CN104059159B - The processing method of Mierocrystalline cellulose Enzymatic Extraction Herba Hedyotidis Diffusae polysaccharide - Google Patents
The processing method of Mierocrystalline cellulose Enzymatic Extraction Herba Hedyotidis Diffusae polysaccharide Download PDFInfo
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Abstract
The invention belongs to active ingredient of Chinese herbs extraction production technology field, be specifically related to a kind of response surface analysis and optimize Mierocrystalline cellulose Enzymatic Extraction Herba Hedyotidis Diffusae polysaccharide method and optimal extract process.The present invention adopts Mierocrystalline cellulose Enzymatic Extraction Herba Hedyotidis Diffusae polysaccharide, by the step such as Mierocrystalline cellulose enzyme process hot water extraction, inactivator, concentrated, alcohol precipitation, deproteinated, alcohol precipitation, depigmentation, alcohol precipitation, extraction yield mensuration, namely according to 1:8 ~ 12 solid-liquid ratio and add about 2% cellulase hot water extraction twice, 80% alcohol precipitation three times, trichoroacetic acid(TCA) deproteinated twice, charcoal absorption decolouring twice.The present invention obtains Herba Hedyotidis Diffusae polysaccharide optimal extract process by response surface three-dimensional plot: about PH7, temperature 55 DEG C, cellulase consumption 2%, Herba Hedyotidis Diffusae polysaccharide extraction yield is 9.83% with this understanding.
Description
Technical field
The invention belongs to active ingredient of Chinese herbs extraction production technology field, be specifically related to one response surface analysis and optimize Mierocrystalline cellulose Enzymatic Extraction Herba Hedyotidis Diffusae polysaccharide method and optimal extract process.
Background technology
Polysaccharide is also called saccharan, by a glycosides chain polymerization class natural high moleculer eompound together by the monose such as aldose or ketose, its molecular weight be generally tens thousand of even reach millions of, extensively be present in animal cell membrane, in plant and microorganism wall, up to the present, existing more than 300 kind of polysaccharide compound is extracted from natural product, and the water-soluble polysaccharide wherein obtained from Chinese medicinal materials is the most important.Research shows: the several functions such as herbal polysaccharide has immunomodulatory, antitumor, anti-inflammatory, antiviral, anti-oxidant, radioprotective, hypoglycemic, reducing blood-fat, protect the liver, wherein the immunoregulatory activity of herbal polysaccharide and antitumor action receive much attention, and are the central topics of herbal polysaccharide research.At present, the main method of Polyose extraction has Hot water extraction, acidleach formulation, alkali method, ultrasonic extraction and microwave loss mechanisms etc., all there is certain deficiency in these methods, as lower in extraction yield, purity is not high, or the too high destruction active polysaccharide of Extracting temperature, or the degraded etc. of polysaccharide molecule can be caused.At present, polysaccharide extracting process mostly is homogeneous design and orthogonal design, these designs adopt to fix other factors, change the single factor exploration method determination processing condition of a factor, can not interaction between investigation factor, also be difficult to investigate the interaction relation between multiple response value and factor, therefore tolerance range is not high simultaneously.In recent years, domesticly start extraction biological enzyme being applied to active ingredient of Chinese herbs, polysaccharide is present in the cell walls of plant, cellulase can make the mass degradation such as Mierocrystalline cellulose, hemicellulose, the cell walls of cracking plant, thus increase effective constituent in cell and improve polysaccharide yield to the diffusion of Extraction medium.Response surface analysis is a kind of optimization method, it is using the function of the response of system as one or more factor, graph technology is used this funtcional relationship to be shown, for the optimal condition that we rely on the observation of intuition to come in Selection experiment design, accurately can find the best of breed of factor and the optimum value of response value on whole region, be desirable method.Responds Surface Methodology overcomes orthogonal experimental design can only process discrete level value, and cannot find out the defect of the optimum value of best of breed on whole region and response value, with this method research Extraction technique, the regression equation tolerance range of trying to achieve is high.Spreading Hedyotis Herb is the dry herb of Rubiaceae (Rubiaceae) plants of Hedyotis Spreading Hedyotis Herb (HerbaHedyotis.), Herba Hedyotidis Diffusae polysaccharide is its important activeconstituents, has antisepsis and anti-inflammation, antitumor and improve the effect such as body's immunity.For improving the extraction yield of Herba Hedyotidis Diffusae polysaccharide, and find out the best of breed of processing condition exactly, the invention provides the technique that response surface analysis optimizes Mierocrystalline cellulose Enzymatic Extraction Herba Hedyotidis Diffusae polysaccharide, the method combined with hot water extraction with enzymolysis extracts Herba Hedyotidis Diffusae polysaccharide, simple process, save time, extraction yield is high; Extraction process adopts response surface analysis design, gained optimal processing parameter is reliable, accurate, for the scale production of Herba Hedyotidis Diffusae polysaccharide and application provide a kind of method of practicality, for Spreading Hedyotis Herb comprehensive development and utilization and improve its economic worth there is practical significance.
