CN108659068A - A kind of isolation and purification method of Cyanidin -3-O- rutinosides and its application - Google Patents
A kind of isolation and purification method of Cyanidin -3-O- rutinosides and its application Download PDFInfo
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Abstract
The invention discloses a kind of isolation and purification method of 3 O rutinosides of Cyanidin and its applications in preparing α glucosidase inhibitors, the isolation and purification method include slightly carry, macroporous resin adsorption, high speed adverse current chromatogram detach and procyanidin, by the way that high speed adverse current chromatogram is combined with solid phase extraction techniques, again by the optimization to technological parameter, the 3 O rutinoside monomers of Cyanidin that high-purity is prepared are detached from mulberry fruit；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 Cyanidin -3-O- rutinosides
Isolation and purification method and its application in preparing alpha-glucosidase restrainer.
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.
Mulberries also known as sorosis, mulberry crow, mulberry jujube etc., are the general designations of Moraceae Mulberry plant ripening fruits.Just there is mulberry early in the Tang Dynasty
The record that Shen is used as medicine.Modern research shows that mulberries are the functional foods for having healthcare function, containing relatively rich sugared, more
The bioactive substances such as phenol have liver-kidney tonifying, fluid dryness, UFA eyesight and other effects.And Mulberry anthocyanin is weight in mulberries
The polyphenols wanted.But since tree peony anthocyanins structure is similar, polarity difference is smaller, leads to high-purity anthocyanin monomer
Isolate and purify it is extremely difficult.But isolate and purify to obtain high-purity anthocyanin monomer currently, having had been reported that.
As 101045741 A of Publication No. CN Chinese patent literature in disclose one kind separation from mulberry fruit prepare it is high
The method of purity anthocyanin monomer extracts the anthocyanin in mulberry fruit using acidic ethanol；With cation exchange resin to mulberry fruit second
Alcohol extracting thing carries out preliminary purification；The anthocyanin in Anthocyanin in Mulberry crude extract, solvent system are detached with countercurrent chromatography
N-butanol, acetonitrile, methyl tertiary butyl ether(MTBE) and trifluoroacetic acid aqueous solution by being in liquid under normal temperature and pressure form, acetonitrile and positive fourth
The volume ratio of alcohol is 1：0.5-1：1.25-3.5, trifluoroacetic acid aqueous solution it is a concentration of per containing 1-10 milliliters of trifluoros in aqueous solution
Acetic acid., can be with tetra- kinds of anthocyanin monomers of isolated C3RG, C7G, C3G and C3Ga through above-mentioned technique, purity is 92% or more.
Although 4 kinds of anthocyanin monomers are only just prepared in the method described in the patent by high speed adverse current chromatogram, its purity is below
98%, it is unable to reach international export standard.
Cyanidin -3-O- rutinosides, structural formula is as follows, is one of main anthocyanin and mulberries flower in mulberries
Color glycosides plays the important composition ingredient of bioactivity.
But do not find that from mulberry fruit separation prepares research and the report of Cyanidin -3-O- rutinoside monomers also at present,
There is not application of the Cyanidin -3-O- rutinoside 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 Cyanidin -3-O- rutinosides
High speed adverse current chromatogram is combined by method with solid phase extraction techniques, then by the optimization to technological parameter, and separation is prepared high-purity
Cyanidin -3-O- rutinoside the monomers of degree；It is found through activity test, the Cyanidin -3-O- rutinoside monomers can
The activity for significantly inhibiting alpha-glucosidase can be used for preparing alpha-glucosidase restrainer.
