CN103910706B - A kind of preparation method of oligomeric procyanidolics of low cost - Google Patents
A kind of preparation method of oligomeric procyanidolics of low cost Download PDFInfo
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Abstract
The invention discloses a kind of preparation method of oligomeric procyanidolics.The method utilizes the solubleness of the pycnogenols of different polymerization degree and the difference of molecular size, adopts the technology such as cold cut, filtration, nanofiltration, spraying dry, obtains the oligomeric procyanidolics that the polymerization degree is 2 ~ 3.Present method simplifies processing step, shortens the production cycle, and avoids a large amount of uses of ethanol and resin, to the input of equipment and the operational requirement of workman all lower, energy-conserving and environment-protective, are suitable for suitability for industrialized production, simplifying the operation, reducing energy consumption, on particularly cost-saving, there is significant contribution.In addition, the pycnogenols high polymer that prior art is separated by present method " is turned waste into wealth ", makes it to be hydrolyzed into pycnogenols oligomer, increases the content of raw material procyanidins oligomer, improves raw material availability, has positive realistic meaning to cost-saving.
Description
Technical field
The present invention relates to a kind of preparation method of oligomeric procyanidolics of low cost, belong to technical field of medical chemistry.
Technical background
Pycnogenols majority commercially available is at present the mixture of the pycnogenols of different polymerization degree, documents and materials show, oligomeric procyanidolics polymer (particularly two, tripolymer) is better than monomer whose in reduction serum cholesterol effect, at anti-oxidant activity and intestinal absorption ability is better than monomer and height gathers pycnogenols polymer, therefore develop the focus that the higher oligomeric procyanidolics of purity is research at present.
The preparation method of the oligomeric procyanidolics that existing bibliographical information is more is resin absorption partition method, such as, publication number be the Chinese patent application " a kind of preparation method of radish procyanidin dimers " of CN103145680A with commercially available radish red pigment for raw material, through dissolving and filtrations, nanofiltration, upper prop and wash-out and lyophilize and obtain radish procyanidin dimers lyophilized powder.Its concrete preparation process is: radish red pigment is dissolved in the ethanol-isopropanol-water mixing solutions of certain polarity by (1), vacuum filtration, collect filtered solution, this filtered solution is radish pycnogenols oligomer solution, main containing radish pycnogenols list aggressiveness, dimer, tripolymer, the tetramer and pentamer, filter residue is radish pycnogenols high polymer, thus achieves the initial gross separation of radish pycnogenols oligomer and high polymer; (2) molecular weight cut-off is used to be 800 ~ 1200 daltonian nanofiltration devices, radish pycnogenols oligomer solution is carried out nanofiltration separation, collect nanofiltration filtered solution, this nanofiltration filtered solution is mainly containing radish pycnogenols list aggressiveness, dimer, be radish pycnogenols oligomer nanofiltration liquid, and nanofiltration trapped fluid is mainly containing radish pycnogenols tripolymer, the tetramer and pentamer; (3) radish pycnogenols oligomer nanofiltration liquid is separated through polyamide resin chromatography post wash-out, obtains radish procyanidin dimers solution; (4) by the lyophilize of radish procyanidin dimers solution, thus radish procyanidin dimers powder is prepared.
Although above-mentioned preparation method of the prior art can obtain the higher oligomeric procyanidolics of purity, its shortcoming still existed is:
1, above-mentioned technology is when initial gross separation pycnogenols oligomer and high polymer, use the ethanol-isopropanol-water mixing solutions determining polarity, not only need with an organic solvent, and the comparison of ingredients being separated the pycnogenols oligomer solution obtained is complicated, organic solvent-resistant film must be used to be separated, and organic solvent-resistant film is expensive at present, apply also immature.2, above-mentioned technology utilizes the method for polyamide resin chromatography post wash-out to realize procyanidin monomers to be separated with dimeric, need to use ethanol and resin in a large number in elution process, and the production cycle is longer, to the input of equipment and the operational requirement of workman all higher, thus make that the energy consumption of whole technique is large, cost is high, both dangerous also not environmentally, and the dimer yield using the method to prepare is also lower.