Summary of the invention
The present invention adopts Mierocrystalline cellulose enzyme process dialogue flower Herba Hedyotidis Diffusae polysaccharide to extract, and adopts response surface design to determine optimum process condition, removes the impurity such as protein, pigment to greatest extent, prepares the metastable Herba Hedyotidis Diffusae polysaccharide of content.
The present invention's method used is adopt Mierocrystalline cellulose enzyme process to extract the processing method of Herba Hedyotidis Diffusae polysaccharide from Spreading Hedyotis Herb.
The present invention adopts the processing method of Mierocrystalline cellulose Enzymatic Extraction Herba Hedyotidis Diffusae polysaccharide to be: pulverized by the Spreading Hedyotis Herb of drying, add cellulase and carry out lixiviate, enzyme-deactivating, concentrated, pure and strong, deproteinated, depigmentation step, specific requirement is, prepare according to Spreading Hedyotis Herb and water weight ratio 1:8 ~ 12 solid-liquid ratio and carry out lixiviate, the cellulase of 1.5 ~ 2.5% is added in above-mentioned feed liquid, with hot water extraction twice, inactivator, 80% alcohol precipitation three times, trichoroacetic acid(TCA) deproteinated twice, charcoal absorption decolouring twice, phend-sulphuric acid measures polysaccharide content and extraction yield.Technique is divided into: add the step such as cellulase hot water extraction, inactivator, concentrated, alcohol precipitation, deproteinated, alcohol precipitation, depigmentation, alcohol precipitation, specifically carry out as follows:
(1) add cellulase hot water extraction: twice lixiviate, Spreading Hedyotis Herb was pulverized 40 mesh sieves.Take Spreading Hedyotis Herb, the solid-liquid ratio by weight 1:8 ~ 12 adds water, mixes; Regulate pH to be about 7 with phosphoric acid buffer, add the cellulase of 1.5 ~ 2.5%, mixing, 55 DEG C of stirring in water bath lixiviates 60 minutes; 90 DEG C of inactivators 10 minutes, suction filtration obtains filtrate; Filter residue is added water by weight the solid-liquid ratio of 1:8 ~ 12, mixes, regulate pH to be about 7 with phosphoric acid buffer, add the cellulase of 1.5 ~ 2.5%, mixing, 55 DEG C of stirring in water bath lixiviates 60 minutes; 90 DEG C of inactivators 10 minutes, suction filtration obtains filtrate; Merge twice filtrate.
