CN102636526A - Electrochemical method for rapidly detecting oxidation resistance of bee product - Google Patents
Electrochemical method for rapidly detecting oxidation resistance of bee product Download PDFInfo
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- CN102636526A CN102636526A CN201210098387XA CN201210098387A CN102636526A CN 102636526 A CN102636526 A CN 102636526A CN 201210098387X A CN201210098387X A CN 201210098387XA CN 201210098387 A CN201210098387 A CN 201210098387A CN 102636526 A CN102636526 A CN 102636526A
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Abstract
The invention provides an electrochemical method for rapidly detecting the oxidation resistance of a bee product. The electrochemical method specifically comprises the following steps of: pre-treating an electrode; extracting phenolic acids substances in the bee product by an ethyl acetate method; taking gallic acid as a standard product to prepare a standard curve; and measuring the content of anti-oxidization substances in the bee product by utilizing a glassy carbon electrode, particularly, determining the content of the anti-oxidization substances by the size of an oxidization reduction current peak value of a solution to be detected. The electrochemical method has the advantages of simplicity in operation and lower cost and the field detection is easy to realize.
Description
Technical field
What the present invention relates to is the detection method of the antioxygenic property of bee product, specifically is a kind of electrochemical method that detects bee product phenolic acid content.
Background technology
Bee products such as honey, melissa powder, propolis have the various biological activity; Like anti-oxidant, anti-inflammation, moisten, antithrombotic, reduction cholesterol, promote cytothesis etc.; Its main cause is to have a large amount of phenolic compounds and ascorbic acid, carotenoid, hydrogen peroxidase, peroxidase etc. in the bee product, and these materials have very strong antioxidation activity.Antioxidation activity can protect biosome to avoid the attack of free radical, can reduce the occurrence probability of cancer, cardiovascular and cerebrovascular disease and diabetes.The phenolic acid that the total antioxygenic property of bee product contains with it is closely related.Because the flower source category is various, each flower source color is different with composition, and the chemical composition and the characteristic of different bee products also are not quite similar, and its antioxidation activity is also variant.
At present, in the analysis bee product there be the main method of antioxygenic property: colourimetry, thin-layered chromatography, vapor-phase chromatography, high performance liquid chromatography and capillary electrophoresis etc.These methods require inspection apparatus accurate, and testing cost is high, complicated operation.In addition, these methods can only be measured the inoxidizability of single structure material usually, but mostly the antioxidant content in the bee product is the potpourri of phenolic acid, so its analysis to the antioxidant content of bee product is not comprehensive.That electrochemical method has is easy and simple to handle, detect fast, the detection sensitivity advantages of higher, and it is used the mensuration to phenolic acid, can avoid the limitation of traditional optical method, analyzes the content of various phenolic acids, thereby measures its antioxygenic property.The report that electrochemical method determining bee product antioxidation activity is not arranged at present, as yet.
Summary of the invention
The object of the present invention is to provide the electrochemical method of antioxidation activity in a kind of highly sensitive, good stability, the simple mensuration bee product, particularly is the electrochemical determination method of the content of the phenolic acid in the bee product.
The present invention realizes through following technical scheme.
A kind of electrochemical method of antioxidation activity of fast detecting bee product comprises the steps:
The pre-service of step a), glass-carbon electrode;
Step b), ethyl acetate method are extracted the phenolic acid in the said bee product;
Step c), be standard items, formulate typical curve with the gallic acid;
Step d), measure the content of the phenolic acid in the said bee product with said glass-carbon electrode.
During practical implementation of the present invention, preferably, said step a) may further comprise the steps: said glass-carbon electrode is used Al on chamois leather
2O
3Powder is polished to minute surface, uses distilled water flushing, uses ultrasonic cleaning 10～30min again; It is that 1: 1 nitric acid, acetone soln respectively soaks 3～15min that said glass-carbon electrode after cleaning is put into volume ratio successively, uses the said glass-carbon electrode of distilled water flushing then; Said glass-carbon electrode is dried under infrared lamp.
