CN101845035A - Method for extracting oligomeric proanthocyanidins - Google Patents
Method for extracting oligomeric proanthocyanidins Download PDFInfo
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Abstract
The invention discloses a method for extracting oligomeric proanthocyanidins, which comprises the following steps of: extracting a plant raw material containing the oligomeric proanthocyanidins with a solvent; enabling a crude extract to pass through a macroporous adsorbent resin column; eluting with water to remove protein and polysaccharide impurities; carrying out gradient elution with water and one or more selecting from alcohol, methyl alcohol and acetone as an eluant; collecting mixed solution with the volume ratio of n-butyl alcohol, acetic acid and water of 4:1:5; standing for demixing, wherein the solution at upper layer is a developing solvent; and carrying out silica gel thin-layer chromatography, wherein the elution part with the Rf value of 0.2-0.5 on a thin-layer chromatography plate is the oligomeric proanthocyanidins extract. Compared with the prior art, the invention has the advantages that the purity and the yield are both remarkably improved, the yield of the oligomeric proanthocyanidins can reach more than 80 percent, the product purity is no less than 50 percent and is basically more than 80 percent, and the method has simple and convenient process, low cost and easy industrialization.
Description
Technical field
The present invention relates to a kind of extracting method, be specifically related to a kind of extracting method of oligomeric procyanidolics.
Background technology
Pycnogenols (Proantho Cyanidins, PC) extensively be present in the plant, its poly-polyphenols that to be a class formed by the flavan-3-alcohol monomer condenses of different quantities mainly is made up of catechin and l-Epicatechol etc., and it is gained the name because of heating under acidic medium generates corresponding cyanidin(e).The simplest pycnogenols is catechin and table catechu monomer.In addition, also have dimer, tripolymer, the tetramer etc., until ten aggressiveness.By polymerization degree size, usually 2～4 aggressiveness are called oligomer (oligomeric procyanidin, OPC), be called more than the pentamer high polymer (polymeric procyanidin, PPC).Oligomeric procyanidolics (oligomeric procyanidins, OPCs) because structure is special, good water solubility, the validity height, bioavailability easily is absorbed by the body more than 90%, becomes the focus of research.The ability that oligomeric procyanidolics has stronger oxidation-resistance, removes the free radical ability and microcirculation in human body is improved is one of present strong, the most effective oxygen free radical scavenger of finding and lipid peroxidation inhibitor.Oligomeric procyanidolics antioxidant effect in vivo is far above vitamin-E and vitamins C, its in vivo resistance of oxidation be about 50 times of vitamin-E, ascorbic 20 times.At present, oligomeric procyanidolics is used widely at cosmetic field and field of health care food.The external oligomeric procyanidolics preparation of producing has become that people often take has and delays senility and health care foods such as beauty treatment.
Reported that multiple separation prepares the method for oligomeric procyanidolics both at home and abroad.These methods mainly are to separate to obtain from plant milk extracts such as Cortex Pini, Semen Vitis viniferae.At present, the extraction and purification process of oligomeric procyanidolics mainly comprises solvent-extraction process and column chromatography.Though solvent-extraction process equipment is simple, process cost is low, only adopts solvent-extraction process, and product purity is lower, can only reach about 50%.Column chromatography can effectively improve product purity.Existing employing column chromatography separates in the report of purification oligomeric procyanidolics, mainly adopts polymeric amide or C
18Bonding phase silica gel is as filler.Wherein, though polymeric amide filler price is lower, but before charging, need raw material is carried out complicated pretreatment, and because the higher oligomeric procyanidolics of the polymerization degree has strong adsorption on polymeric amide, make that the yield of oligomeric procyanidolics is lower, and be easy to make post to imitate and reduce, need to use the alkali lye frequent regeneration, be not suitable for suitability for industrialized production.Adopt C
18Bonding phase silica gel is made filler, though can obtain the higher oligomeric procyanidolics product of purity, it costs an arm and a leg, and is difficult to be applied to large-scale commercial production.Therefore, it is easy to demand seeking a kind of technology urgently, and cost is low, is easy to the novel method of industrialization.
Summary of the invention
Technical problem to be solved by this invention be for the method product purity and the yield that overcome existing oligomeric procyanidolics not ideal enough, or complex operation, or the more high defective of cost, and provide compared with prior art a kind of, product purity and yield all are significantly increased, and technology is easy, and cost is low, is easy to the novel method of the extraction oligomeric procyanidolics of industrialization.
The plant material that method of the present invention comprises the steps: to contain oligomeric procyanidolics is with macroporous adsorptive resins on the crude extract of solvent extraction gained, wash with water and remove deproteinize and polyose impurity, carry out gradient elution with one or more and water in the following organic solvent as eluent more afterwards: ethanol, methyl alcohol and acetone, collection is 4: 1: 5 propyl carbinol with volume ratio, upper solution behind the mixing solutions standing demix of acetic acid and water is a developping agent, the last Rf value of silica gel thin-layer chromatography plate (TLC) is 0.2～0.5 wash-out part, promptly gets the oligomeric procyanidolics extract.
Below, method of the present invention is described in detail:
(1) contains the crude extract of the plant material of oligomeric procyanidolics with the solvent extraction gained
Among the present invention, the described plant material that contains oligomeric procyanidolics can be in the prior art with the crude extract of solvent extraction gained, plant material contains the crude extract of oligomeric procyanidolics through solvent extraction, the present invention is especially preferably by following raw material and the prepared crude extract of method, with the purification step after the better cooperation:
Among the present invention, the described plant material that contains oligomeric procyanidolics can be the various plant materials of reporting in the existing document that contain oligomeric procyanidolics, as Cortex Pini, Semen Vitis viniferae and seed of Fructus Hippophae etc.The higher seed of Fructus Hippophae of the preferred oligomeric procyanidolics content of the present invention better has been extracted seed of Fructus Hippophae dregs behind the Oleum Hippophae for sea-buckthorn, selects this raw material can realize utilization of waste material, and wider than sources such as Cortex Pini and Semen Vitis viniferaes, and cost is lower.
