CN101678077B - Novel antiphlogistic-analgesic agent - Google Patents

Novel antiphlogistic-analgesic agent Download PDF

Info

Publication number
CN101678077B
CN101678077B CN2008800156559A CN200880015655A CN101678077B CN 101678077 B CN101678077 B CN 101678077B CN 2008800156559 A CN2008800156559 A CN 2008800156559A CN 200880015655 A CN200880015655 A CN 200880015655A CN 101678077 B CN101678077 B CN 101678077B
Authority
CN
China
Prior art keywords
lactalbumin
alpha
cox
pain
analgesic agent
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN2008800156559A
Other languages
Chinese (zh)
Other versions
CN101678077A (en
Inventor
山口真
内田胜幸
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Meiji Co Ltd
Meiji Dairies Corp
Original Assignee
Meiji Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Meiji Co Ltd filed Critical Meiji Co Ltd
Publication of CN101678077A publication Critical patent/CN101678077A/en
Application granted granted Critical
Publication of CN101678077B publication Critical patent/CN101678077B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/38—Albumins
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/17—Amino acids, peptides or proteins
    • A23L33/19—Dairy proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Engineering & Computer Science (AREA)
  • Veterinary Medicine (AREA)
  • Medicinal Chemistry (AREA)
  • Public Health (AREA)
  • General Health & Medical Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Organic Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Rheumatology (AREA)
  • Zoology (AREA)
  • Epidemiology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Polymers & Plastics (AREA)
  • Pain & Pain Management (AREA)
  • Orthopedic Medicine & Surgery (AREA)
  • Physical Education & Sports Medicine (AREA)
  • Mycology (AREA)
  • Food Science & Technology (AREA)
  • Nutrition Science (AREA)
  • Endocrinology (AREA)
  • Reproductive Health (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Coloring Foods And Improving Nutritive Qualities (AREA)
  • Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)

Abstract

The object is to provide a novel antiphlogistic-analgesic agent which is highly safe and can be administered over a long period. A study is made on the antiphlogistic-analgesic effect of a-lactalbumin which is one of natural substances contained in foods, and it is found that alpha-lactalbumin exhibits a cyclooxygenase-2(COX-2) inhibition activity and a phospholipase-A2 inhibition activity in vitro. In the in vivo test using a carrageenin food edema model, an adjuvant arthritis model and an acetic acid writhing test, it is also found that alpha-lactalbumin exhibits an anti-inflammatory effect. Further, it is also found that alpha-lactalbumin inhibits COX-2 more selectively compared to COX-1. Therefore, alpha-lactalbumin can be used as an antiphlogistic-analgesic agent having few adverse side effects.

