CN106501419A - The synchronous quantitative detecting method of oxidized form and reduced form glucorphanin in Turnip extractive - Google Patents
The synchronous quantitative detecting method of oxidized form and reduced form glucorphanin in Turnip extractive Download PDFInfo
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Abstract
The invention discloses in Turnip extractive oxidized form and reduced form glucorphanin synchronous quantitative detecting method, comprise the following steps：Oxidized form and reduced form glucorphanin standard substance are dissolved in methanol aqueous solution, dilute series mass concentration standard solution；Take Turnip extractive to be measured and be dissolved in methanol aqueous solution, obtain Turnip extractive sample solution；The standard solution of series mass concentration and Turnip extractive sample solution are carried out high performance liquid chromatography detection analysis respectively, each chromatographic peak area is obtained；The calibration curve equation of oxidized form and reduced form glucorphanin is set up respectively；The chromatographic peak area of oxidized form in sample solution and reduced form glucorphanin is substituted into homologous thread equation respectively, the mass concentration of two kinds of glucorphanins in sample solution is obtained, is calculated the content of oxidized form and reduced form glucorphanin in Turnip extractive.Present invention achieves the synchronous detection by quantitative of oxidized form and reduced form glucorphanin, with quick, efficient, accurate advantage.
Description
Technical field
The invention belongs to plant actives quantitative measurement technology field, and in particular to oxidized form in a kind of Turnip extractive
Synchronous quantitative detecting method with reduced form glucorphanin.
Background technology
Numerous studies show that the active skull cap components contained in brassicaceous vegetable have prevention kinds cancer,
The probability that often crowd of a large amount of edible brassicaceous vegetables suffers from kinds cancer does not substantially less than eat or eats cruciate flower on a small quantity
The crowd of section vegetable.Containing substantial amounts of thioglycoside and myrosin in brassicaceous vegetable, process in people and edible
During brassicaceous vegetable, myrosin can fast hydrolyzing thioglycoside, and be converted into efficient active anticancer
Isothiocyanate, there is anti-cancer and cancer-preventing so as to promote brassicaceous vegetable.Radix Raphani is that a kind of people are daily a large amount of edible
Brassicaceous vegetable, containing abundant thioglycoside and myrosin in Radix Raphani, thioglycoside is mainly oxidized form
Glucorphanin (GRE, chemical constitution are shown in Fig. 1) and reduced form glucorphanin (4-
Methyl mercapto -3- cyclobutenyl glucosinolates, chemical constitution are shown in Fig. 1).During people process and eat Radix Raphani, black mustard
Enzyme can fast hydrolyzing oxidized form and reduced form glucorphanin, and be converted into the oxidized form Radix Raphani sulfur with efficient active anticancer respectively
Plain (4- methylsulfinyl -3- cyclobutenyl isothiocyanates) and reduced form sulforaphen (the different sulfur cyanogen of 4- methyl mercapto -3- cyclobutenyls
Acid esters), so as to promote Radix Raphani to be provided with effect of anti-cancer and cancer-preventing.
Oxidized form and reduced form sulforaphen are the isothiocyanates with II phase detoxification enzyme activity of induction, can not only carry
High human body is may also suppress and kills cancerous cell to carcinogenic Scavenging activity.Research shows, oxidized form and reduced form sulforaphen
The growth of tumor tissues can be hindered by blocking Cancer Cell cycle circulation and promoting cancer cell-apoptosis；Oxidized form and reduction
Type sulforaphen can specifically adjust histiocyte cause as cell carcinogenic metabolic pathway repressor by mechanism and multiple
Cancer metabolic process；Oxidized form and reduced form sulforaphen can efficiently induce the expression of quinone oxidoreductase, accelerate to cause in cell
The metabolism discharge process of cancer material, so as to have functions that cancer chemo-preventive；Oxidized form and reduced form sulforaphen are respectively provided with suppression
Growth of H. pylori propagation processed and the activity of mice gastritis, and can pass through to suppress and eliminate helicobacter pylori infections, so as to
The gastric cancer that effectively prevention helicobacter pylori infections cause.Oxidized form and reduced form sulforaphen are found in Radix Raphani with efficient
The plant actives of active anticancer, are respectively provided with to kinds cancers such as breast carcinoma, pulmonary carcinoma, carcinoma of prostate and intestinal cancer stronger pre-
Anti- and suppression effect.But oxidized form and the equal extreme difference of reduced form sulforaphen stability, degradable, and easily with containing sulfydryl chemical combination
Thing reacts and loses active anticancer, and their precursor substance oxidized form and reduced form glucorphanin stable chemical nature, it is difficult to drop
Solution, and after human body intake, the oxidized form with efficient active anticancer can be converted in the presence of enteric microorganism respectively
With reduced form sulforaphen, therefore, oxidized form and reduced form glucorphanin can be as anticancer and anti-cancer active skull cap components, from trailing plants
The Turnip extractive rich in oxidized form and reduced form glucorphanin is extracted in fore-telling, and adds it to food, functional food, health care
In product and medicine, the development of China's cancer prevention and treatment cause will be advantageously promoted.
