CN105198851B - A kind of purification process of blackberry, blueberry anthocyanidin - Google Patents
A kind of purification process of blackberry, blueberry anthocyanidin Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
- C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
- C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
- C07D311/58—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring other than with oxygen or sulphur atoms in position 2 or 4
- C07D311/60—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring other than with oxygen or sulphur atoms in position 2 or 4 with aryl radicals attached in position 2
- C07D311/62—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring other than with oxygen or sulphur atoms in position 2 or 4 with aryl radicals attached in position 2 with oxygen atoms directly attached in position 3, e.g. anthocyanidins
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Abstract
Two steps are purified the invention discloses a kind of purification process of blackberry, blueberry anthocyanidin, including resin pretreatment, blackberry, blueberry anthocyanidin crude extract, the efficiently purifying of blackberry, blueberry anthocyanidin can be realized, the purity of gained blackberry, blueberry anthocyanidin is high.The purification process of the present invention is from polyamide as adsorbent, and it has a big specific surface area and big granularity, absorption and desorption ability height, low cost, regenerating easily and the preparation method characteristic such as simply.The present invention passes through to the static and dynamic absorption of blackberry, blueberry anthocyanidin and the research of desorption process, optimize purification condition, establish the optimum value of initial concentration, the pH value of adsorption liquid, the pH value of eluent and the concentration of alcohol of blackberry, blueberry anthocyanidin so that the adsorption rate and eluting rate of blackberry, blueberry anthocyanidin are higher.The method of the present invention can realize that blackberry, blueberry anthocyanidin is time saving, efficient purifying, be conducive to saving purification time, cost-effective.
Description
Technical field
The present invention relates to a kind of purification process of blackberry, blueberry anthocyanidin, belong to plant extract technical field of purification.
Background technology
The method that anthocyanidin is extracted from plant is a kind of non-selective extracting method, in addition to pigment, can be produced big
Other compounds of amount, such as carbohydrate, glycitols, organic acid, amino acid, protein will influence the stability of pigment.Therefore,
Purifying is carried out to the anthocyanidin in blackberry extract to be very important, pigment stability can be effectively lifted, and promotes it in food
Application in product processing.In the prior art, the method for purifying anthocyanidin is mainly CO2Method, column chromatography, adsorbent method and double
Aqueous phase abstraction technique.
Absorption is a kind of method of simple and effective purifying biological active component.Have recently been used as purifying from natural plants
The adsorbent of active ingredient (ball pine tree, flavonoids and polyphenol etc.) has macroporous absorbent resin, cationic ion-exchange resin and polyamide
Resin.Wherein, polyamide is a kind of resin with big specific surface area and big granularity.Due to polyamide absorption and
Desorption ability is high, cost is low, regenerating easily, and preparation method is simple, thus causes extensive concern.Polyamide can be from
Most impurity is removed in complicated mixture, so as to improve the content of chemical substance to be purified, is widely used in from natural
Separating effective ingredient in plant.Polyamide has special selectivity for flavones, therefore in plant extract industry, can make
For the superior adsorbent of flavone compound separation and Extraction.But, not yet useful polyamide isolates and purifies blackberry, blueberry flower at present
The relevant report of blue or green element.
The content of the invention
To solve the deficiencies in the prior art, it is an object of the invention to provide a kind of purification process of blackberry, blueberry anthocyanidin, energy
It is enough that efficiently purifying is carried out to blackberry, blueberry anthocyanidin.
In order to realize above-mentioned target, the present invention is adopted the following technical scheme that：
A kind of purification process of blackberry, blueberry anthocyanidin, comprises the following steps：(1) resin is pre-processed：Polyamide is first used
Ethanol boils, then is cleaned with distilled water, standby；
(2) purifying of blackberry, blueberry anthocyanidin crude extract：It will be diluted with water after the concentrated volatilization ethanol of blackberry, blueberry anthocyanidin crude extract,
Adjust after pH value, upper polyamide resin column is first washed with water, then with ethanol elution, is concentrated to give medicinal extract.
Further, foregoing purification process, comprises the following steps：
(1) resin is pre-processed：Polyamide is first boiled 3~5 hours with 60%~95% ethanol, then washed with distillation
Only it is standby to without alcohol taste；
(2) purifying of blackberry, blueberry anthocyanidin crude extract：It will be diluted with water after the concentrated volatilization ethanol of blackberry, blueberry anthocyanidin crude extract
To 0.6~2.99mgmL-1, adjusted with hydrochloric acid behind pH=1~5, with 2~10BV/h speed 1~7BV of upper prop, then use water
Wash 3BV (be washed till almost without sugar), then with pH value for 1~5 40%~100% ethanol with 2~10BV/h speed elution 0.5
~5BV, is concentrated to give medicinal extract.