Specific technical solution is as follows：
A kind of isolation and purification method of Cyanidin -3-O- rutinosides, including：
(1) it slightly carries：Using mulberry fruit as raw material, anthocyanin extract liquor is obtained after alcohol extracting concentration, organic solvent extraction；
(2) macroporous resin adsorption：The anthocyanin extract liquor is injected into macroreticular resin, pattern is obtained through eluting and post-processing
Glucoside extract freeze-dried powder；
(3) high speed adverse current chromatogram detaches：Using n-butanol-methyl tertiary butyl ether(MTBE)-methanol-water-trifluoroacetic acid as two-phase solvent
System obtains anthocyanin concentrate after separation；
The n-butanol, methyl tertiary butyl ether(MTBE), methanol, water and trifluoroacetic acid volume ratio be 2：2：1：5：0.01~0.1；
(4) procyanidin：The anthocyanin concentrate is injected into solid-phase extraction column, carrying out gradient through mobile phase washes
It is de-, then post-treated obtain Cyanidin -3-O- rutinosides；
The step of gradient elution is：First use formic acid-aqueous systems that the concentration expressed in percentage by volume of formic acid is 0.1~1.5%
Elution, then the acidic methanol solution for being respectively 2.5~20% with concentration expressed in percentage by volume carry out gradient elution, and collected volume percentage is dense
The acidic methanol eluent that degree is 10~20%.
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 mulberry fruit 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 at~50 DEG C, obtains anthocyanin crude extract；
The acid ethanol solution is the ethanol solution that the concentration expressed in percentage by volume of acid is 0.1~1.0%；
The acid is selected from least one of hydrochloric acid, formic acid, acetic acid, citric acid；
The concentration expressed in percentage by volume of the ethanol solution is 50~80%；
The mass volume ratio of the mulberry fruit 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 mulberry fruit and acidic ethanol
Volume ratio is 1：8g/mL.
In step (1), the organic solvent extraction is extracted 3~5 times using ethyl acetate as extractant, collects water phase,
The rotary evaporation in vacuo at 40~50 DEG C removes remaining ethyl acetate, obtains anthocyanin extract liquor.
In step (2), the macroporous resin adsorption, specially：
By the anthocyanin extract liquor inject macroreticular resin in, first with deionized water rinse macroreticular resin, then with acidity alcohol
Solution is eluted, and eluent is collected, and after then rotary evaporation in vacuo removes alcohol at 40~50 DEG C, vacuum freeze drying is spent
Color glucoside extract freeze-dried powder；
The specific surface area of the macroreticular resin is 480~520m2/ g, average pore size are 13~14nm, particle size range 0.3
~1.25mm；The optional trade mark such as AB-8, HPD-100 or D101.
The alcoholic solution that the concentration expressed in percentage by volume that the acidity alcohol solution is selected from acid is 0.01~1.5%, the volume of alcoholic solution
Percentage concentration is 20~70%；
The alcoholic solution is selected from methanol solution or ethanol solution, and acid is selected from least one of hydrochloric acid, formic acid, acetic acid.
Further preferably, using 70% ethanol solution containing 0.01% (v/v, similarly hereinafter) hydrochloric acid with 2 times of column volumes (2BV)
It is eluted, collects eluent.
70% ethanol solution for containing 0.01% hydrochloric acid, a concentration of the 70% of the ethanol solution, the hydrochloric acid with
The volume ratio of ethanol solution is 0.01：99.9.
In step (3), 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, and after two-phase reaches balance, the Anthocyanin-rich Extract freeze-dried powder is flowed
Sample introduction after dynamic phased soln collects the efflux for including target product, then dense through being concentrated under reduced pressure to give anthocyanin after liquid phase detects
Contracting liquid.
Preferably, in the two-phase solvent system, n-butanol, methyl tertiary butyl ether(MTBE), methanol, water and trifluoroacetic acid volume
Than being 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 850~950r/min, the mobile phase is pumped into the flow velocity of 3~4mL/min；
The dissolved a concentration of 10~30mg/mL of Anthocyanin-rich Extract freeze-dried powder mobile phase, sampling volume be 1~
15mL。
Preferably, in step (4), the solid-phase extraction column is selected from C18Sep-Pak cartridge solid-phase extraction columns；It is described
Procyanidin, specially：
The anthocyanin concentrate is injected into solid-phase extraction column with the flow velocity of 1~5mL/min, until adsorption volume reaches column
Then the 1/3 of volume carries out gradient elution.
Further preferably, the step of gradient elution is：
Formic acid-the aqueous systems for being first 1.5% with formic acid concentration expressed in percentage by volume elute, and are then with concentration expressed in percentage by volume respectively
2.5%, 5%, 10% and 20% acidic methanol solution elution, most collects 10% and 20% respectively after the flushing of pure methanol afterwards
Acidic methanol eluent after merge；
Acid in the acidic methanol solution is selected from formic acid, and the concentration expressed in percentage by volume of formic acid is 1.5%.
