CN102532338A - Ultrasonic extraction method of glycyrrhiza polysaccharide - Google Patents
Ultrasonic extraction method of glycyrrhiza polysaccharide Download PDFInfo
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Abstract
The invention discloses an ultrasonic extraction method of glycyrrhiza polysaccharide. The method comprises the following steps: screening glycyrrhiza with a 80-mesh pharmacopoeia sieve, and carrying out ultrasonic degreasing for 40 minutes at a ratio of glycyrrhiza to ethanol feed liquid of 1:5 (g/ml); abandoning a degreasing liquid, and washing medicament residue three times with a small amount of 80% ethanol; evaporating a solvent, drying at the temperature of 60 DEG C until alcohol smell disappears; adding 200ml of water in the remained residue, and then carrying out ultrasonic extraction for 30 minutes at the temperature of 75 DEG C, wherein extraction is carried out five times; mixing water extracts, and concentrating to about 15ml; and adding absolute ethyl alcohol the volume of which is 9 times that of a glycyrrhiza polysaccharide liquid, standing for 24 hours at the temperature of 4 DEG C and then carrying out suction filtration, and drying precipitate at the temperature of 60 DEG C until the weight is constant, so as to obtain total glycyrrhiza polysaccharide. By adopting the glycyrrhiza polysaccharide extraction process determined by the method, extraction process flow can be simplified, extraction time is saved, extraction cost is reduced, and the content of the extracted glycyrrhiza polysaccharide reaches 1.992%.
Description
Technical field
The present invention relates to medicine and chemical technology field, in particular a kind of licorice polysaccharide ultrasonic extracting method.
Background technology
Existing licorice polysaccharide process for extracting is numerous and diverse, and used flow process is many, and it is long to adopt the hydro-thermal reflow method to extract licorice polysaccharide extraction time; Though and it is short to adopt ultrasonic extracting method extraction time to reflux than hydro-thermal, extract temperature and to extract power different, in different documents are recorded and narrated; Two kinds of process for extracting solid-liquid ratio proportionings differ bigger; Alcohol precipitating method is disunity also, to the existence of these problems, is necessary to carry out the licorice polysaccharide Study on extraction; To improve the extraction yield of licorice polysaccharide, optimize the licorice polysaccharide process for extracting.
Summary of the invention
Technical problem to be solved by this invention is that the deficiency that is directed against prior art provides a kind of licorice polysaccharide ultrasonic extracting method.
Technical scheme of the present invention is following:
A kind of licorice polysaccharide ultrasonic extracting method, Radix Glycyrrhizae 10g are crossed 80 order pharmacopeia sieve, according to Radix Glycyrrhizae and 1: 5 (g/ml) ultrasonic degreasing of 80% ethanol solid-liquid ratio 40min; Give up degreasing fluid, the medicinal material residue is given a baby a bath on the third day after its birth inferior on a small quantity with 80% ethanol, volatilize solvent; 60 ℃ be dried to do not have the alcohol flavor after, residual residue is added 200ml water at 75 ℃ of supersound extraction 30min, extract altogether 5 times; Merge the water extract, be concentrated into about 15ml, add 9 times of volume absolute ethyl alcohols of licorice polysaccharide liquid; In 4 ℃ leave standstill 24h after suction filtration, precipitate 60 ℃ and be dried to constant weight, the Radix Glycyrrhizae total polysaccharides.
Adopt the determined licorice polysaccharide extraction process of present method, can simplify the extraction flow process, save extraction time, reduce extraction cost, the licorice polysaccharide content of carrying reaches 1.992%.
Description of drawings
Fig. 1 is the licorice polysaccharide typical curve.
Embodiment
Below in conjunction with specific embodiment, the present invention is elaborated.
1 reagent and medicinal material
Reagent: glucose, the vitriol oil, absolute ethyl alcohol, phenol, aluminium flake and NaHCO
3Deng being analytical pure.
