CN103642850A - Determination method of ferulic acid antioxidant activity in vitro - Google Patents
Determination method of ferulic acid antioxidant activity in vitro Download PDFInfo
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- CN103642850A CN103642850A CN201310666357.9A CN201310666357A CN103642850A CN 103642850 A CN103642850 A CN 103642850A CN 201310666357 A CN201310666357 A CN 201310666357A CN 103642850 A CN103642850 A CN 103642850A
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Abstract
The invention aims at providing a determination method of antioxidant activity in vitro of enzymatic hydrolysate ferulic acid of co-expressed feruloyl esterase/xylanase. The ferulic acid is obtained by hydrolyzing wheat bran by using the co-expressed enzyme solution produced through a yeast system (GS115/faeA-xyn11A) of the co-expressed feruloyl esterase A and xylanase, and the antioxidant activity in vitro of the ferulic acid is determined from three aspects of reducing power, DPPH free radical and hydroxyl free radical scavenging rate. The experimental result proves that the ferulic acid has good reducing capacity and free radical scavenging capacity. The determination method is simple and effective, convenient and fast, and has a certain potential value on the antioxidant determination aspect of the ferulic acid in industrial application.
Description
Technical field
The present invention relates to, from reducing power, DPPH free radical and three aspects of hydroxyl radical free radical clearance rate, the enzymolysis product forulic acid of coexpression feruloyl esterase/zytase is carried out to antioxidation activity in vitro mensuration, belong to technical field of bioengineering.
Background technology
Ester bond in feruloyl esterase (EC3.1.1.73) energy catalytic hydrolysis plant cell wall between ferulic acid and its derivatives and polysaccharide, therefrom discharges forulic acid.In recent years, feruloyl esterase is widely used in the fields such as feed, papermaking, food, weaving, medicine, bioenergy, thereby investigator has pushed the development and utilization of feruloyl esterase to a brand-new peak.
Forulic acid is the abundantest hydroxycinnamic acid of content, is the main pharmacodynamics composition of Radix Angelicae Sinensis, Ligusticum wallichii, and is extensively present in the Chinese medicinal materialss such as rattletop, river Chinese herbaceous peony, scouring rush and asafoetide.Forulic acid is a kind of effective natural antioxidants, and security is high, has been widely used in the fields such as food, makeup and new drug development.By one of effective constituent of the healthcare products such as the known Ferulic acid of masses, Sodium Ferulate, be exactly now forulic acid.In addition, forulic acid also has anti-ageing, antibacterial, anti-inflammatory, antiviral, antithrombotic, absorption ultraviolet ray, improves immunizing power, prevention and inhibition cancer, Suppress hyperplasia of prostate, prevention three height, reduces cholesterol, increases a series of physiological activities such as bone density, atherosclerosis and treatment senile dementia.
As a kind of antioxidant, at present, the mensuration of forulic acid anti-oxidation characteristics is paid close attention to by many investigators just gradually, as studied by experiment in many bodies, but these experimentations are comparatively loaded down with trivial details, consuming time longer, along with the continuous expansion of bio-science field, the mensuration work meeting of using animal nutrition to carry out properties in conjunction with simple chemical process is more convenient, and this will speed up the development in more areas such as biological product, biotechnology.
Summary of the invention
The object of this invention is to provide and a kind ofly from reducing power, DPPH free radical and three aspects of hydroxyl radical free radical clearance rate, forulic acid is carried out to the verification method of antioxidation activity in vitro.
Technical scheme of the present invention: utilize the coexpression enzyme liquid hydrolyzed wheat wheat bran that the Yeast system (GS115/faeA-xyn11A) of coexpression feruloyl esterase A and zytase produces to obtain forulic acid, this forulic acid is carried out to the checking of antioxidation activity in vitro from reducing power, DPPH free radical and three aspects of hydroxyl radical free radical clearance rate.
The Yeast system of coexpression feruloyl esterase A and zytase (GS115/faeA-xyn11A) patent applied for (number of patent application: 201210562444.5).