Further preferably, the formic acid-aqueous systems are eluted with 15 times of column volumes (15BV), and the concentration expressed in percentage by volume is
2.5% acidic methanol solution carries out gradient elution, the acid first that the concentration expressed in percentage by volume is 5% with 6 times of column volumes (6BV)
Alcoholic solution carries out gradient elution with 8 times of column volumes (8BV), and the acidic methanol solution that the concentration expressed in percentage by volume is 10% is with 6 times
Column volume (6BV) carries out gradient elution, and the acidic methanol solution that the concentration expressed in percentage by volume is 20% is with 4 times of column volumes (4BV)
Carry out gradient elution.
Formic acid-the aqueous systems only include two component of formic acid and water, therefore, the first that formic acid concentration expressed in percentage by volume is 1.5%
Acid-aqueous systems are -98.5% aqueous systems of 1.5% formic acid.
The concentration of the acidic methanol solution, it is molten with 2.5% acidic methanol that the concentration expressed in percentage by volume of formic acid is 1.5%
For liquid, a concentration of the 2.5% of methanol solution, the volume ratio of formic acid and methanol solution is 1.5：98.5.
After above-mentioned optimum preparation condition, the purity of the Cyanidin -3-O- rutinoside monomers isolated and purified is high
Up to 99%.
Find that the high mallow element -3-O- Arabinosides monomer isolated and purified is to α-Portugal through further activity test
Polyglycoside enzyme has significant inhibition, IC50Value is 0.011mM, 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：
High speed adverse current chromatogram is combined by the present invention with solid phase extraction techniques for the first time, then by the optimization to technological parameter,
Cyanidin -3-O- rutinoside the monomers of high-purity are prepared in separation from mulberry fruit, and purity reaches as high as 99%；This point
Have many advantages, such as that quantity of sample handling is big, reproducible from method, the Cyanidin -3-O- rues of high-purity can largely be prepared
Glucosides monomer enables to realize industrialized production；It is found through further activity test, the Cyanidin -3-O- rutinosides
Monomer 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 Mulberry anthocyanin extract freeze-drying powder in embodiment 1；
Fig. 2 is the high-efficient liquid phase chromatogram of anthocyanin concentrate in embodiment 1；
Fig. 3 is the high-efficient liquid phase chromatogram of final product in embodiment 1；
Fig. 4 is the alpha-glucosaccharase enzyme inhibition activity for the Cyanidin -3-O- rutinosides that embodiment 1 isolates and purifies
Curve (a), and provide the alpha-glucosaccharase enzyme inhibition activity curve (b) of acarbose as a comparison；
Fig. 5 is the high-efficient liquid phase chromatogram of final product in comparative example 4.
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
1kg Mulberry anthocyanins are cleaned, by solid-liquid ratio 1：70% containing 0.1% (v/v) hydrochloric acid is added in 8 (w/v, g/mL)
(volume ratio of ethyl alcohol and water is 70 to ethanol water：30) it, is protected from light ultrasonic extraction 60min at 40 DEG C, is filtered by vacuum later, filters
Slag repeats to extract primary, filtrate merging, and rotary evaporation in vacuo removes ethyl alcohol at 45 DEG C, obtains Mulberry anthocyanin crude extract.
The ethyl acetate that Mulberry anthocyanin crude extract is added to same volume extracts, and extracts 4 times, collects water phase, it
Rotary evaporation in vacuo concentrates at 45 DEG C afterwards, removes remaining ethyl acetate, obtains anthocyanin extract liquor.
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 extract liquor with 0.5BV/
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
The flow velocity of 2BV/h rinses 2h, reuses 70% ethanol water containing 0.01% (v/v) hydrochloric acid and is eluted with the flow velocity of 2BV/h, is received
The acidic ethanol eluent of collection 70%.Vacuum rotating removes ethyl alcohol at 45 DEG C, and then freeze-drying obtains Mulberry anthocyanin and carries
Take object 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, 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, by 300mg Mulberry anthocyanin extract freeze-drying powders
It is dissolved in 15mL mobile phases, sample introduction simultaneously detects under UV detector, collects target peak component and is concentrated under reduced pressure, obtains anthocyanin
Concentrate.
