones, polysaccharide, saponin(e, pyridine dicarboxylic acid, multiprenylmenaquinone in the natto, wherein Nattokinase is subjected to extensive concern because of having good thrombolytic effect, but the glycosidase inhibiting function of natto is not reported always and furthers investigate.
Summary of the invention
One object of the present invention is to disclose a kind of alpha-glucosidase inhibitor; Another object of the present invention is to disclose the new purposes that a kind of natto alpha-glucosidase suppresses; The present invention also aims to disclose the preparation method of this alpha-glucosidase inhibitor.
The present invention seeks to be achieved through the following technical solutions:
To be natto carry or alcohol extracting obtains has the inhibiting activeconstituents of alpha-glucosidase through water alpha-glucosidase inhibitor of the present invention; Described natto is to utilize bacillus natto to carry out solid on for main medium or liquid fermenting is made at soybean or soya products.
The preparation method of alpha-glucosidase inhibitor of the present invention is: get solid fermentation natto 1 weight part, the water, methyl alcohol, 50%-90% ethanol or the propyl alcohol that add the 1-10 parts by volume, stir extraction 2-3 time at 75-95 ℃, or heating and refluxing extraction 2-3 time, extracting solution merges, i.e. the natto reactive site; Decompression or normal pressure are concentrated into the 0.1-1 parts by volume, and be centrifugal, obtains the brown solid powder after the supernatant liquor spraying drying, according to common process, adds conventional auxiliary material, makes the formulation of clinical acceptance; Perhaps get liquid fermenting product 1 parts by volume of natto, normal pressure or be evaporated to the 0.1-1 parts by volume, add its 1-4 volume 75%-95% ethanol doubly and carry out alcohol precipitation in concentrated solution, room temperature was placed 8-24 hour, and is centrifugal, get supernatant liquor, supernatant liquor is waved spraying drying behind the most ethanol, obtains the brown solid powder, according to common process, add conventional auxiliary material, make the formulation of clinical acceptance.Include but not limited to formulations such as granule, pill, tablet, capsule, oral liquid.
The preparation method of alpha-glucosidase inhibitor of the present invention is preferably: get solid fermentation natto 1 weight part, add the water of 5 parts by volume, stir at 90 ℃ and extract 2 times, extracting solution merges, i.e. the natto reactive site; Decompression or normal pressure are concentrated into 0.5 parts by volume, and be centrifugal, obtains the brown solid powder after the supernatant liquor spraying drying, according to common process, adds conventional auxiliary material, makes the formulation of clinical acceptance.Include but not limited to formulations such as granule, pill, tablet, capsule, oral liquid.
The preparation method of alpha-glucosidase inhibitor of the present invention is preferably: get solid fermentation natto 1 weight part, the alcohol heating reflux that adds 3 parts by volume 75% extracts 2 times, and extracting solution merges, i.e. the natto reactive site; Decompression or normal pressure concentrate and wave most ethanol, and be centrifugal, obtains the brown solid powder after the supernatant liquor spraying drying, according to common process, adds conventional auxiliary material, makes the formulation of clinical acceptance.Include but not limited to formulations such as granule, pill, tablet, capsule, oral liquid.
The preparation method of alpha-glucosidase inhibitor of the present invention is preferably: liquid fermenting product 5 parts by volume of getting natto, normal pressure or be evaporated to 0.5 parts by volume, in concentrated solution, add its 4 volume 95% ethanol doubly and carry out alcohol precipitation, room temperature was placed 12 hours, centrifugal, get supernatant liquor, i.e. the natto reactive site; Supernatant liquor is waved spraying drying behind the most ethanol, obtains the brown solid powder, according to common process, adds conventional auxiliary material, makes the formulation of clinical acceptance.Include but not limited to healthcare products or pharmaceutical dosage forms such as granule, pill, tablet, capsule, oral liquid.
