CN1275982C - Method for extraction and purification of garden burnet polysaccharide - Google Patents
Method for extraction and purification of garden burnet polysaccharide Download PDFInfo
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- CN1275982C CN1275982C CN 200510043873 CN200510043873A CN1275982C CN 1275982 C CN1275982 C CN 1275982C CN 200510043873 CN200510043873 CN 200510043873 CN 200510043873 A CN200510043873 A CN 200510043873A CN 1275982 C CN1275982 C CN 1275982C
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- burnet polysaccharide
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Abstract
The present invention discloses a method for separation and purification of garden burnet polysaccharide, which is composed of the following steps that garden burnet is pulverized and is soaked in distilled water according to the quantity of 1: 4 to 5; the garden burnet has a water bath at the temperature of 50 DEG C to 65 DEG C for 15 to 30 hours; the garden burnet is centrifuged by 3000 to 5000 rpm for 10 to 16 minutes; supernatant solution is obtained; activated carbon is used for decoloration for obtaining garden burnet polysaccharide water extracting solution; an organic solvent is used for removing protein in the garden burnet polysaccharide water extracting solution in a conventional mode; a chromatographic column which has the rate of the inner diameter and the height of 1: 25 to 50 is selected; a wave with the length of 280 nm is used for detecting collecting solution by gel filtration chromatography; the garden burnet polysaccharide extracting solution is frozen and dried in vacuum for obtaining garden burnet polysaccharide. The garden burnet polysaccharide prepared by the present invention has more than 90% of purity. The present invention has the advantages of low method requirement, low equipment requirement, low environmental requirement, simple and convenient method, low cost, flexibility and practicality, and is favourable for popularization, development and application.
Description
Technical field
The present invention relates to a kind of in the separation method of pharmaceutically active ingredient, relate in particular to a kind of from the Chinese medicine garden burnet method of separation and purification great burnet polysaccharide.
Background technology
Great burnet polysaccharide is the effective constituent of Chinese medicine garden burnet, be to separate a kind of polysaccharide that obtains from the Chinese medicine garden burnet, great burnet polysaccharide is the inhibitor of alpha-glucosidase, can be used for treatment of diabetes and fat-reducing, little to the human body side effect, with the alpha-glucosidase inhibitor that uses clinically medicine---acarbose (Acarbose, trade(brand)name: glucobay (acarbose) is Bayer A.G's product) phase specific activity is stronger.
α glucuroide (EC3.2.1.20; A-glucosidase), be that a class can be from the general name of the enzyme that contains α glycosidic link substrate catalysis hydrolyzation of glucose base.Mainly the non-reducing end from polysaccharide, disaccharides downcuts glucose on physiological function, and human body all depends on the effect of α glucuroide on the small intestinal mucosa cell brush border to the final digestion of food such as starch.
The α glucosidase inhibitor is a class orally-taken blood sugar reducing medicine of the seventies later stage research and development, its mechanism of action is: competitive inhibition α glucuroide, inhibition is from digestion, the absorption of disaccharide, polysaccharide, oligose, effectively postpone and alleviate the after-dinner blood sugar of diabetes patients especially generation of complication that raises, obtain better therapeutic effect, so far still widespread use.
Great burnet polysaccharide belongs to competitive inhibition to the restraining effect of alpha-glucosidase, so can select various dose according to user's needs, for present fat-reducing main forces, especially young women can be avoided the misery of going on a diet, if can solve the preparation and the prescription of great burnet polysaccharide, will make things convenient for slimmer's use greatly, enlarge the utilization of garden burnet.
By retrieval, the separation purification method about great burnet polysaccharide does not still have open report at present.
Summary of the invention
At the deficiencies in the prior art, the problem to be solved in the present invention provide a kind of from the Chinese medicine garden burnet method of separation and purification great burnet polysaccharide.
