CN102942432A - Refining method of polyphenol substance and use of polyphenol substance extracted by the refining method - Google Patents
Refining method of polyphenol substance and use of polyphenol substance extracted by the refining method Download PDFInfo
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Abstract
The invention provides a separation refining method of a polyphenol substance. The separation refining method comprises the following steps of 1, blending desired polyphenol-containing extract and a developing solvent into a blended solution having a concentration of 1 to 50wt%, 2, feeding the blended solution into a chromatographic column adopting a polar adsorption resin as a chromatographic column stationary phase, carrying out separation by a chromatographic column separation method, and carrying out separation of all components by a high performance liquid chromatography, and 3, through two separation films having different aperture sizes, carrying out further separation refining of a high-desired polyphenol content component obtained by the step 2 to obtain a high-purity desired polyphenol. The refining method can realize refining separation of a specific polyphenol substance from plant extract, has simple and reliable processes and realizes high product purity.
Description
Technical field
The present invention relates to the process for refining and purifying of one or more functional polyphenols, and the application of extracting the functional polyphenol component that obtains according to this process for purification.
Background technology
Polyphenols in antibiotic, antiviral, anti-oxidant, Scavenging active oxygen, reducing cholesterol, smelly eliminating, improve the fields such as enteron aisle, anti-apocleisis, arteriosclerosis, anticancer, antianaphylaxis, check melanin generation, have very widely physiological action, in the raw material of food, food material, foodstuff additive, fodder additives, makeup material, medicine and other fields and batching, also be used widely.Now, can by industrialized utilization mainly be catechin, flavonoid class, chalcones, tanninses etc. are the functional polyphenols of representative, but the Primary product that is also just directly extracted by raw material.Therefore, the present functional polyphenols that is used of major part is the constituent that the compositions such as polymkeric substance by the derivative of polyphenol or Polyphenols mix.Find that by nearest research the in a big way polyphenols of molecular weight distribution is arranged, according to the difference active effect in vivo of molecular size range very big-difference is arranged also.For example usually be considered in the very useful pycnogenols, owing to be difficult to be absorbed, its effect is lower than the effect of 2～3 polymer unit for the large polymkeric substance of molecular weight (for example polymer of 10 above unit).But consider that from stable aspect the polymkeric substance that sometimes molecular weight is larger is more much higher than the polymer of 2～3 unit.
In order to obtain functional higher and more effective active polyphenol constituents, generally all be to cause cost very high with the chromatography of anti-phase system, be difficult to by industrialized utilization.Utilize polystyrene or the polymeric adsorbent of SDVB class, obtain the manufacture method (JP 63-162685 number) of pycnogenols and the manufacture method of polyphenol (special flat 6-300578 number) etc. by in succession open.But these technology are not used for separating separately different types of proanthocyanidin and polyphenols, but the technology of how different types of pycnogenols and polyphenols being extracted together.In addition, the manufacture method of above-mentioned pycnogenols is to be prerequisite for polystyrene adsorption resin, tests according to different alcohol concn, and the extraction yield of pycnogenols and purity are all increased.But how to separate and how to separate according to the kind of each anthocyanidin according to polymerization degree difference, and pycnogenols is not a bit all had to mention in the appeal patent with the process for purification of separating in the relevant blending ingredients of other Polyphenols.Therefore, according to above present situation, research and develop a kind of energy and obtain the stronger purpose polyphenols of functional and active effect refining the separation, and development efficiency is higher, more economical manufacture method obtains the more method of the functional polyphenol substance of high purity content, has very urgent realistic meaning.
In grape and bogberry (being commonly called as cranberry), contain the anthocyanin materials such as pycnogenols class (anthocyanidin B2, anthocyanidin A type etc.) as the catechin polymkeric substance and polymer anthocyanidin (polymers of 7 above unit), outside also have the compositions such as carbohydrate, organic acid, protein.The composition of yellow color pigments mainly is safflower yellow A Flos Carthami yellow pigment B in the petal of safflower in addition, shows that wherein red composition mainly is that carthamin (safflor red) and derivative thereof form.The present industrial carthamin yellow that is utilized, the mixture that basically is comprised of these material compositions wherein also comprises functional polyphenol components.Recently, the functional of each composition becomes more clear, and industrial community has also proposed the different demands to the particular functionality polyphenol.For example a lot of clients wish to obtain the high purity product of the higher anthocyanidin A type of functional result, and the yellow B of the safflower of the safflower yellow A of high purity content or high purity content, are exactly one of them example., consider also there is not in the market suitable product from existing industrialized production cost.