Preferable, before the solvent extraction, above-mentioned plant material is carried out drying and crushing handle.What the exsiccant temperature was preferable is 40～85 ℃.What the particle grain size of pulverizing was preferable is 80 orders.
Among the present invention, the mode of solvent extraction can adopt existing all kinds of SOLVENTS extracting mode, as conventional solvent extraction, the extraction of microwave-assisted solvent, ultrasonic solvent extraction and supercritical fluid extraction etc.Among the present invention, described conventional solvent extraction is meant the solvent-extraction process that does not adopt as microwave and special supplementary means such as ultrasonic.Concrete each condition of above-mentioned each solvent extraction mode can be with reference to state of the art, the present invention is preferably as follows condition especially through optimizing screening: the mixed solvent for one or more and water in the following organic solvent that extracts preferred solvents: ethanol, methyl alcohol, propyl alcohol, butanols, acetoneand ethyl acetate.The concentration of mixed solvent can be selected arbitrarily, and preferable is the aqueous solution of volume percent 10～95% organic solvents.What the consumption of mixed solvent was preferable in each the extraction is 2～20ml/1g plant material, and better is 3～6ml/1g plant material.What the temperature of solvent extraction was preferable is 10～100 ℃, and better is 10～85 ℃.What the number of times of solvent extraction was preferable is 2～5 times, and better is 3 times.What the time of each solvent extraction was preferable is 20 minutes～5 hours, and better is 30～100 minutes.When adopting the microwave-assisted solvent to extract, can adopt permanent power or homothermic microwave exposure mode.When adopting supercritical fluid extraction, that supercutical fluid is preferable is CO
2, entrainment agent is preferable is selected from methyl alcohol, ethanol and the acetone one or more, or does not use entrainment agent.
In the step of the solvent extraction of the present invention's one preferred embodiments, be raw material, extract 3 times under 10～85 ℃ the condition that each 20～100 minutes, extract solvent load was 3～6ml/1g seed of Fructus Hippophae at every turn with the seed of Fructus Hippophae.
After the solvent extraction, according to a conventional method, extracting solution is concentrated into concentrated solution, or the dry solid that gets, described crude extract promptly got.In the crude extract that is made by above-mentioned preferred solvent extracting method, oligomeric procyanidolics content accounts for mass ratio 〉=20% of this crude extract solid content, substantially all more than 35%.Above-mentioned preferred solvent extraction process, the yield of oligomeric procyanidolics content is about more than 85%.
(2) macroporous adsorbent resin column chromatography
Among the present invention, described macroporous adsorbent resin can be polarity or nonpolar macroporous adsorption resin.Grope the macroporous adsorbent resin of the preferred especially following type of the present invention: HP-20, HP-2MG, SP207, SP850, XAD16, XAD1600, XAD7HP, D-101, AB-8, MCI or HPD through a large amount of tests.Adopt above-mentioned preferred macroporous adsorbent resin, the purity of product and yield can obtain improving particularly significantly.What the fineness ratio of chromatography column was preferable is 1: 5～1: 45.Preferred chromatography column specification is φ 12 * 500mm or φ 50 * 500mm.The consumption of macroporous adsorbent resin is preferable is 30～120 times of last sample quality.
After step (1) finishes, with macroporous adsorptive resins on the crude extract of gained.Last quadrat method can be on the conventional wet method sample on sample or the dry method, sample on the preferred wet method.When adopting on the wet method sample, preferably with 0.1～5 times of column volume/hour flow velocity on sample advance post.Preferred 10～60 ℃ of last sample temperature.
Behind the last sample, use water as eluent and carry out wash-out, remove impurity such as deproteinize and polysaccharide.What the consumption of water was preferable is 1～8 times of column volume.The flow velocity of water wash-out is preferable be 1～5 times of column volume/hour.What the temperature of water wash-out was preferable is 10～60 ℃.
Afterwards, carry out gradient elution with one or more and water in the following organic solvent as eluent: ethanol, methyl alcohol and acetone.According to the polarity of selected macroporous adsorbent resin, in the concentration range of volume ratio 0% organic solvent (being water) and volume ratio 100% organic solvent, select gradient.What the consumption of the elutriant of each gradient was preferable is 5～20 column volumes.The flow velocity of wash-out is preferable be 1～5 times of column volume/hour.When selecting different eluents and gradient, the gradient scope of collecting is different, to collect with volume ratio is that upper solution behind 4: 1: 5 the mixing solutions standing demix of propyl carbinol, acetic acid and water is a developping agent, and the Rf value is that 0.2～0.5 wash-out partly is as the criterion on the silica gel thin-layer chromatography plate (TLC).What the temperature of gradient elution was preferable is 10～60 ℃.
In the present invention's one preferred embodiments, select the D101 macroporous adsorbent resin, gradient is followed successively by: 5% aqueous ethanolic solution, 40% aqueous ethanolic solution, straight alcohol, the wash-out part of collecting 40% aqueous ethanolic solution.
In another preferred embodiments of the present invention, select AB-8 type macroporous adsorbent resin, gradient is followed successively by: 40% aqueous acetone solution, pure acetone, the wash-out part of collecting 40% aqueous acetone solution.
In another preferred embodiments of the present invention, select the HP-20 macroporous adsorbent resin, gradient is followed successively by 20% aqueous ethanolic solution, 40% aqueous ethanolic solution, straight alcohol, the wash-out part of collecting 20% aqueous ethanolic solution, and the wash-out part of part 40% methanol aqueous solution.
In another preferred embodiments of the present invention, select the SP207 macroporous adsorbent resin, gradient is followed successively by 10% aqueous ethanolic solution, 50% aqueous ethanolic solution, and straight alcohol is collected the wash-out part of 10% methanol aqueous solution and the wash-out part of part 50% methyl alcohol.
In the above-mentioned preferred embodiments, per-cent is volume percent.
