CN106501419B - Oxidized form quantitative detecting method synchronous with reduced form glucorphanin in Turnip extractive - Google Patents
Oxidized form quantitative detecting method synchronous with reduced form glucorphanin in Turnip extractive Download PDFInfo
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Abstract
The invention discloses the quantitative detecting methods synchronous with reduced form glucorphanin of oxidized form in Turnip extractive, comprising the following steps: oxidized form and reduced form glucorphanin standard items are dissolved in methanol-water solution, dilute series mass concentration standard solution；It takes Turnip extractive to be measured to be dissolved in methanol-water solution, obtains Turnip extractive sample solution；The standard solution of series mass concentration and Turnip extractive sample solution are subjected to high performance liquid chromatography detection analysis respectively, obtain each chromatographic peak area；The calibration curve equation of oxidized form and reduced form glucorphanin is established respectively；The chromatographic peak area of oxidized form in sample solution and reduced form glucorphanin is substituted into homologous thread equation respectively, obtains the mass concentration of two kinds of glucorphanins in sample solution, calculates to obtain the content of oxidized form and reduced form glucorphanin in Turnip extractive.The present invention realizes oxidized form quantitative detection synchronous with reduced form glucorphanin, and it is quick, efficient, accurate to have the advantages that.
Description
Technical field
The invention belongs to plant actives quantitative measurement technology fields, and in particular to oxidized form in a kind of Turnip extractive
Quantitative detecting method synchronous with reduced form glucorphanin.
Background technique
A large number of studies show that the active skull cap components contained in brassicaceous vegetable have effects that prevent kinds cancer,
The probability that the crowd of frequent large amounts of food brassicaceous vegetable suffers from kinds cancer is not substantially less than edible or eats cruciate flower on a small quantity
The crowd of section vegetables.Contain a large amount of glucosinolate and myrosin in brassicaceous vegetable, processes and eat in people
During brassicaceous vegetable, myrosin can fast hydrolyzing glucosinolate, and be converted into efficient anticancer activity
Isothiocyanates, so that brassicaceous vegetable be promoted to have effects that anti-cancer and cancer-preventing.Radish is a kind of daily a large amount of foods of people
Brassicaceous vegetable, glucosinolate and myrosin rich in radish, glucosinolate is mainly oxygen
Change type glucorphanin (4-methylsulfinyl-3-butenyl glucosinolate, chemical structure are shown in Fig. 1) and reduced form radish sulphur
Glycosides (4- methyl mercapto -3- cyclobutenyl glucosinolates, chemical structure are shown in Fig. 1).During people's processing and edible radish,
Myrosin can fast hydrolyzing oxidized form and reduced form glucorphanin, and be converted into the oxidation with efficient anticancer activity respectively
Type sulforaphen (4- methylsulfinyl -3- cyclobutenyl isothiocyanates) and reduced form sulforaphen (4- methyl mercapto -3- butylene
Base isothiocyanates), thus the effect of promoting radish to be provided with anti-cancer and cancer-preventing.
Oxidized form and reduced form sulforaphen can be not only mentioned with the isothiocyanates of II phase detoxification enzyme activity of induction
High human body may also suppress to carcinogenic Scavenging activity and kill cancer cell.Studies have shown that oxidized form and reduced form sulforaphen
It can be by blocking Cancer Cell cycle circulation and promotion cancer cell-apoptosis to hinder the growth of tumor tissues；Oxidized form and reduction
Type sulforaphen can be used as cell carcinogenic metabolic pathway repressor, and histocyte cause is specifically adjusted by mechanism and multiple
Cancer metabolic process；Oxidized form and reduced form sulforaphen can efficiently induce the expression of quinone oxidoreductase, accelerate in cell
The metabolism discharge process of carcinogen, to have effects that cancer chemo-preventive；Oxidized form and reduced form sulforaphen have
Have inhibit growth of H. pylori proliferation and mouse gastritis activity, and can by inhibiting and eliminating helicobacter pylori infections,
To effectively prevent gastric cancer caused by helicobacter pylori infections.Oxidized form and reduced form sulforaphen are the tools found in radish
Have the plant actives of efficient anticancer activity, to the kinds cancers such as breast cancer, lung cancer, prostate cancer and intestinal cancer all have compared with
Strong prevention and inhibition effect.But oxidized form and reduced form sulforaphen stability are very poor, degradable, and Yi Yuhan sulfydryl
Compound reaction and lose anticancer activity, and their precursor substance oxidized form and reduced form glucorphanin chemical property are steady
It is fixed, it is not degradable, and after human body intake, it can be converted into respectively under the action of enteric microorganism with efficient anticancer activity
Oxidized form and reduced form sulforaphen, therefore, oxidized form and reduced form glucorphanin can be used as anticancer and anti-cancer is natural living
Property ingredient, the Turnip extractive for being rich in oxidized form and reduced form glucorphanin is extracted from radish, and adds it to food, function
In energy property food, health care product and drug, China's cancer prevention will be advantageously promoted and treat the development of cause.
