CN104546957A - Water chestnut shell polyphenol extract as well as preparation method and application thereof - Google Patents
Water chestnut shell polyphenol extract as well as preparation method and application thereof Download PDFInfo
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- CN104546957A CN104546957A CN201410810654.0A CN201410810654A CN104546957A CN 104546957 A CN104546957 A CN 104546957A CN 201410810654 A CN201410810654 A CN 201410810654A CN 104546957 A CN104546957 A CN 104546957A
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Abstract
The invention discloses a water chestnut shell polyphenol extract as well as a preparation method and application thereof. The preparation method comprises the following step: by taking water chestnut shells as raw materials, performing alcohol extraction and macroporous resin purification processes to prepare the water chestnut shell polyphenol extract. The water chestnut shell polyphenol extract prepared by using the method disclosed by the invention has significant alpha-glucosidase inhibition effects, and can be further researched and developed to be taken as a health product or a medicine for adjuvant treatment of diabetes.
Description
Technical field
The present invention is specifically related to a kind of Pedicellus et Pericarpium Trapae shell polyphenol extract and preparation method thereof and application, belongs to biological technical field.
Background technology
Alpha-glucosidase in specific manner catalysis contains sucrose or the Maltose hydrolysis of alpha-glucosaccharase, alpha-glucosidase inhibitor can suppress the activity of small intestinal epimere alpha-glucosidase, block carbohydrate breakdown and become single glucose, thus reduce the post-prandial glycemia of diabetic.Alpha-glucosidase inhibitor acts on unique orally-taken blood sugar reducing medicine as a kind of, for employing diet control and unsatisfied 2 diabetes mellitus types of exercise therapy effect, can improve carbohydrate metabolism disturbance, postpones or the generation of the chronic vascular complications that prevents diabetes.
Pedicellus et Pericarpium Trapae is the fruit that Trapaceae Pedicellus et Pericarpium Trapae belongs to annual water plant Pedicellus et Pericarpium Trapae, extensively plants in China's freshwater.Pedicellus et Pericarpium Trapae maturation gather after except on a small quantity with except the edible directly listing of fresh fruit, major part is shelled and is processed into various product with Pedicellus et Pericarpium Trapae core, and shell then directly discards.A large amount of discarded Pedicellus et Pericarpium Trapae shells can be produced every year in the Pedicellus et Pericarpium Trapae course of processing.It is reported that the zoopery of Pedicellus et Pericarpium Trapae shell proves that it has significant hypoglycemic activity (Kharbanda C., Mohammad S. A., Hamid H., et al. Trapa natans L. root extract suppresses hyperglycemic and hepatotoxic effects in STZ-induced diabetic rat model.Journal of Ethnopharmacology, 2014,151,931-936; Yasuda M., Yasutake K., Hino M., et al. Inhibitory effects of polyphenols from water chestnut (Trapa japonica) husk on glycolytic enzymes and postprandial blood glucose elevation in mice.Food Chemistry, 2014,165,42-49).But blood-sugar decreasing active is indefinite in Pedicellus et Pericarpium Trapae shell, need to carry out more extensive research.Containing a large amount of polyphenol compound in Pedicellus et Pericarpium Trapae shell, there is no the report that polyphenol in Pedicellus et Pericarpium Trapae shell is extracted at present.
The object of the invention is the extraction preparation method setting up polyphenol component in a kind of Pedicellus et Pericarpium Trapae shell, extract obtained can be used as adjuvant therapy of diabetes health product or medicine and researching and developing further, the Pedicellus et Pericarpium Trapae shell discarded can be made to be utilized.
Summary of the invention
The object of the invention is to make full use of the natural resources, provide a kind of and there is remarkable Inhibiting α-glucosidase activity thus Pedicellus et Pericarpium Trapae shell polyphenol extract reaching blood sugar decreasing effect and preparation method thereof and the application in prepared by health product or medicine.
