CN1314151A - Use of AFMK in preparing medicine with free radical cloaning function - Google Patents
Use of AFMK in preparing medicine with free radical cloaning function Download PDFInfo
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- CN1314151A CN1314151A CN 01107603 CN01107603A CN1314151A CN 1314151 A CN1314151 A CN 1314151A CN 01107603 CN01107603 CN 01107603 CN 01107603 A CN01107603 A CN 01107603A CN 1314151 A CN1314151 A CN 1314151A
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- afmk
- free radical
- acetyl
- formaldehyde
- methoxyl group
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Abstract
The compound AFMK is prepared into various medicine forms with the action of removing free radical for treating retrogressive nervous system disease, heart disease, rheumarthritis and rheumatoid arthritis, and delaying senility.
Description
The present invention relates to a kind of melatonin deriv and have application in the medicine of free radical scavenging effect in preparation.
Free radical is meant that molecule contains not paired electronics on the outermost track, and the chemical reactivity of this quasi-molecule is very active, and material or the molecule trend of obtaining an electronics from other are arranged.Free radical is an inevitable accessory products in the biological oxidation metabolic process.The biological oxygen that is utilized finally has 2-4% to be finally converted into free radical.As these free radicals biological anti-oxidative defense system of escaping out, comprise antioxidase and interior exogenous small molecule antioxidant, these free radicals will be attacked the biomacromolecule material, as protein, cell membrane, DNA, cause these macromolecular degeneration and damages, finally cause the forfeiture of cell or organism structural damage and function.According to the free-radical theory of accepting extensively for scientist at present, oxygen-derived free radicals and many Pathophysiologys have direct or indirect getting in touch, comprising common clinical frequently-occurring diseases such as aging, alzheimer disease, parkinson disease, heart disease, tumor, rheumatic arthritis, rheumatoid arthritis, heat generation inflammation, gouts.
Melatonin (melatonin) is a N-acetyl group tryptamines, is the intravital endogenous material of human body and mammal, is mainly generated by the pineal gland that is positioned at position, brain center.The discovered in recent years melatonin is present strong endogenous free radical scavenger, and the life-span that can prolong laboratory animal reaches 30%, the damage that the protection central nervous system resists multiple noxious substance, and finding again has adjuvant treatment effect to AIDS.The half-life of melatonin lacks, and only is about 40 minutes, the treatment concentration of the needs that the administration of need repeated multiple times just can be kept.The antioxidation of melatonin is directly to remove free radical, and it has just become side effect by receptor-mediated effect, for example causes drowsiness.Thereby it is short that melatonin has the half-life, and the shortcoming of certain side effect is arranged.
The object of the invention just is to overcome the shortcoming that the melatonin half-life is short, have certain toxic and side effects, provides a kind of chemical compound to have application in the medicine of strong free radical scavenging effect in preparation.
The present invention realizes like this.
The invention provides following chemical compound
N
1-acetyl-N
2-formaldehyde-black the amine of 5-methoxyl group urine;
N
1-acetyl-N
2-formyl-5-methoxykynuramine(AFMK)。
The physicochemical property of AFMK is: tasteless, and faint yellow crystallization, soluble in water and ethanol, its steady chemical structure at room temperature, molecular weight 264.
Tan equals the related content synthetic and that structure is identified with this material in 2000 and is published in (Free Redical Biology ﹠amp on the U.S. magazine; Medicine 2000, and 29:1177-1185), the synthetic method of AFMK is that melatonin is dissolved in the hydrogen peroxide of high concentration that (as (30%), lucifuge, be statically placed under the room temperature 24～48 hours, the melatonin major part is oxidized to AFMK.Use chloroform extraction, collect chloroform, be dissolved in methanol behind the vacuum drying again,, use eluent ethyl acetate by silica gel column chromatography, the collection eluent, vacuum drying can obtain the AFMK (more than 90%) of higher degree, and combined coefficient (product is than reactant) is about 40%.End-product confirms that through electron impact mass spectra analysis and the structural analysis of H1 nuclear magnetic resonance, NMR its structure is AFMK, and AFMK has been found and has been present in the animal body, but reported in literature is not seen in the research of its biological activity and function.
