CN108409805A - A kind of isolation and purification method of delphinidin -3-O- galactosides and its application - Google Patents
A kind of isolation and purification method of delphinidin -3-O- galactosides and its application Download PDFInfo
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Abstract
The invention discloses a kind of isolation and purification method of 3 O galactosides of delphinidin and its applications in preparing α glucosidase inhibitors, the isolation and purification method forms complicated blueberry as raw material using anthocyanin, including alcohol extracting concentration, macroporous resin adsorption, preparative liquid chromatography purifying and high speed adverse current chromatogram separation, by the way that preparative liquid chromatography and high-speed countercurrent chromatography are combined, pass through the optimization to technological parameter again, the 3 O galactoside monomers of delphinidin of high-purity are prepared in separation, and purity is up to 99%；It is found through activity test, which can significantly inhibit the activity of α glucuroides, can be used for preparing α glucosidase inhibitors.
Description
Technical field
Field is isolated and purified the present invention relates to natural products, and in particular to a kind of delphinidin -3-O- galactosides
Isolation and purification method and its application.
Background technology
Diabetes are one of chronic diseases common in world wide, diabetic usually along with hyperglycemic symptoms,
Long-term hyperglycemia can cause the damage of the histoorgans such as nerve, heart, blood vessel and kidney, and cause a variety of acute and chronics concurrent
The generation of disease.It is shown according to the World Health Organization (WHO) statistical data, there is 4.22 hundred million diabetic in the whole world within 2014, wherein 2
Patients with type Ⅰ DM is most commonly seen.Recently as people's eating habit, the acceleration of living-pattern preservation and aging process, China
The illness rate of diabetes rapidly rises, until diabetic's quantity in 2016 end of the year China has reached 1.1 hundred million.Therefore, related sugar
The research of urine disease prevention has particularly important meaning.
Alpha-glucosidase (alpha-D-glucoside glucohydrolase, EC 3.2.1.20) is also known as α-D-
Glucuroide hydrolase is the membrane bound enzyme in glycoside hydrolase GH31 families, including invertase, maltose, different malt
Carbohydrase etc. is primarily present in intestinal villi mucous membrane piglets.After feed, alpha-glucosidase can be by the carbon in food
Hydrate is hydrolyzed to glucose, and glucose enters blood circulation after being absorbed leads to blood glucose rise, therefore, alpha-glucosidase
It is one of the main target enzyme for controlling postprandial blood sugar.Currently, the alpha-glucosidase restrainer of domestic listing mainly have acarbose,
Voglibose and Miglitol would generally cause the adverse reaction of gastrointestinal tract, such as abdominal distension, exhaust using these inhibitor.
Therefore, safely and effectively food function factor, targeted inhibition alpha-glucosidase activity, for reducing the morbidity of diabetes are excavated
Rate and the health status for improving diabetic will be an effective strategies.Current a large amount of researcher both at home and abroad is
Focus on food-borne function factor, it is intended to filter out good activity from food and α-grape of side reaction will not be caused to human body
Glycosidase inhibitor.Currently, it has been reported that the flavone compound in food-borne source, alkaloid, phenolic compound, curcumin
Class compound, terpenoid have a stronger alpha-glucosaccharase enzyme inhibition activity in vitro, and activity better than positive drug Ah
Card wave sugar, but the research report of the alpha-glucosaccharase enzyme inhibition activity in relation to anthocyanin is few, has now been found that Cyanidin -3-
O glucosides have alpha-glucosidase activity.
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.
Therefore, explore a kind of method of the separating high-purity anthocyanin monomers from blueberry to the further investigation of blueberry and
Using being of great significance.But since tree peony anthocyanins structure is similar, polarity difference is smaller, leads to high-purity anthocyanin
Isolating and purifying for monomer is 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 Publication No. CN 106366141A 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 Publication No. CN 106831911A 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
Anthocyanin composition is simple in He Peng Lei, contains only Cyanidin -3-O- glucosides, pelargonin -3-O- glucosides and India
3 kinds of anthocyanin compounds of certain herbaceous plants with big flowers element -3-O- rutinosides, wherein pelargonin -3-O- glucosides account for Anthocyanin content
80% or more.
But for the complicated original of the anthocyanin composition such as such as blueberry (containing the similar anthocyanin compound of at least 12 kinds of structures)
Material, above-mentioned technical solution are then difficult to realize the purifying and preparation of high-purity anthocyanin monomer.Currently, passing through single column chromatography
Or the high-purity anthocyanin that chromatographic technique is prepared 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 104109403 A of Publication No. CN 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.
Delphinidin -3-O- galactosides, structural formula is as follows, is one of main anthocyanin and blueberry flower in blueberry
Color glycosides plays the important composition ingredient of bioactivity.