The separation purification method of great burnet polysaccharide of the present invention, form by following step:
(1) Radix Sangusorbae powder being broken to particle diameter is 200～300 μ m, with water in volume ratio, the amount by 1: 4～5 is dipped in the distilled water, 50～65 ℃ of water-baths 15～30 hours;
(2) with the centrifugal 10～16min of 3000～5000rpm, get supernatant liquor,, get great burnet polysaccharide aqueous extract solution with activated carbon decolorizing;
(3) remove albumen in the great burnet polysaccharide aqueous extract solution in a usual manner with organic solvent;
(4) choosing internal diameter and aspect ratio is 1: 25～50 chromatography column; Add dextrane gel in post, add-on is 60%～90% of a column volume;
(5) gel permeation chromatography step (4), elutriant is a distilled water, and flow velocity is 0.3～0.6ml/min, is that 280nm detects collection liquid with the wavelength, and the elutriant curve has two absorption peaks, collects second peak elutriant, is the extracting solution of great burnet polysaccharide;
(6), promptly get powder---the great burnet polysaccharide of light brown with the extracting solution of ordinary method vacuum lyophilization great burnet polysaccharide.
Wherein, preferably 60 ℃ of the described bath temperatures of step (1), preferably 20～25 hours water-bath time.
Wherein, the described centrifugal speed of step (2) is 4000rpm preferably, and centrifugation time is 10min preferably.
Wherein, the described organic solvent of step (3) ethanol preferably.
Wherein, the internal diameter and the aspect ratio of the described chromatography column of step (4) be preferably 1: 30～40.
Wherein, the described amount with molecular sieve effect that adds in post of step (4) is preferably 70%～80% of column volume.
Wherein, above-mentioned dextrane gel is selected SephadexG-75～SephadexG-200 for use.
Wherein, the preferred SephadexG-150 of above-mentioned dextrane gel.
Wherein, the described flow velocity of step (5) is preferably 0.4ml/min.
Utilize the separation purification method of the great burnet polysaccharide that the present invention proposes, can prepare purity and reach great burnet polysaccharide more than 90%, solve great burnet polysaccharide at all and separated difficulty, purity is low, the unsettled deficiency of quality, and method equipment of the present invention and environmental requirement are low, method is easy, cost is low, and is practical flexibly, is beneficial to popularization, development and application.
The great burnet polysaccharide that adopts separation purification method of the present invention to prepare, confirm through test, the α glucosidase activity there is significant inhibitory effect, test-results shows: the great burnet polysaccharide that the inventive method is prepared reaches 99.9% to the inhibiting rate of α glucosidase activity, and medicine---the acarbose (Acarbose of the alpha-glucosidase inhibitor that uses clinically at present, trade(brand)name: glucobay (acarbose), be Bayer A.G's product) only be 81.5% to the inhibiting rate of α glucosidase activity, illustrate that separation purification method of the present invention is significantly improved than existing method, has possessed commercial applications and promotional value.
Description of drawings
The elution curve of Fig. 1 great burnet polysaccharide gel permeation chromatography
Fig. 2 big white mouse is taken the sugared anti-curve behind the great burnet polysaccharide
Embodiment
Embodiment 1:
(1) Radix Sangusorbae powder being broken to particle diameter is 200～250 μ m, with water by quality ratio, be dipped in the distilled water 50 ℃ of water-baths 20 hours by 1: 4 amount;
(2) with the centrifugal 10min of 4000rpm, get supernatant liquor,, get great burnet polysaccharide aqueous extract solution with activated carbon decolorizing;
(3) remove albumen in the great burnet polysaccharide aqueous extract solution in a usual manner with organic solvent;
(4) choosing internal diameter and aspect ratio is 1: 25 chromatography column; Add the SephadexG-100 dextrane gel in post, add-on is 70% of a column volume;
(5) gel permeation chromatography step (4), elutriant is a distilled water, and flow velocity is 0.5ml/min, is that 280nm detects collection liquid with the wavelength, and the elutriant curve has two absorption peaks, collects second peak elutriant, is the extracting solution of great burnet polysaccharide; (see figure 1)
(6), promptly get powder---the great burnet polysaccharide of light brown with the extracting solution of ordinary method vacuum lyophilization great burnet polysaccharide.
