CN104403014A - High yield preparation method of grifola frondosa polysaccharide - Google Patents
High yield preparation method of grifola frondosa polysaccharide Download PDFInfo
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- CN104403014A CN104403014A CN201410632018.3A CN201410632018A CN104403014A CN 104403014 A CN104403014 A CN 104403014A CN 201410632018 A CN201410632018 A CN 201410632018A CN 104403014 A CN104403014 A CN 104403014A
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- grifola frondosa
- crude polysaccharides
- inorganic salt
- grifolan
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Abstract
The invention discloses a high yield preparation method of grifola frondosa polysaccharide. Proper enzymatic hydrolysis and alcohol precipitation technology are adopted, a polymer/inorganic salt two-water-phase system is utilized, and a macroporous absorption impurity removing technology and an ultra-filtration membrane filtration technology are performed so as to increase the yield of grifola frondosa polysaccharide. The grifola frondosa polysaccharide content of an extract obtained by the provided preparation method is 18% higher than that of an extract obtained through a microwave-assisted extraction technology.
Description
Technical field
The invention belongs to edible mushrooms effective ingredient extraction and separation technology field, in particular to the preparation method of the grifolan of high yield pulp1.
Background technology
Its main active ingredient of Grifola frondosa is Grifola frondosa protein-polysaccharide, based on dextran in polysaccharide component, to have β-(1 → 3) dextran of β-(1 → 6) branch for basic structure, containing a small amount of wood sugar and seminose.Result of study shows, grifolan plays its antitumor action mainly through strengthening cellular immune function, on gene level, this product has stronger chemical resistance mutagenesis, the complex therapy that weak after can be used for chronic hepatitis B and concurrent chemoradiotherapy of malignant tumor, oligoleukocythemia, immunologic function reduce, and have certain effect to the prevention of acquired immune deficiency syndrome (AIDS).Grifolan is effectively oral clinically, safety non-toxic.Grifolan has antitumor, hypertension, reduction blood sugar, anti-obesity, anti-hepatitis drug effect.Show according to the experiment of coming personally of certain cancers special for treating hospital of the U.S., while cancer chemotherapy, more to take effect fruit than chemotherapy with grifolan anticancer.Japan medical science expert carries out antitumor in vivo test with grifolan, and result shows, grifolan inhibiting rate can reach 86.5%, exceeds 32% than the PTS lentinan inhibiting rate of International Certification.
Grifolan is the effective active composition extracted from high-quality Grifola Frondosa sporophore, its fragrant odour, and effect is remarkable, can do the interpolation raw material of all kinds of healthcare products, food.
Aqueous two phase extraction technique starts from the sixties in 20th century, due to aqueous two-phase extraction mild condition, easy amplification, can operate continuously, therefore this technology is successfully applied to the abstraction and purification of the biological products such as protein, nucleic acid and virus, also there is microbiotic, the trial of the separation of small molecuies aspects such as effective components of Chinese medicinal, utilize polymer/inorganic salt double-aqueous phase system also not appear in the newspapers to extract grifolan.