But do not find that from blueberry separation prepares research and the report of delphinidin -3-O- galactoside monomers also at present,
There is not application of the delphinidin -3-O- galactoside monomers in preparing alpha-glucosidase restrainer yet.
Invention content
The present invention is in order to solve the above technical problems, provide a kind of side of isolating and purifying of delphinidin -3-O- galactosides
Liquid chromatography purification is combined by method with high speed adverse current chromatogram isolation technics, then by the optimization to technological parameter, from anthocyanin
Form the delphinidin -3-O- galactoside monomers that high-purity is prepared in separation in complicated blueberry raw material；Through activity test
It was found that the delphinidin -3-O- galactosides monomer can significantly inhibit the activity of alpha-glucosidase, can be used for preparing α-Portugal
Polyglycoside enzyme inhibitor.
Specific technical solution is as follows：
A kind of isolation and purification method of delphinidin -3-O- galactosides, 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
Delphinidin -3-O- galactoside 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 15~16.5min；
(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 delphinidin -3-O- galactoside 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.
In the present invention, the acidity alcohol solution, by taking 2% ethanol solution containing 0.5% hydrochloric acid as an example, ethanol solution
Concentration expressed in percentage by volume is 2%, and the volume ratio of hydrochloric acid and ethanol solution is 0.5：99.5.
In step (3), the Anthocyanin from Blueberry extract freeze-drying powder is first reinjected into preparation solution after deionized water is redissolved
It is purified in chromatography；After purification, the post-processing 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.
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, the delphinidin -3-O- galactosides is thick
The product 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
Delphinidin -3-O- galactoside monomers are obtained after contracting, freeze-drying.
Preferably, the delphinidin -3-O- galactosides 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 25~35 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 delphinidin -3-O- galactosides crude product freeze-dried powder mobile phase.
After above-mentioned optimum preparation condition, the purity of the delphinidin -3-O- galactoside monomers isolated and purified is high
Up to 99%.
Find that the delphinidin -3-O- galactosides monomer isolated and purified is to α-Portugal through further activity test
Polyglycoside enzyme has significant inhibition, IC50Value is 0.11mM, hence it is evident that is better than positive drug acarbose (IC50=
0.52mM), a kind of novel natural alpha-glucosidase restrainer is can be used as, for controlling postprandial blood sugar.
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 delphinidin -3-O- galactoside monomers of isolated high-purity, purity may be up to 99% from blueberry.The separation
Method has many advantages, such as that quantity of sample handling is big, reproducible, and the delphinidin -3-O- galactolipins of high-purity can largely be prepared
Glycosides monomer enables to realize industrialized production；It is found through further activity test, the delphinidin -3-O- galactoside lists
Body can significantly inhibit the activity of alpha-glucosidase, can be used for preparing alpha-glucosidase restrainer.
Description of the drawings
Fig. 1 is the high-efficient liquid phase chromatogram of Anthocyanin from Blueberry extract freeze-drying powder in embodiment 1；
Fig. 2 is high-efficient liquid phase color of the Anthocyanin from Blueberry extract freeze-drying powder through preparative liquid chromatography after purification in embodiment 1
Spectrogram；
Fig. 3 is the high-efficient liquid phase chromatogram of final product in embodiment 1；
Fig. 4 is the alpha-glucosaccharase enzyme inhibition activity curve for the delphinidin -3-O- galactosides that embodiment 1 isolates and purifies
(a), and the alpha-glucosaccharase enzyme inhibition activity curve (b) of acarbose is provided as a comparison；
Fig. 5 is the high-efficient liquid phase chromatogram of final product in comparative example 2；
Fig. 6 is the high-efficient liquid phase chromatogram of final product in comparative example 5；
Fig. 7 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
Extract freeze-drying 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-rich Extract freeze-dried powder deionized water dissolving, its concentration is made to reach 50mg/mL, injection is prepared in liquid phase
Separation, single sample size are 4mL.It is detected under UV detector, collect the peak of 15~16.5min and removing first is concentrated under reduced pressure
Alcohol, freeze-drying, you can obtain delphinidin -3-O- galactoside crude product 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, after two-phase reaches balance in pipeline, 200mg delphinidin -3-O- galactosides is thick
Product 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 and removes
Organic phase, freeze-drying is gone to obtain the delphinidin -3-O- galactoside monomers of 30.1mg, purity 99.47%.
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 15~16.5min, can obtain containing delphinidin -3-O- galactosides and a small amount of impurity
Mixture is detached finally by high speed adverse current chromatogram, you can obtains containing only the monomer pattern of delphinidin -3-O- galactosides
Glycosides, purity are up to 99.47%.
