CN108409807A - A method of separation prepares high mallow element -3-O- glucosides - Google Patents
A method of separation prepares high mallow element -3-O- glucosides Download PDFInfo
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Abstract
The present invention provides a kind of method that separation prepares 3 O glucosides of high mallow element, including alcohol extracting concentration, macroporous resin adsorption, preparative liquid chromatography purifying and high speed adverse current chromatogram separation, is detached in the blueberry raw material complicated from anthocyanin composition and prepare 3 O glucoside monomers of high mallow element.The present invention for the first time combines preparative liquid chromatography and high-speed countercurrent chromatography, and by the optimization to technological parameter, the 3 O glucoside purity monomers of high mallow element of isolated high-purity, purity may be up to 99% from blueberry.
Description
Technical field
Field is isolated and purified the present invention relates to natural products, and in particular to a kind of separation preparation high mallow element -3-O- grapes
The method of glucosides.
Background technology
Anthocyanin is a kind of water colo(u)r being widely present in plant, be by anthocyanidin and one or more glycosyls,
Such as glucose, galactolipin, arabinose combine the polyphenol compound formed by glycosidic bond.Common flower in nature
Green element mainly has 6 kinds, is pelargonin (Pelargonidin), Cyanidin (Cyaniain), delphinidin respectively
(Delphinidin), peonidin (Peonidin), petunidin (Petunidin) and malvidin (Malvidin).In recent years
Come, a large amount of research confirms that natural anthocyanin has anti-oxidant, antitumor, prevention of cardiovascular disease, alleviates diabetes
And the bioactivity such as obesity controlling.With the continuous improvement of people's living standards, people are to natural origin and have no toxic side effect
The cry of function ingredients is also higher and higher, and anthocyanin is as the most representative function factor of natural origin, because its is good
Color function and excellent bioactivity are favored by numerous researchers and food pharmaceutical manufacturer.
But since tree peony anthocyanins structure is similar, polarity difference is smaller, leads to the separation of high-purity anthocyanin monomer
It purifies extremely difficult.But isolate and purify to obtain high-purity anthocyanin monomer currently, having had been reported that.
Pelargonin -3-O- is prepared as the Chinese patent literature of 106366141 A of Publication No. CN discloses a kind of separation
The method of glucoside monomer, it is pure by freeze-drying, alcohol extracting concentration, fractional extraction, AB-8 macroreticular resins using strawberry as raw material
Change is prepared.For another example the Chinese patent literature of 106831911 A of Publication No. CN discloses isolates and purifies in a kind of Cong Peng Lei
The method of pelargonin -3-O- glucoside monomers, including alcohol extracting concentration, ethyl acetate extraction, AB-8 macroreticular resins and high speed
Adverse current chromatogram.
Although the anthocyanin monomer of high-purity has been prepared in above-mentioned technical proposal, main reason is that strawberry and
Anthocyanin composition is simple in Peng Lei, contains only Cyanidin -3-O- glucosides, pelargonin -3-O- glucosides and fish pelargonium
3 kinds of anthocyanin compounds of element -3-O- rutinosides, wherein pelargonin -3-O- glucosides account for Anthocyanin content
80% or more.But for the raw material that anthocyanin composition is complicated, above-mentioned technical solution is then difficult to realize high-purity anthocyanin monomer
Purifying and preparation.
Blueberry, also known as cowberry, blue berry, belong to Ericaceae, and cowberry platymiscium is not only rich in the basal nutrient of needed by human body
Ingredient, but also a variety of different types of anthocyanin are rich in, there is activation retina, hypoglycemic, anti-inflammatory and antitumor action.I
The blueberry cultivation of state is started late, and at present mainly based on directly fresh food consumption, or is processed into the primary such as jam, fruit juice, fruit wine
Product, the blueberry product in relation to high added value are very few in the market at home and abroad.Since the anthocyanin composition in blueberry is multiple
It is miscellaneous, contain the similar anthocyanin compound of at least 12 kinds of structures, causes to prepare by single column chromatography or chromatographic technique at present
Obtained high-purity anthocyanin is anthocyanin mixture rather than high-purity anthocyanin monomer.
As Publication No. CN 106905391A Chinese patent literature in disclose a kind of Anthocyanin from Blueberry extracting and developing
Blueberry is squeezed the juice and is mixed with extractant by purification process, the homogeneous extraction 1~4 under conditions of room temperature, pressure are 100~160MPa
Secondary, filtering, merging filtrate obtain Anthocyanin from Blueberry crude extract, then isolated and purified through HPD600 macroreticular resins.The technical side
Case is discharged blueberry functional component using 80% ethanol solution of pH=1~2 as extractant, using high pressure from biological cell, solution
Blueberry active ingredient of having determined is under the premise of keeping high extraction, the problem of active ingredient is not by high temperature, but what is obtained carries
It is anthocyanin mixture to take object, and the content of anthocyanin is only 46.45%.