Summary of the invention
What the present invention solved is that in prior art, the preparation technology of oligomeric procyanidolics needs to use a large amount of organic solvents and resin, cost higher and production cycle length, complicated operation, the problem that product yield is low, so a kind of simple to operate, with short production cycle, energy-conserving and environment-protective be provided, be suitable for suitability for industrialized production, the preparation method of the oligomeric procyanidolics that with low cost, yield is higher.
The technical scheme that the present invention solves the problems of the technologies described above is:
A preparation method for oligomeric procyanidolics, comprises the steps:
(1) redissolved by the cold water of pycnogenols crude extract with 0 ~ 20 DEG C, obtain pycnogenols suspension liquid, the mass ratio of wherein said pycnogenols crude extract and cold water is 1:5 ~ 1:7;
(2) described pycnogenols suspension liquid is filtered, collect filter cake and filtrate respectively;
(3) be that 900 daltonian nanofiltration membrane carry out nanofiltration separation to described filtrate with molecular weight cut-off, collect trapped fluid;
(4) described trapped fluid is carried out spraying dry, namely prepare oligomeric procyanidolics powder, the polymerization degree of wherein said oligomeric procyanidolics is 2 ~ 3.
In step (1), the temperature of described cold water is 4 ~ 10 DEG C.
In step (1), the purity of described pycnogenols crude extract is more than 70%.
In step (3), working pressure during nanofiltration separation is 1 ~ 2bar.
In step (3), the volume of described trapped fluid is 30 ~ 50% of filtrate original volume.
When carrying out described spraying dry in step (4), the inlet temperature of the spray-drier of use is 140 ~ 160 DEG C, and temperature out is 70 ~ 80 DEG C.
Also comprise: in step (2), described filter cake is dissolved in aqueous ethanolic solution, add the sodium sulfite solution that mass concentration is 4 ~ 6%, stirring reaction 0.5 ~ 1.5 hour at 70 ~ 80 DEG C, reaction solution is concentrated into 70% of the original volume of described reaction solution, the described filtrate that the reaction solution after concentrated and step (2) prepare is merged the nanofiltration separation for step (3).
In described aqueous ethanolic solution, the volumn concentration of ethanol is 5 ~ 15%.
The volume ratio of described sodium sulfite solution and aqueous ethanolic solution is 7:1 ~ 10:1.
The preparation method of oligomeric procyanidolics of the present invention, the cold water of pycnogenols crude extract with 0 ~ 20 DEG C redissolves by step (1), and obtain pycnogenols suspension liquid, the mass ratio also limiting described pycnogenols crude extract and cold water is 1:5 ~ 1:7; And the temperature of further preferred described cold water is 4 ~ 10 DEG C, the mass ratio of described pycnogenols crude extract and cold water is 1:5 ~ 1:7.The present invention is by limiting the temperature of described cold water and consumption, and in the pycnogenols that cold-water solution is obtained, the content of high polymer is few, thus can with follow-up nanofiltration technique with the use of and nanofiltration membrane can't be caused to block.Therefore invention takes the simple process of cold-water solution, natural filtration just to achieve procyanidin monomers, oligomer is separated with high polymer.In addition, the mass percentage of the preferred described pycnogenols crude extract procyanidins of the present invention is more than 70%, therefore the preparation method of oligomeric procyanidolics of the present invention is not only applicable to the refining of commercially available pycnogenols crude extract, is also applicable to the refining of the pycnogenols crude extract directly extracted from plant.
Step (2) is by carrying out natural filtration to described pycnogenols suspension liquid, collect filter cake and filtrate respectively, wherein filtrate is the mixture of procyanidin monomers and oligomer, and filter cake is high poly-pycnogenols, achieves the good separation of procyanidin monomers, oligomer and high polymer.Relative to the ethanol-isopropanol-water mixing solutions determining polarity used in the prior art, this separate mode of cold-water solution of the present invention, natural filtration avoids the use of organic solvent, and the comparison of ingredients being separated the pycnogenols oligomer solution obtained is simple, the polymerization degree of its procyanidins oligomer is 2 ~ 3.
