CN108195952A - A kind of method for detecting genistin in honey vinegar black soya bean - Google Patents
A kind of method for detecting genistin in honey vinegar black soya bean Download PDFInfo
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- CN108195952A CN108195952A CN201711287750.1A CN201711287750A CN108195952A CN 108195952 A CN108195952 A CN 108195952A CN 201711287750 A CN201711287750 A CN 201711287750A CN 108195952 A CN108195952 A CN 108195952A
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- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
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- G01N2030/062—Preparation extracting sample from raw material
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Abstract
The invention discloses a kind of methods for detecting genistin in honey vinegar black soya bean.The honey vinegar black soya bean of drying is crushed, be sieved to obtain honey vinegar black bean powder, adds in methanol solution ultrasound, supernatant is taken after centrifugation, after the supernatant concentration, genistin extracting solution is obtained with methanol solution constant volume；By genistin standard items methanol constant volume, it is ultrasonically treated, genistin standard mother liquor is made, by genistin standard mother liquor methanol constant volume, is made into genistin titer；Will dilution is made in genistin extracting solution, after making it through filter membrane, preserve for use；Obtained sample is subjected to content detection, is methanol, glacial acetic acid and water by mobile phase, so as to detect the content of genistin in honey vinegar black soya bean product.This method is quick, simple result is reliable, convenient and efficient, and genistin content is 134.234~158.321mg/kg in honey vinegar black soya bean product.
Description
Technical field
The invention belongs to isolate and purify genistin technical field, contaminated more particularly, to a kind of detect in honey vinegar black soya bean
The method of the wooden glycosides of material.
Background technology
Black soya bean cares for name conceive be exactly black soybean, the content of 5 kinds of amino acid necessary to human body exists in black soya bean seed
0.244%~2%, fat content contains 1% hereinafter, containing nutrients such as abundant vitamin, minerals in black soya bean in black soya bean
It is more than the several times of common crops there are many trace element, a variety of amino acid, iodine, selenium, amylopectin, soluble pigment etc..
Isoflavones is a kind of secondary metabolite formed in soybean growth course, includes 3 kinds of aglycons and their 9
The derivative of kind B- glucosides.97%~99% is existed in the form of soybean isoflavone glucoside in isoflavones, aglycon shape
Formula is only the 1%~3% of isoflavones total amount.There is isoflavones weak estrogenic activity, antioxidant activity, anti-hemolysis to live
Property and antifungal activity effectively can prevent and inhibit leukaemia, osteoporosis, colon cancer, lung cancer, gastric cancer, breast cancer and preceding
The generation of a variety of diseases such as row gland cancer, women's climacteric syndrome, especially has good prevention to breast cancer and prostate cancer
And therapeutic effect.
Soybean isoflavone compound is yellow including the various member of a variety of chemical constitutions and biological function height, such as soya bean
Element, mangiferin, daidzin and genistin etc..The isoflavones main component extracted from soybean is genistin, is changed
It learns structural formula and sees formula (1).Genistin is one of active ingredient of isoflavones, belongs to phytoestrogen in structure.Research
It was found that genistin cannot directly be absorbed by body, but absorbed in the form of genistein after enzyme hydrolysis in enterobacteriaceae,
Play bioactivity.Genistein is both greater than Daidzein in many aspect activity.
At present, it is studied about the pharmacological activity of genistin and has more report, but to dyewood in its production of black beans
The summary of glycosides detection method is still inadequate with looking forward to.
Invention content
The defects of the purpose of the invention is to overcome the prior art, provides genistin in a kind of detection honey vinegar black soya bean
Method.This method utilizes ultra high efficiency liquid phase color using the genistin in methanol ultrasound assisted extraction method extraction honey vinegar black soya bean
The method that spectrometry instrument is detected its content, through honey vinegar black soya bean extracting solution prepare, standard solution prepare, sample solution prepare and
Ultra Performance Liquid Chromatography instrument detection and etc. obtain the content of genistin in honey vinegar black soya bean.