2 Radix Glycyrrhizae process for extracting are learned and are investigated
2.1 learning, degreasing method investigates
Got certain pharmacopeia sieve Radix Glycyrrhizae, precision takes by weighing 1g and puts in the Erlenmeyer flask, with certain volume and concentration ethanol in 100W ultrasonic degreasing 1～3 time; Reaction times 40min pours out degreasing fluid, and the medicinal material residue is given a baby a bath on the third day after its birth inferior on a small quantity with 90% (V/V) ethanol; Washing lotion merges in the degreasing fluid, the reclaim under reduced pressure degreasing fluid.Residual residue volatilizes ethanol (60 ℃ are dried to nothing alcohol flavor), respectively with 20ml zero(ppm) water 100W (or 45 ℃) supersound extraction twice, 25min/ time; Filtration under diminished pressure, merging filtrate is concentrated into about 1/10 volume with filtrate decompression; In polysaccharide liquid: 1: 4 (v/v) ratio of ethanol adds absolute ethyl alcohol, suction filtration behind 4 ℃ of alcohol precipitation 16h, precipitate 60 ℃ be dried to do not have alcohol and distinguish the flavor of after; It is dissolved fully (on a small quantity repeatedly), constant volume is measured polysaccharide content according to the typical curve method in the 10ml volumetric flask.
The Radix Glycyrrhizae degreasing method adopts orthogonal test to investigate, and factor and level see Table 1, and experimental program sees Table 2, repeats 3 times:
Table 1 Radix Glycyrrhizae degreasing method is investigated factor and level (n=3)
Table 2 orthogonal design table L
9(3
4) (n=3)
Annotate: each level of factor I, II, III in 1,2,3 fingers to finger test in the table.
Table 3 Radix Glycyrrhizae degreasing method is investigated Orthogonal experiment results
Can know that by table 3 the best degreasing condition of extracting licorice polysaccharide is: grinding particle size selects the III level, cross 80 order pharmacopeia sieve; Medicinal material and ethanol solid-liquid ratio select the I level during degreasing, and 1: 5 (g/ml), the degreasing alcohol concn selects the II level, 80% ethanol; The degreasing number of times selects the I level, degreasing 1 time.I and III level are more or less the same in the degreasing solid-liquid ratio, in order to save solvent, select the I level.
The result shows by range analysis, and four investigation factors are followed successively by granularity＞degreasing solid-liquid ratio＞degreasing number of times＞degreasing alcohol concn to the size that influences of Radix Glycyrrhizae degreasing.Degreasing has the greatest impact the Radix Glycyrrhizae granularity to medicinal material; The polysaccharide content extreme difference of surveying reaches 0.03%; The degreasing alcohol concn is little to degreasing influence, and this is because three levels of selected alcohol concn are sayed all higherly to extracting licorice polysaccharide, can not dissolve or dissolve the few reason of licorice polysaccharide that.
2.2 learning, extraction method of polysaccharides investigates
2.2.1 extraction conditions experiment of single factor research
Got 80 order pharmacopeia sieves Radix Glycyrrhizae powder, precision takes by weighing 1g and puts in the Erlenmeyer flask, gets 80% ethanol, and in medicinal material: ethanol=1: 5 (g/ml) ratio adds in the triangular flask with cover, ultrasonic degreasing 1 time, and 40min gives up degreasing fluid.The medicinal material residue is given a baby a bath on the third day after its birth time with 80% concentration ethanol on a small quantity, volatilizes solvent, 60 ℃ be dried to do not have the alcohol flavor after; With zero(ppm) water is solvent, selects solid-liquid ratio, extracts power (or temperature), extraction time and extraction time, and the design experiment of single factor extracts licorice polysaccharide; And polysaccharide extraction liquid is evaporated to about 4～5ml, in polysaccharide liquid: 1: 4 (v/v) ratio of ethanol adds absolute ethyl alcohol, suction filtration behind 4 ℃ of alcohol precipitation 16h; Precipitate 60 ℃ be dried to do not have the alcohol flavor after, it is dissolved fully, constant volume is in the 10ml volumetric flask; Measure polysaccharide content according to the typical curve method, screen the theoretical optimum extraction condition of each single factor.
Concrete EE such as table 4.
