CN101528240A - Activity enhancer for detoxifying enzyme - Google Patents
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- CN101528240A CN101528240A CNA2007800386306A CN200780038630A CN101528240A CN 101528240 A CN101528240 A CN 101528240A CN A2007800386306 A CNA2007800386306 A CN A2007800386306A CN 200780038630 A CN200780038630 A CN 200780038630A CN 101528240 A CN101528240 A CN 101528240A
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Abstract
It is intended to provide a drug, a food or a feed which has an effect of enhancing the activity of a second-phase detoxifying enzyme and an effect of increasing intracellular glutathione content.
Description
Technical field
The present invention relates to a kind of medicine, food or feedstuff with the effect that strengthens the second phase detoxification enzyme activity and glutathion inside cell content.
Background technology
The food that absorbed our every day, water, air and chemicals comprise the disadvantageous composition of organism.In organism, these compositions are identified as the external source object, mainly carry out metabolism then in liver.In the liver xenobiotic metabolism comprise first mutually with second mutually.First mutually in, the xenobiotic material is separated experience oxidation, reduction or hydrolysis under the effect of toxenzyme (for example Cytochrome P450 or monooxygenase) mutually so-called first.Second mutually in, separate mutually under the effect of toxenzyme (for example glutathione S-transferase (being called as " GST " hereinafter sometimes), quinone reductase (being called as " QR " hereinafter sometimes), UDP-glucuronyl transferase (being called as " UGT " hereinafter sometimes), glutathion peroxidase or aryl sulfonic acid transferring enzyme) so-called second, first is combined with other xenobiotic material by metabolic xenobiotic material in mutually or is reduced, and then, promoted the xenobiotic metabolism.
GST is a kind of enzyme that mainly is present in the liver, and GST combines with various electrophilic compounds and reduced glutathion.GST catalysis is from first combination with toxic metabolite of detoxifying mutually in the enzymes metabolism, the perhaps combination of other xenobiotic material (toxicant) of catalysis, and then, promoted detoxification processes.In other words, by strengthening the activity of GST in the organism, can reduce risk by the caused various diseases of toxicant.In the last few years, the active material of GST that can strengthen from natural product was studied.For example, contained germacranolide (germacranolide), one or more plants that are selected from labiate and Myrtaceae Eucalyptus plant or their extract, limonin glycocide (for example patent documentation 1 to 4) such as (limonoidglucoside) in known soybean processed foods or the bay tree.
Glutathion is a kind of tripeptides that is made of cysteine, glutamic acid and glycine, is distributed widely in the organism.As mentioned above, glutathion is the substrate of GST or glutathion peroxidase, and therefore, glutathion is the important component of the shown Detoxication of these enzymes.In addition, glutathion also combines with various harmful substances by the non-enzymatic catalysis mode and shows Detoxication.
QR is that NADH or NADPH a kind of and as coenzyme combine and the enzyme of catalytic reduction quinone or electron acceptor compound, and QR can reduce and detoxify from the oxide in the phasel enzyme metabolism or by active oxygen or oxide that lipid peroxide produced.In the last few years, the active material of QR that can strengthen from natural product was studied.For example, contained indirubin has the active effect of QR (for example patent documentation 5, non-patent literature 1) of enhancing in known sulfur-containing compound (for example ingredient isothiocyanate in Cruciferae (Brassicaceae) plant) and the indigo-blue plant.
UGT is a kind of enzyme that can catalysis forms the reaction (glucuronic acid) of xenobiotic material and glucuronic acid complex (glucuronide), this reaction transfers to glucuronic acid from the metabolite in the phasel enzyme metabolism, perhaps transfers to and uses in UDP-glucuronic acid other xenobiotic material as saccharide donor.Therefore, known UGT can promote substrate molecule is transported in bile or the blood by the water solublity that strengthens substrate molecule, and this causes detoxifcation.In the last few years, the active material of UGT that can strengthen from natural product was studied.For example, contained indigo is known in the indigo-blue plant has an active effect of UGT (for example patent documentation 6) of enhancing.
Agar is a kind of polysaccharide that is made of agarose and agaropectin, and is widely used as the food material use.The low molecular compound agar oligosaccharide (agaro-oligosaccharide) of agarose contains 3 in reduction end, the anhydrous galactopyranose of 6-.Expectation exploitation agar oligosaccharide is as the material of health food.It is reported that the agar oligosaccharide has physiological role, for example anti rheumatism action and anti-inflammatory effect (for example patent documentation 7 to 9).
In addition, L-glycerol-1,5-epoxy-1 α β, 6-dihydroxy-cis-oneself-3-alkene-2-ketone (L-glycero-1,5-epoxy-1 α β, 6-dihydroxy-cis-hexa-3-en-2-one) (DGE) be a kind of by above-mentioned agar oligosaccharide is placed neutral to the alkali condition resulting chemical compound.It is reported that DGE has physiological role, for example anti rheumatism action and anti-inflammatory effect (for example patent documentation 10).
Patent documentation 1:JP-A 10-234326
Patent documentation 2:JP-A 9-234020
Patent documentation 3:JP-A 2006-111585
Patent documentation 4:JP-A 2000-316527
Patent documentation 5:JP-A 2003-40774
Patent documentation 6:JP-A 2003-246734
Patent documentation 7:WO 00/43018
Patent documentation 8:WO 99/24447
Patent documentation 9:WO 2003/086422
Patent documentation 10:WO 99/64424
Non-patent literature 1:Y.Zhang waits the people, Proc Natl Acad Sci USA, 1992, Vol.89, p2399-2403
Summary of the invention
The problem that the present invention solves
Target of the present invention is the material that exploitation has Detoxication, and it can easily be absorbed by safety, and suitable to food material, drug material or feedstuff material, thereby medicine, food or the feedstuff that utilizes this material function is provided.
The method of dealing with problems
Simply describe as this paper, a first aspect of the present invention relates to the reinforcing agent of the second phase detoxification enzyme activity or glutathion inside cell content, comprise and be selected from agar, agarose, contain 3 in reduction end, the chemical compound of the low molecular compound of the agarose of the anhydrous galactopyranose of 6-, following molecular formula (chemical molecular formula 1) representative:
Wherein X and Y are H or CH
2OH is if X is CH
2OH then Y is H, X be H then Y be CH
2OH; And at least a chemical compound in derivant and its salt as active component.In a first aspect of the present invention, contain 3 in reduction end, the example of the low molecular compound of the agarose of the anhydrous galactopyranose of 6-comprises the agar oligosaccharide, particularly preferably is the agar oligosaccharide that comprises the mixture of being made up of Agarobiose., Agarotetraose, Agarohexaose and agarooctaose.In addition, in a first aspect of the present invention, second example of separating toxenzyme mutually comprises glutathione S-transferase, quinone reductase and UDP-glucuronyl transferase.
A second aspect of the present invention relates to the medicine of the reinforcing agent of the second phase detoxification enzyme activity that contains with good grounds first aspect present invention or glutathion inside cell content.
A third aspect of the present invention relates to the food or the feedstuff of the reinforcing agent of the second phase detoxification enzyme activity that contains with good grounds first aspect present invention or glutathion inside cell content.
The invention effect
The invention provides the reinforcing agent of the second phase detoxification enzyme activity or glutathion inside cell content, and the medicine, food or the feedstuff that contain this reinforcing agent, wherein said reinforcing agent comprises and is selected from agar, agarose, contains 3 in reduction end, at least a chemical compound as this compound activity composition in the chemical compound of the low molecular compound of the agarose of the anhydrous galactopyranose of 6-, above-mentioned molecular formula (chemical molecular formula 1) representative and derivant thereof and its salt.The medicine, food or the feedstuff that contain this reinforcing agent can be used for promoting detoxification processes by what strengthen the second phase detoxification enzyme activity or glutathion inside cell content, therefore, it is useful especially being used for the treatment of or treating various diseases as medicine, Foods or drinks, particularly be used to ward off disease as medicine or functional food, this has reduced the risk of various diseases that toxicant causes.