Summary of the invention
The invention provides the high efficiency separation process for purification of one or more functional polyphenols, in particular, functional polyphenol such as the tea polyphenols that contains in the plant, flavonoid class, chalcones, tanninses, carrying out exquisiteness with other compositions such as protein, fat, polyose, inorganics, organism separates, obtain the processing and refining method of the specific polyphenol product of high purity or high purity functional monomer product, the various defectives that exist to overcome prior art.
The present invention realizes by the following technical solutions:
1) will comprise the extracting solution of purpose polyphenol, be adjusted into the modulating liquid of 1～50wt% with developing solvent;
2) use Polar Adsorbent Resin as chromatographic column fixed phase, the modulating liquid in the step 1) by above-mentioned chromatographic column, is adopted the chromatographic column partition method to separate, and separates each component by high performance liquid chromatography; This Polar Adsorbent Resin has the molecular sieving effect;
3) with the component that contains high-content purpose polyphenol through tentatively obtaining, the 2 kind separatory membranes not identical with the aperture carry out further separation and purification, obtain high purity purpose polyphenol.
Described separation and purification step realizes by the following method:
The component (2) that will contain high-content purpose polyphenol flows into wide aperture separatory membrane (4) by refined liquid bucket (6), sees through liquid (H) and flows into circulation barrel (7), and refined liquid is then got back to refined liquid bucket (6) and continued circulation; The liquid (H) that sees through in the circulation barrel (7) flows into small-bore separatory membrane (5), and refined liquid (I) flows into refined liquid bucket (6), and parting liquid is then got back to circulation barrel (7) and continued circulation; The continuous reciprocation cycle of above-mentioned binary cycle system 2～10 hours obtains high purity purpose polyphenol at last in refined liquid bucket (6) or bucket (7).
Described chromatographic column partition method is in the dissolved outlet UV/vis spectrophotometer to be set, to determine the purpose weight polyphenol fraction by high performance liquid chromatography;
Described Polar Adsorbent Resin is styrene resin, selects the aperture greater than the polymeric adsorbent of purpose polyphenol molecular weight;
Consider to select suitable synthetic resins sorption amount according to the physicochemical character of functional purpose polyphenol, the sorption amount of resin with the 10-40 of resin quality doubly for well.
Developing solvent can be water, alkali, acid, ethanol class, ester class, ketone or its mixed solution, preferably according to the characteristic of extracting solution and needs dissolved purpose Polyphenols out, modulates the proportioning of optimal dissolved liquid.For separation and purification obtains purpose polyphenol monoid, extract the polarity of solvent and developing solvent and can select according to the characteristic of the class material of polyphenol.
Following principle is followed in the selection of separatory membrane: in the component that contains the functional purpose polyphenol of high-content, molecular weight and/or the polarity difference of purpose polyphenol and other polyphenols, when the molecular weight of purpose polyphenols less than 6000 the time, the wide aperture separatory membrane must select molecular weight greater than 6000 film, and the small-bore separatory membrane preferably selects molecular weight less than 500 film.
The present invention especially discloses a kind of separation and refining method of A type anthocyanidin, comprises the steps:
A) the cranberry extracting solution that comprises A type anthocyanidin being added mass concentration is the modulating liquid that the ethanol of 50-97wt% is deployed into 15～45wt% concentration;
B) get steps A) in modulating liquid, import the resin tower (3) contain Polar Adsorbent Resin, flow into resin tower (3) with developing solvent and carry out separation and purification;
C) dissolved liquid is separated into 5～7 components in order, analyzes with high performance liquid chromatography, gets the component (2) that contains high-content purpose polyphenol;
D) component (2) that contains high-content purpose polyphenol sees through liquid (H) and flows into circulation barrel (7) by refined liquid bucket (6) inflow wide aperture separatory membrane (4), and refined liquid is then got back to refined liquid bucket (6) and continued circulation; The liquid (H) that sees through in the circulation barrel (7) flows into small-bore separatory membrane (5) again, and refined liquid (I) flows into refined liquid bucket (6), and parting liquid is then got back to circulation barrel (7) and continued circulation; The continuous reciprocation cycle of above-mentioned binary cycle system 2～3 hours obtains the A type anthocyanidin that purity is 90～98wt% at last in refined liquid bucket (6).
