CN102944526A - Method for detecting content difference of aspartyl-enzyme of asparagus plant - Google Patents
Method for detecting content difference of aspartyl-enzyme of asparagus plant Download PDFInfo
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- CN102944526A CN102944526A CN2012105009037A CN201210500903A CN102944526A CN 102944526 A CN102944526 A CN 102944526A CN 2012105009037 A CN2012105009037 A CN 2012105009037A CN 201210500903 A CN201210500903 A CN 201210500903A CN 102944526 A CN102944526 A CN 102944526A
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Abstract
The invention discloses a method for detecting the content difference of aspartyl-enzyme of an asparagus plant. The method comprises the following steps of: extracting the asparagus plant; taking the root, the stem and leaves of the plant to be used as samples; respectively preparing sample solutions by using the samples; carrying out colorimetric analysis on the sample solutions with reference substance solutions by using a colorimetric method; and calculating optical absorbance values and comparing so as to obtain the difference of the contents of aspartyl-enzyme in different parts of the asparagus plant. By utilizing the method, the root, the stem and the leaves of the asparagus plant are used for preparing the sample solutions and are subjected to the colorimetric analysis by using the colorimetric method, and the difference of the contents of the aspartyl-enzyme at the different parts of the asparagus plant can be accurately compared, so that the accurate basis is provided for extracting the aspartyl-enzyme by using the asparagus plant, the efficiency for extracting the aspartyl-enzyme from the asparagus plant is greatly improved, and the problem that the aspartyl-enzyme is short in market is relieved.
Description
Technical field
The present invention relates to the detection method of the N enzyme content difference of a kind of asparagus plant.
Background technology
Asparagus, unique flavor, nutritious more has anti-cancer, anticancer magical effect, have the laudatory title of " vegetables king ".This mainly is because it contains the bioactivator of the health-care effects such as multiple balance the body physiological function, Chronic disease prevention generation.Contained asparaginase in the asparagus all has certain curative effect to breast cancer, lung cancer, the cancer of the esophagus, carcinoma of urinary bladder, cutaneum carcinoma, cancer of the stomach, the carcinoma of the rectum etc.But asparaginase in the market all is the L-ASP by preparations such as Escherichia coli (E.coli) and owen bacterias (Erwinia), but to the difference of asparaginase content in the how to confirm plant, at present there are no the report of related detecting method.
Summary of the invention
The technical problem to be solved in the present invention provides the detection method of the N enzyme content difference of a kind of asparagus plant, can detect the difference of the asparaginase content of asparagus plant different parts, not have at present method can detect the problem of the N enzyme content difference of asparagus thereby solve.
Solve the problems of the technologies described above by the following technical programs:
The embodiment of the invention provides the detection method of N enzyme content difference in a kind of asparagus, comprising:
Extract the asparagus plant, get root, stem and the blade of described plant as sample, make respectively sample solution, described sample solution is carried out colorimetric analysis with reference substance solution by colourimetry respectively, relatively obtain the difference of the N enzyme content at each position of asparagus by calculating absorbance value.
The method that the embodiment of the invention provides, by root, stem and the blade of asparagus plant are made sample solution, utilize colourimetry to carry out colorimetric analysis, can accurately compare the difference of the N enzyme content at each position of asparagus plant, thereby provide accurately foundation for utilizing the asparagus plant to extract the N enzyme, can greatly improve the efficient of from asparagus, extracting the N enzyme, alleviate the N enzyme market problem that supply falls short of demand.
Description of drawings
In order to be illustrated more clearly in the technical scheme of the embodiment of the invention, the accompanying drawing of required use was done to introduce simply during the below will describe embodiment, apparently, accompanying drawing in the following describes only is some embodiments of the present invention, for those of ordinary skill in the art, under the prerequisite of not paying creative work, can also obtain other accompanying drawings according to these accompanying drawings.