During practical implementation of the present invention, preferably, said step b) may further comprise the steps: measure said bee product and be put in the container, the distilled water of adding is stirred to dissolving and forms solution; In said solution, add ethyl acetate, extract three times, merge extraction gained supernatant I; I extracts with distilled water with the gained supernatant, gets supernatant II, adds the ethanolic solution constant volume among the said supernatant II, obtains solution to be measured; Said solution to be measured is placed preservation in the refrigerator.
Further, in the said step b): the amount of measuring of said bee product is 5～10g; The amount of dissolving the distilled water that said bee product adds is 20～30mL; The use amount of said ethyl acetate is 10～20mL; The volumetric concentration of said ethanolic solution is 60～80%, and consumption is 10～20mL; The storage temperature of said solution to be measured in said refrigerator is 0～4 ℃.
During practical implementation of the present invention, preferably, said step c) may further comprise the steps: take by weighing the gallic acid standard items, be dissolved in the ethanolic solution, be configured to the gallic acid titer of variable concentrations; In the electro-chemical test cup, add the HAc-NaAc damping fluid as supporting solution; The gallic acid titer that in the electro-chemical test cup, adds the variable concentrations of preparation then; With the oxidation peak current value of differentiated pulse voltammetry record gallic acid, according to gallic acid concentration value and corresponding oxidation peak current value drawing standard curve.
Further, the volumetric concentration of said ethanol is 60～80%, the HAc-NaAc damping fluid of the 0.1-0.2M that said HAc-NaAc damping fluid is the 5mL of pH4.0 (in the content of NaAc); Described differentiated pulse voltammetry condition is: scanning potential range 0～0.8V, enrichment time 90～120s, sweep velocity 50～100mV/s.
During practical implementation of the present invention; Preferably; Said step d) may further comprise the steps: in the electro-chemical test cup, add the HAc-NaAc damping fluid as supporting solution; In said electro-chemical test cup, add said solution to be measured then and write down the oxidation peak current value of said solution to be measured, calculate the content of phenolic acid in the said solution to be measured, represent with the relative quantity of gallic acid according to said typical curve with the differentiated pulse voltammetry.
Further; The HAc-NaAc damping fluid of the 0.1-0.2M that said HAc-NaAc damping fluid is the 5mL of pH4.0 (in the content of NaAc); The consumption of said solution to be measured is 100 μ L; Described differentiated pulse voltammetry condition is: scanning potential range 0～0.8V, enrichment time 90～120s, sweep velocity 50～100mV/s.
During practical implementation of the present invention, preferably, said bee product is honey, melissa powder or propolis.
The present invention has following beneficial effect: the present invention extracts and measures the phenolic acids in the bee product, and the size of the redox current peak through detecting said solution to be measured is calculated the content of the phenolic acid of bee product according to typical curve.The present invention is easy and simple to handle, and cost is lower, is easy to realize on-the-spot the detection.
Below will combine accompanying drawing that the technique effect of design of the present invention, concrete structure and generation is described further, so that those skilled in the art understands the object of the invention, characteristic and effect fully.
Description of drawings
Fig. 1 is the oxidation peak current value of differentiated pulse voltammetry record gallic acid, and from 1 to 9 concentration is respectively 0 μ g/g, 0.5 μ g/g, 1.0 μ g/g, 1.25 μ g/g, 1.75 μ g/g, 2.25 μ g/g, 2.75 μ g/g, 3.25 μ g/g and 3.75 μ g/g.
Fig. 2 is the typical curve of drawing according to gallic acid concentration value and corresponding oxidation peak current.
Fig. 3 is the oxidation peak current value of differentiated pulse voltammetry record loquat honey.
Fig. 4 is the oxidation peak current value of differentiated pulse voltammetry record mother chrysanthemum and motherwort honey.
Fig. 5 is the oxidation peak current value of differentiated pulse voltammetry record Chinese milk vetch honey.
Embodiment
The experimental technique of unreceipted actual conditions in the following example is usually according to normal condition.