Described Polar Adsorbent Resin is styrene resin, selects the aperture greater than the resin of purpose polyphenol molecular weight; For example diamond IONS OF H P20, HP20SS, HP21, SP206, SP70, SP700, SP207, CHP3C, CHP5C, CHP20P, SP850, all be that Mitsubishi Chemical Industries, Ltd. makes more than the SP825L(), AMBERLITE XAD-1, XAD-2, XAD-4 all is that Japanese ORGANO company makes more than the XAD-2000()
Described developing solvent can be water, alkali, acid, ethanol class, ester class, ketone or its mixed solution, optimization citric acid and alcohol mixeding liquid;
Step B) flow velocity of modulating liquid is 2～20 ml/min in;
The film of wide aperture separatory membrane (4) preferred molecular weight 3000-6000, the film of small-bore separatory membrane (5) preferred molecular weight 300-1000;
Above-mentioned separatory membrane can be selected to be limited-liability company of Asahi Chemical Industry such as SEP-3013, SEP-3023, SEP-3053(are above), character C-02-HR, character C-40-HR(are above is Japanese KURARAY company system), UP00, UP010, be the NADIR system more than the UP020(), Cefilt-UF(Japan insulator limited-liability company) etc.;
Step D) the separation and purification step in is preferably 1～20 liter/min of flow velocity, and pressure 0.2～0.5MPa carries out under the condition that temperature is 15～35 ℃.
The present invention with molecular sieving effect and hydrophobic occluded resin being arranged as chromatographic column fixed phase, separates obtaining functional purpose polyphenols from containing polyphenol and comprising the extracting solution of other compositions.Concrete way is, as synthetic occluded resin, carried out attempting separating with the synthetic occluded resin of styrenic.It obtains the result, as shown in Figure 1, we the molecular sieve effect arranged and hydrophobic occluded resin is arranged as stationary phase, under specific dissolved condition, carry out obtaining in the following sequence various weight polyphenol fractions after chromatographic column separates, obtain successively the high component of the large polarity of molecular weight, large but the low component of polarity of molecular weight, molecular weight is little but component that polarity is high, and molecular weight is little but component that polarity is low.
If but carry out independent separate treatment with above-mentioned synthetic occluded resin treatment process and separatory membrane, the purity of purpose polyphenol does not have significant improvement.This explanation, the inventive method will be synthesized occluded resin treatment process and separatory membrane and be processed the method for carrying out combined treatment and produced significant effect.
The raw material that contains the several functions polyphenol component that uses as the present invention, such as high fruit, leaf, the roots of functional polyphenol content such as grape, bogberry (being commonly called as cranberry), apple also have their squeezeding juice, the extract of plant etc. to use.In the present invention, separation as the purpose polyphenolic substance of specific physiologically active, as the polyphenol monoid that can make with extra care separation, comprise the phenol acids group who consists of by from phenol acids and ester, the polyphenol group who also has polyphenol and organic acid ester to consist of, the polymkeric substance of catechu acids and catechu acids (n≤6) (another name: the polyphenol group who anthocyanidin) consists of, cinnamophenone and its glucoside and or the polyphenol group that consisted of by flavonoid and its glucoside, tannin monoid etc.
The selection of described hydrophobicity occluded resin is extremely important, must consider the intensity of hydrophobicity (being insoluble in water) of the polarity of selected functional purpose polyphenol and molecular weight and synthetic occluded resin and the size in aperture.In other words, the molecular weight of functional purpose polyphenol is less than the aperture of selected synthetic occluded resin, and must select to have certain hydrophobic resin.
Description of drawings
Fig. 1 is the present invention processes each polyphenol component that draws with occluded resin dissolved tendency synoptic diagram.
Fig. 2 is the schematic flow sheet of process for purification of the present invention.
Fig. 3 is the embodiment of the invention 1 and the rayed antioxidant effect comparison diagram that contrasts sample.
Fig. 4 be the embodiment of the invention 2 with the rayed of contrast sample after pigment survival rate figure as a result.
Embodiment
Provide preferred embodiment of the present invention below in conjunction with accompanying drawing, to describe technical scheme of the present invention in detail.
