CN105361185A - Method for separation purification of walnut green seedcase polyphenol substances by macroporous resin - Google Patents
Method for separation purification of walnut green seedcase polyphenol substances by macroporous resin Download PDFInfo
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Abstract
The invention discloses a method for separation purification of walnut green seedcase polyphenol substances by macroporous resin. The method comprises 1, carrying out extraction on walnut green seedcase dry powder at a temperature of 35-40 DEG C through ethanol with content of 50% under ultrasonic action many times, carrying out vacuum filtration on the extract, evaporating the filtrate to obtain walnut green seedcase polyphenol crude extract, diluting the walnut green seedcase polyphenol crude extract until a desired concentration is obtained, and carrying out refrigeration for next use, 2, determining total polyphenol content of walnut green seedcase by a Folin-Ciocaileu method, 3, carrying out macroporous resin pretreatment, 4, carrying out macroporous resin static adsorption and desorption, 5, carrying out macroporous resin dynamic adsorption and desorption, and 6, through a HPLC method, determining free phenol content and composition of the walnut green seedcase polyphenol extract obtained by the macroporous resin separation purification so that the whole separation purification process is finished. The method provides the novel approach for separation purification of walnut green seedcase polyphenol substances, can be operated simply and realizes high separation purification quality and efficiency.
Description
Technical field
The invention belongs to field of food science, be specifically related to the method for polyphenols in a kind of macroreticular resin separation and purification green peel of walnut.
Background technology
In recent years, along with growth in the living standard, the demand of people to health food grows with each passing day.Containing a large amount of polyphenols in plant, these materials have the different physiological roles such as antitumor, anti-oxidant, radioresistance, antihyperglycemic, reducing blood lipid, are the important source material of health food, can obtain polyphenols by separation and purification from plant.But traditional isolation and purification method is as separation by precipitation, solvent extraction etc., there is the low or high in cost of production shortcoming of purity, Amberlyst process can make up these shortcomings, its technique is simple, easy to operate, with low cost, bring considerable economic benefit in the application of food, medicine, cosmetic industry, but not yet occur the application of Amberlyst process in separation and purification green peel of walnut polyphenols at present.
Summary of the invention
The object of the invention is to for above-mentioned the problems of the prior art, the method for polyphenols in a kind of macroreticular resin separation and purification green peel of walnut is provided, improve quality and the efficiency of separation and purification.
To achieve these goals, the technical solution used in the present invention is:
The first step, by green peel of walnut dry powder with 50% ethanol under temperature 35 DEG C ~ 40 DEG C conditions ultrasonic extraction repeatedly, extract vacuum filtration, filtrate evaporates to obtain green peel of walnut polyphenolic extract, is diluted to desired concn, refrigerates for subsequent use;
Second step, employing Folin-Ciocaileu method measure polyphenol total content in green peel of walnut;
3rd step, by macroreticular resin 4%NaOH solution immersion treatment 2 ~ 4h, re-use distilled water flushing to neutral; Then soak 2 ~ 4h with 4%HCl solution, use distilled water flushing to neutral; Finally use 95% alcohol immersion process 2 ~ 4h again, distilled water is washed till without till alcohol taste; For subsequent use;
4th step, macroreticular resin are to the Static Adsorption of green peel of walnut polyphenol dilution and desorb;
5th step, macroreticular resin are to the Dynamic Adsorption of green peel of walnut polyphenol dilution and desorb;
6th step, employing HPLC method measure composition and the content of the green peel of walnut polyphenol extract free phenol after macroreticular resin separation and purification, complete all separation and purification processes.
Described macroreticular resin adopts NKA-9.
In the described first step, the solid-liquid ratio of green peel of walnut dry powder and ethanol is 1g:17mL, ultrasonic extraction twice, and filtrate, by rotary evaporation, obtains green peel of walnut polyphenolic extract.
The detailed process that described Folin-Ciocaileu method measures polyphenol total content in green peel of walnut is: get 0.2mL green peel of walnut polyphenolic extract, join in 10mL volumetric flask, add 0.5mLFC reagent, mix, 1.5mL20% sodium carbonate liquor is added in 1min, mixing, uses deionized water constant volume; Above-mentioned solution after dark placement 1.5 ~ 2h, not add the solution of crude extract as blank, is measured absorbance at 35 ~ 40 DEG C under 760nm wavelength.
