Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
2002-06-24
2004-11-09
Lilling, Herbert J. (Department: 1651)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C435S068100, C435S069520, C435S325000, C435S391000, C435S402000, C530S353000, C264S202000, C424S069000, C424S070140, C424S401000, C424S445000, C514S002600, C514S012200
Reexamination Certificate
active
06815427
ABSTRACT:
TECHNICAL FIELD
The present invention relates to a cell growth promoter containing domestic silkworm sericin, and particularly to a medical material, a cell culture bed material and a cosmetic material.
Further, the present invention relates to a method for obtaining sericin having a function of growing cells from fibers discharged by domestic silkworms, e.g., cocoon filaments or the like.
BACKGROUND ART
Fibers discharged by domestic silkworms, e.g., cocoon filaments or the like, comprise two kinds of proteins, fibroin and sericin. Sericin is present while coat fibroin.
Cocoons are subjected to steps including drying, boiling, thread reeling, etc., followed by degumming (sericin remouing) The silk filaments degummed (fibroin fibers) have been used as silk fabrics.
Further, silk yarn is the only long fiber of the natural fiber and has excellent crystallinity and high strength. Thus, the silk yarn has been used, since old times, also as a thread for operations as well as clothes.
In recent years, silk protein has attracted much attention to its inherent functions and, as a result, has come to be used actively for various applications.
In development of new materials using silk protein, fibroin preceded sericin. In recent years, however, sericin, which is another silk protein, has also become a subject of research and development based on the achievements with respect to fibroin.
For example, there are reports on a cosmetic powder comprising a base the surface of which has been covered with a hydrolysate of sericin (JP-A-10-226626), a cosmetic containing cellulose fiber impregnated with a fibroin dispersion and/or an aqueous sericin solution (JP-A-11-152206), a skin irritation inhibitor containing a sericin hydrolysate as an active component (JP-A-10-245345), a collagen production promoter and a skin preparation for external use for prevention from getting older, both containing a sericin hydrolysate as an active component (JP-A-10-226653), a skin preparation for external use containing a sericin hydrolysate and a degradation product of yeast (JP-A-11-193210) and a cell culture bed comprising a film made up of silk protein(s) (fibroin and/or sericin) of domestic silkworms (Minoura et al., Attachment and growth of cultured fibroblast cells on Silk protein matrices, J. Biomedical Materials Res., Vol.29, 1215-1221(1995)).
On the other hand, when a conventional method in which sericin is recovered from a degumming waste liquor is used as a method for obtaining sericin from cocoon filaments or the like, only products having a low molecular weight such as an average molecular weight of 50,000 or less can be obtained (JP-A-1-168906, JP-A-4-202435, etc.)
In short, since sericin in cocoon filaments are crystallize, it hardly dissolves in water. It, however, has been removed by being degraded in hot alkaline water.
Sericin having a high molecular weight such as an average molecular weight of 100,000 or more has recently become to be obtained efficiently (JP-A-11-92564, JP-A-11-131318, etc.) and a method has been developed in which sericin and the like are eluted from cocoon filaments or the like so that the reduction of molecular weight is controlled (JP-A-11-228837, JP-2000-38514, etc.)
Sericin in domestic silkworms has long been said to be a mixture of proteins having molecular weights of from several tens of thousands to hundreds of thousands. Since it is huge and, therefore, easily forms aggregate or becomes insoluble, it is difficult to handle.
For this reason, the problem that of what types of protein components the fractionation and purification in an undegraded state are constituted has been left unsolved.
Further, in most of the conventional reports in which functions such as a physiological activity are suggested, reduction in molecular weight takes place in an extraction step.
Since we have recently succeeded in extracting and fractionating undegraded sericin, the research of application of these components fractionated has become a main issue.
The object of the present invention is to examine the physiological activities of the components constituting undegraded sericin and to provide new medical materials, cosmetic materials and the like utilizing such functional components.
DISCLOSURE OF THE INVENTION
Means for Solving Problem
For solving the above-described problems, the present inventors first researched the relation between the molecular weight and physiological activity of sericin constituting undegraded sericin. As a result, they obtained information that, of the components constituting undegraded sericin, a sericin having a specific molecular weight has an extremely high cell growth promoting action.
The present invention has been created based on the above information and comprises the following compositions:
Namely, the present invention is present in (1) a cell growth promoter obtainable by elution from a fiber discharged (cocoon filaments or the like) wherein the cell growth promoter comprises sericin having a molecular weight of about 400,000 as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as a main component.
The present invention is present also in (2) a method comprising dissolving sericin contained in a fiber discharged by a domestic silkworm (cocoon filaments or the like) in an aqueous urea solution, and then separating and recovering sericin having a molecular weight of about 400,000 as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
The present invention is present also in (3) the method for separating and recovering according to claim
2
, wherein sericin is dissolved in an aqueous urea solution of a concentration of 4 M or more.
Further, the present invention is present also in (4) a cell growth promoter obtainable by dissolving a fiber discharged by a domestic silkworm (cocoon filaments or the like) in an aqueous solution of a neutral salt, wherein the cell growth promoter contains silk fibroin and sericin having a molecular weight of about 400,000 as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
Further, the present invention is present also in (5) a cell growth promoter, wherein the cell growth promoter is in the form of a powder or a film.
Further, the present invention is present also in (6) a cell growth promoter obtainable by degumming a fiber discharged by a domestic silkworm (cocoon filaments or the like) under mild conditions and removing sericin having low molecular weight, wherein the cell growth promoter comprises a fibrous material in a state where the surface of a fibroin filament is covered with a sericin component having a molecular weight of about 400,000 as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
Further, the present invention is present also in (7) a method for producing a fibrous material in a state where the surface of a fibroin filament is covered with a sericin component having a molecular weight of about 400,000 as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the method comprising degumming a fiber discharged by a domestic silkworm (cocoon filaments or the like) under mild conditions and removing sericin having low molecular weight.
Further, the present invention is present also in (8) a method for producing a fibrous material, wherein scouring by boiling is conducted within mild pH range as close to neutrality as possible.
Further, the present invention is present also in (9) a cell growth promoter obtainable by immersing a liquid silk contained in the middle silk gland and the anterior silk gland in the body of a silkworm, wherein the cell growth promoter comprises sericin having a molecular weight of about 400,000 as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as a substantial main component.
Further, the present invention is present also in (10) a method of using the cell growth promoter described above for a cosmetic material as a skin care material; a wound dressing material, a material for vascular endothe
Takasu Yoko
Tsubouchi Kozo
Yamada Hiromi
Flynn ,Thiel, Boutell & Tanis, P.C.
Lilling Herbert J.
National Institute of Agrobiological Sciences
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