Currently, existing oxidized form and reduced form glucorphanin detection method are mainly high performance liquid chromatography, this method
It is a kind of effective ways for detecting oxidized form and reduced form glucorphanin content, has the characteristics that accuracy and precision are higher,
But existing detection method is all independent quantitative detection oxidized form glucorphanin or reduced form glucorphanin, and is selected in mobile phase
It selects, Detection wavelength selection, accuracy in detection and precision etc. are both needed to be further improved.
Summary of the invention
In the present invention in view of the deficiencies of the prior art, it provides and is aoxidized in quick, efficient, the accurate Turnip extractive of one kind
Type quantitative detecting method synchronous with reduced form glucorphanin.
The purpose of the present invention is what is be achieved through the following technical solutions:
Oxidized form quantitative detecting method synchronous with reduced form glucorphanin in Turnip extractive, comprising the following steps:
(1) oxidized form and reduced form glucorphanin standard items are dissolved in methanol-water solution, as standard items stock solution；
Using methanol-water solution by the standard items stock solution doubling dilution, filtering with microporous membrane obtains the standard items of series mass concentration
Solution；
(2) 75mg Turnip extractive to be measured is weighed, 5mL methanol-water solution is dissolved in, filtering with microporous membrane obtains radish and mentions
Take object sample solution；
(3) Turnip extractive sample obtained by the standard solution of series mass concentration obtained by step (1) and step (2) is molten
Liquid carries out high performance liquid chromatography detection analysis respectively, obtains standard solution, oxidized form and reduced form trailing plants in sample solution respectively
Foretell the chromatographic peak area of sulphur glycosides；
Testing conditions are as follows: 5 μm, the DIKMA Diamodsil C-18 chromatographic column of 4.6 × 250mm, sample volume is 5 μ L,
Column temperature is 30 DEG C；Mobile phase A is trifluoroacetic acid aqueous solution=1/99, v/v that methanol/concentration is 0.1%；Mobile phase B be methanol/
Trifluoroacetic acid aqueous solution=3/7, v/v that concentration is 0.1%；Gradient elution mode, Mobile phase B ratio is from 0% line in 30 minutes
Property increases to 100%；Flow rate of mobile phase is 1mL/min；Mobile phase dosage is 1000mL；UV detection wavelength is 235nm；It adopts
Use quantified by external standard method；
(4) according to testing result obtained by step (3), with oxidized form in serial standards solution and reduced form glucorphanin
Mass concentration X is abscissa, and corresponding chromatographic peak area Y is that ordinate carries out linear regression analysis, establishes oxidized form respectively and goes back
The calibration curve equation of prototype glucorphanin；
(5) according to testing result obtained by step (3), respectively by the color of oxidized form in sample solution and reduced form glucorphanin
Spectral peak area substitutes into corresponding curvilinear equation obtained by step (4), obtains oxidized form and reduced form glucorphanin in sample solution
Mass concentration, calculate obtain Turnip extractive in oxidized form and reduced form glucorphanin content.
The quality of oxidized form and reduced form glucorphanin in the standard solution of series mass concentration in the step (1)
Concentration is 0.0625-2.000mg/mL.
Methanol-water solution in the step (1) and (2) is made from volume ratio of the first alcohol and water according to 1:99；Institute
State organic filter membrane that miillpore filter is 0.45 μm of aperture.
Turnip extractive in (2) is the extract of radish seed, radish sprout or radish rhizome.
The calibration curve equation of oxidized form glucorphanin in the step (4) are as follows: Y=1.2270+48.7148X, R=
0.9994；The calibration curve equation of reduced form glucorphanin are as follows: Y=0.2273+76.9141X, R=0.9999.
The content accounting equation of oxidized form glucorphanin in Turnip extractive in the step (5) are as follows: oxidized form radish
Content=5 of sulphur glycosides × oxidized form glucorphanin mass concentration ÷ Turnip extractive sample quality × 100%；Turnip extractive
The content accounting equation of middle reduced form glucorphanin are as follows: content=5 of reduced form glucorphanin × reduced form glucorphanin matter
Measure concentration ÷ Turnip extractive sample quality × 100%.
Beneficial effects of the present invention:
1. the present invention uses gradient elution mode, elution oxidized form and reduced form glucorphanin can be synchronized, to realize trailing plants
Foretell oxidized form quantitative detection synchronous with reduced form glucorphanin in extract sample, significantly reduces testing cost.
2. the present invention uses gradient elution mode, oxidized form and reduced form glucorphanin not only can be sufficiently eluted, can also be filled
Divide the elution lower impurity component of polarity, so that excessive impurity component be avoided to occur extremely to adsorb in the chromatography column, significantly improves
The service life of chromatographic column.
