CN104940280A - Method for extracting total flavones from radix puerariae employing enzyme preparation - Google Patents
Method for extracting total flavones from radix puerariae employing enzyme preparation Download PDFInfo
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Abstract
The invention relates to a method for extracting total flavones from radix puerariae employing an enzyme preparation. The enzyme preparation adopted by the method is prepared from cellulase, xylanase, protease and amylase. The method comprises the following steps: crushing and sieving dry radix puerariae, and dissolving with distilled water; adding an enzyme preparation, reacting for a period of time, and centrifuging; adding the distilled water, repeatedly leaching, and carrying out vacuum concentration to obtain a puerariae total flavones extract; and adsorbing and eluting the obtained puerariae flavone extract through HPD-600 macroporous resin, concentrating and drying to obtain the puerariae total flavones with the purity of over 95% finally. The method provided by the invention is mild in reaction conditions; the conformation of a natural product can be kept; the stereochemical structure and the biological activity are not destroyed; keeping of the original medicine effect of effective components is facilitated; impurities in a system can be removed; the clarity of an extract liquid is improved; the extraction rate of the effective component of the radix puerariae is improved; the reaction conditions are optimized and combined; and the maximization of the extraction yield is realized.
Description
Technical field
The invention belongs to the application of enzyme preparation, especially a kind of enzyme preparation extracts the method for Radix Puerariae total flavones.
Background technology
The dry root that Radix Puerariae (Radix puerariae) is legume pueraria lobata Pueraria lobata (Wiolld) Ohwi, its sweet in the mouth, pungent, property are put down.Radix Puerariae is a kind of Chinese medicine of China, medical value for Radix Puerariae is just on the books in Shennong's Herbal, its main component is starch, in addition containing have an appointment 12% flavone compound, comprise Semen sojae atricolor (Semen Glycines) glycoside, daidzein, puerarin etc. more than 10 and plant; And containing daucosterol, aminoacid, Coumarins etc.Oneself is regarded as the mountain region plant of medicine-food two-purpose to Radix Puerariae by Ministry of Public Health at present.
Radix Puerariae has that spasmolytic is brought down a fever, promoted the production of body fluid, the function such as rash, yang invigorating antidiarrheal.Similar with other most of flavone compound, Radix Puerariae flavone compounds also has multiple pharmacological function, as improved blood circulation, blood pressure lowering, blood sugar lowering, blood fat reducing, suppression arteriosclerosis, anti-arrhythmia, antiplatelet aggregation, antitumor, anti-hypoxia and antioxidation etc.Because of its significant medical value, oneself is widely used in the fields such as medical industry, food industry, daily-use chemical industry to present Radix Puerariae total flavones.
In recent years, natural plants active ingredient gains great popularity because of features such as its effect is obvious, natural low toxicities, has caused worldwide and has used natural herbs to carry out the upsurge of the various disease of prevention and therapy.Along with carrying out in a deep going way of studying natural plant crude drugs, the research range of Radix Puerariae has had further expansion, wherein the extracting flavonoids research of Radix Puerariae receives extensive concern, the main method of the extraction of current Radix Puerariae total flavones has: (1) Alcohol refluxing method, main using water, ethanol, alkaline alcohol, lime water, methanol, acetone etc. as solvent, reflux.Reflux, extract, 2 ~ 3 times, extracts Radix Puerariae total flavones; (2) alcohol percolation method; (3) infusion process; (4) using microwave-assisted; (5) enzymolysis circumfluence method; (6) ultrasonic counter-current extraction; (7) ion coordination extraction.
Enzyme engineering technology is in recent years for a biotechnology of essential industry.Leniently plant tissue is decomposed by enzyme reaction, the release accelerating effective ingredient is extracted, select corresponding enzyme the impurity affecting liquid preparation clarity can be removed as starch, protein, pectin etc. decompose, and for many containing liposoluble constituent in root, by glucosidase or transglycosylase, make liposoluble constituent change into water-soluble sugar glycoside, enzyme reaction Extracting temperature is low, can plant cell wall be destroyed, promote extracts active ingredients; Change the character of the object composition extracted, strengthen pharmaceutically active; Impurity in removal system, improves extraction system clarity, changes medical material quality.