Preferably, foregoing purification process, comprises the following steps：
(1) resin is pre-processed：Polyamide is first boiled 4 hours with 95% ethanol, then cleaned with distilled water to without alcohol
Taste, it is standby；
(2) purifying of blackberry, blueberry anthocyanidin crude extract：It will be diluted with water after the concentrated volatilization ethanol of blackberry, blueberry anthocyanidin crude extract
To 0.75mgmL-1, adjusted with hydrochloric acid after pH=1, with 5BV/h speed upper prop 5BV, 3BV be then washed with water and (is washed till and does not almost have
Have sugar), then for 3 80% ethanol 3.5BV is eluted with 5BV/h speed with pH value, vacuum distillation is concentrated to give medicinal extract.
In foregoing purification process, the preparation method of the blackberry, blueberry anthocyanidin crude extract is：Blackberry, blueberry is homogenized, blackberry, blueberry is obtained
Slurry is standby；The ethanol of water or any concentration is added, using microwave auxiliary extraction, extract solution is obtained；Extract solution is centrifuged, and collects supernatant
Liquid, is produced.
Further, in foregoing purification process, the preparation method of the blackberry, blueberry anthocyanidin crude extract is：Blackberry, blueberry is carried out even
Slurry, obtains blackberry, blueberry slurry standby；The ratio starched in 5~25mL/1g blackberry, blueberry adds the ethanol of water or any concentration, is extracted using microwave radiation technology
Take, 100~700w of microwave power, 1~9min of microwave treatment time obtains extract solution；Extract solution is centrifuged, and is collected supernatant, is produced.
Preferably, in foregoing purification process, the preparation method of the blackberry, blueberry anthocyanidin crude extract is：Blackberry, blueberry is carried out even
Slurry, obtains blackberry, blueberry slurry standby；The ratio starched in 25mL/1g blackberry, blueberry adds the ethanol that concentration is 52%, micro- using microwave auxiliary extraction
Wave power 469w, microwave treatment time 4min, obtains extract solution；Extract solution is centrifuged, and is collected supernatant, is produced.
In foregoing purification process, extract solution centrifugation is centrifuged 3~8 minutes for extract solution under the conditions of 3500r/min.
Preferably, in foregoing purification process, extract solution centrifugation is centrifuged 5 minutes for extract solution under the conditions of 3500r/min.
In foregoing purification process, polyamide is 60~100 mesh.
In order to ensure blackberry, blueberry anthocyanidin purification process science of the present invention, rationally, inventor has carried out corresponding experimental study
And screening, just it is able to determine technical scheme.Specific experiment content is as follows：
First, experiment material, medicine and instrument
Blackberry, blueberry is provided by polar bear Agro-ecology Co., Ltd sheep Chinese mugwort blackberry, blueberry plantation, and main agents and device information are shown in Table
1 and table 2, other reagents used are that analysis is pure.
The major experimental reagent information of table 1
The device information of table 2
2nd, experiment and conclusion
1st, purification experiment
1.1 purification experiment processes：
(1) resin is pre-processed：
1. ion exchange resin is pre-processed
Ion exchange resin is soaked in water 12h, is then washed with water to non-foam without brown, then carries out wet method dress post.With
5%HCl solution is washed till pH=5 with deionized water again after rinsing resin column, 2h with 2BV/h flow velocity, then with same flow velocity,
PH=10 is washed till with deionized water again after rinsing resin column, 2h with 5%NaOH solution, then with 2BV/h flow velocity, 5%HCl is used
Solution is washed till pH=4~5 with deionized water again after rinsing resin column, 2h, standby.
2. polyamide is pre-processed
Polyamide is first boiled 3~5 hours with 60%~95% ethanol, then cleaned with distilled water to without alcohol taste, it is standby
With；
(2) prepared by blackberry, blueberry anthocyanidin crude extract：
Blackberry, blueberry is homogenized, blackberry, blueberry slurry is obtained standby；The ratio starched in 5~25mL/1g blackberry, blueberry adds water or any concentration
Ethanol, using microwave auxiliary extraction, 100~700w of microwave power, 1~9min of microwave treatment time obtain extract solution；Extract solution
Centrifugation, collects supernatant, produces；
The blackberry, blueberry anthocyanidin crude extract solution be blackberry, blueberry anthocyanidin crude extract it is concentrated volatilization ethanol after be diluted with water
Arrive.
(3) purifying of blackberry, blueberry anthocyanidin crude extract：
0.6~2.99mgmL will be diluted with water to after the concentrated volatilization ethanol of blackberry, blueberry anthocyanidin crude extract-1, adjusted with hydrochloric acid
Save behind pH=1~5, with 2~10BV/h speed 1~7BV of upper prop, 3BV (being washed till almost without sugar) is then washed with water, then use pH
It is worth and 0.5~5BV is eluted with 2~10BV/h speed for 1~5 40%~100% ethanol, is concentrated to give medicinal extract.
