CN104479035A - Polygonatum kingianum polysaccharide extract with sugar and lipid metabolism regulating activity and preparation method thereof - Google Patents
Polygonatum kingianum polysaccharide extract with sugar and lipid metabolism regulating activity and preparation method thereof Download PDFInfo
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Abstract
The invention discloses an extraction method and application of polygonatum kingianum polysaccharide. The extraction method comprises the following steps: cleaning and slicing polygonatum kingianum taken as a raw material, naturally drying in the sun, and then grinding into coarse powder; extracting with distilled water for three times, then concentrating to a certain concentration, and depositing by using anhydrous ethanol to obtain a polygonatum kingianum polysaccharide crude product; dissolving by using distilled water, adding alpha-amylase to remove starch, depositing by using anhydrous ethanol, and then washing by using anhydrous ethanol, acetone and ethyl ether to remove fat-soluble components to obtain yellow powder; removing protein by using a Sevage method after deionized water dissolution, volatilizing an organic solvent, then performing dialysis, and finally freeze-drying to obtain white powder namely polygonatum kingianum polysaccharide. The obtained polygonatum kingianum polysaccharide is used for preparing tablets and has effects of reducing blood pressure, blood fat and blood sugar.
Description
Technical field
The present invention relates to field of traditional Chinese medicine extraction, be specifically related to extract of a kind of P. kingianum polysaccharide and preparation method thereof.
Background technology
Sealwort is the important medicine-food two-purpose resource of China, " Chinese Pharmacopoeia " 2010 editions records the dry rhizome that sealwort is Liliaceae HUANGJING ZANYU CAPSULE P. kingianum Polygonatum kingianum Coll. Et Hemsl. sealwort P. sibiricum Red. or David's-harp P. cyrtonema Hua, practises title " large sealwort " " polygonatum sibiricum Redoute " or " RHIZOMA POLYGONATI ZINGIBERIFORME " respectively.
P. kingianum is large in Yunnan standing stock, its medicinal record is all had in " the southern regions of the Yunnan Province book on Chinese herbal medicine ", " Yunnan plant will " etc., being important " cloud medicine " kind, is also the maximum sealwort kind of output and the market share, has the basis of clinical application widely and good gos deep into DEVELOPMENT PROSPECT.
P. kingianum polysaccharide is considered to chemical composition type main in P. kingianum always, is also the chemical composition specifying to carry out P. kingianum quality control in " Chinese Pharmacopoeia " 2010 editions.Now research shows that P. kingianum polysaccharide has reducing blood sugar and blood fat and strengthens immune function of human body.
After conventional extraction method of polysaccharides is mainly grinding medicinal materials, degreasing, decolouring, obtain polygonatum polysaccharide crude product by the method for water extract-alcohol precipitation, the treating process of polysaccharide is generally ion-exchange, gel-filtration and concentrated after crossing chromatography column, lyophilize and obtains.Above-mentioned polyoses producing method has the following disadvantages: first, is frequently heated in medicinal material skimming processes, and polysaccharide loss is more, affects the extraction yield of polysaccharide; Secondly, degreasing, decolorization complexity, the time is long, and cost is high, is difficult to large-scale production; In addition, the polysaccharide that ordinary method prepares gained is impure more, contains macromolecular substance as protein, starch and aldehydes matter etc., small-molecule substance glucose and fructose etc. more.
Summary of the invention
The raw material that polygonatum polysaccharide of the present invention extracts is P. kingianum, and the raw material of film-making agent is P. kingianum polysaccharide.
This law feature:
1. usually contain the impurity such as the insoluble small organic molecule of macro-molecular protein, inorganic salt, xylogen, pigment and alcohol in carried Crude polysaccharides.Present method adopts a methods combining lyophilize that water extraction adds repeatedly alcohol precipitation, macromolecular substance starch, protein and small-molecule substance etc. are eliminated in process, final step dialysis removing inorganic molecules, flings to the polygonatum polysaccharide that organic substance postlyophilization obtains purer white.
2. propose in treating process the change avoiding polysaccharide color at the beginning of this law, slightly putting forward yield after refining is 13%, and polygonatum polysaccharide is white, and ultraviolet spectrophotometry detects refined polysaccharide 280nm place without absorbing, and does not namely contain albumen.Measuring polysaccharide content with Anthrone-sulfuricacid method is more than 85%.