A kind of wild blueberry anthocyanidin is for another example disclosed in the Chinese patent literature of Publication No. CN 104109403A to carry
It takes, Novel purification method, preparation process includes：It is biological enzymolysis, microwave refluxing extraction, collection, coarse filtration, micro porous filtration, ultrafiltration, true
Vacuum freecing-dry, split-phase, high speed adverse current chromatogram purifying.The technical solution uses non-heating power high efficiency extraction isolation technics, improves
Rate of extraction, but extract, purifying process it is excessively complicated, it is difficult to realize large-scale industrial production, and the extract obtained is still
Purity for anthocyanin mixture, anthocyanin is only up to 42.7%.
(" sephadex chromatography combination high speed adverse current chromatogram extracts anthocyanidin in blueberry ", Guo Danni, the Xiang Can such as Guo Danni
Brightness, Chen Yang et.al, food industry, the 2nd phase in 2016) it tests in conjunction with sephadex chromatography and high speed adverse current chromatogram to wild
Anthocyanidin in blueberry is isolated and purified, and blueberry crude extract first passes through sephadex chromatography initial gross separation, obtains cyanine
The high component of cellulose content, then detached through high speed adverse current chromatogram, with MTBE- n-butanols-acetonitrile-water (1 ︰ of volume ratio, 3 ︰, 1 ︰ 5) for two
Phase solvent system is divided under conditions of flow velocity 0.5m L/min, engine speed 1 860r/min and Detection wavelength 280nm
From disposable isolated two kinds of anthocyanidin, purity are respectively from the sephadex chromatography post separation product of blueberry
65.0% and 90.0%.Although the technical solution discloses its isolated two kinds of anthocyanidin, chromatography is analyzed from the UPLC of its Fig. 2
In figure, only deducibility sample 1 and sample 2 may be anthocyanidin, and can not confirm that two kinds of samples are anthocyanidin for errorless obtaining
Conclusion, it is even more impossible to further confirm that the chemical composition of two kinds of samples.
Therefore, a kind of technique of purifying complex anthocyanin raw material such as blueberry is researched and developed to pushing anthocyanin standard items market
And exploitation blueberry deep processed product is of great significance.
High mallow element -3-O- glucosides (Malvidin-3-O-glucoside), structural formula is as follows, is main in blueberry
One of anthocyanin and Anthocyanin from Blueberry play the important composition ingredient of bioactivity.
But the research of separation preparation high mallow element -3-O- glucoside monomers and report from blueberry are not found also at present.
Invention content
The present invention in order to solve the above technical problems, provide it is a kind of separation prepare high mallow element -3-O- glucosides method,
Preparative liquid chromatography and high-speed countercurrent chromatography are combined, then by the optimization to technological parameter, formed from anthocyanin complicated
Blueberry raw material in separation the high mallow element -3-O- glucoside monomers of high-purity are prepared.
Specific technical solution is as follows：
A method of separation prepares high mallow element -3-O- glucosides, including：
(1) alcohol extracting concentrates：Using blueberry as raw material, Anthocyanin from Blueberry crude extract is concentrated to give through alcohol extracting；
(2) macroporous resin adsorption：The Anthocyanin from Blueberry crude extract is injected into macroreticular resin, is obtained through eluting and post-processing
Anthocyanin from Blueberry extract freeze-drying powder；
(3) preparative liquid chromatography purifies：Using C18 chromatographic columns, gradient elution is carried out through mobile phase, then post-treated is obtained
High mallow element -3-O- glucoside crude product freeze-dried powders；
The mobile phase：Acid-methanol system that A phases are pure methanol or sour concentration expressed in percentage by volume is 0.1~1.5%, B phases
Formic acid-the aqueous systems for being 1.5~5% for formic acid concentration expressed in percentage by volume；
The program of the gradient elution is：The concentration expressed in percentage by volume of A phases keeps 5% constant in 0~5min, 5~
60% is risen to from 5% in 30min, collects the eluate of 22~23min；
(4) high speed adverse current chromatogram detaches：Using n-butanol-methyl tertiary butyl ether(MTBE)-methanol-water-trifluoroacetic acid as two-phase solvent
System is isolated to high mallow element -3-O- glucoside monomers；
The n-butanol, methyl tertiary butyl ether(MTBE), methanol, water and trifluoroacetic acid volume ratio be 2：2：1：5：0.01~0.1.
Unless otherwise instructed, the percentage of all raw materials occurred in the present invention is concentration expressed in percentage by volume.
The various solution occurred in the present invention, unless otherwise instructed, using water as solvent.
In step (1), the alcohol extracting concentration, specially：
Clean blueberry is mixed with acid ethanol solution, ultrasonic extraction filters and collect filtrate afterwards completely, and filtrate is 40
Rotary evaporation in vacuo removes ethyl alcohol and concentrates at~50 DEG C, obtains Anthocyanin from Blueberry crude extract；
The acid ethanol solution is the ethanol solution that the concentration expressed in percentage by volume of acid is 0.1~1.5%；
The acid is selected from least one of hydrochloric acid, formic acid, acetic acid, oxalic acid；
The concentration expressed in percentage by volume of the ethanol solution is 50~95%；
The mass volume ratio (i.e. solid-liquid ratio) of the blueberry and acid ethanol solution is 1：5~12g/mL.
