CN102086185A - Oligomeric proanthocyanidins and method for extracting same - Google Patents

Abstract

The invention provides a method for extracting oligomeric proanthocyanidins (OPC) from OPC-containing plant sources. The method is characterized in that: in the extracting process, conditions of no oxygen or low oxygen and low temperature are kept and a lightproof environment is used. The invention also provides the oligomeric proanthocyanidins extracted by the method; and the oligomeric proanthocyanidins is characterized by being rich in proanthocyanidins oligomers, and the extract can be auto-oxidized under the natural condition. The invention also provides application of proanthocyanidins products prepared by the method in removing superoxide anion radicals O2- and hydroxyl radicals HO.

Description

A kind of oligomeric procyanidolics and extracting method thereof

Technical field

The present invention relates to the method for a kind of oligomeric procyanidolics and extraction thereof, specially refer under lucifuge, deoxidation and cold condition, the method for the high vigor pycnogenols that obtains through solvent extraction and column chromatography and the oligomeric procyanidolics product that obtains by described method.

Background technology

1, the function of pycnogenols

Pycnogenols is a large amount of a kind of flavanol compound polyphenolic compounds that exist in the fruits such as cowberry, Semen Vitis viniferae.It also is most important function composition in the cowberry.Wherein main present-color material is by delphinidin (delphinidin; De), cyanidin (cyanidin; Cy), morning glory pigment (petunidin; Pet), peony pigment (peonidin; Peo) and syringidin (malvidin; Mal) five kinds of numerous anthocyanins of pigment institute deutero-and acetylize anthocyanin are formed, and anthocyanin kind and content are looked the cowberry kind and is different.Main on the cowberry pycnogenols structure by monomer (+)-catechin [(+)-catechin in the Flavonol, C], (-)-l-Epicatechol [(-)-epicatechin, EC] and (-)-L-Epicatechin gallate [(-)-epicatechin-3-O-gallate, EC G] be polymerized, its mean polymerisation degree is up to ten pentamers [7].Usually with two to the tetramer be called oligomer (procyanidolic oligomers, OPC), with more than the pentamer be called high polymer (procyaLnidolicpolymers, PPC).Connect C4-C6 and the two kinds of modes of C4-C8 of mainly containing between each unit of PC, because the difference of monomeric conformation or bonding position, multiple isomer can be arranged, and each polymer and close with polarity between the poly-isomer, so the fractionation of pycnogenols and structural characterization difficulty [1] relatively always.

Pycnogenols is not only the major function composition of cowberry, other multiple important health plant kinds, as ginkgo (leaf), grape (son), black currant, black soya bean, Pale Butterflybush Flower (robinin), Chinese sorghum red pigment, radish red, red rice element, Roselle Calyx is red, mulberry fruit is red, cocoa shell, tealeaves (phenol), its main functional component also is the OPC or derivatives thereof.Nineteen fifty, Masquelier extracts a large amount of OPC from Cortex Pini, and in France Cortex Pini Massonianae extract (wherein contain have an appointment 85% OPC) is registered as medicine, and its commodity are called Pycnogenl, be used to improve the resistibility of blood vessel, reduce the fragility and the permeability [2] of capillary vessel.In practical application subsequently, the doctors in Europe find that OPC is to equally also having tangible curative effect [3] such as diseases such as pollen hypersensitivity, sacroiliitis, stomach ulcer.In Germany, the patented product made from grape pip procyanidin has been used for the treatment of the microcirculation disease, comprises eyes and periphery capillary permeability disease and vein and lymphatic function not complete [4].The Bonaure etc. of France invented with pycnogenols oligomer or monomer be actives (＞1%) the treatment periodontopathy preparation and obtain patent protection [5].France utilizes grape pip procyanidin to make vasoprotector and anti-inflammatory agent [6].Germany develops and is used for the treatment of crapulent pycnogenols preparation and obtains patent protection [7].In Romania, a kind of commodity Elldotelon pycnogenols preparation by name has gone on the market and has been used for the treatment of disease of capillaries [8].France, Italy also develop into makeup with pycnogenols, and protectiving ultraviolet is to the injury effect [9,10] of skin preferably.Japan develops the pycnogenols as medicine, foods and cosmetics antioxidant, and obtains patent protection." in the whole food agent list " listed Semen Vitis viniferae extract in Japanese health ministry in 1996 approval, and with commodity " Gravinal " put goods on the market [11].In the U.S., grape pip procyanidin is directly made formulations such as capsule as the raw material of heath food, becomes one of natural plant ten big best sellers.Because grape pip procyanidin has extremely strong oxidation-resistance composition, therefore be widely used in various bread and cheeses such as cake, cheese etc. the America and Europe, both can be used as nutrition-fortifying agent, can be used as natural antiseptic agent again and use.The cowberry pycnogenols is higher a kind of of quality in all kinds of anthocyanidin, it is reported it have anti-oxidant, anti-inflammatory, promotion Visual purple synthetic, improve immunizing power, different physiological roles [12,13] such as anticancer.

Pycnogenols is brought into play bioactive main mechanism and is comprised following 5 aspects:

1) antioxygenation.Nineteen fifty-one, French Jacques Masquelier has found the antioxidant property of OPC first.Pycnogenols has very strong anti-oxidant activity, and reason is to have electron rich hydroxyl in the pycnogenols molecule, 8 phenolic hydroxyl groups all with two key conjugation, be the donor of hydrogen atom, and the conjugated double bond on the aromatic ring is stablized electronics in molecule.Maffei etc. studies show that Semen Vitis viniferae extract pycnogenols (GSPE), high molecular weight components catechin oligomer can prevent vitamin-E loss in the cytolemma when 0.1-10mmol/L, significantly reduce the membrane lipid peroxidation, obviously postpone hemolytic time of origin, significantly dose-dependently ground is induced at hydroxyl radical free radical and is made vitamin-E regeneration in the snperoxiaized autoxidation mutually of Yelkin TTS, and delaying it and consume, is the physiological regeneration agent of vitamin-E.Research proves that simultaneously grape pip procyanidin suppresses the snperoxiaized effect of Yelkin TTS (PLC) and obviously is better than the contrast catechin, IC50 is respectively 2.5 μ mol/L and 50 μ mol/L. suppress PLC inductive phase and the interim conjugated dienes formation of propagation and increase active IC50 to be respectively 0.1 μ mol/L and 0.5 μ mol/L, obviously be better than the positive controls alpha-tocopherol, its IC50 (the required concentration of 50% free radical inactivation) is respectively 1.5 μ mol/L and 1.25 μ mol/L, but this pycnogenols also noncompetitive ground suppresses to promote that the XOD IC50 of oxonium ion formation is 2.4 μ mol/L, is better than the Zyloric [14] of contrast.Lotito etc. are the lipoid peroxidization resistant of model research pycnogenols with the liposome, when the oxygenizement of lipid originates in water, and the monomer of pycnogenols, dimer, trimerical antioxygenation maximum.When oxidation occurred in the lipid district, pycnogenols high-molecular weight polymer antioxidant capacity was the strongest, and in the LDL oxidation of Cu mediation, the resistance of oxidation of the pycnogenols of different molecular weight does not but have significant difference [15].When researchs such as Koga once give CSPE (25m/kg) fasting rat plasma antioxidant levels is influenced, find that taking the photograph GSPE can improve the resistibility [16] of blood plasma to oxidative stress.The anti-oxidant activity of pycnogenols and its structure have closely related property, and the resistance of oxidation of the pycnogenols of different polymerization degree and configuration is also different.

