CN108822168A - A kind of isolation and purification method of high mallow element -3-O- galactoside and its application - Google Patents
A kind of isolation and purification method of high mallow element -3-O- galactoside and its application Download PDFInfo
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Abstract
The invention discloses a kind of isolation and purification method of high mallow element -3-O- galactoside and its preparing the application in alpha-glucosidase restrainer, the isolation and purification method is using the blueberry of anthocyanin complicated composition as raw material, 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, high mallow element -3-O- galactoside the monomer of high-purity is prepared in separation, and purity is up to 99%；It is found through activity test, the high mallow element -3-O- galactoside monomer can significantly inhibit the activity of alpha-glucosidase, can be used for preparing alpha-glucosidase restrainer.
Description
Technical field
Field is isolated and purified the present invention relates to natural products, and in particular to a kind of point of high mallow element -3-O- galactoside
From purification process and its application.
Background technique
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 the acceleration of people's eating habit, 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 and treatment 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 family, 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, which 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 glucoside has 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 the methods of separating high-purity anthocyanin monomers from blueberry a kind of 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 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 resin 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 monomer, including alcohol extracting concentration, ethyl acetate extraction, AB-8 macroreticular resin 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- glucoside, pelargonin -3-O- glucoside and India
3 kinds of anthocyanin compounds of certain herbaceous plants with big flowers element -3-O- rutinoside, wherein pelargonin -3-O- glucoside accounts for Anthocyanin content
80% or more.
But for the original of the anthocyanin complicated 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, being chromatographed by single column
Or the high-purity anthocyanin that chromatographic technique is prepared is anthocyanin mixture, rather than high-purity anthocyanin monomer.
As 106905391 A of Publication No. CN 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, and homogeneous extracts 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 resin.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 effective component of having determined is under the premise of keeping high extraction, the problem of effective component is not by high temperature, but what is obtained mentions
Taking object is anthocyanin mixture, 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 mention
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 separated through high speed adverse current chromatogram, with MTBE- n-butanol-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 component of two kinds of samples.
High mallow element -3-O- galactoside, structural formula is as follows, is one of main anthocyanin and blueberry pattern in blueberry
The important composition ingredient of glycosides performance bioactivity.
But do not find the research of separation preparation high mallow element -3-O- galactoside monomer and report from blueberry also at present,
There is not the high mallow element -3-O- galactoside monomer preparing the application in alpha-glucosidase restrainer.
Summary of the invention
The present invention in order to solve the above technical problems, provide a kind of isolation and purification method of high mallow element -3-O- galactoside,
Liquid chromatography purification is combined with high speed adverse current chromatogram isolation technics, then by the optimization to technological parameter, from anthocyanin group
High mallow element -3-O- galactoside the monomer of high-purity is prepared at separation in complicated blueberry raw material；It is found through activity test,
The high mallow element -3-O- galactoside monomer can significantly inhibit the activity of alpha-glucosidase, can be used for preparing alpha-glucosaccharase
Enzyme inhibitor.
Specific technical solution is as follows：
A kind of isolation and purification method of high mallow element -3-O- galactoside, including：
(1) alcohol extracting is concentrated：Using blueberry as raw material, Anthocyanin from Blueberry crude extract is concentrated to get through alcohol extracting；
(2) macroporous resin adsorption：The Anthocyanin from Blueberry crude extract is injected into macroreticular resin, is eluted and post-processed to obtain
Anthocyanin from Blueberry extract freeze-drying powder；
(3) preparative liquid chromatography purifies：Using C18 chromatographic column, gradient elution is carried out through mobile phase, then post-treated is obtained
High mallow element -3-O- galactoside crude product freeze-dried powder；
The mobile phase：Acid-methanol system that A phase is pure methanol or sour concentration expressed in percentage by volume is 0.1~1.5%, B phase
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 phase keeps 5% constant in 0~5min, 5~
60% is risen to from 5% in 30min, collects the eluate of 21~22min；
(4) high speed adverse current chromatogram separates：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- galactoside monomer；
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 afterwards completely and collect filtrate, and filtrate is 40
Rotary evaporation in vacuo removes ethyl alcohol and is concentrated 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 guarantee that anthocyanin is extracted completely, 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 acidity alcohol solution is selected from the alcoholic solution that the concentration expressed in percentage by volume of 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：
Concentration after the redissolution is 20~120mg/mL, and sample volume is 1~4mL；
The specification of the C18 chromatographic column is 20mm × 250mm, and temperature is 30 DEG C；
Acid in the A phase is selected from formic acid or trifluoroacetic acid.