Fig. 1 detects N enzymatic activity (U/ml) synoptic diagram in the asparagus root for the method that the embodiment of the invention provides;
Fig. 2 detects N enzymatic activity (U/ml) synoptic diagram in the Asparagus Officinalis L for the method that the embodiment of the invention provides;
Fig. 3 detects N enzymatic activity (U/ml) synoptic diagram in the asparagus leaf for the method that the embodiment of the invention provides.
Embodiment
The below is clearly and completely described the technical scheme in the embodiment of the invention, and obviously, described embodiment only is the present invention's part embodiment, rather than whole embodiment.Based on embodiments of the invention, those of ordinary skills belong to protection scope of the present invention not making the every other embodiment that obtains under the creative work prerequisite.
The embodiment of the invention provides the detection method of the N enzyme content difference of a kind of asparagus plant, may further comprise the steps:
Extract the asparagus plant, (stem can be to get from the long 20cm in stem top to bottom to get root, the stem of described plant, the material of base portion mean diameter 0.5～1.2cm, can be the tender stem that often refers in the industry) and blade as sample, make respectively sample solution, described sample solution is carried out colorimetric analysis with reference substance solution by colourimetry respectively, relatively obtain the difference of the N enzyme content at each position of asparagus by calculating absorbance value.
Further, in the said method, extract before the asparagus plant, also comprise: the asparagus plant is carried out the step that male and female are identified, can detect like this difference of N enzyme content at each position of the asparagus plant of male and female plant.
Further, in the said method, extract before the asparagus plant, also comprise the step that asparagus plant kind is identified, can detect like this difference of N enzyme content at each position of the asparagus plant of different cultivars.Also can carry out simultaneously to the asparagus plant evaluation of kind and male and female plant.
In the said method, make in the following manner sample solution:
Sample (root, stem and the blade obtained from the asparagus plant) is washed, and baking was killed enzyme in 15 minutes under 105 ℃ temperature, oven dry under 70 ℃ temperature, pulverize, sieving with 20 mesh sieves obtains sample dry powder;
Take by weighing the described sample dry powder of 1.5g, be positioned in the apparatus,Soxhlet's behind the filter paper parcel, use absolute ether heating and refluxing extraction 1 hour, diethyl ether solution inclines; After filter paper packet volatilizes ether, place apparatus,Soxhlet's, added the methyl alcohol heating and refluxing extraction 6 hours, behind the recovery methyl alcohol, be transferred to the 100ml measuring bottle, and arrive scale with methanol constant volume, shake up;
Draw sample solution 1ml, put in the 10ml measuring bottle, add the AlCl of mass concentration 5%
3Methanol solution 1ml is diluted to scale with methyl alcohol, shakes up, and leaves standstill 10 minutes, namely obtains sample solution.
Reference substance solution in the said method makes in the following manner, comprising:
Described samples with water is cleaned, and baking was killed enzyme in 15 minutes under 105 ℃ temperature, oven dry under 70 ℃ temperature, pulverize, sieving with 20 mesh sieves obtains sample dry powder;
Take by weighing the described sample dry powder of 1.5g, be positioned in the apparatus,Soxhlet's behind the filter paper parcel, use absolute ether heating and refluxing extraction 1 hour, diethyl ether solution inclines; After filter paper packet volatilizes ether, place apparatus,Soxhlet's, added the methyl alcohol heating and refluxing extraction 6 hours, behind the recovery methyl alcohol, be transferred to the 100ml measuring bottle, and arrive scale with methanol constant volume, shake up;
Draw sample solution 1ml, place the 10ml measuring bottle, be diluted to scale with methyl alcohol, shake up, left standstill 10 minutes, namely obtain reference substance solution.
In the said method, the asparagus plant of extraction is adopted fresh asparagus plant, is no more than 1 hour asparagus plant as adopting plucking time.Can guarantee like this to be fresh as root, stem, the blade of the asparagus plant of sample.
Utilize the method for the embodiment of the invention, the activity that can compare asparaginase in the root, stem, leaf of different cultivars asparagus male and female plant, thereby find the significant difference of asparagus male and female plant aspect bioactivator of different cultivars, provide foundation for from the asparagus plant, extracting the plant source asparaginase.
