CN103923052A - Method for preparing oligomeric proanthocyanidins - Google Patents
Method for preparing oligomeric proanthocyanidins Download PDFInfo
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- CN103923052A CN103923052A CN201410140039.3A CN201410140039A CN103923052A CN 103923052 A CN103923052 A CN 103923052A CN 201410140039 A CN201410140039 A CN 201410140039A CN 103923052 A CN103923052 A CN 103923052A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
- C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
- C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
- C07D311/58—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring other than with oxygen or sulphur atoms in position 2 or 4
- C07D311/60—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring other than with oxygen or sulphur atoms in position 2 or 4 with aryl radicals attached in position 2
- C07D311/62—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring other than with oxygen or sulphur atoms in position 2 or 4 with aryl radicals attached in position 2 with oxygen atoms directly attached in position 3, e.g. anthocyanidins
Abstract
The invention belongs to the technical field of chemical effective component extraction, and particularly relates to a method for preparing oligomeric proanthocyanidins with uniform polymerization. According to the method, 60-70% ethanol is adopted in extraction, and the pH in the extracting process is controlled to be 4.0-4.4, so that monomer, oligomeric proanthocyanidins and polymer proanthocyanidins can be effectively extracted; macroporous resin is used for adsorbing, and elution is performed according to the principle that proanthocyanidins with different polymerization degrees have different soluble properties in ethanol with different concentrations, so that oligomer products with polymerization degree truly concentrated in 2-4 can be further obtained, and the content of the effective component oligomer in proanthocyanidins can be increased.
Description
Technical field
The invention belongs to chemical extracts active ingredients technical field, be specifically related to a kind of method of preparing the oligomeric procyanidolics of polymerization degree homogeneous.
Background technology
Pycnogenols (Proanthocyanidins) is a class Flavonol monomer and polymeric polyphenolic substance thereof, its common feature is that heating all can produce cyanidin(e) in acidic medium, therefore be called again proanthocyanidin, be a kind of pigment composition in plant, be extensively present in each kind of plant.
Grape pip procyanidin (grape seed procyanidine) is that a class is present in the natural phant active substance in Semen Vitis viniferae, and it has good anti-oxidant, the effect of removing free radical, in fields such as food, healthcare products, medicines, has more application.The basic structure of grape pip procyanidin is the polymer being polymerized by the catechin of different quantities or epicatechin monomers, conventionally by what contain 2-4 monomer, be called Procyanidcic Oligomers (oligomersic procyanidine), what contain 4 above monomers is called high polymer pycnogenols (polymersic procyanidine).And generally, Procyanidcic Oligomers is considered to the marrow place of anti-oxidation efficacy, its demand is more extensive.
At present sell and the preparation of the pycnogenols product of application is all generally to take Semen Vitis viniferae as extracting raw material domestic and international market, through steps such as water extraction or organic solvent extraction, concentrated, resin absorption and wash-outs, obtains.But it is not high at ubiquity extraction yield, use the reagent of not environmental protection, the product that wash-out obtains contains the high polymer that a large amount of monomers and the polymerization degree are greater than 4, not only make that whole solubleness is not high, raw material availability is lower, the more important thing is that the whole anti-oxidation efficacy of its product is also received larger impact.
Chinese patent CN101781279A discloses a kind of preparation method of grape pip procyanidin, and it is with the aqueous solution lixiviate Semen Vitis viniferae of 85-95 ℃, and by extract with extraction using alcohol, then through processing such as column chromatography for separation, obtain the pycnogenols of oligomer.Chinese patent CN101597273A discloses a kind of preparation method of Procyanidcic Oligomers equally, it is with 35% ethanolic soln lixiviate Semen Vitis viniferae, and adjust pH alkalescence to separate out throw out extract, subsequently throw out is dissolved in to pH4.0,35% extraction using alcohol, filtrate is obtained Procyanidcic Oligomers throw out through membrane retention.