Description

Novel antiphlogistic-analgesic agent
Technical field
The present invention relates to the diet product and the medical composition that contain alpha-lactalbumin, have anti-inflammatory analgesic action.
Background technology
Nowadays, as the daily prescription drugs that anti-inflammatory analgesic is used, the tens of kinds of synthetic compounds such as the aspirin that is referred to as nonsteroidal antiinflammatory and analgesic agent, indomethacin, ibuprofen, diclofenac are arranged and be commonly called as synthetic glucocorticoid into steroid etc.
Aspirin suppresses the i.e. activity of " epoxidase (COX) " of prostaglandin (PG) synzyme.Because the activity decreased of COX reduces PG, thereby the activity of inhibition of pain evocating substance (Kallidin I) is brought into play the analgesic activity of aspirin thus.Similarly act on medicine that COX brings into play the anti-inflammatory analgesic effect for the steroidal agent different with the mechanism of action distinguishes from aspirin, be called as " nonsteroidal antiinflammatory and analgesic agent (non-steroidal anti-inflammatory drugs:NSAIDs) ".
NSAIDs has the shortcoming that easily causes digestive tract ulcer.Its main cause is, NSAIDs can reduce the PG (PGE that vasodilation and mucous membrane protection are worked 2, PGI 2).And because the activity inhibited of COX, lipoxygenase active hyperfunction, leukotriene increases, the secretion minimizing of gastric juice.Simultaneously, the organized enzyme that destroys gastrointestinal mucosal increases, and causes ulcer.
Take the side effect of the NSAIDs that reduces this type of as purpose, developed Types of Medicine medicine before the loxoprofen etc.But front Types of Medicine medicine has same mechanism of action with in the past NSAIDs, to side effect to alleviate effect limited.
Elimination is the medium-term and long-term research topic of analgesic agent exploitation by the digestive organs infringement that NSAIDs causes.The discovery of the new COX for this reason solution of a difficult problem has brought hope.When finding in 1991 to injure inflammation outside occuring, COX-2 sharply increases.On the other hand, with originally exist in the body, can produce the prostaglandins (PGE that the protection of gastric mucosa is worked 2, PGI 2) type be classified as COX-1.Therefore, the material of the activity of selectivity inhibition COX-2 can become the analgesic agent of few side effects.
As the side effect of NSAIDs, except gastrointestinal damage, also have kidney damage, hepatic injury, skin dermexanthesis etc., in addition, as serious side effect, also have bringing out of asthma attack.If as nonselective NSAIDs in the past, in the inhibition COX-2 as Main Function, suppress the effect of COX-1, then arachidonic metabolism is partial to generate the direction of leukotriene, the activity of lipoxygenase rises, and the leukotriene of bronchoconstriction is increased, and brings out asthma.The asthma that is caused by analgesic agent is called " aspirin asthma ", is not only aspirin, and whole NSAIDs in the past has the danger of bringing out aspirin asthma.And the medicament that only suppresses COX-2 can not make leukotriene increase, and therefore brings out the dangerous little of asthma.Based on this point, very high for the expected value of COX-2 selective depressant.The etodolac of listing in 1994 is strong 10 times to the effect of COX-2, and it is used as the relatively less analgesic agent of Alimentary infringement is used.In addition, meloxicam in 1996 in South Africa, the listing such as France, Britain, also go on the market in Japan in calendar year 2001 subsequently.
Some protein that contain in the food have anti-inflammatory analgesic action.For example, lactoferrin is well known as the protein with anti-inflammatory analgesic action.Lactoferrin is that the molecular weight of finding nineteen thirty-nine is about 80,000 ferrum associativity glycoprotein.At breast milk, especially just the content in Ruzhong is high, can resist the invasion and attack of antibacterial from the health outside, virus etc., have the function of infection defense factor.Lactoferrin is be evaluated as has very high safety, extracts the lactoferrin that obtains and often be added in the diet product for baby such as using adjusting milk powder of giving birth to children from milk.Recently the research with the lactoferrin of various disease associations also gets most of the attention.Reported the function that various Ia biosiss is regulated and control, as: in the experimental animal model to the reinforcement (for example non-patent literature 1) of the organism defense function of pathogenic microorganism and viral infection, in various pathological model anti-inflammatory effect, suppress lgE produce an effect (for example non-patent literature 2), the effect (for example non-patent literature 4) that suppresses to the preventive effect (for example non-patent literature 3) of the cancer that caused by chemical carcinogen, to the angiogenesis that is caused by cancerous cell etc.Clear and definite lactoferrin can strengthen endogenous and exogenous opioid analgesic effect (for example patent documentation 1).
In addition, the alkaline protein with enzymatic activity of lysozyme chloride for being comprised of 18 kinds of 129 aminoacid has antiinflammation.About analgesic activity, reported subcutaneous to mice or oral administration 50～300mg/kg, measure analgesic activity (hot plate method), the result does not find any effect (non-patent literature 5).
In addition, reported also that in recent years the generation fraction of milk surum or milk surum has the activity (patent documentation 7) of inhibition COX-2.
But, alpha-lactalbumin for account for whey protein approximately 25%, molecular weight is 14,000 globular protein.The high homology (non-patent literature 6) that has protein structure similarity and gene order between alpha-lactalbumin and lysozyme.Character as alpha-lactalbumin, known its is calcium binding proteins matter, cystine is high, and known prevention with gastrointestinal motor regulating action (for example patent documentation 2), injury of small intestine or promote repair, antiulcer action (for example non-patent literature 7, patent documentation 5, patent documentation 6).
In addition, reported that alpha-lactalbumin can suppress the symptom of premenstrual syndrome (patent documentation 8).But its mechanism of action is not yet clear and definite.
Patent documentation 1: No. 3806427 communique of Japan Patent
Patent documentation 2: No. 3756449 communique of Japan Patent
Patent documentation 3: Japanese kokai publication hei 5-268879 communique
Patent documentation 4: No. 2916047 communique of Japan Patent
Patent documentation 5: No. 3756449 communique of Japan Patent
Patent documentation 6: No. 3481931 communique of Japan Patent
Patent documentation 7: Japanese Unexamined Patent Application Publication 2007-505078 communique
Patent documentation 8: Japanese Unexamined Patent Application Publication 2007-500755 communique
Non-patent literature 1:Lactoferrin; Structure, Function and Applications.ExcerptaMedica Intern.Congress Series 1195 (2000), pp377-381
Non-patent literature 2:Arthritis Rheum.43 (2000) 2073-2080
Non-patent literature 3:Drug Metab.Pharmacokin.19 (2004) 245-263
Non-patent literature 4:Japan Medical Society in October, 2005 ラ Network ト Off エ リ Application real gives anti-effect と ラ Network ト Off エ リ Application with the 10th time Ga ん chemistry of the new な exhibition Open of change research and pacifies rattan nation hero
Non-patent literature 5: pharmaceuticals イ Application タ PVC ユ one Off オ one system リ Off ラ Star プ＜salt リ ゾ チ Yi ム System drug〉18 pages of Eisai Co., Ltd
Non-patent literature 6:ProcNatl Acad Sci U S is July 1 A.1992; 89 (13): 5887-5891.
Non-patent literature 7:Biosci.Biotechnol.Biochem., 67 (3), 577-583,2003
Summary of the invention
The present invention finishes according to above-mentioned situation, its purpose be to provide a kind of few side effects, safe, can be used as the novel antiphlogistic-analgesic agent that pharmaceuticals or diet product are taken in for a long time.
The present inventors concentrate on studies in order to solve above-mentioned problem.The present inventors consider safety, have meticulously explored the material with antiinflammation in the contained natural materials from food.After the anti-inflammatory analgesic action of alpha-lactalbumin is studied, alpha-lactalbumin external demonstrated active to the inhibition of epoxidase (COX)-2, active to the inhibition of phospholipase A2.In addition, by utilizing the external checking of the swollen model of chondrus ocellatus Holmes colloidality foot, adjuvant-induced arthritis model, acetic acid twisting method, show that acute inflammation, experimental chronic inflammatory disease and pain are had therapeutic effect.The present inventors have further studied the enzyme inhibition activity of alpha-lactalbumin, clear and definite compare with COX-1, more can optionally suppress COX-2, find that alpha-lactalbumin can be used as analgesic agent and uses.The high security of alpha-lactalbumin not only can be contemplated to from the selective inhibitory of COX-2, and edible experience that can also be long-term is as assurance.In the past, about the protein in the food, known lactoferrin had the anti-inflammatory analgesic effect, but the mechanism of action of this effect is not necessarily clear and definite.In addition, lysozyme chloride has the antiinflammatory effect, but does not have analgesic effect.On the other hand, the application's inventor has not only found the alpha-lactalbumin as the protein in the food with anti-inflammatory analgesic effect, and also clear and definite this effect derives from the selective inhibitory of COX-2.Think that the COX-2 selective depressant can solve the side-effect problem of NSAIDs in the past, the present inventors' above-mentioned opinion proof alpha-lactalbumin has great purposes as novel antiphlogistic-analgesic agent.That is, the present invention relates to the new purposes of alpha-lactalbumin, following invention specifically is provided.
