Compositions – Miscellaneous
Reexamination Certificate
1999-06-21
2001-07-24
Warden, Jill (Department: 1743)
Compositions
Miscellaneous
C530S500000, C530S507000, C426S430000, C426S479000, C426S482000, C426S484000, C536S004100, C536S128000
Reexamination Certificate
active
06264853
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to a novel process for purifying a complex containing lignan, phenolic and aliphatic substances from flax and to the novel complex thus obtained.
BACKGROUND OF THE INVENTION
At the present time flaxseed is primarily grown for its oil content for use mainly as an industrial oil. Increasingly flaxseed is also used as a source of edible oil for use in health supplements and margarine. Conventional flaxseed is rich in unsaturated fatty acids in its triglyceride oil and is thus subject to oxidative polymerization.
The principal components of flaxseed that are of commercial importance are oil and meal cake. The oil is used industrially in the manufacture of linoleum and in some paints and varnishes. The meal left after the oil has been substantially removed is utilized as a component in animal feeds. Reports are also available describing a polysaccharide fraction and possible uses. These include the use of flaxseed mucilage as a laxative. Recently, Westcott and Muir (U.S. Pat. No. 5,705,618) have described a process for the extraction, isolation and purification of a chemical belonging to the lignan class of compounds that is contained in flaxseed. Lignans are naturally occurring substances that are formed by dimerization of cinnamoyl alcohols. The principal lignan found in flaxseed is secoisolariciresinol diglucoside, referred to hereinafter as SDG.
The first report of SDG occurring in flaxseed was by Bakke and Klostermann, “A New Diglucoside from Flaxseed”, Proceedings of the N. Dakota Academy of Science 10:18-22 (1956). No physiological activities were reported for this substance at that time. These authors isolated SDG in a multi-step process involving extraction of the defatted seed with ethanol-dioxane, and concentration of the extract to a sirup. The resulting sirup was poured into petroleum ether, causing the separation of a heavy brown plastic material which was subsequently dissolved in aqueous ethanol. This alcoholic solution was then acidified to pH 3.0 and poured into ice-water. The precipitate which formed, coagulated, settled and was collected and dried under reduced pressure. The precipitate has been variously described as ‘tan polymeric powder’ and as a ‘brown, amorphous, gum-like substance’. The precipitate was treated with sodium or barium methoxide in methanol and the liberated SDG was isolated after chromatographic purification.
Considerable interest in SDG has developed following reports of physiological activity of either whole flaxseed or purified SDG. It was demonstrated nearly 30 years ago that when flaxseed was consumed as part of the human diet, increased levels of the so-called mammalian lignans, enterolactone and enterodiol, were found in urine. It has subsequently been reported that flaxseed is very abundant in precursors to these mammalian lignans. Indeed flaxseed or its defatted meal produced 75 times more mammalian lignans than the next source, a seaweed and over 100 times more than more common foodstuffs. It was further observed by Stitch et al., “Excretion, isolation and structure of a new phenolic constituent in female urine”, Nature 287: 738-740 (1980) that the amount of mammalian lignans was greatest in the luteal phase of the reproductive cycle. In addition, Aldercreutz et al, “Excretion of the lignans enterolactone and enterodiol and of equol in omnivorous and vegetarian postmenopausal women and in women with breast cancer”, Lancet 1295-1299 (1982) reported that the levels of mammalian lignans were lower in urine of women with breast cancer than in healthy women.
Within the last ten years numerous researchers have reported on the physiological effects of either flaxseed or SDG. Thompson, “Anticarcinogenic effect of a mammalian lignan precursor from flaxseed”, Proc. 55th Flax Institute of U.S.A., Fargo, N.Dak., 46-50 (1994) has described the effects of flaxseed on the initiation and promotional stages of mammary tumorigenesis. Additional reports have been published on the effect of SDG and flax-oil on mammary tumorigenesis. Other reports from Thompson have detailed the effects of flaxseed or SDG on colon cancer. Clark in U.S. Pat. No. 5,827,256 has also reported on the benefits of SDG to patients suffering from lupus nephritis. Prasad in U.S. Pat. No. 5,846,944 has recently described the effects of SDG in reducing the development of hypercholesterolemic atherosclerosis in animal model experiments. Further, Prasad has also reported that SDG has benefits in diabetes mellitus. Both Thompson and Prasad have separately reported that SDG has antioxidative properties.
A second class of compounds reported to be present in flax seed can generally be referred to as phenolic acids. Specifically, coumaric acid (4-glucosyl-cinnamic acid), caffeic acid (3-hydroxy-4-glucosyl-cinnamic acid) are reported constituents of the complex from flaxseed containing SDG. It is known that phenolic acids of this class of compounds and their derivatives, have in vitro activity as antioxidants. Furthermore compounds of this type have known physiological activities related to tyrosine kinase inhibition. Surprisingly Westcott and Muir (U.S. Pat. No. 5,705,618) have found a third phenolic acid glycoside is also present, namely ferulic acid glycoside(3-methoxy-4-glucosyl-cinnamic acid). Flaxseed contains an additional compound, hydroxy-methyl glutaric acid (HMGA). HMGA, isolated from sources other than flax, has been found by Siddiqi and Beg (U.S. Pat. No. 3,629,449) to have hypocholesteremic properties.
While whole flaxseed or its ground counterpart may be incorporated into the human diet, the amount of such incorporation may be restricted by regulation. Even if not restricted by regulation, the high oil content and the polysaccharide mucilage content would contribute to excessive caloric intake and increased laxation respectively. Further, flaxseed is known to contain cyanide-containing compounds known as cyanogenic glycosides, those found in flaxseed being liminarin, linustatin and neolinustatin. These compounds if consumed in excess over a long period in time can result in goitrogenic problems and damage to other human organs.
It is an object of the present invention to isolate the valuable components of flax without the undesirable components, including cyanogenic glucosides.
SUMMARY OF THE INVENTION
According to one embodiment of the present invention an aqueous aliphatic alcohol extract is obtained from commercial flax, e.g. from flaxseed or flax meal. This aqueous extract is subjected to ultrafiltration whereby low molecular weight species remain with the filtrate and higher molecular weight species are retained. By proper selection of the filtration medium, it has been found that it is possible to retain in substantially pure form a complex comprising secoisolariciresinol diglucoside (SDG), cinnamic acid glucosides and hydroxy methyl glutaric acid (HMGA). The small molecular weight species that remain with the filtrate include amino acids, small peptides, sugars and salts as well as the cyanogenic glucosides, all of which are residual from the metabolic pool of the initial flaxseed.
The ultrafiltration is preferably carried out for a size exclusion of 30,000 Daltons or greater, e.g. 30,000 to 100,000 Daltons. Generally it is in the range of 30,000 to 50,000 Daltons.
A variety of ultrafilters may be used including filters in the form of flat sheets, spiral wound, supported tubes or hollow fiber membranes or other configurations. Such filters are typically made from synthetic polymers, cellulose or ceramics. A preferred ultrafilter for use in the invention is a membrane through which the aqueous extract is passed, e.g. a regenerated cellulose membrane. Particularly good results are obtained when the membrane is in the form of a spiral wound membrane. Ultrafiltration techniques are described, for instance, in U.S. Pat. Nos. 4,963,356 and 4,716,120.
It is also possible to use a column containing a solid support of a type commonly referred to as either size exclusion or gel permeation resins, such as Sephadex LH-20. Such resins
Paton David
Westcott Neil D.
Agriculture and Agri-Food Canada
Cole Monique T.
Warden Jill
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