In addition as preferred, preparation method of the present invention can also comprise following operation in step (2): be dissolved in aqueous ethanolic solution by described filter cake, add the sodium sulfite solution of 4 ~ 6%, stirring reaction 0.5 ~ 1.5 hour at 70 ~ 80 DEG C, reaction solution is concentrated into 70% of the original volume of described reaction solution, by the reaction solution after concentrated and the nanofiltration separation being used for step (3) merging of described filtrate.The present invention carries out separately said hydrolyzed reaction again reason after being first separated by pycnogenols high polymer is: 1. this hydrolysis reaction optionally can not only be hydrolyzed pycnogenols high polymer and not be hydrolyzed oligomer, if first pycnogenols high polymer is not separated, so in hydrolysis reaction, pycnogenols oligomer also can be hydrolyzed into monomer, causes the yield seriously reducing pycnogenols oligomer; 2. be hydrolyzed pycnogenols high polymer separately, then the charging capacity of hydrolysis reaction reduces, and " is turned waste into wealth " by pycnogenols high polymer, improves raw material availability, has saved cost.Because the pycnogenols high polymer solubility property being in ethanol insoluble in cold water is good, but the excessive concentration of ethanol or the too low product of hydrolysis reaction that all can make uneven, polymerization degree deviation is large, the solubility property fluctuation of product and effect is caused to decline, therefore in the preferred described aqueous ethanolic solution of the present invention, the volumn concentration of ethanol is 5 ~ 15%, and the volume ratio of described sodium sulfite solution and aqueous ethanolic solution is 7:1 ~ 10:1.
Described filtrate is carried out nanofiltration separation by step (3), and collect trapped fluid, the main component of this trapped fluid is pycnogenols oligomer, and permeate is mainly containing procyanidin monomers; When nanofiltration, the molecular weight cut-off of preferred nanofiltration membrane is 900 dalton, working pressure is 1 ~ 2bar, compared with the technique of upper prop wash-out after the first nanofiltration of prior art, the present invention makes full use of the molecular size difference of the pycnogenols of different polymerization degree, nanofiltration is only adopted namely to realize being separated of procyanidin monomers and oligomer, avoid a large amount of uses of ethanol and resin, energy-conserving and environment-protective, and with short production cycle, to the input of equipment and the operational requirement of workman all lower, with low cost, be suitable for suitability for industrialized production.For the ease of trapped fluid is carried out follow-up spray drying treatment, same as preferred, the volume of trapped fluid of the present invention is 30 ~ 50% of filtrate original volume.
Described trapped fluid is carried out spray drying treatment by step (4), namely prepares oligomeric procyanidolics powder, and the polymerization degree of wherein said oligomeric procyanidolics is 2 ~ 3; The preferred described spray-dired condition of the present invention is inlet temperature 140 ~ 160 DEG C, temperature out 70 ~ 80 DEG C, feed liquid proportion 1.1 ~ 1.2, peristaltic pump pump speed 33 ~ 44%, air flow quantity 30 ~ 40mm, pumping speed 100%.Under this spray drying condition, the waste of oligomeric procyanidolics is less than 5% of total amount.
In sum, compared with prior art, preparation method's tool of oligomeric procyanidolics of the present invention has the following advantages:
(1) preparation method of oligomeric procyanidolics of the present invention, makes full use of the dissolubility difference of the pycnogenols of different polymerization degree, adopts the technique of cold-water solution, natural filtration, achieves the good separation of procyanidin monomers, oligomer and high polymer.In prior art, those skilled in the art adopt alcohol-water solution etc. to carry out initial gross separation containing organic solvent to pycnogenols, and do not consider water solvent, reason is compared to organic solution, the difference of pure water processing condition can have a strong impact on its solvability to superpolymer, make the polymerization degree deviation amplitude of solute larger, and the excessive effect that not only can affect product of polymerization degree amplitude, also can cause skin care product in shelf-lives, occur the outward appearance wild effects such as muddy.Therefore, to those skilled in the art, water is not suitable for separating oligomeric pycnogenols.But, the present inventor acts in a diametrically opposite way, finding after long-term further investigation, by carrying out strict restriction to the adding proportion of water and the temperature of water, also can realize procyanidin monomers, oligomer is separated with high polymer, and when carrying out follow-up nanofiltration separation, because its composition is simple, substantially not containing high polymer, therefore nanofiltration membrane can not be caused to block, thus cold-water solution technique formerly and posterior nanofiltration technique can be mated, ensure that technique can steady in a long-termly be run.
