Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Glycoprotein – e.g. – mucins – proteoglycans – etc.
Reexamination Certificate
2001-07-30
2004-10-12
Low, Christopher S. F. (Department: 1653)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Glycoprotein, e.g., mucins, proteoglycans, etc.
C530S412000, C530S418000, C530S422000, C530S350000, C514S002600, C514S008100
Reexamination Certificate
active
06803454
ABSTRACT:
BACKGROUND OF THE INVENTION
Field of the Invention
The present invention relates to a new method for extraction and purification of cartilage type proteoglycan.
Description of the Prior Art
One molecule of cartilage type proteoglycan recognized as a conjugated carbohydrate is characterized to have a structure shown in
FIG. 1
, which is a biopolymer having following structural feature. That is, from several to tens of glycosaminoglycan chains (hereinafter shortened to GAG) whose each molecular weight is from several ten thousand to several hundred thousand are bonded to one backbone protein molecule having molecular weight of from several ten thousand to several hundred thousand which is called as a core protein. GAG can be classified to several kinds such as chondroitin sulfate or dermatan sulfate according to the base structure, and, basically is a long chain hetero acidic polysaccharide composed of repeating structures of disaccaride with amino sugar and uronic acid. In said structure, GAG except hyaluronic acid are bonded to a core protein and forms proteoglycan.
In almost all animal organisms, proteoglycan is generally existing as one of the important component of extracellular matrix which exists among cells (refer to FIG.
2
), which is similarly existing with collagen and hyaluronic acid. And, not only it plays the important part of organism construction, but also forms physical circumference surrounding cells and controls various cell activities such as coupling, multiplying or differentiating. Each component of extracellular matrix or GAG individually has some functions such as retaining and supplying of water, antidote or analgesic. When these components bond each other and form macro-molecule structure and each component acts reciprocally, more remarkable effect is displayed.
The cartilage type proteoglycan, which is the object of the present invention, has a huge molecular weight in comparison with collagen, hyaluronic acid or GAG and has a complicated structure. Therefore, even if proteoglycan alone, it has better water retaining and supplying ability than other components in the extracellular matrix, further, can have other functions depending on biological information signal organization of it's GAG portion.
In the meanwhile, in the method for extraction and purification of proteoglycan of nowadays, cartilage of cow or whale is used as a starting material, and extracted and purified by a complicated procedure using toxic or harmful agents such as chloroform, methanol or guanidine hydrochloride. And this method is not recognized as an industrial level. Some kinds of proteoglycan are available in the market by very small amount as a reagent, and the price of them is approximately tens million yen per one gram.
The applicant of the present invention had previously invented a novel mass-producing simplified method for extraction and purification for proteoglycan that can be used as an industrial scale using nasal cartilage of salmon and filed a patent application (Japanese Patent Application 11-331375 filed on Nov. 22, 1999). This method is concretely composed of crushing process of nasal cartilage of salmon, deoiling process, extraction process by solvent and dialysis process. By this method, a method for extraction and purification characterized by mass-producing and low price could be accomplished, however, not only chloroform, methanol and guanidine hydrochloride but also a harmful agent such as hindering agent for protein decomposing enzyme are used, therefore, the possibility for use as the material for medicine took into human body or additives to healthy supporting foods or supplements was difficult, and the use is limited to non-drug chemicals or cosmetics. Further, since the market price of above mentioned chemical agents are relatively expensive, the reducing of extraction and purification cost is limited.
In the meanwhile, since the applicant of this application had presented said low cost proteoglycan, the volition for the development of goods in connection with proteoglycan is enhanced not only in cosmetics industry but also in processed foods industry, healthy supporting foods or supplements industry and medicines industry. However, for the substantial application of proteoglycan to the processed foods industry, healthy supporting foods or supplements industry or medicines industry, the special consideration must be cared for the method for purification of proteoglycan. In the conventional method for extraction and purification of proteoglycan, the use of hydrochloric acid salt of guanidine is common. But, for the new application of proteoglycan, it is strongly required not to use said guanidine hydrochloride further toxic or harmful agents such as chloroform, methanol or hindering agent for protein decomposing enzyme. Still further, the development of more simplified and lower cost method for extraction and purification of proteoglycan had been strongly required.
The inventor of this invention has conduced the intensive study to develop the method for extraction and purification of proteoglycan, in the procedure of which the toxic or harmful agents are not used, further, which is characterized to be more simplified and lower cost, and accomplished the present invention. Namely the object of the present invention is to provide more simplified and lower cost method for extraction and purification for cartilage type proteoglycan.
BRIEF SUMMARY OF THE INVENTION
The invention of claim
1
of the present invention is an extraction method of crude proteoglycan characterizing to use acid as eluting solvent of cartilage. The invention of claim
2
of the present invention is a purification method of crude proteoglycan comprising; extracting crude proteoglycan using acetic acid as eluting solvent of cartilage, filtrating solution containing crude proteoglycan to remove dregs from said solution, centrifuging the solution obtained by said filtrating, adding ethanol saturated with sodium chloride to the supernatant liquid obtained by said centrifuging, and then centrifuging said supernatant liquid added said ethanol saturated with sodium chloride to concentrate said crude proteoglycan in the precipitate. And the invention of claim
3
of the present invention is a further improving method of the purity of crude proteoglycan comprising; extracting crude proteoglycan using acetic acid as eluting solvent of cartilage, filtrating solution containing crude proteoglycan to remove dregs from said solution, centrifuging the solution obtained by said filtrating, adding ethanol saturated with sodium chloride to the supernatant liquid obtained by said centrifuging, centrifuging said supernatant liquid added said ethanol saturated with sodium chloride to concentrate said crude proteoglycan in the precipitate, dissolving said precipitate containing crude proteoglycan using acetic acid as eluting solvent of said crude proteoglycan, and then dialysising.
That is, the important point of the present invention is to use acetic acid, sodium chloride and not-modified ethanol in all processes of extraction and purification of proteoglycan instead of the toxic or harmful agents such as chloroform, methanol or hindering agent for protein decomposing enzyme. These above mentioned agents, that is, acetic acid, sodium chloride and not-modified ethanol are the agents which are used in the ordinary processed foods. For the purpose to accomplish more simplified method for extraction and purification, the substitution process by urea and separation and purification process by DEAE-Sephacel method which are used in above mentioned patent application (JPA 11-331375) are omitted.
REFERENCES:
patent: 2001/0016646 (2001-08-01), Rueger et al.
patent: WO 97/25435 (1997-07-01), None
Miller E. J., Biochemistry 11, 4903-4909.*
Scott, P. G., et al., “Isolation and charatcerization of small proteoglycans from different zones of the porcine knee meniscus” Biochimica et Biophysica Acta vol. 1336, No. 2 Aug. 27, 1997, pp. 254-262.
Kakuhiro Co., Ltd.
Kam Chih-Min
Low Christopher S. F.
Sherman & Shalloway
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