As shown in Figure 2, separation and purification process of the present invention is as follows:
The extracting solution that will comprise the purpose polyphenol is adjusted into the modulating liquid of 1～50wt% with developing solvent; Pour modulating liquid into resin tower (3) afterwards, the developing solvent of bucket in (1) flows into the resin tower (3) that contains the hydrophobicity occluded resin to carry out chromatogram column technique and separates, and the component that will contain high density purpose polyphenol is put into bucket (a 2) recovery.And then the component (2) that will contain the functional purpose polyphenol of high-content is transplanted on refined liquid bucket (6), flow into wide aperture separatory membrane (4) by refined liquid bucket (6), see through liquid (H) and flow into circulation barrel (7), refined liquid is then got back to refined liquid bucket (6) and is continued circulation; The liquid (H) that sees through in the circulation barrel (7) flows into small-bore separatory membrane (5), and refined liquid (I) flows into refined liquid bucket (6), and parting liquid is then got back to circulation barrel (7) and continued circulation; The continuous reciprocation cycle of above-mentioned binary cycle system 2～10 hours obtains the functional purpose polyphenol of high purity at last in refined liquid bucket (6).
Embodiment 1
Below take the separation and purification of A type anthocyanidin as example, to describe technical scheme of the present invention in detail.
The A type anthocyanidin that is rich in cranberry is by the aggretion type tannin, or non-water decomposition class tannin, namely with flavane-3, the 4-glycol is Component units, the macromolecular compound that generates by polymerization.In general, occurring in nature exists all is to be main polymkeric substance (being that polymerization degree n is as 3-30) take 3-30 unit, but have a functional result general all be that 3-5 unit is that master's small molecules polymkeric substance is in the majority.As everyone knows, anthocyanidin A type has hyaluronidase inhibitor effect, antianaphylaxis and anti-inflammatory effect, prevents in addition the urinary tract infection's texts that causes because of bacterium.The method of this patent is exactly from the cranberry extracting solution, simply to obtain efficiently the composition of highly purified refining A type anthocyanidin.
1, the modulating liquid of cranberry
The cranberry extraction step adds the water of the 5-10 weight that contains the 0.2%-0.8% citric acid in fresh cranberry 1 weight, and temperature stirred 2-5 hour in 50-90 ℃ of scope.The cranberry modulating liquid is exactly that above-mentioned extracting liquid filters, and then adds 50-97% ethanol and makes it to become 15-45% concentration, standby rear use.According to the HPLC analytical results as shown in Figure 3, can know and have at least 7 kinds of different Anthocyanins in the cranberry modulating liquid.The purpose of patent of the present invention is exactly the specific functional the strongest purpose composition (the composition NO.7 that marks in the HPLC spectrogram) here simply to be separated and exquisite.
2, process synthetic occluded resin
The device that uses is the glass column with diameter 50mm * long 50cm, filling polystyrene synthetic resins SP-700(Mitsubishi changes into company's system) resin 700ml, with containing aqueous ethanol (concentration the is 20-50%) solution of 0.2%-0.8% citric acid as developing solvent.The speed of 200 milliliters of cranberry modulating liquids with flow velocity 2-20 ml/min, import above pillar under the room temperature.Then carry out separation and purification with above-mentioned developing solvent.By successively stripping order, every 300ml is a component, separates obtaining 7 components.These components are carried out HPLC analyze, the anthocyanidin content of component 4 is maximum, in the component 4 ratio of each contained composition as shown in Figure 3, the contained per-cent of composition 3, composition 6, composition 7 is respectively 12%, 32%, 46%(is with reference to table 1).
The degree of contained each composition in table 1 component 4
3, separatory membrane is processed
Process the component 4 that obtains according to synthetic occluded resin, under following condition, carry out separation and purification with separatory membrane.2 kinds of separatory membranes that the aperture is not identical are respectively FS10-FUST653(Daicen Membrane-Systems LTD and make), molecular weight 6000 specifications, membrane area 0.25m
2) and NPO 030(DaicenMembrane-Systems LTD manufacturing), by molecular weight 300 specifications, Na
2SO
4Elimination factor 80～95%, membrane area 0.25m
2): operational condition is: flow velocity 5-10 liter/min, pressure 0.3-0.5MPa, temperature 25-45 ℃, the filtered solution of various separatory membrane turns back in the maintenance liquid of separatory membrane, circulation 2-5 hour.The refined liquid of the FS10-FUST653 separatory membrane that obtains, the purity of the anthocyanidin of composition NO.7 become very high (with reference to table 1) up to 95%.This part solution rotatory evaporator, 40 ℃, under reduced pressure concentrate, just can obtain the high purity anthocyanidin product of 5g solid.