Summary of the invention
The technical problem that the present invention mainly solves is to provide a kind of method extracting grifolan, by suitable enzymolysis, alcohol precipitation technology, utilize polymer/inorganic salt double-aqueous phase system, macroporous adsorbent resin removal of impurities, ultra-filtration membrane to carry out the extraction yield that step improves grifolan such as filtering, the technical scheme that the present invention adopts for this reason is:
A preparation method for the grifolan of high yield pulp1, comprises the following steps:
(1) being dried by Grifola Frondosa sporophore to moisture content is 10 ~ 20%, then carries out pulverization process, obtains 200 ~ 300 object ash tree pollen, after being mixed into mixed solution with distilled water by the weight ratio of 1:30-50, for subsequent use;
(2) enzymolysis processing: get the cellulase accounting for mixed solution weight 0.05-0.2% and add in mixed solution, reconcile pH value to 4-6, vibration or stir under in 45-55 DEG C of enzymolysis 30-90 minute;
(3) ash tree pollen is carried out microwave-assisted hot water extraction, described microwave power is 500-700W, and extraction time is 3-5 minute; Then proceed ultrasonic assistant hot water extraction, its ultrasonic temperature 20-60 DEG C, power is 600-800W, and ultrasonic time is 20-60min; Then the Grifola frondosa Aqueous extracts of gained is cooled to room temperature, centrifugation obtains supernatant liquor and Grifola frondosa residue,
(4) alcohol precipitation polysaccharide: by step (3) gained supernatant liquor concentrating under reduced pressure, after the dehydrated alcohol adding 2-4 times of volume carries out alcohol precipitation, centrifugal, freezing high and dry after Grifola frondosa Crude polysaccharides;
(5) aqueous two-phase extraction: by water-soluble for Grifola frondosa Crude polysaccharides, obtain Crude polysaccharides solution, adds in polymer/inorganic salt double-aqueous phase system, temperature 30-50 DEG C, shake 30-50 minute under the condition of pH 4-7, concussion speed is 100-200rmin
-1, then leave standstill layering in 20-40 minute; Described polymkeric substance is polyoxyethylene glycol, and inorganic salt are potassiumphosphate; Described Polyethylene glycol is the concentration 15-25% of 10-20%, potassiumphosphate;
(6) lower floor of being rich in inorganic salt is taken out, macroporous adsorbent resin is adopted to carry out wash-out removal of impurities to gained Grifola frondosa Crude polysaccharides with 3-10 times of cylinder ponding with the flow velocity of 1-4BV/h, elution flow rate is 0.5-5BV/h, afterwards, adjust ph scope is 6.5-8, adopts the process of anion-cation exchange resin series connection resin column deproteinated; Described anionite-exchange resin and the mass ratio of resin cation (R.C.) are 1:1 ~ 5.
(7) with interception be 8000 ultra-filtration membrane desalination, molecular weight cut-off scope is 100000-800000D,
Operating pressure is 0.5-0.7Mpa, and temperature is 35-50 DEG C, and the rotating speed of rotor is 400-600r/min, collects and retains concentrated solution, and with distilled water cleaning, merges washing lotion and concentrated solution, dry, obtains grifolan.
The invention has the beneficial effects as follows:
Grifola frondosa coarse polysaccharide extractive rate of the present invention is higher, and purer.
Embodiment
Below preferred embodiment of the present invention is described in detail, can be easier to make advantages and features of the invention be readily appreciated by one skilled in the art, thus more explicit defining is made to protection scope of the present invention.
Embodiment 1
(1) being dried by Grifola Frondosa sporophore to moisture content is 10%, then carries out pulverization process, obtains 300 object ash tree pollen, after being mixed into mixed solution with distilled water by the weight ratio of 1:30, for subsequent use;
(2) enzymolysis processing: get the cellulase accounting for mixed solution weight 0.05% and add in mixed solution, reconciles pH value to 4, vibration or under stirring in 45 DEG C of enzymolysis 90 minutes;
(3) ash tree pollen is carried out microwave-assisted hot water extraction, described microwave power is 500W, and extraction time is 5 minutes; Then proceed ultrasonic assistant hot water extraction, its ultrasonic temperature 20 DEG C, power is 800W, and ultrasonic time is 20min; Then the Grifola frondosa Aqueous extracts of gained is cooled to room temperature, centrifugation obtains supernatant liquor and Grifola frondosa residue,
(4) alcohol precipitation polysaccharide: by step (3) gained supernatant liquor concentrating under reduced pressure, after the dehydrated alcohol adding 2 times of volumes carries out alcohol precipitation, centrifugal, freezing high and dry after Grifola frondosa Crude polysaccharides;
(5) aqueous two-phase extraction: by water-soluble for Grifola frondosa Crude polysaccharides, obtain Crude polysaccharides solution, adds in polymer/inorganic salt double-aqueous phase system, and shake 50 minutes under the condition of temperature 30 DEG C, pH 7, concussion speed is 200rmin
-1, then leave standstill layering in 20 minutes; Described polymkeric substance is polyoxyethylene glycol, and inorganic salt are potassiumphosphate; Described Polyethylene glycol is 10%, the concentration 25% of potassiumphosphate;
(6) lower floor of being rich in inorganic salt is taken out, macroporous adsorbent resin is adopted to carry out wash-out removal of impurities to gained Grifola frondosa Crude polysaccharides with 3 times of cylinder ponding with the flow velocity of 1BV/h, elution flow rate is 0.5BV/h, afterwards, adjust ph scope is 6.5, adopts the process of anion-cation exchange resin series connection resin column deproteinated; Described anionite-exchange resin and the mass ratio of resin cation (R.C.) are 1:1.