3. this law is applicable to the P. kingianum meal with reclaiming after 80% extraction using alcohol saponin(e, and after naturally being dried by the P. kingianum powder of recovery, continue to extract polysaccharide by this law, extraction yield is 9%.Achieve resource reclaim recycling.
4. the polygonatum polysaccharide that obtains of this law is for the preparation of polygonatum polysaccharide tablet, and sugar degree is 10%, and pharmacology proves, polygonatum polysaccharide has reduction triglyceride level (TG), cholesterol (TC), blood sugar and improves the effect of sugar tolerance.
Concrete grammar is as follows:
Take P. kingianum as raw material, clean section, after naturally drying, break into meal; Get 500-1000g, with the distilled water extraction three times of 6-10 times amount, filter, after merging filtrate, be concentrated to the crude drug in whole containing 1.2g/ml, place room temperature; In concentrated solution, add the dehydrated alcohol of 4 times amount, in 4000r/min pelleted by centrifugation 10-20min after standing 24h, sediment separate out, obtains polygonatum polysaccharide crude product; Dissolve with distilled water, add α-amylase destarching; Again by 4 times amount dehydrated alcohol precipitations, add the dehydrated alcohol of 4 times amount, in 4000r/min pelleted by centrifugation 10-20min after standing 24h, sediment separate out, use dehydrated alcohol, acetone, washed with diethylether degrease soluble components respectively, room temperature waves organic solvent, obtains yellow powder; Again with using Sevage method Deproteinization after deionized water dissolving, carry out flowing water after flinging to organic solvent and dialyse 2 days, deionized water is dialysed 1 day, and namely last lyophilize obtains the polygonatum polysaccharide of white powder.
Described destarching step is specially: add α-amylase fully stir after in 50 DEG C of water-baths hydrolyzed starch to the constant basket of liquor kalii iodide; Directly use boiling water bath deactivation α-amylase 5min afterwards; Put to room temperature, centrifugal abandon assorted.
The concrete steps of described Sevage method Deproteinization are: add chloroform-propyl carbinol=4:1 be made into the solution of 5% with deionized water dissolving after, fully after concussion extraction under 4000r/min condition centrifugal 10min, be separated and take out supernatant liquor, removing protein.
Accompanying drawing explanation
Fig. 1 canonical plotting
Fig. 2 cell TC is containing spirogram
Fig. 3 cell TG content
In figure, * * * represents model group P<0.01 compared with normal group; ### represents administration group P<0.01 compared with model group.
CON: normal group MOD: model group GL: polysaccharide low dose group GM: dosage group GH in polysaccharide: polysaccharide high dose group LGLT: dosage group in rosiglitazone
Embodiment
Embodiment 1
Life P. kingianum is cleaned nature dry, drying rear dry product yield is 20%, and P. kingianum is broken into meal, gets 100g powder, and use 12 times amount, 10 times amount, 8 times amount distilled water extraction respectively, extraction time is respectively 1.5h, 1h, 0.5h, and Extracting temperature is set to 80 DEG C.Merge No. three extracting solutions, concentrated concentration is 1g/ml (crude drug in whole).Filter with after 4 times amount dehydrated alcohol precipitation 24h, filter residue distilled water dissolves, add α-amylase destarching, again by 4 times amount dehydrated alcohol precipitations, centrifuging and taking throw out under the same terms, uses dehydrated alcohol, acetone, washed with diethylether degrease soluble components, room temperature air seasoning respectively, wave organic solvent, obtaining yellow powder weight in wet base content is 67.2g.Powder distilled water dissolves, and rear Sevage method removes egg, and carry out flowing water after flinging to organic solvent and dialyse 2 days, deionized water is dialysed 1 day, and the polygonatum polysaccharide content that namely last lyophilize obtains white powder is 13.7g.
Embodiment 2
By the P. kingianum powder reclaimed after use for laboratory 80% extraction using alcohol saponin(e, after naturally drying, take 100g, with above-mentioned same method Hydrolysis kinetics, the polysaccharide weight in wet base 44g obtained after slightly carrying.After refining, lyophilize obtains 9 g.
Embodiment 3
The preparation of P. kingianum polysaccharide tablet: with parts by weight, by kilogram in units of, take respectively: P. kingianum polysaccharide 100 parts, Xylitol 300 parts, 250 parts, dextrin, Microcrystalline Cellulose 250 parts, chitin 50 parts, citric acid 25 parts, Magnesium Stearate 25 parts.