But find in actual product, in the pycnogenols of directly producing in prior art, in its product, include a large amount of former blue and white plain monomer and high polymer, because whole product is evaluated with mean polymerisation degree, although cause its product also substantially to meet the oligopolymer requirement that mean polymerisation degree is 2-4, but, lower in view of really thering is the actual content of pycnogenols of polymerization degree 2-4 of high-efficiency antioxidant function in whole product, and greatly affected the effect of product.For the high polymer product containing in product, be not effectively utilized, not only affect product performance, cause the waste of raw material and the raising of cost yet simultaneously.
Summary of the invention
For this reason, in the Procyanidcic Oligomers that technical problem to be solved by this invention is to produce in prior art, contain a large amount of monomers and high polymer and have a strong impact on the problem of product performance, and then a kind of method of preparing polymerization degree homogeneous, the good Procyanidcic Oligomers of product performance is provided.
The present invention also further provide a kind of by aforesaid method, prepared to the Procyanidcic Oligomers of polymerization degree homogeneous.
The preparation and the process for purification that the invention provides a kind of Procyanidcic Oligomers, comprise following steps:
(1) get containing Procyanidcic Oligomers plant material, be dried and pulverize and sieve, obtain plant drymeal;
(2) with plant drymeal described in the aqueous ethanolic solution lixiviate of volumetric concentration 60-70%, and control reaction pH4.0-4.4, collect extracting solution and obtain containing pycnogenols crude extract;
(3) described crude extract is processed through macroporous resin adsorption, and washed decon;
(4), with the ethanol elution of volumetric concentration 5-10%, collect elutriant and obtain monomer pycnogenols;
(5), with the ethanol elution of volumetric concentration 20-30%, collect elutriant and obtain Procyanidcic Oligomers;
(6) collect containing Procyanidcic Oligomers elutriant, concentrating under reduced pressure is except alcohol, and lyophilize, obtains required Procyanidcic Oligomers.
Further, in described step (3), described macroporous resin is AB-8 macroporous resin.
In described step (2), the parts by weight ratio of the volume parts of described ethanolic soln and described plant drymeal is 6-7:1, and the pass of described parts by weight and described volume parts is the relation of g/ml.
In described step (2), described lixiviate step service temperature is 40-50 ℃.
In described step (2), described lixiviate step repeats 2-3 time, each 30-40 minute.
Described step (3) before, also comprises the step except alcohol by described crude extract concentrating under reduced pressure.
In described step (1), described drying step is lyophilize.
More excellent, described step (5) afterwards, also comprises the step (5 ') with the ethanol elution of 50-60%, collects elutriant and obtains high polymer pycnogenols.
After described step (5 '), also comprise and containing high polymer pycnogenols elutriant, add total amount 0.5%-0.8% dilute hydrochloric acid by described, the step being hydrolyzed, collects hydrolyzed solution, and repeating said steps (3) and (5), obtains containing Procyanidcic Oligomers elutriant.
The dilute hydrochloric acid that adds total amount 0.5%-0.8% in described high polymer elutriant, the concentration of dilute hydrochloric acid is 20% the water diluent containing standard available 37% concentrated hydrochloric acid.
The service temperature of described hydrolysis high polymer pycnogenols step is 60 ℃.
Procyanidcic Oligomers plant material of the present invention includes but not limited to Semen Vitis viniferae, pine tree, grape, apple, pawpaw, lichee, hops, longan, sea-buckthorn, phyllanthus emblica, cassia bark, Chinese sorghum, buckwheat, persimmon, mangosteen, banana, bajiao banana, hawthorn, loquat, cherry, pomegranate, sweet potato, cowberry, the seedpod of the lotus, Kiwifruit, red bayberry, fibert, silk tree, ginkgo, walnut, Queensland nut, garden burnet, cotton, herbage, jujube tree, cocoa tree, Anlnus nepalensis tree, water wax gourd tree, cypress, Bruguiera conjugata tree, Schima superba, birch, eucalyptus, willow, camphor tree, chestnut, robur, in the plants such as black wattle plant one or more.