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 water be sufficiently mixed, ultrasonic extraction 150min, (control temperature at 45 DEG C hereinafter, being protected from light), vacuum after ultrasound
It filtering, obtained filter residue repeats to extract primary, merging filtrate by above-mentioned condition, the rotary evaporation in vacuo removing ethyl alcohol at 45 DEG C,
Obtain 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 fractions.Rotary evaporation in vacuo removes at 50 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
(4%)：Water；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-rich Extract freeze-dried powder deionized water dissolving, its concentration is made to reach 80mg/mL, injection, which is prepared in liquid phase, to divide
From single sample size is 4mL.It is detected under UV detector, collects the peak of 15~16.5min and reduced pressure, be freeze-dried,
It can be obtained delphinidin -3-O- galactoside crude product 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, after two-phase reaches balance in pipeline, 300mg delphinidin -3-O- galactosides is thick
Product 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 147mg delphinidin -3-O- galactosides, purity 98.85%.
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 water be sufficiently mixed, ultrasonic extraction 200min, (control temperature at 45 DEG C hereinafter, being protected from light), vacuum after ultrasound
It filtering, obtained filter residue repeats to extract primary, merging filtrate by above-mentioned condition, the rotary evaporation in vacuo removing ethyl alcohol at 45 DEG C,
Obtain 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
(1.5%)：Water；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-rich Extract freeze-dried powder deionized water dissolving, its concentration is made to reach 120mg/mL, injection is prepared in liquid phase
Separation, single sample size are 4mL.It is detected under UV detector, collects the peak of 15~16.5min and reduced pressure, freezing is dry
It is dry, you can to obtain delphinidin -3-O- galactoside crude product 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, after two-phase reaches balance in pipeline, 400mg delphinidin -3-O- galactosides is thick
Product 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 291.8mg delphinidin -3-O- galactosides, purity 98.64%.
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 water be sufficiently mixed, ultrasonic extraction 200min, (control temperature at 45 DEG C hereinafter, being protected from light), vacuum after ultrasound
It filtering, obtained filter residue repeats to extract primary, merging filtrate by above-mentioned condition, the rotary evaporation in vacuo removing ethyl alcohol at 45 DEG C,
Obtain 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
(0.1%)：Water；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-rich Extract freeze-dried powder deionized water dissolving, its concentration is made to reach 120mg/mL, injection is prepared in liquid phase
Separation, single sample size are 3mL.It is detected under UV detector, collects the peak of 15~16.5min and reduced pressure, freezing is dry
It is dry, you can to obtain delphinidin -3-O- galactoside crude product 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, after two-phase reaches balance in pipeline, 400mg delphinidin -3-O- galactosides is thick
Product 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 315.6mg delphinidin -3-O- galactosides, purity 89.61%.
Comparative example 2
In contrast to embodiment 1, remove the step of preparative liquid chromatography purifies, other steps are constant, after tested, can only obtain
Mixture containing delphinidin -3-O- galactosides is unable to get delphinidin -3-O- galactoside monomers, final product
High-efficient liquid phase chromatogram it is as shown in Figure 5.
Comparative example 3
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)：Methanol：Water：Trifluoroacetic acid presses 1：3：1：5：0.01 volume ratio mixing.After tested, it can not obtain
To delphinidin -3-O- galactoside 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.After tested, Ke Yifen
From obtaining delphinidin -3-O- galactoside monomers, but its purity is only 92.48%, the winged swallow being prepared less than embodiment 1
The purity (99.47%) of careless element -3-O- galactoside monomers.
Comparative example 5
Preparation process is identical as embodiment 1, differs only in：Mobile phase B in preparative liquid chromatography purifying process, will
Formic acid-water solution system replaces with aqueous solution, that is, is not added with formic acid, other steps are constant, after tested, obtained delphinidin -3-
O- galactoside monomer purities are only 71.69%, and the high-efficient liquid phase chromatogram of final product is as shown in Figure 6.
Comparative example 6
Preparation process is identical as embodiment 1, differs only in and changes Fraction collection in preparative liquid chromatography purification process
Time is unable to get delphinidin -3-O- galactoside lists if the Fraction collection time is not located at the range of 15~16.5min
Body, if the Fraction collection time include and be wider than 15~16.5min ranges, obtained delphinidin -3-O- galactoside monomers
Purity is less than 98%, and high-efficient liquid phase chromatogram is as shown in Figure 7.
