CN103558164B - A kind of method of Simultaneously test grape wine procyanidins and resveratrol - Google Patents
A kind of method of Simultaneously test grape wine procyanidins and resveratrol Download PDFInfo
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- CN103558164B CN103558164B CN201310387469.0A CN201310387469A CN103558164B CN 103558164 B CN103558164 B CN 103558164B CN 201310387469 A CN201310387469 A CN 201310387469A CN 103558164 B CN103558164 B CN 103558164B
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Abstract
The present invention relates to Food Inspection and analysis field, be specially a kind of method measuring grape wine procyanidins and resveratrol.The present invention is intended to adopt high performance liquid chromatography series connection secondary array tube detecting device and fluorescence detector, directly measures the procyanidin in grape wine and resveratrol.Wherein fluorescence detector can by the adjustment of excitation wavelength and emission wavelength, reduce the interference of impurity, thus there is high selectivity, its sensitivity is far above UV-detector, can accurately, reliably to substance quantitative analysis, accurately and reliably can detect and analyze procyanidin in grape wine and these two kinds of functional components of resveratrol.
Description
Technical field
The present invention relates to Food Inspection and analysis field, be specially a kind of method measuring grape wine procyanidins and resveratrol.
Background technology
Procyanidin (procyanidins, referred to as PC) is the general name of flavan-3-alcohol derivant, mainly contains the monomer of catechin, dimer, tripolymer to ten dimer combinations form.Its feature is efficient, low toxicity, high bioavailability, and half lethal dose is LD50=3g/kg, is the extremely strong activity in vivo function factor of one of constantly research and development in recent years.Much research shows, PC is the antioxidant that scavenging free radicals is very strong, and it is anti-oxidant, and the ability of scavenging free radicals is 50 times of VE, 20 times of Vc, has caused the very big concern of people.It is not only distributed widely in plant, as ginkgo, Japanese Thuja, North America arborvitae, Turkey arbor-vitae, flower chess pine, white birch boorishly raw thorn certain herbaceous plants with big flowers, Japanese thick grass, ear leaf cassia angustifolia, maritime pine, rheum officinale, larch, lotus pod etc., also be present in some beverage, veterinary antibiotics and grain, as grape, grape wine, apple, cider, beer, tealeaves, barley, Fructus Hordei Germinatus, jowar, legume, manaca, weeds poison, cherry etc.In the numerous natural product related to, the PC rich content in grape and goods thereof, therefore study more.The method for quantitatively determining of this material comprises titrimetry, optical spectroscopy, electrochemical process, chemoluminescence method, Capillary Electrophoresis and the HPLC method on molysite acidified degradation basis at present.Resveratrol (resveratrol), also known as resvertrol, is a Plant Hormone, it should be noted that it can in brewing grape process, because the catalytic reaction of biochemical enzyme generates.The content of high-quality wine resveratrol is higher, and blended liquor (grape juice adds alcohol) Resveratrol content is zero.At present, research shows that it has antitumor, anti-cardiovascular disease, treatment sudden change, anti-oxidant, antimicrobial antiphlogistic, protects the liver, the bio-pharmacology such as cell death inducing and estrogen adjustment is active.The assay method of current this material has high performance liquid chromatography, fluorescence method, chemoluminescence method, capillary electrophoresis, electrochemical process.
Application number be 201210184125.5 Chinese patent once disclosed a kind of method utilizing HPLC to measure 17 kinds of aldehydes matters in grape and citrusfruit, namely adopt high performance liquid chromatography to the gallic acid in grape and citrusfruit, chlorogenic acid, caffeic acid, syringic acid, coumaric acid, rutin, forulic acid, aurantiamarin, aurantiin, salicylic acid, Quercetin, Nobiletin, hesperetin, catechin, epicatechin, benzoic acid, resveratrol measures, by UV-detector qualitative and quantitative analysis, but employing UV-detector, the extraction and purification program of series of complex need be carried out to sample, impurity interference is more, and owing to have employed loaded down with trivial details extraction pretreatment technology, sample is lost, and measuring accuracy is not high.