We find that AFMK is a class resisting oxidation free radical scavenger efficiently.For having powerful protective effect by the lipid DNA damage of free yl induction and the death of hippocampal neurons.Its experimental basis is as follows:
One, the mechanism of AFMK antioxidation, bielectron supply effect
Directly the small molecule antioxidant of oxygen reduction free radical is called free radical scavenger again, can directly provide electronics to remove these deleterious molecules to free radical.Therefore, measure free radical scavenger oxidation---reduction potential can be assessed the radical scavenging activity of these materials.Present a kind of relatively more novel method reaches this purpose with the cyclical voltage analyzer exactly.This experiment is when measuring the sub-ability of power supply of AFMK, and (10% methanol, 90% phosphate buffer, 100 mMs, PH7.4) 1 milliliter of solution places in the measuring chamber of microcirculation voltage determination instrument with the AFMK of 50 millimolar concentrations.Analyzer model: CV-50W (bio-analysis system, westlafayette company, the U.S.), in operating process, the voltage-to-current curve record is (figure one) on the cyclical voltage imager.As seen,, two anelectrode current waves are arranged from the figure one at voltage 456 and 668 millivolts of places.This has shown that AFMK may exist two electron-donating groups.AFMK can provide the feature of two electronics in the cyclical voltage field, pointing out this chemical compound is a high-efficiency antioxidant free radical scavenger, its effect should be better than classical antioxidant vitamin C, vitamin E, glutathion etc., these traditional antioxidants only show an anelectrode electric current in the cyclical voltage field, just can only provide an electronics go in and free radical.
Two, AFMK is to the snperoxiaized protective effect of biomembrane lipid
Lipid peroxidation is that the cell membrane lipid is subjected to the important symbol that oxygen-derived free radicals is attacked damage.In order to detect the protective effect of AFMK cell membrane lipid peroxidation, use following experimental technique.Get the rat hepatocyte and carry out homogenate (100 milligrams of hepatic tissues add 1 milliliter of phosphate buffer PH7.5,50 millimolar concentrations), serosity adds hydrogen peroxide (H
2O
2) and the iron ion (Fe of two valencys
2+) incubation (37 ℃, a hour) together, in the experimental group specimen, add the AFMK of variable concentrations simultaneously.Experimental result shows, H
2O
2And Fe
2+The lipid peroxide of rat hepatocyte homogenate is obviously increased, and its mechanism is that the Tie Tong of divalent crosses Fenton reaction and makes H
2O
2Be reduced into Cytotoxic carboxyl free radical, carboxyl free radical has caused the damage of cell membrane.The lipid peroxide that AFMK can suppress rat hepatocyte homogenate generates, and its inhibitory action is dose-dependence (figure two), thereby proof AFMK is by removing H
2O
2, be reduced into Cytotoxic carboxyl free radical, protected biomembrane by H effectively
2O
2And ferrous ion (Fe
2+) lipid peroxidation that causes.
Three, AFMK is to being caused the protective effect of DNA damage by oxygen-derived free radicals
8-carboxyl NSC 22837 (8-OH-dG) is the important indicator that DNA is subjected to oxidative damage.8-OH-dG is considered to carboxyl free radical and attacks the distinctive product that has that DNA generated, and has been widely used in detecting a kind of effective method of DNA damage.Calf thymus pure dna and H have been adopted in this experiment
2O
2And trivalent chromic ion (Cr
3+) incubation (37 ℃, 60 minutes) together, tervalent chromium ion can provide an electronics to H
2O
2, make it be reduced into Cytotoxic carboxyl free radical, the guanine base that carboxyl free radical is attacked on the DNA chain makes its 8th to go up carboxylated.Add nucleotidase P1 (Nuclease p1) dna digestion then, make DNA be hydrolyzed into the base nucleoside, reuse high-pressure liquid phase (HPLC)-Electrochemical Detection instrument is measured 8-OH-dG result and is shown: 1, H
2O
2And Cr
3+The DNA of incubation together, the content of 8-OH-dG has increased about 20 times than normal control group.2, AFMK can suppress by H
2O
2And Cr
3+The rising of the 8-OH-dG that causes, this inhibition are tangible dose-dependence (figure three), and when the concentration of AFMK was 5 micromoles, the generation of 8-OH-dG had been reduced 80%, and this experimental results show that AFMK has powerful DNA protective effect.