Embodiment 2:
(1) Radix Sangusorbae powder being broken to particle diameter is 250～300 μ m, with water by quality ratio, be dipped in the distilled water 60 ℃ of water-baths 30 hours by 1: 5 amount;
(2) with the centrifugal 16min of 4000rpm, get supernatant liquor,, get great burnet polysaccharide aqueous extract solution with activated carbon decolorizing;
(3) remove albumen in the great burnet polysaccharide aqueous extract solution in a usual manner with organic solvent;
(4) choosing internal diameter and aspect ratio is 1: 40 chromatography column; Add the SephadexG-150 dextrane gel in post, add-on is 80% of a column volume;
(5) gel permeation chromatography step (4) elutriant is a distilled water, and flow velocity is 0.4ml/min; With the wavelength is that 280nm detects collection liquid, and the elutriant curve has two absorption peaks, collects second peak elutriant, is the extracting solution of great burnet polysaccharide; (see figure 1)
(6), promptly get powder---the great burnet polysaccharide of light brown with the extracting solution of ordinary method vacuum lyophilization great burnet polysaccharide.
Embodiment 3:
(1) Radix Sangusorbae powder being broken to particle diameter is 200～250 μ m, with water by quality ratio, be dipped in the distilled water 55 ℃ of water-baths 25 hours by 1: 4 amount;
(2) with the centrifugal 15min of 3000rpm, get supernatant liquor,, get great burnet polysaccharide aqueous extract solution with activated carbon decolorizing;
(3) remove albumen in the great burnet polysaccharide aqueous extract solution in a usual manner with organic solvent;
(4) choosing internal diameter and aspect ratio is 1: 50 chromatography column; Add the SephadexG-200 dextrane gel in post, add-on is 85% of a column volume;
(5) gel permeation chromatography step (4) elutriant is a distilled water, and flow velocity is 0.3ml/min, is that 280nm detects collection liquid with the wavelength, and the elutriant curve has two absorption peaks, collects second peak elutriant, is the extracting solution of great burnet polysaccharide; (see figure 1)
(6), promptly get powder---the great burnet polysaccharide of light brown with the extracting solution of ordinary method vacuum lyophilization great burnet polysaccharide.
Embodiment 4: the determination of activity of great burnet polysaccharide
The α glucosidase activity is measured:
Standard reaction system: 67mmol/L potassium phosphate buffer (pH6.8) 1.7ml, 1mg/ml gsh 50ul, 2mg/ml α glucuroide (Sigma company product) 10ul, 37 ℃ of insulation 10min, add 116mmol/L PNP 300ul, 37 ℃ of 10min add 0.1mol/L yellow soda ash 10ml termination reaction, and 400nm surveys the nitrophenol amount that discharges.
α glucosidase activity unit: pH6.8,37 ℃, it is an activity unit that per minute discharges the 1umol/L nitrophenol;
α glucosidase inhibitor activity unit: pH6.8,37 ℃, making unit of enzyme activity's inactivation is an inhibition unit;
Inhibiting rate: inhibitor vigor ÷ unit of enzyme activity
Through test determination: the great burnet polysaccharide that the inventive method is prepared reaches 99.9% to the inhibiting rate of α glucosidase activity.
Embodiment 5: big white mouse is taken the sugared anti-test behind the great burnet polysaccharide
The great burnet polysaccharide of preparing with the inventive method, with the amount of significant quantity 200mg/time, behind the SD rat oral gavage, feed in 30 minutes, rat blood sugar concentration when measuring 30 minutes after the meal, 60 minutes, 90 minutes respectively, the great burnet polysaccharide prepared of inventive method has digestion, the absorption of obvious suppression disaccharide, polysaccharide, oligose as a result, effectively postpones and slows down the effect that postprandial blood sugar raises.See Fig. 2.