The specific embodiment
The example that can be used for agar of the present invention comprises from the Red seaweeds that belongs to Gelidiaceae (Gelidiaceae) (Amman department Eucheuma gelatinosum (Gelidium amansii) for example, Japan's Eucheuma gelatinosum (Gelidium japonicum), Pacific Ocean Eucheuma gelatinosum (Gelidium pacificum), bedding stone Herba Astragali Sinici (Gelidium subcostatum), chicken feather algae (Pterocladia tenuis) and Acanthopeltis japonica (Acanthopeltis japonica)), the Red seaweeds (for example true Gracilaria tenuistipitata (Gracilaria verrucosa) and thick Gracilaria tenuistipitata (Gracilaria gigas)) that belongs to Gracilaria tenuistipitata section (Gracilariaceae), belong to the Red seaweeds (for example Ceramium kondoi (Ceramiumkondoi) and Campylaephora hypnaeoides (Campylaephora hypnaeoides)) of rosetangle section (Ceramiaceae) and other Red seaweeds as raw material obtained product.Exsiccant Sargassum is often used as raw material under the sun.Fresh Sargassum and dry seaweed can be used among the present invention.Can also use the Sargassum of bleaching former Sargassum that is called as of in dry run, when water spray, being bleached.With hot water extraction raw material Sargassum, cool off so-called to obtain " Tokoroten (gelidium jelly) " then.By the moisture in lyophilization or the compression dehydration removal " Tokoroten ", drying obtains agar then.In the present invention, comprise that from the agar of the Sargassum of various kinds and various forms bar-shaped, banded, tabular, wire and pulverous agar can be used.In addition, the commercially available agar with various intensity can be used.
Agar comprises about 70% agarose and about 30% agaropectin usually.Can use by the known purification methods agarose that purification comes out from agar as agar of the present invention.Can use low-purity or highly purified purified agar sugar with various agarose content.In addition, can also use commercially available agarose.
In the present invention, agar and agarose are defined as having 10000 or more high-molecular weight chemical compound.As described below, molecular weight is lower than 10000 agar or agarose and is defined as its low molecular compound.That is to say, through degradation treatment (for example acidic treatment) still have 10000 or the agar or the agarose of higher molecular weight be agar as used herein or agarose.
In the present invention, can be by to above-mentioned agar, agarose or carry out part degraded preparation as agar or the raw-material ocean of agarose Sargassum and be created in reduction end and contain 3, the low molecular compound of the agarose of the anhydrous galactopyranose of 6-by chemistry, physics and/or enzyme method.The chemistry, physics and/or the enzyme method that are used for the part degraded there is no particular determination, as long as can obtain containing 3 in reduction end, the low molecular compound of the agarose of the anhydrous galactopyranose of 6-gets final product.The example of chemical degradation method is included in acid hydrolysis to neutrallty condition.The example of mechanical degradation method comprises and uses electromagnetic wave or hyperacoustic radiation to cut or degrade.The example of enzymatic degradation method comprises that use hydrolytic enzyme (for example agarase) is hydrolyzed.Contain 3 from effective preparation in reduction end, the viewpoint of the low molecular compound of the agarose of the anhydrous galactopyranose of 6-, particularly preferred example of biodegrading process comprises acidic hydrolysis and the enzyme hydrolysis of using α-agarase to carry out.
In the present invention, contain 3 in reduction end, the example of the low molecular compound of the agarose of the anhydrous galactopyranose of 6-comprises that molecular weight is lower than 10000 low molecular compound agarose, this agarose is preferably gone up by 2-50 sugar, is more preferably by 2-30 sugar and forms, β-D-galactose and 3 wherein, the anhydrous galactopyranose of 6-is alternately arranged.Its more preferred example comprises the agar oligosaccharide.Agar oligosaccharide used herein is meant Agarobiose., Agarotetraose, Agarohexaose, agarooctaose, and two or more mixture of forming that are selected from Agarobiose., Agarotetraose, Agarohexaose and agarooctaose, therefore, the agar oligosaccharide is different with the new agar oligosaccharide that has β-D-galactose in reduction end.
As agar oligosaccharide used in the present invention, Agarobiose., Agarotetraose, Agarohexaose or agarooctaose can use separately, preferably use their mixture.When the agar oligosaccharide that contains Agarobiose., Agarotetraose, Agarohexaose and agarooctaose is used in the present invention, can prepare the agar oligosaccharide according to known method, the preparation method of this method described in the patent WO 00/69285.That is, in the present invention, can use the acid degradation that passes through solid acid and the agar oligosaccharide that contains Agarobiose., Agarotetraose, Agarohexaose and agarooctaose that from raw material agar, obtains.Can also use the commercially available agar oligosaccharide that contains Agarobiose., Agarotetraose, Agarohexaose and agarooctaose (name of product: Agaoligo is made by Takara Bio company limited).
To contain 3 in reduction end, the chemical compound of the anhydrous galactopyranose of 6-etc. places neutral to alkali condition, can obtain the chemical compound of above-mentioned molecular formula of the present invention (chemical molecular formula 1) representative.To in its structure, contain 3 at pH value under less than 7 condition, the chemical compound of the anhydrous galactopyranose of 6-carries out acidic hydrolysis and/or enzymatic degradation, place neutrality to alkali condition acid degradation and/or the enzymatic degradation chemical compound that obtains then, can also obtain the chemical compound of above-mentioned molecular formula (chemical molecular formula 1) representative.Contain 3 in reduction end, the example of the chemical compound of the anhydrous galactopyranose of 6-comprises the agar oligosaccharide, for example Agarobiose., Agarotetraose, Agarohexaose and agarooctaose and κ-angle fork disaccharidase.Contain 3 in its structure, the example of the chemical compound of the anhydrous galactopyranose of 6-comprises agar, agarose, their catabolite, and above-mentionedly contains 3 in reduction end, the low molecular compound of the agarose of the anhydrous galactopyranose of 6-.
When containing 3 in reduction end, the chemical compound of the anhydrous galactopyranose of 6-(for example Agarobiose. or κ-angle fork disaccharidase) be placed in pH value be 7 or higher neutrality to alkali condition following time, be selected from and aforesaidly contain 3 in reduction end, the solution of at least a chemical compound in the chemical compound of the anhydrous galactopyranose of 6-or suspension are used in the reaction, and the composition that is used to carry out this reaction liquid of this reaction there is no particular determination.Preferably, can use alkaline reaction liquid, this alkaline reaction liquid is including, but not limited to inorganic base (for example sodium hydroxide, potassium hydroxide, calcium hydroxide or ammonia) and organic base (for example Tris, ethamine or triethylamine), and these alkali are dissolved in the water (for example distilled water, ion exchange water, tap water etc.) as solvent.The concentration of alkali there is no particular determination.Can use alkali concn to be preferably 0.0001 to 5N, be more preferably 0.001 to 1N reaction liquid.Reaction temperature does not have particular determination, preferably can be 0 to 200 ℃, is more preferably 20 to 130 ℃.Response time there is no particular determination, preferably can be several seconds to several days.Output according to the chemical compound of being expected of the kind of chemical compound and above-mentioned molecular formula (chemical molecular formula 1) representative, can suitably select kind and concentration, reaction temperature and the response time of alkali, and the amount that is dissolved in or is suspended in the raw-material chemical compound of conduct of reaction liquid.The pH value of reaction liquid is generally 7 or higher.But the generation of the chemical compound of above-mentioned molecular formula (chemical molecular formula 1) representative than carrying out soon in low alkali concn, is carried out soon at high temperature than low temperature in the reaction liquid of high alkali concn.For example, by the preparation pH value be 11.5 Agarobiose. or κ-angle fork disaccharidase solution and with this solution remain on 37 ℃ following five minutes, can prepare the chemical compound of above-mentioned molecular formula (chemical molecular formula 1) representative.