Behind the gradient elution,, or be dried to solid, promptly get oligomeric source cyanidin extract the dry simmer down to concentrated solution of collected wash-out part.Oligomeric procyanidolics content accounts for mass ratio 〉=50% of this extract solid content, substantially all more than 80%.The macroporous adsorbent resin column chromatography step, the yield of oligomeric procyanidolics content is about more than 80%.(1) it is about more than 60% that preferred method is produced the total recovery of crude extract and (2) in.
Behind (2) macroporous adsorbent resin column chromatography, preferable also carry out (3) cross gel column separation purification, with further raising product purity.
Among the present invention, what described gel column was preferable is that dextrane gel Sephadex G series (as G-10, G-15, G-25, G-50, G-75, G-100, G-150 or G-200), dextrane gel LH series are (as Sephadex LH-20,), dextrane gel DEAE series (concrete specification such as A25, subfamily such as A50 or FF), dextrane gel T series (concrete specification such as T3, T4, T5, T6, subfamily such as T7 or T9) or agar gel CL series (as agar gel CL-4B, or agar gel CL-6B series.The gel column of the above-mentioned type of all size all is suitable for the present invention, the preferred above-mentioned concrete specification of enumerating.What the fineness ratio of gel column was preferable is 1: 50～1: 150.
Eluent can be selected one or more in water, ethanol, methyl alcohol, acetone and the chloroform, and concentration proportioning can be any, and preferable is the aqueous solution of the organic solvent of volume ratio 10%～40%.What the eluent consumption was preferable is 80～120 times of column volumes.Collection is that the upper solution behind 4: 1: 5 the mixing solutions standing demix of propyl carbinol, acetic acid and water is a developping agent with volume ratio, and it is that 0.2～0.5 wash-out partly is as the criterion that silica gel thin-layer chromatography plate (TLC) is gone up the Rf value.
Gel column with the dry simmer down to concentrated solution of collected wash-out part, or is dried to solid after separating purification, promptly gets oligomeric source cyanidin extract.The mass ratio that oligomeric procyanidolics content accounts for this extract solid content not 〉=80%, substantially all more than 90%.Cross gel column and separate purification step, the yield of oligomeric procyanidolics content is about more than 90%.(1) preferred method is produced the total recovery of crude extract, (2) and (3) more than 60% in.
In the extracting method of the present invention, but the optimum condition arbitrary combination of above steps promptly obtains each preferred embodiments of the present invention.
Agents useful for same of the present invention and raw material are all commercially available to be got.
Positive progressive effect of the present invention is: compared with prior art, the purity and the yield of the oligomeric source cyanidin extract product that extracting method of the present invention makes all are significantly increased, through macroporous adsorbent resin column chromatography, the oligomeric procyanidolics yield can reach more than 80%, product purity 〉=50% is substantially all more than 80%.Even solvent extraction before the macroporous adsorbent resin column chromatography step adds and gel column purification step afterwards, three step total recoverys still can reach more than 60%, and product purity 〉=80% is substantially all more than 90%.And method technology of the present invention is easy, and cost is low, is easy to industrialization.
Embodiment
Further specify the present invention with embodiment below, but the present invention is not limited.
Among the following embodiment, the percentage of solution is volume percent.
Among the following embodiment, the oligomeric procyanidolics content detecting method is in each extract: its content of characteristic color reaction assay of utilization oligomeric proanthocyanidins and hydrochloric acid-propyl carbinol.Preparation contains the propyl carbinol liquid 50ml of volume ratio 30% hydrochloric acid, and is standby.Get oligomeric procyanidolics body reference liquid (purchasing) 1mg/ml 25ul, 50ul, 75ul, 100ul respectively in Tianjin spike company, 4mg/ml 50ul, add 1.4ml hydrochloric acid-butanol solution, adding water makes cumulative volume shake up to 1.5ml, in boiling water, boil 30min, cooling is the 525nm colorimetric at wavelength, the drawing standard curve.With HPLC-MS the extract of gained is carried out composition analysis, choose molecular weight and be lower than 1000 composition, be made into 1mg/ml, get 0.1ml and measure the 525nm optical density by the same method, reference standard curve calculation oligomeric procyanidolics content.
Oligomeric procyanidolics Determination on content method in the plant material is: after the plant material dissolving, be lower than 1000 composition with HPLC-MS isolated molecule amount, survey wherein oligomeric anthocyanidin content by above-mentioned colorimetry.
Among the following embodiment, total recovery is oligomeric procyanidolics content * 100% in oligomeric procyanidolics content/plant material in the product.
Embodiment 1
1) takes by weighing 250g seed of Fructus Hippophae (Qinghai, the place of production), add 60% aqueous ethanolic solution 600ml, stir and extract, extract temperature and be controlled at 40 ℃.For the first time extraction time is 60min, and for the second time and for the third time extraction time is respectively 20min, extracts altogether three times.The each extraction finished after-filtration, and merging filtrate concentrates and obtains the oligomeric procyanidolics concentrated solution.It is 57.6wt% that oligomeric procyanidolics content accounts for this concentrated solution solid content.This step, oligomeric procyanidolics yield are 92.7%.
2) chromatography column is of a size of φ 12 * 500mm, inner filling D101 macroporous adsorbent resin (going up 50 times of sample quality).In the following operation, temperature is controlled at 30 ℃.With the oligomeric procyanidolics concentrated solution with 0.5 times of bed volume/hour flow velocity feed in the chromatography column.First deionized water elution chromatography post after charging is finished with 4 times of column volumes, flow velocity be 2 times of bed volume/hour, carry out gradient elution successively with 5%, 40% aqueous ethanolic solution and straight alcohol respectively then, each gradient consumption is 5 times of column volumes, flow velocity be 2 times of bed volume/hour.Collect the wash-out part of 40% aqueous ethanolic solution, concentrate drying obtains the product that oligomeric procyanidolics content is 91.7wt%.This step, oligomeric procyanidolics yield are 86.0%.
3) column chromatography product dissolve with methanol, (φ 1 * 150cm) by Sephadex G-10 post, with 100 times of column volumes, 20% methanol in water wash-out, reference standards, collection is that the upper solution behind 4: 1: 5 the mixing solutions standing demix of propyl carbinol, acetic acid and water is a developping agent with volume ratio, the Rf value is 0.2～0.5 wash-out part on the silica gel thin-layer chromatography plate, and drying obtains the product that oligomeric procyanidolics content is 97.1wt%.This step, oligomeric procyanidolics yield are 98.6%.