2) remove free radical.Pycnogenols is removed the ability and its molecular structure, the polymerization degree and relevant with the degree of training acid estersization of free radical.And single molecular structure (is example with the dimer) from pycnogenols, per molecule ProcyanidinB-3 (8 phenolic hydroxyl groups are arranged) can catch 8 oxyradicals at most, and molecule Ve (1 phenolic hydroxyl group is arranged) only can catch 1 free radical, and per molecule Vc (2 conjugation hydroxyls are arranged) can pounce on and obtain 2 oxyradicals.So pycnogenols not only can be removed free radical, and can help to preserve and regeneration Vc, Ve, it also can reinforce capillary wall by making elastin and collegen filament simultaneously, thereby further defends the erosion of free radical.According to the literature: pycnogenols retention time in vivo reaches 72h.And the retention time of Vc 3h only.This will strengthen the ability of its oxidation and removing free radicals greatly.Bagchi etc. [17] studies show that pycnogenols has very high biological utilisation, are significantly higher than vitamins C, vitamin-E and beta carotene in Green Tea Extract ability and the protection lipid peroxidation and the anti-DNA damnification ability that cause because of free radical.Castillo etc. [18] studies show that: removing on free radical, the anti-oxidant activity ability pycnogenols＞rutin＞catechin＞foreign coriander glycosides 〉=xitix.[19] such as Saint-Cricq De confirm that with the removing free radical activity of 3 kinds of methods mensuration pycnogenolss its radical scavenging activity depends on its chemical structure and three-dimensional arrangement.Adopt high pressure to separate each oligomer with the low pressure liquid chromatography, even concentration is very low, still have greater activity, can remove free radical effectively, it is closely related to find that these polyphenolic compounds are removed the ability of free radicals and their chemical property and stereochemical structure.

3) anti-mutation, antitumous effect.Cancer and many chronic degenerative diseases are by due to the polygenic mutation.And pycnogenols has certain effect aspect anti-mutation.Experiment in vitro proves: pycnogenols reaches 94% to the inhibiting rate of Trp R2 (mutagenic agent in the food), can suppress cereuisiae fermentum plastosome spontaneous mutation and nucleus by the responsive spontaneous mutation [20] to anti-canavanine of canavanine.And its anti-radioactive rays mutagenesis ability is followed successively by pycnogenols＞rutin＞dimethyl sulfoxide (DMSO) (DMS0)＞xitix＞propylthiouracil (PTU)＞foreign coriander glycosides [18].Former cyanine has inhibition TPA (a kind of skin carcinoma promotor) activity, and anticancer change ability is tripolymer＞dimer＞monomer.Pycnogenols acts on the mitotic division signal conduction of human breast carcinoma MDA-MB468, and the cell cycle is gushed control and apoptotic process, causes that growth of tumour cell suppresses, and programmed death or differentiation have prospect very much on breast cancer treatment.The pycnogenols energy selective induction mankind mastopathy cell apoptosis that Huynh etc. [21] discovery is extracted from Cortex Pini, promptly the apoptosis number that records among the human breast cancer cell with the pycnogenols processing is more much higher than untreated allogenic cell; And in the normal human mammary cell sample, the quantity of pycnogenols and not obvious change apoptotic cell.Agarwal etc. [22] have studied the effect of pycnogenols to human prostata cancer DUI45 cell, they find that cell and the lyase group handled with pycnogenols (10-/L) compare, the growth of DUI45 cell obviously is suppressed, and presents dosage and time-dependent manner. and DUI4S can induce the cell of G1 phase static in the mode of dose-dependently.In other human body prostate cancer cell line. also can be observed the effect of growth of pycnogenols anticancer and cancer cell specific induction of apoptosis.The effect that these results of study explanation pycnogenolss have very strong anti-prostate cancer to take place, this may with its adjusting mitotic division, cell cycle, induce the cell of G1 phase static, effects such as cell growth inhibiting and cancer cell specific induction of apoptosis are relevant.In sum, pycnogenols all has in various degree restraining effect to multiple cancer cells such as skin cancer cell, breast cancer cell, prostate cancer cell etc.

4) cardiovascular protection effect.The effect that pycnogenols has the protection blood vessel is because it has enzyme inhibition activity; can destroy the powerful restraining effect of enzyme generation that collagen protein, elastin, hyaluronic acid etc. constitute blood vessel important composition composition to collagenase, Proteinase, bone marrow serine, Unidasa and glucuronidase etc.; can be by the activity of catching active oxygen and regulating and control above-mentioned enzyme to prevent their destruction to angiogenic substance; also can be hyaluronic complete to protect by the activity that suppresses Unidasa and glucuronidase, make it to keep the macromole that high polymer forms.Fitzpatrick etc. studies have shown that: pycnogenols has the diastole effect to blood vessel.The oral single dose 150mg pycnogenols of clinical research confirmations such as Royer can make varicose vein improve tension force.Maffei etc. [14] have studied the prevention of pycnogenols to ischemia ventricle contracture and ischemic arrhythmia.Pycnogenols also has provide protection to capillary blood vessel, so it is also having certain curative effect aspect the treatment ophthalmic diseases.Boissin and Corbe etc. have studied the influence of pycnogenols to vision respectively, and the result shows that the visual performance of clothes pycnogenols person's eyeball behind strong illumination has clear improvement than control group, shows that pycnogenols may make retina obtain better nutrition.

5) improve the function of eyesight.The cowberry pycnogenols is widely used in improving ophthalmic diseasess such as myopia, long sight, asthenopia, nyctalopia, cataract and the retinitis, is the principal item in the natural product ophthalmic remedy.In World War II, the Great Britain and France pilot is before night flying, and informal dress uses cowberry to strengthen the scotopia ability.Japan doctor Ye Shanlong one adopts the pycnogenols goods that 310 people are carried out clinical observation, asthenopia 30 people wherein, myopia 100 people, long sight 100 people, presbyopia 50 people, macula lutea variability disease 30 people took pycnogenols (early, middle and late respectively obey 25 milligrams) every day continuous three months, and effect is obvious: asthenopia, long sight is efficient is 100%; Myopia, presbyopia are 70%; Macula lutea variability disease is 99% (produce effects is 13%).Liu Chunmin etc. are used for adolescent myopia and eye strain disease with cowberry anthocyanidin, have obtained effect preferably.326～18 years old teenager in Shenzhen carried out one month double-blind method test, and the result shows: 1) anthocyanidin can effectively be alleviated or eliminate because of blurring of vision (77.2%), eyeball that eye strain causes are swollen (84.9%), ophthalmodynia (93.1%), photophobia (50.6) eye dry and astringent (87.3%) and eyeball acid are tired out and felt symptoms such as (81.9%).2) anthocyanidin can effectively improve the distant vision situation of near-sighted vulnerable period, early stage myopia and low myopia, efficient 77.12% (P＜0.05) that reaches.But this improvement is generally between 2～3 row (logarithmic visual acuity chart).May there be eye strain in this with the patient, takes that can to improve or alleviate eye fatigue behind the anthocyanidin relevant.3) anthocyanidin is to the dioptric there was no significant difference (P＞0.05) [23] that influences of near-sighted eyeball before and after the treatment.