By C18Sep-Pak cartridge solid-phase extraction columns are activated with 30mL methanol, use 1.5% aqueous formic acid later
Methanol is rinsed well with the flow velocity of 5mL/min.Anthocyanin concentrate is injected into C18 solid-phase extraction columns with 1mL/min later, directly
Reach the 1/3 of column volume to adsorption volume, then carries out gradient elution.Gradient elution step is：Flow velocity is 0.5BV/min, first
With 1.5% formic acid of 15BV-aqueous solution elution, then washed with 2.5% methanol of 6BV (containing 1.5% formic acid) aqueous solution；Then it uses
8BV5% methanol (containing 1.5% formic acid), solution was washed；Then it is washed with 10% methanol of 6BV (containing 1.5% formic acid) solution, then uses 4BV
20% methanol (containing 1.5% formic acid), solution was washed, and was finally rinsed with pure methanol.Wherein collect 10% and 20% acidic methanol elution
Liquid, 45 DEG C are evaporated under reduced pressure concentration, and freeze-drying can be obtained 100mg Cyanidin -3-O- rutinosides, purity 99.21%.
By the high-efficient liquid phase chromatogram of comparison diagram 1~3 it is found that mulberries are eluted by extraction concentration, extraction, macroreticular resin
Afterwards, the Mulberry anthocyanin extract freeze-drying powder obtained mainly contains 2 anthocyanin (Cyanidin -3-O- glucosides and arrows
Che Jusu -3-O- rutinosides) and the mixture (Fig. 1) of impurity can be obtained further across high speed adverse current chromatogram purifying on a small quantity
To the mixture (Fig. 2) for containing only Cyanidin -3-O- rutinosides and a small amount of impurity, detached finally by solid phase extraction techniques,
The monomer anthocyanin (Fig. 3) for containing only Cyanidin -3-O- rutinosides is can be obtained, and purity is 99.21%.
Embodiment 2
5kg Mulberry anthocyanins are cleaned, by solid-liquid ratio 1：The ethyl alcohol of 70% (v/v) of 1% (v/v) hydrochloric acid is added in 8 (w/v)
(volume ratio of ethyl alcohol and water is 70 to aqueous solution：30) it, is protected from light ultrasonic extraction 90min at 40 DEG C, is filtered by vacuum later, filter residue weight
Multiple extraction is primary, and filtrate merges, and rotary evaporation in vacuo removes ethyl alcohol at 45 DEG C, obtains Mulberry anthocyanin crude extract；
The ethyl acetate that Mulberry anthocyanin crude extract is added to same volume extracts, and extracts 4 times, collects water phase, it
Rotary evaporation in vacuo concentrates at 45 DEG C afterwards, removes remaining ethyl acetate, obtains anthocyanin extract liquor.
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 extract liquor with 0.5BV/
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
The flow velocity of 2BV/h rinses 2h, reuses 70% ethanol water containing 0.01% (v/v) hydrochloric acid and is eluted with the flow velocity of 2BV/h, is received
The acidic ethanol eluent of collection 70%.Vacuum rotating removes ethyl alcohol at 45 DEG C, and then freeze-drying obtains Mulberry anthocyanin and carries
Take object 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, 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 20mL/
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, by 200mg Mulberry anthocyanin extract freeze-drying powders
It is dissolved in 15mL mobile phases, sample introduction simultaneously detects under UV detector, collects target peak component and is concentrated under reduced pressure, obtains anthocyanin
Concentrate.
By C18Sep-Pak cartridge solid-phase extraction columns are activated with 30mL methanol, use 1.5% aqueous formic acid later
Methanol is rinsed well with the flow velocity of 5mL/min.Anthocyanin concentrate is injected into C18 solid-phase extraction columns with 1mL/min later, directly
Reach the 1/3 of column volume to adsorption volume, then carries out gradient elution.Gradient elution step is：Flow velocity is 0.5BV/min, first
It is eluted with 1.5% aqueous formic acids of 15BV, is then washed with 2.5% methanol of 6BV (containing 1.5% formic acid) aqueous solution；Then it uses
8BV5% methanol (containing 1.5% formic acid), solution was washed；Then it is washed with 10% methanol of 6BV (containing 1.5% formic acid) solution, then uses 4BV
20% methanol (containing 1.5% formic acid), solution was washed, and was finally rinsed with pure methanol.Wherein collect 10% and 20% acidic methanol elution
Liquid, 45 DEG C be evaporated under reduced pressure concentration, freeze-drying can 450mg obtain Cyanidin -3-O- rutinosides, purity 98.69%.