Table 4 experiment of single factor condition
| Factor | Solid-liquid ratio | Extract power (or temperature) (℃ or W) | Extraction time (min) | Extraction time |
| Solid-liquid ratio | X 1 | 100W or 45 ℃ | 25 | 2 |
| |
20 | ?X 2 | 25 | 2 |
| |
20 | 100W or 45 ℃ | X 3 | 2 |
| |
20 | 100W or 45 ℃ | 25 | X 4 |
Annotate: X representes the change condition in the experiment of single factor.X
1Expression solid-liquid ratio: medicinal material (g): zero(ppm) water (ml)=1: 10,1: 15,1: 20,1: 25,1: 30.X
2Temperature variable is extracted in expression: 30 ℃, and 45 ℃, 50 ℃, 55 ℃, 60 ℃, 65 ℃, 70 ℃.X
3Expression extraction time: 15min, 20min, 25min, 30min, 35min, 40min, 45min.X
4The expression extraction time: 1 time, 2 times, 3 times, 4 times.
Table 5 licorice polysaccharide extraction conditions list factor is investigated the result
Extract single factor investigation result by table 5 licorice polysaccharide and can know that licorice polysaccharide extracts solid-liquid ratio and selects the A2 level, i.e. the Radix Glycyrrhizae powder: zero(ppm) water=1: 15; Extract temperature and select the B7 level, promptly 70 ℃, extraction time is selected C7, i.e. 45min, and extraction time is 4 times.
2.2.2 extraction conditions orthogonal experiment research
On the 2.2.1 experiment basis, design further checking of four factors, three horizontal quadratures test, the empirical factor level sees Table 4, and each factor level proportioning sees Table 2 in the concrete experiment, and experimental implementation is seen 2.2.1.
Table 6 licorice polysaccharide extraction conditions orthogonal experiment level of factor table
Table 7 licorice polysaccharide extraction conditions Orthogonal experiment results L
9(3
4) (n=3)
Table 8 licorice polysaccharide extraction conditions orthogonal experiment is analyzed
On the experiment of single factor basis, carry out orthogonal test and investigate the Orthogonal experiment results such as the table 7 of licorice polysaccharide extraction conditions; The interpretation result sees table 8; Can know that by table 7,8 extraction conditions of licorice polysaccharide selects the I factor, Radix Glycyrrhizae for extracting solid-liquid ratio: zero(ppm) water=1: 20 (g/ml); Licorice polysaccharide extracts temperature and selects the III level, 75 ℃; Extraction time is selected I level, 30min; Extraction time selects the III level, 5 times.Range analysis is the result show; In each factor different treatment horizontal extent of this The effects; The size order that extracts the licorice polysaccharide influence factor is: extraction time＞extraction time＞extraction temperature＞extraction solid-liquid ratio; To licorice polysaccharide extract influence factor maximum be extraction time, the minimum factor of influence is the extraction solid-liquid ratio.
2.2.3 the selection of licorice polysaccharide alcohol precipitation condition
Got 80 order pharmacopeia sieves Radix Glycyrrhizae powder, precision takes by weighing 25g and puts in the Erlenmeyer flask, gets 80% ethanol, and in medicinal material: ethanol=1: 5 (g/ml) ratio adds in the triangular flask with cover, ultrasonic degreasing 1 time, and 40min gives up degreasing fluid.The medicinal material residue is given a baby a bath on the third day after its birth time with 80% concentration ethanol on a small quantity, volatilizes solvent, 60 ℃ be dried to do not have the alcohol flavor after, be solvent with zero(ppm) water; With Radix Glycyrrhizae: zero(ppm) water=1: 20 (g/ml) ratio adds zero(ppm) water, in 75 ℃ of supersound extraction 30min, extracts altogether 5 times, merges the water extract; Be concentrated into about 15ml, constant volume is got 5 parts of 1ml polysaccharide extraction liquids respectively in the 25ml volumetric flask, respectively 1: 3 by volume (75%); 1: 4 (80%), 1: 6 (85.7%), 1: 9 (90%); The ratio of 1: 19 (95%) adds absolute ethyl alcohol, with these 5 parts of licorice polysaccharides respectively at 4 ℃ of alcohol precipitation 24h after suction filtration, will precipitate dissolving fully, constant volume is in the 10ml volumetric flask respectively, the mensuration polysaccharide content screens best absolute ethyl alcohol alcohol precipitation volume ratio.
After filtering out best alcohol precipitation volume ratio, from above-mentioned 25ml volumetric flask, get 5 parts of 1ml polysaccharide extraction liquids more respectively, by selecting volume ratio to add absolute ethyl alcohol, respectively at 4 ℃ of alcohol precipitation 4h, 8h, 12h, 16h, 24h selects the suitable alcohol precipitation time.