Preferably, the ultrasonic extraction time be 60~240min, the process at 25~49 DEG C, under the conditions of being protected from light into
Row.
To ensure that anthocyanin extraction is complete, the filter residue obtained after extracting for the first time repeats to extract several according still further to the same terms
It is secondary.
Further preferably, the concentration expressed in percentage by volume of the ethanol solution is 60~70%, the quality of blueberry and acidic ethanol
Volume ratio is 1：5~8g/mL.
In step (2), the macroporous resin adsorption, specially：
The Anthocyanin from Blueberry extracting solution is injected in macroreticular resin, first rinses macroreticular resin with deionized water, then use body
The acidity alcohol solution that product percentage concentration is 2~22% carries out gradient elution, the acidity that collected volume percentage concentration is 14~18%
The eluent of alcoholic solution, rotary evaporation in vacuo is except after alcohol, vacuum freeze drying obtains Anthocyanin from Blueberry extract at 40~50 DEG C
Freeze-dried powder；
Preferably, the trade mark of the macroreticular resin is selected from AB-8, HPD-100, D101 or DM-130；
Preferably, the alcoholic solution that the concentration expressed in percentage by volume that the acidity alcohol solution is selected from acid is 0.1~1.5%；
Alcoholic solution is selected from methanol solution or ethanol solution, and concentration expressed in percentage by volume is 2~22%；
Acid is selected from least one of hydrochloric acid, formic acid, acetic acid.
Further preferably, 2%, 6%, 10%, 14%, 18%, 22% containing 0.5% (v/v, similarly hereinafter) hydrochloric acid is respectively adopted
Ethanol solution carry out gradient elutions, 0.5% hydrochloric acid-that collected volume percentage concentration is 14~18% with 2 times of column volumes (2BV)
The eluent of ethanol solution.
The concentration of the acidity alcohol solution, by taking 2% ethanol solution containing 0.5% hydrochloric acid as an example, the volume of ethanol solution
Percentage concentration is 2%, and the volume ratio of hydrochloric acid and ethanol solution is 0.5：99.5.In step (3), first by the Anthocyanin from Blueberry
Extract freeze-drying powder is reinjected in preparative liquid chromatograph and is purified after deionized water is redissolved；After purification, the rear place
Reason includes reduced pressure and vacuum freeze drying.
Preferably：
A concentration of 20~120mg/mL after the redissolution, sample size are 1~4mL；
The specification of the C18 chromatographic columns is 20mm × 250mm, and temperature is 30 DEG C；
Acid in the A phases is selected from formic acid or trifluoroacetic acid.
Further preferably：
A concentration of 50~120mg/mL after the redissolution, sample size are 3~4mL；
The mobile phase：A phases are pure methanol, and B phases are formic acid-aqueous systems that formic acid concentration expressed in percentage by volume is 1.5~5%,
The flow velocity of mobile phase is 5~10mL/min.
The B phases are formic acid-aqueous systems, by taking formic acid concentration expressed in percentage by volume is 1.5% as an example, in particular to formic acid and water
Volume ratio is 1.5：98.5.
In step (4), the high speed adverse current chromatogram separation, specially：
The two-phase solvent system is prepared, upper phase is stationary phase, and lower phase is mobile phase, will with the flow velocity of 20~30mL/min
The stationary phase is pumped into high-speed counter-current chromatograph, 25~35 DEG C, engine speed be 700~1000r/min under conditions of, with
The flow velocity of 1~8mL/min is pumped into the mobile phase, after two-phase reaches balance, by the high mallow element -3-O- glucoside crude products
The freeze-dried powder flowing laggard sample of phased soln collects the efflux for only including target product, then dense through depressurizing after liquid phase detects
High mallow element -3-O- glucoside monomers are obtained after contracting, freeze-drying.
Preferably, the high mallow element -3-O- glucosides crude product freeze-dried powder mobile phase dissolved a concentration of 10~
30mg/mL；
The wavelength of the detection is 280nm.
Further preferably, in the two-phase solvent system, n-butanol-methyl tertiary butyl ether(MTBE)-methanol-water-trifluoroacetic acid
Volume ratio is 2：2：1：5：0.01；
The stationary phase is pumped into high-speed counter-current chromatograph with the flow velocity of 20~30mL/min, in 25~30 DEG C, host
Under conditions of rotating speed is 850~910r/min, the mobile phase is pumped into the flow velocity of 3~4mL/min；
The dissolved a concentration of 13~27mg/mL of the high mallow element -3-O- glucosides crude product freeze-dried powder mobile phase.