(1) a kind of COX-2 selective depressant, it is take alpha-lactalbumin as effective ingredient,
(2) a kind of analgesic agent, it is take alpha-lactalbumin as effective ingredient,
(3) purposes of alpha-lactalbumin in making analgesic agent,
(4) according to above-mentioned (2) described analgesic agent, it is the inflammatory diseases medicine,
(5) according to above-mentioned (4) described analgesic agent, wherein, diseases associated with inflammation is arthritis or rheumarthritis,
(6) according to above-mentioned (2) described analgesic agent, it is the painful diseases curative,
(7) according to above-mentioned (6) described analgesic agent, wherein, painful diseases is dysmenorrhea,
(8) a kind of anti-inflammatory analgesic diet product, it is take alpha-lactalbumin as effective ingredient,
(9) a kind of Therapeutic Method of diseases associated with inflammation, it treats the alpha-lactalbumin of effective dose to mammal,
(10) a kind of Therapeutic Method of painful diseases, it treats the alpha-lactalbumin of effective dose to mammal.
The invention provides a kind of novel analgesic agent.Novel antiphlogistic-analgesic agent selectivity of the present invention suppresses COX-2, so the generation of the side effect such as peptic ulcer, asthma is few, can take in for a long time.Alpha-lactalbumin contains in the milk of newborn class as previously mentioned, is safe from long-term edible experience.In addition, in fact the safety of alpha-lactalbumin has also obtained the science confirmation, for example, report in the patent documentation 5, according to the acute toxicity tests of the alpha-lactalbumin of having used rat, even given the i.e. alpha-lactalbumin of 2,000mg/kg body weight of research on maximum utilized quantity, also identical with blank assay, do not find death.In addition because be the material that originally just is present in the food, so not only as pharmaceuticals, with it as the diet product also Worth Expecting of processing, circulate.And, owing to can from the raw material of the relative low price such as milk, obtain, thereby can produce in a large number.
Lactoferrin in the milk adopts isolation technics to extract from milk to obtain industrial.The content of lactoferrin is according to the difference of the place of production, feedstuff etc., and more or less difference is generally 200mg/kg in Japan's product milk, extremely trace.Therefore, present situation can't be produced in Japan fully for being unable to supply the milk for the required abundance of the lactoferrin that extracts trace in Japan at present, all dependence on import.
In addition, the content of alpha-lactalbumin is with the difference of the place of production, feedstuff etc. difference more or less in the milk, and still, Japan produces milk and is generally 1.2g/kg.Abundanter with lactoferrin phase ratio content, industrial separation is also easy.Therefore, can guarantee stably to obtain, and the supply expense is also cheap.
Description of drawings
Fig. 1 is the figure that is illustrated in the variation of the swollen inhibition test model mesopodium volume of chondrus ocellatus Holmes colloidality foot.Meansigma methods+standard error with one group 6 represents.Control represents matched group, and WPI represents whey protein sepd, and bLG represents beta lactoglobulin, and aLA represents alpha-lactalbumin, and LF represents lactoferrin.*: vs.control, p＜0.05 (check of Dunnet multiple comparisons)
Fig. 2 is the figure of expression adjuvant-induced arthritis (treatment) model mesopodium change in volume.Meansigma methods+standard error with one group 6 represents.Vehicle represents matched group, and bLG represents beta lactoglobulin, and aLA represents alpha-lactalbumin, and LF represents lactoferrin, and Dicro represents diclofenac.*, * *, * * *: vs.Vehicle, p＜0.05,0.01,0.001 (check of Dunnet multiple comparisons)
Fig. 3 is the figure of the number of times of the reaction that pipes in expression adjuvant-induced arthritis (pain) model.Meansigma methods+standard error with one group 6 represents.Vehicle represents matched group, and bLG represents beta lactoglobulin, and aLA represents alpha-lactalbumin, and LF represents lactoferrin, and Dicro represents diclofenac.*, * *: vs.Vehicle, p＜0.05,0.01 (check of Dunnet multiple comparisons)
Fig. 4 is the figure of the number of times of the misery action in the expression acetic acid twisting test model.Meansigma methods+standard error with one group 6 represents.Vehicle represents matched group, and WPI represents whey protein sepd, and bLG represents beta lactoglobulin, and aLA represents alpha-lactalbumin, and LF represents lactoferrin, and Dicro represents diclofenac.*, * *: vs.Vehicle, p＜0.05,0.01 (check of Dunnet multiple comparisons)
Fig. 5 is illustrated in the figure that measures result relevant with the pain degree of dysmenorrhea in the randomized double blinding cross matching that improves effect of alpha-lactalbumin to dysmenorrhea.
Fig. 6 be illustrated in measure alpha-lactalbumin in the randomized double blinding cross matching that improves effect of dysmenorrhea with the figure of dysmenorrhea to the relevant result of the influence degree of daily life.
Fig. 7 is the figure that result relevant with the usage degree of analgesics in the randomized double blinding cross matching that improves effect of alpha-lactalbumin to dysmenorrhea is measured in expression.
The specific embodiment
Below, the present invention is described in detail.But the present invention is not limited to preferred embodiment following, can freely change within the scope of the invention.
The invention relates to the COX-2 selective depressant take alpha-lactalbumin as effective ingredient.Among the present invention so-called COX-2 selective depressant refer to compare with the inhibition to COX-1, the material stronger to the inhibition of COX-2.Although alpha-lactalbumin of the present invention is not particularly limited the source, preferably derive from people and/or non-human mammal (cattle, sheep, goat, horse etc.), more preferably be the alpha-lactalbumin of people or cattle.Aminoacid sequence and the base sequence of these alpha-lactalbumin are known, register in the data bases such as EMBL, DDBJ, NCBI.For example, the cattle alpha-lactalbumin is registered as ACCESSION No.J05147 in DDBJ.The base sequence of cattle alpha-lactalbumin is expressed as sequence numbering: 1, aminoacid sequence is expressed as sequence numbering: 2.Sequence numbering: in the aminoacid sequence of record, the 1st to the 19th is signal peptide in 2.In addition, known to sequence numbering: as to exist the 29th arginine (R) to be replaced as the mutant (" ミ ル ク Gross closes dictionary " be, Chao Warehouse Books shop p.35) of glutamine (Q) in 2 in the aminoacid sequence of record.The alpha-lactalbumin of secreting in the milk is the mature peptide that signal peptide has been cut off.The alpha-lactalbumin of using among the application's the embodiment is above-mentioned cattle mature peptide.
Alpha-lactalbumin can adopt the known technology manufacturings such as ammonium sulfate precipitation method (" the up-to-date original text dairy industry Ji Intraoperative Bian list ” Lao Farming Ji Intraoperative that changes popularizes association, 122-125 page or leaf, 1975), ultrafiltration (patent documentation 3), ion exchange (patent documentation 4) to obtain from the milk of the mammals such as people, cattle, sheep, goat etc.The content of alpha-lactalbumin is with the difference of the place of production, feedstuff etc. difference more or less in the milk, and Japan produces milk and is generally 1.2g/kg, if take milk as making raw material, can prepare a large amount of alpha-lactalbumin.In addition, also can carry out chemosynthesis according to aminoacid sequence and base sequence, perhaps the method manufacturing by the known genetic engineering of those skilled in the art obtains.Can also use more easily commercially available alpha-lactalbumin (such as Davisco company system etc.).