The invention also discloses the Procyanidcic Oligomers that above-mentioned preparation method obtains, its mean polymerisation degree is 2.8-3.2, and wherein, the oligomer content that the polymerization degree is 2-4 is up to more than 95%.
Technique scheme of the present invention has the following advantages compared to existing technology:
1, preparation method of the present invention takes full advantage of oligomeric procyanidolics, monomer also has high polymer different feature of dissolving properties under specific solvent and extraction conditions, ethanol with 60-70% extracts, control described reaction pH4.0-4.4 simultaneously, the oligomer that effect is stronger is separated; Utilize the adsorptive power difference of macroporous resin to different polymerization degree product simultaneously, utilize certain concentration ethanol for elutriant, separated and the collection by the Procyanidcic Oligomers of polymerization degree 2-4, in product, contain hardly monomer and high polymer, not only make the mean polymerisation degree of product in 2.8-3.2 left and right, and in product, the polymerization degree is really for the oligomer of 2-4 has accounted for the more than 95% of integral body, product anti-oxidation efficacy promotes greatly, and the solvability of product in water improves greatly, not tolerant minimizing;
2, preparation method of the present invention takes full advantage of the feature that pycnogenols high polymer can be hydrolyzed, utilize different concentration ethanol for elutriant, in separating-purifying Procyanidcic Oligomers, isolate in a large number high polymer pycnogenols, and will after high polymer hydrolysis, again reclaim oligomer, make full use of raw material, when guaranteeing quality product, improve the yield of product;
3, the method for the invention adopts AB-8 macroporous resin to carry out adsorption treatment, experiment showed, the taking-up best results of extraction effect and the impurity of its effective constituent;
4, the method for the invention is from the pre-treatment of raw material, just rely on the lyophilize of plant material and crushing technology, greatly reduce the loss of pycnogenols, to the processing of final oligomer finished product, also utilized Freeze Drying Technique to reduce the loss of effective constituent, the oligomeric procyanidolics product that makes to obtain has that yield is high, purity is high, good, the anti-oxidation efficacy good technical superiority of solvability in water simultaneously.
Embodiment
Embodiment 1:
Described in the present embodiment, Procyanidcic Oligomers prepares by the following method:
(1) by fresh Semen Vitis viniferae 100g, under-15 ℃～-5 ℃, the condition of 1～10 handkerchief, after lyophilize, pulverize, cross 50 mesh sieves and obtain dry powder;
(2) get the aqueous ethanolic solution of 700ml volumetric concentration 70%, in Semen Vitis viniferae dry powder described in 50 ℃ of lixiviates, and control pH 4.0, extract 3 times each 30 minutes, after united extraction liquid coarse filtration, obtain pycnogenols crude extract;
(3) by said extracted liquid at vacuum tightness 0.09Mpa, under the condition that temperature is 40～50 ℃, be evaporated to proportion 1.10, and AB-8 macroporous resin on the concentrated solution obtaining carried out to adsorption treatment 4h; And wash away impurity with pure water;
(4), with resin described in the ethanol-water solution 2BV wash-out of volumetric concentration 5%, collect elutriant and obtain monomer pycnogenols, and discard;
(5), with resin described in the aqueous ethanolic solution 3BV wash-out of volumetric concentration 30%, collect elutriant and obtain oligomeric procyanidolics;
(5 '), with resin described in the aqueous ethanolic solution 2BV wash-out of volumetric concentration 60%, collects elutriant and obtains high polymer pycnogenols; By described, add total amount 0.5%～0.8% dilute hydrochloric acid containing high polymer pycnogenols elutriant, the step being hydrolyzed, collects hydrolyzed solution, and repeating said steps (3) and (5), and collect that 30% ethanol elution obtains contain oligomeric procyanidolics elutriant;
(6) merge above-mentioned steps (5) and the elutriant of 30% ethanol elution collected in (5 '), at vacuum tightness 0.09Mpa, under the condition that temperature is 40～50 ℃, being evaporated to proportion is 1.25, and lyophilize obtains described Procyanidcic Oligomers product under-15 ℃～-5 ℃, the condition of 1～10 handkerchief.