3. synchronization quantitative detecting method of the invention realizes two kinds of active components of oxidized form and reduced form glucorphanin
Synchronous quantitative detection, significantly improves quantitative detection efficiency, greatly reduces quantitative detection cost；With quick, efficient, accurate
The advantages of, detect it is sensitiveer, the quantitative limit of oxidized form and reduced form glucorphanin respectively up to 0.0045mg/mL and
0.0025mg/mL。
Detailed description of the invention
Fig. 1 is the chemical structure of oxidized form of the invention and reduced form glucorphanin；
In figure: a is oxidized form glucorphanin；B is reduced form glucorphanin；
Fig. 2 is the chromatogram 0.0625mg/mL of oxidized form of the invention and reduced form glucorphanin standard solution；
Fig. 3 is the chromatogram 0.250mg/mL of oxidized form of the present invention and reduced form glucorphanin standard solution；
Fig. 4 is the chromatogram 2.000mg/mL that the present invention is oxidized form and reduced form glucorphanin standard solution；
Fig. 5 is the chromatogram of the Turnip extractive sample solution of the embodiment of the present invention 1；
Fig. 6 is the chromatogram of the Turnip extractive sample solution of the embodiment of the present invention 2；
Fig. 7 is the chromatogram of the Turnip extractive sample solution of the embodiment of the present invention 3.
In Fig. 2-7: peak 1 represents oxidized form glucorphanin；Peak 2 represents reduced form glucorphanin.
Specific embodiment
Below in conjunction with the drawings and specific embodiments, detection technique scheme of the invention is further described, but these
Embodiment does not constitute the limitation to the claimed scope of the invention.
Embodiment 1
Oxidized form quantitative detecting method synchronous with reduced form glucorphanin in Turnip extractive, comprising the following steps:
(1) oxidized form and reduced form glucorphanin standard items are dissolved in methanol-water solution, as standard items stock solution；
Using methanol-water solution by the standard items stock solution doubling dilution, filtering with microporous membrane obtains the standard items of series mass concentration
Solution；
The quality of oxidized form and reduced form glucorphanin in the standard solution of series mass concentration in the step (1)
Concentration is respectively 0.0625mg/mL, 0.125mg/mL, 0.250mg/mL, 0.500mg/mL, 1.000mg/mL and 2.000mg/
mL；
(2) 75mg Turnip extractive to be measured is weighed, 5mL methanol-water solution is dissolved in, filtering with microporous membrane obtains radish and mentions
Take object sample solution；It is arranged and has extracted 3 kinds of Turnip extractive samples to be measured altogether, respectively sample 1, sample 2 as described in table 1
With sample 3；
Methanol-water solution in the step (1) and (2) is made from volume ratio of the first alcohol and water according to 1:99；Institute
State organic filter membrane that miillpore filter is 0.45 μm of aperture.
Turnip extractive in (2) is the extract of radish sprout.
(3) Turnip extractive sample obtained by the standard solution of series mass concentration obtained by step (1) and step (2) is molten
Liquid carries out high performance liquid chromatography detection analysis respectively, obtains standard solution, oxidized form and reduced form trailing plants in sample solution respectively
Foretell the chromatographic peak area of sulphur glycosides；
Testing conditions are as follows: 5 μm, the DIKMA Diamodsil C-18 chromatographic column of 4.6 × 250mm, sample volume is 5 μ L,
Column temperature is 30 DEG C；Mobile phase A is trifluoroacetic acid aqueous solution=1/99, v/v that methanol/concentration is 0.1%；Mobile phase B be methanol/
Trifluoroacetic acid aqueous solution=3/7, v/v that concentration is 0.1%；Gradient elution mode, Mobile phase B ratio is from 0% line in 30 minutes
Property increases to 100%；Flow rate of mobile phase is 1mL/min；Mobile phase dosage is 1000mL；UV detection wavelength is 235nm；It adopts
Use quantified by external standard method；
(4) according to testing result obtained by step (3), oxidized form glucorphanin mass concentration be 0.0625mg/mL,
The corresponding chromatography of the standard solution of 0.125mg/mL, 0.250mg/mL, 0.500mg/mL, 1.000mg/mL and 2.000mg/mL
Peak area is respectively 4.0058,6.4441,12.7776,26.3434,52.0528,97.5527；Reduced form glucorphanin quality
Concentration is the mark of 0.0625mg/mL, 0.125mg/mL, 0.250mg/mL, 0.500mg/mL, 1.000mg/mL and 2.000mg/mL
The corresponding chromatographic peak area of quasi- product solution is respectively 4.9265,9.5765,19.3007,39.2233,77.3107,153.8754；
Using the mass concentration X of oxidized form in serial standards solution and reduced form glucorphanin as abscissa, corresponding chromatographic peak area Y is
Ordinate carries out linear regression analysis, establishes the calibration curve equation of oxidized form and reduced form glucorphanin respectively；
The calibration curve equation of oxidized form glucorphanin in the step (4) are as follows: Y=1.2270+48.7148X, R=
0.9994；The calibration curve equation of reduced form glucorphanin are as follows: Y=0.2273+76.9141X, R=0.9999.