The detailed process of described macroreticular resin Static Adsorption is: take 1.0g resin in 50mL triangular flask, add the 3rd step gained spare resin sample liquid 30mL, sealing is placed on 160 ~ 175r/min shaking table, and vibrate under room temperature 4 ~ 5h, sample from supernatant, measure polyphenol content; Descend formulae discovery adsorbance and adsorption rate according to this, the concentration of investigation sample liquid and pH are on the impact of resin adsorption performance;
qe＝(C0－C1)×V1/W(1)
E(％)＝[(C0－C1)/C0]×100(2)
In formula: qe-adsorbance, mg/g; E-adsorption rate, %; C0-initial concentration, mg/mL;
C1-equilibrium concentration, mg/mL; V1-adsorbent solution volume, mL; The weight of W-resin, g.
The detailed process of the static desorb of described macroreticular resin is: cleaned by the resin distilled water after fully adsorbing, until surperficial n.s liquid remains, use filter paper suck dry moisture again, be placed in triangular flask, add 30mL different concentration ethanol solution, sealing is positioned in 160 ~ 175r/min oscillator, desorb 2 ~ 3h under room temperature, sample from supernatant, survey polyphenol content; Descend formulae discovery desorption efficiency and desorption quantity according to this, the concentration of investigation eluent and pH are on the impact of resin elution performance;
D(％)＝C2×V2×100/(C0－C1)×V1(3)
qd＝C2×V2/W(4)
In formula: D-desorption efficiency, %; Qd-desorption quantity, mg/g; C0-initial concentration, mg/mL;
C1-equilibrium concentration, mg/mL; Polyphenol concentration in C2-stripping liquid, mg/mL; V1-adsorbent solution volume, mL; V2-stripping workshop volume, mL; W-resin taken amount, g.
The detailed process of described macroreticular resin Dynamic Adsorption is: resin wet method good for the 3rd step pretreatment loaded in the chromatographic column of 16mm × 500mm, after distilled water balance, by sample liquid by 1.3mL/min at the uniform velocity loading collect efflux, every 10mL is a pipe, detect and calculate outflow concentration, determine flow velocity, volume conditions, obtain optimal adsorption condition, draw Dynamic Adsorption curve.
The detailed process of described macroreticular resin dynamic desorption is: carry out desorption experiment by adsorbing saturated resin, be washed till n.s liquid with distilled water to remain, at the uniform velocity wash-out is carried out with ethanolic solution by 0.43mL/min, collect eluent, calculate outflow concentration, determine elution flow rate, volume conditions, obtain optimum washing engaging condition, draw dynamic desorption curve.
After adopting HPLC method to measure macroreticular resin separation and purification, in green peel of walnut polyphenol extract, the concrete grammar of free phenol composition and content is: adopt 5 μm, the DiamonsilC18 post of 250mm × 4.6mm; Determined wavelength 280nm; Flow velocity 1.0ml/min; Column temperature 30 DEG C; Sample size 20uL; Mobile phase A is 0.1% formic acid high purity water; Mobile phase B is 0.1% formic acid acetonitrile; Gradient elution program is followed successively by 93%A, 7%B, 0min; 82%A, 18%B, 30min; 78%A, 22%B, 40min; 50%A, 50%B, 45min; 50%A, 50%B, 50min; Sample peak retention time and standard items retention time are contrasted, judges free phenol kind, according to calculated by peak area free phenol content.
Compared with prior art, in the present invention's macroreticular resin separation and purification green peel of walnut, the method for polyphenols has following beneficial effect: macroreticular resin provides the new way of polyphenols separation and purification in green peel of walnut, detect by experiment, be 0.574mg/mL when adsorption conditions is loading polyphenol liquid concentration, pH is 1.0, loading speed is 1.3mL/min, and adsorption rate can reach 90.1%; When the ethanol elution agent that elution requirement is 50%, pH is 3.0, and elution rate 0.43mL/min, desorption efficiency reaches 87.9%.Through HPLC qualification and analysis, green peel of walnut aldehydes matter is primarily of monomer compositions such as former catechin, chlorogenic acid, caffeic acid, epicatechin, forulic acid, rutins.Assorted peak before green peel of walnut polyphenol purifying between retention time 2-5min and 48-50min is lowered after purifying, and before and after purifying, residual peak is retained substantially, illustrates that this kind of resin is applicable to separation and purification green peel of walnut polyphenol thus.The inventive method easy and simple to handle, quality and the efficiency of separation and purification are high.