Compared with prior art, the invention has the advantages that：
The present invention for the first time combines preparative liquid chromatography and high-speed countercurrent chromatography, and by the excellent of technological parameter
Change, the high mallow element -3-O- glucoside monomers of isolated high-purity, purity can be high in the blueberry complicated from anthocyanin composition
Up to 99%.The separation method has many advantages, such as that quantity of sample handling is big, reproducible, and the high mallow of high-purity can largely be prepared
Element -3-O- glucoside monomers, enable realize industrialized production, for develop and use China's blueberry resource provide it is new
Thinking.
Description of the drawings
Fig. 1 is the high-efficient liquid phase chromatogram of Anthocyanin from Blueberry freeze-dried powder in embodiment 1；
Fig. 2 is high performance liquid chromatography of the Anthocyanin from Blueberry freeze-dried powder through preparative liquid chromatography product after purification in embodiment 1
Figure；
Fig. 3 is the high-efficient liquid phase chromatogram of final product in embodiment 1；
Fig. 4 is the high-efficient liquid phase chromatogram of final product in comparative example 2；
Fig. 5 is the high-efficient liquid phase chromatogram of final product in comparative example 5；
Fig. 6 is the high-efficient liquid phase chromatogram of final product in comparative example 6.
Specific implementation mode
With reference to specific embodiment, the invention will be further described, and what is be exemplified below is only the specific implementation of the present invention
Example, but protection scope of the present invention is not limited to that：
Embodiment 1
The fresh blueberries of 1kg are cleaned, according to solid-liquid ratio 1：The ratio of 8 (w/v, g/mL) is added containing 0.1% (v/v) hydrochloric acid
(volume ratio of ethyl alcohol and water is 70 to 70% ethanol water：30) it is sufficiently mixed, ultrasonic extraction 90min, (control temperature is 45
DEG C hereinafter, being protected from light), it is filtered by vacuum after ultrasound, obtained filter residue repeats to extract primary, merging filtrate by above-mentioned condition,
Rotary evaporation in vacuo removes ethyl alcohol at 45 DEG C, obtains anthocyanin crude extract.
AB-8 macroreticular resins are impregnated with ethyl alcohol and are fitted into chromatographic column afterwards for 24 hours, after being washed till no alcohol taste with pure water, use 0.5M
Sodium hydroxide solution 1h is rinsed with the flow velocity of 2BV/h, it is neutrality to be then washed with deionized water to efflux；0.5M is used later
Hydrochloric acid solution 1h is rinsed with the flow velocity of 2BV/h, then rinsed to neutrality with deionized water.By anthocyanin crude extract with 0.4BV/
In the flow velocity injection AB-8 macroreticular resins of h, until adsorption volume reaches the 1/3 of resin total volume.First use deionized water with 2BV/h
Flow velocity rinse resin, then respectively with containing 0.5% (v/v) hydrochloric acid 2%, 6%, 10%, 14%, 18%, 22% ethyl alcohol
Aqueous solution rinses 2h with the flow velocity of 2BV/h, and collects 14%~18%% elution fraction.Rotary evaporation in vacuo removes at 45 DEG C
Ethyl alcohol obtains anthocyanin medicinal extract, and by a small amount of deionized water dissolving of the medicinal extract, freeze-drying later obtains Anthocyanin from Blueberry
Freeze-dried powder.
Using preparation liquid phase column Unitary C18 20mm × 250mm, mobile phase is：A phases：Pure methanol, B phases：Formic acid
(5%)：Water (95%)；Column temperature is 30 DEG C, flow velocity 10mL/min, and gradient is：5% A phases 0~5min, 5%~60%A phase 5
~30min.By anthocyanin freeze-dried powder deionized water dissolving, its concentration is made to reach 50mg/mL, prepared by injection detaches in liquid phase,
Single sample size is 4mL.It is detected under UV detector, collects the peak of 22~23min and reduced pressure, freeze-drying, you can
Obtain high mallow element -3-O- glucoside crude extract freeze-dried powders.
By n-butanol：Methyl tertiary butyl ether(MTBE)：Methanol：Water：Trifluoroacetic acid presses 2：2：1：5：0.01 volume ratio is placed in liquid separation
It in funnel, fully shakes up, after standing 30min, will mutually separate up and down, ultrasound degassing 30min.Using upper phase as stationary phase, lower phase
As mobile phase.After high-speed counter-current chromatograph is started preheating 30min, circulator bath is set as 30 DEG C, by stationary phase with 30mL/
The flow velocity of min is pumped into instrument, is then just connecing rotating forward, starts instrument, makes engine speed to 850r/min.After stabilization of speed, with
The flow pump of 3mL/min enters mobile phase, and after two-phase reaches balance in pipeline, 200mg high mallow element -3-O- glucosides are slightly carried
Object freeze-dried powder is dissolved in 15mL mobile phases, and sample introduction simultaneously detects under UV detector, is collected target peak component and is concentrated under reduced pressure, freezes
It is dry to obtain the high mallow element -3-O- glucoside monomers of 12.8mg, purity 99.30%.
