CN101701030A - Method for extracting toosendanin by ultrasonic field assistant enzyme method - Google Patents
Method for extracting toosendanin by ultrasonic field assistant enzyme method Download PDFInfo
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- CN101701030A CN101701030A CN200910185008A CN200910185008A CN101701030A CN 101701030 A CN101701030 A CN 101701030A CN 200910185008 A CN200910185008 A CN 200910185008A CN 200910185008 A CN200910185008 A CN 200910185008A CN 101701030 A CN101701030 A CN 101701030A
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Abstract
The invention disclose a method for extracting toosendanin by a ultrasonic field assistant enzyme method, which takes pulverized barks, seeds, leaves or roots of bead tree as extracted matter and is characterized that the operational process is as follows: adding extracted matter into extracting solution; then adding biological enzyme into the extracting solution and then placing in the ultrasonic field; finishing ultrasonic enzyme in the ultrasonic field to obtain enzyme solution, sequentially inactivating, centrifuging, filtering, extracting, separating, evaporating and drying the enzyme solution to obtain crystal containing effective constituent toosendanin. The method of the invention has cheap and easily obtained raw materials, simple process, no need of organic reagent usage and low extraction cost.
Description
Technical field
The present invention relates to natural product exploitation and biological technology application, particularly biological enzyme and extract the method and the technology of natural product Kuliansu.
Background technology
Kuliansu (toosendanin) is the tetracyclic triterpenes material that extracts from natural phant China tree or melia toosendan, has certain medical treatment effect, and good broad spectrum disinfestation anti-microbial property arranged, harmless to human body, be easy to degraded, can be used for preventing and treating diseases and pests of agronomic crop, be a kind of botanical pesticide that has potentiality.The Meliaceae effective plant component extracting process comprises solvent extraction method, supercritical fluid extraction and ultrasonic field assistance extraction method, microwave assistant extraction method or the like.
Because Kuliansu polarity is medium, and impurity is more in the bark of Melia azedarach L. or the seeds of trees, very difficult with the method for solvent lixiviate with its whole propositions, and solvent consumption is excessive, the extraction process very complicated; Supercritical carbon dioxide extraction method is suitable under the normal temperature extracting the thermo-sensitivity composition, but the extraction cost height, the equipment complexity, and the material that active component content is lower in, the plant not high to purity requirements such as Kuliansus also is not suitable for; Ultrasonic field assists mixed solvent to extract, and can effectively improve the penetrativity of lixiviate material, improves extraction yield, but consumes more organic solvent, the extraction process very complicated; Microwave can to inside and outside the extractive substance simultaneously evenly, heating promptly, can improve extraction efficiency, but not be suitable for the extraction of thermo-sensitivity composition, and difficultly realize serialization production.
The action principle of enzymolysis process is the cavatition that the high efficiency, specificity and the ultrasonic place that utilize biological enzyme to have has, moiety hydrolysis or degraded with cell walls, destroy cell wall structure, make effective constituent Kuliansu emigrated cell quickly, and dissolving, suspendible or peptization rapidly and efficiently extract a kind of novel extracting method of effective constituent purpose in the cell thereby reach in solvent.Ultrasonic assistance enzymolysis process extract have need not with an organic solvent, reaction conditions gentleness, action time short, product production advantages of higher.Do not see as yet that so far relevant biologic enzymolysis method extracts the effective constituent of Meliaceae plant, especially enzymolysis process extracts the research report of Kuliansu.
Summary of the invention
The method that the purpose of this invention is to provide a kind of extracting toosendanin by ultrasonic field assistant enzyme method is skin, seed, leaf or the root from margosa, assists enzymolysis process extracting effective components Kuliansu with ultrasonic field.Utilize efficient, the specificity of biological enzyme, destroy cell wall structure, under ultrasonic field is assisted, make effective constituent Kuliansu emigrated cell faster, and dissolving, suspendible or peptization are in solution.For Kuliansu provides a kind of new, green method and technology, and the application of having expanded biotechnology.
The characteristics of the method for extracting toosendanin by ultrasonic field assistant enzyme method of the present invention are to operate according to the following procedure:
Carried thing with margosa bark, seed, leaf or the root pulverized, the described thing of being carried is added in the extracting solution; Place ultrasonic field after in described extracting solution, adding biological enzyme; In described ultrasonic field, finish behind the ultrasonic enzymolysis enzymolysis solution, described enzymolysis solution passes through deactivation, centrifugal, filtration, extracting and separating, evaporation drying successively, promptly gets the crystal that contains the effective constituent Kuliansu.
