CN102492666A - Enzyme preparation, application and method for extracting pueraria flavonid by enzyme preparation thereof - Google Patents
Enzyme preparation, application and method for extracting pueraria flavonid by enzyme preparation thereof Download PDFInfo
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- CN102492666A CN102492666A CN2011104455411A CN201110445541A CN102492666A CN 102492666 A CN102492666 A CN 102492666A CN 2011104455411 A CN2011104455411 A CN 2011104455411A CN 201110445541 A CN201110445541 A CN 201110445541A CN 102492666 A CN102492666 A CN 102492666A
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- zymin
- radix puerariae
- total flavones
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- enzyme preparation
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Abstract
The invention provides an enzyme preparation, an application and a method for extracting pueraria flavonid by the enzyme preparation, which belongs to the enzyme preparation application field. The technical scheme is characterized in that the enzyme preparation comprises the following ingredients by weight part: 1-5 parts of cellulose, 1-5 parts of xylanase, 1-5 parts of protease and 1-5 parts of amylase. The invention also provides the application of the enzyme preparation used for extracting the pueraria flavonid, and provides concrete operation steps, the recovery rate of pueraria flavonid can be finally realized to more than 70mg/g. The invention has the beneficial effect that the reaction condition is mild, the conformation of natural products can be kept, a stereo structure and biological activity can not be destroyed, the original drug effect of the active ingredients can be kept; the impurity in a system can be removed, the clarification degree of an extract can be enhanced, and the recovery rate of the effective ingredients of the traditional Chinese medicine can be raised; the optimization combination is carried out on the reaction condition, and the maximization of the extraction rate can be realized.
Description
Technical field
The present invention relates to a kind of zymin and be used to extract the application and the method for Radix Puerariae total flavones.
Background technology
The root of kudzu vine (Puerana labata) is that the pulse family Pueraria lobota belongs to perennial vine; It is a kind of traditional Chinese medicines material; Its total flavones is a main effective constituent in the root of kudzu vine, has that anti-inflammatory is analgesic, an effect of coronary artery dilator blood vessel, coronary blood flow increasing, and the effect that brings high blood pressure down is also arranged simultaneously.As the raw material or the additive of medicine and protective foods, at present, about 500～800 tons/year of Radix Puerariae total flavones international market demand amount, about 150 tons/year of China's demand, the exploitation of Radix Puerariae total flavones more and more causes investigator and developer's concern.
Like decocting method, pickling process, percolation process, circumfluence method etc., there are problems such as effective component extraction rate is not high, the contaminant removal rate is low in traditional extraction and separation method.Enzyme engineering technology is a biotechnology that was used for essential industry in recent years.Leniently plant tissue is decomposed through enzyme reaction, quicken the release of effective constituent and extract, select for use corresponding enzyme can the impurity that influence liquid preparation clarity such as starch, protein, pectin etc. be decomposed removal; And it is many to containing fat-soluble component in the root; Through glucuroide or transglycosylase, make fat-soluble component change into the water-soluble sugar glycoside, it is low that temperature is extracted in enzyme reaction; Can destroy plant cell wall, promote extracts active ingredients; Change the character of the purpose composition that extracts, strengthen pharmaceutical activity; Impurity in the removal system improves extraction system clarity, changes the medicinal material quality.
Summary of the invention
The object of the present invention is to provide a kind of zymin; And it is applied in the extraction Radix Puerariae total flavones; Further provided concrete working method, this method can be under the condition of gentleness be carried out highly selective to effective ingredients in plant and transformed, and not only can overcome in the pure water extracting method commonly used in the industry problems such as effective component extraction rate is low, complex procedures; Improve extraction system clarity, change the medicinal material quality; Can also in extraction, change the structure of original natural component, increase the physiologically active of extract, have the potential industrial application value.