It follows that in the range of mass concentration is 0.0625-2.000mg/mL, oxidized form and reduced form glucorphanin
Standard curve linear relationship it is preferable, corresponding calibration curve equation can be respectively used to calculate Turnip extractive sample solution in
The mass concentration of oxidized form and reduced form glucorphanin.
Illustrate: the content of oxidized form and reduced form glucorphanin is generally in the model of 0.5-12% in conventional Turnip extractive
In enclosing, " precision weighs 75mg Turnip extractive sample to be measured, and 1% methanol-water for being dissolved in 5mL is molten according to of the present invention
In liquid, as Turnip extractive sample solution ", it can satisfy oxidized form and reduced form trailing plants in gained Turnip extractive sample solution
The mass concentration of fore-telling sulphur glycosides is in the range of 0.0625-2.000mg/mL.Therefore, the sample of setting different quality concentration can be passed through
Product solution, being subject to being capable of satisfactory sample quality concentration.
(5) according to testing result obtained by step (3), respectively by the color of oxidized form in sample solution and reduced form glucorphanin
Spectral peak area substitutes into corresponding curvilinear equation obtained by step (4), obtains oxidized form and reduced form glucorphanin in sample solution
Mass concentration, calculate obtain Turnip extractive in oxidized form and reduced form glucorphanin content.
The content accounting equation of oxidized form glucorphanin in Turnip extractive in the step (5) are as follows: oxidized form radish
Content=5 of sulphur glycosides × oxidized form glucorphanin mass concentration ÷ Turnip extractive sample quality × 100%；Turnip extractive
The content accounting equation of middle reduced form glucorphanin are as follows: content=5 of reduced form glucorphanin × reduced form glucorphanin matter
Measure concentration ÷ Turnip extractive sample quality × 100%.
It is specific as follows:
The chromatographic peak area of oxidized form and reduced form glucorphanin is respectively 7.3164 and 119.9826 in sample 1；Sample 2
The chromatographic peak area of middle oxidized form and reduced form glucorphanin is respectively 7.2189 and 123.2129；In sample 3 oxidized form and also
The chromatographic peak area of prototype glucorphanin is respectively 7.4625 and 119.2903.
Be computed, in sample 1-3 in the mass concentration and sample 1-3 of oxidized form and reduced form glucorphanin oxidized form and also
The content of prototype glucorphanin is as shown in table 1.
The assay result of oxidized form and reduced form glucorphanin in 1 Turnip extractive of table
In order to verify the accuracy and practicability of quantitative detecting method, the present embodiment has carried out following test:
1. precision test
0.250 the oxidized form and reduced form glucorphanin standard solution of 3 kinds of mass concentrations are taken respectively: 0.125,
0.500mg/mL, METHOD FOR CONTINUOUS DETERMINATION 3 times under same testing conditions obtain the chromatographic peak of oxidized form and reduced form glucorphanin respectively
Area, and calculate and obtain the RSD value of the chromatographic peak area of oxidized form and reduced form glucorphanin (RSD value, i.e. relative standard are inclined
Difference, the also known as coefficient of variation reflect the index of detection method degree of variation；When RSD value is less than 3%, illustrate detection of the invention
Method degree of variation is smaller).Precision test result is as shown in table 2.
2 precision test measurement result of table
As shown in Table 2, in the standard solution of 3 kinds of mass concentrations, the RSD value point of oxidized form glucorphanin chromatographic peak area
Not Wei 1.06%, 0.55% and 0.63%, the RSD value of reduced form glucorphanin chromatographic peak area is respectively 0.66%, 0.61%
With 0.43%, respectively less than 3%, show that quantitative detecting method precision of the invention is good, degree of variation is smaller.
2. stability test
The oxidized form and reduced form glucorphanin standard solution for drawing 0.500mg/mL, are stored in brown bottle at room temperature
In, under same testing conditions, 0h, 6h and 12h after standard solution preparation, each period METHOD FOR CONTINUOUS DETERMINATION 3 times, point
Not Huo get oxidized form and reduced form glucorphanin chromatographic peak area, and calculate the chromatographic peak of oxidized form and reduced form glucorphanin
The RSD value (when RSD value is less than 3%, illustrating that sample stability of the invention is preferable) of area.Stability test result such as table 3
It is shown.