At present, existing oxidized form and reduced form glucorphanin detection method are mainly high performance liquid chromatography, and the method is
A kind of detection oxidized form and the effective ways of reduced form glucorphanin content, with accuracy and the characteristics of higher precision, but
Some detection methods are all independent detection by quantitative oxidized form glucorphanin or reduced form glucorphanin, and select, detect in mobile phase
The aspects such as wavelength selection, accuracy in detection and precision are both needed to further improve.
Content of the invention
The deficiencies in the prior art are directed in the present invention, there is provided aoxidized in a kind of quick, efficient, accurate Turnip extractive
Type and the synchronous quantitative detecting method of reduced form glucorphanin.
The purpose of the present invention is achieved by the following technical solution：
In Turnip extractive, the synchronous quantitative detecting method of oxidized form and reduced form glucorphanin, comprises the following steps：
(1) oxidized form and reduced form glucorphanin standard substance are dissolved in methanol-water solution, as standard substance storing solution；Profit
With methanol-water solution by the standard substance storing solution doubling dilution, filtering with microporous membrane, the standard substance for obtaining series mass concentration are molten
Liquid；
(2) 75mg Turnip extractives to be measured are weighed, 5mL methanol-water solutions are dissolved in, filtering with microporous membrane obtains Radix Raphani extraction
Thing sample solution；
(3) will be molten to the standard solution and step (2) gained Turnip extractive sample of step (1) gained series mass concentration
Liquid carries out high performance liquid chromatography detection analysis respectively, obtains oxidized form and reduced form Radix Raphani in standard solution, sample solution respectively
The chromatographic peak area of sulfur glycosides；
Testing conditions are as follows：5 μm, the DIKMA Diamodsil C-18 chromatographic columns of 4.6 × 250mm, sample size are 5 μ L,
Column temperature is 30 DEG C；Mobile phase A is trifluoroacetic acid aqueous solution=1/99 that methanol/concentration is 0.1%, v/v；Mobile phase B be methanol/
Concentration is 0.1% trifluoroacetic acid aqueous solution=3/7, v/v；Gradient elution mode, in 30 minutes, Mobile phase B ratio is from 0% line
Property increases to 100%；Flow rate of mobile phase is 1mL/min；Mobile phase consumption is 1000mL；UV detection wavelength is 235nm；Adopt
Use quantified by external standard method；
(4) according to step (3) gained testing result, with oxidized form in serial standards solution and reduced form glucorphanin
Mass concentration X is abscissa, and corresponding chromatographic peak area Y carries out linear regression analyses for vertical coordinate, sets up oxidized form respectively and goes back
The calibration curve equation of prototype glucorphanin；
(5) according to step (3) gained testing result, respectively by oxidized form in sample solution and the color of reduced form glucorphanin
Spectral peak area is substituted in the corresponding curvilinear equation of step (4) gained, obtains oxidized form and reduced form glucorphanin in sample solution
Mass concentration, calculates the content for obtaining oxidized form and reduced form glucorphanin in Turnip extractive.
The quality of oxidized form and reduced form glucorphanin in the standard solution of the series mass concentration in step (1)
Concentration is 0.0625-2.000mg/mL.
Methanol-water solution in step (1) and (2) is first alcohol and water according to 1：Obtained in 99 volume ratio；Institute
State organic filter membrane that microporous filter membrane is 0.45 μm of aperture.
Extract of the Turnip extractive in (2) for radish seed, Radix Raphani sprout or Radix Raphani rhizome.
The calibration curve equation of the oxidized form glucorphanin in step (4) is：Y=1.2270+48.7148X, R=
0.9994；The calibration curve equation of reduced form glucorphanin is：Y=0.2273+76.9141X, R=0.9999.