(2) concentrated: filter vacuum rotating pressure-decreasing being concentrated into concentration is containing about crude drug 0.5 ~ 1 grams per milliliter, puts to room temperature;
(3) first time alcohol precipitation: adding 95% ethanol to determining alcohol in concentrated solution is 80%, limit edged stir, sealing covers tightly, leave standstill 24 ~ 48 hours; Suction filtration, obtains Spreading Hedyotis Herb Crude polysaccharides;
(4) deproteinated: Spreading Hedyotis Herb Crude polysaccharides is added suitable quantity of water and dissolve, Spreading Hedyotis Herb Crude polysaccharides and water ratio w:v are 1:1 ~ 1.5kg/L, add the trichoroacetic acid(TCA) of equal-volume 5%, limit edged stirs, leave standstill 60 minutes, centrifugal 5 minutes of 3000rmp, gets supernatant liquor; Repeat above operation again and carry out second time deproteinated, obtain deproteinated polysaccharide soln;
(5) second time alcohol precipitation: adding 95% ethanol to determining alcohol in deproteinated polysaccharide liquid is 80%, and limit edged stirs, and sealing covers tightly, and leaves standstill 24 ~ 48 hours; Suction filtration, absolute ethanol washing 2-3 time, vacuum-drying, obtains deproteinated polysaccharide;
(6) depigmentation: above gained polysaccharide suitable quantity of water dissolved, Spreading Hedyotis Herb Crude polysaccharides and water ratio w:v are 1:1 ~ 1.5kg/L, add the gac of weight 1%, mixing, is heated to 80 DEG C and stirs 30 minutes, filter, get filtrate.Filtrate again according to above repetitive operation once, obtains depigmentation polysaccharide soln;
(7) third time alcohol precipitation: it is 80% that depigmentation polysaccharide liquid obtained is above added 95% ethanol to determining alcohol, and limit edged stirs, and sealing covers tightly, and leaves standstill 24 hours; Suction filtration, absolute ethanol washing 2-3 time, vacuum-drying, obtains refined polysaccharide.Adopt phend-sulphuric acid to measure polysaccharide content, and calculate polysaccharide yield.
The extraction process of method to polysaccharide that invention applies response surface analysis optimization design is optimized design.
(1) each single factor test is on the impact of Spreading Hedyotis Herb polysaccharide extract rate
1) cellulase consumption is on the impact of polysaccharide extract rate: cellulase consumption on the impact of polysaccharide extract rate in table 1.As can be seen from Table 1, along with the increase of cellulase consumption, the extraction yield of polysaccharide also increases thereupon, and when cellulase consumption reaches 2%, continue to increase cellulase consumption, polysaccharide extract rate substantially no longer increases.
Table 1 cellulase consumption is on the impact of polysaccharide content and polysaccharide extract rate
| Cellulase consumption (%) | 0.5 | 1 | 1.5 | 2 | 2.5 |
| Polysaccharide content (g) | 0.614 | 0.702 | 1.024 | 1.782 | 1.740 |
| Polysaccharide extract rate (%) | 3.07 | 3.51 | 5.12 | 8.91 | 8.70 |
2) Extracting temperature is on the impact of polysaccharide extract rate: Extracting temperature on the impact of polysaccharide extract rate in table 2.As can be seen from Table 2, the rising extraction yield along with Extracting temperature within the scope of 30-50 DEG C also rises thereupon, peaks when about 50 DEG C, and afterwards along with temperature rises, extraction yield declines on the contrary, and this illustrates that the optimum temperuture of cellulase is near 50 DEG C.
Table 2 Extracting temperature is on the impact of polysaccharide content and extraction yield
| Extracting temperature (DEG C) | 40 | 50 | 60 | 70 | 80 |
| Polysaccharide content (g) | 1.016 | 1.224 | 1.150 | 0.782 | 0.794 |
| Polysaccharide extract rate (%) | 5.08 | 6.12 | 5.75 | 3.91 | 3.97 |
3) extraction time is on the impact of polysaccharide extract rate: Extracting temperature on the impact of polysaccharide extract rate in table 3.As can be seen from Table 3, along with the increase of extraction time, polysaccharide extract rate is without considerable change.