Four, AFMK is to cultivating protecting neuron from acute
The hippocampal gyrus neuron is and the closest cell of human impermanent memory relation.One of alzheimer disease pathogenic factor is exactly the hippocampal gyrus neuron degeneration.The selected cultured cell of hippocampal gyrus neurocyte strain (HT22) as this experiment, cultural method is conventional cell culture method, in the DENEM culture fluid, add 10% calf serum, be used as HT22 hippocampal gyrus neuronal cell cultures substrate, then, 37 ℃ of temperature, cultivate in 5% carbon dioxide cell incubator, changed culture fluid once in second day.(4,5-dimethythiazol-zyl)-2,5-diphenyl-tetrazolium bromide (MTT) is used to detect the neuron survival rate to 3-.Concrete grammar is 96 well culture plates, and every hole places about 1500 cells (100 milliliters of culture fluid), and the processed group cell was cultivated 24 hours with AFMK in advance, allows cell attachment grow, and adds cytotoxic substance again and comprises H
2O
2Glutamic acid and β-starch peptide, the common cultivation 16 hours, add MTT (ultimate density 0.5 mg/ml) then and cultivate (37 ℃ together, 4 hours), blue precipitate is dissolved in (lysate: 20%SDS, 50%N in the lysate, N-dimethylformamide is PH4.7) with the survival rate of culture plate reading apparatus (UQuantBio-Tek Instruments In) at 750 nanometers detection cell.All MTT analyze and repeat at least 3 times, and each every group of 7 specimen, all data all are used for meansigma methods plus-minus standard error and represent that (mean ± SE), statistical analysis ANOWA method is checked with StudentNewman-Reuls then.When P value＜0.05, be considered to significant difference.
A, N
1-acetyl-N
2The black amine (AFMK) of-formaldehyde-5 methoxyl group urine is to by H
2O
2The protective effect of inductive hippocampal gyrus nerve cell death
Cell culture condition as mentioned above, the HT22 cell strain was cultivated 24 hours separately or with the AFMK of variable concentrations (1/100 little rub and 1 milli rubs) in advance, added the H of variable concentrations then
2O
2(25,50,100 little rubbing), H
2O
2Be the by-product in the cellular process, when reaching finite concentration or when having transition metal such as divalent iron ion and 1 valency copper ion etc. to exist, can causing the oxidative damage of cell.Experimental result shows H
2O
2Can inductive dose dependent HT22 cell death, AFMK has and can protect H effectively in 100 little rub concentration and the 1 millis concentration of rubbing
2O
2Inductive cultured rat hippocampal stomogastric nerve cell death.Reach 100% (figure four) at high concentration (1 milli rubs) protective rate
B, N
1-acetyl-N
2The black amine (AFMK) of-formaldehyde-5 methoxyl group urine is to the protective effect by the hippocampal gyrus nerve cell death of glutamate induction.
Cell culture condition as mentioned above, HT22 cell strain AFMK independent or variable concentrations cultivated 24 hours in advance, and then the glutamic acid of adding variable concentrations (5 millis rub and 10 millis rub), glutamic acid is exicitatory/amino acid neurotransmitter, when excessive concentration, can cause calcium overload in the neurocyte, free-radical generating finally causes cell injury.Experimental result demonstration glutamic acid can be induced the death of HT22 cell; AFMK has significant protective effect to the death of excitatory amino acid-glutamate induction hippocampal gyrus neurocyte; this protective effect is dose-dependence; protective rate reached 15%, 1 mM and reaches 60% (figure five) when AFMK was 100 micro-molar concentrations.