Embodiment 2:
Described in the present embodiment, Procyanidcic Oligomers prepares by the following method:
(1) by fresh Semen Vitis viniferae 100g, under-15 ℃～-5 ℃, the condition of 1～10 handkerchief, after lyophilize, pulverize, cross 80 mesh sieves and obtain dry powder;
(2) get the aqueous ethanolic solution of 600ml volumetric concentration 70%, in Semen Vitis viniferae dry powder described in 45 ℃ of lixiviates, and control pH in 4.3 left and right, extract 3 times each 40 minutes, after united extraction liquid coarse filtration, obtain pycnogenols crude extract;
(3) said extracted liquid is evaporated to proportion 1.15 under vacuum tightness 0.09Mpa, 40～50 ℃ of conditions of temperature, and AB-8 macroporous resin on the concentrated solution obtaining is carried out to adsorption treatment 3h; And wash away impurity with pure water;
(4), with resin described in the aqueous ethanolic solution 2BV wash-out of volumetric concentration 10%, collect elutriant and obtain monomer pycnogenols, and discard;
(5), with resin described in the aqueous ethanolic solution 3BV wash-out of volumetric concentration 25%, collect elutriant and obtain oligomeric procyanidolics;
(5 '), with resin described in the aqueous ethanolic solution 2BV wash-out of volumetric concentration 55%, collects elutriant and obtains high polymer pycnogenols; By described, add total amount 0.5%～0.8% dilute hydrochloric acid containing high polymer pycnogenols elutriant, the step being hydrolyzed, collects hydrolyzed solution, and repeating said steps (3) and (5), and collect that 25% ethanol elution obtains contain oligomeric procyanidolics elutriant;
(6) merge above-mentioned steps (5) and the elutriant of 25% ethanol elution collected in (5 '), under the condition of 40～50 ℃ of vacuum tightness 0.09Mpa, temperature, being evaporated to proportion is 1.20, and lyophilize obtains described Procyanidcic Oligomers product under-15 ℃～-5 ℃, the condition of 1～10 handkerchief.
Embodiment 3
Described in the present embodiment, Procyanidcic Oligomers prepares by the following method:
(1) by fresh Semen Vitis viniferae 100g, under-15 ℃～-5 ℃, the condition of 1～10 handkerchief, after lyophilize, pulverize, cross 140 mesh sieves and obtain dry powder;
(2) get the aqueous ethanolic solution of 650ml volumetric concentration 30%, in Semen Vitis viniferae dry powder described in 40 ℃ of lixiviates, and control pH 4.4, extract 3 times each 40 minutes, after united extraction liquid coarse filtration, obtain pycnogenols crude extract;
(3), at vacuum tightness 0.09Mpa, under the condition that temperature is 40～50 ℃, be evaporated to proportion 1.15, and AB-8 macroporous resin on the concentrated solution obtaining is carried out to adsorption treatment 3h; And wash away impurity with pure water;
(4), with resin described in the aqueous ethanolic solution 2BV wash-out of volumetric concentration 8%, collect elutriant and obtain monomer pycnogenols, and discard;
(5), with resin described in the aqueous ethanolic solution 3BV wash-out of volumetric concentration 20%, collect elutriant and obtain oligomeric procyanidolics 3BV;
(5 '), with resin described in the aqueous ethanolic solution 2BV wash-out of volumetric concentration 50%, collects elutriant and obtains high polymer pycnogenols 2BV; By described, add total amount 0.5%～0.8% dilute hydrochloric acid containing high polymer pycnogenols elutriant, the step being hydrolyzed, collects hydrolyzed solution, and repeating said steps (3) and (5), and collect that 20% ethanol elution obtains contain oligomeric procyanidolics elutriant;
(6) merge above-mentioned steps (5) and the elutriant of 20% ethanol elution collected in (5 '), under the condition of 40～50 ℃ of vacuum tightness 0.09Mpa, temperature, being evaporated to proportion is 1.20, and lyophilize obtains described Procyanidcic Oligomers product under-15 ℃～-5 ℃, the condition of 1～10 handkerchief.