Table 3 extraction time is on the impact of polysaccharide content and polysaccharide extract rate
| Extraction time (min) | 30 | 60 | 90 | 120 | 150 |
| Polysaccharide content (%) | 0.802 | 0.862 | 0.894 | 1.006 | 1.076 |
| Polysaccharide extract rate (%) | 4.01 | 4.31 | 4.47 | 5.03 | 5.38 |
4) pH is extracted on the impact of polysaccharide extract rate: extract pH to the impact of polysaccharide extract rate in table 4.As can be seen from Table 4, within the scope of pH3-7, the extraction yield increasing Herba Hedyotidis Diffusae polysaccharide along with PH also increases thereupon, and after arriving PH7, extraction yield declines, and illustrates that the optimal pH of cellulase is about 7.
Table 4 extracts the impact of PH on polysaccharide content and polysaccharide extract rate
| pH | 3 | 4 | 5 | 6 | 7 | 8 |
| Polysaccharide content (g) | 0.302 | 0.512 | 0.866 | 1.016 | 1.134 | 0.844 |
| Polysaccharide extract rate (%) | 1.51 | 2.56 | 4.33 | 5.08 | 5.67 | 4.22 |
(2) by response surface analysis optimization design extraction scheme
According to the requirement of response surface design, statistical model finds that 3 influence factors-extraction pH, temperature, cellulase consumption have remarkably influenced to polysaccharide extract rate and content.Therefore on monofactorial basis, carry out the response surface design analysis of Three factors-levels.Experimental factor and level are as following table 5.
Table 5 cellulase response surface optimization extracts the factor and level of also spending Herba Hedyotidis Diffusae
| Level | Extracting temperature (DEG C) | PH value | Cellulase consumption (%) |
| －1 | 40 | 3 | 0.5 |
| 0 | 50 | 7 | 2 |
| 1 | 80 | 8 | 2.5 |
(3) cellulase response surface extracts Herba Hedyotidis Diffusae polysaccharide test-results and significance analysis
According to the design-expert software of response surface design, the face optimization Mierocrystalline cellulose Enzymatic Extraction Herba Hedyotidis Diffusae polysaccharide test-results that meets with a response and regression equation are in table 6.
Table 6 response surface optimization cellulase extracts Herba Hedyotidis Diffusae polysaccharide test-results
(4) cellulase response surface optimization extracts Herba Hedyotidis Diffusae polysaccharide model significance analysis
Cellulase response surface optimization extracts Herba Hedyotidis Diffusae polysaccharide model significance analysis and the results are shown in Table 7.
Table 7 cellulase response surface optimization extracts Herba Hedyotidis Diffusae polysaccharide model significance analysis
| Source | Sum of squares | Degree of freedom | All square | F value | P value |
| Model | 133.02 | 9 | 14.78 | 49.72 | <0.0001 |
| A-Extracting temperature | 0.21 | 1 | 0.21 | 0.72 | 0.4245 |
| B-ph | 9.71 | 1 | 9.71 | 32.66 | 0.0007 |
| C-enzyme dosage | 23.45 | 1 | 23.45 | 78.89 | <0.0001 |
| Ab | 1.11 | 1 | 1.11 | 3.74 | 0.0945 |
| Ac | 0.97 | 1 | 0.97 | 3.27 | 0.1136 |
| Bc | 0.28 | 1 | 0.28 | 0.94 | 0.3656 |
| A 2 | 29.61 | 1 | 29.61 | 99.61 | <0.0001 |
| B 2 | 7.71 | 1 | 7.71 | 25.95 | 0.0014 |
| C 2 | 1.24 | 1 | 1.24 | 4.17 | 0.0804 |
| Residual error | 2.08 | 7 | 0.30 | ||
| Lose and intend | 2.04 | 3 | 0.68 | 60.21 | 0.0009 |
| Error | 0.045 | 4 | 0.011 | ||
| Total mutation | 135.10 | 16 |
Learnt by response surface analysis result, Herba Hedyotidis Diffusae polysaccharide extraction yield trial test result shows that extracting pH value, Extracting temperature and cellulase consumption has remarkably influenced to polysaccharide extract rate, the impact of its cellulase consumption and pH is the most outstanding, and Extracting temperature impact is taken second place relatively.By response surface three-dimensional plot obtain Herba Hedyotidis Diffusae polysaccharide extract optimum process condition be: about pH7., Extracting temperature 55 DEG C, cellulase consumption 2%, Herba Hedyotidis Diffusae polysaccharide extraction yield is 9.83% with this understanding.