3 stability test measurement result of table
As shown in Table 3, after saving 0h, 6h and 12h, the RSD of oxidized form glucorphanin chromatographic peak area in standard solution
Value is respectively 0.63%, 0.47% and 0.53%, the RSD value of reduced form glucorphanin chromatographic peak area is respectively 0.43%,
0.50% and 0.31%, respectively less than 3%, show that sample solution stability of the invention is preferable.
3. reappearance test
3 quantitative detections are repeated to the content of oxidized form and reduced form glucorphanin in Turnip extractive sample 1, are examined
Survey method using method corresponding in embodiment 1 (when RSD value is less than 5%, illustrate detection method reproducibility of the invention compared with
It is good).Reproducible test results is as shown in table 4.
4 reappearance test measurement result of table
As shown in Table 4, by 3 repetition quantitative detections, the content point of oxidized form glucorphanin in Turnip extractive sample 1
Not Wei 0.83%, 0.84% and 0.85%, the content of reduced form glucorphanin is respectively 10.38%, 10.33% and 10.43%.
It is computed, the RSD value of oxidized form glucorphanin chromatographic peak area is 0.69%, the RSD of reduced form glucorphanin chromatographic peak area
Value is 0.48%, respectively less than 5%, illustrates that detection method reproducibility of the invention is preferable.
4. recovery test
Oxidized form and the Turnip extractive sample of reduced form glucorphanin content are appropriate known to accurate preparation, accurately weighed,
It is separately added into oxidized form and reduced form glucorphanin standard items is appropriate, sample introduction measures 5 times, obtains corresponding oxidized form and reduced form
The chromatographic peak area of glucorphanin calculates separately the average recovery rate and RSD value of oxidized form and reduced form glucorphanin, as a result such as
Shown in table 5 and table 6.
5 oxidized form glucorphanin recovery test measurement result (1mL) of table
6 reduced form glucorphanin recovery test measurement result (1mL) of table
By table 5 and table 6 it is found that the average recovery rate of oxidized form and reduced form glucorphanin is distinguished in Turnip extractive sample
For 98.36% and 101.44%, RSD value is respectively 1.14% and 2.61%, shows that the detection method rate of recovery of the invention is preferable.
From testing above: oxidized form quantitative inspection synchronous with reduced form glucorphanin in Turnip extractive of the invention
It is high to survey method precision, stability, detection reproducibility and the rate of recovery are preferable.Using the inventive method, synchronous quantitative detection is obtained
It (is to contain in table 1 that oxidized form and reduced form glucorphanin content, which are respectively 0.83% and 10.38%, in Turnip extractive sample out
The average value of amount).
Embodiment 2
Oxidized form quantitative detecting method synchronous with reduced form glucorphanin in Turnip extractive, comprising the following steps:
(1) oxidized form and reduced form glucorphanin standard items are dissolved in methanol-water solution, as standard items stock solution；
Using methanol-water solution by the standard items stock solution doubling dilution, filtering with microporous membrane obtains the standard items of series mass concentration
Solution；
The quality of oxidized form and reduced form glucorphanin in the standard solution of series mass concentration in the step (1)
Concentration is respectively 0.0625mg/mL, 0.125mg/mL, 0.250mg/mL, 0.500mg/mL, 1.000mg/mL and 2.000mg/
mL；
(2) 75mg Turnip extractive to be measured is weighed, 5mL methanol-water solution is dissolved in, filtering with microporous membrane obtains radish and mentions
Take object sample solution；It is arranged altogether and has extracted 3 kinds of Turnip extractive samples to be measured, respectively the sample 1 as described in table 7, sample 2
With sample 3；
Methanol-water solution in the step (1) and (2) is made from volume ratio of the first alcohol and water according to 1:99；Institute
State organic filter membrane that miillpore filter is 0.45 μm of aperture.
Turnip extractive in (2) is the extract of radish seed.