Above-mentioned P. kingianum polysaccharide, Xylitol, dextrin, Microcrystalline Cellulose, chitin are fully mixed, with water-wet, softwood processed, sieve, dry after whole grain, add citric acid, Magnesium Stearate, compressing tablet after mixing, the heavy 250mg of every sheet, activeconstituents P. kingianum polysaccharide is containing 25mg.
P. kingianum total polysaccharides of the present invention has the effect reducing fat triglyceride level and cholesterol, and have certain α Glycosylase inhibiting rate, result shows, polygonatum polysaccharide can be used for prevention and regulates glucose-lipid metabolism disorder.
Embodiment 4
1. Anthrone-sulfuricacid method measures P. kingianum purity of polysaccharide
The preparation of 1.1 reference substances and sample
The accurately weighed dextrose anhydrous reference substance 50mg being dried to constant weight in 105 DEG C, puts in 100mL volumetric flask, is dissolved in water and is diluted to scale, shaking up, obtain the standard solution of 0.5mg/mL; Precision takes P. kingianum polysaccharide powder 100mg, puts in 100mL volumetric flask, is dissolved in water and is diluted to scale, shaking up, and obtains the sample solution of 1mg/mL.
The formulation of 1.2 typical curves
Precision measures reference substance solution 0.1mL, 0.2mL, 0.3mL, 0.4mL, 0.5mL, 0.6mL are placed in 10mL test tube respectively, each adding distil water is to 2.0mL, shake up, in ice-water bath, slowly drip 0.2% By Anthrone Sulphuric acid solution to scale, shake up, let cool in rearmounted water-bath and be incubated 10 minutes, take out, not add sample for blank, measure absorbancy by ultraviolet spectrophotometry in 582nm place.Result as table 1, Fig. 1.
The mensuration of 1.3 P. kingianum purity of polysaccharide
Method with 1.2 measure respectively P. kingianum polysaccharide content, the concentration of preparation is respectively: 0.01mg/mL, 0.03mg/mL, 0.050mg/mL, 0.07mg/mL.The purity of bringing typical curve formula mensuration P. kingianum polysaccharide according to absorbancy into is more than 85%, result table 2.
2.
p. kingianum polysaccharide alpha-glucoside inhibiting activity
2.1 materials and methods
The preparation of material and P. kingianum total polysaccharides
Alpha-glucosidase (α-Glucosidase, EC 3.2.1.20), 4-nitrophenol-α-D-glucopyranoside (pNPG, lot number: 101324963, CAS:3767-28-0); Foetal calf serum (Foetal Bovine Serum, LOT:1413865); All the other chemical reagent are analytical pure.
Precision takes appropriate P. kingianum polysaccharide, is mixed with 0.1mg/m respectively, and the concentration of 1mg/mL, 10mg/mL, 20mg/mL is as testing sample.
Method
In potassium phosphate buffer (pH6.8) system, temperature of reaction 37 DEG C, is reaction substrate with pNPG, adds alpha-glycosidase, with Na
2cO
3for stop buffer.PNPG solution is colourless, but discharge p-NP (pNP) through glucoside enzymic hydrolysis, pNP is in the basic conditions in yellow-green colour, maximum absorption is had at 400nm place, when adding alpha-glucosidase inhibitor, can the activity of Inhibiting α-glucosidase, reduce the release of pNP, therefore can measure inhibit activities by the change of absorbancy.
Reaction system
Get pH 6.8 phosphate buffered saline buffer 725 μ L, testing sample solution 10 μ L, alpha-glucosidase (0.15U/mL) 100 μ L, mixing, not add testing sample solution as blank (pH 6.8 phosphate buffered saline buffer is supplied), remove as a setting with the reaction system not adding enzyme solution, 37 DEG C of waters bath with thermostatic control are incubated 10 min, 175 μ L 4-nitrophenol-α-D-glucopyranoside (pNPG) are added again in reaction system, after constant temperature 10 min, add the 0.1mol/L Na of 2mL immediately
2cO
3termination reaction.
result
Can find out that in vitro in model, P. kingianum total polysaccharides has certain restraining effect to alpha-glucosidase by table.As table 3.