(7) with interception be 8000 ultra-filtration membrane desalination, molecular weight cut-off scope is 100000-800000D,
Operating pressure is 0.5Mpa, and temperature is 35 DEG C, and the rotating speed of rotor is 400r/min, collects and retains concentrated solution, and with distilled water cleaning, merges washing lotion and concentrated solution, dry, obtains grifolan.
Embodiment 2
(1) being dried by Grifola Frondosa sporophore to moisture content is 15%, then carries out pulverization process, obtains 300 object ash tree pollen, after being mixed into mixed solution with distilled water by the weight ratio of 1:30, for subsequent use;
(2) enzymolysis processing: get the cellulase accounting for mixed solution weight 0.1% and add in mixed solution, reconciles pH value to 5, vibration or under stirring in 50 DEG C of enzymolysis 60 minutes;
(3) ash tree pollen is carried out microwave-assisted hot water extraction, described microwave power is 600W, and extraction time is 4 minutes; Then proceed ultrasonic assistant hot water extraction, its ultrasonic temperature 40 DEG C, power is 700W, and ultrasonic time is 40min; Then the Grifola frondosa Aqueous extracts of gained is cooled to room temperature, centrifugation obtains supernatant liquor and Grifola frondosa residue,
(4) alcohol precipitation polysaccharide: by step (3) gained supernatant liquor concentrating under reduced pressure, after the dehydrated alcohol adding 3 times of volumes carries out alcohol precipitation, centrifugal, freezing high and dry after Grifola frondosa Crude polysaccharides;
(5) aqueous two-phase extraction: by water-soluble for Grifola frondosa Crude polysaccharides, obtain Crude polysaccharides solution, adds in polymer/inorganic salt double-aqueous phase system, and shake 40 minutes under temperature 40 DEG C, pH are the condition of 5.5, concussion speed is 150rmin
-1, then leave standstill layering in 30 minutes; Described polymkeric substance is polyoxyethylene glycol, and inorganic salt are potassiumphosphate; Described Polyethylene glycol is 15%, the concentration 20% of potassiumphosphate;
(6) lower floor of being rich in inorganic salt is taken out, adopt macroporous adsorbent resin to carry out wash-out removal of impurities to gained Grifola frondosa Crude polysaccharides with 6 times of cylinder ponding with the flow velocity of 3BV/h, elution flow rate is 2BV/h, afterwards, adjust ph is 7, adopts the process of anion-cation exchange resin series connection resin column deproteinated; Described anionite-exchange resin and the mass ratio of resin cation (R.C.) are 1:3.
(7) with interception be 8000 ultra-filtration membrane desalination, molecular weight cut-off scope is 100000-800000D,
Operating pressure is 0.6Mpa, and temperature is 40 DEG C, and the rotating speed of rotor is 500r/min, collects and retains concentrated solution, and with distilled water cleaning, merges washing lotion and concentrated solution, dry, obtains grifolan.