The mechanism that pycnogenols improves vision comprises many aspects, as improving darkness adaption [24], promote Visual purple regeneration [25,26], changing transient state refraction [27] etc.Existing discovering; anthocyanidin can effectively improve the human eye macula lutea time of recovery of the identification capacity in the dark situation especially; infer that anthocyanidin can increase the microcirculatory blood flow in eyeground; acceleransstoff metabolism exchange; enhancing is to the provide protection of capillary vessel, and then improves macula lutea time of recovery and night vision.After cyanidin extract that nineteen sixty-eight Bastide P etc. has reported 25%w/w is to Visual purple synthetic promoter action, HitoshiMatsumoto utilized the outer ROS film of frog rod photoreceptor cell in 2003, had detected four kinds of anthocyanidin to Visual purple regenerated promoter action.The result shows that 3-glucosides-Cyanidin can stimulate regeneration, but delphinidin does not have promoter action.Simultaneously in the ROS film, do not observe of the obvious influence of above-mentioned anthocyanidin, show that the main effect of anthocyanidin in the rod sight sensor is the regeneration that promotes Visual purple cGMP-phosphodiester enzyme activity.Even studies show that recently after bleaching, opsin has also kept the ability of certain exciting light transduction path, impels Ca2+ density loss in the born of the same parents, thereby has weakened the sensitivity of light of rod photoreceptor cell.Therefore, will accelerate the recovery process of rod photoreceptor cell sensitivity of light to Visual purple regenerated promoter action, this can explain the castering action of cowberry to the scotopia ability.

2, the quality standard of pycnogenols

Polyphenol content is an important indicator of weighing the anthocyanidin quality, and high-quality Semen Vitis viniferae extract requires its polyphenol content 〉=95%.The biological activity of (two, three, the tetramer) better [28] because oligomer in the pycnogenols, so the content of OPCs is the another key index of quality product.With the Semen Vitis viniferae extract is example, and the component proportions of quality products is polyphenol/pycnogenols/OPCs/ monomer=1/0.82/0.81/0.18 on the world market, and the highest domestic level is polyphenol/pycnogenols/OPCs/ monomer=0.98/0.84/0.46/0.12 at present.In general, the ratio of OPCs/ pycnogenols is high more, and quality is also good more.With regard to detection means, the measuring method of polyphenol content mainly contains 4 kinds, i.e. reference substance UV method, Vanillin method, UV empirical value method and GAE method (Folin-Ciocalteu method).The measuring method of procyanidin content mainly contains 3 kinds, differential technique (measure total polyphenols content with the Vanillin method, measure monomer content with the UV method, get its difference), Bates-Smith method, baud method.The measuring method of OPCs mainly is the HPLC method, is reference substance with the OPCs standard substance promptly, utilizes reversed-phase HPLC accurately to measure monomer, two, three in the anthocyanidin sample, tetrameric content.Along with the development in polyphenolic substance market, the more and more necessary of ingredient standardization improves, and the U.S. has set up the EGCg standard in meeting of anthocyanidin precursor Standardization Research and the green tea extract.As the analytical procedure of polyphenolic substance, green tea generally adopts tartrate iron colorimetry now.Other polyphenolic substances are used the forint denis' method always, and this method is the method for AOAC (AOAC) regulation.Owing to lack the public fixed analytical procedure of each phenolic compound, this method commonly used is method as an alternative.For grasping indivedual component contents, available employing HPLC method.The HPLC method need use a considerable amount of standard substance to measure every kind of composition, and is loaded down with trivial details relatively in operation, but quantitatively is individually necessary for the molecular weight distribution of quantitative assay polyphenolic substance with to every kind of composition.

3, the condition of production of cowberry procyanidin extract

Cowberry is an Ericaceae Vaccinium plant, is called as blueberry, indigo, bilberry etc. respectively in the different places of production.Cowberry is grown in the frozen soil throughout the year, and it is blue that fruit is, and drape layer white fruit powder, the pulp exquisiteness, and fruity is sour-sweet, unique flavor.Its nutrient constituents of fruit is abundant, and nutritive value is high, is rare rare wild fruit, is described as " king of berry " [1].

In Cortex Pini and the grape pip procyanidin product, the content of total polyphenols can reach 95% level on the present world market, and the content of OPCs is then relatively low.Both at home and abroad in the Pericarpium Citri tangerinae extract product content of pycnogenols generally in the 5%-95% scope, different with the quality of product.Wherein, use preparation HPLC refining, the purity of procyanidin monomers can be risen to quite high degree, but its cost is also very expensive, still can not satisfy requirement of massive production.In the Pericarpium Citri tangerinae extract of domestic large-scale production, generally between 5-70%, its quality control index is mainly total polyphenols content to anthocyanidin content, still can not carry out effective Quality Control to the content of monomer and oligomer.

According to domestic and international report, the extraction process of cowberry pycnogenols mainly contains: 1) water extraction method: first water extracts water soluble component, carries out extracting and separating with organic solvent again, and organic solvent is reclaimed in distillation at last, obtains procyanidin extract.2) resin adsorption method: anthocyanidin extracting solution spent ion exchange resin is removed carbohydrate and organic acid.3) membrane separation process: utilize various types of ultra-filtration membranes and reverse osmosis membrane that pigment is made in filtering mode.4) recrystallization method.The anthocyanidin content that uses above-mentioned the whole bag of tricks to obtain can reach about 98%, but wherein the polymerization degree is no more than 50% usually at the content of the oligomeric procyanidolics below 5, because oligomeric procyanidolics is unstable under natural placement condition, the oligomeric procyanidolics product difficulty that therefore obtains high level is very big.

The production unit of China's cowberry procyanidin extract is more, concentrates on zones such as Daxing'an Mountainrange, Heilungkiang, Jilin, Liaoning, Sichuan, Tianjin, and the industrial scale that has is very big, and has possessed Equipment Foundations preferably.But a little less than the research and development relative thin of product, a lot of researchs concentrate on composition, constructional feature, chemical property and the biological function etc. of exploring pigment, and are less relatively about the achievement in research of oligomer scale purifying process.Domestic production technology is based on traditional solvent-extraction process binding resin post fractionation by adsorption, and what have combines newer supercritical extraction.Its product can only be controlled total cyanine (look) element basically, because the difference on notion and the detection method, the product purity of different company's propaganda is widely different, from 25% to 98% have, but in fact all be cowberry anthocyanidin crude extract, high-quality oligomeric procyanidolics product is still rare.