C, N
1-acetyl-N
2The black amine (AFMK) of-formaldehyde-5 methoxyl group urine is to the protective effect by the inductive hippocampal gyrus nerve cell death of beta amyloid peptide
Cell culture condition as mentioned above, the HT22 cell strain was cultivated 24 hours separately or with the AFMK of variable concentrations in advance, and then added the beta amyloid peptide segment (β-25-35) cultivate together of variable concentrations (0.2,2 and 20 micromole).Beta amyloid peptide is considered to the main matter of alzheimer disease neurocyte pathological change, the formation of neural speckle, the proteic deposition of β-starch, cause free radical to form and finally cause nerve cell death, experimental result shows: (β-25-35) can induce the death of HT22 cell to the beta amyloid peptide segment.AFMK is to having protective effect by the inductive nerve cell death of beta amyloid peptide, and this protective effect has significant statistical significance (figure six).
By above-mentioned experimental result, AFMK is in molecule and powerful antioxidation and the free radical scavenging effect of cellular level tool for proof, have potential clinical value, can be processed into peroral dosage form, comprise tablet, slow releasing tablet, capsule, granule, oral liquid, also can be processed into injection, comprise the powder pin, injection also can be processed into exterior-applied formulation, as Transdermal absorption membrane, unguentum, cream, also can be added in the cosmetics or as sunscreen.
Illustrate and conclusion:
N
1-acetyl-N
2The black amine (AFMK) of-formaldehyde-5 methoxyl group urine is an endogenous biogenic amine, is present in naturally in the humans and animals body.The oxidative metabolism of melatonin may be its main source.Up to now, lack research to its biological activity and function.The present invention is by to the further investigation of AFMK, finds first and confirmed that AFMK is a powerful endogenous resisting oxidation free radical scavenger with reliable scientific method.Adopt novel cyclical voltage field technology, can detect that AFMK can form 2 anelectrode current waves in 456 and 668 millivolts voltage field, the chemical constitution of prompting AFMK may exist 2 power supplying groups.Provide electronics in order in and oxygen-derived free radicals be the classical small molecule antioxidant or the essential condition of free radical scavenger antioxidation indispensability.Classical small molecule antioxidant vitamin C or vitamin E only can form an anelectrode current wave in this voltage field.The resisting oxidation free radical scavenging action of saying AFMK from theory significance should be better than traditional antioxidant.
Whether whether biomacromolecule lipid DNA is had protective effect in order to inquire into AFMK, the addition product 8-carboxyl deoxyguanosine of peroxidating of cell membrane lipid and DNA base oxidative damage is chosen testing index.Lipid is the Main Ingredients and Appearance of cell membranes in tissue, plays a crucial role for function of plasma membrane is complete.DNA is the base substance of biological heredity, is the carrier of all bio informations.If these important biomacromolecules sustain damage, will pair cell and biological function generation adverse influence.And oxygen-derived free radicals can cause the damage of lipid to produce lipid peroxide, destroys the integrity of cell function.Oxygen-derived free radicals also can be attacked DNA and make the base degeneration, causes disease or tumor.Experimental result shows that AFMK can protect effectively because H
2O
2With two valency ferrum due to the equal serosity of hepatocyte lipid peroxidation and by H
2O
2With trivalent chromium due to DNA damage, this protective effect is tangible dose-effect relationship.Oxidation is considered to form relevant with carboxyl free radical lipid peroxidation in this experiment with the DNA base.Because at H
2O
2With ferrous iron and H
2O
2With can both generate carboxyl free radical by Fenton reaction in the trivalent chromium system.In order to confirm the resisting oxidation free radical scavenging action of AFMK, selected the HT22 cell strain in the experiment for use at cellular level.The HT22 cell strain originates from rat hippocampal gyrus neurocyte, and the hippocampal gyrus neurocyte is considered to memory function confidential relation is arranged; The dysmnesia of alzheimer disease also may be relevant with the degeneration of patient's hippocampal gyrus neurocyte.In experiment, we have adopted three kinds of different cytotoxic substances, comprise H
2O
2, glutamic acid and amyloid-beta are induced the death of HT22 cell strain.These three kinds of cytotoxic substances think that all the degeneration with neurocyte has confidential relation, and their common ground is exactly to produce the cytotoxicity free radical by different approach.Experimental result shows; but these three kinds of materials are the death of the HT22 hippocampal gyrus neurocyte of inducing culture all; and AFMK can effectively protect the HT22 hippocampal neurons damage that is caused by various toxicant, its protective effect to have tangible dose-effect relationship and significant statistical significance (figure four, five, six).Can confirm that from above experimental result AFMK is a novel and effective endogenous resisting oxidation free radical scavenger, its action principle may for provide electronics with in and free radical.AFMK has the ability to provide 2 electronics, and its resisting oxidation free radical is removed ability may be better than classical antioxidant vitamin C or vitamin E.AFMK can not only protect lipid peroxidation and the oxidation of DNA base at molecular level, and can be in the cellular level protection because the death of the neurocyte that different harmful substances causes.