By the high-efficient liquid phase chromatogram of comparison diagram 1~3 it is found that blueberry passes through extraction concentration, macroreticular resin gradient elution
Afterwards, the Anthocyanin from Blueberry freeze-dried powder obtained is mainly the mixture containing 9 anthocyanin monomers, further across preparation liquid phase color
Spectrum purifying, collects the eluate of 22~23min, can obtain containing high mallow element -3-O- glucosides and other 2 anthocyanin
Mixture is detached finally by high speed adverse current chromatogram, you can obtain containing only the monomer anthocyanin of high mallow element -3-O- glucosides,
And purity is 99.30%.
Embodiment 2
The fresh blueberries of 5kg are cleaned, according to solid-liquid ratio 1：70% containing 0.5% (v/v) hydrochloric acid is added in the ratio of 7 (w/v)
Ethanol solution be sufficiently mixed, ultrasonic extraction 150min, (control temperature at 45 DEG C hereinafter, being protected from light), vacuum is taken out after ultrasound
Filter, obtained filter residue repeat to extract primary, merging filtrate by above-mentioned condition, and rotary evaporation in vacuo removing ethyl alcohol, obtains at 45 DEG C
To anthocyanin crude extract.
AB-8 macroreticular resins are impregnated with ethyl alcohol and are fitted into chromatographic column afterwards for 24 hours, after being washed till no alcohol taste with pure water, use 0.5M
Sodium hydroxide solution 1h is rinsed with the flow velocity of 2BV/h, it is neutrality to be then washed with deionized water to efflux；0.5M is used later
Hydrochloric acid solution 1h is rinsed with the flow velocity of 2BV/h, then rinsed to neutrality with deionized water.By anthocyanin crude extract with 0.4BV/
In the flow velocity injection AB-8 macroreticular resins of h, until adsorption volume reaches the 1/3 of resin total volume.First use deionized water with 2BV/h
Flow velocity rinse resin, then respectively with containing 0.5% (v/v) hydrochloric acid 2%, 6%, 10%, 14%, 18%, 22% acidity
Ethyl alcohol rinses 2h with the flow velocity of 2BV/h, and collects 14%~18%% elution fraction.Rotary evaporation in vacuo removes second at 50 DEG C
Alcohol obtains anthocyanin medicinal extract, and by a small amount of deionized water dissolving of the medicinal extract, freeze-drying later obtains Anthocyanin from Blueberry jelly
Dry powder.
Preparative liquid chromatography purifies：Using preparation liquid phase column Unitary C18 20mm × 250mm, mobile phase is：A phases：
Pure methanol, B phases：Formic acid (4%)：Water；Column temperature is 30 DEG C, flow velocity 10mL/min, and gradient is：5% A phases 0~5min, 5%
5~30min of~60%A phases.By anthocyanin freeze-dried powder deionized water dissolving, its concentration is made to reach 80mg/mL, injects preparation solution
It is detached in phase, single sample size is 4mL.It is detected under UV detector, collects the peak of 22~23min and reduced pressure, freezing
It is dry, you can to obtain high mallow element -3-O- glucoside crude extract freeze-dried powders.
By n-butanol：Methyl tertiary butyl ether(MTBE)：Methanol：Water：Trifluoroacetic acid presses 2：2：1：5：0.01 volume ratio is placed in liquid separation
It in funnel, fully shakes up, after standing 30min, will mutually separate up and down, ultrasound degassing 30min.Using upper phase as stationary phase, lower phase
As mobile phase.After high-speed counter-current chromatograph is started preheating 30min, circulator bath is set as 25 DEG C, by stationary phase with 30mL/
The flow velocity of min is pumped into instrument, is then just connecing rotating forward, starts instrument, makes engine speed to 900r/min.After stabilization of speed, with
The flow pump of 3mL/min enters mobile phase, and after two-phase reaches balance in pipeline, 300mg high mallow element -3-O- glucosides are slightly carried
Object freeze-dried powder is dissolved in 15mL mobile phases, and sample introduction simultaneously detects under UV detector, is collected target peak component and is concentrated under reduced pressure, freezes
It is dry to obtain 62.0mg high mallow element -3-O- glucosides, purity 98.74%.
Embodiment 3
The fresh blueberries of 10kg are cleaned, according to solid-liquid ratio 1：60% containing 0.1% (v/v) hydrochloric acid is added in the ratio of 5 (w/v)
Ethanol solution be sufficiently mixed, ultrasonic extraction 200min, (control temperature at 45 DEG C hereinafter, being protected from light), vacuum is taken out after ultrasound
Filter, obtained filter residue repeat to extract primary, merging filtrate by above-mentioned condition, and rotary evaporation in vacuo removing ethyl alcohol, obtains at 45 DEG C
To anthocyanin crude extract.