Through solvent extraction, column chromatography and three steps of gel, the oligomeric procyanidolics total recovery is 78.5%.
Embodiment 2
1) take by weighing 250g seed of Fructus Hippophae (Qinghai, the place of production), add 70% methanol aqueous solution 800ml, refluxing extraction is extracted temperature and is controlled at 85 ℃.For the first time extraction time is 30min, and for the second time and for the third time extraction time is respectively 15min, extracts altogether three times.The each extraction finished after-filtration, and merging filtrate concentrates and obtains the oligomeric procyanidolics concentrated solution.It is 38.8wt% that oligomeric procyanidolics content accounts for this concentrated solution solid content.This step, the yield of oligomeric procyanidolics are 89.1%.
2) chromatography column is of a size of φ 12 * 500mm, inner filling AB-8 type macroporous adsorbent resin (going up 80 times of sample quality).In the following operation, temperature is controlled at 30 ℃.The oligomeric procyanidolics concentrated solution with 0.3 times of bed volume/hour flow velocity feed in the chromatography column.After charging is finished, earlier with 6 times of column volume deionized water elution chromatography posts, flow velocity be 5 times of bed volume/hour, respectively carry out gradient elution successively with 40% aqueous ethanolic solution and straight alcohol respectively then, the consumption of each gradient is 7 times of column volumes, flow velocity be 5 times of bed volume/hour.Collect the wash-out part of 40% aqueous ethanolic solution, concentrated, drying obtains the product that oligomeric procyanidolics content is 83.6wt%.This step, the yield of oligomeric procyanidolics are 83.7%.
3) column chromatography product dissolve with ethanol, (φ 1 * 150cm) by Sephadex G-25 post, 30% aqueous ethanolic solution wash-out with 80 times of column volumes, reference standards, collection is that the upper solution behind 4: 1: 5 the mixing solutions standing demix of propyl carbinol, acetic acid and water is a developping agent with volume ratio, the Rf value is 0.2～0.5 wash-out part on the silica gel thin-layer chromatography plate, and drying obtains the product that oligomeric procyanidolics content is 92.7wt%.This step, the yield of oligomeric procyanidolics are 94.2%.
Through solvent extraction, column chromatography and three steps of gel, the oligomeric procyanidolics total recovery is 70.3%.
Embodiment 3
1) take by weighing 250g seed of Fructus Hippophae (Qinghai, the place of production), add 70% acetone-water solution 1000ml, supersound extraction is extracted temperature and is controlled at 30 ℃.For the first time extraction time is 90min, and for the second time and for the third time extraction time is respectively 60min, extracts altogether three times.The each extraction finished after-filtration, and merging filtrate concentrates and obtains the oligomeric procyanidolics concentrated solution.It is 47.5wt% that oligomeric procyanidolics content accounts for this concentrated solution solid content.This step, the yield of oligomeric procyanidolics are 91.1%.
2) chromatography column is of a size of φ 50 * 500mm, inner filling HP-20 macroporous adsorbent resin (going up 100 times of sample quality).In the following operation, temperature is controlled at 25 ℃.The oligomeric procyanidolics concentrated solution with 0.5 times of bed volume/hour flow velocity feed in the chromatography column.It is first after charging is finished with 8 times of column volume deionized water elution chromatography posts, flow velocity be 1 times of bed volume/hour, carry out gradient elution successively with 20% aqueous ethanolic solution, 40% aqueous ethanolic solution and straight alcohol then, each gradient consumption is 10 times of column volumes, flow velocity be 1 times of bed volume/hour.(to collect with volume ratio is that upper solution behind 4: 1: 5 the mixing solutions standing demix of propyl carbinol, acetic acid and water is a developping agent to collect the part of the whole of 20% methanol-eluted fractions and 40% methanol-eluted fractions, the Rf value is that 0.2～0.5 wash-out partly is as the criterion on the silica gel thin-layer chromatography plate), concentrate, drying obtains the product that oligomeric procyanidolics content is 87.7wt%.This step, oligomeric procyanidolics yield are 81.8%.
3) column chromatography product dissolve with methanol, (φ 1 * 150cm) by Sephadex DEAE-A25 post, 20% methanol aqueous solution wash-out with 120 times of column volumes, reference standards, collection is that the upper solution behind 4: 1: 5 the mixing solutions standing demix of propyl carbinol, acetic acid and water is a developping agent with volume ratio, the Rf value is 0.2～0.5 wash-out part on the silica gel thin-layer chromatography plate, and drying obtains the product that oligomeric procyanidolics content is 90.6wt%.This step, oligomeric procyanidolics yield are 91.1%.
Through solvent extraction, column chromatography and three steps of gel, the oligomeric procyanidolics total recovery is 67.9%.
Embodiment 4
1) take by weighing 250g seed of Fructus Hippophae (Qinghai, the place of production), the supercritical flow that joins 1000mL is stopped in the extractor, makes supercutical fluid with CO2, flow 1.5L/min.When temperature is raised to 50 ℃, when reaching 15MPa, pressure picks up counting.Keep this condition operation 6 hours, stop then, from separating tank, isolate the supercritical fluid extraction thing.Extracting solution obtains the solvent crude extract after removing solvent, oligomeric procyanidolics content is 45.2wt% in the extract.This step, oligomeric procyanidolics yield are 87.3%.