Further, in NKA-9 macroreticular resin Dynamic Adsorption process of the present invention, if flow velocity is too fast, resin can not fully contact with crude extract, reduces its adsorbance; If flow velocity is excessively slow, though be conducive to the absorption of aldehydes matter, can reduce production efficiency, affect the production cycle, 1.3mL/min is best loading flow velocity.
Further, elution speed of the present invention has desorption effect to be affected significantly, although elution flow rate requires slow, if but flow velocity is excessively slow, desorption time then can be caused to extend, and eluted polyphenol may be adsorbed again in flow process, two alternate meetings re-establish dynamic equilibrium, reduce desorption quantity; And if flow velocity is too fast, then eluant, eluent can be caused fully not contact with the saturated resin of absorption and to flow out fast, and desorption quantity is reduced, and production efficiency declines, and experimental verification 0.43mL/min is best elution flow rate.
Accompanying drawing explanation
Fig. 1 is the effect diagram of pH to polyphenol adsorption rate;
Fig. 2 is variable concentrations polyphenol Static Adsorption curve map;
Fig. 3 is the effect diagram of concentration of alcohol to polyphenol desorption effect;
Fig. 4 is the effect diagram of pH to polyphenol desorption efficiency;
Fig. 5 is the effect diagram of flow velocity to resin adsorption effect;
Fig. 6 is the effect diagram of eluant, eluent flow velocity to resin desorption effect;
Fig. 7 is retention time and peak change figure before green peel of walnut polyphenol purifying;
Fig. 8 is retention time and peak change figure after green peel of walnut polyphenol purifying;
Detailed description of the invention
Below in conjunction with accompanying drawing, the present invention is described in further detail.
One, material and reagent;
Green rind walnut: pluck mid-August, in Lantian County, Shaanxi Province, green rind walnut is got rascal, chopping, grinds after freeze drying, crosses 60 mesh sieves, and sealing splendid attire, in Brown Glass Brown glass bottles and jars only, refrigerates for subsequent use;
Macroreticular resin: NKA-9;
Sodium tungstate, lithium sulfate, sodium molybdate, SPA, concentrated hydrochloric acid, NaOH, absolute ethyl alcohol, natrium carbonicum calcinatum solid, bromine water, methyl alcohol, acetonitrile;
Two, key instrument equipment;
Numerical control ultrasonic cleaner: KQ3200DE type, Kunshan Ultrasonic Instruments Co., Ltd.; Rotary Evaporators: RE-52 type, Town in Shanghai booth laboratory apparatus Co., Ltd; Digital display thermostat water bath: HH-S type, Medical Instruments factory of Jintan City of Jiangsu Province; Ultraviolet-uisible spectrophotometer: UV-1100 type, Shanghai Mei Puda Instrument Ltd.; Constant temperature oscillator: SHZ-82 type, Fuhua Instrument Ltd. of Jintan City; Constant flow pump: HL-2S type, Shanghai Hu Xi analytical instrument Co., Ltd., Factory; Accurate pH meter: PHS-3C type, Shanghai Precision Scientific Apparatus Co., Ltd; Electronic balance: JA1203 type, Shanghai Precision Scientific Apparatus Co., Ltd; Freeze drier: LGJ-18C type, Fourth Ring, Beijing scientific instrument factory.
Three, isolation and purification method;
Embodiment one:
The preparation of 3.1 sample solutions: weigh a certain amount of green peel of walnut dry powder, use 50% ethanol, temperature 35 DEG C, ultrasonic extraction 30min under solid-liquid ratio 1g:17mL condition, extracts 2 times.Extract vacuum filtration, gets filtrate rotary evaporation, obtains green peel of walnut polyphenolic extract, is diluted to respective concentration, refrigerates for subsequent use.
The mensuration of 3.2 polyphenol contents: in green peel of walnut, polyphenol total content measures and adopts Folin-Ciocaileu method, pipette 0.2mL crude extract, join in 10mL volumetric flask, add the FC reagent of 0.5mL, mix, in 1min, add the sodium carbonate liquor of 1.5mL20%, mixing, uses deionized water constant volume;
Above-mentioned solution after dark placement 1.5h, not add the solution of crude extract as blank, is measured absorbance at 35 DEG C under 760nm wavelength.
The pretreatment of 3.3 macroreticular resins: macroreticular resin 4%NaOH solution immersion treatment 2h, distilled water constantly rinses to neutrality; Then soak 2h with 4%HCl solution, distillating washing resin is to neutral; Finally use 95% alcohol immersion process 2h again, distilled water is washed till without till alcohol taste, for subsequent use.