2nd, blackberry, blueberry anthocyanidin content is determined
2.1 blackberry, blueberry anthocyanidin content assay methods：It is to be divided using chlorination Cyanidin -3-O- glucose as standard items
Analysis, determines the anthocyanidin content in blackberry, blueberry cyanine promotor composition.
The drafting of 2.2 standard curves
Accurately weighed 3.34mg anthocyanidin standard items, are dissolved with distilled water, and the constant volume in 10mL volumetric flasks is used as mother liquor.
Mother liquor is diluted to concentration respectively for 33.4,66.8,100.2,133.6,167,200.4,233.8,267.2 μ g/mL, taken respectively
The anthocyanidin standard items (blank control is with 50% ethanol water replacement) of 1.0mL various concentrations are placed in tool plug test tube, are added
5.00mL 1.00% methanol hydrochloride solution, fully shaking, 50 DEG C of water-bath 10min are cooled to room temperature, determine it in 530nm
The absorbance (A530) at place.Using anthocyanidin standard concentration as abscissa (X-axis), absorbance is ordinate (Y-axis), draws mark
Directrix curve.Calibration curve equation is：Y=0.0033X-0.0092, coefficient correlation γ=0.9989, i.e. active ingredient concentration range
In 33.4 μ gmL-1~267.2 μ gmL-1Inside there is good linear relationship.Standard curve is as shown in Figure 1.
The assay of 2.3 blackberry, blueberry anthocyanidin
Appropriate blackberry, blueberry anthocyanidin solution (being obtained by purifying gained medicinal extract dissolving) is taken, suitable concn is diluted to, according to 2.2
" drafting of standard curve " method carries out chromogenic assay, its concentration (c) is determined with standard curve, and then determine blackberry, blueberry anthocyanidin
Content.Shown in specific formula for calculation such as formula (1)：
Note：C：Mass concentration (the μ gmL of blackberry, blueberry anthocyanidin-1)；V：Liquor capacity (mL)；N：Extension rate；M：Sample
Quality (g).
3rd, Static Adsorption and desorption
3.1 Static Adsorption：
Static Adsorption process of the blackberry, blueberry anthocyanidin on resin is to carry out in the following manner：All Staticadsorption experiments
Carried out with batch processing mode at 25 DEG C.By appropriate times of 5mL adsorbent (pretreated resin) and 50mL dilution
Several blackberry, blueberry anthocyanidin crude extract solutions are separately added into 100mL conical flasks, are put on constant temperature oscillator, in 25 DEG C, 150~
24h is vibrated under the conditions of 200r/min.After Static Adsorption, absorbance is surveyed after surplus solution filtering.Resin is calculated by formula (2) to exist
Adsorption capacity (qe-mgmL during balance-1Resin).
Note：qeIt is Static Adsorption ability (mgmL-1Resin)；C0And CeIt is the initial concentration and balance of anthocyanidin respectively
Concentration (mgmL-1)。ViAnd VaIt is the amount of initial sample solution and the amount of initial adsorption agent respectively.
3.2 static desorptions：
Static desorption process is to carry out in the following manner:After ensuring to reach adsorption equilibrium, resin is by deionized water
Cleaning, is then eluted with 50mL ethanol (0.1% hydrochloric acid, v/v).The solution eluted surveys absorbance.Pass through formula (3) and formula
(4) static desorption ability when static desorption is balanced and static desorption efficiency (D, %) are calculated
Adsorption rate is calculated according to formula (9) and obtained
Note：qdIt is static desorption ability (mgmL-1Resin), AoAnd AeIt is the initial absorbance of blackberry, blueberry anthocyanidin respectively
With balance absorbance；ViIt is the amount of initial sample solution, VdIt is the volume (mL) of eluent.
Static Adsorption and static desorption experiment are carried out with different meshes polyamide.As a result it is as shown in Figure 2.From Fig. 2
Understand, 60~100 mesh and 100~200 mesh polyamides show higher quiet relative to the polyamide of other mesh numbers
State is adsorbed and static desorption ability.This is due to that it possesses big surface area.The rate of recovery different meshes of blackberry, blueberry anthocyanidin
Polyamide is tested, as a result as shown in Figure 3.As can be seen from Figure 3, the polyamide correspondence of 60~100 mesh is spent
The rate of recovery of blue or green element is higher than the polyamide of other mesh numbers.The Static Adsorption and steady-state solution of 60~100 mesh polyamides
Energy-absorbing power is respectively 4.41 and 3.29mgmL-1Resin.Because the separating rate of 100~200 mesh polyamides is much smaller than
60~100 mesh polyamides.Therefore, experiment hereafter uses 60~100 mesh polyamides as adsorbent.
4th, adsorption isotherm
The isothermal experiment of equilibrium adsorption of adsorbent is to carry out in the following manner：With 5mL adsorbent and 50mL
The blackberry, blueberry anthocyanidin crude extract solution of various concentrations is separately added into 100mL conical flasks, is placed on constant temperature oscillator, respectively 5
DEG C, 15 DEG C, vibrate 60min under the conditions of 25 DEG C.Determine initial concentration and equilibrium concentration under different temperatures respectively with AAS.