2) chromatography column is of a size of φ 50 * 500mm, inner filling SP207 macroporous adsorbent resin (going up 120 times of sample quality).In the following operation, temperature is controlled at 45 ℃.Use the dissolve with methanol crude extract, with 0.5 times of bed volume/hour flow velocity feed in the chromatography column.It is first after charging is finished with 5 times of column volume deionized water elution chromatography posts, flow velocity be 1 times of bed volume/hour, use 10% aqueous ethanolic solution, 50% aqueous ethanolic solution and straight alcohol then, carry out gradient elution successively, each gradient consumption is 8 times of column volumes, flow velocity be 1 times of bed volume/hour.(to collect with volume ratio is that upper solution behind 4: 1: 5 the mixing solutions standing demix of propyl carbinol, acetic acid and water is a developping agent to collect the part of the whole of 10% methanol-eluted fractions and 50% methanol-eluted fractions, the Rf value is that 0.2～0.5 wash-out partly is as the criterion on the silica gel thin-layer chromatography plate), concentrate, drying obtains the product that oligomeric procyanidolics content is 84.1wt%.This step, oligomeric procyanidolics yield are 85.5%.
3) column chromatography product dissolve with methanol, (φ 1 * 150cm) by Sephadex CL-4B post, 20% methanol aqueous solution wash-out with 110 times of column volumes, reference standards, collection is that the upper solution behind 4: 1: 5 the mixing solutions standing demix of propyl carbinol, acetic acid and water is a developping agent with volume ratio, the Rf value is 0.2～0.5 wash-out part on the silica gel thin-layer chromatography plate, and drying obtains the product that oligomeric procyanidolics content is 92.2wt%.This step, oligomeric procyanidolics yield are 90.6%.
Through solvent extraction, column chromatography and three steps of gel, the oligomeric procyanidolics total recovery is 67.6%.
Embodiment 5
1) takes by weighing the 250g Semen Vitis viniferae, add propyl alcohol-aqueous solution 2500ml of 95%, stir and extract, extract temperature and be controlled at 10 ℃.For the first time extraction time is 5 hours, and for the second time and for the third time extraction time was respectively 3 hours, and the 4th time and the 5th extraction time were respectively 2 hours, extracted altogether 5 times.The each extraction finished after-filtration, and merging filtrate concentrates and obtains the oligomeric procyanidolics concentrated solution.It is 51.3wt% that oligomeric procyanidolics content accounts for this concentrated solution solid content.This step, the yield of oligomeric procyanidolics are 89.5%.
2) chromatography column is of a size of φ 55 * 300mm, inner filling HP-2MG macroporous adsorbent resin (going up 60 times of sample quality).In the following operation, temperature is controlled at 25 ℃.The oligomeric procyanidolics concentrated solution with 5 times of bed volume/hour flow velocity feed in the chromatography column.It is first after charging is finished with 8 times of column volume deionized water elution chromatography posts, flow velocity be 1 times of bed volume/hour, carry out gradient elution successively with 15% aqueous ethanolic solution, 40% aqueous ethanolic solution and straight alcohol then, each gradient consumption is 10 times of column volumes, flow velocity be 1 times of bed volume/hour.(to collect with volume ratio is that upper solution behind 4: 1: 5 the mixing solutions standing demix of propyl carbinol, acetic acid and water is a developping agent to collect the part of the whole of 20% methanol-eluted fractions and 40% methanol-eluted fractions, the Rf value is that 0.2～0.5 wash-out partly is as the criterion on the silica gel thin-layer chromatography plate), concentrate, drying obtains the product that oligomeric procyanidolics content is 85.3wt%.This step, oligomeric procyanidolics yield are 82.1%.
3) column chromatography product dissolve with methanol, (φ 1 * 50cm) by Sephadex G-200 post, water elution with 100 times of column volumes, reference standards, collection is that the upper solution behind 4: 1: 5 the mixing solutions standing demix of propyl carbinol, acetic acid and water is a developping agent with volume ratio, the Rf value is 0.2～0.5 wash-out part on the silica gel thin-layer chromatography plate, and drying obtains the product that oligomeric procyanidolics content is 92.5wt%.This step, oligomeric procyanidolics yield are 93.1%.
Through solvent extraction, column chromatography and three steps of gel, the oligomeric procyanidolics total recovery is 68.4%.
Embodiment 6
1) take by weighing the 250g Cortex Pini, add butanols-aqueous solution 5000ml of 10%, the constant temperature microwave extraction is extracted temperature and is controlled at 100 ℃.Extraction time is 20min for the first time, and extraction time is 20min for the second time, extracts altogether 2 times.The each extraction finished after-filtration, and merging filtrate concentrates and obtains the oligomeric procyanidolics concentrated solution.It is 52.1wt% that oligomeric procyanidolics content accounts for this concentrated solution solid content.This step, the yield of oligomeric procyanidolics are 92.5%.
2) chromatography column is of a size of φ 16 * 700mm, inner filling SP850 macroporous adsorbent resin (going up 50 times of sample quality).In the following operation, temperature is controlled at 25 ℃.The oligomeric procyanidolics concentrated solution with 0.1 times of bed volume/hour flow velocity feed in the chromatography column.It is first after charging is finished with 7 times of column volume deionized water elution chromatography posts, flow velocity be 2 times of bed volume/hour, carry out gradient elution successively with 10% aqueous ethanolic solution, 30% aqueous ethanolic solution and 50% ethanol then, each gradient consumption is 5 times of column volumes, flow velocity be 2 times of bed volume/hour.Collecting with volume ratio is that upper solution behind 4: 1: 5 the mixing solutions standing demix of propyl carbinol, acetic acid and water is a developping agent, the Rf value is 0.2～0.5 wash-out part on the silica gel thin-layer chromatography plate, and concentrated, drying obtains the product that oligomeric procyanidolics content is 82.1wt%.This step, oligomeric procyanidolics yield are 90.3%.
3) column chromatography product dissolve with methanol, (φ 1 * 100cm) by agar gel CL-6B post, methanol-eluted fractions with 120 times of column volumes, reference standards, collection is that the upper solution behind 4: 1: 5 the mixing solutions standing demix of propyl carbinol, acetic acid and water is a developping agent with volume ratio, the Rf value is 0.2～0.5 wash-out part on the silica gel thin-layer chromatography plate, and drying obtains the product that oligomeric procyanidolics content is 93.5wt%.This step, oligomeric procyanidolics yield are 89.5%.
Through solvent extraction, column chromatography and three steps of gel, the oligomeric procyanidolics total recovery is 74.8%.