Also comprise a kind of in the following process for purification refine among the preparation method of alpha-glucosidase inhibitor of the present invention:
A, natto reactive site of the present invention are crossed macroporous resin column, with the ethanol elution of 70%---90%, collect elutriant and promptly get column purification natto reactive site.
B, natto reactive site of the present invention are crossed macroporous resin column, with the ethanol elution of 70%---90%, collect elutriant and promptly get column purification natto reactive site; This crosses column purification natto reactive site elutriant ethyl acetate, n-butanol extraction, and combining extraction liquid promptly got column extracting purifying natto reactive site.
C, natto reactive site of the present invention are crossed macroporous resin column, with the ethanol elution of 70%---90%, collect elutriant and promptly get column purification natto reactive site; This crosses column purification natto reactive site elutriant ethyl acetate, n-butanol extraction, and combining extraction liquid promptly got column extracting purifying natto reactive site; This crosses normal pressure C18 post on the extraction liquid of column extracting purifying natto reactive site, and the 70%-100% methanol-eluted fractions is collected elutriant and promptly got purifying natto reactive site.
The relation of weight part/parts by volume of the present invention is: grams per milliliter
Description of drawings
Fig. 1: AB-19 is to the inhibition curve of thick enzyme
Natto alpha-glucosidase inhibitor of the present invention has the effect that suppresses the blood sugar rising, can make routine clinical formulation, as the protective foods or the medicine of metabolic syndrome patient, diabetic subject or obese people.
Following experimental example and embodiment are used to further specify but are not limited to the present invention.
Experimental example 1 pig intestinal mucosa glucuroide suppresses the foundation test of model
1.1 the preparation process of the thick enzyme of pig intestinal mucosa: get the fresh pig small intestine, clean up, scrape and get small intestinal mucosa, homogenized, placement is spent the night, the high speed frozen centrifugation, merge supernatant liquor, add ammonium sulfate precipitation and make saturation ratio reach 50%, high speed frozen centrifugation 2 times again after refrigeration is placed and spent the night, collecting precipitation, the dialysis desalination, last lyophilize obtains the faint yellow solid powder, the operation of above-mentioned each step is all carried out in 4 ℃ environment, is mixed with 5-10mg/ml solution with phosphate buffered saline buffer during use.
1.2 medicine and reagent
1.2.1 0.05M phosphate buffered saline buffer.
1.2.2 maltose: Shanghai chemical reagents corporation, lot number F20030512.Be mixed with the solution of 56mM (0.02g/ml) with the 0.05M phosphate buffered saline buffer.
1.2.3 Reagent kit of glucose (external diagnosis reagent case): Zhongsheng Beikong Biological Science ﹠ Technology Co., Ltd., lot number 070741.
1.3. laboratory apparatus
Thermostat water bath, Tianjin, island UV-1601PV type spectrophotometer.
1.4 experimental technique
1.4.1 the collection of the carrying out of enzymatic reaction and sample
Get thick enzyme solution 0.1ml and add in the test tube, add glucosidase inhibitor laboratory sample 0.1ml more respectively, substrate maltose solution 0.2ml, 37 ℃ of isothermal reactions 45 minutes.Again above test tube is heated 8 minutes termination reactions in boiling water bath, then in test tube, add 0.6ml distilled water, make cumulative volume reach 1ml, centrifugal 5 minutes, get supernatant liquor and measure glucose content according to the method for test kit.
1.4.2 determination of glucose and evaluation of result in the sample
Assay through glucose in the sample after the enzymatic reaction adopts the liquid of glucose test kit, R1 in the test kit and R2 liquid are pressed 1: 9 mixed, in test tube, add the 1.5ml mixed solution earlier, add sample 30 μ l again, 37 ℃ of reaction 15min, colorimetry 510nm measures light absorption value.