Further preferably：
Concentration after the redissolution is 50~120mg/mL, and sample volume is 3~4mL；
The mobile phase：A phase is pure methanol, and B phase is 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, by the high mallow element -3-O- galactoside crude product
The freeze-dried powder flowing laggard sample of phased soln collects efflux only comprising target product after liquid phase detects, then dense through depressurizing
High mallow element -3-O- galactoside monomer is obtained after contracting, freeze-drying.
Preferably, the high mallow element -3-O- galactoside dissolved concentration of crude product freeze-dried powder mobile phase be 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 revolving speed is 850~910r/min, the mobile phase is pumped into the flow velocity of 3~4mL/min；
High mallow element -3-O- galactoside dissolved the concentration of crude product freeze-dried powder mobile phase is 13~27mg/mL.
After above-mentioned optimum preparation condition, the purity of the high mallow element -3-O- galactoside monomer isolated and purified is up to
99%.
It is found through further activity test, the high mallow element -3-O- galactoside monomer isolated and purified is to α-grape
Glycosidase has significant inhibitory effect, IC50Value is 0.14mM, hence it is evident that is better than positive drug acarbose (IC50=
0.52mM), it can be used as a kind of novel natural alpha-glucosidase restrainer, 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 high mallow element -3-O- galactoside monomer of isolated high-purity, purity may be up to 99% from blueberry.The separation side
Method has many advantages, such as that quantity of sample handling is big, reproducible, and the high mallow element -3-O- galactoside list of high-purity can largely be prepared
Body enables to realize industrialized production；It is found through further activity test, the high mallow element -3-O- galactoside monomer can
The activity for significantly inhibiting alpha-glucosidase can be used for preparing alpha-glucosidase restrainer.
Detailed description of the invention
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 high mallow element -3-O- galactoside 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 embodiment
The invention will be further described combined with specific embodiments below, and what is be exemplified below is only specific implementation of the invention
Example, but protection scope of the present invention is not limited to that：
Embodiment 1
The fresh blueberry of 1kg is 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 resin is impregnated with ethyl alcohol and is fitted into chromatographic column afterwards for 24 hours, after being washed till no alcohol taste with pure water, uses 0.5M
Sodium hydroxide solution 1h is rinsed with the flow velocity of 2BV/h, being then washed with deionized water to efflux is neutrality；0.5M is used later
Hydrochloric acid solution 1h is rinsed with the flow velocity of 2BV/h, then rinsed with deionized water to neutrality.By anthocyanin crude extract with 0.4BV/
In the flow velocity injection AB-8 macroreticular resin of h, until adsorption volume reaches the 1/3 of resin total volume.First with deionized water with 2BV/h
Flow velocity rinse resin, then respectively with contain 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 obtains Anthocyanin from Blueberry later
Extract freeze-drying powder.
Using preparation liquid phase column Unitary C18 20mm × 250mm, mobile phase is：A phase：Pure methanol, B phase：Formic acid
(5%)：Water (95%)；Column temperature is 30 DEG C, flow velocity 10mL/min, and gradient is：5% A phase 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 volume are 4mL.It is detected under UV detector, collect the peak of 21~22min and removing methanol is concentrated under reduced pressure,
Freeze-drying, can be obtained high mallow element -3-O- galactoside crude extract freeze-dried powder.
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, sufficiently shakes up, after standing 30min, will mutually separate up and down, ultrasonic 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- galactoside is slightly mentioned
Object freeze-dried powder is dissolved in 15mL mobile phase, and sample introduction simultaneously detects under UV detector, is collected target peak component and is concentrated under reduced pressure and removes
Organic phase is gone, freeze-drying obtains the high mallow element -3-O- galactoside monomer of 13.8mg, purity 98.92%.
Concentration, macroreticular resin gradient elution are extracted it is found that blueberry passes through by the high-efficient liquid phase chromatogram of comparison diagram 1~3
Afterwards, the Anthocyanin from Blueberry freeze-dried powder obtained is mainly the mixture containing 9 anthocyanin monomers, further across preparation liquid phase color
The eluate of 21~22min is collected in spectrum purifying, available to contain high mallow element -3-O- galactoside and other 3 anthocyanin
Mixture is separated finally by high speed adverse current chromatogram, and the monomer anthocyanin for containing only high mallow element -3-O- galactoside can be obtained,
Purity is up to 98.92%.