The invention provides the preparation method of Pedicellus et Pericarpium Trapae shell polyphenol extract, comprise the following steps:
(1) by Pedicellus et Pericarpium Trapae shell drying and crushing;
(2) be the Pedicellus et Pericarpium Trapae shell dried powder obtained in solvent extraction step (1) with 30-90% ethanol or methanol, extracting solution concentrating under reduced pressure is become extractum;
(3) the extractum water obtained in step (2) or determining alcohol are less than ethanol or the determining alcohol dissolve with methanol that is less than 50% of 50%, filter or centrifugal removing insoluble matter, obtain load solution; The weight of described water or ethanol or methanol is 10-40 times of extractum weight;
(4) get nonpolar or low pole macroporous resin, by the load solution loading that step (3) obtains, carry out gradient elution with water and ethanol or methanol, collect eluent; Described macroporous resin and Pedicellus et Pericarpium Trapae shell weight ratio are 1:0.5-1:10;
(5) eluent concentrating under reduced pressure step (4) collected is dry, obtains Pedicellus et Pericarpium Trapae shell polyphenol extract.
Wherein the extracting method of step (2) is: every kilogram of Pedicellus et Pericarpium Trapae shell adds the ethanol or methanol that 5-40L concentration is 50-80%, hot reflux or dipping or seepage pressure effects 2-4 time, each 1-3h of circumfluence distillation, dipping or each 15 days of seepage pressure effects, merge extractive liquid.Optimum extracting method is: every kilogram of Pedicellus et Pericarpium Trapae shell adds the ethanol that 8L concentration is 80%, seepage pressure effects 2 times, each 15 days, merge extractive liquid.
Wherein the extracting method of step (3) is: the 10-40 of the extractum extractum weight obtained in step (2) water doubly or determining alcohol are less than ethanol or the determining alcohol dissolve with methanol that is less than 50% of 50%, filter or centrifugal removing insoluble matter, obtain load solution.Optimum extracting method is: by 10% dissolve with ethanol of extractum 10 times of extractum weight obtained in step (2), filters or centrifugal removing insoluble matter, obtains load solution.
Wherein the purification process of step (4) is: get nonpolar or low pole macroporous resin column, the weight ratio of macroporous resin and Pedicellus et Pericarpium Trapae shell is 1:1-1:10, by the load solution loading that step (3) obtains, weight is adopted to be that macroporous resin weight 10-20 water doubly carries out eluting, rear employing weight is that macroporous resin weight 8-15 10-50% ethanol doubly carries out eluting, collects ethanol elution.Best purification process is: getting with the ratio of Pedicellus et Pericarpium Trapae shell weight is the low pole macroporous resin of 1:4, by the load solution loading that step (3) obtains, employing weight is that the water of macroporous resin weight 10 times carries out eluting, rear employing weight is that 40% ethanol of macroporous resin weight 8 times carries out eluting, collects the ethanol elution of 40%.
Present invention also offers this application of Pedicellus et Pericarpium Trapae shell polyphenol extract on the health product preparing alpha-glucosidase inhibitor or medicine.
The present invention has the following advantages: the method that after the present invention adopts alcohol extraction, macroporous resin enrichment is refining prepares Pedicellus et Pericarpium Trapae shell polyphenol extract, and technique is simple, cost is low, and the extract polyphenol content obtained is high, reaches more than 90%.Pedicellus et Pericarpium Trapae shell polyphenol extract prepared by the present invention has significant inhibition to alpha-glucosidase, is obviously better than positive control drug acarbose, can be used as the medicine of adjuvant therapy of diabetes or health product and researchs and develops further.
Accompanying drawing explanation
Fig. 1 is that the Pedicellus et Pericarpium Trapae shell polyphenol extract of preparation in the embodiment of the present invention 4 is to the inhibitory action design sketch of alpha-glucosidase activity;
Fig. 2 is the inhibitory action design sketch of positive control drug acarbose to alpha-glucosidase activity.