Below in conjunction with specific embodiment said method is described further.
Concrete steps are: the evaluation of experimental cultivar breeding cultivation → male and female plant → extraction test sample → data analysis, wherein,
(1) breeding cultivation of experimental cultivar:
Select the asparagus kind of ten kinds of common high-qualitys, in the Vegetable Base asparagus garden plantation in the agriculture garden of Changping District, Beijing gold six rings in 2000;
(2) evaluation of male and female plant:
April in 2005, by strain design test Tanaka asparagus plant male and female sex, and make a mark, represent with A1～A10 respectively;
(3) extract test sample:
After the male and female that identify the asparagus plant, randomly draw plant as for the examination material, gather respectively afterwards their stem, blade, root as test sample; As (the thick following root system of fresh mature leaf and 2mm is as sample for the long 20cm of being of stem, base portion mean diameter 0.5～1.2cm) to gather 5 years seedling stems of asparagus;
(3.1) preparation sample solution:
Clean with the tap water flushing, baking 15min kills enzyme under 105 ℃ condition, and it is for subsequent use that dry under 70 ℃ the condition, pulverize, sieve (20 order) obtains dry powder;
Precision takes by weighing the dry powder 1.5g of leaf, stem and the root of asparagus plant, and the filter paper parcel is placed in the apparatus,Soxhlet's, and with absolute ether heating and refluxing extraction 1h, diethyl ether solution inclines; After filter paper packet volatilizes ether, place apparatus,Soxhlet's, add methyl alcohol heating and refluxing extraction 6h, behind the recovery methyl alcohol, be transferred to the 100ml measuring bottle, and arrive scale with methanol constant volume, shake up; The accurate sample solution 1ml that draws puts in the 10ml measuring bottle, adds concentration 5%AlCl
3Methanol solution 1ml is diluted to scale with methyl alcohol, shakes up, and leaves standstill 10min, namely obtains sample solution;
(3.2) reference substance solution preparation is not to add AlCl
3Sample solution product solution in contrast;
(4) determination method:
Adopt colourimetry, generate the principle of asparatate and ammonia according to the hydrolysis of asparagine enzymatic asparagine.
Asparagine+H
2O → asparatate+NH
3The ammonia that generates and Nessler's reagent reaction produce red complex, with the growing amount of colorimetric method for determining ammonia to represent the activity of enzyme, specifically measure absorbance log in the 433nm place, calculate absorbance value, the result is referring to N enzymatic activity (U/ml) in the root of the asparagus of Fig. 1, N enzymatic activity (U/ml) in the stem of the asparagus of Fig. 2, N enzymatic activity (U/ml) in the leaf of the asparagus of Fig. 3.
Contrast by Fig. 1～Fig. 3 can be found out, the enrichment in the root of asparagus of N enzyme, and the difference between male and female plant is different by the difference of each kind.N enzyme content takes second place in the stem of asparagus and the leaf, and in the kind for examination, the difference of N enzyme between male and female plant has no rule.According to these conclusions, can greatly improve the efficient of from asparagus, extracting the N enzyme, thereby alleviate the N enzyme market problem that supply falls short of demand.
The above; only for the better embodiment of the present invention, but protection scope of the present invention is not limited to this, anyly is familiar with those skilled in the art in the technical scope that the present invention discloses; the variation that can expect easily or replacement all should be encompassed within protection scope of the present invention.Therefore, protection scope of the present invention should be as the criterion with the protection domain of claims.
Claims (6)
1. the detection method of the N enzyme content difference of an asparagus plant is characterized in that, comprising:
Extract the asparagus plant, get root, stem and the blade of described plant as sample, make respectively sample solution, described sample solution is carried out colorimetric analysis with reference substance solution by colourimetry respectively, relatively obtain the difference of the N enzyme content at each position of asparagus by calculating absorbance value.
2. method according to claim 1 is characterized in that, before the described extraction asparagus plant, also comprises: the asparagus plant is carried out the step that male and female are identified.