For realizing this purpose, the present invention adopts following technical scheme:
A kind of zymin is characterized in that: the component that comprises following weight part:
Said proteolytic enzyme is selected at least a in papoid, Sumizyme MP, the neutral protease for use.
At least a during said glycase is selected for use in warm AMS and the beta-amylase.
Above-mentioned zymin is used to extract the application of Radix Puerariae total flavones.
Above-mentioned zymin is used to extract the method for Radix Puerariae total flavones, it is characterized in that may further comprise the steps:
(1) root of kudzu vine oven dry is pulverized, sieve, get arrowroot and add dissolved in distilled water;
(2) regulate pH to 4.0-6.0, add a certain amount of zymin, under 40-60 ℃ temperature enzymolysis 1-6 hour;
(3) the high temperature enzyme that goes out;
(4) centrifugal, deposition is extracted 1-3h with 70% alcohol heating reflux, filters, and extracts 2-4 time.
(5) merge supernatant and filtrating, concentrating under reduced pressure reclaims ethanol, and liquid concentrator is a Radix Puerariae total flavones medicinal extract.
In the method for extraction Radix Puerariae total flavones above-mentioned, said plant is crushed to the 40-100 order, and the mass ratio of the zero(ppm) water of vegetable material and adding is 1: 10-30.
The mass ratio of described zymin and zero(ppm) water is 1: 5-15.
Zymin addition described in the step (2) is the 0.1-1% of arrowroot addition.
Enzyme-removal temperature described in the step (3) is 80-100 ℃, time 5-10min.
70% ethanol described in the step (4) and sedimentary mass ratio are 1: 8-15, the refluxing extraction temperature is 60-80 ℃.
The present invention selects for use the principle of this kind zymin to be: cellulase, and can the degrading plant Mierocrystalline cellulose; The xylan of zytase in can hydrolyzing plant; Proteolytic enzyme, the protein in can hydrolysis extract improves the purity of extract; Glycase can hydrolyzed starch.These several kinds of enzyme interactings can destroy the fibrous texture of root of kudzu vine cell walls, even are broken down into small-molecule substance, reduce and extract resistance to mass transfer, effectively discharge Radix Puerariae flavone isoreactivity composition;
Compared with prior art, the present invention has the following advantages:
(1) the present invention adopts the Enzymatic Extraction Radix Puerariae total flavones, and reaction conditions is gentle, can keep the conformation of natural product, does not destroy its three-dimensional arrangement and biological activity, and the original drug effect of composition helps remaining valid.
(2) the present invention selects for use enzyme not only can remove impurity in the system, improves the clarity of extracting solution, has improved the Chinese medicine extraction ratio of effective constituents simultaneously again.
(3) the present invention has carried out optimum combination to proportioning, extraction time, extraction temperature, enzyme concentration, the pH value of zymin, has realized the maximization that Radix Puerariae total flavones extracts yield.
(4) zymin of the present invention is used for extracting the method for Radix Puerariae total flavones, adopt enzyme carry with alcohol extracting bonded condition under, improved the yield of activeconstituents.
(5) adopt the present invention from the Chinese medicine root of kudzu vine, to extract total flavones, the Radix Puerariae total flavones extraction yield is more than 7%, and general water extraction method, extraction yield generally is merely 1-2%, and alcohol extracting is followed the example of, and extraction yield generally is merely 4-5%, so the enzyme formulation is extracted the yield height.
(6) simple, easy to operate, the less investment of technology of the present invention, can not produce noise and pollution, adapt to explained hereafter and use.
Embodiment
Embodiment 1
(1) gets root of kudzu vine oven dry and be crushed to 80 orders, get this powder 10g, add zero(ppm) water 100mL dissolving;
(2) get zymin: cellulase 0.0037g, zytase 0.0056g, papoid 0.0018g, middle temperature AMS 0.0037g add in the root of kudzu vine solution, regulate pH to 4.0, and enzymolysis is 1 hour under 40 ℃ of conditions;
(3) 80 ℃ of enzyme 10min that go out;
(4) centrifugal, deposition adds 70% ethanol of 10 times of volumes, and 80 ℃ of heating and refluxing extraction 1 hour are filtered, and extract 2 times.
