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 alpha-lactalbumin is the COX-2 selective depressant of effective ingredient.Among the present invention so-called COX-2 selective depressant be meant with inhibition effect COX-1 compare, the material stronger to the inhibition effect of COX-2.Though 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.The aminoacid sequence of these alpha-lactalbumin and base sequence are known, register in 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.Excretory alpha-lactalbumin is the mature peptide that signal peptide has been cut off in the milk.The alpha-lactalbumin of using among the application's the embodiment is above-mentioned cattle mature peptide.
Alpha-lactalbumin can adopt 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 known technology manufacturings such as (patent documentations 4) to obtain from the milk of 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 be to make raw material with milk, 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 commercially available alpha-lactalbumin (for example Davisco corporate system etc.) more easily.
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 constitutes by known aminoacid sequence of people, as long as have the COX-2 selective inhibitory activity, 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 sequence of having inserted 1 or a plurality of aminoacid and having obtained, perhaps by following polynucleotide encoded polypeptides, this polynucleotide is the polynucleotide of hybridizing under the condition of strictness 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 the condition of strictness on 1.
Said mutation body polypeptide can by those skilled in the art known technology manufacturing obtain.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 be from animal cell preparation cDNA library, use serves as the probe of basis preparation with the base sequence of natural type alpha-lactalbumin, the polynucleotide that selection and this probe are hybridized under the condition of strictness 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 the hybridization conditions of above-mentioned strictness, 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, be incubated an evening down at 42 ℃ and hybridize.Cleaning mixture in the washing afterwards and temperature conditions can be implemented according to the degree of " 1 * SSC, 0.1%SDS, 37 ℃ ", as harsh more high stringent condition is the degree of " 0.5 * SSC, 0.1%SDS, 42 ℃ ", 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 harsh more, 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 of decision hybridization or other key element (for example concentration and probe concentration, probe length, hybridization time etc.) are carried out appropriate combination.
The polynucleotide encoded polypeptides of utilizing such hybridization technique separation to obtain has high homology with above-mentioned natural type polypeptide usually on aminoacid sequence.So-called high homology is meant to have more than at least 40%, be 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 artificial mutation specifically or randomly, 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 and the inhibition activity of COX-1 and COX-2 be 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, though understand difference to some extent according to different dosage forms, symptom, body weight etc., but the alpha-lactalbumin class as its effective ingredient can give (absorption) about 0.002mg~about 1 in protein conversion every 1kg body weight on the 1st, 000mg preferably gives (absorption) about 0.01mg~about 200mg, most preferably gives (absorption) about 0.2mg~about 40mg.
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~about 50, and 000mg is preferably about 0.5mg~about 10,000mg, most preferably be about 10mg~about 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 patient's age, body weight and the administration purpose of administration 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 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 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 skin (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 for example 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 peptides such as phosphopeptide caseinate, arginine, lysine or aminoacid also can.As saccharic, can enumerate for example saccharide, producing starch (except that dextrin, can also be soluble starch, Britain's starch (Britishstarch), Oxytarch, starch ester, starch ether etc.), dietary fiber etc.As lipid, can enumerate for example 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 for example vitamin A, carotenoid, vitamin B group, vitamin C, D vitamin, vitamin E, K vitamin, Citrin, vitamin Q, nicotinic acid, nicotinic acid, pantothenic acid, biotin, inositol, choline, folic acid etc., as mineral, can enumerate for example calcium, potassium, magnesium, sodium, copper, ferrum, manganese, zinc, selenium etc.As organic acid, can enumerate for example 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 these compositions in a large number 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, administration in every way.As its mode, can enumerate for example oral administration of tablet, capsule, granule, powder, syrup etc., still, also can be processed into 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 used always by adopting usual way in the pharmaceutical preparations technology field in principal agent, be prepared into preparation thus.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 (teenager type, 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 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, teenager type 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 big 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.It is definitely agnogenio, but physical factors, immunology factor, these 3 of environmental factorss are considered to important reasons.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, combines with self joint tissue, causes arthritis.The initial stage symptom mostly is the WA ankylosis, and arthralgia and swelling take place.It is multiple that arthralgia is divided into spontaneous pain when static (), tenderness, kinesalgia etc.In addition, swollen joint, heat, symptom such as rubescent also appear.Arthritis is the synovitis that inflammation has appearred in synovial membrane, how to see brothers' joint symmetrically.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 the joint that is used 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 synthetic glucocorticoid of steroidal class etc.
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 the method that can improve arthritis pain for many years with tight security always.The topica of nearest NSAIDs (topic) is for specificity is suppressed at the inductive COX-2 of inflammation part, (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 big 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 is sprained like that) is meant that a part of health is injured or the caution signals of certain infringement occurs.Be by pain is reacted, to remove reason, to prevent the reaction that worsens.Corresponding, chronic pain is meant that caution signals is inoperative, no matter carries out what kind of treatment, 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 these 3 kinds of COX-1, COX-2, COX-3 now at least as COX.COX-1 is that physiological exists, and it takes on the physiological action of stomach, kidney etc.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, suppress at selectivity under the situation of 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 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, 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 big 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, also can be suitable for above-mentioned disease with the disease of inflammation.
In addition, whole technical literature formerly 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)
Present embodiment has verified that whey protein sepd (WPI) reaches the bullate inhibition effect of various bringing out property of albumen on Carrageenan foots contained in WPI.In addition, WPI is meant with microporous filter (MF), ultrafiltration (UF), cross-flow microfiltration (CFM), ion exchange etc. milk surum is handled Separation of Proteins, improved proteinic concentration 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. corporate system) 1g/kg[WPI group], alpha-lactalbumin (α-LA, derive from cattle, Davisco FoodsInternational, Inc. corporate system) 250mg/kg (25w/w% of WPI) [aLA group], beta lactoglobulin (β-LG, derive from cattle, Davisco Foods International, Inc. corporate system) 500mg/kg (50w/w% of WPI) [bLG group], lactoferrin (LF, derive from cattle, Murray GoulburnIngredients corporate 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 LF use with here shown in the identical material of material.