3. P. kingianum polysaccharide is to the lipometabolic regulating effect of fat L-02 hepatocytes
3.1 materials and methods
Material
Normal people's L-02 hepatocytes, medical fat breast (10% foetal calf serum, 10% medical fat breast and RPMI-1640 nutrient solution), normal nutrient solution (the RPMI-1640 nutrient solution containing 10% foetal calf serum), and G0(is containing the RPMI-1640 nutrient solution of 0.2% foetal calf serum) solution etc.
Method
Modeling: will pass in 6 orifice plates after cell cultures to some amount, except normal group is with except normal nutrient solution cultivation, all the other all use medical fat breast culturing cell 48h, after induction liver L-02 fatization, be divided into model group, P. kingianum polysaccharide low (10 μ g/mL), in (50 μ g/mL), high (100 μ g/mL) three concentration groups and rosiglitazone (50 μm of ol/L) group, often group does three multiple holes.The content of intracellular triglyceride (TG) and cholesterol (TC) is measured after administration 24h.
Statistical method: normal group compares with model group, administration group compares with rosiglitazone and checks with t, compare between administration group and use one-way analysis of variance method.
3. 2 results
From Fig. 2 and Fig. 3, model group cell TC, TG content is significantly higher than normal group, and after giving P. kingianum polysaccharide and rosiglitazone, cell TC, TG content obviously declines, and compared with model group, there is significant difference, and P. kingianum polysaccharide effect there was no significant difference compared with rosiglitazone.Learning that P. kingianum total saponins has thus significantly regulates lipid metabolism active.As Fig. 2, Fig. 3.
In figure, * * * represents model group P<0.01 compared with normal group; ### represents administration group P<0.01 compared with model group.
CON: normal group MOD: model group GL: polysaccharide low dose group GM: dosage group GH in polysaccharide: polysaccharide high dose group LGLT: dosage group in rosiglitazone.
Claims (7)
1. the extracting method of P. kingianum polysaccharide, is characterized in that:
Take P. kingianum as raw material, clean section, after naturally drying, break into meal; Get 500-1000g, with the distilled water extraction three times of 6-10 times amount, filter, after merging filtrate, be concentrated to the crude drug in whole containing 1.2g/ml, place room temperature; In concentrated solution, add the dehydrated alcohol of 4 times amount, in 4000r/min pelleted by centrifugation 10-20min after standing 24h, sediment separate out, obtains polygonatum polysaccharide crude product; Dissolve with distilled water, add α-amylase destarching; Again by 4 times amount dehydrated alcohol precipitations, add the dehydrated alcohol of 4 times amount, in 4000r/min pelleted by centrifugation 10-20min after standing 24h, sediment separate out, use dehydrated alcohol, acetone, washed with diethylether degrease soluble components respectively, room temperature waves organic solvent, obtains yellow powder; Again with using Sevage method Deproteinization after deionized water dissolving, carry out flowing water after flinging to organic solvent and dialyse 2 days, deionized water is dialysed 1 day, and namely last lyophilize obtains the P. kingianum polysaccharide of white powder.
2. the extracting method of P. kingianum polysaccharide as claimed in claim 1, is characterized in that: described destarching step is specially: add α-amylase fully stir after in 50 DEG C of water-baths hydrolyzed starch to the constant basket of liquor kalii iodide; Directly use boiling water bath deactivation α-amylase 5min afterwards; Put to room temperature, centrifugal abandon assorted.
3. the extracting method of P. kingianum polysaccharide as claimed in claim 1, it is characterized in that: the concrete steps of described Sevage method Deproteinization are: add 4:1 chloroform-propyl carbinol be made into the solution of 5% with deionized water dissolving after, after abundant concussion extraction under 4000r/min condition centrifugal 10min, be separated and take out supernatant liquor, removing protein.
4. the P. kingianum polysaccharide of the extracting method acquisition of the P. kingianum polysaccharide described in claim 1-3 any one.
5. a P. kingianum polysaccharide medicine preparation, is characterized in that: P. kingianum polysaccharide according to claim 4 is obtained pharmaceutical preparation as active fraction preparation.
6. a kind of P. kingianum polysaccharide tablet as claimed in claim 5, it is characterized in that: with parts by weight, by kilogram in units of, raw material is P. kingianum polysaccharide 100 parts, Xylitol 300 parts, 250 parts, dextrin, Microcrystalline Cellulose 250 parts, chitin 50 parts, citric acid 25 parts, Magnesium Stearate 25 parts.