Embodiment 2
The fresh blueberry of 5kg is cleaned, according to solid-liquid ratio 1：The ratio of 7 (w/v), which is added, contains the 70% of 0.5% (v/v) hydrochloric acid
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 resin is impregnated with ethyl alcohol and is fitted into chromatographic column afterwards for 24 hours, after being washed till no alcohol taste with pure water, uses 0.5M
Sodium hydroxide solution 1h is rinsed with the flow velocity of 2BV/h, being then washed with deionized water to efflux is neutrality；0.5M is used later
Hydrochloric acid solution 1h is rinsed with the flow velocity of 2BV/h, then rinsed with deionized water to neutrality.By anthocyanin crude extract with 0.4BV/
In the flow velocity injection AB-8 macroreticular resin of h, until adsorption volume reaches the 1/3 of resin total volume.First with deionized water with 2BV/h
Flow velocity rinse resin, then respectively with contain 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 50 DEG C
Ethyl alcohol obtains anthocyanin medicinal extract, and by a small amount of deionized water dissolving of the medicinal extract, freeze-drying obtains Anthocyanin from Blueberry later
Extract freeze-drying powder.
Using preparation liquid phase column Unitary C18 20mm × 250mm, mobile phase is：A phase：Pure methanol, B phase：Formic acid
(4%)：Water；Column temperature is 30 DEG C, flow velocity 10mL/min, and gradient is：5% A phase 0~5min, 5%~60%A phase 5~
30min.By Anthocyanin-rich Extract freeze-dried powder deionized water dissolving, so that its concentration is reached 80mg/mL, divide in injection preparation liquid phase
From single sample volume is 4mL.It is detected under UV detector, collects the peak of 21~22min and reduced pressure, be freeze-dried, i.e.,
High mallow element -3-O- galactoside crude extract freeze-dried powder can be obtained.
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, sufficiently shakes up, after standing 30min, will mutually separate up and down, ultrasonic 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- galactoside is slightly mentioned
Object freeze-dried powder is dissolved in 15mL mobile phase, 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 66.3mg high mallow element -3-O- galactoside, purity 98.49%.
Embodiment 3
The fresh blueberry of 10kg is cleaned, according to solid-liquid ratio 1：The ratio of 5 (w/v), which is added, contains the 60% of 0.1% (v/v) hydrochloric acid
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 resin is impregnated with ethyl alcohol and is fitted into chromatographic column afterwards for 24 hours, after being washed till no alcohol taste with pure water, uses 0.5M
Sodium hydroxide solution 1h is rinsed with the flow velocity of 2BV/h, being then washed with deionized water to efflux is neutrality；0.5M is used later
Hydrochloric acid solution 1h is rinsed with the flow velocity of 2BV/h, then rinsed with deionized water to neutrality.By anthocyanin crude extract with 0.4BV/
In the flow velocity injection AB-8 macroreticular resin of h, until adsorption volume reaches the 1/3 of resin total volume.First with deionized water with 2BV/h
Flow velocity rinse resin, then respectively with contain 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 obtains Anthocyanin from Blueberry later
Extract freeze-drying powder.
Using preparation liquid phase column Unitary C18 20mm × 250mm, mobile phase is：A phase：Pure methanol, B phase：Formic acid
(1.5%)：Water；Column temperature is 30 DEG C, flow velocity 10mL/min, and gradient is：5% A phase 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 volume are 4mL.It is detected under UV detector, collects the peak of 21~22min and reduced pressure, be freeze-dried,
High mallow element -3-O- galactoside crude extract freeze-dried powder can be obtained.
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, sufficiently shakes up, after standing 30min, will mutually separate up and down, ultrasonic 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- galactoside is slightly mentioned
Object freeze-dried powder is dissolved in 15mL mobile phase, 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 130mg high mallow element -3-O- galactoside, purity 98.08%.
Comparative example 1
The fresh blueberry of 10kg is cleaned, according to solid-liquid ratio 1：The ratio of 7 (w/v), which is added, contains the 60% of 0.1% (v/v) hydrochloric acid
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 resin is impregnated with ethyl alcohol and is fitted into chromatographic column afterwards for 24 hours, after being washed till no alcohol taste with pure water, uses 0.5M
Sodium hydroxide solution 1h is rinsed with the flow velocity of 2BV/h, being then washed with deionized water to efflux is neutrality；0.5M is used later
Hydrochloric acid solution 1h is rinsed with the flow velocity of 2BV/h, then rinsed with deionized water to neutrality.By anthocyanin crude extract with 0.4BV/
In the flow velocity injection AB-8 macroreticular resin of h, until adsorption volume reaches the 1/3 of resin total volume.First with deionized water with 2BV/h
Flow velocity rinse resin, then respectively with contain 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 obtains Anthocyanin from Blueberry later
Extract freeze-drying powder.