Embodiment 6:
The great burnet polysaccharide of preparing with the inventive method, amount/sky/rat (220g) with effective therapeutic dose 400mg, to SD rat continuous irrigation stomach 10 days, irritated stomach on the 10th day put to death in 2 hours, get small intestinal mucosa, detect α glucuroide on the small intestinal mucosa cell brush border, recording the great burnet polysaccharide group is 40% of control group (irritating stomach physiological saline) to the inhibiting rate of α glucosidase activity.
External, the great burnet polysaccharide of preparing with the inventive method of various dose acts on big white mouse α glucuroide, the results are shown in Table 1:
Table 1: great burnet polysaccharide is to the inhibiting rate of big white mouse mucous membrane of small intestine α glucosidase activity
| Great burnet polysaccharide (ug) | 8 | 7 | 6 | 5 | 4 | 3 | 2.5 | 2 | 1.5 | 1 |
| Inhibiting rate | 99.9％ | 99.9％ | 99.9％ | 99.9％ | 99.9％ | 94％ | 76％ | 72％ | 62％ | 45％ |
Claims (9)
1. the separation purification method of a great burnet polysaccharide, form by following step:
(1) Radix Sangusorbae powder being broken to particle diameter is 200～300 μ m, with water by quality ratio, the amount by 1: 4～5 is dipped in the distilled water, 50～65 ℃ of water-baths 15～30 hours;
(2) with the centrifugal 10～16min of 3000～5000rpm, get supernatant liquor,, get great burnet polysaccharide aqueous extract solution with activated carbon decolorizing;
(3) remove albumen in the great burnet polysaccharide aqueous extract solution in a usual manner with organic solvent;
(4) choosing internal diameter and aspect ratio is 1: 25～50 chromatography column; Add dextrane gel in post, add-on is 60%～90% of a column volume;
(5) gel permeation chromatography step (4), elutriant is a distilled water, and flow velocity is 0.3～0.6ml/min, is that 280nm detects collection liquid with the wavelength, and the elutriant curve has two absorption peaks, collects second peak elutriant, is the extracting solution of great burnet polysaccharide;
(6), promptly get powder---the great burnet polysaccharide of light brown with the extracting solution of ordinary method vacuum lyophilization great burnet polysaccharide.
2. the separation purification method of great burnet polysaccharide as claimed in claim 1 is characterized in that, the described bath temperature of step (1) is 60 ℃, and the water-bath time is 20～25 hours.
3. the separation purification method of great burnet polysaccharide as claimed in claim 1 is characterized in that, the described centrifugal speed of step (2) is 4000rpm, and centrifugation time is 10min.
4. the separation purification method of great burnet polysaccharide as claimed in claim 1 is characterized in that, the described organic solvent of step (3) is an ethanol.
5. the separation purification method of great burnet polysaccharide as claimed in claim 1 is characterized in that, the internal diameter and the aspect ratio of the described chromatography column of step (4) are 1: 30～40.
6. the separation purification method of great burnet polysaccharide as claimed in claim 1 is characterized in that, the described amount with molecular sieve effect that adds in post of step (4) is 70%～80% of a column volume.
7. the separation purification method of great burnet polysaccharide as claimed in claim 1 is characterized in that, described dextrane gel is selected SephadexG-75～SephadexG-200 for use.
8. the separation purification method of great burnet polysaccharide as claimed in claim 7 is characterized in that, described dextrane gel is selected SephadexG-150 for use.
9. the separation purification method of great burnet polysaccharide as claimed in claim 1 is characterized in that, the described flow velocity of step (5) is 0.4ml/min.
Priority Applications (1)
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| CN 200510043873 CN1275982C (en) | 2005-06-21 | 2005-06-21 | Method for extraction and purification of garden burnet polysaccharide |
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| CN 200510043873 CN1275982C (en) | 2005-06-21 | 2005-06-21 | Method for extraction and purification of garden burnet polysaccharide |
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| CN1699427A CN1699427A (en) | 2005-11-23 |
| CN1275982C true CN1275982C (en) | 2006-09-20 |
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| CN102304191B (en) * | 2011-08-24 | 2013-03-27 | 中国科学院烟台海岸带研究所 | Method for extracting crude polysaccharide from waste internal organs of sea worms |
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