Application examples 1
Delphinidin -3-O- galactoside the water dissolutions that embodiment 1 is prepared, are made into a series of concentration
(0.1mmol/L, 0.2mmol/L, 0.5mmol/L, 0.7mmol/L, 1mmol/L) is used as inhibitor.Alpha-glucosidase is used
It is 0.5U/mL that the phosphate buffer (PBS, pH=6.9) of 0.1mol/L, which is diluted to enzyme activity,.Substrate p-nitrophenyl-α-D- pyrans
Glucoside (PNPG) is configured to a concentration of 1mmol/L. enzymatic reactions with the phosphate buffer (PBS, pH=6.9) of 0.1mol/L
System is the inhibitor mixed of the enzyme and 10 μ L of 20 μ L, and the substrate of the buffer solution and 40 μ L of 130 μ L is added, is reacted at 37 DEG C
30min is added the sodium carbonate liquor of the 1mol/L of 200 μ L, light absorption value is detected under 405nm later.Blank group uses inhibitor
Buffer solution substitutes.Enzyme activity inhibiting rate is calculated according to following formula：Inhibiting rate %=(ABlank-AExperiment)/ABlank* 100%.In the reaction
The IC of the delphinidin -3-O- galactosides measured under system50Value is 0.11mM, and positive control acarbose is 0.52mM.
Application examples 2
It will report that the Cyanidin -3-O- glucosides (commercially available) with alpha-glucosaccharase enzyme inhibition activity are made into a system
Row concentration (0.1mmol/L, 1mmol/L, 2mmol/L, 5mmol/L, 10mmol/L) is used as inhibitor.Alpha-glucosidase is used
It is 0.5U/mL that the phosphate buffer (PBS, pH=6.9) of 0.1mol/L, which is diluted to enzyme activity,.Substrate p-nitrophenyl-α-D- pyrans
Glucoside (PNPG) is configured to a concentration of 1mmol/L. enzymatic reactions with the phosphate buffer (PBS, pH=6.9) of 0.1mol/L
System is the inhibitor mixed of the enzyme and 10 μ L of 20 μ L, and the substrate of the buffer solution and 40 μ L of 130 μ L is added, is reacted at 37 DEG C
30min is added the sodium carbonate liquor of the 1mol/L of 200 μ L, light absorption value is detected under 405nm later.Blank group uses inhibitor
Buffer solution substitutes.Enzyme activity inhibiting rate is calculated according to following formula：Inhibiting rate %=(A blank-A experiments)/A blank * 100%.
The IC of the Cyanidin -3-O- glucosides measured under the reaction system50Value is 1.03mM, and positive control acarbose is
0.52mM。
By comparison it is found that the present invention detaches the delphinidin -3-O- galactosides being prepared to alpha-glucosidase
Inhibition be significantly better than positive control acarbose and Cyanidin -3-O- glucosides.Therefore, system is detached from blueberry
Standby obtained delphinidin -3-O- galactosides can be used as a kind of novel natural alpha-glucosidase restrainer.
Claims (10)
1. a kind of isolation and purification method of delphinidin -3-O- galactosides, 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 flying swallow
Careless element -3-O- galactosides crude product freeze-dried powder；
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 15~16.5min；
(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 delphinidin -3-O- galactoside 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 isolation and purification method of delphinidin -3-O- galactosides according to claim 1, 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 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 of the blueberry and acid ethanol solution is 1：5~12g/mL.
3. the isolation and purification method of delphinidin -3-O- galactosides according to claim 1, 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.
4. the isolation and purification method of delphinidin -3-O- galactosides according to claim 1, which is characterized in that step
(3) in, first by the Anthocyanin from Blueberry extract freeze-drying powder after deionized water is redissolved, reinject in preparative liquid chromatograph into
Row 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.
5. the isolation and purification method of delphinidin -3-O- galactosides according to claim 4, 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.
6. the isolation and purification method of delphinidin -3-O- galactosides according to claim 1, which is characterized in that step
(3) in, the post-processing includes reduced pressure and vacuum freeze drying.
7. the isolation and purification method of delphinidin -3-O- galactosides according to claim 1, 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, after two-phase reaches balance, by the delphinidin -3-O- galactoside 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
Delphinidin -3-O- galactoside monomers are obtained after contracting, freeze-drying.
8. the isolation and purification method of delphinidin -3-O- galactosides according to claim 7, which is characterized in that described
In two-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 25~35 DEG C, engine speed
Under conditions of 850~910r/min, the mobile phase is pumped into the flow velocity of 3~4mL/min.
9. the isolation and purification method of delphinidin -3-O- galactosides according to claim 8, which is characterized in that described
Delphinidin -3-O- galactoside dissolved a concentration of 10~the 30mg/mL of crude product freeze-dried powder mobile phase, sampling volume 1
~15mL；
The wavelength of the liquid phase detection is 280nm.
10. a kind of application of delphinidin -3-O- galactosides in preparing alpha-glucosidase restrainer, which is characterized in that
Delphinidin -3-O- the galactosides isolate and purify to obtain according to any method of claim 1~9.
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