The invention has the beneficial effects as follows: enzyme process is a kind of method of gentleness, can not destroy the structure of polysaccharide, can improve the yield of polysaccharide, and can, on a large scale for suitability for industrialized production, be the high efficiency method obtaining bioactive polysaccharide.The extraction conditions parameter that the optimization of employing Responds Surface Methodology obtains accurately and reliably, has and instructs scale production to be worth, apply and have certain market outlook and corresponding social benefit, economic benefit to production.
Embodiment
Embodiment 1 (1) hot water extraction gets Spreading Hedyotis Herb and pulverizes, and crosses 40 mesh sieves, takes Spreading Hedyotis Herb, mix in the ratio of 8 ~ 12 with water; Regulate pH to be about 7 with phosphoric acid buffer, in feed liquid, add the cellulase of 1.5 ~ 2.5%, mixing, 55 DEG C of stirring in water bath lixiviates 60 minutes;
(2) inactivator: to the solution after extracting 90 DEG C of inactivators 10 minutes, suction filtration obtains filtrate;
(3) add cellulase hot water again to filter residue and carry out second time lixiviate: the filter residue being extracted gained first time, the ratio by weight 1:8 ~ 12 mixes with water; Regulate pH to be about 7 with phosphoric acid buffer, add the cellulase of 1.5 ~ 2.5%, mixing, 55 DEG C of stirring in water bath lixiviates 60 minutes;
(4) inactivator: carry out 90 DEG C of inactivators 10 minutes to secondary filtrate, suction filtration obtains filtrate; Merge twice filtrate.
(5) concentrated: filter vacuum rotating pressure-decreasing to be concentrated into and to be equivalent to crude drug 0.5 ~ 1 grams per milliliter, put to room temperature;
(6) first time alcohol precipitation: adding 95% ethanol to determining alcohol in the concentrated solution of above preparation is 80%, and limit edged stirs, and sealing covers tightly, and leaves standstill 24 hours; Suction filtration, vacuum-drying, obtains Spreading Hedyotis Herb Crude polysaccharides;
(7) first time deproteinated: above obtained Spreading Hedyotis Herb Crude polysaccharides is added appropriate ultrapure water and dissolves, w:v is 1:1 ~ 1.5kg/L, adds the trichoroacetic acid(TCA) of equal-volume 5%, and limit edged stirs, and leave standstill 60 minutes, centrifugal 5 minutes of 3000rmp, gets supernatant liquor;
(8) second time deproteinated: trichoroacetic acid(TCA) supernatant liquor being added again equal-volume 5%, limit edged stirs, and leave standstill 60 minutes, centrifugal 5 minutes of 3000rmp, gets supernatant liquor, obtain deproteinated polysaccharide soln;
(9) second time alcohol precipitation: adding 95% ethanol to determining alcohol in above deproteinated polysaccharide soln is 80%, and limit edged stirs, and sealing covers tightly, and leaves standstill 24 hours; Suction filtration, with absolute ethanol washing 2 ~ 3 times, vacuum-drying, obtains deproteinated polysaccharide;
(10) first time depigmentation: deproteinated polysaccharide obtained is above added appropriate ultrapure water and dissolves, w:v is 1:1 ~ 1.5kg/L, adds 1% gac, stirs evenly, and is heated to 80 DEG C and stirs 30 minutes, filter, get filtrate;
(11) second time depigmentation: above gained filtrate is added 1% gac again and stirs evenly, is heated to 80 DEG C and stirs 30 minutes, filter, get filtrate;
(12) third time alcohol precipitation: it is 80% that depigmentation polysaccharide liquid obtained is above added 95% ethanol to determining alcohol, and limit edged stirs, and sealing covers tightly, and leaves standstill 24 hours; Suction filtration, with absolute ethanol washing 2-3 time, vacuum-drying, obtains Spreading Hedyotis Herb refined polysaccharide.