Under the condition that the alpha-glucosidase inhibitor of different concns exists, the release of glucose during with the unrestraint agent per-cent of glucose represent.Suppressing active calculation formula is;
Sample alpha-glucosaccharase enzyme inhibition rate (%)={ (A contrast-A sample-A blank)/A contrast } * 100
The A contrast: the 0.05M phosphate buffered saline buffer replaces sample liquid through the absorbance under 510nm after the enzymatic reaction;
A sample: through the absorbance of sample liquid under 510nm after the enzymatic reaction;
The A blank: the solvent of preparation sample replaces sample liquid through the absorbance under 510nm after the enzymatic reaction;
Alpha-glucosaccharase enzyme inhibition rate by more different samples, can learn that the high sample of inhibiting rate is strong to the restraining effect of thick enzyme, illustrate that the concentration that contains activeconstituents or activeconstituents in this sample is higher, otherwise when sample is more weak to the restraining effect of thick enzyme, the concentration that does not contain required activeconstituents or activeconstituents in the interpret sample is lower, can monitor the activity of sample in the separation and purification process effectively by this model, thereby the retentive activity composition is removed impurity.
Experimental example 2 is removed dirl butter by alcohol precipitation
Get natto 500 grams (weight in wet base) of solid fermentation, the water that adds 2500ml stirs down at 90 ℃ and extracts twice, and extracted twice liquid merges, normal pressure or concentrating under reduced pressure get concentrated solution 250ml, and the concentrated solution sampling also is labeled as sample A-1, the ethanol of adding 95% carries out alcohol precipitation in concentrated solution then, make determining alcohol reach 75%, refrigerator leaves standstill 2 hours for 4 ℃, and is centrifugal, separation of supernatant and precipitation, two portions carry out drying respectively, and the supernatant liquor position gets dry extract and is labeled as sample A-2, and the precipitation part is A-3; Get natto liquid fermenting gained fermented liquid 2000ml, centrifugal, precipitation is abandoned it, get the supernatant liquor normal pressure or be evaporated to 200ml, sampling also is labeled as sample B-1, and the ethanol of adding 95% carries out alcohol precipitation, makes determining alcohol reach 75%, refrigerator left standstill 2 hours for 4 ℃, centrifugal, separation of supernatant and precipitation, two portions carry out drying respectively, the supernatant liquor position gets dry extract and is labeled as sample B-2, and the precipitation part is B-3; The alpha-glucosidase of each several part sample suppresses active to be measured by the method for experimental example 1, the results are shown in Table 1.
The activity of each several part sample inhibitory enzyme behind table 1 alcohol precipitation
Presentation of results, the natto extracting solution of solid fermentation or fermented liquid mainly are to be positioned at the supernatant liquor part through reactive site behind the alcohol precipitation, the precipitation part does not almost have activity; Can remove impurity such as a large amount of thalline, protein, polypeptide by alcohol precipitation, and activeconstituents is suffered a loss hardly.
Experimental example 3 is removed dirl butter by macroporous adsorbent resin
D3520 non-polar macroporous resin on being dissolved in water the A-2 sample 20g in the experimental example 1 afterwards, also can select resin such as AB-8, the D101 etc. of other model, collect respectively and go up sample effluent liquid, water lotion, 30% ethanol eluate, 50% ethanol eluate, 70% ethanol eluate, 90% ethanol eluate, concentrate and receive cream, be numbered A-4, A-5, A-6, A-7, A-8, A-9 respectively; Get B-2 sample 20g equally and also go up the D3520 macroporous resin, collect sample and concentrate and receive cream, be numbered B-4, B-5, B-6, B-7, B-8, B-9 respectively, the alpha-glucosidase of measuring the each several part sample suppresses activity to be measured, and the results are shown in Table 2.
Table 2 is removed the activity of impurity each several part sample by macroporous adsorbent resin
Annotate: the yield of sample calculates with respect to A-1 and B-1.