Above-mentioned purpose of the present invention is achieved by the following technical programs：
A kind of method for detecting genistin in honey vinegar black soya bean, including step in detail below：
S1. prepared by honey vinegar black soya bean extracting solution：The honey vinegar black soya bean of drying is crushed, be sieved to obtain honey vinegar black bean powder, adds in methanol
Solution A ultrasound, supernatant is taken after centrifugation, after the supernatant concentration, genistin extracting solution is obtained with methanol solution B constant volumes；
S2. genistin standard solution is prepared：By genistin standard items methanol solution C constant volumes, it is ultrasonically treated, is made
Genistin standard mother liquor by genistin standard mother liquor methanol solution C constant volumes, is made into genistin titer；
S3. the preparation of sample solution：Dilution is made with methanol solution C in genistin extracting solution obtained by step S1, is made
After it is by filter membrane, preserve for use；
S4. Ultra Performance Liquid Chromatography instrument detects：The sample that step S3 is obtained carries out content detection, by mobile phase to divide
Pure methanol, glacial acetic acid and water are analysed, detects the content of genistin in honey vinegar black soya bean product.
Preferably, the temperature dried described in step S1 is 60~80 DEG C, and the temperature of the ultrasound is 60~65 DEG C, ultrasound
The aperture of the sieve is 40~60 mesh, and time of the ultrasound is 20~40min, the rate of the centrifugation is 8000~
12000r/min, the time of the centrifugation is 8~12min.
Preferably, a concentration of 8~12% of methanol solution B described in step S1, the concentration of the genistin extracting solution
It is 0.8~1.2%.
Preferably, a concentration of 30~90% of methanol solution A described in step S1, the quality and methanol of the honey vinegar black soya bean
The volume ratio of solution A is 1g：16~20mL.
Preferably, temperature ultrasonic described in step S2 is 30~35 DEG C, and the time of the ultrasound is 5~10min.
Preferably, the quality of genistin standard items described in step S2 and the volume ratio of methanol solution C are 1mg：80~
120mL, a concentration of the 40~60% of the methanol solution C, a concentration of 8~12mg/L of the genistin standard mother liquor, institute
State a concentration of 200~300 μ g/L of genistin titer.
Preferably, methanol described in step S4：Glacial acetic acid：The volume ratio of water is (40~60)：1：(50~60).
Preferably, the condition detected described in step S4 is：The flow velocity of the mobile phase be 0.5~0.8ml/min, column temperature
20~30 DEG C, sample temperature is 12~18 DEG C, and wavelength is 250~260nm.
The present invention is measured the genistin content in honey vinegar black soya bean using ultra performance liquid chromatography (UPLC) method, builds
The vertical UPLC methods for measuring the genistin content in honey vinegar black soya bean, it is intended to which the quality for control honey vinegar black soya bean comprehensively provides ginseng
It examines.It is measured using UPLC methods of the present invention:Genistin is linearly closed in the range of 0.1250~2.0000ng/mL (R=0.9998)
System is good, and precision, reproducibility, the rate of recovery meet the requirements, and this method is quick, easy, reproducible, suitable for measuring honey vinegar
The content of genistin in black soya bean.The content of genistin in honey vinegar Semen sojae atricolor extract is carried out with ultra-performance liquid chromatography with
Track, it is easy to be accurate, it is a kind of effective detection method available for measuring genistin content in honey vinegar black soya bean.
Compared with prior art, the invention has the advantages that：
1. the present invention is measured the genistin content in honey vinegar black soya bean using ultra-performance liquid chromatography, establishes and survey
Determine the ultra performance liquid chromatography method of the genistin content in honey vinegar black soya bean, it is intended to which the quality for control honey vinegar black soya bean comprehensively carries
For reference.It is measured using ultra-performance liquid chromatography of the present invention：Genistin is in 0.1250~2.0000ng/mL (R=
0.9998) linear relationship is good in the range of, and precision, reproducibility, the rate of recovery meet the requirements.
2. the method for the present invention is quick, simple result is reliable, convenient and efficient, reproducible, suitable for measuring honey vinegar black soya bean
The content of genistin, genistin content is 134.234~158.321mg/kg in honey vinegar black soya bean product.
3. the method for the present invention the content of genistin in honey vinegar Semen sojae atricolor extract is carried out with ultra-performance liquid chromatography with
Track, it is easy to be accurate, it is a kind of effective detection method available for measuring genistin content in honey vinegar black soya bean.
Figure of description
Fig. 1 is the titer ultra high efficiency liquid phase test map of 12.50 μ g/L of genistin.
Fig. 2 is the ultra high efficiency liquid phase test map of honey vinegar black soya bean sample.
Specific embodiment
The content further illustrated the present invention with reference to specific embodiment, but should not be construed as limiting the invention.
Unless otherwise specified, the conventional means that technological means used in embodiment is well known to those skilled in the art.Except non-specifically
Illustrate, reagent that the present invention uses, method and apparatus is the art conventional reagent, method and apparatus.