Licorice polysaccharide alcohol precipitation condition is investigated and the results are shown in Table 9.
Table 9 licorice polysaccharide alcohol precipitation condition is investigated the result
Can be known by table 9, after licorice polysaccharide proposes, should be concentrated into small volume, then at polysaccharide liquid: absolute ethyl alcohol=1: 9 time was in 4 ℃ of alcohol precipitations 24 hours, and this moment, the licorice polysaccharide yield was the highest.
2.3 licorice polysaccharide trial-product preparation
80 order pharmacopeia sieve Radix Glycyrrhizae 1g decided in accurate title, with 5ml 80% ethanol ultrasonic degreasing 40min, gives up degreasing fluid.The medicinal material residue is given a baby a bath on the third day after its birth time with 80% ethanol on a small quantity, volatilizes solvent, 60 ℃ be dried to do not have the alcohol flavor after, residual residue is added 20ml zero(ppm) water at 75 ℃ of supersound extraction 30min; Extract altogether 5 times, merge the water extract, be concentrated into about 4～5ml; Add 9 times of volume absolute ethyl alcohols of licorice polysaccharide liquid, in 4 ℃ of alcohol precipitations suction filtration after 24 hours, precipitate 60 ℃ be dried to do not have the alcohol flavor after; It is dissolved fully (on a small quantity repeatedly), constant volume is in the 10ml volumetric flask, and is subsequent use as the polysaccharide trial-product.
2.4 the preparation of Radix Glycyrrhizae total polysaccharides
80 order pharmacopeia sieve Radix Glycyrrhizae 10g decided in accurate title, with 50ml 80% ethanol ultrasonic degreasing 40min, gives up degreasing fluid, and the medicinal material residue is given a baby a bath on the third day after its birth inferior on a small quantity with 80% ethanol; Volatilize solvent, 60 ℃ be dried to do not have the alcohol flavor after, residual residue is added 200ml water at 75 ℃ of supersound extraction 30min, extract altogether 5 times; Merge the water extract, be concentrated into about 15ml, add 9 times of volume absolute ethyl alcohols of licorice polysaccharide liquid; In 4 ℃ leave standstill 24h after suction filtration, precipitate 60 ℃ and be dried to constant weight, the Radix Glycyrrhizae total polysaccharides.
The preparation of 3 phenol solution
Take by weighing phenol 100g with aluminium flake 0.1g and sodium hydrogencarbonate 0.05g air distillation, collect (180 ± 2) ℃ cut.Precision takes by weighing this cut 6g and places brown reagent bottle, adds the 94ml water dissolution, shakes up, and is placed in the refrigerator subsequent use.
The preparation of 4 typical curves
4.1 the preparation of reference liquid
Glucose in right amount in 60 ℃ of baking 1h, is warming up to 105 ℃ more gradually and is dried to constant weight, and precision takes by weighing the glucose 10mg that is dried to constant weight and puts in the 100ml volumetric flask, and adding distil water is settled to scale, mixing, and getting concentration is the glucose reference liquid of 0.1mg/ml.
4.2 the selection of maximum absorption wavelength
Accurately draw 1ml glucose solution (0.1mg/ml) in 10ml tool plug test tube; The phenol 1ml of adding 6% shakes up the back and adds the 5ml vitriol oil rapidly; Fully mixing after placement 30min is cooled to room temperature, is measured absorbancy at 380～580nm; Make blank with the same method of zero(ppm) water 1ml, the selection maximum absorption wavelength is 488nm.
4.3 the drafting of typical curve
The accurate 0.1mg/ml reference liquid 30ml constant volume of drawing in the 50ml volumetric flask, 0.06mg/ml glucose standard solution, respectively precision measure 0.4,0.6,0.8,1.0,1.4,1.6,1.8ml; Place 10ml tool plug test tube respectively, adding distil water is supplemented to 2.0ml, adds the phenol reagent 1ml and the 5ml vitriol oil more respectively; Mixing is placed 30min and is cooled to room temperature, in addition with zero(ppm) water 2.0ml; Add phenol reagent and the same operation of the vitriol oil, measure absorbancy, the standardized solution difference replication of same concentration 3 times in the maximum absorption wave strong point; With the absorbance is ordinate, and glucose microgram (μ g) number is done typical curve (Fig. 1) for abscissa; Getting regression equation is y=154.47x-2.6248, R=0.9992.