In Turnip extractive in step (5), the cubage equation of oxidized form glucorphanin is：Oxidized form glucorphanin
Content=5 × oxidized form glucorphanin mass concentration ÷ Turnip extractive sample quality × 100%；Reduce in Turnip extractive
The cubage equation of type glucorphanin is：The mass concentration ÷ trailing plants of content=5 of reduced form glucorphanin × reduced form glucorphanin
Foretell extract sample quality × 100%.
Beneficial effects of the present invention：
1. the present invention adopts gradient elution mode, can synchronous eluting oxidized form and reduced form glucorphanin, so as to realize Radix Raphani
In extract sample, the synchronous detection by quantitative of oxidized form and reduced form glucorphanin, significantly reduces testing cost.
2. the present invention adopts gradient elution mode, not only can fully eluting oxidized form and reduced form glucorphanin, can also be abundant
, there is dead absorption in the chromatography column so as to avoid excessive impurity component, significantly improve in the relatively low impurity component of eluting polarity
The service life of chromatographic column.
3. the synchronous quantitative detecting method of the present invention achieves the same of two kinds of active components of oxidized form and reduced form glucorphanin
Step detection by quantitative, significantly improves detection by quantitative efficiency, greatly reduces detection by quantitative cost；Have quick, efficient, accurate
Advantage, detection are sensitiveer, and the quantitative limit of oxidized form and reduced form glucorphanin is respectively up to 0.0045mg/mL and 0.0025mg/
mL.
Description of the drawings
Fig. 1 is the chemical constitution of the oxidized form and reduced form glucorphanin of the present invention；
In figure：A is oxidized form glucorphanin；B is reduced form glucorphanin；
Fig. 2 is the chromatogram 0.0625mg/mL of the oxidized form and reduced form glucorphanin standard solution of the present invention；
Fig. 3 is the chromatogram 0.250mg/mL of oxidized form of the present invention and reduced form glucorphanin standard solution；
Fig. 4 is that the present invention is oxidized form and the chromatogram 2.000mg/mL of reduced form glucorphanin standard solution；
Chromatograms of the Fig. 5 for the Turnip extractive sample solution of the embodiment of the present invention 1；
Chromatograms of the Fig. 6 for the Turnip extractive sample solution of the embodiment of the present invention 2；
Chromatograms of the Fig. 7 for 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, the detection technique scheme of the present invention is further described, but these
Embodiment does not constitute the restriction to the claimed scope of the invention.
Embodiment 1
In Turnip extractive, the synchronous quantitative detecting method of oxidized form and reduced form glucorphanin, comprises the following steps：
(1) oxidized form and reduced form glucorphanin standard substance are dissolved in methanol-water solution, as standard substance storing solution；Profit
With methanol-water solution by the standard substance storing solution doubling dilution, filtering with microporous membrane, the standard substance for obtaining series mass concentration are molten
Liquid；
The quality of oxidized form and reduced form glucorphanin in the standard solution of the series mass concentration in 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 extractives to be measured are weighed, 5mL methanol-water solutions are dissolved in, filtering with microporous membrane obtains Radix Raphani extraction
Thing sample solution；Arrange and extracted 3 kinds of Turnip extractive samples to be measured, sample 1 respectively as described in table 1,2 and of sample altogether
Sample 3；
Methanol-water solution in step (1) and (2) is first alcohol and water according to 1：Obtained in 99 volume ratio；Institute
State organic filter membrane that microporous filter membrane is 0.45 μm of aperture.
Extract of the Turnip extractive in (2) for Radix Raphani sprout.
(3) will be molten to the standard solution and step (2) gained Turnip extractive sample of step (1) gained series mass concentration
Liquid carries out high performance liquid chromatography detection analysis respectively, obtains oxidized form and reduced form Radix Raphani in standard solution, sample solution respectively
The chromatographic peak area of sulfur glycosides；
Testing conditions are as follows：5 μm, the DIKMA Diamodsil C-18 chromatographic columns of 4.6 × 250mm, sample size are 5 μ L,
Column temperature is 30 DEG C；Mobile phase A is trifluoroacetic acid aqueous solution=1/99 that methanol/concentration is 0.1%, v/v；Mobile phase B be methanol/
Concentration is 0.1% trifluoroacetic acid aqueous solution=3/7, v/v；Gradient elution mode, in 30 minutes, Mobile phase B ratio is from 0% line
Property increases to 100%；Flow rate of mobile phase is 1mL/min；Mobile phase consumption is 1000mL；UV detection wavelength is 235nm；Adopt
Use quantified by external standard method；
(4) according to step (3) gained testing result, oxidized form glucorphanin mass concentration be 0.0625mg/mL,
The corresponding chromatograph 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 is dense
Spend the standard for 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 product solution is respectively 4.9265,9.5765,19.3007,39.2233,77.3107,153.8754；With
In serial standards solution, mass concentration X of oxidized form and reduced form glucorphanin is abscissa, and corresponding chromatographic peak area Y is vertical
Coordinate carries out linear regression analyses, sets up the calibration curve equation of oxidized form and reduced form glucorphanin respectively；
The calibration curve equation of the oxidized form glucorphanin in step (4) is：Y=1.2270+48.7148X, R=
0.9994；The calibration curve equation of reduced form glucorphanin is：Y=0.2273+76.9141X, R=0.9999.