The production of described forulic acid:
Take pre-treatment wheat bran as substrate, take 0.5g in shaking flask, add a certain amount of coexpression crude enzyme liquid, then add the phosphate buffered saline buffer (1mM, pH5.0) of certain volume to final volume 30mL.Reaction system is obtained to enzymolysis solution at 40 ℃ of insulation 12h, through macroporous resin separation and purification, obtain forulic acid.
Described forulic acid antioxidation activity in vitro verification method:
(1) mensuration of reducing power: add 0.2M PBS damping fluid (pH6.6) in tool plug test tube, the forulic acid solution of 1% potassium ferricyanide solution and different concns, in 50 ℃ of water bath with thermostatic control reaction 20min, cooling immediately, add 10% trichoroacetic acid(TCA) solution, the centrifugal 8min of 4000rpm, get supernatant liquor, add 1% liquor ferri trichloridi and distilled water, vibration mixes, after standing 5min, at 700nm place, measure absorbance (Fig. 1).With distilled water, replace forulic acid solution in contrast.
(2) mensuration of free radical scavenging: add 8 * 10 in tool plug test tube
-5m DPPH ethanolic soln, the forulic acid solution of dehydrated alcohol and different concns, mixes, and room temperature lucifuge is placed 30min, in 517nm place, measures respectively absorbance A
x, it is contrast that the dehydrated alcohol of take replaces forulic acid solution, measures its value for A
o, it is A that ethanol replaces the measured value of DPPH
xo.
DPPH free radical scavenging activity (%)=[A
o-(A
x-A
xo)]/A
o* 100 (Fig. 2)
(3) remove the mensuration of hydroxyl radical free radical activity: in tool plug test tube, add successively 5mM FeSO
4, the forulic acid solution of different concns, 10mM H
2o
2with 10mM Whitfield's ointment, in 37 ℃ of water bath with thermostatic control shaking tables, react 15min, cooling immediately, get supernatant liquor after centrifugal and measure respectively absorbance A in 510nm
x, it is contrast that the Whitfield's ointment of take replaces forulic acid solution, measures its value for A
o, it is A that Whitfield's ointment replaces the measured value of hydrogen peroxide
xo.
Hydroxyl radical free radical clearance rate (%)=[A
o-(A
x-A
xo)]/A
o* 100 (Fig. 2)
Beneficial effect of the present invention: a kind of method that the invention provides novel forulic acid antioxidation activity in vitro checking, under certain condition, by the mensuration to forulic acid reducing power, DPPH free radical and three aspects of hydroxyl radical free radical clearance rate, proof forulic acid has good reducing power and radical scavenging activity, the method is simply effective, convenient and swift, in industrial application, aspect the anti-oxidant mensuration of forulic acid, has certain potential value.
Accompanying drawing explanation
Fig. 1: the reducing power graphic representation of forulic acid
Fig. 2: the removing power curve figure of forulic acid to DPPH free radical and hydroxyl radical free radical
Embodiment
Below in conjunction with specific embodiment, further set forth working method of the present invention.But these embodiment only, for describing the present invention in detail, limit the scope of the invention and be not used in.
The preparation of embodiment 1 forulic acid
Take pre-treatment wheat bran as substrate, take 0.5g in shaking flask, add 3.72mL coexpression crude enzyme liquid, then add phosphate buffered saline buffer (1mM, pH5.0) to final volume 30mL.Reaction system is incubated to 12h at 40 ℃, collect enzymolysis solution, 10mL wheat bran hydrolyzed solution is carried out to dynamic adsorption with the upper macroporous resin column of 1mL/min, after the standing 2h of end upon adsorption, with the slow wash-out of distilled water, flow velocity is 1mL/min, collect elutriant in batches, until no longer containing in water lotion after the impurity such as reducing sugar, pigment, use 50% ethanol instead and carry out wash-out, flow velocity is 1.5mL/min, collects elutriant, being placed in vacuum rotary evaporator, to be concentrated into final volume be 10mL, and to adopt HPLC to detect its ferulaic acid content be 1.75mg.