A kind of electrochemical method of antioxidation activity of fast detecting bee product comprises the steps:
The pre-service of step a), glass-carbon electrode is with activated electrode;
Step b), ethyl acetate method are extracted the phenolic acid in the bee product;
Step c), be standard items, formulate typical curve with the gallic acid;
Step d), measure the content of the phenolic acid in the bee product with glass-carbon electrode.
Step a) may further comprise the steps: glass-carbon electrode is used Al on chamois leather
2O
3Powder is polished to minute surface, uses distilled water flushing, uses ultrasonic cleaning 10～30min again; It is that 1: 1 nitric acid, acetone soln respectively soaks 3～15min that glass-carbon electrode after cleaning is put into volume ratio successively, uses the distilled water flushing glass-carbon electrode then; Glass-carbon electrode is dried under infrared lamp.
Step b) may further comprise the steps: measure 5～10g bee product and be put in the container, add the distilled water of 20～30mL, be stirred to dissolving and form solution; In solution, add 10～20mL ethyl acetate, extract three times, merge extraction gained supernatant I; I extracts with distilled water with the gained supernatant, gets supernatant II, and surplus liquid reclaims ethyl acetate with Rotary Evaporators, and adding volumetric concentration among the supernatant II is 60～80% ethanolic solutions, 10～20mL constant volume, obtains solution to be measured; Solution to be measured is placed the inherent 4 ℃ of preservations of refrigerator.
Step c) may further comprise the steps: take by weighing the gallic acid standard items, be dissolved in volumetric concentration and be in 60～80% the ethanolic solution, be configured to the gallic acid titer of variable concentrations; The HAc-NaAc damping fluid of the 0.1M of the 5mL of adding pH4.0 is as supporting solution in the electro-chemical test cup; The gallic acid titer that in the electro-chemical test cup, adds the variable concentrations of preparation then; With the oxidation peak current value of differentiated pulse voltammetry record gallic acid, according to gallic acid concentration value and corresponding oxidation peak current value drawing standard curve.Differentiated pulse voltammetry condition is: scanning potential range 0～0.8V, enrichment time 90～120s, sweep velocity 50～100mV/s.
Step d) may further comprise the steps: the acetate buffer solution of the 0.1M (pH4.0) of adding 5mL is as supporting solution in the electro-chemical test cup; In the electro-chemical test cup, add 100 μ L solution to be measured then; Write down the oxidation peak current value of said solution to be measured with the differentiated pulse voltammetry; Calculate the content of phenolic acid in the said solution to be measured according to typical curve, represent with the relative quantity of gallic acid.Differentiated pulse voltammetry condition is: scanning potential range 0～0.8V, enrichment time 90～120s, sweep velocity 50～100mV/s.
Bee product is honey, melissa powder or propolis.
Embodiment 1
The pre-service of step a), glass-carbon electrode: glass-carbon electrode is used Al on chamois leather
2O
3Powder is polished to minute surface, uses distilled water flushing, uses ultrasonic cleaning 30min again; It is that 1: 1 nitric acid, acetone soln respectively soaks 15min that glass-carbon electrode after cleaning is put into volume ratio successively, uses the distilled water flushing electrode then; Electrode is dried under infrared lamp.
Step b), the ethyl acetate method is extracted the phenolic acid in the bee product: loquat honey (in November, the 2010 production) 10g of measuring gloomy honeybee garden, Shanghai is put in the beaker, adds the distilled water of 10mL, is stirred to dissolving and forms solution; In solution, add the ethyl acetate of 10mL, extract three times, merge extraction gained supernatant I.Gained supernatant I with 30mL distilled water extraction, is got supernatant II, and surplus liquid reclaims ethyl acetate with 50 ℃ of Rotary Evaporators, 60 commentaries on classics/min, and supernatant II adding volumetric concentration is that 70% ethanol is settled to 20mL, obtains solution to be measured; Solution to be measured is placed the inherent 4 ℃ of preservations of refrigerator.