Using preparation liquid phase column Unitary C18 20mm × 250mm, mobile phase is：A phase：Pure methanol, B phase：Formic acid
(0.1%)：Water；Column temperature is 30 DEG C, flow velocity 10mL/min, and gradient is：5% A phase 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 volume are 3mL.It is detected under UV detector, collects the peak of 21~22min and reduced pressure, be freeze-dried,
High mallow element -3-O- galactoside crude extract freeze-dried powder can be obtained.
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, sufficiently shakes up, after standing 30min, will mutually separate up and down, ultrasonic 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- galactoside is slightly mentioned
Object freeze-dried powder is dissolved in 15mL mobile phase, 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 162mg high mallow element -3-O- galactoside, purity 88.13%.
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 high mallow element -3-O- galactoside is unable to get high mallow element -3-O- galactoside monomer, the height of final product
Effect liquid phase chromatogram figure is as shown in Figure 5.
Comparative example 3
Preparation process is identical as embodiment 1, and difference is only that the dicyandiamide solution by high speed adverse current chromatogram separation replaces with：
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 high mallow element -3-O- galactoside monomer.
Comparative example 4
Preparation process is identical as embodiment 1, and difference is only that the dicyandiamide solution by high speed adverse current chromatogram separation replaces with：
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 high mallow element -3-O- galactoside monomer, but its purity is only 89.31%, the high mallow being prepared lower than embodiment 1
The purity (98.92%) of element -3-O- galactoside monomer.
Comparative example 5
Preparation process is identical as embodiment 1, and difference is only that：Mobile phase B in preparative liquid chromatography purifying process, will
Formic acid-water solution system replaces with aqueous solution, that is, formic acid is not added, other steps are constant, after tested, obtained high mallow element -3-O-
Galactoside monomer purity is only 68.57%, 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, and difference, which is only that, changes Fraction collection in preparative liquid chromatography purification process
Time is unable to get high mallow element -3-O- galactoside monomer if the Fraction collection time is not located at the range of 21~22min, if
The Fraction collection time includes and is wider than 1~22min range, then high mallow element -3-O- galactoside the monomer purity obtained is lower than
98%, high-efficient liquid phase chromatogram is as shown in Figure 7.
Application examples 1
High mallow element -3-O- the galactoside that embodiment 1 is prepared is dissolved with water, is 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
It is 1mmol/L. enzymatic reaction that glucoside (PNPG) phosphate buffer (PBS, pH=6.9) of 0.1mol/L, which is configured to concentration,
System is the enzyme of 20 μ L and the inhibitor mixed of 10 μ L, and the buffer of 130 μ L and the substrate of 40 μ L is added, reacts at 37 DEG C
The sodium carbonate liquor of the 1mol/L of 200 μ L is added later, detects light absorption value under 405nm by 30min.Blank group uses inhibitor
Buffer substitution.Enzyme activity inhibiting rate calculates according to the following formula：Inhibiting rate %=(ABlank-AExperiment)/ABlank* 100%.In the reaction
The IC of the high mallow element -3-O- galactoside measured under system50Value is 0.14mmol/L, and positive control acarbose is
0.52mmol/L。
Application examples 2
It will report that the Cyanidin -3-O- glucoside (commercially available) with alpha-glucosaccharase enzyme inhibition activity is made into a system
Column 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
It is 1mmol/L. enzymatic reaction that glucoside (PNPG) phosphate buffer (PBS, pH=6.9) of 0.1mol/L, which is configured to concentration,
System is the enzyme of 20 μ L and the inhibitor mixed of 10 μ L, and the buffer of 130 μ L and the substrate of 40 μ L is added, reacts at 37 DEG C
The sodium carbonate liquor of the 1mol/L of 200 μ L is added later, detects light absorption value under 405nm by 30min.Blank group uses inhibitor
Buffer substitution.Enzyme activity inhibiting rate calculates according to the following formula：Inhibiting rate %=(A blank-A experiment)/A blank * 100%.?
The IC of the Cyanidin -3-O- glucoside measured under the reaction system50Value is 1.03mmol/L, and positive control acarbose is
0.52mmol/L。
By comparison it is found that the present invention separates the high mallow element -3-O- galactoside being prepared to alpha-glucosidase
Inhibitory effect is significantly better than positive control acarbose and Cyanidin -3-O- glucoside.Therefore, preparation is separated from blueberry
Obtained delphinidin -3-O- galactoside can be used as a kind of novel natural alpha-glucosidase restrainer.