(3) Turnip extractive sample obtained by the standard solution of series mass concentration obtained by step (1) and step (2) is molten
Liquid carries out high performance liquid chromatography detection analysis respectively, obtains standard solution, oxidized form and reduced form trailing plants in sample solution respectively
Foretell the chromatographic peak area of sulphur glycosides；
Testing conditions are as follows: 5 μm, the DIKMA Diamodsil C-18 chromatographic column of 4.6 × 250mm, sample volume is 5 μ L,
Column temperature is 30 DEG C；Mobile phase A is trifluoroacetic acid aqueous solution=1/99, v/v that methanol/concentration is 0.1%；Mobile phase B be methanol/
Trifluoroacetic acid aqueous solution=3/7, v/v that concentration is 0.1%；Gradient elution mode, Mobile phase B ratio is from 0% line in 30 minutes
Property increases to 100%；Flow rate of mobile phase is 1mL/min；Mobile phase dosage is 1000mL；UV detection wavelength is 235nm；It adopts
Use quantified by external standard method；
(4) according to testing result obtained by step (3), oxidized form glucorphanin mass concentration be 0.0625mg/mL,
The corresponding chromatography of the standard solution of 0.125mg/mL, 0.250mg/mL, 0.500mg/mL, 1.000mg/mL and 2.000mg/mL
Peak area is respectively 4.0058,6.4441,12.7776,26.3434,52.0528,97.5527；Reduced form glucorphanin quality
Concentration is the mark of 0.0625mg/mL, 0.125mg/mL, 0.250mg/mL, 0.500mg/mL, 1.000mg/mL and 2.000mg/mL
The corresponding chromatographic peak area of quasi- product solution is respectively 4.9265,9.5765,19.3007,39.2233,77.3107,153.8754；
Using the mass concentration X of oxidized form in serial standards solution and reduced form glucorphanin as abscissa, corresponding chromatographic peak area Y is
Ordinate carries out linear regression analysis, establishes the calibration curve equation of oxidized form and reduced form glucorphanin respectively；
The calibration curve equation of oxidized form glucorphanin in the step (4) are as follows: Y=1.2270+48.7148X, R=
0.9994；The calibration curve equation of reduced form glucorphanin are as follows: Y=0.2273+76.9141X, R=0.9999.
It follows that in the range of mass concentration is 0.0625-2.000mg/mL, oxidized form and reduced form glucorphanin
Standard curve linear relationship it is preferable, corresponding calibration curve equation can be respectively used to calculate Turnip extractive sample solution in
The mass concentration of oxidized form and reduced form glucorphanin.
Illustrate: the content of oxidized form and reduced form glucorphanin is generally in the model of 0.5-12% in conventional Turnip extractive
In enclosing, " precision weighs 75mg Turnip extractive sample to be measured, and 1% methanol-water for being dissolved in 5mL is molten according to of the present invention
In liquid, as Turnip extractive sample solution ", it can satisfy oxidized form and reduced form trailing plants in gained Turnip extractive sample solution
The mass concentration of fore-telling sulphur glycosides is in the range of 0.0625-2.000mg/mL.Therefore, the sample of setting different quality concentration can be passed through
Product solution, being subject to being capable of satisfactory sample quality concentration.
(5) according to testing result obtained by step (3), respectively by the color of oxidized form in sample solution and reduced form glucorphanin
Spectral peak area substitutes into corresponding curvilinear equation obtained by step (4), obtains oxidized form and reduced form glucorphanin in sample solution
Mass concentration, calculate obtain Turnip extractive in oxidized form and reduced form glucorphanin content.
The content accounting equation of oxidized form glucorphanin in Turnip extractive in the step (5) are as follows: oxidized form radish
Content=5 of sulphur glycosides × oxidized form glucorphanin mass concentration ÷ Turnip extractive sample quality × 100%；Turnip extractive
The content accounting equation of middle reduced form glucorphanin are as follows: content=5 of reduced form glucorphanin × reduced form glucorphanin matter
Measure concentration ÷ Turnip extractive sample quality × 100%.
It is specific as follows:
The chromatographic peak area of oxidized form and reduced form glucorphanin is respectively 86.6106 and 8.9186 in sample 1；Sample 2
The chromatographic peak area of middle oxidized form and reduced form glucorphanin is respectively 87.0152 and 8.9955；Oxidized form and reduction in sample 3
The chromatographic peak area of type glucorphanin is respectively 86.3413 and 8.6879.
Calculate in resulting 3 kinds of sample solutions oxygen in the mass concentration and 3 kinds of samples of oxidized form and reduced form glucorphanin
The content of change type and reduced form glucorphanin is as shown in table 7.
The assay result of oxidized form and reduced form glucorphanin in 7 Turnip extractive of table
As shown in Table 7, the synchronization quantitative detection of the present embodiment obtains oxidized form and reduced form trailing plants in Turnip extractive sample
Foretelling sulphur resources is respectively 11.69% and 0.75% (being the average value of content in table 7).
Embodiment 3
Oxidized form quantitative detecting method synchronous with reduced form glucorphanin in Turnip extractive, comprising the following steps:
(1) oxidized form and reduced form glucorphanin standard items are dissolved in methanol-water solution, as standard items stock solution；
Using methanol-water solution by the standard items stock solution doubling dilution, filtering with microporous membrane obtains the standard items of series mass concentration
Solution；
The quality of oxidized form and reduced form glucorphanin in the standard solution of series mass concentration in the step (1)
Concentration is respectively 0.0625mg/mL, 0.125mg/mL, 0.250mg/mL, 0.500mg/mL, 1.000mg/mL and 2.000mg/
mL；
(2) 75mg Turnip extractive to be measured is weighed, 5mL methanol-water solution is dissolved in, filtering with microporous membrane obtains radish and mentions
Take object sample solution；It is arranged altogether and has extracted 3 kinds of Turnip extractive samples to be measured, respectively the sample 1 as described in table 8, sample 2
With sample 3；
Methanol-water solution in the step (1) and (2) is made from volume ratio of the first alcohol and water according to 1:99；Institute
State organic filter membrane that miillpore filter is 0.45 μm of aperture.