Further, monomer is separated with oligomer by nanofiltration by the present invention further, directly can prepare oligomer, the polymerization pycnogenols of more than nanofiltration removing tripolymer is used compared to prior art, remove monomer with resin again, obtain dimer, this law is obviously simple.
In addition, the present invention, compared to separating technology of the prior art, also avoid the use of organic solvent and resin, thus considerably reduces the cost of technique.
(2) preparation method of oligomeric procyanidolics of the present invention, the pycnogenols high polymer that prior art is separated " is turned waste into wealth ", makes it to be hydrolyzed into pycnogenols oligomer, increase the content of raw material procyanidins oligomer, improve raw material availability, save cost.
Embodiment
Below in conjunction with specific embodiment, the preparation method to oligomeric procyanidolics provided by the invention is described in detail.
In the following embodiments, 1 weight part is 1kg.
Embodiment 1
The preparation method of the oligomeric procyanidolics described in the present embodiment, comprises the steps:
(1) the 0 DEG C cold water of pycnogenols crude extract 1 weight part (procyanidin content 85%, wherein monomer 24%, 2 ~ 3 polymerization degree oligomers 43%, high polymer 18%, Xi'an Sen Fu company limited) with 7 weight parts is redissolved, obtain pycnogenols suspension liquid;
(2) use qualitative filter paper to carry out natural filtration to pycnogenols suspension liquid, collect filter cake and filtrate respectively; Measure the pycnogenols in filtrate, wherein monomer accounts for 35.7%, and 2 ~ 3 polymerization degree oligomers account for 64.0%, and high polymer accounts for 0.3%;
(3) by filtrate molecular weight cut-off be 900 daltonian nanofiltration membrane (Ande Membrane Separation Technology & Engineering (Beijing) Co., Ltd., NF-1812) nanofiltration, working pressure is 2bar, and the volume that circulation is concentrated into trapped fluid is 50% of the liquor capacity after merging;
(4) collected trapped fluid is carried out spraying dry under the condition of inlet temperature 140 DEG C, temperature out 70 DEG C, feed liquid proportion 1.1, peristaltic pump pump speed 33%, air flow quantity 30mm, pumping speed 100%, namely oligomeric procyanidolics powder 0.532 weight part is prepared, yield 53.2%, purity 98.1%, mean polymerisation degree 2.3.
Embodiment 2
The preparation method of the oligomeric procyanidolics described in the present embodiment, comprises the steps:
(1) by pycnogenols crude extract 1 weight part (procyanidin content 95%, wherein monomer 27%, 2 ~ 3 polymerization degree oligomers 51%, high polymer 17%, Zhejiang Skyherb Ingredients Co., Ltd.) redissolve with 20 DEG C of cold water of 5 weight parts, obtain pycnogenols suspension liquid;
(2) use qualitative filter paper to carry out natural filtration to pycnogenols suspension liquid, collect filter cake and filtrate respectively; Measure the pycnogenols in filtrate, wherein monomer accounts for 34.1%, and 2 ~ 3 polymerization degree oligomers account for 64.3%, and high polymer accounts for 1.6%;
(3) by filtrate molecular weight cut-off be 900 daltonian nanofiltration membrane (Ande Membrane Separation Technology & Engineering (Beijing) Co., Ltd., NF-1812) nanofiltration, working pressure is 2bar, and the volume that circulation is concentrated into trapped fluid is 50% of the liquor capacity after merging;
(4) by collected trapped fluid spraying dry under the condition of inlet temperature 160 DEG C, temperature out 80 DEG C, feed liquid proportion 1.2, peristaltic pump pump speed 44%, air flow quantity 40mm, pumping speed 100%, namely oligomeric procyanidolics powder 0.592 weight part is prepared, yield 59.2%, purity 97.4%, mean polymerisation degree 2.6.