Detailed description of the invention
The embodiment provided below is only and further illustrates the present invention, description is below only to make those skilled in the art better understand the present invention, but they do not constitute any limitation the present invention.
The instrument and equipment used in each embodiment, chemical reagent and detection method are as follows:
Instrument and equipment: Mettler EL204 electronic balance, Mettler Toledo Inc. of Switzerland; Molecular Devices SpectraMax Plus 384 type microplate reader, Molecular Devices company of the U.S.; The accurate single channel sample injector of Thermo Finnpipette, Thermo Fischer Scient Inc.; Accurate eight passages of Transferpette-8 type move the liquid volley of rifle fire, German general Rand Corporation.
Chemical reagent: gallic acid reference substance (Chinese biological drug inspection office); Acarbose (acarbose, Bayer HealthCare Co produces); Folin-Ciocalteu ' s phenol reagent (Sigma-Aldrich company); Alpha-glucosidase (Sigma-Aldrich company); 4-nitropheny-α-D-glucopyranose(pNPG): (Sigma-Aldrich company); Macroporous resin: D101 type macroporous resin (non-polar macroporous resin, Chemical Plant of Nankai Univ.), HPD722 type macroporous resin (weakness macroporous resin, Bao En chemical plant, Cangzhou, Hebei), LS-305 type macroporous resin (low pole macroporous resin, Xi'an Lan Shen resin company limited), LS-300B type macroporous resin (low pole macroporous resin, Xi'an Lan Shen resin company limited), all the other reagent are analytical pure.
Content assaying method: (ultraviolet spectrophotometry)
Standard curve is set up:
Precision takes in gallic acid reference substance to brown volumetric flask, with water dissolution, is mixed with the standard solution that concentration is 0.1118mg/mL.Gallic acid standard solution 0,5 is added successively, 10,20,30,40,50 μ L in 96 orifice plates.Add 50 μ L10% Folin-Ciocalteu ' s reagent in each sample respectively, after vibration 3min, add 50 μ L 10%Na
2cO
3solution fully mixes, and supplies solvent to 150 μ L with aqueous solution, and after at room temperature placing 1h, 96 orifice plates are put into microplate reader measures each sample light absorption value at 760nm wavelength place, 3 repetitions are done in each detection, get its meansigma methods.(x) draw total phenol content standard curve with light absorption value (y) and gallic acid content: y=0.1995x+0.0788, R=0.9993, gallic acid is good linear relationship within the scope of 0 ~ 5.59 μ g.
Sample size measures:
Precision takes sample powder, dissolves, be mixed with the solution of 1.0mg/mL with pure water.Precision pipettes 5 μ L sample solutions in 96 hole flat boards respectively, by method under standard curve item, add developer reaction, with sample solution not with developer for blank, measure light absorption value, try to achieve the Determination of Polyphenols (total polyphenols is in gallic acid) of each sample according to Galla Turcica (Galla Helepensis) standard curve.
Embodiment 1
Pedicellus et Pericarpium Trapae shell drying and crushing, takes Pedicellus et Pericarpium Trapae shell powder 0.5kg and adds the ethanol that 20L concentration is 50%, circumfluence distillation 3 times, each 1h of circumfluence distillation, merge extractive liquid.Extracting solution is evaporated to extractum, the extractum water dissolution of extractum weight 10 times, crosses and filters insoluble matter, obtain load solution.Get 0.25kg D101 type macroporous resin (non-polar macroporous resin post), by load solution loading, adopt 5L water elution, discard eluent, then adopt 5L10% ethanol to carry out eluting.Collect 10% ethanol elution, concentrating under reduced pressure is dry, obtains Pedicellus et Pericarpium Trapae shell polyphenol extract.
Obtain 5.3g sample by this method, adopting ultraviolet spectrophotometry to record extract polyphenol content is 57.3%.The activity of this polyphenol extract to alpha-glucosidase has certain inhibitory action after measured, and when sample concentration is 1.0mg/mL, inhibition percentage is 61.2%.