Turnip extractive in (2) is the extract of radish rhizome.
(3) Turnip extractive sample obtained by the standard solution of series mass concentration obtained by step (1) and step (2) is molten
Liquid carries out high performance liquid chromatography detection analysis respectively, obtains standard solution, oxidized form and reduced form trailing plants in sample solution respectively
Foretell the chromatographic peak area of sulphur glycosides；
Testing conditions are as follows: 5 μm, the DIKMA Diamodsil C-18 chromatographic column of 4.6 × 250mm, sample volume is 5 μ L,
Column temperature is 30 DEG C；Mobile phase A is trifluoroacetic acid aqueous solution=1/99, v/v that methanol/concentration is 0.1%；Mobile phase B be methanol/
Trifluoroacetic acid aqueous solution=3/7, v/v that concentration is 0.1%；Gradient elution mode, Mobile phase B ratio is from 0% line in 30 minutes
Property increases to 100%；Flow rate of mobile phase is 1mL/min；Mobile phase dosage is 1000mL；UV detection wavelength is 235nm；It adopts
Use quantified by external standard method；
(4) according to testing result obtained by step (3), oxidized form glucorphanin mass concentration be 0.0625mg/mL,
The corresponding chromatography of the standard solution of 0.125mg/mL, 0.250mg/mL, 0.500mg/mL, 1.000mg/mL and 2.000mg/mL
Peak area is respectively 4.0058,6.4441,12.7776,26.3434,52.0528,97.5527；Reduced form glucorphanin quality
Concentration is the mark of 0.0625mg/mL, 0.125mg/mL, 0.250mg/mL, 0.500mg/mL, 1.000mg/mL and 2.000mg/mL
The corresponding chromatographic peak area of quasi- product solution is respectively 4.9265,9.5765,19.3007,39.2233,77.3107,153.8754；
Using the mass concentration X of oxidized form in serial standards solution and reduced form glucorphanin as abscissa, corresponding chromatographic peak area Y is
Ordinate carries out linear regression analysis, establishes the calibration curve equation of oxidized form and reduced form glucorphanin respectively；
The calibration curve equation of oxidized form glucorphanin in the step (4) are as follows: Y=1.2270+48.7148X, R=
0.9994；The calibration curve equation of reduced form glucorphanin are as follows: Y=0.2273+76.9141X, R=0.9999.
It follows that in the range of mass concentration is 0.0625-2.000mg/mL, oxidized form and reduced form glucorphanin
Standard curve linear relationship it is preferable, corresponding calibration curve equation can be respectively used to calculate Turnip extractive sample solution in
The mass concentration of oxidized form and reduced form glucorphanin.
Illustrate: the content of oxidized form and reduced form glucorphanin is generally in the model of 0.5-12% in conventional Turnip extractive
In enclosing, " precision weighs 75mg Turnip extractive sample to be measured, and 1% methanol-water for being dissolved in 5mL is molten according to of the present invention
In liquid, as Turnip extractive sample solution ", it can satisfy oxidized form and reduced form trailing plants in gained Turnip extractive sample solution
The mass concentration of fore-telling sulphur glycosides is in the range of 0.0625-2.000mg/mL.Therefore, the sample of setting different quality concentration can be passed through
Product solution, being subject to being capable of satisfactory sample quality concentration.
(5) according to testing result obtained by step (3), respectively by the color of oxidized form in sample solution and reduced form glucorphanin
Spectral peak area substitutes into corresponding curvilinear equation obtained by step (4), obtains oxidized form and reduced form glucorphanin in sample solution
Mass concentration, calculate obtain Turnip extractive in oxidized form and reduced form glucorphanin content.
The content accounting equation of oxidized form glucorphanin in Turnip extractive in the step (5) are as follows: oxidized form radish
Content=5 of sulphur glycosides × oxidized form glucorphanin mass concentration ÷ Turnip extractive sample quality × 100%；Turnip extractive
The content accounting equation of middle reduced form glucorphanin are as follows: content=5 of reduced form glucorphanin × reduced form glucorphanin matter
Measure concentration ÷ Turnip extractive sample quality × 100%.
It is specific as follows:
The chromatographic peak area of oxidized form and reduced form glucorphanin is respectively 6.0498 and 19.1482 in sample 1；Sample 2
The chromatographic peak area of middle oxidized form and reduced form glucorphanin is respectively 6.1232 and 19.3020；Oxidized form and reduction in sample 3
The chromatographic peak area of type glucorphanin is respectively 6.1472 and 18.9174.