4, analytical procedure
Analyze anthocyanidin A, directly connect with UV detector and fluorimetric detector, HPLC(Shimadzu Seisakusho Ltd. system that can while display monitoring on screen) analyze.Analysis condition is as follows: Solvent A: 0.1% trifluoroacetic acid water, and Solvent B: 50% acetonitrile, flow: 0.8ml/min, post: ODS (C-18), column temperature: 40 ℃, injection rate: 20 μ l, working method: Solvent A; 0% (0 minute) → Solvent B; 100% (30 minutes) are detected: detect wavelength 276nm, wavelength of fluorescence 316nm.
Application Example
In recent years, from the security of soft drink tinting material, and the impression aspect such as natural tendency considered that manufacturer and human consumer preferred to use natural pigment.24 hours convenience stores slowly popularizing in large-and-medium size cities particularly, use the painted beverage of natural pigment to need for a long time by irradiations such as luminescent lamps, the light stability of the natural pigment that exists in the beverage and the problem such as how to prevent from fading have become very urgently with important.Now, though existing part producer brings into use the oxidation-resistance agent such as rutin preparation and chlorogenic acid preparation to improve the light stability of natural pigments, but on the market or can the produce effect appearance of better natural pigment stablizer of expectation.
This application case is to have used the Anthocyanins that separation and purification obtains in the foregoing invention, and the light stability of cabbage red natural pigment has been carried out simultaneous test.Use cabbage haematochrome (market sell goods, the specification of the strong 530nm=800 of extinction), the colourless soda pop that the market is sold carries out painted.Control substance of plant drug as the stable experiment of natural pigment has adopted chlorogenic acid preparation and two kinds in rutin preparation (with light drugmaker system).The component made from extra care separating obtained maximum concentration anthocyanidin among control substance of plant drug and the present invention, add respectively 100,50,25ppm is to colourless soda pop, add again the cabbage haematochrome concentration (absorbancy under the 530nm is controlled at about 0.8) that regulates, put into each 3 pipe Glass Containers (100ml).Be determined at respectively the absorbancy of three samples of preservation after 7 days under the sunlight with extinction photometer.Using maximum absorbing wavelength is (530nm), as 100%, measures the mean value of the absorbancy survival rate after processing with the absorbancy before processing.Contrast to have with this and add and without the antioxidant effect that adds the anthocyanidin constituent.
(A: without any interpolation as shown in Figure 3, B: refining anthocyanidin 25ppm adds among the present invention, C: refining anthocyanidin 50ppm adds among the present invention, D: refining anthocyanidin 100ppm adds among the present invention, E: commercially available rutin antioxidant 100ppm adds, F: commercially available chlorogenic acid antioxidant 100ppm adds) be the result of solar light irradiation after 7 days.Can find out significantly that from Fig. 3 add district (B, C, D) at refining anthocyanidin, along with the increase of anthocyanidin concentration, the light stability of cabbage haematochrome is significantly improved.In addition, contrast also will be got well with reference to the Antioxidant Rutin preparation in district and the effect of chlorogenic acid preparation.From then on test-results can illustrate, separation and purification and the anthocyanidin constituent have the effect that the natural cabbage pigment that well prevents light direct beam and cause fades, also can predict thus in the photoxidation that prevents of natural pigment and stablize the field good application prospect is arranged.And, also can be with a wide range of applications in fields such as finished product and work in-process medicine and makeup.
Contain monomer anthocyanidin in the purple grape pigment, catechin, the compositions such as the polymer cyanine element polymkeric substance pigment of tannin and flavonoid and anthocyanidin condensation, the anthocyania pigment tone of monomer is brighter, is subjected to easily the pH value of solution, temperature, the impact such as concentration and metal ion, stability is not good enough.What but the tone of the anthocyanidin of polymkeric substance showed is the color of original grape, shows slightly comparatively dim, and partially red, and stability is very good.General food color can separately use these two kinds of pigments if can make a thorough investigation of application target, and the range of application of that anthocyanidin just can be promoted greatly.Utilize the present invention just can be simple and effective in the purple grape extracting solution, monomer anthocyanidin be separated efficiently with polymkeric substance anthocyanidin and is made with extra care.
1. the modulation of purple grape extracting solution
Purple grape inspissated juice (5 times of concentrated solutions) 1 weight adds and contains 0.The water 10-30 weight of 1-0.5% citric acid after stirring, is filtered and is obtained the purple grape extracting solution.The device that uses is the glass column with diameter 50mm * long 50cm, filling polystyrene synthetic resins XAD-4(ORUGANO company system) resin 400ml, under flow velocity 3-15ml/ minute condition, carry out chromatographic separation, again with after successively washing with the water of 800ml, again with containing the concentration citric acid of 0.1-0.5% and the ethanolic soln of 30-70% concentration, carry out the elution chromatography post under the room temperature, obtain the 800ml dissolution fluid and be the needed purple grape extracting solution that contains monomer anthocyanidin and polymkeric substance anthocyanidin.