Step c); The formulation of gallic acid typical curve: take by weighing the gallic acid standard items; Be dissolved in volumetric concentration and be in 70% the ethanolic solution, be configured to the gallic acid titer that concentration is respectively 0 μ g/g, 0.5 μ g/g, 1.0 μ g/g, 1.25 μ g/g, 1.75 μ g/g, 2.25 μ g/g, 2.75 μ g/g, 3.25 μ g/g and 3.75 μ g/g; The HAc-NaAc damping fluid of the 0.1M of the 5mL of adding pH4.0 is as supporting solution in the electro-chemical test cup; In the electro-chemical test cup, add above-mentioned gallic acid titer then respectively; Oxidation peak current value with differentiated pulse voltammetry record gallic acid; Scanning potential range 0～0.8V, enrichment time 120s, sweep velocity 50mV/s.According to gallic acid concentration value and corresponding oxidation peak current value drawing standard curve (Fig. 2), equation of linear regression be I (μ A)=-0.1908-0.8916C (μ g/g), r
2=0.9946. I represents oxidation peak current, and C represents gallic acid concentration, r
2Represent linearly dependent coefficient.
Step d); The acetate buffer solution of the 0.1M (pH4.0) of adding 5mL is as supporting solution in the electro-chemical test cup; In the electro-chemical test cup, add 100 μ L solution to be measured then, write down the oxidation peak current value of solution to be measured, scan potential range 0～0.8V with the differentiated pulse voltammetry; Enrichment time 120s, sweep velocity 50mV/s.The content that calculates phenolic acid in the loquat honey according to typical curve is 105.28 ± 5.60 μ g/g.
Embodiment 2
The pre-service of step a), glass-carbon electrode: with embodiment 1.
Step b), ethyl acetate method are extracted the phenolic acid in the bee product: mother chrysanthemum honey (in November, 2010 production) and motherwort honey (in October, 2010 production) each 10g of measuring gloomy honeybee garden, Shanghai are put in the beaker respectively; The distilled water that adds 10mL is stirred to dissolving and forms solution.In solution, add the ethyl acetate of 10mL, extract three times, merge extraction gained supernatant I.Gained supernatant I with 30mL distilled water extraction, is got supernatant II, and surplus liquid reclaims ethyl acetate with Rotary Evaporators, and it is that 70% ethanol is settled to 20mL that supernatant II adds volumetric concentration respectively, obtains solution to be measured.Solution to be measured is placed the inherent 4 ℃ of preservations of refrigerator.
The formulation of step c), gallic acid typical curve: with embodiment 1.
Step d), in the electro-chemical test cup, add 5mL the acetate buffer solution of 0.1M (pH4.0) as supporting solution; In the electro-chemical test cup, add 100 μ L solution to be measured then; Write down the oxidation peak current value of solution to be measured with the differentiated pulse voltammetry; Scanning potential range 0～0.8V, enrichment time 120s, sweep velocity 50mV/s.Calculate according to typical curve, the content of phenolic acid is 140.55 ± 6.21 μ g/g in the mother chrysanthemum nectar, and the content of phenolic acid is 237.27 ± 10.59 μ g/g in the motherwort honey.
Embodiment 3
The pre-service of step a), glass-carbon electrode: with embodiment 1.
Step b), ethyl acetate method are extracted the phenolic acid in the bee product: longan honey (in September, the 2010 production) 10g of measuring Wang Shi honeybee garden, Jiangxi is put in the beaker, adds the distilled water of 10mL, is stirred to dissolving and forms solution.In solution, add the ethyl acetate of 10mL, extract three times, merge extraction gained supernatant I.Gained supernatant I with the extraction of 30mL distilled water, is got supernatant II, and surplus liquid reclaims ethyl acetate with Rotary Evaporators, and it is that 70% ethanol is settled to 20mL that supernatant II adds volumetric concentration, obtains solution to be measured.The solution for preparing is placed the inherent 4 ℃ of preservations of refrigerator.
The formulation of step c), gallic acid typical curve: with embodiment 1.