Claims (10)
1. a kind of isolation and purification method of high mallow element -3-O- galactoside, which is characterized in that including：
(1) alcohol extracting is concentrated：Using blueberry as raw material, Anthocyanin from Blueberry crude extract is concentrated to get through alcohol extracting；
(2) macroporous resin adsorption：The Anthocyanin from Blueberry crude extract is injected into macroreticular resin, is eluted and post-processed to obtain blueberry
Anthocyanin-rich Extract freeze-dried powder；
(3) preparative liquid chromatography purifies：Using C18 chromatographic column, gradient elution is carried out through mobile phase, then post-treated obtains high mallow
Element -3-O- galactoside crude product freeze-dried powder；
The mobile phase：Acid-methanol system that A phase is pure methanol or sour concentration expressed in percentage by volume is 0.1~1.5%, B phase is 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 phase keeps 5% constant in 0~5min, in 5~30min
60% is risen to from 5%, collects the eluate of 21~22min；
(4) high speed adverse current chromatogram separates：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- galactoside monomer；
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 high mallow element -3-O- galactoside 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 afterwards completely and collect filtrate, and filtrate is 40~50
Rotary evaporation in vacuo removes ethyl alcohol and is concentrated 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 high mallow element -3-O- galactoside 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
The acidity alcohol solution that concentration is 2~22% 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 acidity alcohol solution is selected from the alcoholic solution that the concentration expressed in percentage by volume of acid is 0.1~1.5%, and alcoholic solution is selected from methanol solution
Or ethanol solution, acid are selected from least one of hydrochloric acid, formic acid, acetic acid.
4. the isolation and purification method of high mallow element -3-O- galactoside according to claim 1, 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；
Concentration after the redissolution is 20~120mg/mL, and sample volume is 1~4mL；
The specification of the C18 chromatographic column is 20mm × 250mm, and temperature is 25~30 DEG C；
Acid in the A phase is selected from formic acid or trifluoroacetic acid.
5. the isolation and purification method of high mallow element -3-O- galactoside according to claim 4, it is characterised in that：
Concentration after the redissolution is 50~120mg/mL；
The mobile phase：A phase is pure methanol, and B phase is 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 high mallow element -3-O- galactoside 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 high mallow element -3-O- galactoside 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, and after two-phase reaches balance, the high mallow element -3-O- galactoside crude product is frozen
The dry powder flowing laggard sample of phased soln collects efflux only comprising target product after liquid phase detects, then be concentrated under reduced pressure,
High mallow element -3-O- galactoside monomer is obtained after freeze-drying.
8. the isolation and purification method of high mallow element -3-O- galactoside according to claim 7, 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 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 high mallow element -3-O- galactoside according to claim 8, which is characterized in that the brocade
Certain herbaceous plants with big flowers element -3-O- galactoside dissolved the concentration of crude product freeze-dried powder mobile phase be 10~30mg/mL, sampling volume be 1~
15mL；
The wavelength of the liquid phase detection is 280nm.
10. a kind of high mallow element -3-O- galactoside is preparing the application in alpha-glucosidase restrainer, which is characterized in that institute
Stating high mallow element -3-O- galactoside, any method isolates and purifies to obtain according to claim 1~9.
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| CN110256514A (en) * | 2019-06-05 | 2019-09-20 | 吉林农业大学 | A kind of stable state high mallow -3,5-O- glucosulfone glycoside derivates and the preparation method and application thereof |
| CN112321656A (en) * | 2020-09-14 | 2021-02-05 | 浙江大学 | Method for separating and preparing acylated anthocyanin |
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| CN102875515A (en) * | 2012-11-07 | 2013-01-16 | 江苏省农业科学院 | Method for extracting malvidin from blueberry |
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| KR20120037201A (en) * | 2010-10-11 | 2012-04-19 | 강원대학교산학협력단 | Composition for prevention and treatment of obesity comprising liriope seed extracts |
| CN102875515A (en) * | 2012-11-07 | 2013-01-16 | 江苏省农业科学院 | Method for extracting malvidin from blueberry |
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| CN110256514A (en) * | 2019-06-05 | 2019-09-20 | 吉林农业大学 | A kind of stable state high mallow -3,5-O- glucosulfone glycoside derivates and the preparation method and application thereof |
| CN112321656A (en) * | 2020-09-14 | 2021-02-05 | 浙江大学 | Method for separating and preparing acylated anthocyanin |
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