Equilibrium adsorption is described by Lang Gemiaoer and Freundlich model.Lang Gemiaoer thermoisopleths are widely used in monolayer adsorption,
Shown in empirical equation such as formula (5)：
Note：KLAnd qmIt is adsorption equilibrium costant and empirical respectively, can be by drawing Ce/qeAnd CeLinear graph and phase
The regression coefficient (γ) answered is worth to.
Freundlich model is widely used in nonideal adsorbed solution, shown in empirical equation such as formula (6)：
Note：KFIt is Freundlich constant (adsorption capacity)；1/n (adsorption strength) is an empirical, Ke Yitong
Cross drafting logqeAnd logCeLinear graph and corresponding regression coefficient (γ) value obtain.
The regularity of distribution of the blackberry, blueberry anthocyanidin between liquid phase and solid-phase media is that balance institute is reached in measurement adsorption process
The time needed, come what is determined, is stated by Lang Gemiaoer and Freundlich thermoisopleth.Respectively 15 DEG C, 25 DEG C, 35
Analyzed at DEG C.The constant of Lang Gemiaoer and Freundlich equation uses slope and intercept meter in Fig. 4 and Fig. 5 respectively
Calculate, as a result as shown in table 3.Lang Gemiaoer and Freundlich thermoisopleth are most common moulds in research desorption balance data
Type.The surface of Freundlich model assumption resin is inherently multiphase, is characterized in different energy point absorption.
It describes the absorption behavior of a monolayer and polymolecular layer.Lang Gemiaoer models describe one with as far as possible identical
The absorption behavior that the monolayer adsorption of adsorption site and adsorbed molecule are not interacted each other.Correlation coefficient charts in table 3
Bright Freundlich model is more suitable for describing equilibrium adsorption.The adsorption strength value (n) of Freundlich model is 0<n<10
In the range of, this shows that the absorption of blackberry, blueberry anthocyanidin is good.It can see from table, KFIncrease with the reduction of temperature, table
The absorption of bright blackberry, blueberry anthocyanidin is reduced as temperature rises.Thermo-responsive anthocyanidin is degraded at higher temperatures can explain this
A little results.In addition, the coefficient correlation of Freundlich model is far above Lang Gemiaoer models.It can therefore be concluded that, blackberry, blueberry flower
The absorption of blue or green element has preferably relevance with Freundlich equation, especially under the conditions of 15 DEG C.
Table 3
Note：qmax- maximum adsorption ability；γ-regression coefficient；KL- Lang Gemiaoer constants；KF- Freundlich constant；n–
Adsorption strength.
5th, adsorption equilibrium and dynamics
The blackberry, blueberry anthocyanidin crude extract of 5mL adsorbents (pretreated resin) and 50mL dilution suitable multiple is molten
Liquid is separately added into 100mL conical flasks, puts and 60min is vibrated on constant temperature oscillator.1000 μ L solution are taken to survey absorbance every 5min, with
This judges that adsorption process reaches the time of balance.
Pass through the absorption of pseudo- First order dynamic model and pseudo-second-order equation model analysis blackberry, blueberry anthocyanidin.Experimental data and
Uniformity between the predicted value of adsorption capacity is expressed with the coefficient correlation (γ values) of model.Of a relatively high γ values show this
Model successfully describes the adsorption dynamics adsorption kinetics of anthocyanidin.
Pseudo- First order dynamic model, as shown in formula (7)：
ln(qe-qt)=lnqe-K1t (7)
Formula (7) can be exchanged into
Pseudo-second-order equation model, as shown in formula (8)：
Note：K1And K2It is the speed constant of pseudo- First order dynamic model and pseudo-second-order equation model respectively；qtIt is in the time
Blackberry, blueberry anthocyanidin adsorbance (mgmL during for t-1)；qeIt is the adsorption capacity in adsorption equilibrium.
Absorption is a physical and chemical processes, is related to mass transfer of a large amount of solutes (adsorbate) from liquid phase to adsorbent surface.Absorption
Dynamic (dynamical) research can provide the information on absorption mechanism, can be used for the efficiency for assessing adsorption process.Adsorption dynamics adsorption kinetics
Research describe solute infiltration rate in solid-liquid interface.