(5) merge supernatant and filtrating, concentrating under reduced pressure reclaims ethanol, and liquid concentrator is a Radix Puerariae total flavones medicinal extract.
Adopt the spectrophotometry general flavone content, the Radix Puerariae total flavones extraction yield is 7.34%.
Embodiment 2
(1) gets root of kudzu vine oven dry and be crushed to 80 orders, get this powder 10g, add zero(ppm) water 150mL dissolving;
(2) get zymin: cellulase 0.0062g, zytase 0.0093g, Sumizyme MP 0.0031g, middle temperature AMS 0.0062g adds in the root of kudzu vine solution, regulates pH to 4.5, and enzymolysis is 2 hours under 45 ℃ of conditions;
(3) 85 ℃ of enzyme 10min that go out;
(4) centrifugal, deposition adds 70% ethanol of 12 times of volumes, 70 ℃ of refluxing extraction 3 hours, and filtered while hot is extracted 3 times;
(5) merge supernatant and filtrating, concentrating under reduced pressure reclaims ethanol, and liquid concentrator is a Radix Puerariae total flavones medicinal extract.
Adopt the spectrophotometry general flavone content, the Radix Puerariae total flavones extraction yield is 7.49%.
Embodiment 3:
(1) gets root of kudzu vine oven dry and be crushed to 100 orders, get this powder 15g, add zero(ppm) water 400ml dissolving;
(2) get zymin: cellulase 0.0125g, zytase 0.0375g, neutral protease 0.0325g, beta-amylase 0.065g add in the root of kudzu vine solution, regulate pH to 5.0, and enzymolysis is 3 hours under 50 ℃ of conditions;
(3) 85 ℃ of enzyme 10min that go out;
(4) centrifugal, deposition adds 70% ethanol of 10 times of volumes, 75 ℃ of refluxing extraction 2 hours, and filtered while hot is extracted 2 times;
(5) merge supernatant and filtrating, concentrating under reduced pressure reclaims ethanol, and liquid concentrator is a Radix Puerariae total flavones medicinal extract.
Adopt the spectrophotometry general flavone content, the Radix Puerariae total flavones extraction yield is 7.82%.
Embodiment 4
(1) gets root of kudzu vine oven dry and be crushed to 80 orders, get this powder 10g, add zero(ppm) water 300mL dissolving;
(2) get zymin: cellulase 0.02g, zytase 0.01g, neutral protease 0.02, middle temperature AMS 0.03g add in the root of kudzu vine solution, regulate pH to 5.0, and enzymolysis is 1.5 hours under 40 ℃ of conditions;
(3) 85 ℃ of enzyme 10min that go out;
(4) centrifugal, deposition adds 70% ethanol of 8 times of volumes, 75 ℃ of refluxing extraction 3 hours, and filtered while hot is extracted 2 times;
(5) merge supernatant and filtrating, concentrating under reduced pressure reclaims ethanol, and liquid concentrator is a Radix Puerariae total flavones medicinal extract.
Adopt the spectrophotometry general flavone content, the Radix Puerariae total flavones extraction yield is 7.82%.
Embodiment 5:
(1) gets root of kudzu vine oven dry and be crushed to 80 orders, get this powder 10g, add zero(ppm) water 100mL dissolving;
(2) get zymin (cellulase: zytase: papoid: middle temperature AMS=2: 2: 3: 1) 0.05g, regulate pH to 6.0, enzymolysis is 1 hour under 60 ℃ of conditions;
(3) 85 ℃ of enzyme 10min that go out;
(4) centrifugal, deposition adds 70% ethanol of 10 times of volumes, 85 ℃ of refluxing extraction 3 hours, and filtered while hot is extracted 3 times;
(5) merge supernatant and filtrating, concentrating under reduced pressure reclaims ethanol, and liquid concentrator is a Radix Puerariae total flavones medicinal extract.