Embodiment 4
Described in the present embodiment, Procyanidcic Oligomers prepares by the following method:
(1) by fresh Semen Vitis viniferae 100g, under-15 ℃～-5 ℃, the condition of 1～10 handkerchief, after lyophilize, pulverize, cross 50 mesh sieves and obtain dry powder;
(2) get the aqueous ethanolic solution of 600ml volumetric concentration 70%, in Semen Vitis viniferae dry powder described in 45 ℃ of lixiviates, and control pH 4.4, extract 3 times each 40 minutes, after united extraction liquid coarse filtration, obtain pycnogenols crude extract;
(3), at vacuum tightness 0.09Mpa, under the condition that temperature is 40～50 ℃, be evaporated to proportion 1.10, and AB-8 macroporous resin on the concentrated solution obtaining is carried out to adsorption treatment 3h; And wash away impurity with pure water;
(4), with resin described in the aqueous ethanolic solution 2BV wash-out of volumetric concentration 5%, collect elutriant and obtain monomer pycnogenols, and discard;
(5), with resin described in the aqueous ethanolic solution 3BV wash-out of volumetric concentration 25%, collect elutriant and obtain oligomeric procyanidolics 3BV;
(6) merge the elutriant of 25% ethanol elution of collecting in above-mentioned steps (5), under the condition of 40～50 ℃ of vacuum tightness 0.09Mpa, temperature, being evaporated to proportion is 1.20, and lyophilize obtains described Procyanidcic Oligomers product under-15 ℃～-5 ℃, the condition of 1～10 handkerchief.
Comparative example 1
(1) fresh Semen Vitis viniferae is pulverized, crossed 80 mesh sieves;
(2) with 8 times of amount 70% ethanol, pH is controlled at 4.2 left and right, and 45 ℃ are extracted Semen Vitis viniferae and do; Powder 3 times each 40 minutes, obtains pycnogenols crude extract after united extraction liquid coarse filtration;
(3) said extracted liquid is evaporated to proportion 1.15;
(4) spraying is dry.
Comparative example 2
(1) fresh Semen Vitis viniferae is pulverized, crossed 80 mesh sieves;
(2) with 8 times of amount 35% ethanol, pH is controlled at 4.2 left and right, and 45 ℃ are extracted Semen Vitis viniferae and do; Powder 3 times each 40 minutes, obtains pycnogenols crude extract after united extraction liquid coarse filtration;
(3) said extracted liquid is evaporated to proportion 1.15;
(4) spraying is dry.
Comparative example 3
(1) fresh Semen Vitis viniferae is pulverized, crossed 80 mesh sieves;
(2) with 8 times of amount 95% ethanol, pH is controlled at 4.2 left and right, and 45 ℃ are extracted Semen Vitis viniferae and do;
Powder 3 times each 40 minutes, obtains pycnogenols crude extract after united extraction liquid coarse filtration;
(3) said extracted liquid is evaporated to proportion 1.15;
(4) spraying is dry.
Experimental example
Measure each polymerization degree product content of above-described embodiment 1-4 and comparative example 1-3 products obtained therefrom, concrete content assaying method adopts vanillin-sulfuric acid method; Polymerization degree measurement adopts improvement vanillin method to measure.
Mean polymerisation degree calculates
Principle: traditional vanillin method is to utilize phenol formaldehyde condensation between each flavan-3-ol unit of Vanillin and pycnogenols to react to measure the quality of pycnogenols.Butler etc. studies show that, when adopting glacial acetic acid to make solvent, Vanillin only with end flavan-3-alcohol generation condensation reaction, by red product absorbancy, record pycnogenols amount of substance concentration.With traditional vanillin method, measure the quality of pycnogenols on the one hand, comprise monomer and polymer composition; Take glacial acetic acid on the other hand as solvent, make Vanillin only react to measure the amount of substance of pycnogenols with pycnogenols end flavan-3-alcohol, both in conjunction with so that obtain the mean polymerisation degree of pycnogenols.