The demand of market to Radix Puerariae flavone is increasing.But due to the deficiency of input in science and technology, domestic Radix Puerariae flavone preparation technique backwardness relatively, and the extracting method of Radix Puerariae flavone mostly is organic solvent extraction, production efficiency is low, energy consumption is large, environmental pollution is serious, impurity content is high, and product lacks the market competitiveness.Enzyme process is applied in the extraction of Radix Puerariae flavone, the method can carry out high selectivity conversion to effective ingredients in plant under mild conditions, not only can overcome the problems such as effective component extraction rate in alcohol water extracting method conventional in industry is low, complex procedures, improve extraction system clarity, change medical material quality, the structure of original natural component can also be changed in extraction, increase the physiologically active of extract, there is potential industrial application value.
Summary of the invention
The object of the invention is to overcome the problems such as effective component extraction rate in existing extracting method is low, complex procedures, provide a kind of and apply the method that enzyme preparation extracts Radix Puerariae total flavones.
The object of the invention is to be achieved through the following technical solutions:
(1) Radix Puerariae of drying is pulverized, sieve, get powder of Radix Puerariae and add distilled water dissolving;
(2) regulate pH to 4.0-6.0, add a certain amount of enzyme preparation, enzymolysis 1-3 hour at the temperature of 40-60 DEG C;
(3) high temperature enzyme denaturing;
(4) centrifugal, supernatant is for subsequent use, and precipitation adds distilled water 60 DEG C of hot dipping 10h of equivalent again, 2 times repeatedly.
(5) merge supernatant and filtrate, concentrating under reduced pressure is Radix Puerariae total flavones extractum.
(6) the Radix Puerariae flavone extractum water-soluble liquid obtained carries out adsorbing through HPD-600 macroporous resin, eluting.First wash away the water-solubility impurities such as saccharide, protein, tannin with distilled water during eluting, then use 60% ethanol elution instead, eluent is concentrated and reclaims ethanol, concentrated solution drying is obtained the Radix Puerariae flavone that purity is greater than 95%.
In described step (1), powder of Radix Puerariae is broken to 40-100 order, and Radix Puerariae is 1:10-30 with the solid-liquid ratio of the distilled water added.
The weight portion of step (2) enzyme preparation consists of: cellulase 1-5 part, xylanase 1-5 part, protease 1-5 part, amylase 1-5 part.Wherein protease selects at least one in papain, alkaline protease, neutral protease.Amylase selects at least one in mesophilicα-diastase and beta amylase.Enzyme preparation addition is the 0.1-1% of powder of Radix Puerariae addition
Enzyme-removal temperature described in step (3) is 80-100 DEG C, time 5-10min.
The condition of the HPD-600 macroporous resin described in step (6) is: blade diameter length ratio is 1:6, sample concentration 50mg/mL, and maximum applied sample amount is 3BV, and flow velocity is 1mL/min, eluting agent 5BV.
Advantage of the present invention and good effect are:
The present invention's application enzyme preparation extracts Radix Puerariae total flavones, and reaction condition is gentle, can keep the conformation of natural product, not destroy its stereochemical structure and biological activity, be conducive to the original drug effect of composition of remaining valid; Impurity in system can be removed, improve the clarity of extracting solution, improve the extraction ratio of kudzu vine root.
Detailed description of the invention
Below in conjunction with embodiment, the present invention is further described, and following embodiment is illustrative, is not determinate, can not limit protection scope of the present invention with following embodiment.