Embodiment 3
(1) being dried by Grifola Frondosa sporophore to moisture content is 20%, then carries out pulverization process, obtains 200 object ash tree pollen, after being mixed into mixed solution with distilled water by the weight ratio of 1:50, for subsequent use;
(2) enzymolysis processing: get the cellulase accounting for mixed solution weight 0.2% and add in mixed solution, reconciles pH value to 6, vibration or under stirring in 55 DEG C of enzymolysis 30 minutes;
(3) ash tree pollen is carried out microwave-assisted hot water extraction, described microwave power is 700W, and extraction time is 3 minutes; Then proceed ultrasonic assistant hot water extraction, its ultrasonic temperature 60 DEG C, power is 600W, and ultrasonic time is 60min; Then the Grifola frondosa Aqueous extracts of gained is cooled to room temperature, centrifugation obtains supernatant liquor and Grifola frondosa residue,
(4) alcohol precipitation polysaccharide: by step (3) gained supernatant liquor concentrating under reduced pressure, after the dehydrated alcohol adding 4 times of volumes carries out alcohol precipitation, centrifugal, freezing high and dry after Grifola frondosa Crude polysaccharides;
(5) aqueous two-phase extraction: by water-soluble for Grifola frondosa Crude polysaccharides, obtain Crude polysaccharides solution, adds in polymer/inorganic salt double-aqueous phase system, and shake 50 minutes under the condition of temperature 50 C, pH 4, concussion speed is 200rmin-
1, then leave standstill layering in 20 minutes; Described polymkeric substance is polyoxyethylene glycol, and inorganic salt are potassiumphosphate; Described Polyethylene glycol is 20%, the concentration 15% of potassiumphosphate;
(6) lower floor of being rich in inorganic salt is taken out, macroporous adsorbent resin is adopted to carry out wash-out removal of impurities to gained Grifola frondosa Crude polysaccharides with 10 times of cylinder ponding with the flow velocity of 4BV/h, elution flow rate is 5BV/h, afterwards, adjust ph scope is 8, adopts the process of anion-cation exchange resin series connection resin column deproteinated; Described anionite-exchange resin and the mass ratio of resin cation (R.C.) are 1:5.
(7) with interception be 8000 ultra-filtration membrane desalination, molecular weight cut-off scope is 100000-800000D,
Operating pressure is 0.7Mpa, and temperature is 35 DEG C, and the rotating speed of rotor is 600r/min, collects and retains concentrated solution, and with distilled water cleaning, merges washing lotion and concentrated solution, dry, obtains grifolan.
By the grifolan content extracted through the present invention with contrast with the grifolan content that microwave radiation exaraction grifolan obtains merely, grifolan content on average increases by 18%.
The foregoing is only embodiments of the invention; not thereby the scope of the claims of the present invention is limited; every utilize description of the present invention to do equivalent structure or equivalent flow process conversion; or be directly or indirectly used in other relevant technical fields, be all in like manner included in scope of patent protection of the present invention.
Claims (1)
1. a preparation method for the grifolan of high yield pulp1, is characterized in that, comprises the following steps:
(1) being dried by Grifola Frondosa sporophore to moisture content is 10 ~ 20%, then carries out pulverization process, obtains 200 ~ 300 object ash tree pollen, after being mixed into mixed solution with distilled water by the weight ratio of 1:30-50, for subsequent use;
(2) enzymolysis processing: get the cellulase accounting for mixed solution weight 0.05-0.2% and add in mixed solution, reconcile pH value to 4-6, vibration or stir under in 45-55 DEG C of enzymolysis 30-90 minute;
(3) ash tree pollen is carried out microwave-assisted hot water extraction, described microwave power is 500-700W, and extraction time is 3-5 minute; Then proceed ultrasonic assistant hot water extraction, its ultrasonic temperature 20-60 DEG C, power is 600-800W, and ultrasonic time is 20-60min; Then the Grifola frondosa Aqueous extracts of gained is cooled to room temperature, centrifugation obtains supernatant liquor and Grifola frondosa residue;