It follows that being oxidized form and reduced form glucorphanin in the range of 0.0625-2.000mg/mL in mass concentration
Standard curve linear relationship preferable, corresponding calibration curve equation can be respectively used to calculate Turnip extractive sample solution in
Oxidized form and the mass concentration of reduced form glucorphanin.
Explanation：In conventional Turnip extractive, the content of oxidized form and reduced form glucorphanin is typically in the scope of 0.5-12%
Interior, " precision weighs 75mg Turnip extractive samples to be measured, is dissolved in 1% methanol-water solution of 5mL according to of the present invention
In, as Turnip extractive sample solution ", disclosure satisfy that oxidized form and reduced form Radix Raphani sulfur in gained Turnip extractive sample solution
The mass concentration of glycosides is in the range of 0.0625-2.000mg/mL.Therefore, the sample that can pass through to arrange different quality concentration is molten
Liquid, by can satisfactory sample quality concentration be defined.
(5) according to step (3) gained testing result, respectively by oxidized form in sample solution and the color of reduced form glucorphanin
Spectral peak area is substituted in the corresponding curvilinear equation of step (4) gained, obtains oxidized form and reduced form glucorphanin in sample solution
Mass concentration, calculates the content for obtaining oxidized form and reduced form glucorphanin in Turnip extractive.
In Turnip extractive in step (5), the cubage equation of oxidized form glucorphanin is：Oxidized form glucorphanin
Content=5 × oxidized form glucorphanin mass concentration ÷ Turnip extractive sample quality × 100%；Reduce in Turnip extractive
The cubage equation of type glucorphanin is：The mass concentration ÷ trailing plants of content=5 of reduced form glucorphanin × reduced form glucorphanin
Foretell extract sample quality × 100%.
Specific as follows：
In sample 1, the chromatographic peak area of oxidized form and reduced form glucorphanin is respectively 7.3164 and 119.9826；Sample 2
The chromatographic peak area of middle oxidized form and reduced form glucorphanin is respectively 7.2189 and 123.2129；Oxidized form and reduction in sample 3
The chromatographic peak area of type glucorphanin is respectively 7.4625 and 119.2903.
It is computed, oxidized form and also in the mass concentration and sample 1-3 of oxidized form and reduced form glucorphanin in sample 1-3
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 that accuracy and the practicality of quantitative detecting method, the present embodiment have carried out following test：
1. precision test
The oxidized form and reduced form glucorphanin standard solution of 3 kind mass concentrations are taken respectively：0.125、0.250、
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 the chromatographic peak area for obtaining oxidized form and reduced form glucorphanin RSD values (RSD values, i.e. relative standard deviation,
Also known as the coefficient of variation, reflect the index of detection method degree of variation；When RSD values are less than 3%, the detection method of the present invention is described
Degree of variation is less).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 values point of oxidized form glucorphanin chromatographic peak area
Not Wei 1.06%, 0.55% and 0.63%, the RSD values of reduced form glucorphanin chromatographic peak area are respectively 0.66%, 0.61% and
0.43%, respectively less than 3%, show that the quantitative detecting method precision of the present invention is good, degree of variation is less.
2. stability test
The oxidized form and reduced form glucorphanin standard solution of 0.500mg/mL is drawn, is stored under room temperature in brown bottle,
Under same testing conditions, 0h, 6h and 12h after standard solution is prepared, each time period METHOD FOR CONTINUOUS DETERMINATION 3 times, respectively
The chromatographic peak area of oxidized form and reduced form glucorphanin is obtained, and calculates the chromatographic peak area of oxidized form and reduced form glucorphanin
RSD values (when RSD values are less than 3%, illustrate that sample stability of the invention is preferable).Stability test result is as shown in table 3.