According to purpose, the alkaline solution that contains above-mentioned molecular formula (chemical molecular formula 1) representative chemical compound of preparation can use after neutralization, perhaps uses to being used as acid solution less than 7 by regulating pH value.The same with the mode of the chemical compound that obtains above-mentioned molecular formula (chemical molecular formula 1) representative, when pH value less than 7 condition under in structure, containing 3, the chemical compound of the anhydrous galactopyranose of 6-carries out acidic hydrolysis and/or enzymatic degradation, and then remain on neutral during to alkali condition, acidic hydrolysis can be realized by for example following process: use aqueous solution to be used as reaction liquid, preferred use 0.001 to 5N acid to comprise mineral acid (hydrochloric acid for example, sulphuric acid or nitric acid) and organic acid (citric acid for example, formic acid, acetic acid, lactic acid or ascorbic acid usp/bp), an amount of material compound is dissolved in or is suspended in the reaction liquid, and then keeping reaction liquid being preferably under 0-200 ℃ the reaction temperature, the preferred reaction time is several seconds to several days.Solid acid also can be used as acid.Under the condition of enzymatic degradation, this reaction can be used under reaction liquid identical with acidic hydrolysis and the identical reaction condition and carry out, for example use an amount of α-agarase, for example use the α-agarase under the condition that is showing enzymatic activity described in the patent WO 00/50578.
Can come the chemical compound of above-mentioned molecular formula contained in the purification reaction solution (chemical molecular formula 1) representative by the known means of purification that comprises chemical method and physical method.Can come this chemical compound of purification by the combination of purification process, purification process comprises the gel filtration method, utilize molecular weight fraction film level separating method, the solution extracting method of (molecular weight-fractioning membrane), and the various chromatographic processes that make spent ion exchange resin etc.For example, X is CH in the chemical compound of above-mentioned molecular formula (chemical molecular formula 1) representative
2When OH, Y are H, L-glycerol-1,5-epoxy-1 α β, 6-dihydroxy-cis-oneself-3-alkene-2-ketone (being known as DGE after this sometimes) is that purification comes the resulting product of Agarobiose. by handling under from neutrality to alkali condition.X is H in the chemical compound of above-mentioned molecular formula (chemical molecular formula 1) representative, and Y is CH
2During OH, D-glycerol-1,5-epoxy-1 α β, 6-dihydroxy-cis-oneself-(use κ-DGE) after this sometimes is that purification comes κ-resulting product of angle fork disaccharidase by handling under from neutrality to alkali condition to 3-alkene-2-ketone.Herein, DGE is considered to a kind of chemical compound that is produced (Jpn.J.Phycol. (Sorui) 48:13-19, March 10,2000) when above-mentioned agar oligosaccharide is brought in the organism.The structure of DGE is shown in following molecular formula (chemical molecular formula 2).
In addition, the derivant of above-claimed cpd also can be used as active component and is used for the present invention.The example of derivant comprises the chemical compound that connects various substituted radicals.But derivant there is no particular determination, as long as derivant can reach the effect of expectation.The example of substituted radical comprises aliphatic group (straight chain aliphatic group (methyl for example; ethyl and n-pro-pyl) and side chain aliphatic group (isopropyl for example; isobutyl group; prenyl and geranyl)); aromatic group (phenyl for example; naphthyl; xenyl; pyrrole radicals and indyl); aromatic-aliphatic group (for example benzyl and phenethyl); hydroxyl; carboxyl; sulfate; phosphate; mercapto; amino; nitro; alkoxyl (for example methoxyl group); acyloxy (for example acetyl group); halogen (chlorine for example; bromine and fluorine); aminoacid and peptide.In addition, as described below, this derivant can be the derivant as the chemical compound of prodrug.
Derivant as active component of the present invention, agar, agarose or contain 3 in reduction end, the derivant of the low molecular compound of the agarose of the anhydrous galactopyranose of 6-(for example agar oligosaccharide) comprises but is not confined to, is preferably sulfating product and methylate especially.The preferred example of the derivant of the chemical compound of above-mentioned molecular formula (chemical molecular formula 1) representative is by this chemical compound and the derivant that reaction produced that contains the SH group compound.The structure of such derivant is shown in following molecular formula (chemical molecular formula 3).
Wherein R is that the chemical compound that contains the SH group removes the residue that the SH group is obtained.
The chemical compound of the employed SH of containing group there is no particular determination, as long as this chemical compound contains at least one SH group.In above-mentioned molecular formula (chemical molecular formula 3), R is in the reaction of the chemical compound of chemical compound that contains the SH group and above-mentioned molecular formula (chemical molecular formula 1) representative, when the chemical compound of above-mentioned molecular formula (chemical molecular formula 1) representative combine with the chemical compound that contains the SH group post consumption fall a SH group after remaining residue.Therefore, when the chemical compound that contains the SH group contained two or more SH group, one or more SH groups appeared in the residue by the R representative.The example that contains the chemical compound of SH group comprises methanthiol, butyl mercaptan, mercaptoethanol, contains the aminoacid of SH group, and the amino acid derivativges that contains the SH group.
The amino acid whose example that contains the SH group comprises cysteine and homocysteine.The example that contains the amino acid derivativges of SH group comprises above-mentioned amino acid whose derivant, for example cysteine derivative, contain the peptide of cysteine and contain the peptide of cysteine derivative.The example of cysteine derivative comprises amino-compound, acetyl compounds and the ester compounds of cysteine ester.The peptide that contains cysteine there is no particular determination, as long as this peptide contains cysteine as its constituent.The peptide that contains cysteine comprises oligopeptide, low molecular peptide (for example glutathion) and the macromolecule peptide of being made up of polypeptide (for example albumen).In addition, in the present invention, by merging the condition (for example reduction is handled) that above-mentioned reaction and the peptide that will contain cysteine or homocystine are converted to the peptide that contains cysteine or homocysteine, the peptide that contains cystine or homocystine can also be used as the peptide that contains cysteine or homocysteine.It is identical substantially with the above-mentioned peptide that contains cysteine to contain the included material of the example of peptide of cysteine derivative, and difference only is to replace cysteine with cysteine derivative.The example that contains the peptide of cysteine also comprises the peptide that contains cysteine that contains carbohydrate, lipid etc.In addition, can use the salt, anhydride, ester etc. of above-mentioned various materials.
The preparation of the chemical compound of above-mentioned molecular formula (chemical molecular formula 3) representative there is no particular determination, for example can carry out according to the method described in the patent WO 99/64424.
The salt of employed above-claimed cpd pharmaceutically acceptable salt preferably can obtain by known method for transformation in the present invention.The salt that the example of salt comprises the salt that forms with mineral acid (for example hydrochloric acid, hydrobromic acid, hydroiodic acid and sulphuric acid), forms with organic acid (for example formic acid, acetic acid, oxalic acid, malonic acid and succinic acid), and ammonium salt by obtaining with halogenated hydrocarbons (for example methyl iodide, benzyl halide etc.) reaction.
In addition, employed chemical compound can form derivant (prodrug) among the present invention, and this derivant can be hydrolyzed at an easy rate in vivo to reach the effect of expectation, and for example chemical compound can be esterified.This prodrug can prepare in accordance with known methods.
In addition, various isomers (for example optical isomer of above-claimed cpd, keto-enol tautomerization body and geometric isomer) also can be used as active component of the present invention.In addition, active component can also be isolating isomer or mixture of isomers.
The invention provides the reinforcing agent (reinforcing agent that is called as second phase detoxification enzyme activity of the present invention or glutathion inside cell content hereinafter sometimes) of the second phase detoxification enzyme activity or glutathion inside cell content, this reinforcing agent comprises and is selected from agar, agarose, contains 3 in reduction end, at least a chemical compound as active component (being called as active component of the present invention hereinafter sometimes) in the chemical compound of the agarose low molecular compound of the anhydrous galactopyranose of 6-, above-mentioned molecular formula (chemical molecular formula 1) representative and derivant thereof and its salt.
As used herein second example of separating toxenzyme mutually comprises glutathione S-transferase (GST), quinone oxidase (QR), UDP-glucuronyl transferase (UGT), glutathion peroxidase or aryl sulfonic acid transferring enzyme.More preferred example is GST, QR and UGT.Shown in following embodiment 1 to 7, can be by for example but be not confined to measure the enzymatic activity of GST, QR or UGT especially by the active potentiation of GST, QR that active component of the present invention produced or UGT, the gene expression amount of perhaps measuring GST, QR or UGT improves.