Embodiment 7
1) take by weighing seed of Fructus Hippophae dregs after the 250g sea-buckthorn has been extracted Oleum Hippophae, add ethyl acetate-aqueous solution 1000ml of 50%, the constant temperature microwave extraction is controlled at 50 ℃.For the first time extraction time is 40min, and for the second time and for the third time extraction time is respectively 30min, extracts altogether 3 times.The each extraction finished after-filtration, and merging filtrate concentrates and obtains the oligomeric procyanidolics concentrated solution.It is 53.5wt% that oligomeric procyanidolics content accounts for this concentrated solution solid content.This step, the yield of oligomeric procyanidolics are 90.5%.
2) chromatography column is of a size of φ 50 * 500mm, inner filling XAD16 macroporous adsorbent resin (going up 80 times of sample quality).In the following operation, temperature is controlled at 60 ℃.The oligomeric procyanidolics concentrated solution with 2 times of bed volume/hour flow velocity feed in the chromatography column.It is first after charging is finished with 8 times of column volume deionized water elution chromatography posts, flow velocity be 4 times of bed volume/hour, carry out gradient elution successively with 15% aqueous ethanolic solution, 35% aqueous ethanolic solution and 55% ethanol then, each gradient consumption is 20 times of column volumes, flow velocity be 1 times of bed volume/hour.Collecting with volume ratio is that upper solution behind 4: 1: 5 the mixing solutions standing demix of propyl carbinol, acetic acid and water is a developping agent, the Rf value is 0.2～0.5 wash-out part on the silica gel thin-layer chromatography plate, and concentrated, drying obtains the product that oligomeric procyanidolics content is 85.5wt%.This step, oligomeric procyanidolics yield are 79.5%.
3) column chromatography product dissolve with methanol, (φ 1 * 150cm) by dextran T6 post, ethanol elution with 120 times of column volumes, reference standards, collection is that the upper solution behind 4: 1: 5 the mixing solutions standing demix of propyl carbinol, acetic acid and water is a developping agent with volume ratio, the Rf value is 0.2～0.5 wash-out part on the silica gel thin-layer chromatography plate, and drying obtains the product that oligomeric procyanidolics content is 95.5wt%.This step, oligomeric procyanidolics yield are 89.5%.
Through solvent extraction, column chromatography and three steps of gel, the oligomeric procyanidolics total recovery is 64.4%.
Embodiment 8
1) take by weighing seed of Fructus Hippophae dregs after the 250g sea-buckthorn has been extracted Oleum Hippophae, add ethyl acetate-aqueous solution 1000ml of 50%, the constant temperature microwave extraction is controlled at 50 ℃.For the first time extraction time is 40min, and for the second time and for the third time extraction time is respectively 30min, extracts altogether 3 times.The each extraction finished after-filtration, and merging filtrate concentrates and obtains the oligomeric procyanidolics concentrated solution.It is 45.5wt% that oligomeric procyanidolics content accounts for this concentrated solution solid content.This step, the yield of oligomeric procyanidolics are 92.1%.
2) chromatography column is of a size of φ 16 * 300mm, inner filling XAD1600 macroporous adsorbent resin (going up 30 times of sample quality).In the following operation, temperature is controlled at 25 ℃.The oligomeric procyanidolics concentrated solution with 4 times of bed volume/hour flow velocity feed in the chromatography column.It is first after charging is finished with 4 times of column volume deionized water elution chromatography posts, flow velocity be 3 times of bed volume/hour, carry out gradient elution successively with 10% aqueous ethanolic solution, 60% aqueous ethanolic solution and straight alcohol then, each gradient consumption is 10 times of column volumes, flow velocity be 5 times of bed volume/hour.Collecting with volume ratio is that upper solution behind 4: 1: 5 the mixing solutions standing demix of propyl carbinol, acetic acid and water is a developping agent, the Rf value is 0.2～0.5 wash-out part on the silica gel thin-layer chromatography plate, and concentrated, drying obtains the product that oligomeric procyanidolics content is 83.2wt%.This step, oligomeric procyanidolics yield are 84.5%.
3) column chromatography product dissolve with methanol, (φ 1 * 150cm) by agar gel CL-4B post, acetone wash-out with 120 times of column volumes, reference standards, collection is that the upper solution behind 4: 1: 5 the mixing solutions standing demix of propyl carbinol, acetic acid and water is a developping agent with volume ratio, the Rf value is 0.2～0.5 wash-out part on the silica gel thin-layer chromatography plate, and drying obtains the product that oligomeric procyanidolics content is 90.5wt%.This step, oligomeric procyanidolics yield are 88.5%.
Through solvent extraction, column chromatography and three steps of gel, the oligomeric procyanidolics total recovery is 68.8%.
Embodiment 9
1) take by weighing seed of Fructus Hippophae dregs after the 250g sea-buckthorn has been extracted Oleum Hippophae, add ethyl acetate-aqueous solution 1000ml of 50%, the constant temperature microwave extraction is controlled at 50 ℃.For the first time extraction time is 40min, and for the second time and for the third time extraction time is respectively 30min, extracts altogether 3 times.The each extraction finished after-filtration, and merging filtrate concentrates and obtains the oligomeric procyanidolics concentrated solution.It is 48.4wt% that oligomeric procyanidolics content accounts for this concentrated solution solid content.This step, the yield of oligomeric procyanidolics are 89.5%.
2) chromatography column is of a size of φ 50 * 500mm, inner filling XAD7HP macroporous adsorbent resin (going up 100 times of sample quality).In the following operation, temperature is controlled at 25 ℃.The oligomeric procyanidolics concentrated solution with 0.5 times of bed volume/hour flow velocity feed in the chromatography column.It is first after charging is finished with 3 times of column volume deionized water elution chromatography posts, flow velocity be 4 times of bed volume/hour, carry out gradient elution successively with 10% aqueous ethanolic solution, 30% aqueous ethanolic solution, 50% aqueous ethanolic solution straight alcohol then, each gradient consumption is 10 times of column volumes, flow velocity be 1 times of bed volume/hour.Collecting with volume ratio is that upper solution behind 4: 1: 5 the mixing solutions standing demix of propyl carbinol, acetic acid and water is a developping agent, the Rf value is 0.2～0.5 wash-out part on the silica gel thin-layer chromatography plate, and concentrated, drying obtains the product that oligomeric procyanidolics content is 88.1wt%.This step, oligomeric procyanidolics yield are 82.5%.