3.4 macroreticular resin Static Adsorption and desorbs:
(1) Staticadsorption experiment: take 1.0g resin in 50mL triangular flask, add sample liquid 30mL, sealing is placed on 160r/min shaking table, and vibrate under room temperature 4h, samples from supernatant, measures polyphenol content.Descend formulae discovery adsorbance and adsorption rate according to this, investigation sample liquid concentration and pH are on the impact of resin adsorption performance:
qe＝(C0－C1)×V1/W，(1)
E(％)＝[(C0－C1)/C0]×100(2)
In formula: qe-adsorbance, mg/g; E-adsorption rate, %; C0-initial concentration, mg/mL;
C1-equilibrium concentration, mg/mL; V1-adsorbent solution volume, mL; The weight of W-resin, g.
(2) static desorption experiment: the resin distilled water after fully adsorbing is cleaned, until surperficial n.s liquid remains, use filter paper suck dry moisture again, be placed in triangular flask, add 30mL different concentration ethanol solution, sealing is positioned in 160r/min oscillator, desorb 2h under room temperature, sample from supernatant, survey polyphenol content.Descend formulae discovery desorption efficiency and desorption quantity according to this, investigation eluate concentration and pH are on the impact of resin elution performance:
D(％)＝C2×V2×100/(C0－C1)×V1(3)
qd＝C2×V2/W(4)
In formula: D-desorption efficiency, %; Qd-desorption quantity, mg/g; C0-initial concentration, mg/mL;
C1-equilibrium concentration, mg/mL; Polyphenol concentration in C2-stripping liquid, mg/mL; V1-adsorbent solution volume, mL; V2-stripping workshop volume, mL; W-resin taken amount, g.
3.5 macroreticular resin Dynamic Adsorption and desorbs:
Resin wet method good for pretreatment is loaded in the chromatographic column of 16mm × 500mm, after distilled water balance, sample liquid is collected efflux with certain speed loading, every 10mL is a pipe, detect and calculate outflow concentration, determine the condition such as flow velocity, volume, obtain optimal adsorption condition, draw Dynamic Adsorption curve.
Desorption experiment is carried out by adsorbing saturated resin, be washed till n.s liquid with distilled water to remain, wash-out is carried out with certain flow velocity debita spissitudo ethanolic solution, collect eluent, calculate outflow concentration, determine the condition such as elution flow rate, volume, obtain optimum washing engaging condition, draw dynamic desorption curve.
In 3.6 green peel of walnut, the composition of polyphenols and content HPLC analyze:
HPLC method is adopted to measure composition and the content of monomer whose phenol by the green peel of walnut polyphenol extract after macroreticular resin separation and purification.Concrete grammar is: DiamonsilC18 post (5 μm, 250mm × 4.6mm); Determined wavelength 280nm; Flow velocity 1.0ml/min; Column temperature 30 DEG C; Sample size 20uL; Mobile phase A (0.1% formic acid high purity water) and Mobile phase B (0.1% formic acid acetonitrile); Gradient elution program: 93%A, 7%B, 0min; 82%A, 18%B, 30min; 78%A, 22%B, 40min; 50%A, 50%B, 45min; 50%A, 50%B, 50min.Sample peak retention time and standard items retention time contrast, and judge free phenol kind, according to calculated by peak area free phenol content.
Embodiment two:
The preparation of 3.1 sample solutions: weigh a certain amount of green peel of walnut dry powder, use 50% ethanol, temperature 37.5 DEG C, ultrasonic extraction 32.5min under solid-liquid ratio 1g:17mL condition, extracts 2 times.Extract vacuum filtration, gets filtrate rotary evaporation, obtains green peel of walnut polyphenolic extract, is diluted to respective concentration, refrigerates for subsequent use.
The mensuration of 3.2 polyphenol contents: in green peel of walnut, polyphenol total content measures and adopts Folin-Ciocaileu method, pipette 0.2mL crude extract, join in 10mL volumetric flask, add the FC reagent of 0.5mL, mix, in 1min, add the sodium carbonate liquor of 1.5mL20%, mixing, uses deionized water constant volume;
Above-mentioned solution after dark placement 1.75h, not add the solution of crude extract as blank, is measured absorbance at 37.5 DEG C under 760nm wavelength.
The pretreatment of 3.3 macroreticular resins: macroreticular resin 4%NaOH solution immersion treatment 3h, distilled water constantly rinses to neutrality; Then soak 3h with 4%HCl solution, distillating washing resin is to neutral; Finally use 95% alcohol immersion process 3h again, distilled water is washed till without till alcohol taste, for subsequent use.