Embodiment 1
1. prepared by honey vinegar black soya bean extracting solution：A certain amount of honey vinegar black soya bean produced according to standard Q/LQ00025-2017 is weighed,
60 DEG C of low temperature dryings, spare in baking oven, weighs the honey vinegar black soya bean of 100g drying, is crushed with Multifunction food pulverizer, with 50
The polished standard screen is sieved to obtain honey vinegar black bean powder, weighs 2.5g (being accurate to 0.01g) honey vinegar black bean powders in 250ml conical flask with stopper
In, 30% methanol solution of 45ml is added in, 60 DEG C of extraction 30min in ultrasonic extraction instrument is placed in, in centrifuge, uses 10000r/
The speed centrifugation 10min of min, then takes supernatant, is transferred in the concentrate bottle of 100ml, in a rotary evaporator, setting 60
DEG C, about 20ml is concentrated into, concentrate is transferred in 50mL volumetric flasks, is rinsed concentrate bottle with 10% methanol solution and is settled to scale,
Obtain honey vinegar black soya bean extracting solution.
2. standard solution is prepared：Precision weighs genistin standard items 1mg and is dissolved in 100ml volumetric flasks, with 50% methanol
It is settled to graduation mark, the genistin standard mother liquor of 10mg/L is made in 30 DEG C of hydrotropy 5min of ultrasonic wave, then accurate draws 10mg/L
Genistin standard mother liquor 2.5ml, with 30% methanol constant volume to graduation mark, is made into 250 μ g/L dyewoods in 100ml volumetric flasks
Glycosides titer.
3. the preparation of sample solution：Accurate measurement 1ml step 1 gained honey vinegar black soya bean extracting solutions are dilute in 100ml volumetric flasks
100 times are released, takes the extracting solution after 1ml dilutions, after 0.45 μm of filter membrane, is preserved for use, every part of sample need to prepare 3 parts and put down respectively
Row sample solution carries out assay for Ultra Performance Liquid Chromatography instrument.
4. chromatographic condition：Ultra Performance Liquid Chromatography instrument (U.S. UPLCH-CLASS), CORTECSC18Col μm of n (4.6mm ×
100mm, 2.7 μm) chromatographic column (Ireland)；Mobile phase is methanol：Glacial acetic acid：Water=45：1：55：Flow velocity is 0.5ml/min；Column
25 DEG C of temperature；15 DEG C of sample temperature；Detection wavelength 259nm.
5. Specification Curve of Increasing：Respectively prepared by absorption 8.00,4.00,2.00,1.00,0.50ml (1.4.3.2) step " 2 "
250 μ g/L genistins titers in 10ml volumetric flasks, with 50% methanol constant volume to graduation mark, successively it is a concentration of
200.00th, the titer of 100.00,50.00,25.00,12.50 μ g/L respectively with 10 μ l sample introductions, measures peak area and chromatogram
(Fig. 1)；Using reference substance solution sample size X as abscissa, integrating peak areas value Y draws standard curve for ordinate.
6. sample size measures：10 μ l of sample solution prepared by accurate aspiration step 3 enter liquid chromatograph, in 259nm
Under chromatographic condition, its peak area and chromatogram (Fig. 2) are measured, compared with standard series, measuring genistin content in sample is
134.234mg/kg。
Embodiment 2
1. prepared by honey vinegar black soya bean extracting solution：A certain amount of honey vinegar black soya bean produced according to standard Q/LQ00025-2017 is weighed,
70 DEG C of low temperature dryings, spare in baking oven, weighs the honey vinegar black soya bean of 100g drying, is crushed with Multifunction food pulverizer, with 40
The polished standard screen is sieved to obtain honey vinegar black bean powder, weighs 2.5g (being accurate to 0.01g) honey vinegar black bean powders in 250ml conical flask with stopper
In, 30% methanol solution of 45ml is added in, 60 DEG C of extraction 30min in ultrasonic extraction instrument is placed in, in centrifuge, uses 8000r/
The speed centrifugation 10min of min, then takes supernatant, is transferred in the concentrate bottle of 100ml, in a rotary evaporator, setting 60
DEG C, about 20ml is concentrated into, concentrate is transferred in 50mL volumetric flasks, is rinsed concentrate bottle with 10% methanol solution and is settled to scale,
Obtain honey vinegar black soya bean extracting solution.