7. a P. kingianum polysaccharide tablet, is characterized in that: comprise P. kingianum polysaccharide and pharmaceutically acceptable auxiliary material.
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Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106511278A (en) * | 2016-12-06 | 2017-03-22 | 四川农业大学 | Polygonatum polysaccharide granules and preparation method thereof |
| CN107259429A (en) * | 2017-06-18 | 2017-10-20 | 合肥睿联生物科技有限公司 | A kind of wonderful taste longan of Siberian solomonseal rhizome polysaccharide and preparation method thereof |
| CN107827995A (en) * | 2017-12-18 | 2018-03-23 | 大理大学 | A kind of method for extraction and purification of P. kingianum polysaccharide |
| CN107822133A (en) * | 2017-12-01 | 2018-03-23 | 安徽省旌德博仕达农业科技有限公司 | A kind of plant polyphenol is modified the preparation method of sealwort dietary fiber |
| CN108543014A (en) * | 2018-05-28 | 2018-09-18 | 云南中医学院 | Application of the P. kingianum in preparing disorders of lipid metabolism and adjusting drug or health-oriented products |
| CN110326731A (en) * | 2019-07-23 | 2019-10-15 | 陕西师范大学 | A kind of polygonatum kingianurn auxiliary hyperglycemic solid beverage and preparation method thereof |
| CN111227224A (en) * | 2018-11-10 | 2020-06-05 | 江西乐迪医疗科技有限公司 | Medical formula food helpful for protecting kidney function of diabetic nephropathy patient |
| CN111533819A (en) * | 2020-05-11 | 2020-08-14 | 四川金方生物医药科技有限公司 | Method for extracting polygonatum polysaccharide for beverage |
| CN114703241A (en) * | 2021-12-31 | 2022-07-05 | 安徽中医药大学 | Preparation method and application of polygonatum cyrtonema polysaccharide processed with wine |
| CN115043955A (en) * | 2022-06-29 | 2022-09-13 | 西南林业大学 | Polygonatum polysaccharide extract and preparation method thereof |
| CN116284488A (en) * | 2023-05-05 | 2023-06-23 | 曲靖医学高等专科学校 | Extraction method of Polygonatum kingianum polysaccharide |
| CN116874634A (en) * | 2023-08-24 | 2023-10-13 | 安徽中医药大学 | Polygonatum cyrtonema Fabricius degradation polysaccharide and preparation method and application thereof |
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| CN107259429A (en) * | 2017-06-18 | 2017-10-20 | 合肥睿联生物科技有限公司 | A kind of wonderful taste longan of Siberian solomonseal rhizome polysaccharide and preparation method thereof |
| CN107822133A (en) * | 2017-12-01 | 2018-03-23 | 安徽省旌德博仕达农业科技有限公司 | A kind of plant polyphenol is modified the preparation method of sealwort dietary fiber |
| CN107827995A (en) * | 2017-12-18 | 2018-03-23 | 大理大学 | A kind of method for extraction and purification of P. kingianum polysaccharide |
| CN108543014A (en) * | 2018-05-28 | 2018-09-18 | 云南中医学院 | Application of the P. kingianum in preparing disorders of lipid metabolism and adjusting drug or health-oriented products |
| CN111227224A (en) * | 2018-11-10 | 2020-06-05 | 江西乐迪医疗科技有限公司 | Medical formula food helpful for protecting kidney function of diabetic nephropathy patient |
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| CN110326731B (en) * | 2019-07-23 | 2022-09-23 | 陕西师范大学 | Polygonatum kingianum and rhizoma polygonati solid beverage assisting in reducing blood sugar and preparation method thereof |
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| CN114703241A (en) * | 2021-12-31 | 2022-07-05 | 安徽中医药大学 | Preparation method and application of polygonatum cyrtonema polysaccharide processed with wine |
| CN114703241B (en) * | 2021-12-31 | 2024-05-10 | 安徽中医药大学 | Preparation method and application of wine-processed polygonatum sibiricum polysaccharide |
| CN115043955A (en) * | 2022-06-29 | 2022-09-13 | 西南林业大学 | Polygonatum polysaccharide extract and preparation method thereof |
| CN116284488A (en) * | 2023-05-05 | 2023-06-23 | 曲靖医学高等专科学校 | Extraction method of Polygonatum kingianum polysaccharide |
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