Embodiment 2:
Pedicellus et Pericarpium Trapae shell drying and crushing, takes Pedicellus et Pericarpium Trapae shell powder 0.5kg and adds the methanol that 50L concentration is 80%, circumfluence distillation 3 times, each 1h of circumfluence distillation, merge extractive liquid.Extracting solution is evaporated to extractum, extractum 50% dissolve with ethanol of extractum weight 40 times, crosses and filters insoluble matter, obtain load solution.Get 5.0kg HPD722 type macroporous resin (low pole macroporous resin column), by load solution loading, adopt 5L water elution, discard eluent, then adopt 5L 30% ethanol to carry out eluting.Collect 30% ethanol elution, concentrating under reduced pressure is dry, obtains Pedicellus et Pericarpium Trapae shell polyphenol extract.
Obtain 10.1g sample by this method, adopting ultraviolet spectrophotometry to record extract polyphenol content is 82.9%.The activity of this polyphenol extract to alpha-glucosidase has certain inhibitory action after measured, and when sample concentration is 0.01mg/mL, inhibition percentage is 50.1%.
Embodiment 3:
Pedicellus et Pericarpium Trapae shell drying and crushing, takes Pedicellus et Pericarpium Trapae shell powder 0.5kg and adds the ethanol that 5L concentration is 80%, Soakage extraction 2 times, each 15 days, merge extractive liquid.Extracting solution is evaporated to extractum, and extractum 10% dissolve with ethanol of extractum weight 10 times, centrifugal removing insoluble matter, obtains load solution.Get 2.0kg LS-305 type macroporous resin (low pole macroporous resin column), by load solution loading, adopt 5L water elution, discard eluent, then adopt 4L 50% ethanol to carry out eluting.Collect 40% ethanol elution, concentrating under reduced pressure is dry, obtains Pedicellus et Pericarpium Trapae shell polyphenol extract.
Obtain 16.7g sample by this method, adopting ultraviolet spectrophotometry to record extract polyphenol content is 80.2%.The activity of this polyphenol extract to alpha-glucosidase has certain inhibitory action after measured, and when sample concentration is 0.01mg/mL, inhibition percentage is 48.1%.
Embodiment 4:
Pedicellus et Pericarpium Trapae shell drying and crushing, take Pedicellus et Pericarpium Trapae shell powder 0.5kg and add the ethanol that 5L concentration is 80%, diafiltration extracts 2 times, each 15 days, merge extractive liquid.Extracting solution is concentrated into extractum, and extractum 10% dissolve with ethanol of extractum weight 10 times, centrifugal removing insoluble matter, obtains load solution.Get 2.0kg LS-300B type macroporous resin (low pole macroporous resin column), by load solution loading, adopt 5L water elution, discard eluent, then adopt 4L40% ethanol to carry out eluting, collect ethanol elution.Collect 40% ethanol elution, concentrating under reduced pressure is dry, obtains Pedicellus et Pericarpium Trapae shell polyphenol extract.
Obtain 15.2g sample by this method, adopting ultraviolet spectrophotometry to record extract polyphenol content is 93.1%.
Embodiment 5:
The alpha-glucosidase inhibition embodiment that Pedicellus et Pericarpium Trapae shell extract is right.
Take the Pedicellus et Pericarpium Trapae shell extract powder of preparation in embodiment 4, add dimethyl sulfoxide (DMSO), be mixed with the solution of concentration about 1 mg/ml, then be diluted to series concentration, as test sample, measure variable concentrations protein sample to the inhibitory action of alpha-glucosidase activity with 96 orifice plates.
Enzyme activity determination: add 1 μ l enzyme liquid, 60 μ l phosphate buffer (67 mmol/L in reacting hole successively, pH6.8, lower same), at 37 DEG C of reaction 10min, add 10 μ l reaction substrates (0.1 mmol/L pNPG, phosphate buffered saline, lower with), after reaction 10min, measure the absorbance (A at 405 nm places
enzyme is lived).