(13) determination of polysaccharide: adopt phend-sulphuric acid to measure polysaccharide content, and calculate polysaccharide yield.
The present invention is according to the optimum extraction condition of response surface image analysis gained: adding cellulase consumption in Extracting temperature 55 DEG C, extraction PH7, feed liquid is 2%, carry out 3 times to extract, the results are shown in Table 8, Herba Hedyotidis Diffusae polysaccharide extraction yield is respectively: 8.81%, 8.92% and 8.94%, less with model prediction difference, illustrate that this model can reflect the optimal conditions that Herba Hedyotidis Diffusae polysaccharide extracts well.
Table 8 cellulase response surface optimization extracts the concrete result of implementation of Herba Hedyotidis Diffusae polysaccharide
| Concrete mensuration number of times | For the first time | For the second time | Third time 5--> |
| Polysaccharide content (g) | 1.76 | 1.78 | 1.79 |
| Polysaccharide extract rate (%) | 8.81 | 8.92 | 8.95 |
Embodiment 2 (1) hot water extraction gets Spreading Hedyotis Herb and pulverizes, and crosses 40 mesh sieves, takes Spreading Hedyotis Herb 20 grams, add water to 200 milliliters, mix; Regulate pH to be about 7 with phosphoric acid buffer, in feed liquid, add the cellulase of 2%, mixing, 55 DEG C of stirring in water bath lixiviates 60 minutes; Second time lixiviate is carried out to filter residue, adds water to 200 milliliters, mix, regulate pH to be about 7 with phosphoric acid buffer, add the cellulase of 2%, mixing, 55 DEG C of stirring in water bath lixiviates 60 minutes, suction filtration obtains filtrate, merge twice filtrate, inactivator, 90 DEG C of inactivators are carried out 10 minutes to filtrate.
(2) concentrated: filter vacuum rotating pressure-decreasing to be concentrated into 30 ~ 40 milliliters, to put to room temperature;
(3) first time alcohol precipitation: adding 95% ethanol to determining alcohol in the concentrated solution of above preparation is 80%, and limit edged stirs, and sealing covers tightly, and leaves standstill 24 hours; Suction filtration, vacuum-drying, obtains Spreading Hedyotis Herb Crude polysaccharides;
(4) first time deproteinated: above obtained Spreading Hedyotis Herb Crude polysaccharides is added 20 ~ 30 milliliters of ultrapure waters and dissolves, add the trichoroacetic acid(TCA) of equal-volume 5%, limit edged stirs, and leave standstill 60 minutes, centrifugal 5 minutes of 3000rmp, gets supernatant liquor;
(5) second time deproteinated: trichoroacetic acid(TCA) supernatant liquor being added again equal-volume 5%, limit edged stirs, and leave standstill 60 minutes, centrifugal 5 minutes of 3000rmp, gets supernatant liquor, obtain deproteinated polysaccharide soln;
(6) second time alcohol precipitation: adding 95% ethanol to determining alcohol in above deproteinated polysaccharide soln is 80%, limit edged stirs, sealing covers tightly, leave standstill 24 hours, suction filtration, with dehydrated alcohol 8 ~ 20 milliliters washing 2 ~ 3 times, deproteinated polysaccharide and dehydrated alcohol w:v are 1:3 ~ 5kg/L, vacuum-drying, obtains deproteinated polysaccharide;
(7) first time depigmentation: deproteinated polysaccharide obtained is above added 20 ~ 30 milliliters of ultrapure waters and dissolves, add weight 1% gac, stir evenly, be heated to 80 DEG C and stir 30 minutes, filter, get filtrate;
(8) second time depigmentation: above gained filtrate is added weight 1% gac again and stirs evenly, is heated to 80 DEG C and stirs 30 minutes, filter, get filtrate;
(9) third time alcohol precipitation: it is 80% that depigmentation polysaccharide liquid obtained is above added 95% ethanol to determining alcohol, and limit edged stirs, and sealing covers tightly, and leaves standstill 24 hours; Suction filtration, with absolute ethanol washing 2-3 time, vacuum-drying, obtains Spreading Hedyotis Herb refined polysaccharide 1.76 ~ 1.79 grams.