In order to assess applicability of the pseudo- First order dynamic model for the absorption of anthocyanidin, log (q are depictede-qt) with t's
Linear graph, as shown in Figure 6.qeValue (being obtained from model) and K1(being obtained from the intercept and slope in figure) is as shown in table 4 respectively.t/
qtWith t linear graph, as shown in Figure 7.Equilibrium adsorption capacities and pseudo-second-order equation constant K2(obtained from the intercept and slope in figure
) as shown in table 4 respectively.As can be seen from Table 4, the γ (0.9314) of pseudo- First order dynamic model is far below pseudo- second motive force
Learn the γ (0.9979) of model.It is possible thereby to infer, pseudo-second-order equation model is more suitable for the absorption for describing blackberry, blueberry anthocyanidin.It is pseudo-
First order dynamic model describes the starting stage of adsorption process.Pseudo-second-order equation model was entirely adsorbed to prediction and description
Journey is more particularly suitable.Pseudo-second-order equation model is more suitable for describing suction of the blackberry, blueberry anthocyanidin on resin than pseudo- First order dynamic model
Attached dynamics.
Table 4
Note：K1- puppet first order kinetics speed constant；K2- pseudo-second-order equation speed constant；qeThe absorption of-kinetic model
Ability；γ-regression coefficient.
6th, the condition optimizing for adsorbing and eluting
Determine after adsorption equilibrium and dynamics, the initial ranges of purifying process parameter are obtained using single factor experiment, including
Initial concentration (mgmL-1), the pH value of adsorption liquid, the pH value and concentration of alcohol (%) of eluent.
Determine optimal initial concentration in the following way：Take 5mL resin sorbent and the anthocyanidin of 20mL various concentrations
Crude extract solution is separately added into conical flask, is put and is vibrated 1h on 15 DEG C of constant temperature oscillators.Determine blackberry, blueberry anthocyanidin not to be adsorbed
Absorbance.
Determine the pH value of optimal adsorption liquid in the following way：Take the suction of 5mL resin sorbent pH value different with 20mL
Attached liquid is separately added into conical flask, is put and is vibrated 1h on 15 DEG C of constant temperature oscillators.Determine the extinction of blackberry, blueberry anthocyanidin not to be adsorbed
Degree.
It is determined that the pH value of optimal eluent is in the following way：Take the 5mL resin sorbent for having adsorbed blackberry, blueberry anthocyanidin
The eluent of pH value different with 50mL is separately added into conical flask, is put and is vibrated 1h on 15 DEG C of constant temperature oscillators.Measure is eluted
Blackberry, blueberry anthocyanidin absorbance.
It is determined that optimal concentration of alcohol is in the following way：Take 5mL the resin sorbent for having adsorbed blackberry, blueberry anthocyanidin and
The ethanol of 50mL various concentrations is separately added into conical flask, is put and is vibrated 1h on 15 DEG C of constant temperature oscillators.It is black that measure is eluted
The absorbance of certain kind of berries anthocyanidin.
The influence that 6.1 initial concentrations are adsorbed to blackberry, blueberry anthocyanidin
In order to study influence of the initial concentration to absorption of anthocyanidin, using in 0.6~2.99mgmL-1In the range of it is different
The mode of influence of the initial concentration to blackberry, blueberry anthocyanidin adsorption rate is stated.Keep pH value=3 of adsorption liquid, the pH value of eluent
=4 and concentration of alcohol be 75%.As shown in Figure 8, adsorption rate with initial concentration from 2.99 to 0.75mgmL-1Reduction and increase
Plus, but initial concentration is further reduced, the increase of adsorption rate is not obvious.Therefore, preferred initial dense of the absorption of blackberry, blueberry anthocyanidin
Spend for 0.75mgmL-1。
The influence that the pH value of 6.2 adsorption liquids is adsorbed to blackberry, blueberry anthocyanidin
As shown in figure 9, adsorption rate is reduced with the pH value of adsorption liquid from 1 to 5 increase, therefore pH value=1 of adsorption liquid
When, most beneficial for the absorption of blackberry, blueberry anthocyanidin.
The influence that the pH value of 6.3 eluents is eluted to blackberry, blueberry anthocyanidin
In order to study the influence of the pH value of eluent to the elution of anthocyanidin, using the pH value in eluent from 1 to 5 scopes
The mode of the interior influence to desorption efficiency is stated.Holding initial concentration is 0.75mgmL-1, adsorption liquid pH value=1 and ethanol it is dense
Spend for 75%.As shown in Figure 10, desorption efficiency (eluting rate) increases with the pH value of eluent from 1 to 3 increase, in eluent
PH value be 3 when peak, further improve eluent pH value, cause desorption efficiency to reduce.Therefore the pH value of eluent
Optimum value is 3.
The influence that 6.4 concentration of alcohol are eluted to blackberry, blueberry anthocyanidin
As shown in figure 11, with the increase (from 20% to 100%) of concentration of alcohol, desorption efficiency (eluting rate) is also increasing,
After concentration of alcohol reaches 80%, desorption efficiency is again on a declining curve.Therefore the optimal concentration of ethanol is 80%.