2. standard solution is prepared：Precision weighs genistin standard items 1mg and is dissolved in 100ml volumetric flasks, with 50% methanol
It is settled to graduation mark, the genistin standard mother liquor of 10mg/L is made in 30 DEG C of hydrotropy 5min of ultrasonic wave, then accurate draws 10mg/L
Genistin standard mother liquor 2.5ml, with 50% methanol constant volume to graduation mark, is made into 200 μ g/L dyewoods in 100ml volumetric flasks
Glycosides titer.
3. the preparation of sample solution：Accurate measurement 1ml step 1 gained honey vinegar black soya bean extracting solutions are dilute in 100ml volumetric flasks
100 times are released, takes the extracting solution after 1ml dilutions, after 0.45 μm of filter membrane, is preserved for use, every part of sample need to prepare 3 parts and put down respectively
Row sample solution carries out assay for Ultra Performance Liquid Chromatography instrument.
4. chromatographic condition：Ultra Performance Liquid Chromatography instrument (U.S. UPLCH-CLASS), CORTECSC18Col μm of n (4.6mm ×
100mm, 2.7 μm) chromatographic column (Ireland)；Mobile phase is methanol：Glacial acetic acid：Water=45：1：55：Flow velocity is 0.5ml/min；Column
25 DEG C of temperature；15 DEG C of sample temperature；Detection wavelength 259nm.
5. Specification Curve of Increasing：Respectively prepared by absorption 8.00,4.00,2.00,1.00,0.50ml (1.4.3.2) step 2
250 μ g/L genistins titers are in 10ml volumetric flasks, with 50% methanol constant volume to graduation mark, successively it is a concentration of
200.00th, the titer of 100.00,50.00,25.00,12.50 μ g/L respectively with 10 μ l sample introductions, measures peak area and chromatography
Figure；Using reference substance solution sample size X as abscissa, integrating peak areas value Y draws standard curve for ordinate.
6. sample size measures：10 μ l of sample solution prepared by accurate aspiration step 3 enter liquid chromatograph, in 259nm
Under chromatographic condition, its peak area and chromatogram are measured, compared with standard series, measuring genistin content in sample is
158.321mg/kg。
Embodiment 3
1. prepared by honey vinegar black soya bean extracting solution：A certain amount of honey vinegar black soya bean produced according to standard Q/LQ00025-2017 is weighed,
80 DEG C of low temperature dryings, spare in baking oven, weighs the honey vinegar black soya bean of 100g drying, is crushed with Multifunction food pulverizer, with 60
The polished standard screen is sieved to obtain honey vinegar black bean powder, weighs 2.5g (being accurate to 0.01g) honey vinegar black bean powders in 250ml conical flask with stopper
In, 90% methanol solution of 45ml is added in, 60 DEG C of extraction 30min in ultrasonic extraction instrument is placed in, in centrifuge, uses 12000r/
The speed centrifugation 10min of min, then takes supernatant, is transferred in the concentrate bottle of 100ml, in a rotary evaporator, setting 60
DEG C, about 20ml is concentrated into, concentrate is transferred in 50mL volumetric flasks, is rinsed concentrate bottle with 10% methanol solution and is settled to scale,
Obtain honey vinegar black soya bean extracting solution.
2. standard solution is prepared：Precision weighs genistin standard items 1mg and is dissolved in 100ml volumetric flasks, with 50% methanol
It is settled to graduation mark, the genistin standard mother liquor of 10mg/L is made in 30 DEG C of hydrotropy 5min of ultrasonic wave, then accurate draws 10mg/L
Genistin standard mother liquor 2.5ml, with 90% methanol constant volume to graduation mark, is made into 300 μ g/L dyewoods in 100ml volumetric flasks
Glycosides titer.
3. the preparation of sample solution：It is accurate to measure honey vinegar black soya bean extracting solution obtained by 1ml steps " 1 ", in 100ml volumetric flasks
100 times of dilution takes the extracting solution after 1ml dilutions, after 0.45 μm of filter membrane, preserve for use, every part of sample need to prepare 3 parts respectively
Parallel sample solution carries out assay for Ultra Performance Liquid Chromatography instrument.