Summary of the invention
For the present invention that solves the problem is intended to adopt high performance liquid chromatography series connection secondary array tube detecting device and fluorescence detector, directly measure the procyanidin in grape wine and resveratrol.Wherein fluorescence detector by the adjustment of excitation wavelength and emission wavelength, can reduce the interference of impurity, thus has high selectivity, its sensitivity far above UV-detector, can accurately, reliably to substance quantitative analysis.
Concrete technical scheme is as follows: a kind of method measuring grape wine procyanidins and resveratrol, comprises the following steps:
(1) sample preparation
Get the organic filter membrane standby of the appropriate mistake of grape wine and be used as testing sample;
(2) mensuration of high performance liquid chromatograph system
Getting appropriate testing sample adds in high performance liquid chromatograph, mobile phase is utilized to carry out gradient elution to it, then diode array detector is utilized to carry out qualitative analysis to the procyanidin in sample and resveratrol, the principle of work of diode array detector enters injection device again after polychromatic light is absorbed by compositional selecting by sample cell, being radiated on diode array device makes the light intensity of each nano wave length be transformed into corresponding electrical signal intensity, namely the absorption spectrum of component is obtained, thus obtain the structural information of specific components, the structure contributing to unknown component or complex component is determined, therefore, can according to the chromatographic retention (R of object
t) more preliminary qualitative to compound with standard substance retention time, utilize object more qualitative with the absorption spectrum of standard substance simultaneously, dual qualitative more accurately and reliably, finally recycle fluorescence detector and carry out quantitative test to the procyanidin in sample and resveratrol, fluorescence detector utilizes the compound with special construction to receive ultraviolet excitation (E
ex) after can launch the light (E longer than excitation source wavelength
em) fluorescent characteristic to detect, many organic compounds have natural fluoresence activity, wherein with the compound of aromatic group, there is fluorescence activity very strong, at certain condition, fluorescence intensity is directly proportional to material concentration, can be determined the concentration of material by the intensity size of fluorescence, it is particularly suitable for condensed-nuclei aromatics, steride etc., and procyanidin and resveratrol all have condensed-nuclei aromatics structure.Fluorescence detector is highly sensitive, selectivity good, exceeds 2-3 the order of magnitude than UV-detector;
(3) quantitative by typical curve
Take appropriate procyanidin and resveratrol standard items respectively, methyl alcohol is utilized to dissolve and constant volume, configure the mixed solution of two standard items successively, wherein, procyanidin concentration is followed successively by 0.0100, 0.0500, 0.100, 0.150, 0.200, 0.250mg/ml, the concentration of resveratrol is followed successively by 1.00, 5.00, 10.0, 15.0, 20.0, 25.0g/ml, mixed solution is repeated step (2) to measure, the data obtained take peak area as ordinate, concentration is horizontal ordinate, obtain peak area-concentration standard curve, curve determines the content of sample procyanidins and resveratrol thus.
During high performance liquid chromatograph system measurement, liquid phase chromatogram condition is: chromatographic column is ultimateXB-C18 chromatographic column, and its specification is 150mm × 4.6mm, i.d..5 μm; Mobile phase A is 0.1% formic acid, and Mobile phase B is acetonitrile, gradient elution program: 0min-10min, 10%B, 10min-11min, 10%B → 25%B, 11min-23min, 25%B, 23min-24min, 25%B → 10%B, 24min-30min, 10%B; Flow velocity is 1ml/min, and column temperature is 30 DEG C, and sample size is 20 μ l; The scanning wavelength of diode array detector is 210nm-600nm; Fluorescence detector condition is: excitation wavelength during 0min-11min and emission wavelength are 280nm, 305nm respectively, and the excitation wavelength of 11min-30min and emission wavelength are 320nm, 370nm respectively.
By technique scheme, assay method provided by the present invention has high selectivity, high sensitivity, accurately and reliably can detect and analyze procyanidin in grape wine and these two kinds of functional components of resveratrol.