Enzyme blank determination: add 1 μ l enzyme liquid, 60 μ l phosphate buffers in reacting hole successively, 37 DEG C of reactions 10 minutes, adds 10 μ l phosphate buffers, after reaction 10min, measures the absorbance (A at 405 nm places
enzyme is blank).
Sample determination: add 1 μ l enzyme liquid, 50 μ l phosphate buffers, 10 μ l samples in reacting hole successively, 37 DEG C of reactions 10 minutes, adds 10 μ l reaction substrates, after reaction 10min, measures the absorbance (A at 405 nm places
sample).
Sample blank measures: in reacting hole, add 61 μ l phosphate buffers, at 37 DEG C of reaction 10min, adds 10 μ l reaction substrates, after reaction 10min, measures the absorbance (A at 405 nm places
sample blank).
Above all samples and blank reaction all repeat 3 times, average as experimental data.
Calculate: test sample is to the inhibition percentage=[(A of alpha-glucosidase
enzyme is lived-A
enzyme is blank)-(A
sample-A
sample blank)]/(A
enzyme is lived-A
enzyme is blank) × 100%; Half-inhibition concentration (IC
50): take protein concentration as abscissa, it is vertical coordinate to the inhibition percentage of alpha-glucosidase, mapping, obtain sample dose effect curve, the protein concentration corresponding when inhibition percentage is 50% is the half-inhibition concentration (IC of sample to alpha-glucosidase
50).
Be simultaneously positive control drug with acarbose, measure it to the inhibition percentage of alpha-glucosidase and IC
50.
Fig. 1 shows Pedicellus et Pericarpium Trapae shell polyphenol extract to the inhibitory action of alpha-glucosidase activity.The IC of Pedicellus et Pericarpium Trapae shell polyphenol extract for alpha-glucosidase is drawn by Fig. 1
50be 0.268 μ g/mL.
Fig. 2 represents the inhibitory action of positive control drug acarbose to alpha-glucosidase activity.The IC of acarbose for alpha-glucosidase is drawn by Fig. 2
50be 0.56 mg/mL.
Visible, Pedicellus et Pericarpium Trapae shell polyphenol extract has significant inhibitory action to alpha-glucosidase, and successful is higher than positive control drug acarbose.
Claims (7)
1. the preparation method of kind of Pedicellus et Pericarpium Trapae shell polyphenol extract, is characterized in that: the method comprises the following steps:
(1) by Pedicellus et Pericarpium Trapae shell drying and crushing;
(2) be the Pedicellus et Pericarpium Trapae shell dried powder obtained in solvent extraction step (1) with 30-90% ethanol or methanol, extracting solution concentrating under reduced pressure is become extractum;
(3) the extractum water obtained in step (2) or determining alcohol are less than ethanol or the determining alcohol dissolve with methanol that is less than 50% of 50%, filter or centrifugal removing insoluble matter, obtain load solution; The weight of described water or ethanol or methanol is 5-40 times of extractum weight;
(4) get nonpolar or low pole macroporous resin, by the load solution loading that step (3) obtains, carry out gradient elution with water and ethanol or methanol, collect eluent; Described macroporous resin and Pedicellus et Pericarpium Trapae shell weight ratio are 1:0.5-1:10;
(5) eluent concentrating under reduced pressure step (4) collected is dry, obtains Pedicellus et Pericarpium Trapae shell polyphenol extract.
2. the preparation method of Pedicellus et Pericarpium Trapae shell polyphenol extract according to claim 1, is characterized in that: in step (4), eluting adopts ethanol or methanol concentration to be 10-50%.
3. according to the preparation method of Pedicellus et Pericarpium Trapae shell polyphenol extract according to claim 1, it is characterized in that: in step (2), extracting method is the ethanol or methanol that every kilogram of Pedicellus et Pericarpium Trapae shell adds that 5-40L concentration is 50-80%, hot reflux or dipping or seepage pressure effects 2-4 time, the each 1-3h of circumfluence distillation, dipping or each 15 days of seepage pressure effects, merge extractive liquid.
