Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
2001-06-06
2003-11-18
Low, Christopher S. F. (Department: 1653)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C514S773000, C514S775000, C426S491000, C426S271000, C426S587000, C426S588000, C426S590000, C426S041000, C426S042000, C426S656000, C426S657000, C426S002000
Reexamination Certificate
active
06649590
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a method of producing a fraction containing a high concentration of milk-derived basic cystatin from a milk-derived basic protein fraction.
The present invention also relates to a method of producing a decomposition product of the resulting fraction containing a high concentration of milk-derived basic cystatin by decomposing said fraction containing a high concentration of milk-derived basic cystatin using a protease.
Further, the present invention relates to a drink, food product, medicine and feed to which the fraction containing a high concentration of milk-derived basic cystatin and/or the decomposition product of the fraction containing a high concentration of milk-derived basic cystatin milk-derived basic cystatin are admixed, and if necessary, calcium and/or vitamins are additionally admixed, for the prevention and treatment of various bone diseases such as osteoporosis, bone joint diseases such as rheumatism, and periodontal disease.
2. Description of the Related Art
In recent years, the number of people suffering from various bone diseases, such as bone fractures and lumbago has been on the rise because of the aging population. Bone formation and bone resorption continuously take place in bone tissue and are well balanced in early life, but the balance is lost with an increase in bone resorption with aging for various reasons. If this unbalance continues for a long period of time, the bone tissue becomes fragile, which results in various bone diseases such as osteoporosis, bone fractures and lumbago. It is believed that if this uncoupling leaning toward bone resorption can be impeded, various bone diseases can be prevented.
Conventional methods to prevent and treat various bone diseases include (1) dietary supplementation of calcium, (2) moderate exercise, (3) sunbathing, and (4) administration of medicines. For dietary supplementation, calcium salts such as calcium carbonate and calcium phosphate, natural calcium supplements such as bovine bone powders, eggshells and fish bone powders are used. For moderate exercise, moderate running or walking are highly recommended. However, even moderate exercise is difficult for people who are physically weak and, not to mention, almost impossible for elderly people who are confined to bed. The third method, sunbathing, is good for the supplementation of activated vitamin D
3
, but not sufficient by itself. For the last method, administration of medicines, 1&agr;-hydroxy vitamin D
3
, calcitonin preparations or the like are known to be effective to cure and treat osteoporosis. It is believed that bone joint diseases, such as rheumatism, and periodontal disease can be treated by suppressing bone resorption since they are ultimately associated with the bone resorption.
The present inventors intensively searched for a milk whey protein fraction having an osteoblast growth stimulating factor, a bone resorption suppressing factor and bone strengthening activity, in order to obtain a material which is effective for the prevention and treatment of various bone diseases, bone joint diseases and periodontal disease. Namely, the present inventors fractionated proteins in milk, in particular milk whey, in an attempt to obtain a fraction having a suppressing activity on osteoclastic bone resorption. As a result, the present inventors found that a bone strengthening activity in a protein-peptide mixture which was obtained by treating a water soluble fraction of whey proteins with a reverse osmotic membrane or electrodialysis to remove salts (Japanese Patent Application Laid-open No. H4-183371). Furthermore, the present inventors found that a fraction obtained by treating an aqueous solution of this protein-peptide mixture with ethanol, heat, salts or an ultrafiltration membrane has a bone strengthening activity (Japanese Patent Application Laid-open No. H5-176715, Japanese Patent Application Laid-open No. H5-320066). The present inventors also found that basic proteins present in milk in trace amounts have a collagen synthesis stimulating activity in osteoblasts and bone resorption preventing activity (Japanese Patent Application Laid-open No. H7-207509).
Cystatin, a cysteine protease inhibitor, is a substance which inhibits proteolytic activity of cysteine proteases having an SH group in the active center and is found in animal tissues, cells, blood and urine. Further, a virus growth inhibiting activity is recognized as a useful activity of cystatin (Biochem. Biophys. Res. Commun., Vol. 127, p. 1072, 1985).
In recent years, the number of people suffering from osteoporosis caused by osteoclastic bone resorption has been rapidly increasing. Currently, a calcitonin drug is known as a medicine to suppress this osteoclastic bone resorption. However, a calcitonin drug is a hormonal agent used as a medicine, and the safety of its use as a food material has not been examined to this day. Further, production of calcitonin in bulk from animal tissues, cells, blood or urine for use as a food material has not been attained.
SUMMARY OF THE INVENTION
The present inventors tried to isolate and purify an active substance having a bone resorption suppressing activity from a basic protein fraction having a bone resorption suppressing activity, identified the resulting isolated and purified substance, and confirmed that this substance is a milk basic cystatin. Furthermore, the present inventors found that the milk basic cystatin has a suppressing activity more specifically on osteoclastic bone resorption, as compared with other kinds or types of cystatins from origins other than milk (Japanese Patent Laid-open No. 2000-281587, published Oct. 10, 2000, which is after the priority date of this application).
Under these circumstances, a method of producing a milk-derived basic cystatin usable as a food material in bulk and at a low cost is in need. Accordingly, an object of the present invention is to provide a method of producing a fraction containing a high concentration of basic cystatin from a milk-derived basic protein fraction.
Further, an object of the present invention is to provide a method of producing a decomposition product of the fraction containing a high concentration of milk basic cystatin since a protease decomposition product of the fraction containing a high concentration of milk basic cystatin is also known to have a bone resorption suppressing activity.
Furthermore, another object of the present invention is to provide a drink, food product, medicine or feed to which the fraction containing a high concentration of milk-derived basic cystatin and/or the decomposition product of the fraction containing a high concentration of milk-derived basic cystatin are admixed for the prevention and treatment of various bone diseases such as osteoporosis, bone joint diseases such as rheumatism, and periodontal disease.
The process for extracting basic cystatin from milk is broadly divided into (1) a process to extract a basic protein fraction from milk, and (2) a process to extract basic cystatin from the basic protein fraction. As explained below, a basic protein fraction can be extracted in great volume from milk volume by, for example, contacting milk with a cation exchange resin, and then eluting a fraction adsorbed on the resin with an elute having a salt concentration of 0.1-1.0 M. However, a further treatment is required for recovering basic cystatin at a high concentration such as 1% or higher by dry weight. Separating basic cystatin from other basic proteins can be accomplished to a certain degree by a method utilizing its physico-chemical characteristics, such as steps of heating a solution at 80° or higher, recovering a supernatant by removing the resultant precipitate, adding alcohol to the supernatant, and removing the resultant precipitate. However, it is difficult to obtain basic cystatin at a high concentration by the above method. The present invention provides a method of extracting and recovering basic cystatin at a high concentration from a basic pr
Kawakami Hiroshi
Matsuoka Yasuhiro
Serizawa Atsushi
Takada Yukihiro
Toba Yasuhiro
Kam Chih-Min
Knobbe Martens Olson & Bear LLP
Low Christopher S. F.
Snow Brand Milk Products Co. Ltd.
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