For impure pycnogenols, its mean polymerisation degree DP=m/(M*n).Wherein m is pycnogenols quality, μ g; N is pycnogenols μ mol; M is the relative molecular mass of monomer (+)-catechin.For example, by content method, first measuring procyanidin content in 10mg sample is m, then measures 10mg sample pycnogenols amount of substance n by amount of substance method for measurement of concentration, brings data into and can try to achieve mean polymerisation degree.
One, assay
Vanillin-sulfuric acid method
1.1. material and instrument
Catechin mark product (99%), pycnogenols sample, methyl alcohol (AR), the vitriol oil (AR), Vanillin (AR).
1.2. instrument
Glassware: volumetric flask (10ml, 50ml, 100ml), transfer pipet (0.5ml, 5ml), graduated cylinder (50ml), appearance is surrounded by the tool plug test tube (10ml) of tinfoil paper.
Spectrophotometer, water-bath, electronic balance.
2. experimental principle
Higher based on pycnogenols A ring chemically reactive, the hydroxyl on it can react with Vanillin generation phenol formaldehyde condensation, forms coloured carbonium ion under concentrated acid effect, and the absorbancy of carbonium ion can be measured the content of pycnogenols accordingly.Hydrochloric acid or sulfuric acid all can be used as the catalyzer of reaction process.
3. experimental procedure
3.1 reagent preparation
(1) preparation sample solution: take a certain amount of pycnogenols sample, with distilled water, be settled to certain volume, ultrasonic 10min dissolution, being made into concentration is the pycnogenols sample solution of 0.01-0.1mg/mL.
(2) preparation developer
Reagent A: 4% Vanillin-methanol solution: 4g Vanillin arrives 100ml by methanol constant volume;
The reagent B:30% vitriol oil-methanol solution: measure the vitriol oil of 30ml, by methanol constant volume to 100ml.
3.2 colorimetric conditions
Get the 0.5ml sample solution+2.5ml4% Vanillin-methanol solution+2.5ml30% vitriol oil-methanol solution, join in the test tube that 10ml appearance is surrounded by tinfoil paper, add a cover and shake up, 30 ℃ of lucifuges reaction 15min of water-bath, using methyl alcohol as blank, and 500nm measures absorbancy.
3.3. the formulation of typical curve (linearity range is 0.01-0.1mg/mL)
(1) take 5mg catechin mark product distilled water and be settled in 50ml volumetric flask, make the trial-product of 0.1mg/ml.
(2) from trial-product, pipette respectively 2,4,6,8,10ml in 10ml volumetric flask, be made into the solution that concentration is respectively 0.02,0.04,0.06,0.08,0.1mg/ml. pipette respectively 0.5ml and survey absorbancy according to above-mentioned colorimetric condition, do typical curve.
3.4. samples contg is measured:
Get the sample solution that 0.5ml prepared and survey absorbancy according to above-mentioned colorimetric condition, the absorbancy that sample is estimated is brought typical curve into can calculate samples contg.
Two. amount of substance is measured
Improvement vanillin method
1. material and instrument
1.1 reagent
Pycnogenols sample, catechin mark product (99%), Vanillin (AR), glacial acetic acid (AR),
Methyl alcohol (AR).
1.2 instrument
2.1 glassware:
Volumetric flask (10ml, 50ml, 100ml), transfer pipet (0.5ml, 5ml), graduated cylinder (50ml), appearance is surrounded by the tool plug test tube (10ml) of tinfoil paper.
2.2 spectrophotometer
2.3 water-bath
2.4 electronic balance
2. principle
Traditional vanillin method is to utilize phenol formaldehyde condensation between each flavan-3-ol unit of Vanillin and pycnogenols to react to measure the quality amount of pycnogenols.Butler etc. studies show that, when adopting glacial acetic acid to make solvent, Vanillin only with end flavan-3-alcohol generation condensation reaction by, red product absorbancy records pycnogenols amount of substance concentration.With traditional vanillin method, measure the quality of pycnogenols on the one hand, comprise monomer and polymer composition; Take glacial acetic acid on the other hand as solvent, make Vanillin only react to measure the amount of substance of pycnogenols with pycnogenols end flavan-3-alcohol, both in conjunction with so that obtain the mean polymerisation degree of pycnogenols.