3.4 macroreticular resin Static Adsorption and desorbs:
(1) Staticadsorption experiment: take 1.0g resin in 50mL triangular flask, add sample liquid 30mL, sealing is placed on 170r/min shaking table, and vibrate under room temperature 4.5h, samples from supernatant, measures polyphenol content.Descend formulae discovery adsorbance and adsorption rate according to this, investigation sample liquid concentration and pH are on the impact of resin adsorption performance:
qe＝(C0－C1)×V1/W，(1)
E(％)＝[(C0－C1)/C0]×100(2)
In formula: qe-adsorbance, mg/g; E-adsorption rate, %; C0-initial concentration, mg/mL;
C1-equilibrium concentration, mg/mL; V1-adsorbent solution volume, mL; The weight of W-resin, g.
(2) static desorption experiment: the resin distilled water after fully adsorbing is cleaned, until surperficial n.s liquid remains, use filter paper suck dry moisture again, be placed in triangular flask, add 30mL different concentration ethanol solution, sealing is positioned in 170r/min oscillator, desorb 2.5h under room temperature, sample from supernatant, survey polyphenol content.Descend formulae discovery desorption efficiency and desorption quantity according to this, investigation eluate concentration and pH are on the impact of resin elution performance:
D(％)＝C2×V2×100/(C0－C1)×V1(3)
qd＝C2×V2/W(4)
In formula: D-desorption efficiency, %; Qd-desorption quantity, mg/g; C0-initial concentration, mg/mL;
C1-equilibrium concentration, mg/mL; Polyphenol concentration in C2-stripping liquid, mg/mL; V1-adsorbent solution volume, mL; V2-stripping workshop volume, mL; W-resin taken amount, g.
3.5 macroreticular resin Dynamic Adsorption and desorbs:
Resin wet method good for pretreatment is loaded in the chromatographic column of 16mm × 500mm, after distilled water balance, sample liquid is collected efflux with certain speed loading, every 10mL is a pipe, detect and calculate outflow concentration, determine the condition such as flow velocity, volume, obtain optimal adsorption condition, draw Dynamic Adsorption curve.
Desorption experiment is carried out by adsorbing saturated resin, be washed till n.s liquid with distilled water to remain, wash-out is carried out with certain flow velocity debita spissitudo ethanolic solution, collect eluent, calculate outflow concentration, determine the condition such as elution flow rate, volume, obtain optimum washing engaging condition, draw dynamic desorption curve.
In 3.6 green peel of walnut, the composition of polyphenols and content HPLC analyze:
HPLC method is adopted to measure composition and the content of monomer whose phenol by the green peel of walnut polyphenol extract after macroreticular resin separation and purification.Concrete grammar is: DiamonsilC18 post (5 μm, 250mm × 4.6mm); Determined wavelength 280nm; Flow velocity 1.0ml/min; Column temperature 30 DEG C; Sample size 20uL; Mobile phase A (0.1% formic acid high purity water) and Mobile phase B (0.1% formic acid acetonitrile); Gradient elution program: 93%A, 7%B, 0min; 82%A, 18%B, 30min; 78%A, 22%B, 40min; 50%A, 50%B, 45min; 50%A, 50%B, 50min.Sample peak retention time and standard items retention time contrast, and judge free phenol kind, according to calculated by peak area free phenol content.
Embodiment three:
The preparation of 3.1 sample solutions: weigh a certain amount of green peel of walnut dry powder, use 50% ethanol, temperature 40 DEG C, ultrasonic extraction 35min under solid-liquid ratio 1g:17mL condition, extracts 2 times.Extract vacuum filtration, gets filtrate rotary evaporation, obtains green peel of walnut polyphenolic extract, is diluted to respective concentration, refrigerates for subsequent use.
The mensuration of 3.2 polyphenol contents: in green peel of walnut, polyphenol total content measures and adopts Folin-Ciocaileu method, pipette 0.2mL crude extract, join in 10mL volumetric flask, add the FC reagent of 0.5mL, mix, in 1min, add the sodium carbonate liquor of 1.5mL20%, mixing, uses deionized water constant volume;
Above-mentioned solution after dark placement 2h, not add the solution of crude extract as blank, is measured absorbance at 40 DEG C under 760nm wavelength.