4.4 the mensuration of conversion factor
Precision takes by weighing 60 ℃ of licorice polysaccharide 25.6mg that are dried to constant weight and places the 50ml volumetric flask, is dissolved in water and is diluted to scale, gets 2ml then respectively and places 5 25ml volumetric flasks, is diluted with water to scale.Press typical curve preparation item its absorbancy of method mensuration down respectively; Go out glucose content and MV in 5 parts of licorice polysaccharides by regression equation calculation; (C * D) calculates RSD (n=5), and W is polysaccharide quality (mg) in the formula to calculate conversion factor f=W/ again; C is glucose concn in the polysaccharide soln (mg/ml), and D is the extension rate of polysaccharide.
Table 10 licorice polysaccharide conversion factor calculation result
5 polysaccharide determination methodological studies
5.1 stability test
Precision takes by weighing 6 parts in same sample; Process need testing solution, measure licorice polysaccharide content respectively one time at a distance from 0h, 6h, 12h, 18h, 24h, 48h, calculate RSD with " 6 trial-products mensuration " is every; It is stable in 48h to investigate test stability (table 6) this sample, and RSD is 3.68%.
5.2 replica test
Precision takes by weighing same sample and processes need testing solution for 6 parts, measures licorice polysaccharide content with " 6 trial-products mensuration " in accordance with the law, calculates RSD, investigation test stability (table 6), and this test repeatability is better, and sample determination RSD is 3.883%.
5.3 average recovery experiment
Precision takes by weighing 6 parts in the Radix Glycyrrhizae sample of known content, add 1mg/ml glucose reference liquid 5ml after, process and supply examination
Article solution is measured licorice polysaccharide content with " 6 trial-products mensuration " in accordance with the law, measures the average recovery rate of glucose, calculates RSD (table 6), and this tests average average recovery is 94.043%, and RSD is 4.024%.
Table 11 stability, circulation ratio and average recovery experimental result
6 trial-products are measured
The accurate need testing solution 1.0ml that draws adds phenol reagent 1ml respectively, vitriol oil 5ml in tool plug test tube; Mixing is placed 30min, is cooled to room temperature; Other gets zero(ppm) water 1.0ml and is contrast; With the method operation, measure absorbancy in 488nm, be 55.945 μ g/ml (n=6) according to glucose concn in the typical curve calculating licorice polysaccharide need testing solution.
7 cubages
7.1 glucose content in the sample
Glucose content calculation formula in the sample:
Glucose content (%)=C * D * 10
-6/ W * 100%
C is surveyed sugared concentration (μ g/ml) by the typical curve regression equation in the formula, and D is the extension rate (250 times) of test liquid, and W is trial-product weight (g).
Glucose content is 1.381% in the sample
7.2 polysaccharide content in the sample
Polysaccharide content calculation formula in the sample:
Polysaccharide content (%)=C * D * f/W * 100%
In the formula: C: the concentration of glucose in the polysaccharide soln (μ g/ml); D: the extension rate of polysaccharide; F: conversion factor; The quality of W testing sample (μ g).
Polysaccharide content in the sample (%)=55.945 μ g/ml * 0.000001 * 250 * 1.443/1.013 * 100=1.992%
Polysaccharide content is 1.992% in the Radix Glycyrrhizae
Proteinic removing in 8 licorice polysaccharides (Sevag method)
Get the licorice polysaccharide powder 10mg of 2.4 following alcohol precipitation after drying; Soak at room temperature is (on a small quantity repeatedly) in the about 10ml of zero(ppm) water, and after the dissolving, (mixed solution of chloroform and propyl carbinol (5: 1v/v)) adds licorice polysaccharide solution in the ratio of 1: 5 (v/v) with Sevag liquid fully; According to behind the strong jolting 20min; The centrifugal 10min of 5000r/min discards denatured protein layer and organic layer, gets supernatant.6 times so repeatedly, and working sample solution is at the absorbance of 260nm, 280nm respectively, like table 12, after protein removed 6 times continuously in the licorice polysaccharide, absorbance was basicly stable.
