Process for purification of proanthocyanidin oligomer

Details Process for purification of proanthocyanidin oligomer Process for purification of proanthocyanidin oligomer

2001-10-19

2004-10-05

Leary, Louise N. (Department: 1654)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

C435S018000, C435S019000

Reexamination Certificate

active

06800433

ABSTRACT:

TECHNICAL FIELD
The present invention relates to a process for efficiently purifying proanthocyanidin oligomers with high purity which have a variety of biological activities including antitumor, anti-inflammatory, anti-aging, antioxidant, antiallergy, antibacterial, and hair growth activities, and can usefully be applied to foods, cosmetics, drugs or the like.
BACKGROUND ART
Generally, proanthocyanidin, which is known as a biophylaxis substance of higher plants, is a generic name for polymers in the form of a dimer or higher order which are polymerized by binding formats, such as 4&bgr;→6, 4&bgr;→8, 4&bgr;→8·2&bgr;O→7, with flavan-7-ol as a constitutional unit. These are also called condensed tannins, (“E. Steinegger & R. Hänsel, Pharmacognosy [1
st
vol.], Approach to Chemistry and Pharmacology” (Translated by Shuji Itokawa et al., Hirokawa Publishing Co.), 204-208 (1977); Porter L. J., Flavans and proanthocyanidins, In: Harborne, J. B. (ed.), “The Flavonoids, Advances in Research Science 1986”, Chapman & Hall, 23-55 (1994)). These are generally called proanthocyanidin because they produce anthocyanidin and turn red by acid treatment. Proanthocyanidins are known to show a variety of biological activities. The activities that have been reported include antitumor, anti-inflammatory, anti-aging, antioxidant, antiallergy, antibacterial, and hair growth activities [Bart Schwitters/Jack Masquelier, “21
st
century Biophylaxis Substance OPC”, translated by Akira Sasaki, Fragrance Journal, 50-135 (1997); Tomoya Takahashi, et al., Journal of Investigative Dermatology, 112, 310-316 (1999)]. Not all the relationships between structure and activity, between these biological activities and the degree of polymerization of proanthocyanidins have been clarified. For example, regarding hair growth activity, dimeric to pentameric proanthocyanidin oligomers (especially dimer and trimer) in proanthocyanidins have been reported to have the highest activity (WO96/00561).
Regarding separation and purification of proanthocyanidins from plant bodies, attempts have been made to separate and purify proanthocyanidins from various plant bodies including grape seeds, pine barks, ginkgo leaves, peanuts, and cocoa beans. Examples of industrial extraction from raw materials in these include the extraction from grape seeds (Japanese Published Unexamined Application No.3-200781,W097/39632, U.S. Pat. No. 5,484,594), pine bark (U.S. Pat. No. 4,698,360,W097/44407) or the like. In the method according to Japanese Published Unexamined Application No.3-200781, a pretreatment is performed by allowing white grape seeds to contact with water at less than 70° C., followed by extraction by hot water. The resulting extract is applied to a Sephadex LH-20™ column, and then eluted with 70% ethanol, thereby obtaining proanthocyanidins-containing powder with a purity of approximately 90%. In the method according to U.S. Pat. No. 4,698,360, 1 ton of pinaster bark is subjected to hot water extraction under pressure, and then ethyl acetate elution and precipitation by addition of chloroform are repeated, so that proanthocyanidins-containing powder is obtained. However, none of the purified products resulting from the above methods contains 90% or more of dimeric to pentameric proanthocyanidin oligomers only. All of these purified products also contain monomers, hexameric or higher order polymers, or other organic acids.
As a process for purifying proanthocyanidins using the counter current liquid-liquid distribution method, for example, a method using water and ethyl acetate is described in Andrew G. H. Lea, J. Sci. Fd. Agric., 29, 471-477 (1978), in Japanese Published Unexamined Application No.61-16982 and the like.
As a process for purifying proanthocyanidins using solid-liquid extraction, for example, a method using ethyl acetate is described in Japanese Published Unexamined Application No.8-176137 and EP0707005.For example, 100 kg of crushed grapes are extracted with a mixed solvent of water (1650L), sodium chloride (300 kg) and acetone (550L). Next, acetone is removed by distillation so as to obtain the residue. The residue is then subjected to solid-liquid extraction using ethyl acetate, and then dichloroethane (45L) is added thereto, thereby obtaining 1.5 kg to 2.5 kg of a proanthocyanidin precipitate (EP0707005)
Further, known methods for purifying proanthocyanidins using chromatography include a method using the above Sephadex LH-20™ column (a method for extraction from grape seeds, Japanese Published Unexamined Application No.3-200781), and a method using polystyrene-based adsorption resin (a method for extraction from red beans, Japanese Published Examined Application No. 7-62014). For example, polystyrene polystyrene-based resin “Sepabeads SP-850™ “(MITSUBISHI CHEMICAL CORPORATION) is added to water obtained by immersing dried red beans therein and the mixture is stirred, thereby allowing proanthocyanidins to adsorb thereto. Then, the resin is dried at less than 70° C., and then eluted with 80% (v/v) ethanol at 70° C., so that crude proanthocyanidins-containing powder with a purity of approximately 60% can be obtained.
However, all of these processes are for purifying proanthocyanidin mixtures independent of polymerization degree. That is, these processes are not for efficiently and selectively obtaining dimeric to pentameric proanthocyanidin oligomers. Their recovery rate of dimeric to pentameric proanthocyanidin oligomers is low.
Regarding separation of proanthocyanidins by polymerization degree, a method using normal phase silica gel liquid chromatography is known (A method for extraction from cocoa beans: J. Rigaud et al., J. Chromatogr. A, 654, 255-260 (1993), A method for extraction from grape seeds: Corine Prieur et al., Phytochemistry, 36, 781-784 (1994)). The former method comprises loading a sample solution containing proanthocyanidins, which has been obtained by methanol extraction from cocoa beans, into a silica gel column, followed by gradient elution using a mixed solvent of dichloromethane:methaol:formic acid:water [(41→5):(7→43):1:1] as a mobile phase. The latter method comprises loading a sample solution containing proanthocyanidins, which has been obtained by methanol extraction from grape seeds, into a silica gel column, followed by gradient elution using a mixed solvent of dichloromethane:methanol:water:trifluoroacetic acid [(82→10):(18→86):2:0.05] as a mobile phase.
However, these methods involve problems such that recovery and reuse of solvents are difficult because of the use of a solvent containing chlorine and the complication of a solvent composition. Further, gradient elution to apply concentration gradients to a mobile phase is required. Therefore, these methods are not appropriate for mass purification-oriented industrial separation methods in view of safety and economy.
DISCLOSURE OF THE INVENTION
The object of the present invention is to provide processes for efficiently purifying dimeric to pentameric proanthocyanidin oligomers with high purity from raw materials containing proanthocyanidins or crude purification products therefrom.
A combination of conventional techniques is not good enough to remove substances other than the target components being dimeric to pentameric proanthocyanidin oligomers, for example monomers constituting proanthocyanidins, such as flavonoids, catechin or epicatechin, hexameric or higher order high polymeric proanthocyanidin polymers, or other contaminants. That is, it is difficult to efficiently obtain with high purity dimeric to pentameric proanthocyanidin oligomers, which are target components of this invention. In addition, most of known methods are not appropriate for industrial processes in view of the complication of a solvent composition used, economy, safety or the like.
As a result of thorough studies to solve these problems, we have completed a process for efficiently purifying dimeric to pentameric proanthocyanidin oligomers with high purity.
The first embodiment is

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