Adopt the spectrophotometry general flavone content, the Radix Puerariae total flavones extraction yield is 8.59%.
Embodiment 6:
(1) gets root of kudzu vine oven dry and be crushed to 100 orders, get this powder 10g, add zero(ppm) water 200mL dissolving;
(2) get zymin (cellulase: zytase: Sumizyme MP: middle temperature AMS=2: 3: 1: 2) 0.08g, regulate pH to 4.5, enzymolysis is 2 hours under 45 ℃ of conditions;
(3) 90 ℃ of enzyme 10min that go out;
(4) centrifugal, deposition was with 80 ℃ of refluxing extraction of 75% ethanol 3 hours, and filtered while hot is extracted 3 times;
(5) merge supernatant and filtrating, concentrating under reduced pressure reclaims ethanol, and liquid concentrator is a Radix Puerariae total flavones medicinal extract.
Adopt the spectrophotometry general flavone content, the Radix Puerariae total flavones extraction yield is 7.55%.
Embodiment 7:
(1) gets root of kudzu vine oven dry and be crushed to 80 orders, get this powder 10g, add zero(ppm) water 200ml dissolving;
(2) get zymin (cellulase: zytase: neutral protease: middle temperature AMS=1: 2: 2: 5) 0.1g, regulate pH to 4.5, enzymolysis is 3 hours under 45 ℃ of conditions;
(3) 85 ℃ of enzyme 10min that go out;
(4) centrifugal, deposition adds 70% ethanol of 10 times of volumes, 65 ℃ of refluxing extraction 80mim, and filtered while hot is extracted 3 times;
(5) merge supernatant and filtrating, concentrating under reduced pressure reclaims ethanol, and liquid concentrator is a Radix Puerariae total flavones medicinal extract.
Adopt the spectrophotometry general flavone content, the Radix Puerariae total flavones extraction yield is 7.73%.
Embodiment 8:
(1) gets root of kudzu vine oven dry and be crushed to 80 orders, get this powder 10g, add zero(ppm) water 150ml dissolving;
(2) get zymin (cellulase: zytase: neutral protease: beta-amylase=1: 1: 3: 2) 0.22g, regulate pH to 5.0, enzymolysis is 3 hours under 60 ℃ of conditions;
(3) 100 ℃ of enzyme 10min that go out;
(4) centrifugal, deposition is with 75 ℃ of refluxing extraction 80mim of 75% ethanol, and filtered while hot is extracted 3 times;
(5) merge supernatant and filtrating, concentrating under reduced pressure reclaims ethanol, and liquid concentrator is a Radix Puerariae total flavones medicinal extract.
Adopt the spectrophotometry general flavone content, the Radix Puerariae total flavones extraction yield is 7.25%.
Claims (8)
1. zymin is characterized in that: the component that comprises following weight part:
2. the described a kind of zymin of claim 1 is characterized in that: said proteolytic enzyme is selected at least a in papoid, Sumizyme MP, the neutral protease for use.
3. the described a kind of zymin of claim 1 is characterized in that: at least a during said glycase is selected for use in warm AMS and the beta-amylase.
4. the described zymin of claim 1 is used to extract the method for Radix Puerariae total flavones, it is characterized in that may further comprise the steps:
(1) root of kudzu vine oven dry is pulverized, sieve, get arrowroot and add dissolved in distilled water;
(2) regulate pH to 4.0-6.0, add a certain amount of zymin, under 40-60 ℃ temperature enzymolysis 1-6 hour;
(3) the high temperature enzyme that goes out;
(4) centrifugal, deposition is extracted 1-3h with 70% alcohol heating reflux, filters, and extracts 2-4 time.