Shown in following embodiment 8, active component of the present invention can be by for example but be not that the content that is confined to measure glutathion inside cell especially improves to the potentiation of the glutathion inside cell content that produced.
The reinforcing agent of second phase detoxification enzyme activity of the present invention or glutathion inside cell content strengthens second activity of separating toxenzyme (for example above-mentioned GST, QR and UGT) mutually, also further increase the amount of glutathion, glutathion can be used as some second substrates of separating toxenzyme mutually, be present in the cell, therefore can promote in the organism especially metabolism at liver toxic material, and the function of liver.Therefore, the reinforcing agent of second phase detoxification enzyme activity of the present invention or glutathion inside cell content can reduce the risk of the various diseases that is caused by various toxic substances, therefore is particularly suited for using as following medicine and functional food.The toxicant that its metabolism can be promoted by GST, QR, UGT and glutathion is not to be confined to specific chemical compound especially, also comprises multiple different allogenic material, for example carcinogen, agricultural chemicals, environmental contaminants and have the medicine of side effect.That is to say that the disease of the reinforcing agent role of second phase detoxification enzyme activity of the present invention or glutathion inside cell content is not limited to specific disease.In addition, shown in the comparing embodiment described as follows, in its reduction end contains the new agar oligosaccharide of β-D-galactose, finding by this potentiation of the second phase detoxification enzyme activity that active component of the present invention produced.
According to the present invention, provide the medicine (being called as medicine of the present invention hereinafter sometimes) of the reinforcing agent that contains second phase detoxification enzyme activity of the present invention or glutathion inside cell content.Medicine of the present invention can promote the metabolic liver function of toxicant by strengthening the second phase detoxification enzyme activity or glutathion inside cell content.Medicine of the present invention is useful for treatment or prevention various diseases, and this disease is accompanied by the degeneration of liver function, for example hepatitis, liver cirrhosis, hepatocarcinoma, fatty liver and ethanol hepatopathy disease.Medicine of the present invention is suitable as a kind of preventive medicine that these diseases take place that is used to reduce very much, and is main because the enhancing second phase detoxification enzyme activity that active component of the present invention produced or the effect of glutathion inside cell content.In addition, when medicine of the present invention when having the medication combined use of infringement liver side effect because the metabolic facilitation of the toxicant of medicine of the present invention, medicine of the present invention can reduce the hepar damnification after the medication.In the case, active component of the present invention and other medicine can mix preparation becomes a kind of dosage form, and perhaps can prepare separately becomes independent dosage form, uses simultaneously then.
In addition, except above-mentioned various diseases, medicine of the present invention can be used to prevent and treat the disease that is caused by various toxicants.The example of these diseases comprises but is not confined to cancer, arteriosclerosis, Alzheimer (Alzheimer) disease, obesity (metabolic syndrome) and dermatosis especially.
The example of medicine of the present invention comprises by mixing above-mentioned active component and mixes the preparation that is obtained with the known drug carrier that wherein said active component is used to the reinforcing agent of second phase detoxification enzyme activity of the present invention or glutathion inside cell content.The employed medicine of this paper comprises accurate medicine (quasi drugs).In addition, medicine of the present invention can also be united use with other composition that has same use with active component of the present invention (promptly second separating toxenzyme mutually), perhaps with known have strengthen second mutually other composition of detoxification enzyme activity unite use.Known example with other composition that strengthens the second phase detoxification enzyme activity comprises but is not confined to isothiocyanate and curcumin especially.
Medicine of the present invention can also be united use with glutathion or the composition that contains a large amount of glutathion.The Detoxication of medicine of the present invention can also further can be used as second glutathion that detoxifies zymolyte mutually by adding and strengthen, and second activity of separating toxenzyme mutually can assign to strengthen by active component of the present invention or the one-tenth that can strengthen the amount of glutathion inside cell.The example that can increase the composition of glutathion inside cell comprises but is not confined to isothiocyanate especially.
Medicine of the present invention can be made by following step usually: above-mentioned active component is mixed with pharmaceutically acceptable liquid or solid carrier; Randomly adding solvent, dispersant, emulsifying agent, buffer, stabilizing agent, excipient, binding agent, distintegrant, lubricant waits mixture preparation becoming solid dosage forms for example tablet, granule, powder, powder agent or capsule, perhaps liquid dosage form (for example traditional liquid agent, suspension or emulsion).In addition, medicine of the present invention can be formulated into and be dryed product, and being used for became liquid form again with suitable carrier or with external goods (external preparation) before using.
Can select pharmaceutical carrier according to the mode of administration and the dosage form of medicine.When medicine of the present invention is the oral drugs of solid composite, the example of the oral drugs of solid composite comprises tablet, pill, capsule, powder, granula subtilis and granule, and the example of employed pharmaceutical carrier comprises starch, lactose, mannitol (mannite), carboxymethyl cellulose, corn starch and inorganic salt.In preparation oral drugs process, can add binding agent, distintegrant, surfactant, lubricant, flow promoter, correctives, coloring agent, spice etc.For example, when the oral medicine thing was tablet or pill, if expectation, oral drugs can wrap by sugar-coat or the intestinal or the stomach soluble film of sucrose, gel, hydroxypropyl cellulose etc.When medicine of the present invention was the oral drugs of liquid component, the example of the oral drugs of liquid component comprised pharmaceutically acceptable emulsion, solution, suspension and syrup, and the example of the pharmaceutical carrier that can use comprises purified water and ethanol.In the process of preparation liquid component oral drugs, if auxiliary reagent (for example wetting agent or suspending agent), sweeting agent, flavoring agent, antiseptic etc. can be further added in expectation.
When medicine of the present invention is parenteral drug (parenteral drug), can be by above-mentioned active component of the present invention being dissolved or suspended in the diluent (for example distilled water for injection, normal saline solution, liquid glucose solution, injection vegetable oil, Oleum sesami, Oleum Arachidis hypogaeae semen, soybean oil, Semen Maydis oil, propylene glycol or Polyethylene Glycol), optionally wait and make this medicine to wherein adding antibacterial, stabilizing agent, degree of rising agent (tonicity), the agent of releiving.The intestines and stomach external used medicine can also be made into solid composite, can be dissolved in sterile water for injection or sterilization solvent before use.
The example of external goods comprises solid, semisolid or the liquid formulations that is used for transdermal administration or saturating mucosa (oral cavity or intranasal) administration.In addition, also comprise suppository etc.The object lesson of external goods comprises emulsion (for example emulsifying agent or washing liquid), flowing product (external-use tincture or the liquid reagent that for example are used for transmucosal administration), ointment (for example oily ointment or hydrophilic ointment) and is used for the paster (for example thin film, adhesive tape or compress) of transdermal administration or transmucosal administration.
The above-mentioned various dosage forms of medicine can use known drug carrier etc. suitably to make according to traditional method.The content of active component changes according to dosage form, medication etc. in the medicine, but there is no particular determination.Preferably, the content of active constituents of medicine is a kind of like this amount, so that can be in the amount of the administration of the active component in the following dosage scope.The content of active constituents of medicine of the present invention is typically about 1 weight % to 100 weight %.
Medicine of the present invention is by the medication administration of suitable its pharmaceutical dosage form.Medication there is no particular determination.For example, medicine of the present invention can be by inside, outside or drug administration by injection.When medicine of the present invention or prophylactic agent during, can pass through vein, muscle, subcutaneous, Intradermal or similar approach administration by drug administration by injection.When medicine of the present invention during, can pass through for example suitable medication administration of the external goods of suppository by outside administration.
According to dosage form, medication and intended use thereof, and age, body weight and symptom that administration is tried patient suitably select drug dose of the present invention, so drug dose is not fixed.The contained active component dosage of medicine is preferably body weight every day 0.005 to 5000mg/kg usually for the adult, is more preferably 0.05 to 500mg/kg body weight, is more preferably 0.5 to 50mg/kg body weight again.Certainly, therefore dosage can be lower than above-mentioned dosage range or can surpass above-mentioned dosage range according to various conditions and difference.Can be in ideal dosage range once a day or carry out administration every day several times.The term administration is arbitrarily.Medicine of the present invention can by oral administration himself, perhaps can also add in any food that absorbs every day.