The feature of the inventive method also is:
Described extracting solution is a kind of or multiple solution of blended or buffered soln, and the solvent of regulating described extracting liquid pH value is hydrochloric acid, sulfuric acid or acetum or damping fluid.
Described biological enzyme is a kind of or blended plurality of enzymes preparation or enzyme powder; Described biological enzyme is cellulase, hemicellulase, polygalacturonase, proteolytic enzyme, driselase or the macerozyme that the pair cell wall has Degradation.
The consumption of described biological enzyme is 0.1mgml
-1～10mgml
-1, the temperature of described ultrasonic enzymolysis is 20～80 ℃, described ultrasonic enzymolysis is to finish in 1～12 hour under steady temperature.
The described thing of being carried is pulverized by 10～100 purpose granularity requirements, and the described mass volume ratio of being carried thing and extracting solution is 1: 5～1: 100gml
-1, the pH of described extracting solution is 3～6.5.
Extraction agent in the described extracting and separating is one or more in propyl carbinol, ethyl acetate and the trichloromethane.
The temperature of described evaporation drying is set to 30～120 ℃.
Described ultrasonic field is built-in or external, and the ultrasonic power of described ultrasonic field is 10W～400W, and ultrasonic frequency is 10KHz～100KHz.
Compared with the prior art, beneficial effect of the present invention is embodied in:
1, the present invention adopts ultrasonic field to assist enzymolysis process, has utilized high efficiency, the specificity of biological enzyme, can effectively crack the China tree cell walls, makes the acceleration stripping of effective constituent under ultrasonication, realizes high-level efficiency, the low-cost extraction.
2, under the identical extraction conditions, the extraction yield of the inventive method has improved more than 2 times even 2 times than conventional solvent extraction, and extraction effect is obvious.
3, be more than 1.5% through Kuliansu extraction yield after the inventive method; After separating postmenstruation, the purity of products obtained therefrom reaches more than 85%.
Description of drawings
Fig. 1 extracts the high-efficient liquid phase chromatogram of Kuliansu crude product for the inventive method.
Fig. 2 extracts the infrared spectrogram of Kuliansu crude product for the inventive method.
Below pass through embodiment, and the invention will be further described in conjunction with the accompanying drawings.
Embodiment
Embodiment 1:
Dried bark of Melia azedarach L. is crushed to 60 orders, gets the tool plug triangular flask that 5g places 250ml, add 100ml pH and be 5, temperature is 50 ℃ acetum, adds biochemical level cellulase 0.02g, enzyme work 〉=20000ug
-1, be that 40W, frequency are in the 28KHz supersound extraction device at power, temperature is 45 ℃ of constant temperature enzymolysis 2 hours; After enzymolysis finished, heating made enzyme deactivation in 5 minutes in boiling water; Filter after centrifugal 10 minutes with 4000rpm; Filtrate is used 50ml n-butanol extraction 24 hours, repeats twice, combining extraction liquid; The reduction vaporization drying obtains the pale yellow powder product.Because this composition is not volatile, reduction vaporization is in order to accelerate rate of drying, as long as pressure is lower than barometric point, the boiling point that temperature is lower than propyl carbinol under this pressure all can.
Extract after testing, the extraction yield of Kuliansu is 1.74%.
In the present embodiment, the solution before extracting, promptly extracting solution is the slightly acidic acetum, and enzymolysis need just can carry out under the certain pH environment, and the liquid that extracts behind the enzymolysis after the effective constituent is enzymolysis solution.
Embodiment 2:
The dried China tree seeds of trees are crushed to 60 orders, get 5g and place 250ml tool plug triangular flask, adding 100ml pH is 4.5, and temperature is 50 ℃ a hydrochloric acid soln, adds biochemical level hemicellulase 0.02g, enzyme work 〉=2000ug
-1, be that 60W, frequency are in the 40KHz supersound extraction device at power, temperature is 45 ℃ of constant temperature enzymolysis 2 hours; After enzymolysis finished, heating made enzyme deactivation in 5 minutes in boiling water; Filter after centrifugal 10 minutes with 4000rpm; Filtrate repeats twice, combining extraction liquid with 50ml n-butanol extraction 24h; The reduction vaporization drying obtains the pale yellow powder product.
Extract after testing, the extraction yield of Kuliansu is 1.855%.