Accompanying drawing explanation
Fig. 1 is standard items diode array detector chromatogram;
Fig. 2 is sample diode array detector chromatogram;
Fig. 3 is standard items diode array detector spectrogram;
Fig. 4 is sample diode array detector spectrogram;
Fig. 5 is standard items fluorescence detector chromatogram;
Fig. 6 is fluorescent detecting device chromatogram;
Fig. 7 is procyanidin typical curve;
Fig. 8 is resveratrol typical curve.
Embodiment
One, measure adopt equipment and medicine as follows: Waters2695 type high performance liquid chromatograph system, Waters2996 type diode array detector, waters2475 fluorescence detector, Empower chromatographic work station and data handling system; Procyanidin (> 95%, the natural research and development company limited of Tianjin spike); Resveratrol (Nat'l Pharmaceutical & Biological Products Control Institute, lot number: 111535); Saintcoeur board grape wine one bottle.
Two, determination step is as follows:
(1) sample preparation
Respectively get commercially available saintcoeur board grape wine 1ml cross 0.22 μm of organic filter membrane after organic filter membrane standby for subsequent use be used as testing sample;
(2) mensuration of high performance liquid chromatograph system
Getting 1ml testing sample adds in high performance liquid chromatograph, and utilize mobile phase to carry out gradient elution to it, liquid phase chromatogram condition is: chromatographic column is ultimateXB-C18 chromatographic column, and its specification is 150mm × 4.6mm, i.d..5 μm; Mobile phase A is 0.1% formic acid, and Mobile phase B is acetonitrile, gradient elution program: 0min-10min, 10%B, 10min-11min, 10%B → 25%B, 11min-23min, 25%B, 23min-24min, 25%B → 10%B, 24min-30min, 10%B; Flow velocity is 1ml/min, and column temperature is 30 DEG C, and sample size is 20 μ l.
Then diode array detector is utilized to carry out qualitative analysis to the procyanidin in sample and resveratrol, when this measures, the scanning wavelength 210nm-600nm of diode array detector; Finally recycle fluorescence detector and quantitative test is carried out to the procyanidin in sample and resveratrol.When this measures, the condition of fluorescence detector is: the excitation wavelength (Eex) during 0min-11min and emission wavelength (Eem) are 280nm, 305nm respectively, and excitation wavelength (Eex) and the emission wavelength (Eem) of 11min-30min are 320nm, 370nm respectively.
(3) quantitative by typical curve
Precision takes procyanidin 0.0300g in 10ml volumetric flask, dissolves and constant volume with methyl alcohol.Obtain 3.00mg/ml procyanidin standard solution.Precision takes 0.0150g resveratrol standard items in 10ml volumetric flask, dissolves and constant volume, join to obtain 1.50mg/ml resveratrol standard reserving solution with methyl alcohol, then obtains standard solution 0.300mg/ml with methanol dilution 5 times.With methanol dilution, configure the mixed solution of two standard items successively.Wherein, procyanidin concentration is 0.0100,0.0500,0.100,0.150,0.200, the standard working solution of 0.250mg/ml, the concentration of resveratrol is 1.00,5.00,10.0,15.0,20.0,25.0 μ g/ml, mixed solution is repeated step (2) to measure, the data obtained take peak area as ordinate, concentration is horizontal ordinate, obtains peak area-concentration standard curve.
(4) data analysis
As Fig. 1 and Fig. 2 contrasts known, on diode array detector chromatogram, the retention time of standard items procyanidin and resveratrol is respectively 8.489 minutes and 20.108 minutes, and the retention time of sample procyanidins and resveratrol is respectively 8.504 minutes and 20.504 minutes, this and standard items are close; As shown in Figure 3 and Figure 4, on diode array detector spectrogram, the maximum absorption wavelength of standard items procyanidin and resveratrol is 280nm and 306nm respectively, and the maximum absorption wavelength of sample procyanidins and resveratrol is respectively 279nm and 306nm, this and standard items are close, therefore, these two kinds of compositions of qualitative analysis sample are carried out by spectrum and chromatogram.