Calculate in resulting 3 kinds of sample solutions oxygen in the mass concentration and 3 kinds of samples of oxidized form and reduced form glucorphanin
The content of change type and reduced form glucorphanin is as shown in table 8.
The assay result of oxidized form and reduced form glucorphanin in 8 Turnip extractive of table
As shown in Table 8, the synchronization quantitative detection of the present embodiment obtains oxidized form and reduced form trailing plants in Turnip extractive sample
Foretelling sulphur resources is respectively 0.67% and 1.64% (being the average value of content in table 8).
Above specific embodiments of the present invention have been described, but the present invention is not limited to above description.For this
For the technical staff in field, oxidized form quantitative detecting method synchronous with reduced form glucorphanin in any pair of Turnip extractive
The equal modification and substitution of progress are all within the scope of the invention.Therefore, without departing from the spirit and scope of the invention
The equal transformation and modification made, all should be contained within the scope of the invention.
Claims (6)
1. oxidized form quantitative detecting method synchronous with reduced form glucorphanin in Turnip extractive, which is characterized in that including with
Lower step:
(1) oxidized form and reduced form glucorphanin standard items are dissolved in methanol-water solution, as standard items stock solution；It utilizes
Methanol-water solution is by the standard items stock solution doubling dilution, filtering with microporous membrane, and the standard items for obtaining series mass concentration are molten
Liquid；
(2) 75mg Turnip extractive to be measured is weighed, 5mL methanol-water solution is dissolved in, filtering with microporous membrane obtains Turnip extractive
Sample solution；
(3) by Turnip extractive sample solution obtained by the standard solution of series mass concentration obtained by step (1) and step (2) point
Not carry out high performance liquid chromatography detection analysis, obtain standard solution, oxidized form and reduced form radish sulphur in sample solution respectively
The chromatographic peak area of glycosides；
Testing conditions are as follows: 5 μm, the DIKMA Diamodsil C-18 chromatographic column of 4.6 × 250mm, sample volume is 5 μ L, column temperature
It is 30 DEG C；Mobile phase A is trifluoroacetic acid aqueous solution=1/99, v/v that methanol/concentration is 0.1%；Mobile phase B is methanol/concentration
For 0.1% trifluoroacetic acid aqueous solution=3/7, v/v；Gradient elution mode, Mobile phase B ratio linearly increases from 0% in 30 minutes
Add to 100%；Flow rate of mobile phase is 1mL/min；Mobile phase dosage is 1000mL；UV detection wavelength is 235nm；Using outer
Mark standard measure；
(4) according to testing result obtained by step (3), with the quality of oxidized form and reduced form glucorphanin in serial standards solution
Concentration X is abscissa, and corresponding chromatographic peak area Y is that ordinate carries out linear regression analysis, establishes oxidized form and reduced form respectively
The calibration curve equation of glucorphanin；
(5) according to testing result obtained by step (3), respectively by the chromatographic peak of oxidized form in sample solution and reduced form glucorphanin
Area substitutes into corresponding curvilinear equation obtained by step (4), obtains the matter of oxidized form and reduced form glucorphanin in sample solution
Concentration is measured, the content for obtaining oxidized form and reduced form glucorphanin in Turnip extractive is calculated.
2. synchronous quantitative detecting method according to claim 1, which is characterized in that the series mass in the step (1)
The mass concentration of oxidized form and reduced form glucorphanin is 0.0625-2.000mg/mL in the standard solution of concentration.
3. synchronous quantitative detecting method according to claim 1, which is characterized in that the first in the step (1) and (2)
Alcohol-water solution is made from volume ratio of the first alcohol and water according to 1:99；The miillpore filter has machine filter for 0.45 μm of aperture
Film.
4. synchronous quantitative detecting method according to claim 1, which is characterized in that the radish in the step (2) is extracted
Object is the extract of radish seed, radish sprout or radish rhizome.
5. synchronous quantitative detecting method according to claim 1, which is characterized in that the oxidized form trailing plants in the step (4)
Foretell the calibration curve equation of sulphur glycosides are as follows: Y=1.2270+48.7148X, R=0.9994；The standard curve of reduced form glucorphanin
Equation are as follows: Y=0.2273+76.9141X, R=0.9999.
6. synchronous quantitative detecting method according to claim 1, which is characterized in that the radish in the step (5) is extracted
The content accounting equation of oxidized form glucorphanin in object are as follows: content=5 of oxidized form glucorphanin × oxidized form glucorphanin
Mass concentration ÷ Turnip extractive sample quality × 100%；The content accounting equation of reduced form glucorphanin in Turnip extractive
Are as follows: content=5 of reduced form glucorphanin × reduced form glucorphanin mass concentration ÷ Turnip extractive sample quality ×
100%.
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