3) column chromatography product dissolve with methanol, (φ 1 * 150cm) by Sephadex LH-20 post, chloroform wash-out with 120 times of column volumes, reference standards, collection is that the upper solution behind 4: 1: 5 the mixing solutions standing demix of propyl carbinol, acetic acid and water is a developping agent with volume ratio, the Rf value is 0.2～0.5 wash-out part on the silica gel thin-layer chromatography plate, and drying obtains the product that oligomeric procyanidolics content is 94.5wt%.This step, oligomeric procyanidolics yield are 88.5%.
Through solvent extraction, column chromatography and three steps of gel, the oligomeric procyanidolics total recovery is 65.3%.
Embodiment 10
1) take by weighing seed of Fructus Hippophae dregs after the 250g sea-buckthorn has been extracted Oleum Hippophae, add ethyl acetate-aqueous solution 1000ml of 50%, the constant temperature microwave extraction is controlled at 50 ℃.For the first time extraction time is 40min, and for the second time and for the third time extraction time is respectively 30min, extracts altogether 3 times.The each extraction finished after-filtration, and merging filtrate concentrates and obtains the oligomeric procyanidolics concentrated solution.It is 51.7wt% that oligomeric procyanidolics content accounts for this concentrated solution solid content.This step, the yield of oligomeric procyanidolics are 87.5%.
2) chromatography column is of a size of φ 50 * 500mm, inner filling MCI macroporous adsorbent resin (going up 90 times of sample quality).In the following operation, temperature is controlled at 25 ℃.The oligomeric procyanidolics concentrated solution with 0.5 times of bed volume/hour flow velocity feed in the chromatography column.It is first after charging is finished with 5 times of column volume deionized water elution chromatography posts, flow velocity be 2 times of bed volume/hour, carry out gradient elution successively with 30% aqueous ethanolic solution, 40% aqueous ethanolic solution and 50% ethanol then, each gradient consumption is 10 times of column volumes, flow velocity be 1 times of bed volume/hour.Collecting with volume ratio is that upper solution behind 4: 1: 5 the mixing solutions standing demix of propyl carbinol, acetic acid and water is a developping agent, the Rf value is 0.2～0.5 wash-out part on the silica gel thin-layer chromatography plate, and concentrated, drying obtains the product that oligomeric procyanidolics content is 83.5wt%.This step, oligomeric procyanidolics yield are 86.5%.
3) column chromatography product dissolve with methanol, (φ 1 * 150cm) by Sephadex DEAE-A50 post, 40% methanol aqueous solution wash-out with 120 times of column volumes, reference standards, collection is that the upper solution behind 4: 1: 5 the mixing solutions standing demix of propyl carbinol, acetic acid and water is a developping agent with volume ratio, the Rf value is 0.2～0.5 wash-out part on the silica gel thin-layer chromatography plate, and drying obtains the product that oligomeric procyanidolics content is 91.5wt%.This step, oligomeric procyanidolics yield are 92.9%.
Through solvent extraction, column chromatography and three steps of gel, the oligomeric procyanidolics total recovery is 70.3%.
Embodiment 11
1) take by weighing seed of Fructus Hippophae dregs after the 250g sea-buckthorn has been extracted Oleum Hippophae, add ethyl acetate-aqueous solution 1000ml of 50%, the constant temperature microwave extraction is controlled at 50 ℃.For the first time extraction time is 40min, and for the second time and for the third time extraction time is respectively 30min, extracts altogether 3 times.The each extraction finished after-filtration, and merging filtrate concentrates and obtains the oligomeric procyanidolics concentrated solution.It is 50.3wt% that oligomeric procyanidolics content accounts for this concentrated solution solid content.This step, the yield of oligomeric procyanidolics are 92.4%.
2) chromatography column is of a size of φ 50 * 500mm, inner filling HPD macroporous adsorbent resin (going up 90 times of sample quality).In the following operation, temperature is controlled at 10 ℃.The oligomeric procyanidolics concentrated solution with 0.5 times of bed volume/hour flow velocity feed in the chromatography column.It is first after charging is finished with 1 times of column volume deionized water elution chromatography post, flow velocity be 1 times of bed volume/hour, carry out gradient elution successively with 10% aqueous ethanolic solution, 40% aqueous ethanolic solution and 70% ethanol then, each gradient consumption is 10 times of column volumes, flow velocity be 1 times of bed volume/hour.Collecting with volume ratio is that upper solution behind 4: 1: 5 the mixing solutions standing demix of propyl carbinol, acetic acid and water is a developping agent, the Rf value is 0.2～0.5 wash-out part on the silica gel thin-layer chromatography plate, and concentrated, drying obtains the product that oligomeric procyanidolics content is 84.3wt%.This step, oligomeric procyanidolics yield are 80.5%.
3) column chromatography product dissolve with methanol, (φ 1 * 150cm) by Sephadex DEAE-A25 post, 10% methanol aqueous solution wash-out with 120 times of column volumes, reference standards, collection is that the upper solution behind 4: 1: 5 the mixing solutions standing demix of propyl carbinol, acetic acid and water is a developping agent with volume ratio, the Rf value is 0.2～0.5 wash-out part on the silica gel thin-layer chromatography plate, and drying obtains the product that oligomeric procyanidolics content is 92.1wt%.This step, oligomeric procyanidolics yield are 87.5%.
Through solvent extraction, column chromatography and three steps of gel, the oligomeric procyanidolics total recovery is 65.0%.