In addition, in the present invention, spendable alpha-lactalbumin be not limited in aforesaid people or non-human mammal as natural type by the known alpha-lactalbumin that is consisted of by known aminoacid sequence of people, as long as have the COX-2 selective inhibitory activity, it also can be the polypeptide that in above-mentioned aminoacid sequence, has the mutant of sudden change.Example as such polypeptide, can enumerate the mutant of natural type, homologue, artificial mutant etc., particularly, can enumerate in the alpha-lactalbumin aminoacid sequence that is included in natural type increases, disappearance, displacement, the polypeptide of the sequence of having inserted 1 or a plurality of aminoacid and having obtained, perhaps by the polypeptide of following polymerized nucleoside acid encoding, this polynucleotide is the polynucleotide of hybridizing under strict condition with the complementary strand of the polynucleotide that natural alpha-lactalbumin is encoded, more specifically, can enumerate and be included in sequence numbering: increase in the aminoacid sequence of record on 2, disappearance, displacement, the sequence of having inserted 1 or a plurality of aminoacid and having obtained, polypeptide with COX-2 selective inhibitory activity, in addition, as other examples, can also enumerate the polypeptide with COX-2 selective inhibitory activity by following polymerized nucleoside acid encoding, this polynucleotide is and is included in sequence numbering: the polynucleotide that the complementary strand of the polynucleotide of the coding region of the base sequence of record is hybridized under strict condition on 1.
Said mutation body polypeptide can be obtained by the known technology manufacturing of those skilled in the art.For example, can be from the zooblast of the expression alpha-lactalbumin of mammary gland etc. the total mRNA of preparation, use with the base sequence of natural type alpha-lactalbumin and carry out RT-PCR as the primer of basic engineering, the DNA that obtains imported to suitable promoter make its expression in the host-vector system, can obtain mutant polypeptide thus.In addition, can prepare cDNA library from zooblast, use is take the base sequence of the natural type alpha-lactalbumin probe as the basis preparation, the polynucleotide that selection and this probe are hybridized under strict condition from this cDNA library, this polynucleotide is incorporated in the suitable host, make its expression, can obtain mutant polypeptide.About above-mentioned promoter and host-vector system, so long as those skilled in the art can suitably select from known promoter and host-vector system.
About above-mentioned strict hybridization conditions, so long as those skilled in the art can suitably select.As an example, comprising 25% Methanamide, under exacting terms more, be in the hybridization solution of 50% Methanamide, 4 * SSC, 50mM Hepes pH7.0,10 * Denhart ' s solution, 20 μ g/ml denatured salmon sperm dnas, after carrying out the prehybridization of a whole night under 42 ℃, add label probe, hybridize for one evening 42 ℃ of lower insulations.Cleaning mixture in the washing afterwards and temperature conditions can be implemented according to the degree of " 1 * SSC, 0.1%SDS, 37 ℃ ", being the degree of " 0.5 * SSC, 0.1%SDS, 42 ℃ " as harsher high stringent condition, is the degree of " 0.2 * SSC, 0.1%SDS, 65 ℃ " as the high stringent condition of further harshness.The wash conditions of such hybridization is harsher, is expected to isolate the polynucleotide that has high homology with probe sequence.But, above-mentioned SSC, SDS and temperature conditions be combined as example, so long as those skilled in the art, just can realize and above-mentioned same stringency by the above-mentioned key element of the stringency that determines hybridization or other key element (such as concentration and probe concentration, probe length, hybridization time etc.) are carried out appropriate combination.
The polypeptide that utilizes such hybridization technique to separate the polymerized nucleoside acid encoding that obtains has high homology with above-mentioned natural type polypeptide at aminoacid sequence usually.So-called high homology refers to have more than at least 40%, is preferably more than 60%, more preferably more than 80%, more preferably more than 90%, further be preferably at least 95% homology.
In addition, also can in the base sequence of natural type alpha-lactalbumin, adopt the cassette mutagenesis method or utilize the positions such as sudden change method of PCR to import specifically or randomly artificial mutation, use suitable host-vector system that this polynucleotide is expressed, can obtain mutant polypeptide thus.Whether the above-mentioned polypeptide that makes has the COX-2 selective inhibitory activity, can measure this polypeptide according to known method the inhibition activity of COX-1 and COX-2 is confirmed, for example, the method described in the application's that can adopt in the back the embodiment is confirmed.The aminoacid sequence of the mutant polypeptide with COX-2 selective inhibitory activity like this and the aminoacid sequence of natural type have the homology of height, for example, homology is more than 80% or more than 85%, more preferably more than 90%, more preferably more than 95%.
In the present embodiment, as described later shown in the embodiment, in order to reach anti-inflammatory analgesic action, although understand to some extent difference according to different dosage forms, symptom, body weight etc., but can give (absorption) approximately 0.002mg～approximately 1 as the alpha-lactalbumin class of its effective ingredient in convert every 1kg body weight on the 1st of protein, 000mg preferably gives (absorption) approximately 200mg of 0.01mg～approximately, most preferably gives (absorption) approximately 40mg of 0.2mg～approximately.
Dosage as the alpha-lactalbumin of the effective ingredient of analgesic agent of the present invention, generally according to the effective ingredient meter, each day is about 0.1mg～approximately 50, and 000mg is preferably approximately 0.5mg～approximately 10,000mg, most preferably be approximately 10mg～approximately 2,000mg, once or gradation, the patient who uses compositions of the present invention to treat for needs can be before the meal, after the meal, carry out administration in the meal and/or before going to bed.Dosage is determined according to the patient's of administration age, body weight and administration purpose respectively.
Analgesic agent of the present invention can use with any form of pharmaceuticals or diet product.For example, can be used as the direct administration of pharmaceuticals, the food or the trophic function food that perhaps also can be used as the special purposes such as specific health food are directly taken in, and can be expected to thus various inflammation, pain are treated and/or prevented.In addition, though the forms such as aqueous, pasty state, solid, powder all can, be added in the various food (milk, refreshment drink, fermentation milk, yoghourt, cheese, bread, cookies, crispbread, pizza crust (pizza crust), formula milk, liquid food, patient with food, nutraceutical, frozen food, processed food and other delicatessen foods etc.) absorption and also can.
As the food that contains analgesic agent of the present invention, can mix making water, protein, saccharic, lipid, vitamins, minerals, organic acid, organic base, fruit juice, flavouring agent class etc.As protein, can enumerate of animal or plant nature protein, their analytes such as whole milk powder, defatted milk powder, partially skimmed milk powder, casein, whey powder, whey protein, whey protein concentrate, whey protein sepd, alpha-casein, beta-casein, κ-casein, beta lactoglobulin, lactoferrin, soybean protein, egg protein, meat protein; The various compositions of milk etc. that come from such as butter, whey mineral, butter, milk surum, non-protein type nitrogen, sialic acid, phospholipid, lactose.Also can contain the peptides such as phosphopeptide caseinate, arginine, lysine or aminoacid also can.As saccharic, can enumerate for example saccharide, producing starch (except dextrin, can also be soluble starch, Britain's starch (Britishstarch), Oxytarch, starch ester, starch ether etc.), dietary fiber etc.As lipid, can enumerate such as Adeps Sus domestica, fish oil etc., they distillate oil, hydrogenated oil and fat, ester exchange wet goods animal raw fat; Petiolus Trachycarpi oil, Oleum Helianthi, Semen Maydis oil, Oleum Brassicae campestris, cocos nucifera oil, their distillate oil, hydrogenated oil and fat, ester exchange wet goods vegetative grease etc.As vitamins, can enumerate such as vitamin A, carotenoid, vitamin B group, vitamin C, D vitamin, vitamin E, K vitamin, Citrin, CoenzymeQ10, nicotinic acid, nicotinic acid, pantothenic acid, biotin, inositol, choline, folic acid etc., as mineral, can enumerate such as calcium, potassium, magnesium, sodium, copper, ferrum, manganese, zinc, selenium etc.As organic acid, can enumerate such as malic acid, citric acid, lactic acid, tartaric acid etc.In the manufacturing of the diet product that contain analgesic agent of the present invention, these compositions can be composites, also can derive from natural materials, and/or can use the food that contains in a large number these compositions as raw material.These compositions can be used in combination more than 2 kinds.As the form of food, solid, liquid all can.In addition, also can be gel etc.
When analgesic agent of the present invention uses as pharmaceuticals, in every way administration.As its mode, can enumerate the oral administration such as tablet, capsule, granule, powder, syrup etc., still, also can be processed into the preparations such as injection or solution and adopt through medium other the administering mode of pipe, quiet notes, Intradermal, muscle.Above-mentioned various preparation can add the known adjuvant that excipient, bonding agent, disintegrating agent, lubricant, correctives, cosolvent, suspensoid, coating materials, solvent, isotonic agent etc. are commonly used by adopting usual way in the pharmaceutical preparations technology field in principal agent, be prepared into thus preparation.In addition, also can contain an amount of calcium.Can also further add an amount of vitamin, mineral, organic acid, saccharide, aminoacid, peptide class etc.