According to the present invention, provide the food or the feedstuff (hereinafter being called as food of the present invention or feedstuff sometimes) of the reinforcing agent that contains second phase detoxification enzyme activity of the present invention or glutathion inside cell content.Identical with the model of action of medicine of the present invention, food of the present invention or feedstuff can strengthen the second phase detoxification enzyme activity or glutathion inside cell content, therefore can be used as food or the feedstuff that strengthens the second phase detoxification enzyme activity or glutathion inside cell content.Food of the present invention and feedstuff can be used for treatment or prevention various diseases, and this disease is accompanied by the degeneration of liver function, for example hepatitis, liver cirrhosis, hepatocarcinoma, fatty liver and ethanol hepatopathy disease.Food of the present invention or feedstuff are very suitable for as reducing the functional food that these diseases take place, and this is mainly by the second phase detoxification enzyme activity that active component of the present invention produced or the potentiation of glutathion inside cell content.
In addition, except above-mentioned various diseases, food of the present invention or feedstuff also can be used for preventing or treat the disease that the toxicant by bioaccumulation causes.The example of such disease comprises but is not confined to cancer, arteriosclerosis, Alzheimer's disease, obesity (metabolic syndrome) and dermatosis especially.In addition, the absorption of food of the present invention or feedstuff has caused the health that caused by Detoxication to worsen the improvement of (for example pachylosis and tired).
An aspect of food of the present invention is for having Detoxication, i.e. the food of detoxification.The example of this food comprises and is used to reduce functional food (food that is used for health maintenance) that above-mentioned disease risks takes place, is used for promoting in the organism that detoxifcation prevents the antidotal functional food of aging phenomenon, and the functional food that is used for the prevention of being still drank after a night (hangover prevention) before or after the ethanol picked-up.This functional food comprises specific health food, label shows that active component of the present invention participates in the function of food on it, and this food can reduce the occurrence risk of above-mentioned disease, has Detoxication, has anti-aging effects or has the preventive effect of being still drank after a night.
Identical with the model of action of medicine of the present invention, food of the present invention or feedstuff can also use with mixing of other composition, this composition is applicable to the purpose identical with active component of the present invention, and promptly known second of the second phase detoxification enzyme activity that can strengthen is separated toxenzyme or other composition mutually.In addition, identical with the model of action of medicine of the present invention, food of the present invention or feedstuff can also with known have strengthen first of its active function and separate the mixing of toxenzyme or composition mutually and use.In addition, food of the present invention or feedstuff can also with glutathion or contain a large amount of glutathion composition mix two uses.Preferably, the phase-splitting of preferably using as food material in food of the present invention and the above-mentioned blendable composition that becomes mixes, for example Broccoli, Rhizoma Curcumae Longae, Rhizoma Wenyujin Concisum, yeast, fresh water clam extract, Concha Ostreae extract, milk Ji extract, fucoidan, angelica keiskei koidzumi and converted products thereof.
Term " comprises " that the meaning when being used for food of the present invention or feedstuff is to comprise, add and/or dilute.In this article, term " comprises " and is meant that active component used among the present invention is included in food or the feedstuff.Term " interpolation " is meant that active component used among the present invention is added in food or the feedstuff raw material.Term " dilution " is meant that the raw material of food or feedstuff is added in the active component used among the present invention.Food of the present invention also comprises the food product that active component is added as food additive.
The method that is used to make food of the present invention or feedstuff there is no particular determination, as long as food or feedstuff comprise active component of the present invention.Processes such as for example, mixing, the cooking, processing can be carried out according to those processes that is used for bread and cheese or feedstuff.Food of the present invention or feedstuff can be made by the manufacture method of common food or feedstuff.
The example of food of the present invention comprises but is not confined to comprise the machined valley series products (milling product for example of active component of the present invention especially, starch product, the premixing product, noodles, macaroni, bread, bean sauce, noodle prepared from buckwheat, gluten bread, rice flour, the rice cake of gel noodles and packing), (for example plastics are fatty and oily for processing fats and oils product, it bran sieve oil, salad oil, mayonnaise and flavoring agent), soybean processing series products (bean curd for example, miso and fermented soybean), manufactured meat series products (Petaso for example, Baconic, compacting Petaso and sausage), process extra large series products (for example freezing demersal fish, Boiled fish beans, tubulose Boiled fish beans bar, ground fish cake, fried fish beans pie, the fish ball, tendon, fish ham or sausage, dried oceanic bonito, processing fish roe product, canned marine product and sweet soy sauce Boiled fish), milk series products (for example former milk, butter, yoghourt, butter, cheese, the concentrated milk, powdery milk and ice cream), vegetable for processing and fruit product (are for example stuck with paste, beans, Pickles, fruice, vegetable beverage and bland), confection class (chocolate for example, cookies, sweet circle, cake, sweet rice cake and sweet rice), alcoholic beverage (rice wine for example, China white wine, wine, Whiskey, liquor, Little water., brandy, gin, rum, medicated beer, soft alcoholic beverage, Eaux-De-Vie and liqueur), luxurious beverage (green tea for example, tea, oolong tea, coffee, soft drink and lactic acid beverage), seasoning class (soy sauce for example, beans, vinegar and sweet rice wine), canned, (for example various cooked foods (for example are stamped through the beef of the cooking and the rice of vegetable for bottled or packed food, meat and vegetable cooking rice in the canister, steam Semen Phaseoli rice and curry)), half-dried or reserve ration (liver pat for example, other tablespread (spread), noodle prepared from buckwheat or Wu Dongtang and condensed soup), dry food (instant noodle for example, the instant curry, instant coffee, powder fruit juice, powder soup, the instant miso shiru, food through the cooking, beverage and soup through the cooking) through cooking, frozen food (sukiyaki for example, teacup steams, Broiled River Eel, Hamburg steak, steamed dumpling with the dough gathered at the top, Carnis Sus domestica falls into dumpling, various bar and fruit cocktail), solid food, liquid food (for example soup), add industrial or agricultural or forestry food (for example spice), process tame livestock products and processing marine product.The employed food of this paper comprises beverage.For example, according to the present invention, this beverage can be by being dissolved in the water the agar oligosaccharide, and suitably add the existing employed composition of beverage therein and make.
Food of the present invention can be any form, but comprise oral absorpting form (for example powder type, sheet form, particle form and capsule form), if one or more active component are comprised, are added and/be diluted in the food of the present invention, and the content of this active component need reach the amount of the effect that is enough to demonstrate enhancing second phase detoxification enzyme activity or glutathion inside cell.Food of the present invention also comprises above-mentioned active component self of the present invention, and active component and suitable emulsifying agent, excipient etc. are by the mixture of proper proportion.These food can self and be eaten, perhaps also can be mixed with water, absorb as beverage then.
Active component content in the food of the present invention there is no particular determination, can suitably select according to the functional and activity that they show.For example, the content of the active component of food of the present invention is preferably 0.0001 to 100 weight %, is more preferably 0.001 to 60 weight %, is more preferably 0.01 to 30 weight % again.
Food of the present invention can be absorbed under such amount, for the adult, every day, active component dosage of the present invention can absorbed amount be preferably 0.005 to 5000mg/kg body weight usually, was more preferably 0.05 to 500mg/kg body weight, was more preferably 0.5 to 50mg/kg body weight again.
In addition, the invention provides and be used to have the organism feedstuff that strengthens the second phase detoxification enzyme activity or the active effect of glutathion inside cell, this feedstuff comprises, just comprises, adds or dilute above-mentioned active component.Another aspect of the present invention provides the method for cultivating organism, and this method comprises the above-mentioned active component of administration organism.The organism nutrient chemical that comprises above-mentioned active component that further provides on the other hand of the present invention.
The example of the employed organism of this paper comprises but is not to be confined to letting animals feed and pet animals especially.The example of letting animals feed comprises livestock (for example horse, cattle, pig, sheep, goat, camel and lamb), animal for research class (for example mice, rat, Cavia porcellus and rabbit), poultry (for example chicken, duck, turkey and Ostriches), Fish, shell-fish and shell.The example of pet animals comprises Canis familiaris L. and cat.As feedstuff, example is the feedstuff that is used to keep and/or improve health.As the organism nutrient chemical, example is wetting agent, feed additive and beverage additive.