Embodiment 2 forulic acid antioxidation activity in vitro checkings
(1) mensuration of reducing power
In tool plug test tube, add 1mL0.2M PBS damping fluid (pH6.6), the forulic acid sample solution of 2mL1% potassium ferricyanide solution and 1mL different concns, in 50 ℃ of water bath with thermostatic control reaction 20min, cooling immediately, add 1.0mL10% trichoroacetic acid(TCA) solution, the centrifugal 8min of 4000rpm, get 2mL supernatant liquor, add 1mL1% liquor ferri trichloridi and 2.0mL distilled water, vibration mixes, after standing 5min, at 700nm place, measure respectively absorbance, the reducing power of result forulic acid presents stable rising tendency along with the rising of concentration, and present certain dose-effect relationship, the reducing power of 20 μ g/mL forulic acid is 5.29 times of 4 μ g/mL forulic acid.With distilled water, replace forulic acid solution in contrast.
(2) mensuration of free radical scavenging
In tool plug test tube, add 3mL8 * 10
-5m DPPH ethanolic soln, the forulic acid solution of 1mL dehydrated alcohol and 1mL different concns, mixes, and room temperature lucifuge is placed 30min, in 517nm place, measures respectively absorbance A
x, it is contrast that the dehydrated alcohol of take replaces forulic acid solution, measures its value for A
o, it is A that ethanol replaces the measured value of DPPH
xo.Result shows that the resistance of oxidation of forulic acid strengthens along with increasing of concentration, and when forulic acid concentration is 8 μ g/mL, its free radical scavenging activity is 25.3%, and when forulic acid concentration is 16 μ g/mL, its DPPH clearance rate rises to 42.6%.
DPPH free radical scavenging activity (%)=[A
o-(A
x-A
xo)]/A
o* 100
(3) remove the mensuration of hydroxyl radical free radical activity
In tool plug test tube, add successively 2mL5mM FeSO
4, the forulic acid solution of 1mL different concns, 1mL10mMH
2o
2with 1mL10mM Whitfield's ointment, in 37 ℃ of water bath with thermostatic control shaking tables, react 15min, cooling immediately, get supernatant liquor after centrifugal and measure respectively absorbance A in 510nm
x, it is contrast that the Whitfield's ointment of take replaces forulic acid solution, measures its value for A
o, it is A that Whitfield's ointment replaces the measured value of hydrogen peroxide
xo.Result shows, the forulic acid of lower concentration just shows good hydroxyl radical free radical clearance rate, and along with the increase of forulic acid concentration, corresponding hydroxyl radical free radical is removed ability and also presented stable rising tendency.When forulic acid concentration is 20 μ g/mL, its hydroxyl radical free radical clearance rate reaches 92%.
Hydroxyl radical free radical clearance rate (%)=[A
o-(A
x-A
xo)]/A
o* 100.
Claims (2)
1. the production method of forulic acid:
Take pre-treatment wheat bran as substrate, take 0.5g in shaking flask, add 3.72mL coexpression crude enzyme liquid, then add phosphate buffered saline buffer (1mM, pH5.0) to final volume 30mL, reaction system is incubated to 12h at 40 ℃, collect enzymolysis solution, 10mL wheat bran hydrolyzed solution is carried out to dynamic adsorption with the upper macroporous resin column of 1mL/min, after the standing 2h of end upon adsorption, with the slow wash-out of distilled water, flow velocity is 1mL/min, collect elutriant in batches, treat no longer to contain in water lotion reducing sugar, after the impurity such as pigment, use 50% ethanol instead and carry out wash-out, flow velocity is 1.5mL/min, collect elutriant, being placed in vacuum rotary evaporator, to be concentrated into final volume be 10mL.