Analgesic agent of the present invention can be used for the treatment of diseases associated with inflammation.Analgesic agent of the present invention for example is applicable to arthritis, rheumarthritis (juvenile, chronic), ulcerative colitis, Crow engler (Crohn ' s) sick (Crohn disease), but not only be confined to these, all can be suitable for the disease of inflammation.
There are the kinds such as infectivity, immunity, crystallinity in the arthritis.Particularly, known have tuberculosis, gout, beaevais' disease, morphotropism joint disease, acute rheumatic fever, chronic eczema arthritis, juvenile rheumarthritis, still disease, Reiter syndrome, ankylosing spondylitis etc. (the abundant husband in well village, tail shape please youth, high Jiu Shi Mo, the clear youth of well of hanging down compiles, " up-to-date internal medicine is large to be the 74th Juan Seki Festival illness＜Gu Seki Festival illness 2〉", the distribution of middle mountain bookstore, p.19, nineteen ninety-five).As being used for the treatment of one of medicine of these diseases, can use the NSAIDs of COX inhibitor etc.
Rheumarthritis is the diseases associated with inflammation that is caused by multiple arthritis.Its exact cause is not clear, but physical factors, immunology factor, these 3 of environmental factorss are considered to important reason.As the immunology factor, think to secrete the material that is known as cytokine, the lymphocyte function that plays an important role in immunity is unusual, consequently, unusual antibody occurs, is combined with self joint tissue, causes arthritis.The initial stage symptom mostly is the WA ankylosis, and arthralgia and swelling occur.It is multiple that arthralgia is divided into spontaneous pain when static (), tenderness, kinesalgia etc.In addition, swollen joint, heat, the symptom such as rubescent also appear.Arthritis is the synovitis that inflammation has appearred in synovial membrane, how to see symmetrically brothers' joint.There is brothers' finger, hands, foot, elbow, knee joint, shoulder etc. in the normal joint that occurs, and development goes down to occur distortion.In case suffer from rheumarthritis, be difficult to cure fully, the patient will follow this disease all one's life.
Nowadays, as the inflammatory pain of the inflammatory pain in joint that be used for to relax beaevais' disease etc. or bone junction at prescription drug used in everyday, the synthetic compound of tens of kinds of the aspirin that is known as NSAIDs, indomethacin, ibuprofen, diclofenacs etc. is arranged and is known as the synthetic glucocorticoid etc. of steroid.
Transmitting inflammation pain be prostaglandin, make the biosynthesis of prostaglandin reduce/stop if suppressing COX by NSAIDs, then pain relaxes.But, as previously mentioned, if this class medicine of life-time service then might bring out to altofrequency peptic ulcer.Therefore, for fear of bringing out peptic ulcer, seeking for many years the method that can improve arthritis pain with tight security always.The topica of nearest NSAIDs (topic) is for specificity is suppressed at COX-2 that inflammation part induces, (the abundant husband in well village, tail shape are pleased youth, high Jiu Shi Mo, the clear youth of well of hanging down compiles to reduce cox 2 inhibitor as the peptic ulcer of the side effect of NSAIDs, " up-to-date internal medicine is large to be the 74th Juan Seki Festival illness＜Gu Seki Festival illness 2〉", the distribution of middle mountain bookstore, p.19, nineteen ninety-five, and Fukushima Athens compiles, " メ Le Network マ ニ ユ ア Le medical science feelings Reported home edition ", Nikkei BP company, p.233-235,1999).
In addition, analgesic agent of the present invention also has alleviation effects to the pain without inflammation.Pain is divided into these two kinds of acute pain and chronic pains.Acute pain (for example ankle from sprains is such) refers to that a part of health is injured or the caution signals of certain infringement occurs.By pain is reacted, to remove reason, to prevent the reaction that worsens.Corresponding, chronic pain refers to that caution signals is inoperative, no matter carries out what kind for the treatment of, the situation that pain does not also disappear.As chronic pain, neuropathic pain, nociceptive pain or their mixed type etc. are arranged.
Epoxidase (COX) is known by people as the relevant enzyme of prostaglandin (PG) biosynthesis.There is report to have at least now these 3 kinds of COX-1, COX-2, COX-3 as COX.COX-1 is that physiological exists, and it takes on the physiological action of stomach, kidney etc.The stimulation such as its concentration and LPS or inflammation are irrelevant, do not change.On the other hand, COX-2 is relevant with inflammation and cell injury etc.Do not express under the normal condition, the stimulation by LPS, IL-1 etc. or inflammation etc. are mainly newly produced by wandering cell, and its generation can be suppressed by dexamethasone.That is, in the situation that selectivity suppresses COX-2, be expected to obtain the few side effects to harmonization of the stomach kidney etc., the effect that inflammation and tissue injury etc. is improved.
Indication as the COX inhibitor, known have: (1) beaevais' disease, osteoarthrisis deformans knee, congealed shoulder, neck shoulder wrist syndrome, lumbago, tenosynovitis, rheumatism or the organ of locomotion diseases such as gout, (2) pain after the postoperative wound, cancerous pain, the pain in tooth section field, the disease neuralgia, the calculus pain, the painful diseases such as dysmenorrhea, (3) various infection diseases, malignant tumor, collagen etc. are followed the disease of heating, (4) cerebral infarction, transient ischemic attack, ischemic heart desease, mucocutaneous lymphnode syndrome, albuminuria, cancerometastasis etc. utilize the indication of antithrombotic or antiplatelet effects, (5) endotoxin shock, patent ductus arteriosus, hypotension, the Bartter syndrome, masculine sterility, immunosuppressant, the reinforcements of immunotherapy etc. mainly utilize (the abundant husbands in well village such as the synthetic inhibiting indication of PG, tail shape is pleased the youth, high Jiu Shi Mo, the clear youth of well of hanging down compiles, " up-to-date internal medicine is large to be the 74th Juan Seki Festival illness＜Gu Seki Festival illness 2〉", the distribution of middle mountain bookstore, p.77-78, nineteen ninety-five).Therefore, have COX-2 and suppress active analgesic agent of the present invention and be not limited to treat all with the disease of inflammation, also can be suitable for above-mentioned disease.
In addition, whole formerly technical literature of quoting in this description is all incorporated in this description as a reference.
Embodiment
Below, the present invention will be described to enumerate embodiment, but the present invention is not limited to these.
[embodiment 1] (the swollen inhibition test of chondrus ocellatus Holmes colloidality foot is to acutely inflamed effect)
The inhibition that the present embodiment has been verified whey protein sepd (WPI) and contained various bringing out property of albumen on Carrageenan foots swell in WPI.In addition, WPI refers to microporous filter (MF), ultrafiltration (UF), cross-flow microfiltration (CFM), ion exchange etc. milk surum be processed Separation of Proteins, improved the concentration of protein and the material that obtains.
(method)
The be through with Wistar rat (male, 6 ages in week, 1 group 6,5 groups) of above domestication of 1 week of use carries out the oral administration of various substances.As substances, whey protein sepd (the WPI of normal saline will be dissolved in, derive from cattle, Davisco Foods International, Inc. company's system) 1g/kg[WPI group], alpha-lactalbumin (α-LA, derive from cattle, Davisco FoodsInternational, Inc. company's system) 250mg/kg (25w/w% of WPI) [aLA group], beta lactoglobulin (β-LG, derive from cattle, Davisco Foods International, Inc. company's system) 500mg/kg (50w/w% of WPI) [bLG group], lactoferrin (LF, derive from cattle, Murray GoulburnIngredients company system) 10mg/kg (1w/w% of WPI) [LF group], and normal saline [matched group], come administration with the consumption indicated separately by the capacity of 4ml/kg respectively.The oral administration of substances is after 1 hour, left the carrageenin solution 0.1ml of limb foot plantar subcutaneous injection 1w/w%.At the time point determining foot volume of carrageenin administration after 3 hours, estimate antiinflammation.In addition, in the following embodiments, WPI, α-LA, β-LG and the LF use material identical with the material here.
(result)
As the main component among the WPI, there are alpha-lactalbumin (accounting for the 25w/w% of WPI), beta lactoglobulin (accounting for the 50w/w% of WPI), lactoferrin (accounting for the 1w/w% of WPI).Current in order to study the active component among the WPI, contain the proportional administration of having carried out according to each material.Consequently, only have alpha-lactalbumin to show the swollen inhibition of on Carrageenan foot.From then on result, the swollen inhibition of WPI on Carrageenan foot derives from alpha-lactalbumin (Fig. 1).