The result proves, the reactive site that obtains from solid natto or fermented liquid separates by macroporous resin, its upper prop effluent liquid and the equal non-activity of water lotion, the activity of 30% and 50% ethanol eluate is very low, and 70% and 90% ethanol eluate is active higher, hence one can see that mainly concentrates on 70% and 90% ethanol eluate by reactive site behind the macroporous resin, and this two portions sample is merged, and it is numbered A-10 and B-10.
Experimental example 4 is by the test of the extracting and separating reactive site of opposed polarity organic solvent
A-8 in the experimental example 3 and A-9 two portions sample are merged into A-10 (2.0g), B-8 and B-9 two portions sample are merged into B-10 (4.0g), behind A-10 and B-10 water suspendible, ultrasonic 5 minutes, use chloroform respectively, ethyl acetate, propyl carbinol extracts, A-10, B-10 is through its aqueous solution of extraction back, chloroform extraction liquid, acetic acid ethyl acetate extract and butanol extraction liquid are numbered A-11 respectively through behind the concentrate drying, A-12, A-13, A-14 and B-11, B-12, B-13, B-14, the each several part sample dissolves with 20% dimethyl sulfoxide (DMSO) (DMSO), dissolve in 20% DMSO as sample and can add a small amount of tween 80 hydrotropy when bad, the determination of activity of each sample sees Table 3.
The activity of each position sample behind the table 3 opposed polarity organic solvent extraction
Annotate: the yield of sample calculates with respect to A-1 and B-1.
The result proves, extraction through three kinds of organic solvent chloroforms, ethyl acetate, propyl carbinol, aqueous solution part and chloroform extraction liquid partly do not have activity or activity very weak, and activeconstituents mainly is present in the extraction liquid of ethyl acetate, propyl carbinol, carry out next step purifying after these two portions are merged.
The normal pressure column chromatography for separation reactive site test of experimental example 5 C18 posts
The extracting solution of natto fermented liquid or solid natto passes through alcohol precipitation, upward most of impurity has been removed in the processing of steps such as macroporous resin and extraction, use dissolve with methanol so in this experiment A-13, A-14, B-13, B-14 four sample segments are merged back (3.2g), last C18 normal pressure post is further purified.The filler granularity is 50u, the C18 filler before the dress post earlier with the abundant swelling of methyl alcohol, before using with 30% methyl alcohol balance pillar; Sample is admixed a small amount of silica gel dry method upper prop after with dissolve with methanol, methyl alcohol with 30%, 50%, 70%, 100% carries out gradient elution successively, collect each component, and be numbered AB-15, AB-16, AB-17, AB-18, the each several part sample dissolves (can add the tween 80 hydrotropy) with 20% dimethyl sulfoxide (DMSO) (DMSO) after flinging to solvent, and measure activity, experimental result sees Table 4.
The activity at each position of normal pressure column chromatography of table 4 C18 post
The result proves, on the sample behind the C18 with the methanol-eluted fractions of different concns, activeconstituents mainly be distributed in 70% and the elutriant of pure methyl alcohol in.AB-17 and AB-18 two duplicate samples are merged, be numbered AB-19 (1.23g).
Experimental example 6 sample AB-19 are to the vitro inhibition curve of the thick enzyme of chitterlings
Sample AB-19 with 20% DMSO dissolving, can be added a small amount of tween 80 hydrotropy, and is diluted to different concentration, measure vitro inhibition curve, the results are shown in Table 5 and accompanying drawing 1 the thick enzyme of chitterlings.
Table 5
The result proves that sample AB-19 has tangible dose-effect relationship in external inhibition to thick enzyme, and along with the increase of sample concentration, its inhibiting rate also increases thereupon, IC
50About ≈ 20ug/ml, interpret sample AB-19 has tangible alpha-glucosidase to suppress active.
Experimental example 7
The sample of embodiment 1-5 with 20% DMSO dissolving (can add a small amount of tween 80 hydrotropy when sample dissolution is bad), is measured the inhibition activity of each sample to thick enzyme by the method for experimental example 1, the results are shown in Table 6.