The present invention is directed to the problem that exists in the pycnogenols extraction, a kind of more excellent method of extracting oligomeric procyanidolics from cowberry is provided.Studies show that, under dark, deoxidation (and low temperature) condition, adopt the methacrylate resin chromatography can access high-load small molecules pycnogenols.This technology is for producing high-quality cowberry activity extract, and enlarging China's Pericarpium Citri tangerinae extract provides important references in the development space of food, makeup, field of medicaments.For other multiple natural phant that contains OPC, as grape (seed), ginkgo (leaf), black currant, black soya bean, Pale Butterflybush Flower (robinin), Chinese sorghum red pigment, radish red, red rice element, Roselle Calyx is red, mulberry fruit is red, cocoa shell, tealeaves (phenol), betel nut, pomegranate rind, Herba Agrimoniae etc. all help to improve the output of low polymerization degree OPC by using this kind method.

Summary of the invention

In one aspect of the invention, provide a kind of method of extracting oligomeric procyanidolics from the plant material that contains OPC, described method feature is to carry out under low temperature environment, and keeps anaerobic or hypoxemia and lucifuge environment in purification process.

In one aspect of the invention, provide a kind of method of extracting oligomeric procyanidolics from the plant material that contains OPC, described method is included in carries out the following step under the low temperature environment:

(a) extraction step: get the plant material that contains OPC organic solvent lixiviate, collect supernatant and filter, concentrate, concentrated solution is added non-polar solvent extract;

(b) purification step: the concentrated extract that obtains in the step (a) is carried out purifying by macroporous resin, and keep anaerobic or low-oxygen environment and lucifuge environment in this purification process;

With purified product concentrate, freeze-drying promptly obtains desired product.

In one embodiment, described (b) step is specially: macroporous resin column on the concentrated extract that obtains in the step (a) is saturated to macroporous resin adsorption, and wash described resin column to washing fluid with de-oxygenised water again and do not have obvious color; Then described macroporous resin column is washed with eluting solvent, eluted product is concentrated, finished product will promptly be obtained after the concentrated solution freeze-drying, wherein before with the concentrated extract upper prop, earlier described pillar is handled to get rid of oxygen in the post with de-oxygenised water, form oxygen-free environment, and in elution process, in eluting solvent, keep feeding nitrogen to keep the deoxidation environment always.

In one embodiment, the plant material of the described OPC of containing can be but be not limited to cranberry.For other multiple natural phant that contains OPC, as grape (seed), ginkgo (leaf), black currant, black soya bean, Pale Butterflybush Flower (robinin), jowar red pigment, radish red, red rice element, Roselle Calyx is red, mulberry fruit is red, cocoa shell, tealeaves (phenol), betel nut, pomegranate rind, Herba Agrimoniae etc. all help to improve the output of low polymerization degree OPC by using this kind method.

In one embodiment, with the column chromatography path of purification usefulness in (b) step with lighttight material as parcel such as masking foil or with as described in step in the darkroom, operate, to form the lucifuge environment.

In one embodiment, described (a) extraction step is also carried out under the lucifuge environment.

In one embodiment, the present invention extracts the process of oligomeric procyanidolics and carries out under 0-25 ℃ envrionment temperature, preferably under 4-16 ℃ envrionment temperature, more preferably carries out under 4 ℃ envrionment temperature.

In one embodiment, macroporous resin used in the present invention is a methacrylate resin, comprises HP-2MG resin or XAD resin.

In one embodiment, in the post elution process, keep sour environment.In one embodiment, use the eluting solvent of pH=3-6 that resin column is carried out wash-out.

In one embodiment, extraction solvent used in the present invention can be methyl alcohol, ethanol or acetone, the acetone of especially preferred 70% (v/v), and the acetone of preferred precooling.

In one embodiment, non-polar solvent used in the present invention is sherwood oil and chloroform.

In one embodiment, eluting solvent used in the present invention is an ethanol.In one embodiment, eluting solvent used in the present invention is the ethanol of 10%-50%-75%-95%.

In one embodiment, upward column temperature of the present invention is 4-10 ℃, can use room temperature in case of necessity, but be no more than 30 ℃.

In one embodiment, described liquid material can be 1: 1 than (ratio of extraction solvent and raw material), and 2: 1,3: 1,4: 1,5: 1, preferred 1: 1 to 3: 1, most preferably the liquid material was than 3: 1.

In one embodiment, described liquid material is preferably the ratio of 70% acetone and cowberry pomace than the ratio for acetone and cowberry pomace.

In one embodiment, the extraction time in described (a) step is 1 hour.

In one embodiment, described de-oxygenised water is the deoxidation distilled water of pH 3.0.

Aspect another, provide the pycnogenols product that is obtained by aforesaid method in the present invention, it is characterized by, be rich in the pycnogenols oligomer, extract places under the natural condition, and the autoxidation phenomenon can take place.

In one embodiment, the main component of wherein said pycnogenols product is the following composition of 4 aggressiveness.

In one embodiment, the anthocyanidin content of wherein said pycnogenols product reaches 98% (w/w), and pycnogenols (the containing monomer) content below 4 aggressiveness is up to more than 90% (w/w).

In another aspect of the present invention, provide the pycnogenols product that obtains by aforesaid method to be used to remove ultra-oxygen anion free radical O ₂ ^-, the application of hydroxyl radical free radical HO.

The preferred embodiment of the invention describes in detail

Especially, the invention provides a kind of method of extracting oligomeric procyanidolics from cranberry, particular content is as follows.

Following operating process is all carried out under 4-16 ℃ envrionment temperature, preferably carries out under 4 ℃.Get clean Fructus vaccini vitis-idaeae, with the homogenate stirrer with its pulping, the centrifugal 30min of 4000rpm, fruit juice reserves for other use, get pomace with organic solvent (being generally the acetone of the 70%v/v) lixiviate of precooling 1 hour, the weight ratio of pomace and extraction solvent is 1: 3, temperature at room temperature, the centrifugal 30min of 4000rpm, the collection supernatant filters, vacuum concentration to organic solvent all steams (organic solvent of recovery can reuse), concentrated solution adding volume ratio is 4: 1 a mixing non-polar solvent (sherwood oil and chloroform), equal-volume extraction 2～3 times.