According to these inventions, according to the enhancing second phase detoxification enzyme activity of active component of the present invention or the effect of glutathion inside cell, to invent the shown effect of employed above-mentioned organism identical with these in the effect that can estimate medicine of the present invention.That is, the caused various diseases of noxious substance in the organism can be treated or prevent to feedstuff of the present invention, and for example, this feedstuff can reduce the risk of the caused generation various diseases of toxicant.
For the biological subject body, employed above-mentioned active component according to being preferably 0.005 to 5000mg/kg body weight, is more preferably 0.05 to 500mg/kg body weight usually among the present invention's every day, is more preferably the amount administration of 0.5 to 50mg/kg body weight again.Administration can realize below for example: add and the mixed active composition in the raw material of the artificial mixed fodder for the treatment of administration biological subject body, perhaps by active component is mixed mutually with the powdery raw material of artificial mixed fodder, and then further add mixture in other raw material, and mixture has been mixed with other raw material.The content of active component there is no particular determination in the feedstuff, can suitably select according to the purpose of expectation.For example, the content of active component is preferably 0.0001 to 100 weight % in the feedstuff, is more preferably 0.001 to 60 weight %, is more preferably 0.01 to 30 weight % again.
The method that is used to make feedstuff of the present invention there is no particular determination, and the mixture of this feedstuff also can be made according to normal diet, as long as contain above-mentioned active component of the present invention in the feedstuff of manufacturing.Also can make the organism nutrient chemical according to same way as.
According to the present invention, livestock animals, animal for research, poultry, the health of animals such as pet animals can maintain under the good condition or be enhanced, this is by for example allowing the biological subject body to absorb to comprise the feedstuff of above-mentioned active component used in the present invention, above-mentioned active component has the potentiation of enhancing II detoxification enzyme activity used in the present invention or the effect of glutathion inside cell, or by the biological subject body is soaked in the liquid of employed above-mentioned active component (for example, by resulting liquid that wetting agent is dissolved in the water) in containing the present invention.These aspects are part aspects of organism side's culture method among the present invention.
When the above-mentioned active component of the present invention that is used for of effective dosage is brought into play its time spent of doing for organism, do not find that being used for above-mentioned active component of the present invention has toxicity.For example, under case of oral administration, when according to the single dose administration mice Agarobiose. of 2000mg/kg body weight, Agarotetraose, Agarohexaose, agarooctaose and composition thereof or DGE, find the case that causes death.In addition, when the single dose according to the 2000mg/kg body weight passes through the above-mentioned active component of oral administration administration to rat, find the case that causes death.
Embodiment
With reference to the following example the present invention is more specifically described, wherein the present invention is not limited to the following example.In an embodiment, remove specified otherwise, the meaning of " % " is " volume % ".
Preparation embodiment 1: the preparation of Agarobiose.
(AGAR NOBLE) is suspended among the HCl of 0.1N with agar, is made into 10% concentration, then 100 ℃ of heating 19 minutes.The above-mentioned sample of 10ml is installed on the ToyopearlHW40C that water balance crosses (being made by the TOSOH company) chromatographic column (volume is 4.4cm * 85cm).Make water utilize gel filtration chromatography to separate as mobile phase, flow velocity is per minute 1.4ml.The material of eluting is detected by the differential refractometer, collects 7 milliliters fraction at every turn.
The peak appears respectively when elution time is 406 minutes, 435 minutes, 471 minutes and 524 minutes.Get fraction, at silica gel 60 F corresponding to each peak
254Carry out a plate on the plate (being made by Merck (Merck) company), use the 1-butanols of 5: 5: 1 ratios: ethanol: water launches, and uses orcin-sulphuric acid method to analyze then.The result is an Agarobiose. for the peak that comes out at 524 minutes eluting.This fraction obtains the 140mg Agarobiose. after by lyophilizing.
Preparation embodiment 2:L-glycerol-1,5-epoxy-1 α β, the preparation of 6-dihydroxy-cis-own-3-alkene-2-ketone (DGE)
With the suspension of the commercially available agar of 2.5g (AGAR NOBLE) in 50ml 0.1N HCl, heating obtained solution in 13 minutes under 100 ℃ temperature.This solution is cooled to room temperature, uses NaOH to regulate pH value to 12, and then this solution that neutralizes.
Use following positive HPLC to carry out purification neutralized reaction product.Collect each peak, carry out drying under the decompression, and then be dissolved in the water.Suppress active by the growth of cancer cells that uses the HL-60 cell to measure each fraction.Find that retention time is that 4.05 and 4.16 fraction has growth of cancer cells and suppresses active.
Then, collect retention time in a large number and be 4.05 and 4.16 fraction, and it is carried out structural analysis.It is found that the result is a L-glycerol-1 for this fraction, 5-epoxy-1 α β, 6-dihydroxy-cis-oneself-3-alkene-2-ketone (DGE).The condition that is used for positive HPLC shows below.
Post: PALPAK S type (4.6mm * 250mm is made by TAKARA SHUZO company limited)
Mobile phase A: 90% acetonitrile solution
Mobile phase B: 50% acetonitrile solution
Flow velocity: 1ml/min
Eluting: mobile phase A (10 minutes) → mobile phase A is to linear concentration gradient (40 minutes) → Mobile phase B (10 minutes) of Mobile phase B
Detect: at the absorbance of 195nm
Column temperature: 40 ℃
Embodiment 1: strengthen the assessment (1) of the active effect of glutathione S-transferase (GST)
Hepa1c1c7 cell (ATCC CRL-2026) is suspended in the DMEM (Dulbecco modifiedEagle medium) (by the Sigma manufacturing) of the penicillin-streptomycin (being made by the NacalaiTesque company limited) that contains 10% hyclone (being made by MP Biomedicals company) and 1%, and cell concentration is 4 * 10
5Individual cell/ml.In each hole in 96 hole microwell plates (microtiter plate), add the cell suspending liquid of 0.2ml, when having 5% carbon dioxide, 37 ℃ of overnight incubation.Then, change culture medium in each hole with DMEM.In each hole, add the aqueous solution of 0.4 μ l test substances, cultivated then 24 hours.Preparation embodiment 1 resulting Agarobiose. and commercially available agar oligosaccharide (trade name: Agaoligo is made by Takara Bio company limited, all contains 20 to 25% Agarobiose., Agarotetraose, Agarohexaose and agarooctaose) are used as test substances.In negative control, add entry and replace test substances.After cultivation is finished, remove culture medium, use phosphate buffer to wash this cell.Then, add the cytolysis solution (10mM Tris-HCl (pH value is 7.4)) of 0.1ml, 38.5mM KCl and 1mM EDTA 1%NP-40), were hatched 10 minutes at 37 ℃, obtained enzymatic solution.In the enzymatic solution of 25 μ l, add the reaction solution (0.13M buffer solution of potassium phosphate (pH value is 6.5), 1.3mM glutathion) of 155 μ l.Before closing on measurement, add the CDNB (2,4-chloronitrobenzene by TOKYO CHEMICAL INDUSTRY company limited made) of 20 μ l as the 10mM of reaction substrate, and the variation of measuring absorbance at the 340nm place.Carry out three times activity analysiss.Use is measured protein content with enzymatic solution and MicroBCA analysis of protein test kit (being made by PIERCE company) that phosphate buffer carries out 50 times of dilutions.The amount that adds test substances need reach the shown concentration of following table.GST is active in the GST activity with respect to contrast, and calculates according to following formula.
GST activity=(protein content that adds the maximum rate coefficient/adding test substances part of test substances part)/(protein content that adds the maximum rate coefficient/adding water section of water section)
The result is as shown in table 1.Table 1 has shown GST activity in the cell that adds after the various test substances.Find that adding agar oligosaccharide or Agarobiose. have caused the active remarkable increase of GST.