Step d), in the electro-chemical test cup, add 5mL the acetate buffer solution of 0.1M (pH4.0) as supporting solution; In the electro-chemical test cup, add 100 μ L solution to be measured then; Write down the oxidation peak current value of solution to be measured with the differentiated pulse voltammetry; Scanning potential range 0～0.8V, enrichment time 120s, sweep velocity 50mV/s.Calculate according to typical curve, the content of phenolic acid is 162.21 ± 5.36 μ g/g in the longan honey.
Checking embodiment
Adopt the Folin-Ciocalteu method to measure the content of phenolic acid in the bee product, checking the inventive method.Its reaction principle is that phenolic compound can generate blue compound with the acid of the tungsten in Folin-Ciocalteu reagent reduction under alkali condition, and the depth of color and phenol content are proportionate, and absorption maximum is arranged about 760nm.
Accurately pipette the prepared solution to be measured of step b) among the 1mL embodiment 1～3; The Folin-Ciocalteu reagent that adds 1mL, mixing adds the sodium carbonate liquor of 5mL 1mol/L then; Be settled to 10mL with distilled water; Fully mixing at dark condition held 1h, is to measure OD value under the 760nm at wavelength.The reference substance gallic acid is configured to the standard solution of 0.1mg/mL, formulates typical curve according to the method described above, among the embodiment 1～3 in the prepared solution to be measured of step b) the content of phenolic acid in typical curve, check in corresponding value.
Measure the result and show that the content of the phenolic acid in loquat, mother chrysanthemum, motherwort and the longan honey is respectively 108.05 ± 9.00 μ g/g, 145.87 ± 9.26 μ g/g, 201.32 ± 0.39 μ g/g and 162.41 ± 9.52 μ g/g.The content of the phenolic acid in loquat, mother chrysanthemum, motherwort and the longan honey that records according to embodiment 1～3; The content value of the phenolic acid that records with this checking embodiment; Through the SPSS16.0 software analysis; Two kinds of methods and results there was no significant differences explain that method of the present invention can record the content of the phenolic acid of bee product exactly.
More than describe preferred embodiment of the present invention in detail.The ordinary skill that should be appreciated that this area need not creative work and just can design according to the present invention make many modifications and variation.Therefore, all technician in the art all should be in the determined protection domain by claims under this invention's idea on the basis of existing technology through the available technical scheme of logical analysis, reasoning, or a limited experiment.
Claims (9)
1. the electrochemical method of the antioxidation activity of a fast detecting bee product is characterized in that, comprises the steps:
The pre-service of step a), glass-carbon electrode;
Step b), ethyl acetate method are extracted the phenolic acid in the said bee product;
Step c), be standard items, formulate typical curve with the gallic acid;
Step d), measure the content of the phenolic acid in the said bee product with said glass-carbon electrode.
2. the electrochemical method of the antioxidation activity of fast detecting bee product as claimed in claim 1, wherein, said step a) may further comprise the steps: said glass-carbon electrode is used Al on chamois leather
2O
3Powder is polished to minute surface; Use distilled water flushing; Use ultrasonic cleaning 10～30min again; It is that 1: 1 nitric acid, acetone soln respectively soaks 3～15min that said glass-carbon electrode after cleaning is put into volume ratio successively, uses the said glass-carbon electrode of distilled water flushing then, and said glass-carbon electrode is dried under infrared lamp.
3. the electrochemical method of the antioxidation activity of detection bee product according to claim 1, wherein, said step b) may further comprise the steps: measure said bee product and be put in the container, the distilled water of adding is stirred to dissolving and forms solution; In said solution, add ethyl acetate, extract three times, merge extraction gained supernatant I; I extracts with distilled water with the gained supernatant, gets supernatant II, adds the ethanolic solution constant volume among the said supernatant II, obtains solution to be measured; Said solution to be measured is placed preservation in the refrigerator.