The pretreatment of 3.3 macroreticular resins: macroreticular resin 4%NaOH solution immersion treatment 4h, distilled water constantly rinses to neutrality; Then soak 4h with 4%HCl solution, distillating washing resin is to neutral; Finally use 95% alcohol immersion process 4h again, distilled water is washed till without till alcohol taste, for subsequent use.
3.4 macroreticular resin Static Adsorption and desorbs:
(1) Staticadsorption experiment: take 1.0g resin in 50mL triangular flask, add sample liquid 30mL, sealing is placed on 175r/min shaking table, and vibrate under room temperature 5h, samples from supernatant, measures polyphenol content.Descend formulae discovery adsorbance and adsorption rate according to this, investigation sample liquid concentration and pH are on the impact of resin adsorption performance:
qe＝(C0－C1)×V1/W，(1)
E(％)＝[(C0－C1)/C0]×100(2)
In formula: qe-adsorbance, mg/g; E-adsorption rate, %; C0-initial concentration, mg/mL;
C1-equilibrium concentration, mg/mL; V1-adsorbent solution volume, mL; The weight of W-resin, g.
(2) static desorption experiment: the resin distilled water after fully adsorbing is cleaned, until surperficial n.s liquid remains, use filter paper suck dry moisture again, be placed in triangular flask, add 30mL different concentration ethanol solution, sealing is positioned in 175r/min oscillator, desorb 3h under room temperature, sample from supernatant, survey polyphenol content.Descend formulae discovery desorption efficiency and desorption quantity according to this, investigation eluate concentration and pH are on the impact of resin elution performance:
D(％)＝C2×V2×100/(C0－C1)×V1(3)
qd＝C2×V2/W(4)
In formula: D-desorption efficiency, %; Qd-desorption quantity, mg/g; C0-initial concentration, mg/mL;
C1-equilibrium concentration, mg/mL; Polyphenol concentration in C2-stripping liquid, mg/mL; V1-adsorbent solution volume, mL; V2-stripping workshop volume, mL; W-resin taken amount, g.
3.5 macroreticular resin Dynamic Adsorption and desorbs:
Resin wet method good for pretreatment is loaded in the chromatographic column of 16mm × 500mm, after distilled water balance, sample liquid is collected efflux with certain speed loading, every 10mL is a pipe, detect and calculate outflow concentration, determine the condition such as flow velocity, volume, obtain optimal adsorption condition, draw Dynamic Adsorption curve.
Desorption experiment is carried out by adsorbing saturated resin, be washed till n.s liquid with distilled water to remain, wash-out is carried out with certain flow velocity debita spissitudo ethanolic solution, collect eluent, calculate outflow concentration, determine the condition such as elution flow rate, volume, obtain optimum washing engaging condition, draw dynamic desorption curve.
In 3.6 green peel of walnut, the composition of polyphenols and content HPLC analyze:
HPLC method is adopted to measure composition and the content of monomer whose phenol by the green peel of walnut polyphenol extract after macroreticular resin separation and purification.Concrete grammar is: DiamonsilC18 post (5 μm, 250mm × 4.6mm); Determined wavelength 280nm; Flow velocity 1.0ml/min; Column temperature 30 DEG C; Sample size 20uL; Mobile phase A (0.1% formic acid high purity water) and Mobile phase B (0.1% formic acid acetonitrile); Gradient elution program: 93%A, 7%B, 0min; 82%A, 18%B, 30min; 78%A, 22%B, 40min; 50%A, 50%B, 45min; 50%A, 50%B, 50min.Sample peak retention time and standard items retention time contrast, and judge free phenol kind, according to calculated by peak area free phenol content.
See Fig. 1-6, it is to be noted that
1. in the leaching process of the thick polyphenol of green peel of walnut, temperature chooses 35 ~ 40 DEG C, is best with 35 DEG C.
2. during thick polyphenol rotary evaporation, temperature chooses 40 ~ 45 DEG C.
3. soak time 2 ~ 4h during macroreticular resin pretreatment, with 4h immersion for Best Times, the time is too short causes the impurity in resin can not effectively stripping.
4. crude extract pH value is adjusted to respectively the Optimal pH that 1.0 ~ 7.0, pH1.0 is the absorption of crude extract polyphenol.
5. preparing polyphenol concentration is 0.246 ~ 0.708mg/mL crude extract, and mensuration adsorption concentration is 0.574mg/mL.
6. stripping liquid pH value is adjusted to the Optimal pH of 1.0 ~ 7.0, pH3.0 as stripping liquid respectively.