1. separatory membrane is processed
Process the 800ml purple grape pigment extract that obtains according to synthetic occluded resin, under following condition, carry out separation and purification with separatory membrane.2 kinds of separatory membranes that the aperture is not identical, being respectively FS10-FUST653(Daicen Membrane-Systems LTD makes), molecular weight 6000 specifications, membrane area 0.25m2) and NPO030(Daicen Membrane-Systems LTD make), by molecular weight 300 specifications, Na2SO4 elimination factor 80～95%, membrane area 0.25m2): operational condition is: flow velocity 3-10 liter/min, pressure 0.2-0.5MPa, temperature 25-45 ℃, the filtered solution of various separatory membrane turns back in the maintenance liquid of separatory membrane, circulation 5-10 hour.The refined liquid of the FS10-FUST653 separatory membrane that obtains (polymkeric substance anthocyanidin), and the refined liquid composition of NPO030 separatory membrane (monomer anthocyanidin) (with reference to table 2).This two portions solution is used respectively rotatory evaporator, and 40 ℃, under reduced pressure concentrate, just can obtain the anthocyanidin product of two kinds of specifications, monomer anthocyanidin 40 grams, polymkeric substance anthocyanidin 35 grams.
3. interpretation of result
HPLC(Shimadzu Seisakusho Ltd. that the UV detector is arranged is adopted in the analysis of purple grape pigment composition) carry out under the following conditions: Solvent A: 0.1% trifluoroacetic acid water, Solvent B: 50% acetonitrile, flow: 0.8ml/min, post: ODS (C-18), column temperature: 40 ℃, injection rate: 20 μ l, working method: Solvent A; 0% (0 minute) → Solvent B; 100% (30 minutes) are detected: detect wavelength 530nm.
Three index lightness (L) are adopted in the analysis of tone, and colored degree (Chroma) and colourity (Hue) compare analysis, and the result is as shown in table 2.In three kinds of compositions, the variation of lightness L value is little, but the difference of colored degree (Chroma) and colourity (Hue) is just very obvious.The colored degree of monomer anthocyanidin is maximum, and the expression color is the most bright-coloured, the comparison dull colors that just show that polymkeric substance anthocyanidin is relative.
The refining colorimetric analysis contrast that separates the purple grape composition that obtains of table 2. the present invention
Application case
In the refreshment drink, like the popularity of grape juice beverage more and more higher, convenience store and various supermarket have grape juice beverage selling, this type of grape juice beverage generally all is added as tinting material with the purple grape haematochrome, but because because the packing of beverage generally all is to use transparent or semitransparent grog bottle to be the master, so the requirement to the light stability of the natural pigment in the grape juice beverage is more and more higher, the purple grape pigment is very unstable to light in the Sucus Vitis viniferae of present situation.Therefore also can there be a novel appearance to the comparatively stable purple grape haematochrome of light in market very much.The product that utilizes the present invention to obtain just can satisfy the requirement in this market.
Implementation method:
Utilize the present invention from raw material Sucus Vitis viniferae extracting solution, to make with extra care resulting monomer anthocyanidin and polymerization anthocyanidin by membrane sepn, follow the Sucus Vitis viniferae extracting solution of processing without film to carry out the contrast of photostability test.Three kinds of specification pigments of appeal, becoming absorbancy under the 503nm with the modulation of the damping fluid of pH about 3.0 respectively is 0.8 solution, again the solution of adjusting is respectively charged in the transparent glass test tube of 3 100ml, under sunlight, shine respectively 3, after 5 and 10 days, measure respectively again absorbing wavelength and be the absorbancy under the 530nm, as 100%, calculate the mean value of the survival rate (%) of postradiation absorbancy with the absorbancy of pre-irradiation.As shown in Figure 4 (A: the purple grape pigment that is untreated, B: the monomer anthocyania pigment, C: the polymkeric substance anthocyania pigment, every group represents respectively the result of irradiation after 3,5,10 days from left to right).After the solar light irradiation 10 days, almost faded fully with the red extracting solution of purple grape of processing without film and to have compared, the pigment survival rate of monomer anthocyanidin and polymkeric substance anthocyanidin still also has 30% and 80%.The monomer anthocyanidin that this explanation utilizes manufacture method gained of the present invention is compared with undressed grape haematochrome with polymerization anthocyanidin has very good light stability.