[embodiment 2] (adjuvant-induced arthritis (treatment) model is to effect of subacute inflammation (experimental chronic inflammatory disease))
Make the experimental animal model of CFA induced adjuvant arthritis in rats as the animal model of rheumatic arthritis in the present embodiment, to verify that various albumen contained among the WPI are to arthritic therapeutic effect.
(method)
To the Freund's complete adjuvant of the left limb foot plantar subcutaneous injection 0.1mL of the Wistar rat (male, 6 ages in week) of the above domestication of 1 week that is through with, to bring out adjuvant-induced arthritis.The Freund's complete adjuvant that uses contains the liquid paraffin of 8.5mL, the surfactant sorbester p17 of 1.5mL and the Bacillus tuberculosis H37Rv of 7.5mg deactivation in 10mL.After giving adjuvant and beginning 14, filter out the arthritic rat of abundant trouble, divide into groups take the sufficient volume of the left limb foot sole of the foot as benchmark (1 group 6,5 groups).
With the substances (alpha-lactalbumin [aLA group], beta lactoglobulin [bLG group], lactoferrin [LF group]) that is dissolved in normal saline separately with the consumption of 300mg/kg, will be as the diclofenac (and the pure pharmaceutical worker's industry of light company system) [positive controls] of nonsteroidal antiinflammatory and analgesic agent (NSAIDs) with the consumption of 50mg/kg, between the 14th～16 day that gives that adjuvant begins 3 days, carry out continuously oral administration by the capacity of 4ml/kg.In addition, group in contrast, the normal saline to same capability carries out continuous oral administration continuously between the 14th～16 day that gives that adjuvant begins 3 days.
Give the 14th (after just having given test substance) that adjuvant begins, measuring the sufficient volume of the left limb foot sole of the foot on the 15th, 17, the research therapeutic effect.
(result)
For the edema of the left limb foot that has given adjuvant, alpha-lactalbumin has suppressed the deterioration of edema.In addition, by giving lactoferrin, sufficient volume reduces, and has obtained therapeutic effect (Fig. 2).
[embodiment 3] (adjuvant-induced arthritis (pain) model is to effect of chronic pain)
The Freund's complete adjuvant 0.1mL identical to the left limb foot plantar subcutaneous injection of the Wistar rat (male, 6 ages in week) of the above domestication of 1 week that is through with and embodiment 2 is to bring out adjuvant-induced arthritis.After giving adjuvant and beginning 16, filter out the arthritic rat of abundant trouble, divide into groups take the sufficient volume of the left limb foot sole of the foot as benchmark (1 group 6,5 groups).
With the substances (alpha-lactalbumin [aLA group], beta lactoglobulin [bLG group], lactoferrin [LF group]) that is dissolved in normal saline separately with the consumption of 300mg/kg, will be as the diclofenac [positive controls] of analgesic agent with the consumption of 50mg/kg, carry out oral administration by the capacity of 4ml/kg.Giving substances after 1 hour, mensuration has or not the reaction that pipes to the podarthral crooked stimulation pain that stretches of left hind.The number of times that will pipe when implementing 10 crooked stretching, extensions quantizes, and estimates thus the pain inhibition.
(result)
Give alpha-lactalbumin and single gives in the lactoferrin at single, all have the inhibition (Fig. 3) to arthritis ache.
[embodiment 4] (the acetic acid twisting test is to effect of pain)
Verified the therapeutic effect of contained various bringing out property of albumen Dichlorodiphenyl Acetate pain among the WPI in the present embodiment.
The be through with mice of above domestication of 1 week of use is (male, 6 ages in week, 1 group 6,5 groups), with the whey protein sepd (WPI) [WPI group], alpha-lactalbumin [aLA group], beta lactoglobulin [bLG group], lactoferrin [LF group] that are dissolved in normal saline separately with the consumption of 300mg/kg, will be as the diclofenac [positive control (Dicro) group] of analgesic agent with the consumption of 50mg/kg, carry out oral administration according to the capacity of 4ml/kg.In addition, (Vehicle) organizes in contrast, and per os gives the normal saline of same capability.After the oral administration of substances begins 1 hour, give the acetum 0.2mL of 0.6v/v% to intraperitoneal.To quantize from giving the painful action frequency that acetic acid begins after 10 minutes between till 30 minutes 20 minutes, estimate thus the pain inhibition.
(result)
The result as shown in Figure 4.The visible significant analgesic effect of alpha-lactalbumin and lactoferrin.
[embodiment 5] (enzyme suppresses experiment)
Verified in the present embodiment that substances (alpha-lactalbumin, WPI) is active to the various enzymeinhibitions relevant with inflammation.
(method)
(1) determination of activity (n=2) of using the enzyme standard substance of epoxidase-1 (COX-1) to carry out
With the enzyme standard substance of the substances (alpha-lactalbumin or WPI) of each concentration and epoxidase-1 (EC 1.14.99.1, derive from human blood platelets) HBSS (Hanks ' Balanced SaltSolutions (Hanks ' balanced salt solution), 15mM HEPES; PH 7.4) in 37 ℃ of lower precultures after 15 minutes, the mode take ultimate density as 100 μ M added arachidonic acid, 37 ℃ of lower reactions 15 minutes.After the reaction, utilize EIA (enzyme immuno assay, EIA enzyme immunoassay) to measure PGE 2Concentration.In addition, setting the sample that does not add substances (alpha-lactalbumin or WPI) organizes in contrast.The meansigma methods of the measured value of matched group is made as enzyme inhibition rate 100%, calculates the enzyme inhibition rate of alpha-lactalbumin and WPI, to suppress 50% concentration as IC 50Value.
(2) determination of activity (n=2) of using the enzyme standard substance of cyclooxygenase-2 (COX-2) to carry out
Substances (alpha-lactalbumin or WPI) and cyclooxygenase-2 (EC 1.14.99.1, people's restructuring with each concentration; The Sf21 insect cell) enzyme standard substance in the Tris-HCl buffer (pH7.7) of the 100mM that contains 1mM glutathion, 1 μ M hematin, 500 μ M phenol 37 ℃ of lower precultures after 15 minutes, mode take ultimate density as 0.3 μ M added arachidonic acid, 37 ℃ of lower reactions 5 minutes.After the reaction, utilize EIA to measure PGE 2Concentration.In addition, setting the sample that does not add substances (alpha-lactalbumin or WPI) organizes in contrast.The meansigma methods of the measured value of matched group is made as enzyme inhibition rate 100%, calculates the enzyme inhibition rate of alpha-lactalbumin and WPI, to suppress 50% concentration as IC 50Value.
(3) determination of activity (n=2) of using the enzyme standard substance of inducible nitric oxide synthase (iNOS) to carry out
Substances (alpha-lactalbumin or WPI) and inducible nitric oxide synthase (EC 1.14.13.39 with each concentration, derive from mouse macrophage) the enzyme standard substance containing the NADPH of 4mM (nicotinamide adenine dinucleotide (nicotinamide adenine dinucleotide) reduced form), 8 μ M (6R)-5,6,7, the biological dish purine of 8-tetrahydrochysene-L-, the FAD of 8 μ M, in the Tris-HCl buffer (pH7.9) of the 100mM of the DTT of 6mM (dithiothreitol, DTT (Dithiothreitol)) 37 ℃ of lower precultures after 15 minutes, mode take ultimate density as 100 μ M added arginine, 37 ℃ of lower reactions 2 hours.After the reaction, utilize spectrophotography to NO 2-Carry out quantitatively.In addition, setting the sample that does not add substances (alpha-lactalbumin or WPI) organizes in contrast.Take the meansigma methods of the quantitative values of matched group as enzyme inhibition rate 100%, calculate the enzyme inhibition rate of alpha-lactalbumin and WPI, to suppress 50% concentration as IC 50Value.
(4) determination of activity (n=2) of using every standard substance of phospholipase A2 (PLA2) to carry out
With the enzyme standard substance of the substances (alpha-lactalbumin or WPI) of each concentration and phospholipase A2-I (EC 3.1.1.4, derive from Pancreas Sus domestica) or phospholipase A2-II (EC 3.1.1.4, derive from western Pedicellus et Pericarpium Trapae speckle rattle snake) in the glycine of the 0.1M of the EDTA that contains 20 μ M-NaOH buffer (pH 9.0) 37 ℃ of lower precultures after 15 minutes, the mode take ultimate density as 0.03 μ Ci adds 1-palmityl-2-[1- 14C] oleoyl-L-3-phosphatidylcholine, 37 ℃ of lower reactions 5 minutes.Remove unreacted 1-palmityl-2-[1- 14C] behind oleoyl-L-3-phosphatidylcholine, utilize the radioactivity of liquid flashing counter measuring oleate fraction.In addition, setting the sample that does not add substances (alpha-lactalbumin or WPI) organizes in contrast.The meansigma methods of the radioactivity (Ci) of matched group is made as enzyme inhibition rate 100%, calculates the enzyme inhibition rate of alpha-lactalbumin and WPI, to suppress 50% concentration as IC 50Value.
(result)
The result is shown in table 1 and table 2.Alpha-lactalbumin has high activity to cyclooxygenase-2, phospholipase A2-I, phospholipase A2-II.Especially optionally to the high activity of cyclooxygenase-2 performance.In addition, alpha-lactalbumin is compared with WPI and is also had high cyclooxygenase-2 activity.