The methacrylic ester macroporous resin is adorned post with wet method, standby after the pre-treatment (pre-treatment step is seen below).Before with above-mentioned concentrated extract upper prop, earlier pillar is walked 1～2 volume with de-oxygenised water (feeding the oxygen that nitrogen can be removed in the solution in 5-20 minute in distilled water), to get rid of oxygen in the post, form oxygen-free environment, simultaneously with whole column chromatography path (peristaltic pump, emulsion tube, glass fibre reinforced plastic prop etc.) with the masking foil parcel, form no luminous environment.The concentrated extract upper prop is saturated to macroporous resin adsorption, and being eluted to washing fluid with acid de-oxygenised water does not again have obvious color (the OPC acidic conditions is down for red, be eluted to do not have explanation OPC is adsorbed on the resin column substantially when obviously red).And then use the organic solvent (ethanol) of 10%-50%-75%-95% to wash respectively, it is 3～6 that each concentration wash-out organic solvent is all adjusted pH.In elution process, in eluting solvent (ethanol), keep feeding nitrogen, to keep the deoxidation environment always.With eluting solvent with 0.5～4 column volume/hour speed cross post.Collected the liquid behind the post.During the effluent liquid very slight color, stop eluting solvent and cross post.Write down each eluted product volume.10%, 50%, 75%, 95% the eluted product of collecting is concentrated at 40～70 ℃ with Rotary Evaporators, will obtain finished product after the concentrated solution freeze-drying that obtain.

The pre-treatment step of resin:

The macroporous resin that uses among the present invention is a methacrylate resin, comprises XAD resin or HP-2MG resin, and resin is crossed the 100-200 mesh sieve in advance, spends the night with 95% alcohol immersion, washes with water to no oyster white again; Hydrochloric acid soln with 4% soaked 4 hours, the water flushing; Aqueous slkali soaking with 4% 4 hours, water flushing again; Sodium phosphate dibasic-citrate buffer solution with pH=5 is washed, again water wash to pH between 5-6; Dress post, compacting is standby.

Description of drawings:

Fig. 1: the RP-HPLC figure that extracts the cowberry pycnogenols product that obtains with the inventive method.

Fig. 2: different extraction solvents are to the influence of pycnogenols extraction yield.

Fig. 3: different acetone concentration are to the influence of pycnogenols extraction effect.

Fig. 4: the varying environment temperature is to the influence of pycnogenols extraction effect.

Fig. 5: the influence of different liquid material comparison pycnogenols extraction effects.

Fig. 6: different extraction times are to the influence of pycnogenols extraction effect.

Fig. 7: antioxidant kind and concentration thereof are to the influence of ultra-oxygen anion free radical clearance rate.

Fig. 8: antioxidant kind and concentration thereof are to the influence of hydroxyl radical free radical clearance rate.

Fig. 9: OPC sample anti-oxidant vigor is along with the decay of time.

Figure 10: the pycnogenols product that obtains under the different condition relatively.

The cowberry oligomeric procyanidolics product that A prepares with the embodiment of the invention 1 method.Described product presents red-purple metalluster under natural light, crystalline particle is arranged.

B. do not apply deoxidation, lucifuge, temperature control condition, with the cowberry pycnogenols product of HP-2MG resins, color is purple black.

C. the cowberry pycnogenols product that Japanese KND Co., Ltd. purifies (common commercially available prod at present), anthocyanidin content 25% is Powdered.

Embodiment

Embodiment 1 extractsPycnogenols in the cowberry (4 ℃ envrionment temperatures)

Operating process in the present embodiment 1 is all carried out under 4 ℃ envrionment temperature (adopting the air regulator temperature control).

After Daxing'an Mountainrange bog bilberry squeezing removed juice, take by weighing pomace 100g, the 70%v/v acetone 300ml that adds precooling (4 ℃) extracts 1h, the centrifugal 30min of 4000rpm, collect the filtering filtrate of supernatant, vacuum concentration, (the pillar filler is selected the HP-2MG resin for use with the concentrated solution upper prop then, 4 ℃), wherein on pillar, wash 1～2 column volume with the de-oxygenised water (distilled water) that feeds nitrogen before the sample.The concentrated solution upper prop is saturated to the HP-2MG resin absorption, last sample finishes, be eluted to washing fluid with the deoxidation distilled water of pH=3.0 again and do not have obvious redness (for red, be eluted to does not have that explanation OPC is adsorbed on the resin column substantially when obviously red to OPC under acidic conditions).10%-50%-75%-95% ethanol with pH=3.0 washes described chromatography column respectively then, with eluting solvent with 0.5 column volume/hour speed cross post.In eluting solvent, feed nitrogen during the wash-out, to keep the stability of system always.Collected the eluting liquid behind the post.When the effluent liquid very slight color, stop wash-out, write down each eluted product volume.50% and 75% cut of collecting is carried out vacuum concentration with Rotary Evaporators (RE-52A rotation vacuum-drying instrument, Shanghai Yarong Biochemical Instrument Plant) at 40 ℃.The concentrated solution freeze-drying is got the product that red-purple has metalluster, and after testing, total polyphenols content reaches following pycnogenols (the containing monomer) content of 98%, 4 aggressiveness up to 90% in the described product.

In above-mentioned column chromatography process,, make sample in the chromatography process, be in the lucifuge state by in the darkroom, operating.

The pre-treatment step of resin: described HP-2MG resin resin is crossed the 100-200 mesh sieve in advance, spend the night, wash with water again to no oyster white with 95% alcohol immersion; Hydrochloric acid soln with 4% soaked 4 hours, the water flushing; Aqueous slkali soaking with 4% 4 hours, water flushing again; Sodium phosphate dibasic-citrate buffer solution with pH=5 is washed, again water wash to pH between 5-6; Dress post, compacting is standby.

A) HPLC chromatographic separation result:

The HPLC chromatographic condition

Adopt Alltima C ₁₈Reverse-phase chromatographic column (250mm * 4.6mm, 5 μ m) column temperature: 25 ℃; Moving phase is 2% aqueous formic acid and 2% formic acid methanol solution, behind the filtering with microporous membrane of 0.45 μ m, divide 2 gradients to carry out wash-out: during 1～5min graded for from 95: 5 to 95: 5, and during 5～50min from 95: 5 to 0: 100. flow velocity: 1ml/min; Detect wavelength: 278nm; Sample size: 20 μ l, Semen Vitis viniferae standard substance sample size are 100 μ l.

The mass spectrum condition

With the lasting sample introduction of the speed of 1 μ l/min, the ion detection mode is single reaction ion detection (SRM) with syringe; Ion polarity is positive ion (positive); The ionization mode is electro-spray ionization (ESI); Capillary voltage is 2.0kV; Ion source temperature is 200 ℃; Sweep limit m/z 75-2000; Collision energy 35.0V.

Cowberry procyanidin extract by embodiment 1 preparation the results are shown in Figure 1 after chromatographic column is separated, each peak appearance time and relative content see Table 1, and with calculated by peak area, topmost 8 elution peaks are below 4 aggressiveness, and its area integral content reaches 90%.Compare with 95% grape pip procyanidin extract, go out peak number amount and appearance time and all exist than big-difference, the HPLC separating resulting shows that two kinds of products are obviously different on composition.