Table 1
Embodiment 2: strengthen the assessment (2) of the active effect of glutathione S-transferase (GST)
According to embodiment 1, determine to use the active effect of the preparation embodiment caused GST of 2 resulting DGE.The each measurement repeated three times.GST is active in the GST activity with respect to contrast, and according to embodiment 1 in identical formula calculate.
The result is as shown in table 2.Table 2 has shown the activity of the interior GST of cell behind the adding DGE.Find, add DGE and caused the active remarkable increase of GST.
Table 2
Embodiment 3: the active measurement of quinone reductase (QR)
With reference to people such as Hans J.Prochaska, Analytical Biochemistry 169, the improved method of the part of method described in the 328-336 (1988) is measured the activity of QR.With the DMEM of Hepa1c1c7 cell suspension in the penicillin-streptomycin that contains 10% hyclone and 1%, cell concentration is 4 * 10
5Individual cell/ml.In each hole in 96 hole microwell plates, add the cell suspending liquid of 0.2ml, when having 5% carbon dioxide, 37 ℃ of overnight incubation.Then, change culture medium in each hole with DMEM.In each hole, add the aqueous solution of 0.4 μ l test substances, cultivated then 24 hours.Will be by (the trade name: of resulting Agarobiose. and commercially available agar oligosaccharide among the preparation embodiment 1 Agaoligo) as test substances.In negative control, add entry and replace test substances.After cultivation is finished, remove culture medium, use phosphate buffer to come washed cell.Then, the cytolysis solution of adding 0.1ml (1%NP-40), hatched 10 minutes at 37 ℃, obtains enzymatic solution by 2mM EDTA (pH value is 7.8).In the enzymatic solution of 25 μ l, add the reaction solution (25mM Tris-HCl (pH value is 7.4), 0.67%BSA, 0.01%Tween 20,5 μ MFAD, 1mM G6P, 30 μ M NADP, 0.3mg/ml MTT, 2U/ml G6PDH (making)) of 100 μ l by Sigma.At this moment, add or do not add substrate.When adding substrate, be that 50 μ M add menadione (being made by Sigma) in reaction solution further with ultimate density.After 30 minutes, the sodium carbonate that adds 75 μ l 2N comes cessation reaction in incubated at room, and measures its absorbance at the 590nm place.Carry out three times activity analysiss.Use is measured protein content with enzymatic solution and MicroBCA analysis of protein test kit that phosphate buffer carries out 50 times of dilutions.Add test substances amount to reach the shown concentration of following table.QR is active in the QR activity with respect to contrast, and calculates according to following formula.
QR activity={ [(absorbance that adds the test substances part when substrate is arranged)-(absorbance that adds the test substances part during no substrate)]/(protein content that adds the test substances part) }/[(absorbance that adds water section when substrate is arranged)-(absorbance of adding water section during no substrate)]/(protein content of adding water section)) }
The result is as shown in table 3.Table 3 has shown and has added intracellular QR activity after the various test substances.Find, add agar oligosaccharide or Agarobiose. and caused the active remarkable increase of QR.
Table 3
Embodiment 4: the assessment of inducing the effect of glutathione S-transferase (GST) mRNA expression
With the DMEM of Hepa1c1c7 cell suspension in the penicillin-streptomycin that contains 10% hyclone and 1%, cell concentration is 4 * 10
5Individual cell/ml.In each hole of 6 orifice plates, add the cell suspending liquid of 5ml, when having 5% carbon dioxide, 37 ℃ of overnight incubation.Then, change culture medium in each hole with DMEM.In each hole, (trade name: Agaoligo), ultimate density is 100 μ g/ml, cultivates then 16 hours to add the commercially available agar oligosaccharide that is used as test substances.In negative control, add entry and replace test substances.After cultivation is finished, remove culture medium, add the RNA iso (making) of 0.5ml by Takara Bio company limited.Cell is recovered in the microcentrifugal tube (Eppendorf micro-tube) of 1.5ml, and placed room temperature 5 minutes.Again to the chloroform that wherein adds 0.1ml.With the mixed liquor mixing until becoming milky.Mixed liquor was placed room temperature 5 minutes, under 4 ℃ centrifugal 15 minutes with 10000rpm.Shift supernatant to the another one microcentrifugal tube.Again to the isopropyl alcohol that wherein adds 0.25ml, mix homogeneously.Mixed liquor was placed in room temperature 10 minutes, obtained precipitate in centrifugal 10 minutes with 10000rpm under 4 ℃.Use 75% ethanol of 0.5ml to come washing precipitate, under 4 ℃ centrifugal 5 minutes, dry then with 10000rpm.Precipitate is dissolved in the 20 μ l waters for injection, obtains the solution of total RNA in water.Use EXScript RT-PCR test kit (making) to carry out reverse transcription reaction and PCR in real time by Takara Bio company limited.In PCR in real time, use the Auele Specific Primer of GST and the Auele Specific Primer of TfR in contrast (Tfrc).Use Smart Cycler II System (making) to measure by Cepheid company.Carry out twice activity analysis.The expression of GST mRNA is as the amount with respect to the GST mRNA of contrast, and calculates according to following formula.
The expression of GST mRNA=[(expression that adds GST mRNA in the test substances part)/(expression that adds Tfrc mRNA in the test substances part)]/[(expression that adds GSTmRNA in the water section)/(expression that adds Tfrc mRNA in the water section)]
The result is as shown in table 4.Table 4 has shown and has added behind the agar oligosaccharide expression of GST mRNA in the cell.Find that the agar oligosaccharide has the activity of inducing GST mRNA to express significantly.
Table 4
Embodiment 5: the assessment of inducing the effect of quinone reductase (QR) mRNA expression
Measure the agar oligosaccharide for the activity of inducing QR mRNA to express according to the method described in the embodiment 4.Commercially available agar oligosaccharide (trade name: Agaoligo) be used as test substances and use.Carry out twice activity analysis.The expression of QR mRNA is as the amount with respect to the QR mRNA of contrast, and calculates according to following formula.
The expression of QR mRNA=[(expression that adds QR mRNA in the test substances part)/(expression that adds Tfrc mRNA in the test substances part)]/[(expression that adds QRmRNA in the water section)/(expression that adds Tfrc mRNA in the water section)]
The result is as shown in table 5.Table 5 has shown and has added behind the agar oligosaccharide expression of QR mRNA in the cell.Find that the agar oligosaccharide has the activity of inducing QR mRNA to express significantly.
Table 5
Embodiment 6: strengthen the assessment of the active effect of UDP-glucuronyl transferase (UGT)
With reference to B.Burchell, people such as P.Weatherill, Methods in Enzymology 77, the improved method of the part of method described in the p169 (1981) is measured the UGT activity.With the DMEM of Hepa1c1c7 cell suspension in the penicillin-streptomycin that contains 10% hyclone and 1%, cell concentration is 4 * 10
5Individual cell/ml.In each hole of 12 orifice plates, add the cell suspending liquid of 2ml, when having 5% carbon dioxide, 37 ℃ of overnight incubation.Then, change culture medium in each hole with DMEM.In each hole, add 4 μ l be used as test substances commercially available agar oligosaccharide (trade name: aqueous solution Agaoligo), cultivated then 24 hours.In negative control, add entry and replace test substances.After cultivation is finished, remove culture medium, use phosphate buffer to wash this cell.After cell is frozen and thaws, to the reaction solution that wherein adds 0.2ml (0.1M Tris-HCl (pH value is 7.4), 1mM MgCl
2, 0.02%Triton X-100,0.15mM p-nitrophenols (PNP is made by Nacalai Tesque company limited)), stir, in ice, hatch and obtained enzymatic solution in 30 minutes.80 μ l enzymatic solution are transferred in the 96 hole microplates.In each hole, add or do not add the glucuronic acid (making) of 20 μ l 20mM by Wako Pure ChemicalIndustries company limited.Hatched this plate 1 hour at 37 ℃.
In addition, use the PNP of concentration known to make calibration curve.Then, add the glycine buffer (pH value is 10.4) of 100 μ l 2M, measure its absorbance at the 405nm place.Carry out three times activity analysiss.The amount of the test substances that is added is according to the concentration that is shown in the following table.UGT is active in the amount with respect to the bonded PNP that contrasts, and calculates according to following formula.