[table 1]
IC 50 The enzyme inhibition rate of the alpha-lactalbumin of 1w/w%
Epoxidase-1 Can't measure 39％
Cyclooxygenase-2 0.0558w/w％ -
Inducible nitric oxide synthase Can't measure 13％
Phospholipase A2-I 0.375w/w％ -
Phospholipase A2-II 0.108w/w％ -
(the enzyme inhibition of table 1 alpha-lactalbumin)
[table 2]
IC 50 The enzyme inhibition rate of the WPI of 1w/w%
Epoxidase-1 Can't measure 45％
Cyclooxygenase-2 0.285w/w％ -
Inducible nitric oxide synthase Can't measure 15％
Phospholipase A2-I Can't measure Promote 21% enzymatic activity
Phospholipase A2-II 0.194w/w％ -
(the enzyme inhibition of table 2WPI)
[embodiment 6] (to people's test of dysmenorrhea (pain))
In the present embodiment, verified the improve effect of alpha-lactalbumin to dysmenorrhea by randomized double blinding cross matching.
(experimenter)
Subtend entrust cooperation the bulletin application 70 women of cooperation carried out prior investigation, 30 of the women (22～50 years old of following condition are satisfied in choice, average 36.4 years old) as the experimenter: menstrual cycle stabilizes, it is 28 ± 3, the answer of the nearest self-recording formula questionnaire about menstruation (table 3: the pain degree of dysmenorrhea, dysmenorrhea are to the influence degree of daily life, the usage degree of analgesics) is: the pain degree of dysmenorrhea is more than 1, and dysmenorrhea score (dysmenorrhea is to the influence degree of daily life, the usage degree of analgesics) adds up to more than 2.
(tested material)
The identical tablet A of outward appearance, B have been prepared.A kind of for containing the tablet (containing alpha-lactalbumin 150mg in 1) of alpha-lactalbumin, another kind does not contain alpha-lactalbumin (placebo tablet) fully.Experimenter and estimator are not knowing before the off-test which kind of sheet is to begin test under the state of alpha-lactalbumin tablet.
(the absorption method of tested material)
In putting down into 3 menstrual cycle of 19 years 9～Decembers, implemented test.The tested material that will be set as from 1 menstrual cycle that first menstruation end finishes to next menstruation the 1st time is taken in the cycle.The 1st time tested material is taken in ensuing 1 menstrual cycle of cycle (menstruation finishes to finish to next menstruation) for not taking in the lay-off period of tested material.Then, ensuing 1 menstrual cycle of lay-off period (menstruation finishes to finish to next menstruation) is the 2nd time tested material absorption cycle.
The experimenter is divided into 2 groups, 1 group (tablet A takes in phase-lay-off period-tablet B and takes in the phase, n=13) and 2 groups (tablet B takes in phase-lay-off period-tablet A and takes in the phase, n=17) has set respectively the different order of absorption tablet A, tablet B.
The tested material absorption phase is that 1 tested material (tablet A or tablet B) of 2 is taken in continuously at early, middle and late 3 times.
In addition, reach duration of test before on-test and can freely use analgesics.
(evaluation methodology)
1 intermenstrual period before on-test, the experimenter has filled in self-recording formula questionnaire (table 3).And take in phase and the lay-off period in tested material, the experimenter is required to fill in continuously self-recording formula questionnaire 1 time on the 1st.The content of this self-recording formula questionnaire is relevant with dysmenorrhea and QOL.Intermenstrual period be recorded to from fill out the degree of the dysmenorrhea in the formula questionnaire, on the impact of daily life and the usage degree of analgesics, adopt oral account to describe point system (verbal rating scale) and quantize, adopt each experimenter during in maximum estimate.In addition, the dysmenorrhea of evaluation object is defined as lumbago and hypogastric region pain.
Further ask the result's of the menstruation of calculating before on-test and lay-off period meansigma methods, this is worth as non-absorption time value.
(statistical disposition)
Obtain meansigma methods and the standard error of each measured value, statistical software is adopted in the check of significant difference, tests so that corresponding group to be arranged in the non parametric tests method.(with the rank test of Wilcoxson symbol)
(result)
Compare with the non-absorption phase, measure the oral account of the pain degree of dysmenorrhea and describe score value significantly (p＜0.01) reduction (Fig. 5) by the absorption of alpha-lactalbumin tablet.
Compare with the non-absorption phase, estimate dysmenorrhea the oral account of the influence degree (QOL) of daily life is described tendency (p＜0.10) that score value has a minimizing by the absorption of alpha-lactalbumin tablet (Fig. 6).
For the usage degree of analgesics, the number of taking analgesics because of dysmenorrhea before on-test is 21 people (70%), still, takes in interim 10 people (all 33%) that are reduced to by take in phase and placebo tablet at the alpha-lactalbumin tablet.Compare with the non-absorption phase, the oral account of measuring the usage degree of analgesics is described score value and is taken at tablet arbitrarily and interimly remarkable reduction (p＜0.01) is all arranged (Fig. 7).
Can be clear and definite from above result, take in alpha-lactalbumin 300mg by 3 times on the 1st (early, middle and late), then the usage ratio of analgesics significantly reduces, and dysmenorrhea is relaxed simultaneously, and QOL is also had the effect of improvement.
[table 3]
The pain degree of dysmenorrhea
Degree Score
Painless 0
Mild pain 1
Moderate pain 2
Have an intense pain 3
Extreme pain 4
The usage degree of analgesics
Degree Content Score
Nothing Nothing
0
Slightly Intermenstrual period has 1 and has used analgesics 1
Moderate Intermenstrual period has 2 and has used analgesics 2
Severe Intermenstrual period has more than 3 days and has used analgesics 3
Dysmenorrhea is to the influence degree of daily life
Degree Content Score
Nothing Nothing
0
Slightly Work (study housework) etc. has some obstacles 1
Moderate Reach the degree of wanting to lie down and having a rest, work (study housework) is impacted 2
Severe Be unable to leave the bed to work (study housework) more than 1 day 3
(content of table 3 self-recording formula questionnaire)
Alpha-lactalbumin class as effective ingredient of the present invention can be produced in a large number by cheap raw materials such as milk.In addition, few side effects, safe, can take in for a long time, therefore, by sneaking in the pharmaceuticals or taking in as food, can obtain the anti-inflammatory analgesic effect.And, by daily administration or absorption, can carry out sufficient nutritional care, therefore, can also be for the manufacture of the purposes for functional food of the patient of malnutrition etc.
Sequence table
＜110〉Mingzhi Dairy Co., Ltd
＜120〉novel antiphlogistic-analgesic agent
<130>M1-A0704P
<150>JP 2007-127286
<151>2007-05-11
<160>2
<170>PatentIn version 3.4
<210>1
<211>724
<212>DNA
<213>Bos taurus
<220>
<221>CDS
<222>(28)..(456)
<220>
<221>sig_peptide
<222>(28)..(84)
<220>
<221>mat_peptide
<222>(85)..(453)
<400>1
atttcagaat cttggggggt aaccaaa atg atg tcc ttt gtc tct ctg ctc ctg 54
Met Met Ser Phe Val Ser Leu Leu Leu
-15
gta ggc atc cta ttc cat gcc acc cag gct gaa cag tta aca aaa tgt 102
Val Gly Ile Leu Phe His Ala Thr Gln Ala Glu Gln Leu Thr Lys Cys
-10 -5 -1 1 5
gag gtg ttc cgg gag ctg aaa gac ttg aag ggc tac gga ggt gtc agt 150
Glu Val Phe Arg Glu Leu Lys Asp Leu Lys Gly Tyr Gly Gly Val Ser
10 15 20
ttg cct gaa tgg gtc tgt acc acg ttt cat acc agt ggt tat gac aca 198
Leu Pro Glu Trp Val Cys Thr Thr Phe His Thr Ser Gly Tyr Asp Thr
25 30 35
caa gcc ata gta caa aac aat gac agc aca gaa tat gga ctc ttc cag 246
Gln Ala Ile Val Gln Asn Asn Asp Ser Thr Glu Tyr Gly Leu Phe Gln
40 45 50
ata aat aat aaa att tgg tgc aaa gac gac cag aac cct cac tca agc 294
Ile Asn Asn Lys Ile Trp Cys Lys Asp Asp Gln Asn Pro His Ser Ser
55 60 65 70
aac atc tgt aac atc tcc tgt gac aag ttc ctg gat gat gat ctt act 342
Asn Ile Cys Asn Ile Ser Cys Asp Lys Phe Leu Asp Asp Asp Leu Thr
75 80 85
gat gac att atg tgt gtc aag aag att ctg gat aaa gta gga att aac 390
Asp Asp Ile Met Cys Val Lys Lys Ile Leu Asp Lys Val Gly Ile Asn
90 95 100
tac tgg ttg gcc cat aaa gca ctc tgt tct gag aag ctg gat cag tgg 438
Tyr Trp Leu Ala His Lys Ala Leu Cys Ser Glu Lys Leu Asp Gln Trp
105 110 115
ctc tgt gag aag ttg tgaacacctg ctgtctttgc tgcttctgtc ctctttctgt 493
Leu Cys Glu Lys Leu
120
tcctggaact cctctgcccc gtggctacct cgttttgctt ctttgtaccc ccttgaagct 553
aactcgtctc tgagccctgg gccctgtagt gacaatggac atgtaaggac taatctccag 613
gggtgcatga atggcgctct ggacttttga cccttsstcg atgtccctga tggcgctttt 673
aatgcaacag tacatattcc acttttgtcc cgaataaaaa gcctgatttt g 724
<210>2
<211>142
<212>PRT
<213>Bos taurus
<400>2
Met Met Ser Phe Val Ser Leu Leu Leu Val Gly Ile Leu Phe His Ala
-15 -10 -5
Thr Gln Ala Glu Gln Leu Thr Lys Cys Glu Val Phe Arg Glu Leu Lys
-1 1 5 10
Asp Leu Lys Gly Tyr Gly Gly Val Ser Leu Pro Glu Trp Val Cys Thr
15 20 25
Thr Phe His Thr Ser Gly Tyr Asp Thr Gln Ala Ile Val Gln Asn Asn
30 35 40 45
Asp Ser Thr Glu Tyr Gly Leu Phe Gln Ile Asn Asn Lys Ile Trp Cys
50 55 60
Lys Asp Asp Gln Asn Pro His Ser Ser Asn Ile Cys Asn Ile Ser Cys
65 70 75
Asp Lys Phe Leu Asp Asp Asp Leu Thr Asp Asp Ile Met Cys Val Lys
80 85 90
Lys Ile Leu Asp Lys Val Gly Ile Asn Tyr Trp Leu Ala His Lys Ala
95 100 105
Leu Cys Ser Glu Lys Leu Asp Gln Trp Leu Cys Glu Lys Leu
110 115 120