3 stability test measurement result of table
As shown in Table 3, after preserving 0h, 6h and 12h, the RSD values of oxidized form glucorphanin chromatographic peak area in standard solution
Respectively 0.63%, 0.47% and 0.53%, the RSD values of reduced form glucorphanin chromatographic peak area are respectively 0.43%, 0.50%
With 0.31%, respectively less than 3%, show that the sample solution stability of the present invention is preferable.
3. reappearance test
Repeat 3 detection by quantitative to the content of oxidized form and reduced form glucorphanin in Turnip extractive sample 1, detect
Method is using corresponding method in embodiment 1 (when RSD values are less than 5%, illustrating that the detection method repeatability of the present invention is preferable).
Reproducible test results are as shown in table 4.
4 reappearance test measurement result of table
As shown in Table 4, by 3 repetition detection by quantitative, the content difference of oxidized form glucorphanin in Turnip extractive sample 1
For 0.83%, 0.84% and 0.85%, the content of reduced form glucorphanin is respectively 10.38%, 10.33% and 10.43%.Through
Calculate, the RSD values of oxidized form glucorphanin chromatographic peak area are 0.69%, and the RSD values of reduced form glucorphanin chromatographic peak area are
0.48%, respectively less than 5%, illustrate that the detection method repeatability of the present invention is preferable.
4. recovery test
Known to accurately preparing, the Turnip extractive sample of oxidized form and reduced form glucorphanin content is appropriate, accurately weighed, point
Not Jia Ru oxidized form and reduced form glucorphanin standard substance appropriate, sample introduction is determined 5 times, obtains corresponding oxidized form and reduced form Radix Raphani
The chromatographic peak area of sulfur glycosides, calculates average recovery rate and the RSD values of oxidized form and reduced form glucorphanin respectively, as a result such as 5 He of table
Shown in table 6.
5 oxidized form glucorphanin recovery test measurement result (1mL) of table
6 reduced form glucorphanin recovery test measurement result (1mL) of table
From table 5 and table 6, in Turnip extractive sample, the average recovery rate of oxidized form and reduced form glucorphanin is respectively
98.36% and 101.44%, RSD value be respectively 1.14% and 2.61%, show the present invention the detection method response rate preferable.
From above test：The synchronous detection by quantitative of oxidized form and reduced form glucorphanin in the Turnip extractive of the present invention
Method precision is high, and stability, detection repeatability and the response rate are preferable.Using the inventive method, synchronous detection by quantitative draws
In Turnip extractive sample, oxidized form and reduced form glucorphanin content are respectively 0.83% and 10.38% and (are content in table 1
Meansigma methodss).
Embodiment 2
In Turnip extractive, the synchronous quantitative detecting method of oxidized form and reduced form glucorphanin, comprises the following steps：
(1) oxidized form and reduced form glucorphanin standard substance are dissolved in methanol-water solution, as standard substance storing solution；Profit
With methanol-water solution by the standard substance storing solution doubling dilution, filtering with microporous membrane, the standard substance for obtaining series mass concentration are molten
Liquid；
The quality of oxidized form and reduced form glucorphanin in the standard solution of the series mass concentration in 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 extractives to be measured are weighed, 5mL methanol-water solutions are dissolved in, filtering with microporous membrane obtains Radix Raphani extraction
Thing sample solution；Arrange and extracted 3 kinds of Turnip extractive samples to be measured altogether, respectively such as the sample 1 described in table 7,2 and of sample
Sample 3；
Methanol-water solution in step (1) and (2) is first alcohol and water according to 1：Obtained in 99 volume ratio；Institute
State organic filter membrane that microporous filter membrane is 0.45 μm of aperture.
Extract of the Turnip extractive in (2) for radish seed.