7th, Dynamic Adsorption and desorption
7.1 Dynamic Adsorptions and desorption experiment：
Dynamic Adsorption and desorption experiment are carried out on chromatographic column (20mm × 400mm).The bed volume (BV) of adsorbent is
40mL, it is highly 12.7cm to load adsorbent.Different flow velocitys (2BV/h, 5BV/h, 10BV/h) are compared to blackberry, blueberry anthocyanidin
The influence of absorption.Collected once every 0.5BV, carry out absorbance measurement.Reach after adsorption saturation, the adsorbent in post spend from
Sub- water (2BV) cleans up, then with ethanol (pH=3,80%, v/v) under (2BV/h, 5BV/h, 10BV/h) different in flow rate
Elution.
7.2 Dynamic Adsorptions and desorption curve
For the absorption liquid measure needed for specification and elution liquid measure, go to calculate sample (2BV/ under different in flow rate by experiment
H, 5BV/h and 10BV/h) leak point.Therefore, by the volume and the concentration of solute of adsorption liquid, the poly- of 60~100 mesh numbers is obtained
The dynamic leakage curve of amide resin.
As shown in figure 12.When flow velocity is 5BV/h, absorption property reaches most preferably.When in low flow velocity, absorbate point
Son is more with the adsorbent interphase interaction time, although and during high flow rate required time less, the residence time of absorbate
It is short, adsorption capacity can be impacted, leakage critical point also will faster reach.Generally, outflow concentration reaches adsorption liquid concentration
5% be defined as leak point.To sum up, absorption liquid measure be 5BV, absorption flow velocity be 5BV/h be Dynamic Adsorption optimum condition.
Drawn by the volume and solute concentration of stripping workshop and obtain dynamic desorption curve, as shown in figure 13.It is 3 with pH value
80% ethanol elution anthocyanidin, keeps flow velocity in 2BV/h, 5BV/h and 10BV/h respectively.As can be seen from the figure, in eluent
Consumption is 3.5BV, and flow velocity can elute anthocyanidin completely when being 5BV/h.Therefore, eluent consumption is 3.5BV, and flow velocity is 5BV/
H is optimal desorption condition.
8th, influence of the different resins for the purifying of blackberry, blueberry anthocyanidin
Ion exchange resin, macroporous absorbent resin and polyamide have been selected respectively, and research different resins are for blackberry, blueberry
The influence of the purifying of anthocyanidin, the results are shown in Table 5.Adsorbed and contained in obtained blackberry, blueberry anthocyanidin medicinal extract by polyamide
432mg·g-1Anthocyanidin, this content is higher than the anthocyanidin purity (160mgg obtained by cationic exchange resin adsorption-1)
With the anthocyanidin purity (176mgg obtained by macroporous absorbent resin-1).Therefore, polyamide is optimal selection.
Table 5
The present invention is advantageous in that：The invention provides a kind of purification process of blackberry, blueberry anthocyanidin, it can realize black
The efficiently purifying of certain kind of berries anthocyanidin, the purity of gained blackberry, blueberry anthocyanidin is high.The purification process of the present invention selects 60~100 mesh polyamide
Resin is as adsorbent, and it has a big specific surface area and big granularity, absorption and desorption ability height, low cost, regenerating easily and
The characteristic such as preparation method is simple.The present invention is excellent by the static and dynamic absorption of blackberry, blueberry anthocyanidin and the research of desorption process
Change purification condition, establish initial concentration, the pH value of adsorption liquid, the pH value of eluent and the concentration of alcohol of blackberry, blueberry anthocyanidin
Optimum value so that the adsorption rate and eluting rate of blackberry, blueberry anthocyanidin are higher.The method of the present invention can realize blackberry, blueberry anthocyanidin
Time saving, efficient purifying, is conducive to saving purification time, cost-effective.
Brief description of the drawings
Fig. 1 is the standard curve that blackberry, blueberry anthocyanidin content is determined；
Fig. 2 is the absorption of blackberry, blueberry anthocyanidin and eluting power figure using different meshes polyamide；
Fig. 3 is the blackberry, blueberry anthocyanidin rate of recovery figure using different meshes polyamide；
Fig. 4 is C under different temperaturese/qeAnd CeLinear graph；
Fig. 5 is logq under different temperatureseAnd logCeLinear graph；
Fig. 6 is log (qe-qt) with t linear graph；
Fig. 7 is t/qtWith t linear graph；
Fig. 8 is blackberry, blueberry anthocyanidin adsorption rate with initial concentration variation diagram；
Fig. 9 is blackberry, blueberry anthocyanidin adsorption rate with adsorption liquid pH value variation diagram；
Figure 10 is blackberry, blueberry anthocyanidin eluting rate with eluent pH value variation diagram；
Figure 11 is blackberry, blueberry anthocyanidin eluting rate with concentration of alcohol variation diagram；
Figure 12 is dynamic adsorption curve map；
Figure 13 is dynamic desorption curve map.
Embodiment
The present invention is further introduced below in conjunction with specific embodiment.