Oxygen-derived free radicals is with old and feeble, and the relation of oxygen-derived free radicals and numerous disease has obtained the support of more and more experimental basis.Think that according to comparatively popular old and feeble theory at present the accumulated damage of radical pair cell is a major reason in the aging course.Oxygen free radical injury also is one of pathology cause of disease of numerous disease.Comprising nervous system degenerative disease (alzheimer disease, parkinson disease), heart disease, tumor, rheumatic arthropathy, rheumatoid joint disease, gout and mistake disease property pain.Because AFMK has powerful resisting oxidation free radical scavenging action, is again an endogenous material (prompting may have no side effect), for the clinical above-mentioned disease of slow down aging and prevention and treatment that is used for potential using value is arranged.
The result estimates that the effective dose of human body is 2～10mg/ days by experiment, every day 1-4 time.
The invention will be further described below in conjunction with embodiment.
AFMK's is synthetic.
Take by weighing melatonin 5 gram, be dissolved in 100 milliliters of 30% the hydrogen peroxide, lucifuge, left standstill 24～48 hours, add the chloroform extraction secondary, each 400 milliliters, merge extractive liquid, reclaims chloroform, residue dissolves with 5 ml methanol, cross the silicic acid column chromatography, use eluent ethyl acetate, collect the part that has comprised AFMK with the fraction collection instrument, vacuum drying obtains the AFMK2 gram of purity more than 90%.
The preparation of the granule of AFMK.
It is an amount of to get AFMK, adds starch, Icing Sugar, makes granule.
The preparation of the tablet of AFMK.
It is an amount of to get AFMK, adds starch, Icing Sugar granulation, adds magnesium stearate, Pulvis Talci, by the tablet machine tabletting.
The invention is not restricted to the foregoing description.
Claims (6)
1, N
1-acetyl-N
2The black amine of-formaldehyde-5 methoxyl group urine has application in the medicine of free radical scavenging effect in preparation, and its structural formula is,
2, application according to claim 1 is characterized in that N
1-acetyl-N
2The dosage form that the black amine of-formaldehyde-5 methoxyl group urine is processed into is peroral dosage form, comprises tablet, slow releasing tablet, capsule, granule or oral liquid, or is processed into injection, comprises powder pin or injection, be processed into exterior-applied formulation, comprise the Transdermal absorption membrane, unguentum or cream, or be processed into cosmetics.
3, application according to claim 1 is characterized in that N
1-acetyl-N
2The black amine of-formaldehyde-5 methoxyl group urine is used to prepare the medicine of treatment and prevention nervous system degenerative disease.
4, application according to claim 1 is characterized in that N
1-acetyl-N
2The black amine of-formaldehyde-5 methoxyl group urine is used to prepare the medicine of treatment and prevention of cardiac.
5, application according to claim 1 is characterized in that N
1-acetyl-N
2The medicine that the black amine of-formaldehyde-5 methoxyl group urine is used to prepare treatment and prevents rheumatic arthropathy, rheumatoid joint disease, gout, mistake disease property pain.
6, application according to claim 1 is characterized in that N
1-acetyl-N
2The black amine of-formaldehyde-5 methoxyl group urine is used to prepare the medicine of slow down aging.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US10266482B2 (en) * | 2017-05-12 | 2019-04-23 | National University Corporation Tokyo Medical And Dental University | Long-term memory inducing agent |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US10266482B2 (en) * | 2017-05-12 | 2019-04-23 | National University Corporation Tokyo Medical And Dental University | Long-term memory inducing agent |
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