2. forulic acid antioxidation activity in vitro checking:
(1) mensuration of reducing power: add 1mL0.2M PBS damping fluid (pH6.6) in tool plug test tube, the forulic acid sample solution of 2mL1% potassium ferricyanide solution and 1mL different concns, in 50 ℃ of water bath with thermostatic control reaction 20min, cooling immediately, add 1.0mL10% trichoroacetic acid(TCA) solution, the centrifugal 8min of 4000rpm, get 2mL supernatant liquor, add 1mL1% liquor ferri trichloridi and 2.0mL distilled water, vibration mixes, after standing 5min, at 700nm place, measure respectively absorbance, the reducing power of result forulic acid presents stable rising tendency along with the rising of concentration, and present certain dose-effect relationship, the reducing power of 20 μ g/mL forulic acid is 5.29 times of 4 μ g/mL forulic acid, with distilled water, replace forulic acid solution in contrast,
(2) mensuration of free radical scavenging: add 3mL8 * 10 in tool plug test tube
-5m DPPH ethanolic soln, the forulic acid solution of 1mL dehydrated alcohol and 1mL different concns, mixes, and room temperature lucifuge is placed 30min, in 517nm place, measures respectively absorbance A
x, it is contrast that the dehydrated alcohol of take replaces forulic acid solution, measures its value for A
o, it is A that ethanol replaces the measured value of DPPH
xo, DPPH free radical scavenging activity (%)=[A
o-(A
x-A
xo)]/A
o* 100; Result shows that the resistance of oxidation of forulic acid strengthens along with increasing of concentration, and when forulic acid concentration is 8 μ g/mL, its free radical scavenging activity is 25.3%, and when forulic acid concentration is 16 μ g/mL, its DPPH clearance rate rises to 42.6%;
(3) remove the mensuration of hydroxyl radical free radical activity: in tool plug test tube, add successively 2mL5mM FeSO
4, the forulic acid solution of 1mL different concns, 1mL10mM H
2o
2with 1mL10mM Whitfield's ointment, in 37 ℃ of water bath with thermostatic control shaking tables, react 15min, cooling immediately, get supernatant liquor after centrifugal and measure respectively absorbance A in 510nm
x, it is contrast that the Whitfield's ointment of take replaces forulic acid solution, measures its value for A
o, it is A that Whitfield's ointment replaces the measured value of hydrogen peroxide
xo, hydroxyl radical free radical clearance rate (%)=[A
o-(A
x-A
xo)]/A
o* 100; Result shows, the forulic acid of lower concentration just shows good hydroxyl radical free radical clearance rate, and along with the increase of forulic acid concentration, corresponding hydroxyl radical free radical is removed ability and also presented stable rising tendency, when forulic acid concentration is 20 μ g/mL, its hydroxyl radical free radical clearance rate reaches 92%.
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Cited By (6)
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| 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 |
| CN104949966A (en) * | 2014-03-26 | 2015-09-30 | 爱科来株式会社 | Reducing power analysis method and reducing power analysis reagent |
| CN106959280A (en) * | 2017-04-24 | 2017-07-18 | 刘涛瑞 | A kind of anti-oxidant development test method of apple young fruit OPC |
| CN111747852A (en) * | 2020-07-31 | 2020-10-09 | 济南大学 | Cis-ferulic acid amyl ester compound and preparation method and application thereof |
| CN115025017A (en) * | 2022-06-17 | 2022-09-09 | 齐鲁工业大学 | Ligusticum wallichii and cimicifugae foetidae mixed enzymatic hydrolysate and application thereof |
| CN115025016A (en) * | 2022-06-17 | 2022-09-09 | 齐鲁工业大学 | Ligusticum wallichii enzymatic hydrolysate and application thereof |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104949966A (en) * | 2014-03-26 | 2015-09-30 | 爱科来株式会社 | Reducing power analysis method and reducing power analysis reagent |
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| 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 |
| CN106959280A (en) * | 2017-04-24 | 2017-07-18 | 刘涛瑞 | A kind of anti-oxidant development test method of apple young fruit OPC |
| CN111747852A (en) * | 2020-07-31 | 2020-10-09 | 济南大学 | Cis-ferulic acid amyl ester compound and preparation method and application thereof |
| CN115025017A (en) * | 2022-06-17 | 2022-09-09 | 齐鲁工业大学 | Ligusticum wallichii and cimicifugae foetidae mixed enzymatic hydrolysate and application thereof |
| CN115025016A (en) * | 2022-06-17 | 2022-09-09 | 齐鲁工业大学 | Ligusticum wallichii enzymatic hydrolysate and application thereof |
| CN115025017B (en) * | 2022-06-17 | 2023-11-14 | 齐鲁工业大学 | Ligusticum wallichii and cimicifuga foetida mixed enzymolysis liquid and application thereof |
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Application publication date: 20140319 |