7. make stripping liquid with ethanol, with 50% ethanol for best desorb concentration.
In 8.NKA-9 macroreticular resin Dynamic Adsorption process, if flow velocity is too fast, resin can not fully contact with crude extract, reduces its adsorbance; If flow velocity is excessively slow, though be conducive to the absorption of aldehydes matter, can production efficiency be reduced, affect the production cycle, therefore select 1.3mL/min to be best loading flow velocity.
9. in general, elution speed has desorption effect affects significantly.Elution flow rate requires slow, if but flow velocity is excessively slow, then and desorption time can be caused to extend, and eluted polyphenol may be adsorbed again in flow process, two alternate meetings re-establish dynamic equilibrium, reduce desorption quantity; If flow velocity is too fast, then eluant, eluent can be caused fully not contact with the saturated resin of absorption and to flow out fast, and desorption quantity is reduced, and production efficiency declines, therefore selects 0.43mL/min to be best elution flow rate.
Comparison diagram 7 and Fig. 8, result shows that NKA-9 resin is the resin of best separation and purification green peel of walnut polyphenols, and adsorption conditions is sample solution polyphenol concentration 0.574mg/mL, pH1.0, loading speed 1.3mL/min, and adsorption rate can reach 90.1%; Elution requirement is the ethanol elution agent of 50%, and pH3.0, elution rate 0.43mL/min, desorption efficiency reaches 87.9%.Through HPLC qualification and analysis, green peel of walnut aldehydes matter is primarily of monomer compositions such as former catechin, chlorogenic acid, caffeic acid, epicatechin, forulic acid, rutins.Assorted peak before green peel of walnut polyphenol purifying between retention time 2-5min and 48-50min is lowered after purifying, and before and after purifying, residual peak is retained substantially, illustrates that this kind of resin is applicable to separation and purification green peel of walnut polyphenol.
Claims (9)
1., by a method for polyphenols in macroreticular resin separation and purification green peel of walnut, it is characterized in that:
The first step, by green peel of walnut dry powder with 50% ethanol under temperature 35 DEG C ~ 40 DEG C conditions ultrasonic extraction repeatedly, extract vacuum filtration, filtrate evaporates to obtain green peel of walnut polyphenolic extract, is diluted to desired concn, refrigerates for subsequent use;
Second step, employing Folin-Ciocaileu method measure polyphenol total content in green peel of walnut;
3rd step, by macroreticular resin 4%NaOH solution immersion treatment 2 ~ 4h, re-use distilled water flushing to neutral; Then soak 2 ~ 4h with 4%HCl solution, use distilled water flushing to neutral; Finally use 95% alcohol immersion process 2 ~ 4h again, distilled water is washed till without till alcohol taste; For subsequent use;
4th step, macroreticular resin are to the Static Adsorption of green peel of walnut polyphenol dilution and desorb;
5th step, macroreticular resin are to the Dynamic Adsorption of green peel of walnut polyphenol dilution and desorb;
6th step, employing HPLC method measure composition and the content of the green peel of walnut polyphenol extract free phenol after macroreticular resin separation and purification, complete all separation and purification processes.
2. the method for polyphenols in macroreticular resin separation and purification green peel of walnut according to claim 1, is characterized in that: macroreticular resin adopts NKA-9.
3. the method for polyphenols in macroreticular resin separation and purification green peel of walnut according to claim 1, it is characterized in that: in the described first step, the solid-liquid ratio of green peel of walnut dry powder and ethanol is 1g:17mL, ultrasonic extraction twice, filtrate, by rotary evaporation, obtains green peel of walnut polyphenolic extract.
4. the method for polyphenols in macroreticular resin separation and purification green peel of walnut according to claim 1, it is characterized in that, the detailed process that described Folin-Ciocaileu method measures polyphenol total content in green peel of walnut is: get 0.2mL green peel of walnut polyphenolic extract, join in 10mL volumetric flask, add 0.5mLFC reagent, mix, in 1min, add 1.5mL20% sodium carbonate liquor, mixing, uses deionized water constant volume; Above-mentioned solution after dark placement 1.5 ~ 2h, not add the solution of crude extract as blank, is measured absorbance at 35 ~ 40 DEG C under 760nm wavelength.