Table 6
Following embodiment all can realize the effect of above-mentioned experimental example.
Embodiment
Embodiment 1:
Get solid fermentation natto 500g, the water that adds 2500ml stirs for 90 ℃ and extracts 2 times, and extracted twice liquid merges, and normal pressure is concentrated into 250ml, and is centrifugal, obtains the brown solid powder after the supernatant liquor spraying drying, according to common process, adds conventional auxiliary material, makes granule.
Embodiment 2:
Get natto fermented liq 5L, fling to the concentrated solution that obtains 500ml behind most of water through concentrating under reduced pressure, 95% ethanol that adds 2L in concentrated solution carries out alcohol precipitation, and room temperature was placed 12 hours, centrifugal, get supernatant liquor, supernatant liquor is waved behind the most ethanol through spraying drying, brown ceramic powder 75g, can directly use as glycosidase inhibitor, or, add conventional auxiliary material according to common process, make tablet.
Embodiment 3:
Get solid fermentation natto 10Kg, 75% the alcohol heating reflux that adds 30L extracts 2 times, and extracted twice liquid merges, volume was 550ml after concentrating under reduced pressure was waved most ethanol, and is centrifugal, and supernatant liquor is through getting brown ceramic powder 828g after the spraying drying, according to common process, add conventional auxiliary material, make pill.
Embodiment 4:
Get the pressed powder 50g of embodiment 2, the water that adds 500ml, made its even suspendible in ultrasonic 10 minutes, use ethyl acetate and n-butanol extraction three times respectively, the consumption of each organic solvent is 200ml, and acetic acid ethyl acetate extract and butanol extraction liquid are flung to the solvent merging respectively, obtain alpha-glucosidase and suppressed active extract 28g, according to common process, add conventional auxiliary material, make capsule.
Embodiment 5:
Get natto fermented liq 50L, fling to the concentrated solution that obtains 5L behind most of water through normal pressure or concentrating under reduced pressure, 95% ethanol that adds 20L in concentrated solution carries out alcohol precipitation, room temperature was placed 12 hours, centrifugal, get supernatant liquor, supernatant liquor is gone up the AB-8 nonpolar macroporous adsorption resin after waving most ethanol, ethanol elution with 70% and 90%, the ethanol elution part of collection 70% and 90%, to fling to ethanol after this two portions elutriant merging, adding water to cumulative volume is 2.5L, made its even suspendible in ultrasonic 10 minutes, respectively extract three times with ethyl acetate and propyl carbinol respectively, the consumption of each organic solvent is 2.5L, acetic acid ethyl acetate extract and butanol extraction liquid are flung to the solvent merging respectively, add small amount of methanol and made its dissolving in ultrasonic 10 minutes, admix sample on a small amount of silica gel dry method then, with 70%, 100% methanol-eluted fractions, collect 70% and 100% meoh eluate two portions sample and, obtained alpha-glucosidase after the drying and suppressed active extract 19g, according to common process with its merging, add conventional auxiliary material, make granule.
Embodiment 6:
Get solid fermentation natto 3Kg, 75% the alcohol heating reflux that adds 3000ml extracts 2 times, extracted twice liquid merges, and volume was 550ml after concentrating under reduced pressure was waved most ethanol, and is centrifugal, D3520 nonpolar macroporous adsorption resin on the supernatant liquor, ethanol elution with 70% and 90%, concentrate drying gets pressed powder 12.8g after merging two portions elution samples, according to common process, add conventional auxiliary material, make oral liquid.