Instantiation of the present invention:
embodiment 1
(1) get dry powder of Radix Puerariae and be broken to 80 orders, get this powder 10g, add distilled water 100mL and dissolve;
(2) get enzyme preparation: cellulase 0.0037g, xylanase 0.0056g, papain 0.0018g, mesophilicα-diastase 0.0037g adds in Radix Puerariae solution, regulates pH to 4.0, enzymolysis 1 hour under 40 DEG C of conditions;
(3) 80 DEG C of enzyme denaturing 10min;
(4) centrifugal, precipitation adds 100mL distilled water, and 60 DEG C of heating extraction 10 hours are filtered, and repeatedly extract 2 times;
(5) merge supernatant and filtrate, concentrating under reduced pressure is Radix Puerariae total flavones extractum.Adopt spectrophotometry general flavone content, Radix Puerariae total flavones extraction ratio is 7.32%;
(6) the Radix Puerariae flavone extractum water-soluble liquid obtained carries out adsorbing through HPD-600 macroporous resin, eluting.The condition of HPD-600 macroporous resin is: blade diameter length ratio is 1:6, sample concentration 50mg/mL, and maximum applied sample amount is 3BV, and flow velocity is 1mL/min, eluting agent 5BV.First wash away the water-solubility impurities such as saccharide, protein, tannin with distilled water during eluting, then use 60% ethanol elution instead, eluent is concentrated and reclaims ethanol, concentrated solution is dry, adopt high effective liquid chromatography for measuring to obtain Radix Puerariae flavone purity and be greater than 95%.
embodiment 2
(1) get dry powder of Radix Puerariae and be broken to 80 orders, get this powder 10g, add distilled water 150mL and dissolve;
(2) get enzyme preparation: cellulase 0.0062g, xylanase 0.0093g, alkaline protease 0.0031g, mesophilicα-diastase 0.0062g, add in Radix Puerariae solution, regulate pH to 4.5, enzymolysis 2 hours under 45 DEG C of conditions;
(3) 85 DEG C of enzyme denaturing 10min;
(4) centrifugal, precipitation adds 150mL distilled water, and 60 DEG C of heating extraction 10 hours are filtered, and repeatedly extract 2 times;
(5) supernatant and filtrate is merged, concentrating under reduced pressure Radix Puerariae total flavones extractum.Adopt spectrophotometry general flavone content, Radix Puerariae total flavones extraction ratio is 7.48%;
(6) the Radix Puerariae flavone extractum water-soluble liquid obtained carries out adsorbing through HPD-600 macroporous resin, eluting.The condition of HPD-600 macroporous resin is: blade diameter length ratio is 1:6, sample concentration 50mg/mL, and maximum applied sample amount is 3BV, and flow velocity is 1mL/min, eluting agent 5BV.First wash away the water-solubility impurities such as saccharide, protein, tannin with distilled water during eluting, then use 60% ethanol elution instead, eluent is concentrated and reclaims ethanol, concentrated solution is dry, adopt high effective liquid chromatography for measuring to obtain Radix Puerariae flavone purity and be greater than 95%.
embodiment 3:
(1) get dry powder of Radix Puerariae and be broken to 100 orders, get this powder 15g, add distilled water 400ml and dissolve;
(2) get enzyme preparation: cellulase 0.0125g, xylanase 0.0375g, neutral protease 0.0325g, beta amylase 0.065g adds in Radix Puerariae solution, regulates pH to 5.0, enzymolysis 3 hours under 50 DEG C of conditions;
(3) 85 DEG C of enzyme denaturing 10min;
(4) centrifugal, precipitation adds 400mL distilled water, and 60 DEG C of heating extraction 10 hours are filtered, and repeatedly extract 2 times;
(5) merge supernatant and filtrate, concentrating under reduced pressure is Radix Puerariae total flavones extractum.Adopt spectrophotometry general flavone content, Radix Puerariae total flavones extraction ratio is 7.80%;
(6) the Radix Puerariae flavone extractum water-soluble liquid obtained carries out adsorbing through HPD-600 macroporous resin, eluting.The condition of HPD-600 macroporous resin is: blade diameter length ratio is 1:6, sample concentration 50mg/mL, and maximum applied sample amount is 3BV, and flow velocity is 1mL/min, eluting agent 5BV.First wash away the water-solubility impurities such as saccharide, protein, tannin with distilled water during eluting, then use 60% ethanol elution instead, eluent is concentrated and reclaims ethanol, concentrated solution is dry, adopt high effective liquid chromatography for measuring to obtain Radix Puerariae flavone purity and be greater than 95%.