5. the method for polyphenols in macroreticular resin separation and purification green peel of walnut according to claim 1, it is characterized in that, the detailed process of described macroreticular resin Static Adsorption is: take 1.0g resin in 50mL triangular flask, add the 3rd step gained spare resin sample liquid 30mL, sealing is placed on 160 ~ 175r/min shaking table, vibrate under room temperature 4 ~ 5h, samples, measure polyphenol content from supernatant; Descend formulae discovery adsorbance and adsorption rate according to this, the concentration of investigation sample liquid and pH are on the impact of resin adsorption performance;
qe＝(C0－C1)×V1/W(1)
E(％)＝[(C0－C1)/C0]×100(2)
In formula: qe-adsorbance, mg/g; E-adsorption rate, %; C0-initial concentration, mg/mL;
C1-equilibrium concentration, mg/mL; V1-adsorbent solution volume, mL; The weight of W-resin, g.
6. the method for polyphenols in macroreticular resin separation and purification green peel of walnut according to claim 1, it is characterized in that, the detailed process of the static desorb of described macroreticular resin is: cleaned by the resin distilled water after fully adsorbing, until surperficial n.s liquid remains, then uses filter paper suck dry moisture, be placed in triangular flask, add 30mL different concentration ethanol solution, sealing is positioned in 160 ~ 175r/min oscillator, desorb 2 ~ 3h under room temperature, sample from supernatant, survey polyphenol content; Descend formulae discovery desorption efficiency and desorption quantity according to this, the concentration of investigation eluent and pH are on the impact of resin elution performance;
D(％)＝C2×V2×100/(C0－C1)×V1(3)
qd＝C2×V2/W(4)
In formula: D-desorption efficiency, %; Qd-desorption quantity, mg/g; C0-initial concentration, mg/mL;
C1-equilibrium concentration, mg/mL; Polyphenol concentration in C2-stripping liquid, mg/mL; V1-adsorbent solution volume, mL; V2-stripping workshop volume, mL; W-resin taken amount, g.
7. the method for polyphenols in macroreticular resin separation and purification green peel of walnut according to claim 1, it is characterized in that, the detailed process of described macroreticular resin Dynamic Adsorption is: resin wet method good for the 3rd step pretreatment loaded in the chromatographic column of 16mm × 500mm, after distilled water balance, by sample liquid by 1.3mL/min at the uniform velocity loading collect efflux, every 10mL is a pipe, detect and calculate outflow concentration, determine flow velocity, volume conditions, obtain optimal adsorption condition, draw Dynamic Adsorption curve.
8. the method for polyphenols in macroreticular resin separation and purification green peel of walnut according to claim 1, it is characterized in that, the detailed process of described macroreticular resin dynamic desorption is: carry out desorption experiment by adsorbing saturated resin, be washed till n.s liquid with distilled water to remain, at the uniform velocity carry out wash-out with ethanolic solution by 0.43mL/min, collect eluent, calculate outflow concentration, determine elution flow rate, volume conditions, obtain optimum washing engaging condition, draw dynamic desorption curve.
9. the method for polyphenols in macroreticular resin separation and purification green peel of walnut according to claim 1, it is characterized in that, after adopting HPLC method to measure macroreticular resin separation and purification, in green peel of walnut polyphenol extract, the concrete grammar of free phenol composition and content is: adopt 5 μm, the DiamonsilC18 post of 250mm × 4.6mm; Determined wavelength 280nm; Flow velocity 1.0ml/min; Column temperature 30 DEG C; Sample size 20uL; Mobile phase A is 0.1% formic acid high purity water; Mobile phase B is 0.1% formic acid acetonitrile; Gradient elution program is followed successively by 93%A, 7%B, 0min; 82%A, 18%B, 30min; 78%A, 22%B, 40min; 50%A, 50%B, 45min; 50%A, 50%B, 50min; Sample peak retention time and standard items retention time are contrasted, judges free phenol kind, according to calculated by peak area free phenol content.
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| CN107163616A (en) * | 2017-05-22 | 2017-09-15 | 郭良 | A kind of method and its purification process for extracting walnut green husk pigment |
| CN107468731A (en) * | 2017-07-12 | 2017-12-15 | 浙江理工大学 | Total polyphenols separation purifying technique in a kind of Bai le blade |
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| CN110229054A (en) * | 2019-07-19 | 2019-09-13 | 商洛学院 | A kind of preparation method of green peel of walnut juglone extract and its application in the prevention and treatment of Chinese medicine root rot |
| CN113559146A (en) * | 2021-07-28 | 2021-10-29 | 西北农林科技大学 | Method for efficiently extracting walnut green husk polyphenol substances by electron beam irradiation |
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