3. experimental procedure
3.1 solution preparation
(1) preparation sample solution: the pycnogenols sample that takes 10mg left and right, add the pure methyl alcohol dissolution (4%%-6% that add-on is constant volume of 4-6ml,), then use acetic acid constant volume in 100ml volumetric flask, ultrasonic 10min dissolution, is made into the sample solution that amount of substance concentration is 0.025-0.25umol/mL.
(2) preparation developer: 4% hydrochloric acid and 1% Vanillin glacial acetic acid solution: take 1g Vanillin and add 4ml hydrochloric acid, use acetic acid constant volume to 100ml.
3.2 colorimetric conditions
1ml sample solution+5ml4% hydrochloric acid and 1% Vanillin glacial acetic acid solution) join in the test tube that 10ml appearance is surrounded by tinfoil paper, add a cover and shake up, 20 ℃ of reaction 10min, using acetic acid as blank, and 500nm measures absorbancy.
3.3. formulate typical curve (linearity range 0.025-0.25umol/mL)
(1) take 5mg catechin table mark for product acetic acid (monomer is dissolved in acetic acid, does not need methyl alcohol dissolution) be settled to 50ml volumetric flask, make the trial-product of 0.032umol/ml.
(2) from trial-product, pipette respectively 1,2,3,4,5,6ml is in 10ml volumetric flask, and being made into respectively amount of substance concentration is 0.032,0.064,, 0.064,0.128, .0.16umol/ml solution, pipettes respectively 1ml, according to above-mentioned colorimetric condition, measures absorbancy at 500nm place, does typical curve.3.4 amount of substance concentration determinations
Pipette the sample solution 1ml having prepared and according to colorimetric condition, at 500nm place, measure absorbancy, substitution typical curve is tried to achieve amount of substance concentration.
In conjunction with nanofiltration and ultrafiltration, the aqueous solution of prior art and the finished product of the present invention is carried out respectively separated, intercepting polymerization degree sample of (600-2000) between the corresponding molecular weight area of the oligomeric procyanidolics of 2-4, collect the sample in small molecules amount and macromolecule interval simultaneously, then three's vacuum concentration is extremely dry, measure three's solid masses and obtain the ratio of three in former state, then we are again multiple soluble in water by three, can obtain a three about other data of poorly water-soluble, finally, the three minutes aqueous solution is done respectively to anti-oxidant experiment, can obtain the difference of three's effect.Concrete numerical value is as following table:
The oligomeric procyanidolics mean polymerisation degree that employing the method for the invention obtains is at 2.8-3.2, and the target oligomer content that wherein polymerization degree is 2-4 accounts for the more than 95% of whole product, and the whole 2-3% of monomer pycnogenols, does not almost have high polymer in product.With respect to containing a large amount of monomers (reaching 15%-25%) in a lot of other mean polymerisation degree in the prior art also oligomeric procyanidolics within the scope of 2-4, with high polymer (reaching 15%-25%), the situation that mean polymerisation degree is lower is compared, its active constituent content increases greatly, and the solubleness of product and validity all significantly promote.
Obviously, above-described embodiment is only for example is clearly described, and the not restriction to embodiment.For those of ordinary skill in the field, can also make other changes in different forms on the basis of the above description.Here exhaustive without also giving all embodiments.And the apparent variation of being extended out thus or change are still among the protection domain in the invention.