4. according to the preparation method of Pedicellus et Pericarpium Trapae shell polyphenol extract according to claim 1, it is characterized in that: step (2) concrete grammar is as follows: every kilogram of Pedicellus et Pericarpium Trapae shell adds the ethanol that 8L concentration is 80%, seepage pressure effects 2 times, each 15 days, merge extractive liquid.
5. according to the preparation method of Pedicellus et Pericarpium Trapae shell polyphenol extract according to claim 1, it is characterized in that: step (3) concrete grammar is as follows: by 10% dissolve with ethanol of extractum 10 times of extractum weight obtained in step (2), filter or centrifugal removing insoluble matter, obtain load solution.
6. according to the preparation method of Pedicellus et Pericarpium Trapae shell polyphenol extract according to claim 1, it is characterized in that: concrete grammar is as follows in step (4): getting with the ratio of Pedicellus et Pericarpium Trapae shell weight is the low pole macroporous resin of 1:4, by the load solution loading that step (3) obtains, employing weight is that the water of macroporous resin weight 10 times carries out eluting, rear employing weight is that 40% ethanol of macroporous resin weight 8 times carries out eluting, collects the ethanol elution of 40%.
7. profit requires the application of Pedicellus et Pericarpium Trapae shell polyphenol extract on the health product preparing Inhibiting α-glucosidase or medicine described in 1.
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| CN105287663A (en) * | 2015-10-27 | 2016-02-03 | 湖北荆楚理工科技开发有限公司 | Extraction process for effective components of trapa bispinosa roxb leaves |
| CN107501228A (en) * | 2017-09-04 | 2017-12-22 | 武汉轻工大学 | A kind of lotus rhizome section polyphenol extract and its preparation method and application |
| CN108236618A (en) * | 2018-01-12 | 2018-07-03 | 江苏省中国科学院植物研究所 | A kind of preparation method and application of water chestnut shell extract |
| CN108542929A (en) * | 2018-07-05 | 2018-09-18 | 吉林化工学院 | Bluish dogbane total polyphenol extract and preparation method thereof |
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| CN113662974A (en) * | 2021-07-20 | 2021-11-19 | 嘉兴学院 | Preparation and purification method of water chestnut shell polyphenol extract |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105287663A (en) * | 2015-10-27 | 2016-02-03 | 湖北荆楚理工科技开发有限公司 | Extraction process for effective components of trapa bispinosa roxb leaves |
| CN107501228A (en) * | 2017-09-04 | 2017-12-22 | 武汉轻工大学 | A kind of lotus rhizome section polyphenol extract and its preparation method and application |
| CN107501228B (en) * | 2017-09-04 | 2020-04-07 | 武汉轻工大学 | Lotus rhizome node polyphenol extract and preparation method and application thereof |
| CN108236618A (en) * | 2018-01-12 | 2018-07-03 | 江苏省中国科学院植物研究所 | A kind of preparation method and application of water chestnut shell extract |
| CN110652505A (en) * | 2018-06-28 | 2020-01-07 | 嘉兴学院 | Application of Trapa acornis nakai shell polyphenol extract in preparation of medicine for resisting HER-2 over-expression type breast cancer |
| CN110652505B (en) * | 2018-06-28 | 2022-05-31 | 嘉兴学院 | Application of Trapa acornis nakai shell polyphenol extract in preparation of medicine for resisting HER-2 over-expression type breast cancer |
| CN108542929A (en) * | 2018-07-05 | 2018-09-18 | 吉林化工学院 | Bluish dogbane total polyphenol extract and preparation method thereof |
| CN113662974A (en) * | 2021-07-20 | 2021-11-19 | 嘉兴学院 | Preparation and purification method of water chestnut shell polyphenol extract |
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