UGT activity=[(the PNP amount of the adding test substances part of no glucuronic acid)-(the PNP amount that the adding test substances part of glucuronic acid is arranged)]/[(the PNP amount of the adding water section of no glucuronic acid)-(the PNP amount that the adding water section of glucuronic acid is arranged)]
The result is as shown in table 6.Table 6 has shown and has added behind the agar oligosaccharide activity of UGT in the cell.Find, add the agar oligosaccharide and caused the active remarkable increase of UGT.
Table 6
Embodiment 7: the assessment of inducing the effect of UDP-glucuronyl transferase (UGT) mRNA expression
Measure the activity that the agar oligosaccharide induces UGT mRNA to express according to method described in the embodiment 4.Commercially available agar oligosaccharide (trade name: Agaoligo) be used as test substances.Carry out twice activity analysis.The expression of UGT mRNA is as the amount with respect to the UGT mRNA of contrast, and calculates according to following formula.
The expression of UGT mRNA=[(expression that adds UGT mRNA in the test substances part)/(expression that adds Tfrc mRNA in the test substances part)]/[(expression that adds UGTmRNA in the water section)/(expression that adds Tfrc mRNA in the water section)]
The result is as shown in table 7.The amount that UGT mRNA expresses in the cell behind the table 7 demonstration adding agar oligosaccharide.Find that the agar oligosaccharide has the activity of significantly inducing UGT mRNA to express.
Table 7
Embodiment 8: for the assessment of glutathion inside cell (GSH) content raising effect
With reference to people such as Clarissa Gerhauser, Cancer Research 57, the improved method of the part of method described in the 272-278 (1997) is measured GST content.With the DMEM of Hepa1c1c7 cell suspension in the penicillin-streptomycin that contains 10% hyclone and 1%, cell concentration is 4 * 10
5Individual cell/ml.In each hole of 96 hole microplates, add the cell suspending liquid of 0.2ml, when having 5% carbon dioxide, 37 ℃ of overnight incubation.Then, change culture medium in each hole with DMEM.In each hole, add the aqueous solution of 0.4 μ l test substances, cultivated then 24 hours.By preparation embodiment 1 resulting Agarobiose. and commercially available agar oligosaccharide (trade name: Agaoligo) be used to test substances.In negative control, add entry and replace test substances.After cultivation is finished, remove culture medium, use phosphate buffer to wash this cell.Remove solution, repeat three times the freeze-thaw cell processes.Obtain cell lysates to wherein adding 0.1ml buffer A (125 μ M sodium phosphate buffers (pH value is 7.5), 6.3mM EDTA).Adding 100 μ l reaction solutions in 25 μ l cell lysates (25mM Tris-HCl (pH value is 7.4), 1mM G6P, 30 μ M NADP, 2U/ml G6PDH, 0.25U/ml glutathion reductase (making) by Sigma, 0.6mMDTNB).After 5 minutes, measure its absorbance in incubated at room at the 405nm place.Carry out three times activity analysiss.Simultaneously, the GST of the 2-200 μ l of a series of 2 times of dilutions replaces cell lysates as standard testing.Use is measured protein content with cell lysates and MicroBCA analysis of protein test kit that phosphate buffer carries out 50 times of dilutions.Add test substances according to concentration shown in the following table.The concentration of GSH is as the concentration with respect to the GSH of contrast, and calculates according to following formula.
The concentration of GSH=[(concentration that adds GSH in the test substances part)/(adding proteic concentration in the test substances part)]/[(concentration that adds GSH in the water section)/(adding proteic concentration in the water section)]
The result is as shown in table 8.Table 8 has shown and has added after the various test substances content of GSH in the cell.Find, add the remarkable increase that agar oligosaccharide or Agarobiose. have caused GSH content.
Table 8
Comparing embodiment: new agar oligosaccharide is for the assessment that strengthens glutathione S-transferase (GST) activity and the active effect of quinone reductase (QR)
Use Neoagarohexaose active and active effect of QR according to the assessment of the same procedure among embodiment 1 and the embodiment 3 respectively as the resulting enhancing of agar oligosaccharide GST.The result is as shown in table 9.Table 9 has shown and has added behind the Neoagarohexaose activity of GST and the activity of QR in the cell.Find, add Neoagarohexaose and do not cause the active and active remarkable increase of QR of GST.
Table 9
Industrial applicability
According to the present invention, provide the enhancing of glutathione content in II detoxification enzyme activity or the cell Agent comprises the fine jade that is selected from agar, agarose, contains the anhydrous galactopyranose of 3,6-in reduction end The low molecular compound of lipolysaccharide, the compound that is represented by above-mentioned molecular formula (chemical molecular formula 1) And at least a compound as active component in derivative and its salt, and contain reinforcing agent Medicine, food or feed. The medicine, food or the feed that contain reinforcing agent can be by strengthening the II detoxifcation In enzymatic activity or the cell glutathione content be used for promoting detoxification processes, therefore, be particularly suitable for Medicine, Foods or drinks as treatment or prevention various diseases especially are suitable as the reduction disease The medicine of prevention of risk disease or functional food.
Claims (6)
1. the reinforcing agent of the second phase detoxification enzyme activity or glutathion inside cell content, it comprises and is selected from agar, agarose, contains 3 in reduction end, the low molecular compound of the agarose of the anhydrous galactopyranose of 6-, by the chemical compound of following molecular formula (chemical molecular formula 1) representative:
Wherein X and Y are H or CH
2OH, condition is, when X is CH
2Y is H during OH, and Y is CH when X is H
2OH; And at least a chemical compound in derivant and its salt as active component.
2. the reinforcing agent of second phase detoxification enzyme activity as claimed in claim 1 or glutathion inside cell content wherein contains 3 in reduction end, and the agarose low molecular compound of the anhydrous galactopyranose of 6-is the agar oligosaccharide.
3. the reinforcing agent of second phase detoxification enzyme activity as claimed in claim 2 or glutathion inside cell content, wherein the agar oligosaccharide is the mixture that comprises Agarobiose., Agarotetraose, Agarohexaose and agarooctaose.
4. as the reinforcing agent of each described second phase detoxification enzyme activity or glutathion inside cell content in the claim 1 to 3, wherein second to separate toxenzyme mutually be glutathione S-transferase, quinone reductase or UDP-glucuronyl transferase.
5. contain medicine just like the reinforcing agent of each described second phase detoxification enzyme activity or glutathion inside cell content in the claim 1 to 4.
6. contain food or feedstuff just like the reinforcing agent of each described second phase detoxification enzyme activity or glutathion inside cell content in the claim 1 to 4.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2006281731 | 2006-10-16 | ||
| JP281731/2006 | 2006-10-16 | ||
| JP004610/2007 | 2007-01-12 |
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| Publication Number | Publication Date |
|---|---|
| CN101528240A true CN101528240A (en) | 2009-09-09 |
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ID=41095673
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNA2007800386306A Pending CN101528240A (en) | 2006-10-16 | 2007-10-11 | Activity enhancer for detoxifying enzyme |
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| Country | Link |
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| CN (1) | CN101528240A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108371663A (en) * | 2018-01-24 | 2018-08-07 | 山东大学 | Application of the agaropectin oligose in preparing the drug that treatment cognitive function fails relevant the nervous system disease |
| KR20220104496A (en) * | 2021-01-18 | 2022-07-26 | 한국과학기술연구원 | Composition for relieving hangover based on soybean curd residue micro-powder |
-
2007
- 2007-10-11 CN CNA2007800386306A patent/CN101528240A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108371663A (en) * | 2018-01-24 | 2018-08-07 | 山东大学 | Application of the agaropectin oligose in preparing the drug that treatment cognitive function fails relevant the nervous system disease |
| KR20220104496A (en) * | 2021-01-18 | 2022-07-26 | 한국과학기술연구원 | Composition for relieving hangover based on soybean curd residue micro-powder |
| KR102642811B1 (en) | 2021-01-18 | 2024-03-05 | 한국과학기술연구원 | Composition for relieving hangover based on soybean curd residue micro-powder |
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