Claims (12)
1. a preparation method for Procyanidcic Oligomers, is characterized in that, comprises following steps:
(1) get containing Procyanidcic Oligomers plant material, be dried and pulverize and sieve, obtain plant drymeal;
(2) with plant drymeal described in the aqueous ethanolic solution lixiviate of volumetric concentration 60-70%, and to control pH value in reaction be 4.0-4.4, collects extracting solution and obtain containing pycnogenols crude extract;
(3) described crude extract is processed through macroporous resin adsorption, and washed decon;
(4) with the aqueous ethanolic solution wash-out of volumetric concentration 5-10%, collect the elutriant obtaining containing monomer pycnogenols;
(5) with the aqueous ethanolic solution wash-out of volumetric concentration 20-30%, collect the elutriant obtaining containing Procyanidcic Oligomers;
(6) get containing Procyanidcic Oligomers elutriant, concentrating under reduced pressure is except alcohol, and lyophilize, obtains required Procyanidcic Oligomers.
2. the preparation method of Procyanidcic Oligomers according to claim 1, is characterized in that:
In described step (3), described macroporous resin is AB-8 macroporous resin.
3. the preparation method of Procyanidcic Oligomers according to claim 1 and 2, is characterized in that:
In described step (2), the parts by weight ratio of the volume parts of described ethanolic soln and described plant drymeal is 6-7:1, and the pass of described parts by weight and described volume parts is the relation of g/ml.
4. according to the preparation method of the arbitrary described Procyanidcic Oligomers of claim 1-3, it is characterized in that:
In described step (2), described lixiviate step service temperature is 40-50 ℃.
5. the preparation method of Procyanidcic Oligomers according to claim 4, is characterized in that:
In described step (2), described lixiviate step repeats 2-3 time, each 30-40 minute.
6. according to the preparation method of the arbitrary described Procyanidcic Oligomers of claim 1-5, it is characterized in that:
Described step (3) before, also comprises the step except alcohol by described crude extract concentrating under reduced pressure.
7. according to the preparation method of the arbitrary described Procyanidcic Oligomers of claim 1-6, it is characterized in that:
In described step (1), described drying step is lyophilize.
8. according to the preparation method of the arbitrary described Procyanidcic Oligomers of claim 1-7, it is characterized in that:
Described step (5) afterwards, also comprises the step (5 ') with the ethanol elution of 50-60%, collects elutriant and obtains high polymer pycnogenols.
9. the preparation method of Procyanidcic Oligomers according to claim 8, is characterized in that:
After described step (5 '), also comprise and containing high polymer pycnogenols elutriant, add total amount 0.5%-0.8% dilute hydrochloric acid by described, the step being hydrolyzed, collects hydrolyzed solution, and repeating said steps (3) and (5), obtains containing Procyanidcic Oligomers elutriant.
10. the preparation method of Procyanidcic Oligomers according to claim 9, is characterized in that:
The dilute hydrochloric acid that adds total amount 0.5%-0.8% in described high polymer elutriant, the concentration of dilute hydrochloric acid is 20% the water diluent containing standard available concentrated hydrochloric acid.
11. according to the preparation method of the Procyanidcic Oligomers described in claim 9 or 10, it is characterized in that:
The service temperature of described hydrolysis high polymer pycnogenols step is 60 ℃.
12. Procyanidcic Oligomers that prepare according to the arbitrary described method of claim 1-11.
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| CN104177863A (en) * | 2014-09-09 | 2014-12-03 | 山东省林业科学研究院 | Method for extracting red pigment from green pin needles |
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| CN105294636A (en) * | 2015-11-16 | 2016-02-03 | 大兴安岭林格贝寒带生物科技股份有限公司 | Preparation method of oligomeric proanthocyanidin |
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| CN106381319A (en) * | 2016-08-31 | 2017-02-08 | 山东省葡萄研究院 | High-efficiency extraction and separation method of grape seed proanthocyanidin oligomer |
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| CN106381319B (en) * | 2016-08-31 | 2020-07-14 | 山东省葡萄研究院 | Efficient extraction and separation method for grape seed procyanidin oligomers |
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| CN109943605B (en) * | 2017-12-20 | 2023-03-24 | 威海惠安康生物科技有限公司 | Preparation method of oligomeric proanthocyanidins with uniform components |
| CN109943605A (en) * | 2017-12-20 | 2019-06-28 | 威海惠安康生物科技有限公司 | A kind of preparation method of homogeneous components glucosidase procyanidins |
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