Case study on implementation 3
The safflower that obtains take the petal of safflower as the raw material extracting is yellow, is applied in widely beverage as natural pigment, snack categories, and the field such as flour products.Along with the development to the research of carthamin, the yellow color component of finding carthamin mainly is that safflower yellow A Flos Carthami yellow pigment B forms.But in fact, except the pigment of these two kinds of yellow colors, show in addition dark brown Polyphenols pigment composition and show red carthamin composition, and their presoma composition is included in the extract the inside.
Carthamin yellow composition A and B are the very bright-coloured yellow pigment compositions of energy Show Color, but because comprise dark brown Polyphenols and red carthamin composition in the extract, so different according to extracting condition and raw material and manufacturing process, the carthamin yellow color of producing each time can be very different.The method according to this invention can obtain highly purified bright-colored safflower yellow A and B composition by simple and effective separation and purification, and dark brown polyphenol components.Just can be according to different characteristics and performance more effective taking full advantage of in addition.
2. the modulation of Flos Carthami extract
Dry safflower petal 1 weight adds water 10-40 weight, stirred extracting in 4-7 hour, filtration obtains Flos Carthami extract, this extracting solution carries out chromatographic separation with the glass column (being filled with the occluded resin 1500ml of ORUGANOXAD-4) of 8cmX60cm under flow velocity 10-40ml/ minute condition, again with after successively washing with the water of 1500ml, use the concentration ethanol solution of 20-50% again, carry out the elution chromatography post under the room temperature, dissolution fluid is needed carthamin yellow extracting solution.
3. separatory membrane is processed
Process the carthamin yellow extracting solution that obtains according to synthetic occluded resin, under following condition, carry out separation and purification with separatory membrane.2 kinds of separatory membranes that the aperture is not identical, being respectively FS10-FUST653(Daicen Membrane-Systems LTD makes), molecular weight 6000 specifications, membrane area 0.25m2) and NPO030(Daicen Membrane-Systems LTD make), by molecular weight 300 specifications, Na2SO4 elimination factor 80～95%, membrane area 0.25m2): operational condition is: flow velocity 5-10 liter/min, pressure 0.3-0.5MPa, temperature 25-45 ℃, the filtered solution of various separatory membrane turns back in the maintenance liquid of separatory membrane, circulation 4-7 hour.The refined liquid of the FS10-FUST653 separatory membrane that obtains, and the refined liquid composition of NPO030 separatory membrane, (with reference to table 3).This two portions solution is used respectively rotatory evaporator, and 40 ℃, under reduced pressure concentrate, just can obtain the carthamin yellow product of two kinds of specifications.
4. interpretation of result
HPLC(Shimadzu Seisakusho Ltd. that the UV detector is arranged is adopted in the analysis of sailor yellow colour content) carry out under the following conditions: Solvent A: 0.1% trifluoroacetic acid water, Solvent B: 50% acetonitrile, flow: 0.8ml/min, post: ODS (C-18), column temperature: 40 ℃, injection rate: 20 μ l, working method: Solvent A; 0% (0 minute) → Solvent B; 100% (30 minutes) are detected: detect wavelength 404nm.Shown in the analytical results breast table 3, (Y value is according to three primary color theory to the Y value of the carthamin yellow extracting solution of processing without film, an index that represents yellow colour brightness, Y value is larger, the brightness that represents yellow color is larger, that is to say that color is more bright-coloured), be 50.57, after the film processing, the Y value of the refined liquid after the NPO030 film is processed brings up to 64.03, and the Y value of the refined liquid after the processing of FS10-FUST653 film is 32.50, the boldness that the color of the carthamin yellow that obtains is described has had very large difference, obtain after the macromole film is processed be dark brown be main safflower polyphenol components, mainly be brightly painted safflower yellow A and B composition product and the small molecules film obtains after processing.
The refining Y value of separating the carthamin yellow product that obtains of table 3. the present invention
| Y value | |
| Carthamin yellow extracting solution without the film processing | 50．57 |
| NPO030, the refined liquid after film is processed | 64．03 |
| Refined liquid after the FA10-FUST653 film is processed | 32．50 |
According to the present invention, as food, food material, foodstuff additive, fodder additives, the makeup material, medicine etc. are made raw material, and have antibiotic, antiviral, anti-oxidant, the active acid element is eliminated, and cholesterol reduces, and is anticancer, antianaphylaxiies etc. are physiological action very widely, from the effect of this Polyphenols, according to using required components group, not only excellent efficiency and economical but also easy method are arranged with not only safety, obtain best earning rate, the polyphenol components thing is effectively utilized.