(3) will be molten to the standard solution and step (2) gained Turnip extractive sample of step (1) gained series mass concentration
Liquid carries out high performance liquid chromatography detection analysis respectively, obtains oxidized form and reduced form Radix Raphani in standard solution, sample solution respectively
The chromatographic peak area of sulfur glycosides；
Testing conditions are as follows：5 μm, the DIKMA Diamodsil C-18 chromatographic columns of 4.6 × 250mm, sample size are 5 μ L,
Column temperature is 30 DEG C；Mobile phase A is trifluoroacetic acid aqueous solution=1/99 that methanol/concentration is 0.1%, v/v；Mobile phase B be methanol/
Concentration is 0.1% trifluoroacetic acid aqueous solution=3/7, v/v；Gradient elution mode, in 30 minutes, Mobile phase B ratio is from 0% line
Property increases to 100%；Flow rate of mobile phase is 1mL/min；Mobile phase consumption is 1000mL；UV detection wavelength is 235nm；Adopt
Use quantified by external standard method；
(4) according to step (3) gained testing result, oxidized form glucorphanin mass concentration be 0.0625mg/mL,
The corresponding chromatograph 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 is dense
Spend the standard for 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 product solution is respectively 4.9265,9.5765,19.3007,39.2233,77.3107,153.8754；With
In serial standards solution, mass concentration X of oxidized form and reduced form glucorphanin is abscissa, and corresponding chromatographic peak area Y is vertical
Coordinate carries out linear regression analyses, sets up the calibration curve equation of oxidized form and reduced form glucorphanin respectively；
The calibration curve equation of the oxidized form glucorphanin in step (4) is：Y=1.2270+48.7148X, R=
0.9994；The calibration curve equation of reduced form glucorphanin is：Y=0.2273+76.9141X, R=0.9999.
It follows that being oxidized form and reduced form glucorphanin in the range of 0.0625-2.000mg/mL in mass concentration
Standard curve linear relationship preferable, corresponding calibration curve equation can be respectively used to calculate Turnip extractive sample solution in
Oxidized form and the mass concentration of reduced form glucorphanin.
Explanation：In conventional Turnip extractive, the content of oxidized form and reduced form glucorphanin is typically in the scope of 0.5-12%
Interior, " precision weighs 75mg Turnip extractive samples to be measured, is dissolved in 1% methanol-water solution of 5mL according to of the present invention
In, as Turnip extractive sample solution ", disclosure satisfy that oxidized form and reduced form Radix Raphani sulfur in gained Turnip extractive sample solution
The mass concentration of glycosides is in the range of 0.0625-2.000mg/mL.Therefore, the sample that can pass through to arrange different quality concentration is molten
Liquid, by can satisfactory sample quality concentration be defined.
(5) according to step (3) gained testing result, respectively by oxidized form in sample solution and the color of reduced form glucorphanin
Spectral peak area is substituted in the corresponding curvilinear equation of step (4) gained, obtains oxidized form and reduced form glucorphanin in sample solution
Mass concentration, calculates the content for obtaining oxidized form and reduced form glucorphanin in Turnip extractive.
In Turnip extractive in step (5), the cubage equation of oxidized form glucorphanin is：Oxidized form glucorphanin
Content=5 × oxidized form glucorphanin mass concentration ÷ Turnip extractive sample quality × 100%；Reduce in Turnip extractive
The cubage equation of type glucorphanin is：The mass concentration ÷ trailing plants of content=5 of reduced form glucorphanin × reduced form glucorphanin
Foretell extract sample quality × 100%.
Specific as follows：
In sample 1, the chromatographic peak area of oxidized form and reduced form glucorphanin is respectively 86.6106 and 8.9186；In sample 2
The chromatographic peak area of oxidized form and reduced form glucorphanin is respectively 87.0152 and 8.9955；Oxidized form and reduced form in sample 3
The chromatographic peak area of glucorphanin is respectively 86.3413 and 8.6879.
Calculate in 3 kinds of sample solutions of gained and aoxidize in the mass concentration and 3 kinds of samples of oxidized form and reduced form glucorphanin
The content of 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 synchronous detection by quantitative of the present embodiment draws oxidized form and reduced form Radix Raphani in Turnip extractive sample
Sulphur resources are respectively 11.69% and 0.75% (being the meansigma methodss of content in table 7).
Embodiment 3
In Turnip extractive, the synchronous quantitative detecting method of oxidized form and reduced form glucorphanin, comprises the following steps：
(1) oxidized form and reduced form glucorphanin standard substance are dissolved in methanol-water solution, as standard substance storing solution；Profit
With methanol-water solution by the standard substance storing solution doubling dilution, filtering with microporous membrane, the standard substance for obtaining series mass concentration are molten
Liquid；
The quality of oxidized form and reduced form glucorphanin in the standard solution of the series mass concentration in 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 extractives to be measured are weighed, 5mL methanol-water solutions are dissolved in, filtering with microporous membrane obtains Radix Raphani extraction
Thing sample solution；Arrange and extracted 3 kinds of Turnip extractive samples to be measured altogether, respectively such as the sample 1 described in table 8,2 and of sample
Sample 3；
Methanol-water solution in step (1) and (2) is first alcohol and water according to 1：Obtained in 99 volume ratio；Institute
State organic filter membrane that microporous filter membrane is 0.45 μm of aperture.