(4) alcohol precipitation polysaccharide: by step (3) gained supernatant liquor concentrating under reduced pressure, after the dehydrated alcohol adding 2-4 times of volume carries out alcohol precipitation, centrifugal, freezing high and dry after Grifola frondosa Crude polysaccharides;
(5) aqueous two-phase extraction: by water-soluble for Grifola frondosa Crude polysaccharides, obtain Crude polysaccharides solution, adds in polymer/inorganic salt double-aqueous phase system, temperature 30-50 DEG C, shake 30-50 minute under the condition of pH 4-7, concussion speed is 100-200rmin
-1, then leave standstill layering in 20-40 minute; Described polymkeric substance is polyoxyethylene glycol, and inorganic salt are potassiumphosphate; Described Polyethylene glycol is the concentration 15-25% of 10-20%, potassiumphosphate;
(6) lower floor of being rich in inorganic salt is taken out, macroporous adsorbent resin is adopted to carry out wash-out removal of impurities to gained Grifola frondosa Crude polysaccharides with 3-10 times of cylinder ponding with the flow velocity of 1-4BV/h, elution flow rate is 0.5-5BV/h, afterwards, adjust ph scope is 6.5-8, adopts the process of anion-cation exchange resin series connection resin column deproteinated; Described anionite-exchange resin and the mass ratio of resin cation (R.C.) are 1:1 ~ 5;
(7) with interception be 8000 ultra-filtration membrane desalination, molecular weight cut-off scope is 100000-800000D, operating pressure is 0.5-0.7Mpa, temperature is 35-50 DEG C, and the rotating speed of rotor is 400-600r/min, collects and retains concentrated solution, and clean with distilled water, merge washing lotion and concentrated solution, dry, obtain grifolan.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105669876A (en) * | 2016-03-02 | 2016-06-15 | 漳州片仔癀药业股份有限公司 | Extraction process and application of grifolan |
| CN105732836A (en) * | 2016-03-02 | 2016-07-06 | 漳州片仔癀药业股份有限公司 | Extraction process and application of polysaccharide of grifola frondosa to preparation of medicament for repairing gastric mucosa damage |
| CN105777931A (en) * | 2016-03-02 | 2016-07-20 | 漳州片仔癀药业股份有限公司 | Grifolan extracting method and pharmaceutical application thereof |
| CN105949338A (en) * | 2016-05-24 | 2016-09-21 | 江苏江大源生态生物科技股份有限公司 | Method using microwaves to extract grifola frondosa polysaccharides |
| CN106008027A (en) * | 2016-06-30 | 2016-10-12 | 江苏挚信花卉发展有限公司 | Green environment-friendly nutrient solution beneficial to plant growth |
| CN108676107A (en) * | 2018-06-05 | 2018-10-19 | 广东药科大学 | A method of purifying Schizophyllum commune Fr polysaccharides using Bi-aqueous extraction |
| CN109400744A (en) * | 2018-11-21 | 2019-03-01 | 湖南中医药大学 | A kind of oxidation resistant southern reed bamboo shoot polyoses producing method |
| CN110066313A (en) * | 2019-05-05 | 2019-07-30 | 中国科学院兰州化学物理研究所 | A kind of preparation method of high-purity Lycium chinense glycopeptide |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105669876A (en) * | 2016-03-02 | 2016-06-15 | 漳州片仔癀药业股份有限公司 | Extraction process and application of grifolan |
| CN105732836A (en) * | 2016-03-02 | 2016-07-06 | 漳州片仔癀药业股份有限公司 | Extraction process and application of polysaccharide of grifola frondosa to preparation of medicament for repairing gastric mucosa damage |
| CN105777931A (en) * | 2016-03-02 | 2016-07-20 | 漳州片仔癀药业股份有限公司 | Grifolan extracting method and pharmaceutical application thereof |
| CN105949338A (en) * | 2016-05-24 | 2016-09-21 | 江苏江大源生态生物科技股份有限公司 | Method using microwaves to extract grifola frondosa polysaccharides |
| CN106008027A (en) * | 2016-06-30 | 2016-10-12 | 江苏挚信花卉发展有限公司 | Green environment-friendly nutrient solution beneficial to plant growth |
| CN108676107A (en) * | 2018-06-05 | 2018-10-19 | 广东药科大学 | A method of purifying Schizophyllum commune Fr polysaccharides using Bi-aqueous extraction |
| CN109400744A (en) * | 2018-11-21 | 2019-03-01 | 湖南中医药大学 | A kind of oxidation resistant southern reed bamboo shoot polyoses producing method |
| CN110066313A (en) * | 2019-05-05 | 2019-07-30 | 中国科学院兰州化学物理研究所 | A kind of preparation method of high-purity Lycium chinense glycopeptide |
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Application publication date: 20150311 |