Claims (2)
1. a processing method for Mierocrystalline cellulose Enzymatic Extraction Herba Hedyotidis Diffusae polysaccharide, is characterized in that: prepare extracting solution according to the solid-liquid ratio of Spreading Hedyotis Herb and water weight ratio 1:8 ~ 12, add cellulase hot water extraction in extracting solution, 80% alcohol precipitation, deproteinated, depigmentation, final refining forms;
Specifically carry out as follows:
(1) hot water extraction: pulverized by Spreading Hedyotis Herb, crosses 40 mesh sieves, adds water be mixed with Spreading Hedyotis Herb extracting solution by the solid-liquid ratio of 1:8 ~ 12 weight ratio; Regulate pH to be 7 with phosphoric acid buffer, add the cellulase of 1.5 ~ 2.5%, mixing, 55 ° of C stirring in water bath heat extraction 60 minutes, and suction filtration obtains filtrate; Filter residue is added water by the solid-liquid ratio of 1:8 ~ 12 weight ratio, pH is regulated to be 7 with phosphoric acid buffer, add the cellulase of 1.5 ~ 2.5% again, mixing, 55 ° of C stirring in water bath heating extractions are extracted once for 60 minutes again, suction filtration obtains filtrate, merges twice filtrate, carries out 90 ° of C inactivators 10 minutes to filtrate;
(2) concentrated: filter vacuum rotating pressure-decreasing to be concentrated into concentration for containing crude drug 0.5 ~ 1 grams per milliliter, to put to room temperature;
(3) first time alcohol precipitation: in concentrated solution, add 95% ethanol to determining alcohol is 80%, limit edged stir, sealing covers tightly, leave standstill 24 ~ 48 hours; Suction filtration, obtains Spreading Hedyotis Herb Crude polysaccharides;
(4) deproteinated: Spreading Hedyotis Herb Crude polysaccharides is added suitable quantity of water and dissolve, add the trichoroacetic acid(TCA) of equal-volume 5%, limit edged stirs, leave standstill 60 minutes, centrifugal 5 minutes of 3000rmp, gets supernatant liquor, repeat above operation again and carry out second time deproteinated, obtain deproteinated polysaccharide soln;
(5) second time alcohol precipitation: adding 95% ethanol to determining alcohol in the Herba Hedyotidis Diffusae polysaccharide liquid after deproteinated is 80%, and limit edged stirs, and sealing covers tightly, and leaves standstill 24 ~ 48 hours; Suction filtration, with absolute ethanol washing 2-3 time, vacuum-drying, obtains Herba Hedyotidis Diffusae polysaccharide;
(6) depigmentation: Herba Hedyotidis Diffusae polysaccharide suitable quantity of water dissolved, adds the gac of weight 1%, and mixing, is heated to 80 DEG C of temperature and stirs 30 minutes, filters, collects filtrate; Filtrate is added the gac of weight 1%, mixing, is heated to 80 DEG C of temperature and stirs 30 minutes, filters, obtains depigmentation Herba Hedyotidis Diffusae polysaccharide solution;
(7) third time alcohol precipitation: it is 80% that depigmentation Herba Hedyotidis Diffusae polysaccharide liquid is added 95% ethanol to determining alcohol, limit edged stir, sealing covers tightly, leave standstill 24 hours; Suction filtration, with absolute ethanol washing 2-3 time, vacuum-drying, obtains refining Herba Hedyotidis Diffusae polysaccharide.