Protein removes the number of times investigation in table 12 licorice polysaccharide
Should be understood that, concerning those of ordinary skills, can improve or conversion, and all these improvement and conversion all should belong to the protection domain of accompanying claims of the present invention according to above-mentioned explanation.
Claims (1)
1. a licorice polysaccharide ultrasonic extracting method is characterized in that, Radix Glycyrrhizae 10g crosses 80 order pharmacopeia sieve, according to Radix Glycyrrhizae and 1: 5 (g/ml) ultrasonic degreasing of 80% ethanol solid-liquid ratio 40min; Give up degreasing fluid, the medicinal material residue is given a baby a bath on the third day after its birth inferior on a small quantity with 80% ethanol, volatilize solvent; 60 ℃ be dried to do not have the alcohol flavor after, residual residue is added 200ml water at 75 ℃ of supersound extraction 30min, extract altogether 5 times; Merge the water extract, be concentrated into about 15ml, add 9 times of volume absolute ethyl alcohols of licorice polysaccharide liquid; In 4 ℃ leave standstill 24h after suction filtration, precipitate 60 ℃ and be dried to constant weight, the Radix Glycyrrhizae total polysaccharides.
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| CN103172758A (en) * | 2013-03-14 | 2013-06-26 | 内蒙古医科大学 | Method for extracting polysaccharides from fringed pinks |
| CN107987176A (en) * | 2017-11-29 | 2018-05-04 | 马斌祥 | A kind of technique using radix glycyrrhizae extraction high-purity liquorice polysaccharide |
| CN108752492A (en) * | 2018-05-23 | 2018-11-06 | 阜阳师范学院 | The extracting method of total reducing sugar in snake gourd fruit pulp |
| CN109957038A (en) * | 2019-03-16 | 2019-07-02 | 莎车县富源甘草制品有限公司 | A kind of preparation method of Radix Glycyrrhizae Thick many candies |
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| CN115677875A (en) * | 2022-11-23 | 2023-02-03 | 深圳市恒云生物科技有限公司 | A method for extracting Glycyrrhiza polysaccharide, glycyrrhiza polysaccharide and essence |
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| CN103172758A (en) * | 2013-03-14 | 2013-06-26 | 内蒙古医科大学 | Method for extracting polysaccharides from fringed pinks |
| CN103172758B (en) * | 2013-03-14 | 2015-12-02 | 内蒙古医科大学 | The method of polysaccharide is extracted from fringed pink |
| CN107987176A (en) * | 2017-11-29 | 2018-05-04 | 马斌祥 | A kind of technique using radix glycyrrhizae extraction high-purity liquorice polysaccharide |
| CN108752492A (en) * | 2018-05-23 | 2018-11-06 | 阜阳师范学院 | The extracting method of total reducing sugar in snake gourd fruit pulp |
| CN109957038A (en) * | 2019-03-16 | 2019-07-02 | 莎车县富源甘草制品有限公司 | A kind of preparation method of Radix Glycyrrhizae Thick many candies |
| CN110343622A (en) * | 2019-07-26 | 2019-10-18 | 兰州理工大学 | The Radix Glycyrrhizae endophyte and its institute's producing enzyme of cellulase-producing are in the application for extracting Radix Glycyrrhizae acid |
| CN110343622B (en) * | 2019-07-26 | 2021-01-01 | 兰州理工大学 | Glycyrrhiza endophyte for producing cellulase and application of produced enzyme in extraction of glycyrrhizic acid in glycyrrhiza |
| CN114249842A (en) * | 2020-09-25 | 2022-03-29 | 广西中医药大学 | Method for extracting sugarcane leaf polysaccharide |
| CN114249842B (en) * | 2020-09-25 | 2023-07-21 | 广西中医药大学 | Method for extracting sugarcane leaf polysaccharide |
| CN115677875A (en) * | 2022-11-23 | 2023-02-03 | 深圳市恒云生物科技有限公司 | A method for extracting Glycyrrhiza polysaccharide, glycyrrhiza polysaccharide and essence |
| CN115677875B (en) * | 2022-11-23 | 2024-01-26 | 深圳市恒云生物科技有限公司 | Licorice polysaccharide extraction process, licorice polysaccharide and essence |
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Application publication date: 20120704 |