Embodiment 3
The preparation method of the oligomeric procyanidolics described in the present embodiment, comprises the steps:
(1) the 4 DEG C cold water of pycnogenols crude extract 1 weight part (procyanidin content 85%, wherein monomer 24%, 2 ~ 3 polymerization degree oligomers 43%, high polymer 18%, Xi'an Sen Fu company limited) with 6 weight parts are redissolved, obtain pycnogenols suspension liquid;
(2) use qualitative filter paper to carry out natural filtration to pycnogenols suspension liquid, collect filter cake and filtrate respectively; With the aqueous ethanolic solution dissolving filter cake of 15%, add the sodium sulfite solution of 6% of 10 times of volumes, at 80 DEG C of stirring in water bath reaction 1.5h, rotary evaporation concentration of reaction solution until reaction solution original volume 70%, merge concentrated after reaction solution and filtrate;
(3) by merge after solution molecular weight cut-off be 900 daltonian nanofiltration membrane (Ande Membrane Separation Technology & Engineering (Beijing) Co., Ltd., NF-1812) nanofiltration, working pressure is 2bar, and the volume that circulation is concentrated into trapped fluid is 50% of the liquor capacity after merging;
(4) by collected trapped fluid spraying dry under the condition of inlet temperature 160 DEG C, temperature out 80 DEG C, feed liquid proportion 1.2, peristaltic pump pump speed 44%, air flow quantity 40mm, pumping speed 100%, namely oligomeric procyanidolics powder 0.583 weight part is prepared, yield 58.3%, purity 99.1%, mean polymerisation degree 2.5.
Embodiment 4
The preparation method of the oligomeric procyanidolics described in the present embodiment, comprises the steps:
(1) by pycnogenols crude extract 1 weight part (procyanidin content 95%, wherein monomer 27%, 2 ~ 3 polymerization degree oligomers 51%, high polymer 17%, Zhejiang Skyherb Ingredients Co., Ltd.) redissolve with 10 DEG C of water of 5.5 weight parts, obtain pycnogenols suspension liquid;
(2) use qualitative filter paper to carry out natural filtration to pycnogenols suspension liquid, collect filter cake and filtrate respectively; With the aqueous ethanolic solution dissolving filter cake of 5%, add the sodium sulfite solution of 4% of 7 times of volumes, at 70 DEG C of stirring in water bath reaction 0.5h, rotary evaporation concentration of reaction solution until reaction solution original volume 70%, merge concentrated after reaction solution and filtrate;
(3) by merge after solution molecular weight cut-off be 900 daltonian nanofiltration membrane (Ande Membrane Separation Technology & Engineering (Beijing) Co., Ltd., NF-1812) nanofiltration, working pressure is 1bar, and the volume that circulation is concentrated into trapped fluid is 30% of the liquor capacity after merging;
(4) collected trapped fluid is carried out spraying dry under the condition in inlet temperature 150 DEG C, temperature out 75 DEG C, feed liquid proportion 1.1, peristaltic pump pump speed 40%, air flow quantity 35mm, pumping speed 100%, namely oligomeric procyanidolics powder 0.623 weight part is prepared, yield 62.3%, purity is 98.4%, the polymerization degree 2.6.