AB-8 macroreticular resins are impregnated with ethyl alcohol and are fitted into chromatographic column afterwards for 24 hours, after being washed till no alcohol taste with pure water, use 0.5M
Sodium hydroxide solution 1h is rinsed with the flow velocity of 2BV/h, it is neutrality to be then washed with deionized water to efflux；0.5M is used later
Hydrochloric acid solution 1h is rinsed with the flow velocity of 2BV/h, then rinsed to neutrality with deionized water.By anthocyanin crude extract with 0.4BV/
In the flow velocity injection AB-8 macroreticular resins of h, until adsorption volume reaches the 1/3 of resin total volume.First use deionized water with 2BV/h
Flow velocity rinse resin, then respectively with containing 0.5% (v/v) hydrochloric acid 2%, 6%, 10%, 14%, 18%, 22% acidity
Ethyl alcohol rinses 2h with the flow velocity of 2BV/h, and collects 14%~18%% elution fraction.Rotary evaporation in vacuo removes second at 45 DEG C
Alcohol obtains anthocyanin medicinal extract, and by a small amount of deionized water dissolving of the medicinal extract, freeze-drying later obtains Anthocyanin from Blueberry jelly
Dry powder.
Preparative liquid chromatography purifies：Using preparation liquid phase column Unitary C18 20mm × 250mm, mobile phase is：A phases：
Pure methanol, B phases：Formic acid (1.5%)：Water；Column temperature is 30 DEG C, flow velocity 10mL/min, and gradient is：5% A 0~5min of phase,
5%~60%A phases, 5~30min.By anthocyanin freeze-dried powder deionized water dissolving, its concentration is made to reach 120mg/mL, injection system
It is detached in standby liquid phase, single sample size is 4mL.It is detected under UV detector, collects the peak of 22~23min and reduced pressure,
Freeze-drying, you can obtain high mallow element -3-O- glucoside crude extract freeze-dried powders.
By n-butanol：Methyl tertiary butyl ether(MTBE)：Methanol：Water：Trifluoroacetic acid presses 2：2：1：5：0.01 volume ratio is placed in liquid separation
It in funnel, fully shakes up, after standing 30min, will mutually separate up and down, ultrasound degassing 30min.Using upper phase as stationary phase, lower phase
As mobile phase.After high-speed counter-current chromatograph is started preheating 30min, circulator bath is set as 35 DEG C, by stationary phase with 30mL/
The flow velocity of min is pumped into instrument, is then just connecing rotating forward, starts instrument, makes engine speed to 910r/min.After stabilization of speed, with
The flow pump of 4mL/min enters mobile phase, and after two-phase reaches balance in pipeline, 400mg high mallow element -3-O- glucosides are slightly carried
Object freeze-dried powder is dissolved in 15mL mobile phases, and sample introduction simultaneously detects under UV detector, is collected target peak component and is concentrated under reduced pressure, freezes
It is dry to obtain 122.2mg high mallow element -3-O- glucosides, purity 98.26%.
Comparative example 1
The fresh blueberries of 10kg are cleaned, according to solid-liquid ratio 1：60% containing 0.1% (v/v) hydrochloric acid is added in the ratio of 7 (w/v)
Ethanol solution be sufficiently mixed, ultrasonic extraction 200min, (control temperature at 45 DEG C hereinafter, being protected from light), vacuum is taken out after ultrasound
Filter, obtained filter residue repeat to extract primary, merging filtrate by above-mentioned condition, and rotary evaporation in vacuo removing ethyl alcohol, obtains at 45 DEG C
To anthocyanin crude extract.
AB-8 macroreticular resins are impregnated with ethyl alcohol and are fitted into chromatographic column afterwards for 24 hours, after being washed till no alcohol taste with pure water, use 0.5M
Sodium hydroxide solution 1h is rinsed with the flow velocity of 2BV/h, it is neutrality to be then washed with deionized water to efflux；0.5M is used later
Hydrochloric acid solution 1h is rinsed with the flow velocity of 2BV/h, then rinsed to neutrality with deionized water.By anthocyanin crude extract with 0.4BV/
In the flow velocity injection AB-8 macroreticular resins of h, until adsorption volume reaches the 1/3 of resin total volume.First use deionized water with 2BV/h
Flow velocity rinse resin, then respectively with containing 0.5% (v/v) hydrochloric acid 2%, 6%, 10%, 14%, 18%, 22% acidity
Ethyl alcohol rinses 2h with the flow velocity of 2BV/h, and collects 14%~18%% elution fraction.Rotary evaporation in vacuo removes second at 45 DEG C
Alcohol obtains anthocyanin medicinal extract, and by a small amount of deionized water dissolving of the medicinal extract, freeze-drying later obtains Anthocyanin from Blueberry jelly
Dry powder.
Prepare liquid phase purifying：Using preparation liquid phase column Unitary C18 20mm × 250mm, mobile phase is：A phases：Pure first
Alcohol, B phases：Formic acid (0.1%)：Water；Column temperature is 30 DEG C, flow velocity 10mL/min, and gradient is：5% A phases 0~5min, 5%~
5~30min of 60%A phases.By anthocyanin freeze-dried powder deionized water dissolving, its concentration is made to reach 120mg/mL, injects preparation solution
It is detached in phase, single sample size is 3mL.It is detected under UV detector, collects the peak of 22~23min and reduced pressure, freezing
It is dry, you can to obtain high mallow element -3-O- glucoside crude extract freeze-dried powders.