The purification process of the blackberry, blueberry anthocyanidin of embodiment 1, comprises the following steps：
(1) resin is pre-processed：Polyamide is first boiled 5 hours with 60% ethanol, then cleaned with distilled water to without alcohol
Taste, it is standby；
(2) blackberry, blueberry anthocyanidin crude extract (is prepared, the content of blackberry, blueberry anthocyanidin in the crude extract medicinal extract by art methods
About 2.8%) purifying：Blackberry, blueberry anthocyanidin crude extract is diluted with water to 0.6mgmL-1, adjusted with hydrochloric acid after pH=5, with
Polyamide resin column 7BV in 2BV/h speed, is then washed with water 3BV (be washed till almost without sugar), then with pH value for 1 40%
Ethanol elutes 5BV with 2BV/h speed, is concentrated to give medicinal extract.
Using this purifying process, the adsorption rate of blackberry, blueberry anthocyanidin is 81%, and eluting rate is 70%, black in purifying gained medicinal extract
The content of certain kind of berries anthocyanidin is 62%.
The purification process of the blackberry, blueberry anthocyanidin of embodiment 2, comprises the following steps：
(1) resin is pre-processed：60~100 mesh polyamides are first boiled 3 hours with 80% ethanol, then washed with distillation
Only it is standby to without alcohol taste；
(2) purifying of blackberry, blueberry anthocyanidin crude extract：It will be diluted with water after the concentrated volatilization ethanol of blackberry, blueberry anthocyanidin crude extract
To 2.99mgmL-1, adjusted with hydrochloric acid after pH=3, with polyamide resin column 1BV in 10BV/h speed, 3BV be then washed with water
(be washed till almost without sugar), then with pH value for 5 100% ethanol with 10BV/h speed elution 0.5BV, be concentrated to give medicinal extract.Adopt
This purifying process is used, the adsorption rate of blackberry, blueberry anthocyanidin is 78%, and eluting rate is 65%, blackberry, blueberry anthocyanidin in purifying gained medicinal extract
Content is 60%.
Blackberry, blueberry anthocyanidin crude extract is according to said method prepared in step (2)：Blackberry, blueberry is homogenized, blackberry, blueberry slurry is obtained standby；Press
The ratio of 5mL/1g blackberry, blueberry slurry adds absolute ethyl alcohol, using microwave auxiliary extraction, microwave power 700w, microwave treatment time
1min, obtains extract solution；Extract solution is centrifuged 8 minutes under the conditions of 3500r/min, is collected supernatant, is produced.The crude extract is condensed into
The content of blackberry, blueberry anthocyanidin is 3.2% after medicinal extract.
The purification process of the blackberry, blueberry anthocyanidin of embodiment 3, comprises the following steps：
(1) resin is pre-processed：80 mesh polyamides are first boiled 4 hours with 95% ethanol, then cleaned with distilled water to
It is standby without alcohol taste；
(2) purifying of blackberry, blueberry anthocyanidin crude extract：It will be diluted with water after the concentrated volatilization ethanol of blackberry, blueberry anthocyanidin crude extract
To 0.75mgmL-1, adjusted with hydrochloric acid after pH=1, with polyamide resin column 5BV in 5BV/h speed, 3BV be then washed with water
(be washed till almost without sugar), then with pH value for 3 80% ethanol with 5BV/h speed elution 3.5BV, vacuum distillation is concentrated to give leaching
Cream.Using this purifying process, the adsorption rate of blackberry, blueberry anthocyanidin is 85%, and eluting rate is 78%, and blackberry, blueberry is spent in purifying gained medicinal extract
The content of blue or green element is 66%.
Blackberry, blueberry anthocyanidin crude extract is according to said method prepared in step (2)：Blackberry, blueberry is homogenized, blackberry, blueberry slurry is obtained standby；Press
The ratio of 25mL/1g blackberry, blueberry slurry adds the ethanol that concentration is 52%, using microwave auxiliary extraction, microwave power 469w, at microwave
Reason time 4min, obtains extract solution；Extract solution is centrifuged 5 minutes under the conditions of 3500r/min, is collected supernatant, is produced.The crude extract
The content for being condensed into blackberry, blueberry anthocyanidin after medicinal extract is 5.2%.
The purification process of the blackberry, blueberry anthocyanidin of embodiment 4, comprises the following steps：
(1) resin is pre-processed：60~70 mesh polyamides are first boiled 4.5 hours with 75% ethanol, then use distilled water
Clean to without alcohol taste, it is standby；
(2) purifying of blackberry, blueberry anthocyanidin crude extract：It will be diluted with water after the concentrated volatilization ethanol of blackberry, blueberry anthocyanidin crude extract
To 1.5mgmL-1, adjusted with hydrochloric acid after pH=2, with polyamide resin column 4BV in 8BV/h speed, 3BV be then washed with water
(be washed till almost without sugar), then with pH value for 4 90% ethanol with 4BV/h speed elution 3BV, vacuum distillation is concentrated to give leaching
Cream.Using this purifying process, the adsorption rate of blackberry, blueberry anthocyanidin is 80%, and eluting rate is 67%, and blackberry, blueberry is spent in purifying gained medicinal extract
The content of blue or green element is 61%.