Embodiment 5
The preparation method of the oligomeric procyanidolics described in the present embodiment, comprises the steps:
(1) the 15 DEG C cold water of pycnogenols crude extract 1 weight part (procyanidin content 85%, wherein monomer 24%, 2 ~ 3 polymerization degree oligomers 43%, high polymer 18%, Xi'an Sen Fu company limited) with 6.5 weight parts are redissolved, obtain pycnogenols suspension liquid;
(2) use qualitative filter paper to carry out natural filtration to pycnogenols suspension liquid, collect filter cake and filtrate respectively; With the aqueous ethanolic solution dissolving filter cake of 10%, add the sodium sulfite solution of 5% of 8.5 times of volumes, at 75 DEG C of stirring in water bath reaction 1h, rotary evaporation concentration of reaction solution until reaction solution original volume 70%, merge concentrated after reaction solution and filtrate;
(3) by merge after solution molecular weight cut-off be 900 daltonian nanofiltration membrane (Ande Membrane Separation Technology & Engineering (Beijing) Co., Ltd., NF-1812) nanofiltration, working pressure is 1.5bar, and the volume that circulation is concentrated into trapped fluid is 40% of the liquor capacity after merging;
(4) by collected trapped fluid spraying dry under the condition of inlet temperature 160 DEG C, temperature out 80 DEG C, feed liquid proportion 1.2, peristaltic pump pump speed 44%, air flow quantity 40mm, pumping speed 100%, namely oligomeric procyanidolics powder 0.523 weight part is prepared, yield 52.3%, purity is 95.7%, the polymerization degree 2.5.
Experimental technique
In the above embodiment of the present invention 1-2, step (2) measures filtrate procyanidins monomer, 2 ~ 3 polymerization degree oligomers and high polymer content, and the experimental technique adopted when measuring the purity of described oligomeric procyanidolics product and the polymerization degree in the step of above-described embodiment 1-5 (4) is:
One, oligomeric procyanidolics product purity measuring method
When measuring the purity of described oligomeric procyanidolics product, the method for employing is vanillin-sulfuric acid method, the reagent that the method uses and instrument and test procedure as follows:
1, reagent and instrument
1.1 reagent:
Catechin mark product (99%), oligomeric procyanidolics product, methyl alcohol (AR), the vitriol oil (AR), Vanillin (AR).
1.2 instruments:
Volumetric flask (10ml, 50ml, 100ml), transfer pipet (0.5ml, 5ml), graduated cylinder (50ml), appearance are surrounded by tool plug test tube (10ml), water-bath, electronic balance, the spectrophotometer of tinfoil paper.
2, test procedure
2.1 preparation sample solutions:
Take a certain amount of oligomeric procyanidolics product, be settled to certain volume with distilled water, ultrasonic 10min dissolution, be made into the pycnogenols sample solution that concentration is 0.01-0.1mg/mL.
2.2 colorimetric conditions:
The Vanillin methanol solution of 2.5ml massfraction 4% and the vitriol oil methanol solution of 2.5ml volume fraction 30% are joined 10ml appearance to be surrounded by the test tube of tinfoil paper, add 0.5ml pycnogenols sample solution again, add a cover and shake up, water-bath 30 DEG C of lucifuge reaction 15min, using methyl alcohol as blank, measure absorbancy at 500nm place.
The formulation (linearity range is 0.01-0.1mg/mL) of 2.3 typical curves:
1. take 5mg catechin mark product, be settled in 50ml volumetric flask with distilled water, be mixed with the trial-product of 0.1mg/ml;
2. from trial-product, pipette 2 respectively, 4,6,8,10ml is in 5 10ml volumetric flasks, with distilled water constant volume be respectively 0.02 to be made into concentration, 0.04,0.06,0.08, the need testing solution of 0.1mg/ml, pipette the above-mentioned need testing solution of 0.5ml respectively, absorbancy is measured, drawing standard curve according to above-mentioned colorimetric condition.
2.4 samples contgs measure:
Get sample solution that 0.5ml prepared according to the absorbancy of above-mentioned colorimetric condition working sample, substituted into the content that typical curve can calculate oligomeric procyanidolics product, and then calculate the purity of oligomeric procyanidolics product.
Two, the measuring method of the oligomeric procyanidolics product polymerization degree
When measuring the polymerization degree of described oligomeric procyanidolics product, the method for employing is improvement vanillin method, the reagent that the method uses and instrument and test procedure as follows:
1, reagent and instrument
1.1 reagent:
Oligomeric procyanidolics product, catechin mark product (99%), Vanillin (AR), glacial acetic acid (AR), methyl alcohol (AR).