Embodiment 3:
Dried bark of Melia azedarach L. is crushed to 40 orders, gets 3g and place 250ml tool plug triangular flask, adding 100ml pH is 4.5, and temperature is 50 ℃ a hydrochloric acid soln, adds biochemical stage pectase 0.02g, enzyme work 〉=30000ug
-1, be that 80W, frequency are in the 45KHz supersound extraction device at power, temperature is 50 ℃ of constant temperature enzymolysis 2.5h; After enzymolysis finished, heating 5min made enzyme deactivation in boiling water; To filter behind the centrifugal 10min of 6000rpm; Filtrate is used 50ml n-butanol extraction 24 hours, repeats twice, combining extraction liquid; The reduction vaporization drying obtains the pale yellow powder product.
Extract after testing, the extraction yield of Kuliansu is 2.085%.
Embodiment 4:
Dried bark of Melia azedarach L. is crushed to 40 orders, gets 3g and place 250ml tool plug triangular flask, adding 100ml pH is 4.5, and temperature is 50 ℃ a sulphuric acid soln, adds biochemical stage pectase, each 0.01g of hemicellulase, and enzyme is lived all 〉=20000ug
-1, be that 120W, frequency are in the 48KHz supersound extraction device at power, temperature is 50 ℃ of constant temperature enzymolysis 2 hours; After enzymolysis finished, heating made enzyme deactivation in 5 minutes in boiling water; Filter after centrifugal 10 minutes with 6000rpm; Filtrate repeats twice, combining extraction liquid with 50ml n-butanol extraction 24h; The reduction vaporization drying obtains the pale yellow powder product.
Extract after testing, the extraction yield of Kuliansu is 2.140%.
Embodiment 5:
Dried bark of Melia azedarach L. is crushed to 40 orders, get 4g and place 250ml tool plug triangular flask, adding 100ml pH is 4.5, and temperature is 50 ℃ acetic acid one sodium-acetate buffer, add biochemical level cellulase, polygalacturonase, each 0.01g of hemicellulase, enzyme is lived all 〉=20000ug
-1, be that 200W, frequency are in the 28KHz supersound extraction device at power, temperature is 50 ℃ of constant temperature enzymolysis 2h; After enzymolysis finished, heating 5min made enzyme deactivation in boiling water; Filter after centrifugal 10 minutes with 6000rpm; Filtrate is used 50ml n-butanol extraction 24 hours, repeats twice, combining extraction liquid; The reduction vaporization drying obtains the pale yellow powder product.
Extract after testing, the extraction yield of Kuliansu is 2.213%.
Embodiment 6:
Dried bark of Melia azedarach L. is crushed to 80 orders, gets 4g and place 250ml tool plug triangular flask, adding 100ml pH is 5, and temperature is 50 ℃ a phosphate buffered saline buffer, adds biochemical level cellulase, each 0.01g of driselase, and enzyme work is 〉=20000ug
-1, be that 200W, frequency are in the 40KHz supersound extraction device at power, temperature is 50 ℃ of constant temperature enzymolysis 2h; After enzymolysis finished, heating 5min made enzyme deactivation in boiling water; Filter after centrifugal 10 minutes with 6000rpm; Filtrate is used 50ml n-butanol extraction 24 hours, repeats twice, combining extraction liquid; The reduction vaporization drying obtains the pale yellow powder product.
Extract after testing, the extraction yield of Kuliansu is 2.313%.
Figure 1 shows that the high-efficient liquid phase chromatogram of the inventive method extraction Kuliansu crude product.
Figure 2 shows that the infrared spectrogram of the inventive method extraction Kuliansu crude product.
Claims (8)
1. the method for extracting toosendanin by ultrasonic field assistant enzyme method is characterized in that operating according to the following procedure:
Carried thing with margosa bark, seed, leaf or the root pulverized, the described thing of being carried is added in the extracting solution; Place ultrasonic field after in described extracting solution, adding biological enzyme; In described ultrasonic field, finish behind the ultrasonic enzymolysis enzymolysis solution, described enzymolysis solution passes through deactivation, centrifugal, filtration, extracting and separating, evaporation, drying successively, promptly gets the crystal that contains the effective constituent Kuliansu.
2. the method for extracting toosendanin by ultrasonic field assistant enzyme method according to claim 1, it is characterized in that described extracting solution is a kind of or multiple solution of blended or buffered soln, the solvent that is used to regulate described extracting liquid pH value is hydrochloric acid, sulfuric acid or acetum or damping fluid.