Extract of the Turnip extractive in (2) for Radix Raphani rhizome.
(3) will be molten to the standard solution and step (2) gained Turnip extractive sample of step (1) gained series mass concentration
Liquid carries out high performance liquid chromatography detection analysis respectively, obtains oxidized form and reduced form Radix Raphani in standard solution, sample solution respectively
The chromatographic peak area of sulfur glycosides；
Testing conditions are as follows：5 μm, the DIKMA Diamodsil C-18 chromatographic columns of 4.6 × 250mm, sample size are 5 μ L,
Column temperature is 30 DEG C；Mobile phase A is trifluoroacetic acid aqueous solution=1/99 that methanol/concentration is 0.1%, v/v；Mobile phase B be methanol/
Concentration is 0.1% trifluoroacetic acid aqueous solution=3/7, v/v；Gradient elution mode, in 30 minutes, Mobile phase B ratio is from 0% line
Property increases to 100%；Flow rate of mobile phase is 1mL/min；Mobile phase consumption is 1000mL；UV detection wavelength is 235nm；Adopt
Use quantified by external standard method；
(4) according to step (3) gained testing result, oxidized form glucorphanin mass concentration be 0.0625mg/mL,
The corresponding chromatograph 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 is dense
Spend the standard for 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 product solution is respectively 4.9265,9.5765,19.3007,39.2233,77.3107,153.8754；With
In serial standards solution, mass concentration X of oxidized form and reduced form glucorphanin is abscissa, and corresponding chromatographic peak area Y is vertical
Coordinate carries out linear regression analyses, sets up the calibration curve equation of oxidized form and reduced form glucorphanin respectively；
The calibration curve equation of the oxidized form glucorphanin in step (4) is：Y=1.2270+48.7148X, R=
0.9994；The calibration curve equation of reduced form glucorphanin is：Y=0.2273+76.9141X, R=0.9999.
It follows that being oxidized form and reduced form glucorphanin in the range of 0.0625-2.000mg/mL in mass concentration
Standard curve linear relationship preferable, corresponding calibration curve equation can be respectively used to calculate Turnip extractive sample solution in
Oxidized form and the mass concentration of reduced form glucorphanin.
Explanation：In conventional Turnip extractive, the content of oxidized form and reduced form glucorphanin is typically in the scope of 0.5-12%
Interior, " precision weighs 75mg Turnip extractive samples to be measured, is dissolved in 1% methanol-water solution of 5mL according to of the present invention
In, as Turnip extractive sample solution ", disclosure satisfy that oxidized form and reduced form Radix Raphani sulfur in gained Turnip extractive sample solution
The mass concentration of glycosides is in the range of 0.0625-2.000mg/mL.Therefore, the sample that can pass through to arrange different quality concentration is molten
Liquid, by can satisfactory sample quality concentration be defined.
(5) according to step (3) gained testing result, respectively by oxidized form in sample solution and the color of reduced form glucorphanin
Spectral peak area is substituted in the corresponding curvilinear equation of step (4) gained, obtains oxidized form and reduced form glucorphanin in sample solution
Mass concentration, calculates the content for obtaining oxidized form and reduced form glucorphanin in Turnip extractive.
In Turnip extractive in step (5), the cubage equation of oxidized form glucorphanin is：Oxidized form glucorphanin
Content=5 × oxidized form glucorphanin mass concentration ÷ Turnip extractive sample quality × 100%；Reduce in Turnip extractive
The cubage equation of type glucorphanin is：The mass concentration ÷ trailing plants of content=5 of reduced form glucorphanin × reduced form glucorphanin
Foretell extract sample quality × 100%.
Specific as follows：
In sample 1, the chromatographic peak area of oxidized form and reduced form glucorphanin is respectively 6.0498 and 19.1482；In sample 2
The chromatographic peak area of oxidized form and reduced form glucorphanin is respectively 6.1232 and 19.3020；Oxidized form and reduced form in sample 3
The chromatographic peak area of glucorphanin is respectively 6.1472 and 18.9174.