Embodiment 3
10kg Mulberry anthocyanins are cleaned, by solid-liquid ratio 1：8 (w/v) are added 70% (v/v's) of 1.5% (v/v) hydrochloric acid
(volume ratio of ethyl alcohol and water is 70 to ethanol water：30) it, is protected from light ultrasonic extraction 70min at 40 DEG C, is filtered by vacuum later, filters
Slag repeats to extract primary, filtrate merging, and rotary evaporation in vacuo removes ethyl alcohol at 45 DEG C, obtains Mulberry anthocyanin crude extract；
The ethyl acetate that Mulberry anthocyanin crude extract is added to same volume extracts, and extracts 4 times, collects water phase, it
Rotary evaporation in vacuo concentrates at 45 DEG C afterwards, removes remaining ethyl acetate, obtains anthocyanin extract liquor.
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 extract liquor 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
The flow velocity of 2BV/h rinses 2h, reuses 70% ethanol water containing 0.01% (v/v) hydrochloric acid and is eluted with the flow velocity of 2BV/h, is received
The acidic ethanol eluent of collection 70%.Vacuum rotating removes ethyl alcohol at 45 DEG C, and then freeze-drying obtains Mulberry anthocyanin and carries
Take object 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, 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 950r/min.After stabilization of speed, with
The flow pump of 3mL/min enters mobile phase, after two-phase reaches balance in pipeline, by 400mg Mulberry anthocyanin extract freeze-drying powders
It is dissolved in 15mL mobile phases, sample introduction simultaneously detects under UV detector, collects target peak component and is concentrated under reduced pressure, obtains anthocyanin
Concentrate.
By C18Sep-Pak cartridge solid-phase extraction columns are activated with 30mL methanol, use 1.5% aqueous formic acid later
Methanol is rinsed well with the flow velocity of 5mL/min.Anthocyanin concentrate is injected into C18 solid-phase extraction columns with 1mL/min later, directly
Reach the 1/3 of column volume to adsorption volume, then carries out gradient elution.Gradient elution step is：Flow velocity is 0.5BV/min, first
It is eluted with 1.5% aqueous formic acids of 15BV, is then washed with 2.5% methanol of 6BV (containing 1.5% formic acid) aqueous solution；Then it uses
8BV5% methanol (containing 1.5% formic acid), solution was washed；Then it is washed with 10% methanol of 6BV (containing 1.5% formic acid) solution, then uses 4BV
20% methanol (containing 1.5% formic acid), solution was washed, and was finally rinsed with pure methanol.10% and 20% meoh eluate is wherein collected,
45 DEG C are evaporated under reduced pressure concentration, and freeze-drying can be obtained 800mg Cyanidin -3-O- rutinosides, purity 98.35%.
Comparative example 1
Preparation process is same as Example 1, differs only in and replaces with the dicyandiamide solution of high speed adverse current chromatogram：Positive fourth
Alcohol：Methyl tertiary butyl ether(MTBE)：Methanol：Water：Trifluoroacetic acid presses 1：3：1：5：0.01 volume ratio mixing, remaining condition is constant, adverse current
System retention rate is too low, leads to not obtain Cyanidin -3-O- rutinoside monomers.
Comparative example 2
Preparation process is same as Example 1, differs only in and replaces with the dicyandiamide solution of high speed adverse current chromatogram：Positive fourth
Alcohol：Methyl tertiary butyl ether(MTBE)：Methanol：Water：Trifluoroacetic acid presses 2：2：1：5：0.2 volume ratio mixing, remaining condition is constant, can obtain
To Cyanidin -3-O- rutinoside monomers, but its purity is only 75.9%.