The HPLC retention time and the nucleo plasmic relation of table 1 anthocyanidin elution peak

B) polyphenol content is measured

With 10 milliliters of dissolve with ethanol 0.5000 gram gallic acid, 100 milliliters of constant volumes, pipette 0.0,1.0,2.0,3.0,5.0 respectively, 10.0mL in the 100mL volumetric flask, use the distilled water constant volume.Add forint-Xue and block many reagent colour developments, under the 765nm wavelength, measure absorbancy.Each concentration do two parallel, average, the production standard curve, its regression equation is: Y=9.4607X-0.024, R ²=0.9993

Extracting sample solution 1mL, according to above-mentioned standard curve making method, measure its absorbancy (each sample do two parallel, average), polyphenol content in the calculation sample.

Polyphenol content=(C * V * n/M) * 100% is C wherein: gallic acid concentration (mg/mL); V: liquor capacity (ml); N: extension rate; M: sampling amount (mg).

The result records that polyphenol content is 90% (w/w) in the described procyanidin extract

C) procyanidin content measuring method

Flavonol is the basic framework molecule of anthocyanidin, use the Vanillin method can working sample in the content of total Flavonol.(taking by weighing the 1.000g Vanillin is dissolved in the methanol solution with 1% Vanillin methanol solution, last constant volume is to 100mL) and 8% hydrochloric acid methanol liquid (getting the 8mL concentrated hydrochloric acid is dissolved in the methyl alcohol, be settled to 100mL) with 1: 1 ratio preparation, can obtain developer (necessary matching while using).

The catechin methanol solution of preparation normal concentration, colour developing under the 500nm wavelength, is measured its absorbancy, and the typical curve equation that obtains is that Y=0.0016x 1, wherein R ²=0.9959.

Accurately take by weighing pycnogenols powder 1.0g after the freeze-drying that is obtained by embodiment 1 method, add 10mL methyl alcohol, the vortex concussion was extracted 20 minutes.With the centrifugal 15min of extracting solution 10000rpm.Get the supernatant liquor constant volume to certain volume (procyanidin content and decide) per sample, get 1mL constant volume liquid again, add the 5mL developer, carry out color reaction, measure absorbancy at 500nm wavelength place, calculate procyanidin content.

Pycnogenols (%)=(m ₁/ m) * (V ₂/ V ₁) * 10 ^-6* 100%

m ₁For the establishing criteria opisometer is calculated procyanidin content in the test solution (mg); M is the quality (g) of sample; V ₁Be 1mL; V ₂Constant volume (mL) for sample.

The result records that procyanidin content is 98% (w/w) in the described procyanidin extract.

Be the effect that shows that this patent inventive point is brought, now show the picture (see figure 10) of the product that obtains under three kinds of different conditions.Contrast shows that it is purplish red adopting the resulting product colour of embodiment 1 condition, presents metalluster under the natural light, and crystalline particle (seeing Figure 10 A) is arranged, and shows that wherein the purity of target component is higher.And it is purple black not carry out the resulting product colour of deoxidation, lucifuge, temperature control (all the other conditions are described identical with embodiment 1), shows that wherein quinones content increases, and oxidized degree is dark (seeing Figure 10 B).The like product that Japan provides then presents Powdered (seeing Figure 10 C).

Embodiment 2 extractsPycnogenols in the cowberry (16 ℃ envrionment temperatures)

Operating process in the present embodiment 2 is all carried out under 16 ℃ envrionment temperature (adopting the air regulator temperature control).Other conditions and step are fully consistent with embodiment 1 with raw material.After testing, resulting cowberry pycnogenols product and embodiment 1 basically identical, procyanidin content is 95% (w/w), following pycnogenols (the containing monomer) content of 4 aggressiveness reaches 90% (w/w).

Embodiment 3

Used extraction pycnogenols method condition step and raw material in the present embodiment, mensuration total polyphenols, pycnogenols are consistent with embodiment 1 with the method for total Flavonol.

1.1 definite (except the solvent difference that adopts, all the other method conditions and raw material are all identical with embodiment 1) of extraction solvent

Methyl alcohol, ethanol, acetone are three kinds of aldehydes matter extraction agents commonly used.Select for use ethanol, methyl alcohol, the acetone of same concentrations to extract, determine best extraction solvent, the result as seen from Figure 2, methyl alcohol and acetone are better than ethanol to the extraction effect of polyphenol, and be difficult for to introduce impurity.Methyl alcohol toxicity is bigger.Therefore, this experimental selection acetone is as the extraction solvent of the best.

1.2 experiment of single factor

Select extraction solvent concentration, extract envrionment temperature, solid-liquid ratio (raw material such as cowberry pomace, Semen Vitis viniferae dry powder, Ginkgo Leaf etc.) and soak the ratio of body solvent (as acetone)), factor such as extraction time investigates the influence to total phenol extraction rate and pycnogenols extraction yield, to determine that correlative factor and scope thereof are (except described factor changes, all the other method conditions are all identical with embodiment 1), result such as Fig. 3-6.

By Fig. 3 (variable is that acetone concentration is by 40%-100% (v/v)) as can be known, the pycnogenols extraction yield maximum of 70% (v/v) acetone.Pure acetone contains carbonyl, can produce hydrogen bond association with the pycnogenols molecule.Because acetone polarity is less, and is lower to the dissolving power of pycnogenols molecule.By adding the polarity size that the bigger water of polarity can be regulated acetone solvent, along with acetone soln polarity increases, the pycnogenols extraction yield obviously improves.When acetone soln concentration reaches 70%, the extraction yield maximum of pycnogenols.

As can be known, in the time of 4 ℃ (envrionment temperature), the extraction yield of cowberry pycnogenols is the highest by Fig. 4 (variable is an envrionment temperature, is respectively 0 ℃, 4 ℃ and 25 ℃).Because pycnogenols contains poly-hydroxy, in the presence of illumination and oxygen, its less stable, the temperature height is extracted in easily oxidation, polymerization, and the pycnogenols extraction yield is descended.

Among Fig. 5, the liquid material was than (ratio of the acetone of 70%v/v and cowberry pomace) 1 group 1: 1,2 groups 2: 1,3 groups 3: 1,4 groups 4: 1,5 groups 5: 1, as seen from Figure 5, liquid material ratio is 1: 1 to 3: 1 o'clock, and the pycnogenols massfraction significantly increases, the liquid material was pycnogenols massfraction vertex than 3: 1, continued to increase the liquid material and slightly descended than pycnogenols massfraction.

As seen from Figure 6, along with the prolongation of extraction time, the pycnogenols massfraction is on a declining curve, thinks when 1h and extracts fully, so the selection extraction time is 1h.

1.3 the selection of macroporous adsorbent resin

By 6 kinds of absorption with macroporous adsorbent resin cowberry pycnogenolss are tested, by following table 2 as seen, the Semi-polarity resin is higher to the pycnogenols adsorption rate, and polar resin takes second place, and non-polar resin is minimum; Nonpolar bigger to the pycnogenols desorption efficiency with the Semi-polarity resin, polar resin is less.This may be relevant with structure (containing polar hydroxyl and nonpolar phenyl ring) and the polarity and the structure of resin of pycnogenols, take all factors into consideration absorption and desorb situation, select methacrylate type resin purification cowberry pycnogenols, be fixed to the HP-2MG macroporous adsorbent resin that MIT produces.