embodiment 4
(1) get dry powder of Radix Puerariae and be broken to 80 orders, get this powder 10g, add distilled water 300 mL and dissolve;
(2) get enzyme preparation: cellulase 0.02g, xylanase 0.01g, neutral protease 0.02, mesophilicα-diastase 0.03g adds in Radix Puerariae solution, regulates pH to 5.0, enzymolysis 1.5 hours under 40 DEG C of conditions;
(3) 85 DEG C of enzyme denaturing 10min;
(4) centrifugal, precipitation adds 300mL distilled water, and 60 DEG C of heating extraction 10 hours are filtered, and repeatedly extract 2 times;
(5) merge supernatant and filtrate, concentrating under reduced pressure is Radix Puerariae total flavones extractum.Adopt spectrophotometry general flavone content, Radix Puerariae total flavones extraction ratio is 7.82%;
(6) the Radix Puerariae flavone extractum water-soluble liquid obtained carries out adsorbing through HPD-600 macroporous resin, eluting.The condition of HPD-600 macroporous resin is: blade diameter length ratio is 1:6, sample concentration 50mg/mL, and maximum applied sample amount is 3BV, and flow velocity is 1mL/min, eluting agent 5BV.First wash away the water-solubility impurities such as saccharide, protein, tannin with distilled water during eluting, then use 60% ethanol elution instead, eluent is concentrated and reclaims ethanol, concentrated solution is dry, adopt high effective liquid chromatography for measuring to obtain Radix Puerariae flavone purity and be greater than 95%.
embodiment 5:
(1) get dry powder of Radix Puerariae and be broken to 80 orders, get this powder 10g, add distilled water 100mL and dissolve;
(2) get enzyme preparation (cellulase: xylanase: papain: mesophilicα-diastase=2:2:3:1) 0.05g, regulate pH to 6.0, enzymolysis 1 hour under 60 DEG C of conditions;
(3) 85 DEG C of enzyme denaturing 10min;
(4) centrifugal, precipitation adds 100mL distilled water, and 60 DEG C of heating extraction 10 hours are filtered, and repeatedly extract 2 times;
(5) merge supernatant and filtrate, concentrating under reduced pressure is Radix Puerariae total flavones extractum.Adopt spectrophotometry general flavone content, Radix Puerariae total flavones extraction ratio is 8.60%;
(6) the Radix Puerariae flavone extractum water-soluble liquid obtained carries out adsorbing through HPD-600 macroporous resin, eluting.The condition of HPD-600 macroporous resin is: blade diameter length ratio is 1:6, sample concentration 50mg/mL, and maximum applied sample amount is 3BV, and flow velocity is 1mL/min, eluting agent 5BV.First wash away the water-solubility impurities such as saccharide, protein, tannin with distilled water during eluting, then use 60% ethanol elution instead, eluent is concentrated and reclaims ethanol, concentrated solution is dry, adopt high effective liquid chromatography for measuring to obtain Radix Puerariae flavone purity and be greater than 95%.
embodiment 6:
(1) get dry powder of Radix Puerariae and be broken to 100 orders, get this powder 10g, add distilled water 200mL and dissolve;
(2) get enzyme preparation (cellulase: xylanase: alkaline protease: mesophilicα-diastase=2:3:1:2) 0.08g, regulate pH to 4.5, enzymolysis 2 hours under 45 DEG C of conditions;
(3) 90 DEG C of enzyme denaturing 10min;
(4) centrifugal, precipitation adds 200mL distilled water, and 60 DEG C of heating extraction 10 hours are filtered, and repeatedly extract 2 times;
(5) merge supernatant and filtrate, concentrating under reduced pressure is Radix Puerariae total flavones extractum.Adopt spectrophotometry general flavone content, Radix Puerariae total flavones extraction ratio is 7.57%;
(6) the Radix Puerariae flavone extractum water-soluble liquid obtained carries out adsorbing through HPD-600 macroporous resin, eluting.The condition of HPD-600 macroporous resin is: blade diameter length ratio is 1:6, sample concentration 50mg/mL, and maximum applied sample amount is 3BV, and flow velocity is 1mL/min, eluting agent 5BV.First wash away the water-solubility impurities such as saccharide, protein, tannin with distilled water during eluting, then use 60% ethanol elution instead, eluent is concentrated and reclaims ethanol, concentrated solution is dry, adopt high effective liquid chromatography for measuring to obtain Radix Puerariae flavone purity and be greater than 95%.