(5) merge supernatant and filtrating, concentrating under reduced pressure reclaims ethanol, and liquid concentrator is a Radix Puerariae total flavones medicinal extract.
5. the described zymin of claim 3 is used to extract the method for Radix Puerariae total flavones, it is characterized in that the plant described in the step (1) is crushed to the 40-100 order, and the mass ratio of the zero(ppm) water of vegetable material and adding is 1: 10-30.
6. the described zymin of claim 3 is used to extract the method for Radix Puerariae total flavones, it is characterized in that: the zymin addition described in the step (2) is the 0.1-1% of arrowroot addition.
7. the described zymin of claim 3 is used to extract the method for Radix Puerariae total flavones, it is characterized in that: the enzyme-removal temperature described in the step (3) is 80-100 ℃, time 5-10min.
8. the described zymin of claim 3 is used to extract the method for Radix Puerariae total flavones, it is characterized in that: 70% ethanol described in the step (4) and sedimentary mass ratio are 1: 8-15, the refluxing extraction temperature is 60-80 ℃.
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| CN2011104455411A CN102492666A (en) | 2011-12-28 | 2011-12-28 | Enzyme preparation, application and method for extracting pueraria flavonid by enzyme preparation thereof |
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| CN2011104455411A CN102492666A (en) | 2011-12-28 | 2011-12-28 | Enzyme preparation, application and method for extracting pueraria flavonid by enzyme preparation thereof |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103789366A (en) * | 2014-02-21 | 2014-05-14 | 王健鹏 | Method for extracting puerarin through compound enzyme hydrolysis |
| CN103923737A (en) * | 2014-05-08 | 2014-07-16 | 龚意科 | Method for extracting maca flavone oiling agent from complex enzyme |
| CN106473995A (en) * | 2016-12-14 | 2017-03-08 | 杨攀 | The extracting method of Herba Bidentis Bipinnatae effective ingredient and its purposes preparing facial film |
| CN107245483A (en) * | 2017-07-19 | 2017-10-13 | 江苏农林职业技术学院 | A kind of complex enzyme for being used to extract kudzu root flavone and the method extracted using the complex enzyme |
| CN107595893A (en) * | 2017-08-25 | 2018-01-19 | 广西中医药大学 | A kind of enzyme treated prepared slices of Chinese crude drugs and preparation method thereof |
| CN109078042A (en) * | 2018-08-14 | 2018-12-25 | 齐鲁工业大学 | A kind of method that compound bio enzymatic hydrolysis extracts active constituent in pueraria lobata |
-
2011
- 2011-12-28 CN CN2011104455411A patent/CN102492666A/en active Pending
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103789366A (en) * | 2014-02-21 | 2014-05-14 | 王健鹏 | Method for extracting puerarin through compound enzyme hydrolysis |
| CN103923737A (en) * | 2014-05-08 | 2014-07-16 | 龚意科 | Method for extracting maca flavone oiling agent from complex enzyme |
| CN106473995A (en) * | 2016-12-14 | 2017-03-08 | 杨攀 | The extracting method of Herba Bidentis Bipinnatae effective ingredient and its purposes preparing facial film |
| CN107245483A (en) * | 2017-07-19 | 2017-10-13 | 江苏农林职业技术学院 | A kind of complex enzyme for being used to extract kudzu root flavone and the method extracted using the complex enzyme |
| CN107595893A (en) * | 2017-08-25 | 2018-01-19 | 广西中医药大学 | A kind of enzyme treated prepared slices of Chinese crude drugs and preparation method thereof |
| CN109078042A (en) * | 2018-08-14 | 2018-12-25 | 齐鲁工业大学 | A kind of method that compound bio enzymatic hydrolysis extracts active constituent in pueraria lobata |
| CN109078042B (en) * | 2018-08-14 | 2022-05-31 | 齐鲁工业大学 | Method for extracting active ingredients from radix puerariae through compound biological enzymolysis |
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Application publication date: 20120613 |