Comparative example 3
Preparation process is same as Example 1, differs only in and replaces with the dicyandiamide solution of high speed adverse current chromatogram：Positive fourth
Alcohol：Methyl tertiary butyl ether(MTBE)：Acetonitrile：Water：Trifluoroacetic acid presses 2：2：1：5：0.01 volume ratio mixing, remaining condition is constant, can be with
Cyanidin -3-O- rutinoside monomers are obtained, but its purity is only 91.5%.
Comparative example 4
Preparation process is same as Example 1, differs only in and does not add formic acid in Solid Phase Extraction solvent gradient, i.e., first
It is washed, then is washed successively with 2.5% methanol aqueous solutions of 6BV, 5% methanol solutions of 8BV, 10% methanol solutions of 6BV with 15BV aqueous solutions
It elutes, is finally rinsed with pure methanol, remaining condition is constant, obtained Cyanidin -3-O- rues with 20% methanol solutions of 4BV
Glucosides monomer purity is 82.3%, and high-efficient liquid phase chromatogram is as shown in Figure 5.
Application examples 1
Cyanidin -3-O- rutinoside the water dissolutions that embodiment 1 is isolated and purified, 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 Cyanidin -3-O- rutinosides measured under system50Value is 0.34mmol/L, and positive control acarbose is
0.52mmol/L。
Application examples 2
It will report that the anthocyanin (Cyanidin -3-O- glucosides) with alpha-glucosaccharase enzyme inhibition activity is made into one
Series concentration (0.1mmol/L, 1mmol/L, 2mmol/L, 5mmol/L, 10mmol/L) is used as inhibitor.By alpha-glucosidase
It is 0.5U/mL to be diluted to enzyme activity with the phosphate buffer (PBS, pH=6.9) of 0.1mol/L.Substrate p-nitrophenyl-α-D- pyrroles
It is anti-that glucopyranoside glycosides (PNPG) phosphate buffer (PBS, pH=6.9) of 0.1mol/L is configured to a concentration of 1mmol/L. enzymatics
It is the inhibitor mixed of the enzyme and 10 μ L of 20 μ L to answer system, 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 1.03mmol/L, and positive control acarbose is
0.52mmol/L。
By comparison it is found that the Cyanidin -3-O- rutinosides that isolate and purify of the present invention are to alpha-glucosidase
Inhibition (IC50=0.34mmol/L) it is significantly better than positive control acarbose and Cyanidin -3-O- glucosides.Cause
This, the Cyanidin -3-O- rutinosides being prepared are detached from mulberries and can be used as a kind of more effectively natural α -
Glucosidase inhibitor.
Claims (10)
1. a kind of isolation and purification method of Cyanidin -3-O- rutinosides, which is characterized in that including：
(1) it slightly carries：Using mulberry fruit as raw material, anthocyanin extract liquor is obtained after alcohol extracting concentration, organic solvent extraction；
(2) macroporous resin adsorption：The anthocyanin extract liquor is injected into macroreticular resin, is carried through eluting and post-processing to obtain anthocyanin
Take object freeze-dried powder；
(3) high speed adverse current chromatogram detaches：Using n-butanol-methyl tertiary butyl ether(MTBE)-methanol-water-trifluoroacetic acid as two phase solvent system,
Anthocyanin concentrate is obtained after separation；
The n-butanol, methyl tertiary butyl ether(MTBE), methanol, water and trifluoroacetic acid volume ratio be 2：2：1：5：0.01~0.1；
(4) procyanidin：The anthocyanin concentrate is injected into solid-phase extraction column, gradient elution is carried out through mobile phase, then
It is post-treated to obtain Cyanidin -3-O- rutinosides；
The step of gradient elution is：Formic acid-aqueous systems elution that the concentration expressed in percentage by volume of formic acid is 0.1~1.5% is first used,
The acidic methanol solution for being respectively again 2.5~20% with concentration expressed in percentage by volume carries out gradient elution, and collected volume percentage concentration is
10~20% acidic methanol eluent.
2. the isolation and purification method of Cyanidin -3-O- rutinosides according to claim 1, which is characterized in that step
(1) in, the alcohol extracting concentration, specially：
Clean mulberry fruit 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 at DEG C, obtains anthocyanin crude extract；
The acid ethanol solution is the ethanol solution that the concentration expressed in percentage by volume of acid is 0.1~1.0%；
The acid is selected from least one of hydrochloric acid, formic acid, acetic acid, citric acid；
The concentration expressed in percentage by volume of the ethanol solution is 50~80%；
The mass volume ratio of the mulberry fruit and acid ethanol solution is 1：5~12g/mL.