embodiment 7:
(1) get dry powder of Radix Puerariae and be broken to 80 orders, get this powder 10g, add distilled water 200ml and dissolve; ;
(2) get enzyme preparation (cellulase: xylanase: neutral protease: mesophilicα-diastase=1:2:2:5) 0.1g, regulate pH to 4.5, enzymolysis 3 hours under 45 DEG C of conditions;
(3) 85 DEG C of enzyme denaturing 10min;
(4) centrifugal, precipitation adds 200mL distilled water, and 60 DEG C of heating extraction 10 hours are filtered, and repeatedly extract 2 times;
(5) merge supernatant and filtrate, concentrating under reduced pressure is Radix Puerariae total flavones extractum.Adopt spectrophotometry general flavone content, Radix Puerariae total flavones extraction ratio is 7.75%;
(6) the Radix Puerariae flavone extractum water-soluble liquid obtained carries out adsorbing through HPD-600 macroporous resin, eluting.The condition of HPD-600 macroporous resin is: blade diameter length ratio is 1:6, sample concentration 50mg/mL, and maximum applied sample amount is 3BV, and flow velocity is 1mL/min, eluting agent 5BV.First wash away the water-solubility impurities such as saccharide, protein, tannin with distilled water during eluting, then use 60% ethanol elution instead, eluent is concentrated and reclaims ethanol, concentrated solution is dry, adopt high effective liquid chromatography for measuring to obtain Radix Puerariae flavone purity and be greater than 95%.
embodiment 8:
(1) get dry powder of Radix Puerariae and be broken to 80 orders, get this powder 10g, add distilled water 150ml and dissolve;
(2) get enzyme preparation (cellulase: xylanase: neutral protease: beta amylase=1:1:3:2) 0.22g, regulate pH to 5.0, enzymolysis 3 hours under 60 DEG C of conditions;
(3) 100 DEG C of enzyme denaturing 10min;
(4) centrifugal, precipitation adds 150mL distilled water, and 60 DEG C of heating extraction 10 hours are filtered, and repeatedly extract 2 times;
(5) merge supernatant and filtrate, concentrating under reduced pressure is Radix Puerariae total flavones extractum.Adopt spectrophotometry general flavone content, Radix Puerariae total flavones extraction ratio is 7.27%;
(6) the Radix Puerariae flavone extractum water-soluble liquid obtained carries out adsorbing through HPD-600 macroporous resin, eluting.The condition of HPD-600 macroporous resin is: blade diameter length ratio is 1:6, sample concentration 50mg/mL, and maximum applied sample amount is 3BV, and flow velocity is 1mL/min, eluting agent 5BV.First wash away the water-solubility impurities such as saccharide, protein, tannin with distilled water during eluting, then use 60% ethanol elution instead, eluent is concentrated and reclaims ethanol, concentrated solution is dry, adopt high effective liquid chromatography for measuring to obtain Radix Puerariae flavone purity and be greater than 95%.
Claims (6)
1. apply the method that enzyme preparation extracts Radix Puerariae total flavones, it is characterized in that:
Step is:
(1) Radix Puerariae of drying is pulverized, sieve, get powder of Radix Puerariae and add distilled water dissolving;
(2) regulate pH to 4.0-6.0, add a certain amount of enzyme preparation, enzymolysis 1-3 hour at the temperature of 40-60 DEG C;
(3) high temperature enzyme denaturing;
(4) centrifugal, supernatant is for subsequent use, and precipitation adds distilled water 60 DEG C of hot dipping 10h of equivalent again, 2 times repeatedly.