Embodiment 7:
Get natto fermented liq 50L, fling to the concentrated solution that obtains 5L behind most of water through normal pressure or concentrating under reduced pressure, 95% ethanol that adds 20L in concentrated solution carries out alcohol precipitation, room temperature was placed 12 hours, centrifugal, get supernatant liquor, supernatant liquor is gone up the AB-8 nonpolar macroporous adsorption resin after waving most ethanol, the ethanol elution with 70% and 90%, the ethanol elution part of collection 70% and 90%, to fling to ethanol after this two portions elutriant merging, adding water to cumulative volume is 2.5L, makes its even suspendible, and respectively extracts three times with ethyl acetate and propyl carbinol respectively in ultrasonic 10 minutes, the consumption of each organic solvent is 2.5L, acetic acid ethyl acetate extract and butanol extraction liquid are flung to the solvent merging respectively, add small amount of methanol and made its dissolving, the last sample of C18 in ultrasonic 10 minutes, with 70%, 100% methanol-eluted fractions, collect 70% and 100% meoh eluate two portions sample and, obtained alpha-glucosidase after the drying and suppressed active extract 19g, according to common process with its merging, add conventional auxiliary material, make tablet.
Claims (10)
1. alpha-glucosidase inhibitor is characterized in that this alpha-glucosidase inhibitor made by following method:
Get solid fermentation natto 1 weight part, add water, methyl alcohol, 50%-90% ethanol or the propyl alcohol of 1-10 parts by volume, stir at 75-95 ℃ and extract 2-3 time, or heating and refluxing extraction 2-3 time, extracting solution merging, i.e. natto reactive site; Decompression or normal pressure are concentrated into the 0.1-1 parts by volume, and be centrifugal, obtains pale brown look after the supernatant liquor spraying drying to the brown solid powder, according to common process, adds conventional auxiliary material, makes the formulation of clinical acceptance;
Wherein, the method for natto reactive site purification refine is one of following method:
A, mistake macroporous resin column with the ethanol elution of 70%-90%, are collected elutriant and were promptly got column purification natto reactive site;
B, further with method A cross column purification natto reactive site elutriant with ethyl acetate, n-butanol extraction, combining extraction liquid promptly got column extracting purifying natto reactive site;
C, further with C18 post on the extraction liquid of crossing column extracting purifying natto reactive site of method B, the 70%-100% methanol-eluted fractions is collected elutriant and is promptly got purifying natto reactive site.
2. alpha-glucosidase inhibitor as claimed in claim 1 is characterized in that this alpha-glucosidase inhibitor made by following method:
Get solid fermentation natto 1 weight part, add the water of 5 parts by volume, stir at 90 ℃ and extract 2 times, extracting solution merges, i.e. the natto reactive site; Decompression or normal pressure are concentrated into 0.5 parts by volume, and be centrifugal, obtains pale brown look after the supernatant liquor spraying drying to the brown solid powder, according to common process, adds conventional auxiliary material, makes granule, pill, tablet, capsule or oral liquid.
3. alpha-glucosidase inhibitor as claimed in claim 1 is characterized in that this alpha-glucosidase inhibitor made by following method:
Get solid fermentation natto 1 weight part, the alcohol heating reflux that adds 3 parts by volume 75% extracts 2 times, and extracting solution merges, i.e. the natto reactive site; Decompression or normal pressure concentrate and wave most ethanol, and be centrifugal, obtains pale brown look after the supernatant liquor spraying drying to the brown solid powder, makes granule, pill, tablet, capsule or oral liquid;
Wherein, the method for natto reactive site purification refine:
Cross macroporous resin column,, collect elutriant and promptly got column purification natto reactive site with the ethanol elution of 70%-90%.
4. alpha-glucosidase inhibitor is characterized in that this alpha-glucosidase inhibitor made by following method:
Get liquid fermenting product 5 parts by volume of natto, normal pressure or be evaporated to 0.5 parts by volume adds its 4 volume 95% ethanol doubly and carries out alcohol precipitation in concentrated solution, and room temperature was placed 12 hours, and is centrifugal, gets supernatant liquor, i.e. the natto reactive site; Supernatant liquor is waved most ethanol spraying drying, obtains pale brown look to the brown solid powder, makes granule, pill, tablet, capsule or oral liquid;
Wherein, the method for natto reactive site purification refine:
Cross macroporous resin column,, collect elutriant and promptly got column purification natto reactive site with the ethanol elution of 70%-90%.