2. the processing method of Mierocrystalline cellulose Enzymatic Extraction Herba Hedyotidis Diffusae polysaccharide according to claim 1, is characterized in that: (1) is got Spreading Hedyotis Herb and pulverized, and crosses 40 mesh sieves, takes Spreading Hedyotis Herb 20 grams, add water to 200 milliliters, mix; Regulate pH to be 7 with phosphoric acid buffer, in feed liquid, add the cellulase of 2%, mixing, 55 ° of C stirring in water bath lixiviate 60 minutes; Second time hot water extraction is carried out to filter residue, adds water to 200 milliliters, mix, regulate pH to be 7 with phosphoric acid buffer, add the cellulase of 2%, mixing, 55 ° of C stirring in water bath lixiviate 60 minutes, suction filtration obtains filtrate, merges secondary filtrate and carries out 90 ° of C inactivators 10 minutes;
(2) concentrated: filter vacuum rotating pressure-decreasing to be concentrated into 30 ~ 40 milliliters, to put to room temperature;
(3) first time alcohol precipitation: adding 95% ethanol to determining alcohol in the concentrated solution of above preparation is 80%, and limit edged stirs, and sealing covers tightly, and leaves standstill 24 hours; Suction filtration, vacuum-drying, obtains Spreading Hedyotis Herb Crude polysaccharides;
(4) first time deproteinated: above obtained Spreading Hedyotis Herb Crude polysaccharides is added 20 ~ 30 milliliters of ultrapure waters and dissolves, add the trichoroacetic acid(TCA) of equal-volume 5%, limit edged stirs, and leave standstill 60 minutes, centrifugal 5 minutes of 3000rmp, gets supernatant liquor;
(5) second time deproteinated: trichoroacetic acid(TCA) supernatant liquor being added again equal-volume 5%, limit edged stirs, and leave standstill 60 minutes, centrifugal 5 minutes of 3000rmp, gets supernatant liquor, obtain deproteinated polysaccharide soln;
(6) second time alcohol precipitation: adding 95% ethanol to determining alcohol in above deproteinated polysaccharide soln is 80%, and limit edged stirs, and sealing covers tightly, and leaves standstill 24 hours; Carry out suction filtration, with absolute ethanol washing 2 ~ 3 times, deproteinated polysaccharide and dehydrated alcohol w:v are 1:3 ~ 5kg/L, and vacuum-drying obtains deproteinated polysaccharide;
(7) first time depigmentation: deproteinated polysaccharide obtained is above added 20 ~ 30 milliliters of ultrapure waters and dissolves, add gac according to 1% weight, stir evenly, be heated to 80 DEG C and stir 30 minutes, filter, get filtrate;
(8) second time depigmentation: above gained filtrate is added gac according to 1% concentration again and stirs evenly, is heated to 80 DEG C and stirs 30 minutes, filter, get filtrate;
(9) third time alcohol precipitation: it is 80% that depigmentation polysaccharide liquid obtained is above added 95% ethanol to determining alcohol, and limit edged stirs, and sealing covers tightly, and leaves standstill 24 hours; Suction filtration, absolute ethanol washing 2-3 time, vacuum-drying, obtains Spreading Hedyotis Herb refined polysaccharide 1.76 ~ 1.79 grams.
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| CN109988250A (en) * | 2019-04-18 | 2019-07-09 | 浙江中医药大学 | A method of extracting Veronicastrum Herb Thick many candies from Veronicastrum Herb |
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| CN101654485A (en) * | 2009-09-04 | 2010-02-24 | 山东中医药大学附属医院 | Spreading hedvotis herb polysaccharide for curing malignant tumor and preparation method thereof |
| CN103626883A (en) * | 2012-08-29 | 2014-03-12 | 奇复康药物研发(苏州)有限公司 | Extraction method for hedyotis diffusa polysaccharide |
| CN103083352A (en) * | 2013-01-24 | 2013-05-08 | 西南民族大学 | Application of oldenlandia diffusa extract to preparation of medicament for promoting growth and development of animals |
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