(5) merge supernatant and filtrate, concentrating under reduced pressure is Radix Puerariae total flavones extractum.
(6) the Radix Puerariae flavone extractum water-soluble liquid obtained carries out adsorbing through HPD-600 macroporous resin, eluting.First wash away the water-solubility impurities such as saccharide, protein, tannin with distilled water during eluting, then use 60% ethanol elution instead, eluent is concentrated and reclaims ethanol, concentrated solution drying is obtained the Radix Puerariae flavone that purity is greater than 95%.
2. application enzyme preparation according to claim 1 extracts the method for Radix Puerariae total flavones, it is characterized in that: in described step (1), powder of Radix Puerariae is broken to 40-100 order, and Radix Puerariae is 1:10-30 with the solid-liquid ratio of the distilled water added.
3. application enzyme preparation according to claim 1 extracts the method for Radix Puerariae total flavones, it is characterized in that: the weight portion of described step (2) enzyme preparation consists of: cellulase 1-5 part, xylanase 1-5 part, protease 1-5 part, amylase 1-5 part.Wherein protease selects at least one in papain, alkaline protease, neutral protease.Amylase selects at least one in mesophilicα-diastase and beta amylase.
4. application enzyme preparation according to claim 1 extracts the method for Radix Puerariae total flavones, it is characterized in that: in described step (2), enzyme preparation addition is the 0.1-1% of powder of Radix Puerariae addition.
5. application enzyme preparation according to claim 1 extracts the method for Radix Puerariae total flavones, it is characterized in that: in described step (3), enzyme-removal temperature is 80-100 DEG C, time 5-10min.
6. application enzyme preparation according to claim 1 extracts the method for Radix Puerariae total flavones, it is characterized in that: in described step (6), the condition of HPD-600 macroporous resin is: blade diameter length ratio is 1:6, sample concentration 50mg/mL, maximum applied sample amount is 3BV, flow velocity is 1mL/min, eluting agent 5BV.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106491451A (en) * | 2016-12-14 | 2017-03-15 | 杨攀 | The extracting method of root of Broussonetiapapyrifera(I)vent effective ingredient and its prepare the purposes of cosmetics |
| CN107156864A (en) * | 2017-04-26 | 2017-09-15 | 湖北省农业科学院中药材研究所 | A kind of method that water-soluble fibre is prepared from root of kudzu vine waste material |
| CN109315657A (en) * | 2018-09-29 | 2019-02-12 | 广州天天有财付网络科技有限公司 | A kind of pueraria lobata mulse and preparation method thereof |
| CN112384234A (en) * | 2019-01-31 | 2021-02-19 | 邦泰生物工程(深圳)有限公司 | A method for preparing radix Puerariae extract from radix Puerariae leftover |
| CN112402482A (en) * | 2020-12-07 | 2021-02-26 | 北京美康堂医药科技有限公司 | Method for extracting and purifying flavonoid components in traditional Chinese medicinal materials |
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2014
- 2014-03-24 CN CN201410110802.8A patent/CN104940280A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106491451A (en) * | 2016-12-14 | 2017-03-15 | 杨攀 | The extracting method of root of Broussonetiapapyrifera(I)vent effective ingredient and its prepare the purposes of cosmetics |
| CN107156864A (en) * | 2017-04-26 | 2017-09-15 | 湖北省农业科学院中药材研究所 | A kind of method that water-soluble fibre is prepared from root of kudzu vine waste material |
| CN109315657A (en) * | 2018-09-29 | 2019-02-12 | 广州天天有财付网络科技有限公司 | A kind of pueraria lobata mulse and preparation method thereof |
| CN112384234A (en) * | 2019-01-31 | 2021-02-19 | 邦泰生物工程(深圳)有限公司 | A method for preparing radix Puerariae extract from radix Puerariae leftover |
| CN112402482A (en) * | 2020-12-07 | 2021-02-26 | 北京美康堂医药科技有限公司 | Method for extracting and purifying flavonoid components in traditional Chinese medicinal materials |
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