8 kinds of macroporous resins of table 2 are to the comparison of pycnogenols adsorptive capacity

Embodiment 4

Cowberry pycnogenols and grape pip procyanidin are to ultra-oxygen anion free radical O ^2- Scavenging(action)

Ultra-oxygen anion free radical O ₂ ^-Forming the earliest in vivo, is the predecessor of other oxyradical, and its toxicity is the major cause of body generation oxygen intoxication.Utilize pyrogallol-luminol,3-aminophthalic acid cyclic hydrazide luminescence system to study the scavenging(action) of cowberry proanthocyanidin, grape pip procyanidin to superoxide anion.Under alkaline condition, the pyrogallol autoxidation produces ultra-oxygen anion free radical, this free radical stimulate luminous agent luminol,3-aminophthalic acid cyclic hydrazide makes it to be in the excited state negative ion, emit energy and get back to ground state, send the chemical cold light of wavelength X=430nm, in certain concentration range, luminous intensity and ultra-oxygen anion free radical quantity are linear.Can adopt the biochemiluminescence instrument to detect luminous intensity, so in the investigation system antioxidant to the removing ability [16] of ultra-oxygen anion free radical.

From Fig. 7 as seen, X-coordinate 1 is represented 20 μ g/mL, and 2 represent 40 μ g/mL, and 3 represent 80 μ g/mL, and the ability of antioxidant for clearing ultra-oxygen anion free radical (being clearance rate) size order is cowberry pycnogenols＞grape pip procyanidin＞Vc.And for a kind of antioxidant, along with its concentration is elevated to 80 μ g/mL by 20 μ g/mL, it removes the also corresponding raising of ability of ultra-oxygen anion free radical. to the 47.08 μ g/mL that are about of the 503nhibiting concentration Vc of ultra-oxygen anion free radical, grape pip procyanidin be about 37.55 μ g/mL, the cowberry pycnogenols be about 28.35 μ g/mL, 503nhibiting concentration is more little, and its ability of removing ultra-oxygen anion free radical is strong more.This shows that the cowberry pycnogenols is to O ₂ ^-The removing ability significantly be better than grape pip procyanidin and Vc.

Cowberry pycnogenols and grape pip procyanidin are to the scavenging(action) of hydroxyl radical free radical HO

Hydroxy radical qiao is the strongest to organism toxicity in the active oxygen known at present, that harm is maximum a kind of free radical.Its reactivity is extremely strong, and the life-span is very short, almost can react with all cells composition, and is very harmful to body, but its steam circle is little, only can with contiguous molecular reaction.[9]

As seen from Figure 8, X-coordinate 1 is represented 5 μ g/mL, and 2 represent 10 μ g/mL, and 3 represent 20 μ g/mL, and 4 represent 40 μ g/mL, and antioxidant knows that the ability size order of hydroxy radical qiao is cowberry pycnogenols＞Vc＞grape pip procyanidin.And for a kind of antioxidant, along with concentration raises, the corresponding raising of ability of its removing hydroxy radical qiao.Calculate as can be known, to the 12.70 μ g/mL that are about of the 503nhibiting concentration Vc of hydroxy radical qiao, grape pip procyanidin be about 14.03 μ g/mL, the cowberry pycnogenols be about 11.32 μ g/mL, 503nhibiting concentration is more little, its ability of removing hydroxyl radical free radical is strong more.This shows that the cowberry pycnogenols is better than grape pip procyanidin and Vc to the removing ability of hydroxy radical qiao.

OPC sample anti-oxidant vigor is along with the decay of time

Under deoxidation and the dark condition, prepared OPC sample has better anti-oxidant vigor, and when leaving standstill in air, anti-oxidant vigor can be decayed gradually.Compare with the product under the collating condition, its time-the vigor curve is significantly different.(X as shown in Figure 9; Hour; The OD value of Y:760nm):

Reference

[1]http://www.blueberry.net.cn

[2]Bagchi?D，Krohn?RL，Bagchi?M，et?al.Oxygen?free?radical?scavengingabilities?of?vitamins?Cand?E，and?a?grape?seed?Proanthocyanidin?extract?invitro.Res?Commum?Mol?Pathol?Pharmacol[J].1997，95，179-189.

[3]Shantaram?S.J.，Charles?A.K，et?al.Chemopreventive?effects?of?grape?seedproanthocyanidin?extract?on?Chang?liver?cell.Toxicology[J].2000，153(1-3)，83-90.

[4]Brasseur，T.Flavonoids?as?medicines(Medicaments?renfermant?desflavonoides).J.de?Pharmaciede?Belgique[J].1989，44，403-410.

[5]Bonnaure，Mallet，Martine，Andress.Monomeric?and?oligomericprocyanidines?for?the?treatment?of?parodontal?diseases.IPC?A61K31/35.EPO301975.1989.

[6]Bernhart?claude(FR).Process?for?preparing?pyrrole?derivatives.Applicant；SANFI.IpcC07D207/327.KR8500431.1985.

[7]Brekhman，mudzhiri，et?al.Alcoholic?drinks?with?additive?mixture?to?combatdrunkenness.Applicant：INST?Biolog?Morya?Dalnevostoch.IPC?C12GIT1214054，1990.

[8]Kontek?Armand(RO)，et?al.Weakly?alcholic?effervescent?beverage?obtainingprocess.Applicant：INST?DE?CERCETARIPENTRU?VITIC(RO).RO100432.1992.

[9]Sudheer?K.M.and?Santosh?K.K.Grape?seed?proanthocyanidins?inhibite?UVradiation?induced?oxidative?stress?and?activation?of?MAPK?and?NFKBsignaling?in?human?epidermal?keratinocytes.Free?Radical?Biology?andMedicine[J].Available?online?26January，2006.

[10]Bombardelli?Ezio(IT)，Sabadie?Michel(FR).Phospholipid?complexes?ofextracts?of?vitis?vinifera，their?preparation?process?and?pharmaceutical?andcosmetic?compositions?containing?them.Applicant：INDENA?SPA(IT)，SANOFISA(FA).IPCA61K31/685.ECA61K8/55C.US4963527，1990.

[11]Masquelier，J.Vinetatherosclerose.Action?protectrice?des?procyanidines.?Compte?rendu?de?Simposium?Internationale?Lalimentation?et?laconsomation?du?vin，Verone，Italie，1982，147-155.