1.2 instruments:
Volumetric flask (10ml, 50ml, 100ml), transfer pipet (0.5ml, 5ml), graduated cylinder (50ml), appearance are surrounded by tool plug test tube (10ml), water-bath, electronic balance, the spectrophotometer of tinfoil paper.
2, test procedure
2.1 preparation sample solutions:
Take 10mg oligomeric procyanidolics product, be added in 4-6ml methyl alcohol, be settled in 100ml volumetric flask with acetic acid, ultrasonic 10min dissolution, be made into the sample solution that substance withdrawl syndrome is 0.025-0.25umol/mL.
2.2 colorimetric conditions:
The glacial acetic acid solution 5ml being dissolved with 4% hydrochloric acid and 1% Vanillin is joined 10ml appearance be surrounded by the test tube of tinfoil paper, then add 1ml sample solution, add a cover and shake up, 20 DEG C of reaction 10min, using acetic acid as blank, measure absorbancy at 500nm place.
2.3 drawing standard curves (linearity range 0.025-0.25umol/mL):
1. take 5mg catechin mark product, be settled in 50ml volumetric flask with acetic acid, make the trial-product that substance withdrawl syndrome is 0.032umol/ml;
2. from trial-product, pipette 1 respectively, 2,3,4,5,6ml is in 6 10ml volumetric flasks, with acetic acid be made into respectively substance withdrawl syndrome be 0.0032,0.0064,0.0096,0.0128,0.016,00192umol/ml need testing solution, pipette the above-mentioned need testing solution of 1ml respectively, absorbancy is measured, drawing standard curve according to above-mentioned colorimetric condition.
2.4 substance withdrawl syndrome measure
Pipette the sample solution 1ml that the prepared absorbancy according to above-mentioned colorimetric condition working sample solution, substituted into the substance withdrawl syndrome that typical curve calculates oligomeric procyanidolics product, the content of the oligomeric procyanidolics product then obtained in conjunction with first part, the molecular weight of product can be drawn as calculated, and then the polymerization degree of oligomeric procyanidolics product can be obtained.
Three, first the assay of filtrate procyanidins monomer, 2 ~ 3 polymerization degree oligomers and high polymer carries out separating treatment to the monomer of the pycnogenols in filtrate, 2 ~ 3 polymerization degree oligomers and high polymer, treatment process is: adopt AB-8 resin to carry out column chromatography for separation to filtrate, applied sample amount is every milliliter of resin 0.05g, after first using 4 times of column volume distilled water flushings, through 10% ethanol elution of 3 times of column volumes, obtain procyanidin monomers; Use 25% ethanol elution of 4 times of volumes again, obtain pycnogenols oligomer, finally use 60% ethanol elution of 4 times of volumes, obtain pycnogenols high polymer; Respectively the polymerization degree of procyanidin monomers, oligomer and high polymer is verified according to the measuring method of the above-mentioned polymerization degree.Elutriant is removed, by weighing the content of the monomer, 2 ~ 3 polymerization degree oligomers and the high polymer that obtain filtrate procyanidins respectively through aftertreatment.
Obviously, above-described embodiment is only for clearly example being described, and the restriction not 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 the need to also giving all embodiments.And thus the apparent change of extending out or variation be still among the protection domain of the invention.
Claims (12)
1. a preparation method for oligomeric procyanidolics, is characterized in that, comprises the steps:
(1) redissolved by the cold water of pycnogenols crude extract with 0 ~ 20 DEG C, obtain pycnogenols suspension liquid, the mass ratio of wherein said pycnogenols crude extract and cold water is 1:5 ~ 1:7;
(2) described pycnogenols suspension liquid is filtered, collect filter cake and filtrate respectively;
(3) be that 900 daltonian nanofiltration membrane carry out nanofiltration separation to described filtrate with molecular weight cut-off, collect trapped fluid;
(4) described trapped fluid is carried out spraying dry, namely prepare oligomeric procyanidolics powder, the polymerization degree of wherein said oligomeric procyanidolics is 2 ~ 3.