Claims (10)
1. the separation and refining method of a polyphenols is characterized in that, comprises the steps:
1) will comprise the extracting solution of purpose polyphenol, be adjusted into the modulating liquid of 1～50wt% with developing solvent;
2) use Polar Adsorbent Resin as chromatographic column fixed phase, the modulating liquid in the step 1) by above-mentioned chromatographic column, is adopted the chromatographic column partition method to separate, and separates each component by high performance liquid chromatography;
3) with the component that contains high-content purpose polyphenol through tentatively obtaining, the 2 kind separatory membranes not identical with the aperture carry out further separation and purification, obtain high purity purpose polyphenol.
2. the method for claim 1, it is characterized in that, the described separation and purification step of step 3) comprises: the component (2) that will contain high-content purpose polyphenol flows into wide aperture separatory membrane (4) by refined liquid bucket (6), see through liquid (H) and flow into circulation barrel (7), refined liquid is then got back to refined liquid bucket (6) and is continued circulation; The liquid (H) that sees through in the circulation barrel (7) flows into small-bore separatory membrane (5), and refined liquid (I) flows into refined liquid bucket (6), and parting liquid is then got back to circulation barrel (7) and continued circulation; The continuous reciprocation cycle of above-mentioned binary cycle system 2～10 hours obtains high purity purpose polyphenol at last in refined liquid bucket (6) or bucket (7).
3. the method for claim 1 is characterized in that, described Polar Adsorbent Resin is styrene resin, selects the aperture greater than the polymeric adsorbent of purpose polyphenol molecular weight.
4. the method for claim 1, it is characterized in that, described developing solvent is selected from water, alkali, acid, ethanol class, ester class, ketone or its mixed solution, when the molecular weight of purpose polyphenols less than 6000 the time, the wide aperture separatory membrane must select molecular weight greater than 6000 film, and the small-bore separatory membrane preferably selects molecular weight less than 500 film.
5. such as each described method of claim 1～4, it is characterized in that described polyphenol is the functional polyphenol of tea polyphenols, flavonoid class, chalcones or tannins.
6. the separation and refining method of an A type anthocyanidin is characterized in that, comprises the steps:
A) the cranberry extracting solution that comprises A type anthocyanidin being added mass concentration is the modulating liquid that the ethanol of 50-97wt% is deployed into 15～45wt% concentration;
B) get steps A) in modulating liquid, import the resin tower (3) contain Polar Adsorbent Resin, flow into resin tower (3) with developing solvent and carry out separation and purification;
C) dissolved liquid is separated into 5～7 components in order, analyzes with high performance liquid chromatography, gets the component (2) that contains high-content purpose polyphenol;
D) component (2) that contains high-content purpose polyphenol sees through liquid (H) and flows into circulation barrel (7) by refined liquid bucket (6) inflow wide aperture separatory membrane (4), and refined liquid is then got back to refined liquid bucket (6) and continued circulation; The liquid (H) that sees through in the circulation barrel (7) flows into small-bore separatory membrane (5) again, and refined liquid (I) flows into refined liquid bucket (6), and parting liquid is then got back to circulation barrel (7) and continued circulation; The continuous reciprocation cycle of above-mentioned binary cycle system 2～3 hours obtains the A type anthocyanidin that purity is 90～98wt% at last in refined liquid bucket (6).
7. method as claimed in claim 6 is characterized in that, described Polar Adsorbent Resin is styrene resin, selects the aperture greater than the polymeric adsorbent of purpose polyphenol molecular weight; Described developing solvent is citric acid and alcohol mixeding liquid.
8. method as claimed in claim 6 is characterized in that, wide aperture separatory membrane (4) is the film of molecular weight 3000-6000, and small-bore separatory membrane (5) is the film of molecular weight 300-1000.
9. method as claimed in claim 6 is characterized in that, step B) in the flow velocity of modulating liquid be 2～20 ml/min, step D) in the separation and purification step be 1～20 liter/min of flow velocity, pressure 0.2～0.5MPa carries out under the condition that temperature is 15～35 ℃.
10. the application of the A type anthocyanidin that obtains of each method is characterized in that according to claim 6～9, can add in food, makeup, pharmaceuticals or medicine intermediate, field of health care products as colorant stabilizer or tinting material and use.
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