(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, have only alpha-lactalbumin to show the bullate inhibition effect of on Carrageenan foot.From then on result, the bullate inhibition effect 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 rat assist agent arthritis model in the present embodiment, to verify that various albumen contained among the WPI are to arthritic therapeutic effect as the animal model of rheumatic arthritis.
(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, filtering out the arthritic rat of abundant trouble, is benchmark divide into groups (1 group 6,5 groups) with the sufficient volume of the left limb foot sole of the foot.
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 corporate 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 oral administration continuously 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, be benchmark divide into groups (1 group 6,5 groups) with the sufficient volume of the left limb foot sole of the foot.
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 pain thus and suppresses effect.
(result)
Give alpha-lactalbumin and single gives in the lactoferrin at single, all have inhibition effect (Fig. 3) arthritis ache.
[embodiment 4] (the acetic acid twisting test is to effect of pain)
Various bringing out property of albumen Dichlorodiphenyl Acetate treatment of pain effects contained among the WPI have been verified 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, and estimate pain thus and suppress effect.
(result)
The result as shown in Figure 4.The visible significant analgesic effect of alpha-lactalbumin and lactoferrin.
[embodiment 5] (enzyme suppresses experiment)
Verified the inhibition activity of substances (alpha-lactalbumin, WPI) in the present embodiment to the various enzymes 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 ℃ down pre-cultivate 15 minutes after, be that the mode of 100 μ M adds arachidonic acid with ultimate density, 37 ℃ of reactions 15 minutes down.After the reaction, utilize EIA (enzyme immuno assay, EIA enzyme immunoassay) to measure the concentration of PGE2.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 being made as enzyme inhibition rate 100%, calculating the enzyme inhibition rate of alpha-lactalbumin and WPI, is IC to suppress 50% concentration
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 reorganization with each concentration; The Sf21 insect cell) enzyme standard substance descend pre-cultivation after 15 minutes at 37 ℃ in the Tris-HCl buffer (pH7.7) of the 100mM that contains 1mM glutathion, 1 μ M hematin, 500 μ M phenol, with ultimate density is that the mode of 0.3 μ M adds arachidonic acid, 37 ℃ of reactions 5 minutes down.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 being made as enzyme inhibition rate 100%, calculating the enzyme inhibition rate of alpha-lactalbumin and WPI, is IC to suppress 50% concentration
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, descend pre-cultivation after 15 minutes at 37 ℃ in the Tris-HCl buffer (pH7.9) of the 100mM of the DTT of 6mM (dithiothreitol, DTT (Dithiothreitol)), with ultimate density is that the mode of 100 μ M adds arginine, 37 ℃ of reactions 2 hours down.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.Meansigma methods with the quantitative values of matched group is an enzyme inhibition rate 100%, calculates the enzyme inhibition rate of alpha-lactalbumin and WPI, is IC to suppress 50% concentration
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 glycine-NaOH buffer (pH 9.0) of the 0.1M of the EDTA that contains 20 μ M 37 ℃ pre-down cultivate 15 minutes after, be that the mode of 0.03 μ Ci adds 1-palmityl-2-[1-with ultimate density
14C] oleoyl-L-3-phosphatidylcholine, reacted 5 minutes down at 37 ℃.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 being made as enzyme inhibition rate 100%, calculating the enzyme inhibition rate of alpha-lactalbumin and WPI, is IC to suppress 50% concentration
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% |
??- |
(enzyme of table 1 alpha-lactalbumin suppresses effect)
[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% |
??- |
(enzyme of table 2WPI suppresses effect)
[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.
(being tried thing)
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.
(being tried the absorption method of thing)
In putting down into 3 menstrual cycle of 19 years 9~Decembers, implemented test.To finish to be set at the 1st time being tried the thing absorption cycle from first menstruation to 1 menstrual cycle that next menstruation finishes.The 1st time the thing that tried is taken in ensuing 1 menstrual cycle of cycle (menstruation finishes to finish to next menstruation) for not taking in the lay-off period that is tried thing.Then, ensuing 1 menstrual cycle of lay-off period (menstruation finishes to finish to next menstruation) is the 2nd time being tried the thing 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 the different order of absorption tablet A, tablet B respectively.
Tried the thing absorption phase and be 1 thing (tablet A or tablet B) that tried 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 in being tried thing absorption phase and lay-off period, the experimenter is required to fill in self-recording formula questionnaire continuously 1 time on the 1st.The content of this self-recording formula questionnaire is relevant with dysmenorrhea and QOL.Intermenstrual period record from fill out the degree of the dysmenorrhea in the formula questionnaire, to the influence 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 the 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.(rank test of band Wilcoxson symbol)
(result)
Compare with the non-absorption phase, measure the oral account of the pain degree of dysmenorrhea and describe score value (p<0.01) reduction (Fig. 5) significantly 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 obtains relaxing 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 |
Do not have |
Do not have |
??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 |
Do not have |
Do not have |
??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 by cheap raw material mass production such as milk.In addition, few side effects, safe, can take in for a long time, therefore,, can obtain the anti-inflammatory analgesic effect by sneaking in the pharmaceuticals or taking in as food.And, by daily administration or absorption, can carry out sufficient nutrition management, therefore, can also be used for the purposes of manufacturing needles to the patient's of malnutrition functional food etc.