By n-butanol：Methyl tertiary butyl ether(MTBE)：Methanol：Water：Trifluoroacetic acid presses 2：2：1：5：0.1 volume ratio is placed in liquid separation leakage
It in bucket, fully shakes up, after standing 30min, will mutually separate up and down, ultrasound degassing 30min.Using upper phase as stationary phase, lower phase is made
For mobile phase.After high-speed counter-current chromatograph is started preheating 30min, circulator bath is set as 35 DEG C, by stationary phase with 30mL/
The flow velocity of min is pumped into instrument, is then just connecing rotating forward, starts instrument, makes engine speed to 910r/min.After stabilization of speed, with
The flow pump of 4mL/min enters mobile phase, and after two-phase reaches balance in pipeline, 400mg high mallow element -3-O- glucosides are slightly carried
Object freeze-dried powder is dissolved in 15mL mobile phases, and sample introduction simultaneously detects under UV detector, is collected target peak component and is concentrated under reduced pressure, freezes
It is dry to obtain 145.8mg high mallow element -3-O- glucosides, purity 89.29%.
Comparative example 2
In contrast to embodiment 1, remove the step of preparative liquid chromatography purifies, other steps are constant, gained final product
High-efficient liquid phase chromatogram as shown in figure 4, it is found that the comparative example can only obtain the mixture containing high mallow element -3-O- glucosides,
It is unable to get high mallow element -3-O- glucoside monomers.
Comparative example 3
Preparation process is same as Example 1, differs only in and replaces with the dicyandiamide solution that high speed adverse current chromatogram detaches：Just
Butanol：Methyl tertiary butyl ether(MTBE)：Methanol：Water：Trifluoroacetic acid presses 1：3：1：5：0.01 volume ratio mixing.After tested, it is unable to get
High mallow element -3-O- glucoside monomers.
Comparative example 4
Preparation process is identical as embodiment 1, differs only in and replaces with the dicyandiamide solution that high speed adverse current chromatogram detaches：
N-butanol：Methyl tertiary butyl ether(MTBE)：Acetonitrile：Water：Trifluoroacetic acid presses 2：2：1：5：0.01 volume ratio mixing.It after tested, although can
To obtain high mallow element -3-O- glucoside monomers, but its purity is 90.57%, the high mallow being prepared far below embodiment 1
The purity (99.30%) of element -3-O- glucoside monomers.
Comparative example 5
Preparation process is same as Example 1, differs only in：It, will in Mobile phase B in preparative liquid chromatography purifying process
Formic acid-water solution system replaces with aqueous solution, that is, is not added with formic acid, other steps are constant, the high-efficient liquid phase chromatogram of final product
As shown in figure 5, it is high mallow element -3-O- glucoside monomers to obtain, but purity is only 71.83%.
Comparative example 6
Preparation process is same as Example 1, when differing only in Fraction collection in change preparative liquid chromatography purification process
Between, if the Fraction collection time is not located at the range of 22~23min, high mallow element -3-O- glucoside monomers are unable to get, if group
Point acquisition time includes and is wider than 22~23min ranges, then the purity of isolated high mallow element -3-O- glucoside monomers is low
In 98%, the high-efficient liquid phase chromatogram of product is as shown in Figure 6.
Claims (10)
1. a kind of method that separation prepares high mallow element -3-O- glucosides, which is characterized in that including：
(1) alcohol extracting concentrates：Using blueberry as raw material, Anthocyanin from Blueberry crude extract is concentrated to give through alcohol extracting；
(2) macroporous resin adsorption：The Anthocyanin from Blueberry crude extract is injected into macroreticular resin, blueberry is obtained through eluting and post-processing
Anthocyanin-rich Extract freeze-dried powder；
(3) preparative liquid chromatography purifies：Using C18 chromatographic columns, gradient elution is carried out through mobile phase, then post-treated obtains high mallow
Element -3-O- glucoside crude product freeze-dried powders；
The mobile phase：Acid-methanol system that A phases are pure methanol or sour concentration expressed in percentage by volume is 0.1~1.5%, B phases are first
Formic acid-aqueous systems that sour concentration expressed in percentage by volume is 1.5~5%；
The program of the gradient elution is：The concentration expressed in percentage by volume of A phases keeps 5% constant in 0~5min, in 5~30min
60% is risen to from 5%, collects the eluate of 22~23min；
(4) high speed adverse current chromatogram detaches：Using n-butanol-methyl tertiary butyl ether(MTBE)-methanol-water-trifluoroacetic acid as two phase solvent system,
It is isolated to high mallow element -3-O- glucoside monomers；
The n-butanol, methyl tertiary butyl ether(MTBE), methanol, water and trifluoroacetic acid volume ratio be 2：2：1：5：0.01~0.1.