[12]Anna?Rizzolo，Renato?C?Nani，Daniela?Viscardi，et?al.Modification?ofglass?transition?temperature?through?carbohydrates?addition?andanthocyanin?and?soluble?phenol?stability?of?frozen?blueberryjuices[J].Journal?of?Food?Fngineering，2003，56(2-3)：229-231

[13]Sharma?SD，Katiyar?SK.Dietary?grape-seed?proanthocyanidin?inhibitionof?ultraviolet?B-induced?immune?suppression?is?associated?with?inductionof?IL-12[J].Carcinogenesis，2006，27(1)：95-102

[14]Maffei?Facino?R，Carini?M，Aldini?C?et?al.Sparing?effect?to?ptocyanidinsfrom?Vitia?vinifera?on?vitaminE.Planta?Med，1998，61(1)：343

[15]Lotito?SB，Actis-Goretta?L，Renart?ML，et?al.Influence?of?oligomer?chainlength?on?the?antioridant?activity?of?procyanidins.Biochem?Res?Comun，2000，276(3)：945-951

[16]Koga?T.，Moro?K.，Nakumori?K，et?al.Icrease?of?antioxideative?protential?ofrat?plasma?by?oral?administration?Of?proanthocyanidin-rich?extract?fromgrape?seeds.J?Agric?Food?Chem，1999，47(5)：1892.

[17]Bagchi?D，Bagchi?M，Stohs?SJ，etal.Free?radicals?and?grape?seedproanthocyanidin?extract：importance?in?human?health?and?diseaseprevention.Toxicology，2000，148(2-3)：187-197.

[18]Castiuo?J，Benarente-Garcia?O，Corente?J，etal.Antioxidant?activity?andradioprotective?effects?against?chromosomal?damage?induced?in?vivo?byX-rays?of?flavah-3-ols?from?grape?seed：comparative?study?versua?otherphenolic?and?organic?compounds.J?Agric?Food?Chem，2000，48(5)：1738-1745.

[19]Saint?Cricq?De?GanlejacN，Provoabc，Vivas?N.et?al.Polyphenol?profiles?ofFrench?eider?apple?varieties.J?Agric?Food?Chem，1999，7(2)：4255

[20] state plants, Xu Li. pycnogenols: tool plant amedica with broad prospects for development. and World Notes on Plant Medicine, 1996,11 (5): 196-204.

[21]Huynh?TH.Teel?RW.Selective?induction?of?apoptoois?in?human?mommary?cancer?cell[MCF7]by?pyonogenol.Anticancer?Res.2000，20(4)：2417

[22]Agamal?C?Sharma?Y，Agarwal?R.Studies?of?procyanidins?on?DUI45?cellsOf?human?prostatic?Cancer.Mol?Carcinog.2000，28(3)：129

[23] Liu Chunmin, Zhou Ling, Wang Kangmei. anthocyanidin is to the influence of adolescent myopia eyesight and eye strain symptom. Chinese prescription drugs, 2004,8 (29): 72-73.

[24]Scialdone，D.L’Azione?delle?antocianine?sul?senso?luminoso.Ann.Oftalmol.Clin.Ocul.1966，92，43-51.

[25]Bastide，P.；Rouher，F.；Tronche，P.Rhodopsin?et?anthocyanosides.Bull.Soc.D’Ophtalmologie?Fr.1968，9，801-807.

[26]Hitoshi?Matsumoto，Yuko?Nakamura，Shuji?Tachibahaki，SatoruKawamura，Masao?Hirayama.Stimulatory?Effect?of?Cyanidin?3-Glycosideson?the?Regeneration?of?Rhodopsin.J.Agric.Food?Chem.2003，51，3560-3563.

[27]Nakaishi，H.；Matsumoto，H.；Tominaga，S.；Hirayama，M.Effects?ofblackcurrant?anthocyanosides?intake?on?dark?adaptation?and?VDTwork-induced?transient?refractive?alternation?in?healthy?humans.Altern.Med.ReV.2000，5，553-562.

[28]packler?L，rimbach?g，virgili?f.Antioxidant?activity?and?biologic?propertiesof?a?procyoniding-rich?extract?form?pine?bark，pycnogenol.Free?rad?boilmed，1999(27)：704-724.

Claims (18)

1. method of extracting oligomeric procyanidolics from contain the OPC plant material, described method feature is to carry out under low temperature environment, and keeps anaerobic or hypoxemia and lucifuge environment in purification process.

2. the method for claim 1, described method is included in carries out the following step under the low temperature environment:

(a) extraction step: get the plant material that contains OPC organic solvent lixiviate, collect supernatant and filter, concentrate, concentrated solution is added non-polar solvent extract;

(b) purification step: the concentrated extract that obtains in the step (a) is carried out purifying by macroporous resin, and keep anaerobic or low-oxygen environment and lucifuge environment in this purification process;

With purified product concentrate, freeze-drying promptly obtains desired product.

3. the method for claim 2, wherein said (b) step is specially: macroporous resin column on the concentrated extract that obtains in the step (a) is saturated to macroporous resin adsorption, wash described resin column to washing fluid with de-oxygenised water again and do not have obvious color; Then described macroporous resin column is washed with eluting solvent, eluted product is concentrated, finished product will promptly be obtained after the concentrated solution freeze-drying, wherein before with the concentrated extract upper prop, earlier described pillar is handled to get rid of oxygen in the post with de-oxygenised water, form oxygen-free environment, and in elution process, in eluting solvent, keep feeding nitrogen to keep the deoxidation environment always.

4. claim 1 or 2 method, the plant material of the wherein said OPC of containing is a cranberry, grape (seed), ginkgo (leaf), black currant, black soya bean, Pale Butterflybush Flower (robinin), Chinese sorghum red pigment, radish red, red rice element, Roselle Calyx fibre, mulberry fruit are red, cocoa shell, tealeaves (phenol), betel nut, pomegranate rind or Herba Agrimoniae.

5. each method of claim 1-4 is wherein operated with lighttight material parcel or with described step the column chromatography path of purification usefulness in (b) step, to form the lucifuge environment in the darkroom.

6. the method for claim 2, wherein said (a) extraction step is also carried out under the lucifuge environment.

7. claim 1 or 2 method, the process of described extraction oligomeric procyanidolics is carried out under 0-25 ℃ envrionment temperature.

8. the method for claim 7, described envrionment temperature is 4-16 ℃.

9. the method for claim 2, wherein used macroporous resin is a methacrylate resin.

10. the method for claim 9, wherein used methacrylate resin is HP-2MG resin or XAD resin.

11. the method for claim 2 wherein keeps sour environment in the post elution process.

12. the method for claim 2, wherein employed extraction solvent is the acetone of 70% (v/v) in (a) step.

13. the method for claim 2, wherein the ratio of extraction solvent and raw material is 3: 1 described in (a) step.

14. the method for claim 2, the extraction time in wherein said (a) step is 1 hour.

15. a pycnogenols product that is obtained by each method of claim 1-14 is characterized by, and is rich in the pycnogenols oligomer, extract places under the natural condition, and the autoxidation phenomenon can take place.

16. the pycnogenols product of claim 15, the main component of wherein said pycnogenols product are the following composition of 4 aggressiveness.

17. the pycnogenols product of claim 16, the anthocyanidin content of wherein said pycnogenols product reach 98% (w/w), pycnogenols (the containing monomer) content below 4 aggressiveness is up to more than 90% (w/w).

18. each pycnogenols product is used to remove ultra-oxygen anion free radical O among the claim 16-17 ₂ ^-, the application of hydroxyl radical free radical HO.

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