3. the isolation and purification method of Cyanidin -3-O- rutinosides according to claim 1, which is characterized in that step
(1) in, the organic solvent extraction, using ethyl acetate as extractant, extraction several times, collects water phase, at 40~50 DEG C
Rotary evaporation in vacuo removes remaining ethyl acetate, obtains anthocyanin extract liquor.
4. the isolation and purification method of Cyanidin -3-O- rutinosides according to claim 1, which is characterized in that step
(2) in, the macroporous resin adsorption, specially：
The anthocyanin extract liquor is injected in macroreticular resin, first rinses macroreticular resin with deionized water, then use acidity alcohol solution
It is eluted, collects eluent, after then rotary evaporation in vacuo removes alcohol at 40~50 DEG C, vacuum freeze drying obtains anthocyanin
Extract freeze-drying powder；
The specific surface area of the macroreticular resin is 480~520m2/ g, average pore size be 13~14nm, particle size range be 0.3~
1.25mm；
The alcoholic solution that the concentration expressed in percentage by volume that the acidity alcohol solution is selected from acid is 0.01~1.5%, the volume basis of alcoholic solution
A concentration of 20~70%；
The alcoholic solution is selected from methanol solution or ethanol solution, and acid is selected from least one of hydrochloric acid, formic acid, acetic acid.
5. the isolation and purification method of Cyanidin -3-O- rutinosides according to claim 1, which is characterized in that step
(3) 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 Anthocyanin-rich Extract freeze-dried powder mobile phase
Sample introduction after dissolving collects the efflux for including target product, then through being concentrated under reduced pressure to give anthocyanin concentration after liquid phase detects
Liquid.
6. the isolation and purification method of Cyanidin -3-O- rutinosides according to claim 5, 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 850~950r/min, the mobile phase is pumped into the flow velocity of 3~4mL/min；
The dissolved a concentration of 10~30mg/mL of Anthocyanin-rich Extract freeze-dried powder mobile phase, sampling volume be 1~
15mL。
7. the isolation and purification method of Cyanidin -3-O- rutinosides according to claim 1, which is characterized in that step
(4) in, the solid-phase extraction column is selected from C18Sep-Pak cartridge solid-phase extraction columns.
8. the isolation and purification method of Cyanidin -3-O- rutinosides according to claim 1, which is characterized in that step
(4) in, the procyanidin, specially：
The anthocyanin concentrate is injected into solid-phase extraction column with the flow velocity of 1~5mL/min, until adsorption volume reaches column volume
1/3, then carry out gradient elution.
9. the isolation and purification method of Cyanidin -3-O- rutinosides according to claim 8, which is characterized in that described
The step of gradient elution is：
Formic acid-the aqueous systems for being first 1.5% with formic acid concentration expressed in percentage by volume elute, and are then with concentration expressed in percentage by volume respectively
2.5%, 5%, 10% and 20% acidic methanol solution elution, most collects 10% and 20% respectively after the flushing of pure methanol afterwards
Acidic methanol eluent after merge；
Acid in the acidic methanol solution is selected from formic acid, and the concentration expressed in percentage by volume of formic acid is 1.5%.
10. a kind of application of Cyanidin -3-O- rutinosides in preparing alpha-glucosidase restrainer, which is characterized in that
Cyanidin -3-O- the rutinosides isolate and purify to obtain according to any method of claim 1~9.
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| CN113684235A (en) * | 2021-08-19 | 2021-11-23 | 江苏科技大学 | Method for converting mulberry red pigment component by double aqueous phase whole cell catalysis |
| CN116173090A (en) * | 2021-11-26 | 2023-05-30 | 上海医药工业研究院 | Red ginseng refined product, preparation method thereof and application of red ginseng extract |
| CN116138248A (en) * | 2023-02-22 | 2023-05-23 | 西北农林科技大学 | Preparation method and application of diluent for freezing preservation of semen of dairy sheep |
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