Claims (2)

1. the application of alpha-lactalbumin in making the COX-2 selective depressant.
2. the application of alpha-lactalbumin in making the dysmenorrhea treatment medicine.
CN2008800156559A 2007-05-11 2008-05-09 Novel antiphlogistic-analgesic agent Active CN101678077B (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
JP2007127286 2007-05-11
JP127286/2007 2007-05-11
PCT/JP2008/058631 WO2008140041A1 (en) 2007-05-11 2008-05-09 Novel antiphlogistic-analgesic agent

Publications (2)

Publication Number Publication Date
CN101678077A CN101678077A (en) 2010-03-24
CN101678077B true CN101678077B (en) 2013-02-20

Family

ID=40002237

Family Applications (1)

Application Number Title Priority Date Filing Date
CN2008800156559A Active CN101678077B (en) 2007-05-11 2008-05-09 Novel antiphlogistic-analgesic agent

Country Status (5)

Country Link
JP (2) JP5683106B2 (en)
KR (1) KR20100017708A (en)
CN (1) CN101678077B (en)
HK (1) HK1142277A1 (en)
WO (1) WO2008140041A1 (en)

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103431276A (en) * 2013-08-31 2013-12-11 山东卫康生物医药科技有限公司 Mixed-grain total-nutrition formulation food for patients with inflammatory bowel diseases
JP6038416B1 (en) * 2015-04-07 2016-12-07 株式会社明治 Fire suppression agent
JP2016204356A (en) * 2015-04-23 2016-12-08 株式会社明治 Hepatic fibrogenesis inhibitor
AU2016366635A1 (en) * 2015-12-07 2018-06-21 Benevolentai Cambridge Limited VAP-1 inhibitors for treating pain
CN113876944A (en) * 2021-10-18 2022-01-04 南京京达生物技术有限公司 Preparation method of immunologic adjuvant

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2006219458A (en) * 2005-02-14 2006-08-24 Meiji Milk Prod Co Ltd Agent for inhibiting ischemic enteropathy

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP4534316B2 (en) * 2000-07-14 2010-09-01 株式会社玄米酵素 Nutritional supplement manufacturing method and nutritional supplement
JP2003088329A (en) * 2001-09-19 2003-03-25 Yamagami Chizuko Analgesic health supplement
US7144590B2 (en) * 2003-01-09 2006-12-05 Lipoprotein Technologies, Inc. Bioactive compositions derived from humulus lupulus
JP4578821B2 (en) * 2004-02-19 2010-11-10 明治乳業株式会社 Pharmaceutical composition for the treatment of nephritis

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2006219458A (en) * 2005-02-14 2006-08-24 Meiji Milk Prod Co Ltd Agent for inhibiting ischemic enteropathy

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
ROBERT J.et al.Changes in Lactoferrin, Immunoglobulin G, Bovine SerumAlbumin, and a-Lactalbumin During Acute Experimentaland Natural Coliform Mastitis in Cows.《INFECTION AND IMMUNITY》.1976,第13卷(第2期),第533-542页. *
山口真.ホエイタンパク質およびホエイペプチドの特長と抗.《Milk Scicence》.2005,第54卷(第2期),1-8. *

Also Published As

Publication number Publication date
WO2008140041A1 (en) 2008-11-20
JP5797234B2 (en) 2015-10-21
JPWO2008140041A1 (en) 2010-08-05
KR20100017708A (en) 2010-02-16
JP2013241418A (en) 2013-12-05
CN101678077A (en) 2010-03-24
HK1142277A1 (en) 2010-12-03
JP5683106B2 (en) 2015-03-11

Similar Documents

Publication Publication Date Title
JP3128244B2 (en) Casein phosphopeptide, casein containing the same, and methods for producing them
JP5306188B2 (en) Preparations containing whey proteins and hydrolysates for enhancing muscle recovery
CA2778647A1 (en) Nutritional compositions including a high protein component and exogenous nucleotides
CN101678077B (en) Novel antiphlogistic-analgesic agent
JP5465834B2 (en) Liver function protectant
KR20100105598A (en) Sense-improving agent
KR101094693B1 (en) Skin conditioning agent
KR101413207B1 (en) Pharmaceutical composition and health functional food for preparing or treating inflammatory disease
CN101360508B (en) Pharmaceutical composition, food or drink, or feed for intestinal disease
JP4452569B2 (en) Foods based on peach blossoms and lactoferrin
JP2008184428A (en) Skin-beautifying agent
WO2012077076A1 (en) Treating or preventing sensitivity to milk allergens
AU2016272477B2 (en) Enzyme-treated milk product, method for producing same, composition, and product
JP6579605B2 (en) Skin improver
JP6579604B2 (en) Hair growth / hair growth promoter
KR20140072893A (en) Sensation-improving agent
JP2007055979A (en) Recovery promoter from diarrhea by rotavirus infection
JP6555738B2 (en) Preventive / ameliorating agent for diseases with fatigue
JP2008115169A (en) PHARMACEUTICAL AND NUTRITIONAL COMPOSITION FOR INCREASING TOTAL IgA LEVEL AND/OR ANTIGEN-SPECIFIC IgA ANTIBODY TITER IN MOTHER&#39;S MILK
JP2009126787A (en) Sense-improving agent
JPH1028553A (en) Immunoactivation liquid food
JP7397562B2 (en) Lower back pain reliever
JP5995803B2 (en) Catechol-O-methyltransferase inhibitor
JP2024121661A (en) Blood sugar improver
JP2007230998A (en) Oral anti-inflammatory agent

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
REG Reference to a national code

Ref country code: HK

Ref legal event code: DE

Ref document number: 1142277

Country of ref document: HK

C53 Correction of patent for invention or patent application
CB02 Change of applicant information

Address after: Tokyo, Japan, Japan

Applicant after: Meiji Co., Ltd.

Address before: Tokyo, Japan, Japan

Applicant before: Meiji Dairies Corporation

COR Change of bibliographic data

Free format text: CORRECT: APPLICANT; FROM: MEIJI DAIRIES CORPORATION TO: MEIJI KK

C14 Grant of patent or utility model
GR01 Patent grant
REG Reference to a national code

Ref country code: HK

Ref legal event code: GR

Ref document number: 1142277

Country of ref document: HK