Calculate in 3 kinds of sample solutions of gained and aoxidize in the mass concentration and 3 kinds of samples of oxidized form and reduced form glucorphanin
The content of 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 synchronous detection by quantitative of the present embodiment draws oxidized form and reduced form Radix Raphani in Turnip extractive sample
Sulphur resources are respectively 0.67% and 1.64% (being the meansigma methodss of content in table 8).
Above the specific embodiment of the present invention is described, but the present invention is not limited to above description.For this
For the technical staff in field, any synchronous quantitative detecting method to oxidized form in Turnip extractive and reduced form glucorphanin enters
Capable equal modifications and substitutions are all within the scope of the invention.Therefore, institute without departing from the spirit and scope of the invention
The impartial conversion that makes and modification, should all cover within the scope of the invention.
Claims (6)
1. in Turnip extractive oxidized form and reduced form glucorphanin synchronous quantitative detecting method, it is characterised in that including following
Step：
(1) oxidized form and reduced form glucorphanin standard substance are dissolved in methanol-water solution, as standard substance storing solution；Using first
The standard substance storing solution doubling dilution, filtering with microporous membrane are obtained the standard solution of series mass concentration by alcohol-water solution；
(2) 75mg Turnip extractives to be measured are weighed, 5mL methanol-water solutions are dissolved in, filtering with microporous membrane obtains Turnip extractive sample
Product solution；
(3) by the standard solution of step (1) gained series mass concentration and step (2) gained Turnip extractive sample solution point
High performance liquid chromatography detection analysis is not carried out, oxidized form and reduced form glucorphanin in standard solution, sample solution is obtained respectively
Chromatographic peak area；
Testing conditions are as follows：5 μm, the DIKMA Diamodsil C-18 chromatographic columns of 4.6 × 250mm, sample size be 5 μ L, column temperature
For 30 DEG C；Mobile phase A is trifluoroacetic acid aqueous solution=1/99 that methanol/concentration is 0.1%, v/v；Mobile phase B is methanol/concentration
For 0.1% trifluoroacetic acid aqueous solution=3/7, v/v；Gradient elution mode, in 30 minutes, Mobile phase B ratio is from 0% linear increasing
Add to 100%；Flow rate of mobile phase is 1mL/min；Mobile phase consumption is 1000mL；UV detection wavelength is 235nm；Using outer
Mark standard measure；
(4) according to step (3) gained testing result, with the quality of oxidized form and reduced form glucorphanin in serial standards solution
Concentration X is abscissa, and corresponding chromatographic peak area Y carries out linear regression analyses for vertical coordinate, sets up oxidized form and reduced form respectively
The calibration curve equation of glucorphanin；
(5) according to step (3) gained testing result, respectively by oxidized form in sample solution and the chromatographic peak of reduced form glucorphanin
Area is substituted in the corresponding curvilinear equation of step (4) gained, obtains the quality of oxidized form and reduced form glucorphanin in sample solution
Concentration, calculates the content for obtaining oxidized form and reduced form glucorphanin in Turnip extractive.
2. synchronous quantitative detecting method according to claim 1, it is characterised in that the series mass in step (1)
In the standard solution of concentration, the mass concentration of oxidized form and reduced form glucorphanin is 0.0625-2.000mg/mL.
3. synchronous quantitative detecting method according to claim 1, it is characterised in that the first in step (1) and (2)
Alcohol-water solution is first alcohol and water according to 1：Obtained in 99 volume ratio；The microporous filter membrane has machine filter for 0.45 μm of aperture
Film.
4. synchronous quantitative detecting method according to claim 1, it is characterised in that the Turnip extractive in (2) is trailing plants
Foretell the extract of seed, Radix Raphani sprout or Radix Raphani rhizome.
5. synchronous quantitative detecting method according to claim 1, it is characterised in that the oxidized form Radix Raphani in step (4)
The calibration curve equation of sulfur glycosides is：Y=1.2270+48.7148X, R=0.9994；The calibration curve equation of reduced form glucorphanin
For：Y=0.2273+76.9141X, R=0.9999.
6. synchronous quantitative detecting method according to claim 1, it is characterised in that the Turnip extractive in step (5)
The cubage equation of middle oxidized form glucorphanin is：The quality of content=5 of oxidized form glucorphanin × oxidized form glucorphanin is dense
Degree ÷ Turnip extractive sample quality × 100%；In Turnip extractive, the cubage equation of reduced form glucorphanin is：Reduced form
Mass concentration ÷ Turnip extractive sample quality × 100% of content=5 of glucorphanin × reduced form glucorphanin.
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