4. chromatographic condition：Ultra Performance Liquid Chromatography instrument (U.S. UPLCH-CLASS), CORTECSC18Col μm of n (4.6mm ×
100mm, 2.7 μm) chromatographic column (Ireland)；Mobile phase is methanol：Glacial acetic acid：Water=45：1：55：Flow velocity is 0.5ml/min；Column
25 DEG C of temperature；15 DEG C of sample temperature；Detection wavelength 259nm.
5. Specification Curve of Increasing：Respectively prepared by absorption 8.00,4.00,2.00,1.00,0.50ml (1.4.3.2) step 2
250 μ g/L genistins titers are in 10ml volumetric flasks, with 50% methanol constant volume to graduation mark, successively it is a concentration of
200.00th, the titer of 100.00,50.00,25.00,12.50 μ g/L respectively with 10 μ l sample introductions, measures peak area and chromatography
Figure；Using reference substance solution sample size X as abscissa, integrating peak areas value Y draws standard curve for ordinate.
6. sample size measures：10 μ l of sample solution prepared by accurate aspiration step 3 enter liquid chromatograph, in 259nm
Under chromatographic condition, its peak area and chromatogram are measured, compared with standard series, measuring genistin content in sample is
138.141mg/kg。
Above-described embodiment is the preferable embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any Spirit Essences without departing from the present invention with made under principle change, modification, replacement, combine and simplification,
Equivalent substitute mode is should be, is included within protection scope of the present invention.
Claims (8)
- A kind of 1. method for detecting genistin in honey vinegar black soya bean, which is characterized in that including step in detail below：S1. prepared by honey vinegar black soya bean extracting solution：The honey vinegar black soya bean of drying is crushed, be sieved to obtain honey vinegar black bean powder, adds in methanol solution A Ultrasound takes supernatant after centrifugation, and after the supernatant concentration, genistin extracting solution is obtained with methanol solution B constant volumes；S2. genistin standard solution is prepared：By genistin standard items methanol solution C constant volumes, it is ultrasonically treated, dyestuff is made Wooden glycosides standard mother liquor by genistin standard mother liquor methanol solution C constant volumes, is made into genistin titer；S3. the preparation of sample solution：Dilution is made with methanol solution C in genistin extracting solution obtained by step S1, makes it logical After filter membrane, preserve for use；S4. Ultra Performance Liquid Chromatography instrument detects：The sample that step S3 is obtained carries out content detection, pure to analyze by mobile phase Methanol, glacial acetic acid and water detect the content of genistin in honey vinegar black soya bean product.
- 2. the method for genistin in detection honey vinegar black soya bean according to claim 1, which is characterized in that described in step S1 The temperature of drying is 60~80 DEG C, and the temperature of the ultrasound is 60~65 DEG C, and the aperture of the ultrasound sieve is 40~60 mesh, described The time of ultrasound, the rate of the centrifugation was 8000~12000r/min for 20~40min, time of the centrifugation for 8~ 12min。
- 3. the method for genistin in detection honey vinegar black soya bean according to claim 1, which is characterized in that described in step S1 A concentration of the 8~12% of methanol solution B, a concentration of the 0.8~1.2% of the genistin extracting solution.
- 4. the method for genistin in detection honey vinegar black soya bean according to claim 1, which is characterized in that described in step S1 A concentration of the 30~90% of methanol solution A, the quality of the honey vinegar black soya bean and the volume ratio of methanol solution A are 1g：16~20mL.
- 5. the method for genistin in detection honey vinegar black soya bean according to claim 1, which is characterized in that described in step S2 The temperature of ultrasound is 30~35 DEG C, and the time of the ultrasound is 5~10min.
- 6. the method for genistin in detection honey vinegar black soya bean according to claim 1, which is characterized in that described in step S2 The quality of genistin standard items and the volume ratio of methanol solution C are 1mg：80~120mL, the methanol solution C's is a concentration of 40~60%, a concentration of 8~12mg/L of the genistin standard mother liquor, a concentration of the 200 of the genistin titer ~300 μ g/L.
- 7. the method for genistin in detection honey vinegar black soya bean according to claim 1, which is characterized in that described in step S4 Methanol：Glacial acetic acid：The volume ratio of water is (40~60)：1：(50~60).
- 8. the method for genistin in detection honey vinegar black soya bean according to claim 1, which is characterized in that described in step S4 The condition of detection is：The flow velocity of the mobile phase is 0.5~0.8ml/min, and 20~30 DEG C of column temperature, sample temperature is 12~18 DEG C, wavelength is 250~260nm.
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Cited By (1)
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| CN115266975A (en) * | 2022-07-25 | 2022-11-01 | 四川新绿色药业科技发展有限公司 | Method for measuring content of genistin in endothelium corneum gigeriae galli and processed decoction pieces, standard decoction and formula granules thereof |
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