4. the electrochemical method of the antioxidation activity of detection bee product according to claim 3, wherein, in the said step b): the amount of measuring of said bee product is 5～10g; The amount of dissolving the distilled water that said bee product adds is 20～30mL; The use amount of said ethyl acetate is 10～20mL; The volumetric concentration of said ethanolic solution is 60～80%, and consumption is 10～20mL; The storage temperature of said solution to be measured in said refrigerator is 0～4 ℃.
5. the electrochemical method of the antioxidation activity of detection bee product according to claim 1, wherein, said step c) may further comprise the steps: take by weighing the gallic acid standard items, be dissolved in the ethanolic solution, be configured to the gallic acid titer of variable concentrations; In the electro-chemical test cup, add the HAc-NaAc damping fluid as supporting solution; The gallic acid titer that in the electro-chemical test cup, adds the variable concentrations of preparation then; With the oxidation peak current value of differentiated pulse voltammetry record gallic acid, according to gallic acid concentration value and corresponding oxidation peak current value drawing standard curve.
6. the electrochemical method of the antioxidation activity of detection bee product according to claim 5, wherein, the volumetric concentration of said ethanol is 60～80%, the HAc-NaAc damping fluid of the 0.1-0.2M of the 5mL that said HAc-NaAc damping fluid is pH4.0; Described differentiated pulse voltammetry condition is: scanning potential range 0～0.8V, enrichment time 90～120s, sweep velocity 50～100mV/s.
7. the electrochemical method of the antioxidation activity of detection bee product according to claim 3; Wherein, Said step d) may further comprise the steps: in the electro-chemical test cup, add the HAc-NaAc damping fluid as supporting solution; In said electro-chemical test cup, add said solution to be measured then and write down the oxidation peak current value of said solution to be measured, calculate the content of phenolic acid in the said solution to be measured according to said typical curve with the differentiated pulse voltammetry.
8. the electrochemical method of the antioxidation activity of detection bee product according to claim 7; Wherein, The HAc-NaAc damping fluid of the 0.1-0.2M of the 5mL that said HAc-NaAc damping fluid is pH4.0, the consumption of said solution to be measured are 100 μ L, and described differentiated pulse voltammetry condition is: scanning potential range 0～0.8V; Enrichment time 90～120s, sweep velocity 50～100mV/s.
9. the electrochemical method of the antioxidation activity of detection bee product according to claim 1, wherein, said bee product is honey, melissa powder or propolis.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104713990A (en) * | 2015-02-11 | 2015-06-17 | 江苏出入境检验检疫局动植物与食品检测中心 | Method for identifying Manuka Honey |
| CN108982640A (en) * | 2018-06-28 | 2018-12-11 | 杭州电子科技大学 | A kind of evaluation method of the total antioxidant capacity based on cross-linked hydrogel |
| CN109521068A (en) * | 2018-12-06 | 2019-03-26 | 杭州电子科技大学 | A method of detection honey oxidation resistance |
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| RU2238554C1 (en) * | 2003-07-25 | 2004-10-20 | Закрытое акционерное общество "АП Химавтоматика" | Method for determining total antioxidation activity of biologically active substances |
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2012
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| RU2238554C1 (en) * | 2003-07-25 | 2004-10-20 | Закрытое акционерное общество "АП Химавтоматика" | Method for determining total antioxidation activity of biologically active substances |
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| KRYSTYNA PYRZYNSKA ET AL: "Analysis of phenolic acids and flavonoids in honey", 《TRENDS IN ANALYTICAL CHEMISTRY》 * |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104713990A (en) * | 2015-02-11 | 2015-06-17 | 江苏出入境检验检疫局动植物与食品检测中心 | Method for identifying Manuka Honey |
| CN108982640A (en) * | 2018-06-28 | 2018-12-11 | 杭州电子科技大学 | A kind of evaluation method of the total antioxidant capacity based on cross-linked hydrogel |
| CN109521068A (en) * | 2018-12-06 | 2019-03-26 | 杭州电子科技大学 | A method of detection honey oxidation resistance |
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Application publication date: 20120815 |