5. alpha-glucosidase inhibitor is characterized in that this alpha-glucosidase inhibitor made by following method:
Get natto fermented liq 50L, fling to the concentrated solution that obtains 5L behind most of water through normal pressure or concentrating under reduced pressure, 95% ethanol that adds 20L in concentrated solution carries out alcohol precipitation, room temperature was placed 12 hours, centrifugal, get supernatant liquor, supernatant liquor is gone up the AB-8 nonpolar macroporous adsorption resin after waving most ethanol, ethanol elution with 70% and 90%, the ethanol elution part of collection 70% and 90%, to fling to ethanol after this two portions elutriant merging, adding water to cumulative volume is 2.5L, made its even suspendible in ultrasonic 10 minutes, respectively extract three times with ethyl acetate and propyl carbinol respectively, the consumption of each organic solvent is 2.5L, acetic acid ethyl acetate extract and butanol extraction liquid are flung to the solvent merging respectively, add small amount of methanol and made its dissolving in ultrasonic 10 minutes, admix sample on a small amount of silica gel dry method then, with 70%, 100% methanol-eluted fractions, collect 70% and 100% meoh eluate two portions sample and, obtained alpha-glucosidase after the drying and suppressed active extract 19g, according to common process with its merging, add conventional auxiliary material, make granule, pill, tablet, capsule or oral liquid.
6. the preparation method of an alpha-glucosidase inhibitor is characterized in that this method is:
Get solid fermentation natto 1 weight part, add water, methyl alcohol, 50%-90% ethanol or the propyl alcohol of 1-10 parts by volume, stir at 75-95 ℃ and extract 2-3 time, or heating and refluxing extraction 2-3 time, extracting solution merging, i.e. natto reactive site; Decompression or normal pressure are concentrated into the 0.1-1 parts by volume, and be centrifugal, obtains pale brown look after the supernatant liquor spraying drying to the brown solid powder, according to common process, adds conventional auxiliary material, makes the formulation of clinical acceptance;
Wherein, the method for natto reactive site purification refine is one of following method:
A, mistake macroporous resin column with the ethanol elution of 70%-90%, are collected elutriant and were promptly got column purification natto reactive site;
B, further with method A cross column purification natto reactive site elutriant with ethyl acetate, n-butanol extraction, combining extraction liquid promptly got column extracting purifying natto reactive site;
C, further with C18 post on the extraction liquid of crossing column extracting purifying natto reactive site of method B, the 70%-100% methanol-eluted fractions is collected elutriant and is promptly got purifying natto reactive site.
7. preparation method as claimed in claim 6 is characterized in that this method is:
Get solid fermentation natto 1 weight part, add the water of 5 parts by volume, stir at 90 ℃ and extract 2 times, extracting solution merges, i.e. the natto reactive site; Decompression or normal pressure are concentrated into 0.5 parts by volume, and be centrifugal, obtains pale brown look after the supernatant liquor spraying drying to the brown solid powder, according to common process, adds conventional auxiliary material, makes granule, pill, tablet, capsule or oral liquid.
8. preparation method as claimed in claim 6 is characterized in that this method is:
Get solid fermentation natto 1 weight part, the alcohol heating reflux that adds 3 parts by volume 75% extracts 2 times, and extracting solution merges, i.e. the natto reactive site; Decompression or normal pressure concentrate and wave most ethanol, and be centrifugal, obtains pale brown look after the supernatant liquor spraying drying to the brown solid powder, makes granule, pill, tablet, capsule or oral liquid.