Claims (15)
1. method of extracting oligomeric procyanidolics, it is characterized in that its plant material that comprises the steps: to contain oligomeric procyanidolics is with macroporous adsorptive resins on the crude extract of solvent extraction gained, wash with water and remove deproteinize and polyose impurity, carry out gradient elution with one or more and water in the following organic solvent as eluent more afterwards: ethanol, methyl alcohol and acetone, collection is 4: 1: 5 propyl carbinol with volume ratio, upper solution behind the mixing solutions standing demix of acetic acid and water is a developping agent, the Rf value is 0.2～0.5 wash-out part on the silica gel thin-layer chromatography plate, promptly gets the oligomeric procyanidolics extract.
2. the method for claim 1, it is characterized in that: the described plant material that contains oligomeric procyanidolics is Cortex Pini, Semen Vitis viniferae or seed of Fructus Hippophae.
3. method as claimed in claim 2 is characterized in that: described seed of Fructus Hippophae is the seed of Fructus Hippophae dregs after sea-buckthorn has been extracted Oleum Hippophae.
4. the method for claim 1 is characterized in that: the mode of described solvent extraction is that conventional solvent extraction, microwave-assisted solvent extract, ultrasonic solvent extraction or supercritical fluid extraction.
5. as claim 1 or 4 described methods, it is characterized in that:
In the described solvent extraction, extract solvent and be the mixed solvent of one or more and water in the following organic solvent: ethanol, methyl alcohol, propyl alcohol, butanols, acetoneand ethyl acetate; The concentration of mixed solvent is the aqueous solution of volume percent 10～95% organic solvents; In each the extraction, the consumption of mixed solvent is 2～20ml/1g plant material; The temperature of solvent extraction is 10～100 ℃; The number of times of solvent extraction is 2～5 times; The time of each solvent extraction is 20 minutes～5 hours;
When adopting the microwave-assisted solvent to extract, adopt permanent power or homothermic microwave exposure mode;
When adopting supercritical fluid extraction, supercutical fluid is CO
2, entrainment agent is selected from one or more in methyl alcohol, ethanol and the acetone, or does not use entrainment agent.
6. the method for claim 1, it is characterized in that: described macroporous adsorbent resin is the macroporous adsorbent resin of following type: HP-20, HP-2MG, SP207, SP850, XAD16, XAD1600, XAD7HP, D-101, AB-8, MCI or HPD.
7. the method for claim 1, it is characterized in that: the fineness ratio of described macroporous adsorbent resin is 1: 5～1: 45; The consumption of macroporous adsorbent resin is 30～120 times of last sample quality.
8. the method for claim 1, it is characterized in that: with the last quadrat method of macroporous adsorptive resins on the crude extract is sample on the wet method, with 0.1～5 times of column volume/hour flow velocity on sample advance post, last sample temperature is 10～60 ℃.
9. the method for claim 1, it is characterized in that: described washing with water in the step that removes deproteinize and polyose impurity, the consumption of water is 1～8 times of column volume, the flow velocity of water wash-out be 1～5 times of column volume/hour, the temperature of water wash-out is 10～60 ℃.
10. the method for claim 1, it is characterized in that: the gradient of described gradient elution is selected in the concentration range of volume ratio 0% organic solvent and volume ratio 100% organic solvent, the consumption of the elutriant of each gradient is 5～20 column volumes, the flow velocity of wash-out be 1～5 times of column volume/hour, the temperature of gradient elution is 10～60 ℃.
11. the method for claim 1 is characterized in that:
Described macroporous adsorptive resins is the D101 macroporous adsorbent resin, and in the described gradient elution, gradient is followed successively by: 5% aqueous ethanolic solution, 40% aqueous ethanolic solution, straight alcohol, the wash-out part of collecting 40% aqueous ethanolic solution;
Perhaps, described macroporous adsorptive resins is an AB-8 type macroporous adsorbent resin, and in the described gradient elution, gradient is followed successively by: 40% aqueous acetone solution, pure acetone, the wash-out part of collecting 40% aqueous acetone solution;
Perhaps, described macroporous adsorptive resins is the HP-20 macroporous adsorbent resin, in the described gradient elution, gradient is followed successively by 20% methanol aqueous solution, 40% methanol aqueous solution, pure methyl alcohol, collect the wash-out part of 20% methanol aqueous solution, and the wash-out part of part 40% methanol aqueous solution;
Perhaps, described macroporous adsorptive resins is the SP207 macroporous adsorbent resin, and in the described gradient elution, gradient is followed successively by 10% aqueous ethanolic solution, 50% aqueous ethanolic solution, straight alcohol is collected the wash-out part of 10% methanol aqueous solution and the wash-out part of part 50% methyl alcohol;
Per-cent is volume percent.
12. the method for claim 1, it is characterized in that: the prepared oligomeric procyanidolics extract of the method for claim 1 is carried out gel column again separate purification, with in water, ethanol, methyl alcohol, acetone and the chloroform one or more is eluent, collection is that the upper solution behind 4: 1: 5 the mixing solutions standing demix of propyl carbinol, acetic acid and water is a developping agent with volume ratio, the Rf value is 0.2～0.5 wash-out part on the silica gel thin-layer chromatography plate, promptly gets the oligomeric procyanidolics extract after further purifying.
13. method as claimed in claim 12 is characterized in that: described gel column is sephadex G series, dextrane gel LH series, dextrane gel DEAE series, dextrane gel T series or agar gel CL series.
14. method as claimed in claim 12 is characterized in that: the fineness ratio of described gel column is 1: 50～1: 150.
15. method as claimed in claim 12 is characterized in that: described eluent is that concentration proportioning is the aqueous solution of the organic solvent of volume ratio 10%～40%, and the eluent consumption is 80～120 times of column volumes.
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| CN109731030A (en) * | 2019-03-18 | 2019-05-10 | 泉州师范学院 | A kind of preparation method of Bauhinia blakeana flower procyanidine |
| CN109820924A (en) * | 2019-03-20 | 2019-05-31 | 汤臣倍健股份有限公司 | A kind of preparation method of the separation and Extraction effective component from grape pip |
| CN114031590A (en) * | 2021-11-17 | 2022-02-11 | 中北大学 | Method for efficiently preparing high-purity oligomeric proanthocyanidins from sea buckthorn seed meal |
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