Blackberry, blueberry anthocyanidin crude extract is according to said method prepared in step (2)：Blackberry, blueberry is homogenized, blackberry, blueberry slurry is obtained standby；Press
The ratio of 15mL/1g blackberry, blueberry slurry adds water, and using microwave auxiliary extraction, microwave power 100w, microwave treatment time 9min must be carried
Take liquid；Extract solution is centrifuged 3 minutes under the conditions of 3500r/min, is collected supernatant, is produced.The crude extract is condensed into black after medicinal extract
The content of certain kind of berries anthocyanidin is 4.1%.
Claims (7)
1. a kind of purification process of blackberry, blueberry anthocyanidin, it is characterised in that：Comprise the following steps：
(1) resin is pre-processed：Polyamide is first boiled 3~5 hours with 60%~95% ethanol, then cleaned with distilled water to
It is standby without alcohol taste；Wherein, polyamide is 60~100 mesh；
(2) purifying of blackberry, blueberry anthocyanidin crude extract：0.6 will be diluted with water to after the concentrated volatilization ethanol of blackberry, blueberry anthocyanidin crude extract
~2.99mgmL-1, adjusted with hydrochloric acid behind pH=1~5, with 2~10BV/h speed 1~7BV of upper prop, 3BV be then washed with water,
0.5~5BV is eluted with 2~10BV/h speed for 1~5 40%~100% ethanol with pH value again, medicinal extract is concentrated to give.
2. the purification process of blackberry, blueberry anthocyanidin according to claim 1, it is characterised in that：Comprise the following steps：
(1) resin is pre-processed：Polyamide is first boiled 4 hours with 95% ethanol, then cleaned with distilled water to without alcohol taste, it is standby
With；
(2) purifying of blackberry, blueberry anthocyanidin crude extract：It will be diluted with water to after the concentrated volatilization ethanol of blackberry, blueberry anthocyanidin crude extract
0.75mg·mL-1, adjusted with hydrochloric acid after pH=1, with 5BV/h speed upper prop 5BV, be then washed with water 3BV, then with pH value be 3
80% ethanol 3.5BV is eluted with 5BV/h speed, vacuum distillation is concentrated to give medicinal extract.
3. the purification process of the blackberry, blueberry anthocyanidin according to any one of claim 1~2, it is characterised in that：The blackberry, blueberry flower
The preparation method of blue or green element crude extract is：Blackberry, blueberry is homogenized, blackberry, blueberry slurry is obtained standby；The ethanol of water or any concentration is added, is adopted
With microwave auxiliary extraction, extract solution is obtained；Extract solution is centrifuged, and is collected supernatant, is produced.
4. the purification process of blackberry, blueberry anthocyanidin according to claim 3, it is characterised in that：The blackberry, blueberry anthocyanidin crude extract
Preparation method be：Blackberry, blueberry is homogenized, blackberry, blueberry slurry is obtained standby；The ratio starched in 5~25mL/1g blackberry, blueberry adds water or any
The ethanol of concentration, using microwave auxiliary extraction, 100~700w of microwave power, 1~9min of microwave treatment time obtain extract solution；Carry
Take liquid to centrifuge, collect supernatant, produce.
5. the purification process of blackberry, blueberry anthocyanidin according to claim 4, it is characterised in that：The blackberry, blueberry anthocyanidin crude extract
Preparation method be：Blackberry, blueberry is homogenized, blackberry, blueberry slurry is obtained standby；It is 52% that the ratio starched in 25mL/1g blackberry, blueberry, which adds concentration,
Ethanol, using microwave auxiliary extraction, microwave power 469w, microwave treatment time 4min obtain extract solution；Extract solution is centrifuged, and is received
Collect supernatant, produce.
6. the purification process of blackberry, blueberry anthocyanidin according to claim 3, it is characterised in that：The extract solution centrifugation is extraction
Liquid is centrifuged 3~8 minutes under the conditions of 3500r/min.
7. the purification process of blackberry, blueberry anthocyanidin according to claim 6, it is characterised in that：The extract solution centrifugation is extraction
Liquid is centrifuged 5 minutes under the conditions of 3500r/min.
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| Optimization of the microwave-assisted extraction and antioxidant activities of anthocyanins from blackberry using a response surface methodology;Yao Wen等;《RSC Advances》;20150122;第5卷;第19687页左栏2.2 * |
| 蓝莓花青素的提取及抗氧化性研究;陈健等;《北京林业大学学报》;20110331;第33卷(第2期);第127页左栏1.1.2、右栏1.2.6，第129页右栏3 * |
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