2. the method that separation according to claim 1 prepares high mallow element -3-O- glucosides, which is characterized in that step (1)
In, the alcohol extracting concentration, specially：
Clean blueberry is mixed with acid ethanol solution, ultrasonic extraction filters and collect filtrate afterwards completely, and filtrate is 40~50
Rotary evaporation in vacuo removes ethyl alcohol and concentrates at DEG C, obtains Anthocyanin from Blueberry crude extract；
The acid ethanol solution is the ethanol solution that the concentration expressed in percentage by volume of acid is 0.1~1.5%；
The mass volume ratio of the blueberry and acid ethanol solution is 1：5~12g/mL.
3. the method that separation according to claim 2 prepares high mallow element -3-O- glucosides, which is characterized in that the acid
Selected from least one of hydrochloric acid, formic acid, acetic acid, oxalic acid；
The concentration expressed in percentage by volume of the ethanol solution is 50~95%.
4. the method that separation according to claim 1 prepares high mallow element -3-O- glucosides, which is characterized in that step (2)
In, the macroporous resin adsorption, specially：
The Anthocyanin from Blueberry extracting solution is injected in macroreticular resin, first rinses macroreticular resin with deionized water, then with volume hundred
A concentration of 2~22% acidity alcohol solution is divided to carry out gradient elution, the acid alcohol that collected volume percentage concentration is 14~18% is molten
The eluent of liquid, rotary evaporation in vacuo is except after alcohol, vacuum freeze drying obtains the freeze-drying of Anthocyanin from Blueberry extract at 40~50 DEG C
Powder；
The trade mark of the macroreticular resin is selected from AB-8, HPD-100, D101 or DM-130；
The alcoholic solution that the concentration expressed in percentage by volume that the acidity alcohol solution is selected from acid is 0.1~1.5%, alcoholic solution are selected from methanol solution
Or ethanol solution, acid is selected from least one of hydrochloric acid, formic acid, acetic acid.
5. the method that separation according to claim 1 prepares high mallow element -3-O- glucosides, which is characterized in that step (3)
In, first the Anthocyanin from Blueberry extract freeze-drying powder is reinjected in preparative liquid chromatograph and carried out after deionized water is redissolved
Purifying；
A concentration of 20~120mg/mL after the redissolution, sample size are 1~4mL；
The specification of the C18 chromatographic columns is 20mm × 250mm, and temperature is 25~30 DEG C；
Acid in the A phases is selected from formic acid or trifluoroacetic acid.
6. the method that separation according to claim 5 prepares high mallow element -3-O- glucosides, it is characterised in that：
A concentration of 50~120mg/mL after the redissolution；
The mobile phase：A phases are pure methanol, and B phases are formic acid-aqueous systems that formic acid concentration expressed in percentage by volume is 1.5~5%, flowing
The flow velocity of phase is 5~10mL/min.
7. the method that separation according to claim 1 prepares high mallow element -3-O- glucosides, which is characterized in that step (3)
In, the post-processing includes reduced pressure and vacuum freeze drying.
8. the method that separation according to claim 1 prepares high mallow element -3-O- glucosides, which is characterized in that step (4)
In, the high speed adverse current chromatogram separation, specially：
The two-phase solvent system is prepared, upper phase is stationary phase, and lower phase is mobile phase, will be described with the flow velocity of 20~30mL/min
Stationary phase is pumped into high-speed counter-current chromatograph, 25~35 DEG C, engine speed be 700~1000r/min under conditions of, with 1~
The flow velocity of 8mL/min is pumped into the mobile phase, and after two-phase reaches balance, the high mallow element -3-O- glucoside crude products are frozen
The dry powder flowing laggard sample of phased soln is collected after liquid phase detects and only include the efflux of target product, then through reduced pressure,
High mallow element -3-O- glucoside monomers are obtained after freeze-drying.
9. the method that separation according to claim 8 prepares high mallow element -3-O- glucosides, which is characterized in that described two
In phase solvent system, n-butanol, methyl tertiary butyl ether(MTBE), methanol, water and trifluoroacetic acid volume ratio be 2：2：1：5：0.01；
The stationary phase is pumped into high-speed counter-current chromatograph with the flow velocity of 20~30mL/min, in 30~35 DEG C, engine speed
Under conditions of 850~910r/min, the mobile phase is pumped into the flow velocity of 3~4mL/min.
10. the method that separation according to claim 9 prepares high mallow element -3-O- glucosides, which is characterized in that the brocade
Certain herbaceous plants with big flowers element -3-O- glucoside dissolved a concentration of 10~the 30mg/mL of crude product freeze-dried powder mobile phase, sampling volume be 1~
15mL；
The wavelength of the liquid phase detection is 280nm.
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| CN112321656A (en) * | 2020-09-14 | 2021-02-05 | 浙江大学 | Method for separating and preparing acylated anthocyanin |
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