9. preparation method as claimed in claim 6 is characterized in that this method is:
Get liquid fermenting product 5 parts by volume of natto, normal pressure or be evaporated to 0.5 parts by volume adds its 4 volume 95% ethanol doubly and carries out alcohol precipitation in concentrated solution, and room temperature was placed 12 hours, and is centrifugal, gets supernatant liquor, i.e. the natto reactive site; Supernatant liquor is waved spraying drying behind the most ethanol, obtains pale brown look to the brown solid powder, makes granule, pill, tablet, capsule or oral liquid;
Wherein, the method for natto reactive site purification refine:
Cross macroporous resin column,, collect elutriant and promptly got column purification natto reactive site with the ethanol elution of 70%-90%.
10. the preparation method of an alpha-glucosidase is characterized in that this method:
Get natto fermented liq 50L, fling to the concentrated solution that obtains 5L behind most of water through normal pressure or concentrating under reduced pressure, 95% ethanol that adds 20L in concentrated solution carries out alcohol precipitation, room temperature was placed 12 hours, centrifugal, get supernatant liquor, supernatant liquor is gone up the AB-8 nonpolar macroporous adsorption resin after waving most ethanol, ethanol elution with 70% and 90%, the ethanol elution part of collection 70% and 90%, to fling to ethanol after this two portions elutriant merging, adding water to cumulative volume is 2.5L, made its even suspendible in ultrasonic 10 minutes, respectively extract three times with ethyl acetate and propyl carbinol respectively, the consumption of each organic solvent is 2.5L, acetic acid ethyl acetate extract and butanol extraction liquid are flung to the solvent merging respectively, add small amount of methanol and made its dissolving in ultrasonic 10 minutes, admix sample on a small amount of silica gel dry method then, with 70%, 100% methanol-eluted fractions, collect 70% and 100% meoh eluate two portions sample and, obtained alpha-glucosidase after the drying and suppressed active extract 19g, according to common process with its merging, add conventional auxiliary material, make granule, pill, tablet, capsule or oral liquid.
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| CN102648757A (en) * | 2012-05-14 | 2012-08-29 | 北京工商大学 | Preparation method of fermented soybeans with alpha-glucosaccharase inhibitory activity |
| CN106361994B (en) * | 2016-10-25 | 2019-07-05 | 四川大学 | Alpha-glucosidase activity constituents for suppressing in Habenaria Ciliolaris Kranzl and its preparation method and application |
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|---|---|---|---|---|
| EP0834262A1 (en) * | 1996-04-10 | 1998-04-08 | Nichimo Company Limited | Substance containing health-promoting component and process for the production thereof |
| CN1275083A (en) * | 1998-08-26 | 2000-11-29 | 日本合成化学工业株式会社 | Alpha-glucosidase inhibitor |
-
2007
- 2007-08-24 CN CN2007101207165A patent/CN101371869B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0834262A1 (en) * | 1996-04-10 | 1998-04-08 | Nichimo Company Limited | Substance containing health-promoting component and process for the production thereof |
| CN1275083A (en) * | 1998-08-26 | 2000-11-29 | 日本合成化学工业株式会社 | Alpha-glucosidase inhibitor |
Non-Patent Citations (5)
| Title |
|---|
| Fujita Hiroyukia,etc.Efficacy and safety of Touchi Extract, an a-glucosidase inhibitorderived from fermented soybeans, in non-insulin-dependentdiabetic mellitus.《THE JOURNAL OF Nutritional Biochemistry》.2001,第12卷351-356. * |
| Hiroyuki Fujita.etc.Fermented soybean-derived Touchi-extract with anti-diabetic effect via α-glucosidase inhibitory action in a long-term administration study with KKAy mice.《Life Sciences》.2001,第70卷219-227. * |
| JP特开2000－72687A 2000.03.07 |
| 孙丽艳等.发酵罐制备大豆异黄酮糖苷转化酶(β-糖苷酶)条件的研究.《大豆通报》.2006,(第3期), * |
| 郭瑞华等.豆豉多糖的提纯及成分分析.《时珍国医国药》.2006,第17卷(第4期),511-512. * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101371869A (en) | 2009-02-25 |
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