3. method according to claim 1 and 2 is characterized in that, makes in the following manner sample solution:
Described samples with water is cleaned, and baking was killed enzyme in 15 minutes under 105 ℃ temperature, oven dry under 70 ℃ temperature, pulverize, sieving with 20 mesh sieves obtains sample dry powder;
Take by weighing the described sample dry powder of 1.5g, be positioned in the apparatus,Soxhlet's behind the filter paper parcel, use absolute ether heating and refluxing extraction 1 hour, diethyl ether solution inclines; After filter paper packet volatilizes ether, place apparatus,Soxhlet's, added the methyl alcohol heating and refluxing extraction 6 hours, behind the recovery methyl alcohol, be transferred to the 100ml measuring bottle, and arrive scale with methanol constant volume, shake up;
Draw sample solution 1ml, put in the 10ml measuring bottle, add the AlCl3 methanol solution 1ml of mass concentration 5%, be diluted to scale with methyl alcohol, shake up, left standstill 10 minutes, namely obtain sample solution.
4. method according to claim 1 and 2 is characterized in that, described reference substance solution makes in the following manner, comprising:
Described samples with water is cleaned, and baking was killed enzyme in 15 minutes under 105 ℃ temperature, oven dry under 70 ℃ temperature, pulverize, sieving with 20 mesh sieves obtains sample dry powder;
Take by weighing the described sample dry powder of 1.5g, be positioned in the apparatus,Soxhlet's behind the filter paper parcel, use absolute ether heating and refluxing extraction 1 hour, diethyl ether solution inclines; After filter paper packet volatilizes ether, place apparatus,Soxhlet's, added the methyl alcohol heating and refluxing extraction 6 hours, behind the recovery methyl alcohol, be transferred to the 100ml measuring bottle, and arrive scale with methanol constant volume, shake up;
Draw sample solution 1ml, place the 10ml measuring bottle, be diluted to scale with methyl alcohol, shake up, left standstill 10 minutes, namely obtain reference substance solution.
5. method according to claim 1 and 2 is characterized in that, the asparagus plant of described extraction is the asparagus plant that plucking time was no more than 1 hour.
6. method according to claim 1 and 2 is characterized in that, the stem of described plant is from the long 20cm in stem top to bottom, the material of base portion mean diameter 0.5～1.2cm.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106596428A (en) * | 2016-12-15 | 2017-04-26 | 南京市儿童医院 | Kit for determining vitality of PEG-asparaginase in patient body with acute leukemia |
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| CN1709415A (en) * | 2005-06-16 | 2005-12-21 | 江西汇仁药业有限公司 | Asparagus extract and its preparing method |
| US20070178219A1 (en) * | 2002-09-19 | 2007-08-02 | Eric Boudreaux | Method for Reducing Acrylamide Formation |
| CN102181405A (en) * | 2011-01-25 | 2011-09-14 | 湖州市农业科学研究院 | Recombinant virus and method for producing L-asparaginase II by using virus |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20070178219A1 (en) * | 2002-09-19 | 2007-08-02 | Eric Boudreaux | Method for Reducing Acrylamide Formation |
| CN1709415A (en) * | 2005-06-16 | 2005-12-21 | 江西汇仁药业有限公司 | Asparagus extract and its preparing method |
| CN102181405A (en) * | 2011-01-25 | 2011-09-14 | 湖州市农业科学研究院 | Recombinant virus and method for producing L-asparaginase II by using virus |
Non-Patent Citations (2)
| Title |
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| HAN YINGYAN ET AL: "Comparative Studies on Bioactive Compounds in Different Varieties of Asparagus officinalis L.", 《ACTA HORTICULTURAE》, 31 December 2008 (2008-12-31) * |
| 付婕等: "石刁柏中总黄酮含量的测定分析", 《安徽农业科学》, 31 December 2006 (2006-12-31) * |
Cited By (1)
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| CN106596428A (en) * | 2016-12-15 | 2017-04-26 | 南京市儿童医院 | Kit for determining vitality of PEG-asparaginase in patient body with acute leukemia |
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Application publication date: 20130227 |