3. the method for extracting toosendanin by ultrasonic field assistant enzyme method according to claim 1 is characterized in that described biological enzyme is a kind of or blended plurality of enzymes preparation or enzyme powder; Described biological enzyme is cellulase, hemicellulase, polygalacturonase, proteolytic enzyme, driselase or the macerozyme that the pair cell wall has Degradation.
4. according to the method for claim 1 or 3 described extracting toosendanin by ultrasonic field assistant enzyme method, the consumption that it is characterized in that described biological enzyme is 0.1mgml
-1～10mgml
-1, the temperature of described ultrasonic enzymolysis is 20～80 ℃, described ultrasonic enzymolysis is to finish in 1～12 hour under steady temperature.
5. the method for extracting toosendanin by ultrasonic field assistant enzyme method according to claim 1 is characterized in that the described thing of being carried pulverizes by 10～100 purpose granularity requirements, and the described mass volume ratio of being carried thing and extracting solution is 1: 5～1: 100gml
-1, the pH of described extracting solution is 3～6.5.
6. the method for extracting toosendanin by ultrasonic field assistant enzyme method according to claim 1 is characterized in that extraction agent in the described extracting and separating is one or more in propyl carbinol, ethyl acetate and the trichloromethane.
7. the method for extracting toosendanin by ultrasonic field assistant enzyme method according to claim 1 is characterized in that the temperature of described evaporation drying is set to 30～120 ℃.
8. the method for extracting toosendanin by ultrasonic field assistant enzyme method according to claim 1 is characterized in that described ultrasonic field is built-in or external, and the ultrasonic power of described ultrasonic field is 10W～400W, and ultrasonic frequency is 10KHz～100KHz.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107488515A (en) * | 2017-09-30 | 2017-12-19 | 江苏农林职业技术学院 | A kind of extract solution and the method using extract solution extraction essential oil of the flower of Camellia sinensis |
| CN107722103A (en) * | 2017-10-24 | 2018-02-23 | 江苏金太阳纺织科技股份有限公司 | A kind of extracting method of margosine |
| CN108653395A (en) * | 2018-06-21 | 2018-10-16 | 南京林业大学 | A kind of artificial forest bark active constituent Coupled |
| CN108704002A (en) * | 2018-06-21 | 2018-10-26 | 南京林业大学 | A kind of artificial forest bark active constituent enzymatic treatment assists Coupled with ultrasonic wave |
| CN108836994A (en) * | 2018-07-25 | 2018-11-20 | 南京林业大学 | A kind of extracting method of artificial forest bark saponin(e active component |
-
2009
- 2009-10-28 CN CN200910185008A patent/CN101701030A/en active Pending
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107488515A (en) * | 2017-09-30 | 2017-12-19 | 江苏农林职业技术学院 | A kind of extract solution and the method using extract solution extraction essential oil of the flower of Camellia sinensis |
| CN107488515B (en) * | 2017-09-30 | 2020-03-31 | 江苏农林职业技术学院 | Extracting solution and method for extracting tea tree flower essential oil by using same |
| CN107722103A (en) * | 2017-10-24 | 2018-02-23 | 江苏金太阳纺织科技股份有限公司 | A kind of extracting method of margosine |
| WO2019080571A1 (en) * | 2017-10-24 | 2019-05-02 | 江苏金太阳纺织科技股份有限公司 | Method for extracting toosendanin |
| CN108653395A (en) * | 2018-06-21 | 2018-10-16 | 南京林业大学 | A kind of artificial forest bark active constituent Coupled |
| CN108704002A (en) * | 2018-06-21 | 2018-10-26 | 南京林业大学 | A kind of artificial forest bark active constituent enzymatic treatment assists Coupled with ultrasonic wave |
| CN108653395B (en) * | 2018-06-21 | 2020-12-22 | 南京林业大学 | Coupling extraction method for active ingredients of bark of artificial forest |
| CN108704002B (en) * | 2018-06-21 | 2021-05-11 | 南京林业大学 | Artificial forest bark active ingredient enzyme treatment and ultrasonic-assisted coupling extraction method |
| CN108836994A (en) * | 2018-07-25 | 2018-11-20 | 南京林业大学 | A kind of extracting method of artificial forest bark saponin(e active component |
| CN108836994B (en) * | 2018-07-25 | 2021-06-18 | 南京林业大学 | Method for extracting saponins active ingredients from bark of artificial forest |
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Open date: 20100505 |