As shown in Figure 5 and Figure 6, the retention time of standard items procyanidin and resveratrol is respectively 8.489 minutes and 20.108 minutes, and the retention time of sample procyanidins and resveratrol is respectively 8.517 minutes and 20.237 minutes, as shown in Figure 7 and Figure 8, gained peak area-concentration standard curve is respectively:
Procyanidin: Y=6.00*10
8x+3.54*10
5, coefficient R=0.9989;
Resveratrol: Y=3.00*10
9x+5.60*10
6, coefficient R=0.9991;
The content recording 1mlsaintcoeur board grape wine procyanidins and resveratrol is thus 1.326mg/ml and 8.585ug/ml.
Carry out methodological study to the method that this measures procyanidin and resveratrol, acquired results is as following table:
As seen from the above table, this method measuring procyanidin and resveratrol is highly sensitive, and separating power is strong, the qualitative analysis is accurate, detectability is low, can detect the content of grape wine procyanidins and resveratrol rapidly simultaneously.
Claims (3)
1. a method for Simultaneously test grape wine procyanidins and resveratrol, is characterized in that, comprises the following steps:
(1) sample preparation
Get the organic filter membrane standby of the appropriate mistake of grape wine and be used as testing sample;
(2) mensuration of high performance liquid chromatograph system
Getting appropriate testing sample adds in high performance liquid chromatograph, mobile phase is utilized to carry out gradient elution to it, then utilize diode array detector to carry out qualitative analysis to the procyanidin in sample and resveratrol, finally recycle fluorescence detector and quantitative test is carried out to the procyanidin in sample and resveratrol;
(3) quantitative by typical curve
Take appropriate procyanidin and resveratrol standard items respectively, methyl alcohol is utilized to dissolve and constant volume, configure the mixed solution of two standard items successively, wherein, procyanidin concentration is followed successively by 0.0100, 0.0500, 0.100, 0.150, 0.200, 0.250mg/ml, the concentration of resveratrol is followed successively by 1.00, 5.00, 10.0, 15.0, 20.0, 25.0g/ml, mixed solution is repeated step (2) to measure, the data obtained take peak area as ordinate, concentration is horizontal ordinate, obtain peak area-concentration standard curve, curve determines the content of sample procyanidins and resveratrol thus, wherein during high performance liquid chromatograph system measurement, liquid phase chromatogram condition is: chromatographic column is ultimateXB-C18 chromatographic column, its specification is 150mm × 4.6mm, i.d.5 μm, mobile phase A is 0.1% formic acid, and Mobile phase B is acetonitrile, gradient elution program: 0min-10min, 10%B, 10min-11min, 10%B → 25%B, 11min-23min, 25%B, 23min-24min, 25%B → 10%B, 24min-30min, 10%B, flow velocity is 1ml/min, and column temperature is 30 DEG C, and sample size is 20 μ l.
2. a kind of method of Simultaneously test grape wine procyanidins and resveratrol as claimed in claim 1, it is characterized in that, the scanning wavelength of diode array detector is 210nm-600nm.
3. a kind of method of Simultaneously test grape wine procyanidins and resveratrol as claimed in claim 1, it is characterized in that, fluorescence detector condition is: excitation wavelength during 0min-11min and emission wavelength are 280nm, 305nm respectively, and the excitation wavelength of 11min-30min and emission wavelength are 320nm, 370nm respectively.
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| CN109682669A (en) * | 2018-12-24 | 2019-04-26 | 久为利华（天津）文化发展有限公司 | The detection method of anthocyanidin content in grape wine |
| CN111579507A (en) * | 2020-05-22 | 2020-08-25 | 国珍健康科技（北京）有限公司 | Sulfuric acid-vanillin method for detecting content of procyanidine |
| CN112033941B (en) * | 2020-06-30 | 2021-03-30 | 中南民族大学 | Method for rapidly detecting resveratrol in food |
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