2. preparation method as claimed in claim 1, it is characterized in that, in step (1), the temperature of described cold water is 4 ~ 10 DEG C.
3. preparation method as claimed in claim 1, it is characterized in that, in step (1), the purity of described pycnogenols crude extract is more than 70%.
4. the preparation method as described in as arbitrary in claim 1-3, it is characterized in that, in step (3), working pressure during nanofiltration separation is 1 ~ 2bar.
5. preparation method as claimed in claim 4, it is characterized in that, in step (3), the volume of described trapped fluid is 30 ~ 50% of filtrate original volume.
6. preparation method as claimed in claim 1, it is characterized in that, when carrying out described spraying dry in step (4), the inlet temperature of the spray-drier of use is 140 ~ 160 DEG C, and temperature out is 70 ~ 80 DEG C.
7. the preparation method as described in claim 1,2,3,5 or 6, it is characterized in that, also comprise: in step (2), described filter cake is dissolved in aqueous ethanolic solution, add the sodium sulfite solution that mass concentration is 4 ~ 6%, stirring reaction 0.5 ~ 1.5 hour at 70 ~ 80 DEG C, reaction solution is concentrated into 70% of the original volume of described reaction solution, the described filtrate that the reaction solution after concentrated and step (2) prepare is merged the nanofiltration separation for step (3).
8. preparation method as claimed in claim 4, it is characterized in that, also comprise: in step (2), described filter cake is dissolved in aqueous ethanolic solution, add the sodium sulfite solution that mass concentration is 4 ~ 6%, at 70 ~ 80 DEG C, stirring reaction 0.5 ~ 1.5 hour, is concentrated into 70% of the original volume of described reaction solution by reaction solution, and the described filtrate that the reaction solution after concentrated and step (2) prepare is merged the nanofiltration separation for step (3).
9. preparation method as claimed in claim 7, it is characterized in that, in described aqueous ethanolic solution, the volumn concentration of ethanol is 5 ~ 15%.
10. preparation method as claimed in claim 8, it is characterized in that, in described aqueous ethanolic solution, the volumn concentration of ethanol is 5 ~ 15%.
11. preparation methods as claimed in claim 7, is characterized in that, the volume ratio of described sodium sulfite solution and aqueous ethanolic solution is 7:1 ~ 10:1.
12. preparation methods as described in claim 8,9 or 10, it is characterized in that, the volume ratio of described sodium sulfite solution and aqueous ethanolic solution is 7:1 ~ 10:1.
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| CN101597273A (en) * | 2009-06-26 | 2009-12-09 | 青岛大学 | A kind of preparation method of oligomer grape seed procyanidin |
| CN102432580A (en) * | 2011-12-23 | 2012-05-02 | 晨光生物科技集团股份有限公司 | Method for degrading high polymer proanthocyanidin |
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| CN101597273A (en) * | 2009-06-26 | 2009-12-09 | 青岛大学 | A kind of preparation method of oligomer grape seed procyanidin |
| CN102432580A (en) * | 2011-12-23 | 2012-05-02 | 晨光生物科技集团股份有限公司 | Method for degrading high polymer proanthocyanidin |
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| 葡萄籽原花青素分离提纯、组分鉴定及抗氧化性研究;吴朝霞;《中国博士学位论文全文数据库 工程科技I辑》;20060415(第04期);B024-2 * |
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Effective date of registration: 20190428 Address after: 528400 No. 8, Perfect Road, South China Modern Chinese Medicine City, Nanlang Town, Zhongshan City, Guangdong Province Co-patentee after: Yangzhou perfect daily necessities Co., Ltd. Patentee after: Perfect (Guangdong) Commodity Co., Ltd. Co-patentee after: Perfect (China) Co., Ltd. Address before: 528402 Dongming North Road, Shiqi District, Zhongshan City, Guangdong Province (Private Science Park) Patentee before: Perfect (China) Co., Ltd. |