CN1281742C - Polycondensed amino-acid coordinated SOD simulatives - Google Patents
Polycondensed amino-acid coordinated SOD simulatives Download PDFInfo
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- CN1281742C CN1281742C CN 02124983 CN02124983A CN1281742C CN 1281742 C CN1281742 C CN 1281742C CN 02124983 CN02124983 CN 02124983 CN 02124983 A CN02124983 A CN 02124983A CN 1281742 C CN1281742 C CN 1281742C
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Abstract
The present invention relates to a method of an amino acid condensation product used as a ligand and different divalent metal ions in a water solution for preparing polycondensing amino acid metal compositions--a polycondensing ferrous aspartate composition and a polycondensing aspartic acid manganese composition. Firstly, after polycondensing aspartic acid is dissolved in ammonia water, a manganese sulphate solution is added to react in a reflux way to obtain the polycondensing aspartic acid manganese composition when the pH is adjusted to a neutral condition; ferrous sulfate and hydroxylamine hydrochloride with the same mol as the ferrous sulfate are added under the acidic condition, and the polycondensing ferrous aspartate composition is obtained by a reflux reaction. The chemical constitution and the structural characterization representation for the compositions are carried out by element analysis and infrared spectrums, and the SOD sample activity of a catalyzed O2[. -] disproportionation reaction in the present invention is measured by using an improved nitroblue tetrazolium (NBT) illumination method. The SOD simulacrum has the characteristics of moderate molecular weight, good water solubility, nontoxicity, high activity, etc. and has wide application prospects.
Description
Technical field
The present invention relates to a kind of superoxide-dismutase (SOD) stand-in.
Background technology
People have done a large amount of research with regard to natural superoxide-dismutase (SOD), and it can remove biological intravital ultra-oxygen anion free radical (O single-mindedly
2 -), make it change nontoxic material rapidly into, thus but prophylactic activity oxygen intoxication, anti-ageing and reduce the generation of diseases such as cancer.But, since natural SOD self molecular weight big, be difficult for permeate through cell membranes, in vivo the transformation period short and isoantigen is arranged, shortcoming such as cost an arm and a leg, in actual application, be very limited.
From the active centre of SOD, forming title complex with metal ions such as all kinds of parts such as each seed amino acid, Schiff alkali and Cu, Fe, Mn, to come Simulating SOD be the focus of current research.Developed into whole simulation in the work of Simulating SOD from simple analog SOD active centre, whole mimic advantage is to provide the hydrophobic microenvironment that is similar to enzyme, improves the activity of SOD with this.Amino acid molecular is modified, cooperated with metal ion again, obtained having the SOD simulation substance of greater activity as on the amino acid molecular nitrogen-atoms, introducing hydrophobic long chain alkyl group lauroyl.The research work of a large amount of SOD stand-in, filtered out many in the laboratory and clinical experimental stage have the active material of SOD sample.
Yet the SOD stand-in also are not applied among the reality veritably at present.Its reason mainly is that prepared stand-in differ far away apart from practical application, all need such as work such as the toxicity inspection of stand-in, carcinogenic, teratogenesis, mutagenic test and pharmacokinetics and to carry out, only possess these reliable data, can progressively enter the application stage.In addition, the solvability of SOD stand-in, stability have also limited its range of application.
In the research process of numerous SOD stand-in, show the compound that cupric ion and all kinds of part form, the active simulation substance that generally all is higher than iron, manganese, zine ion and part formation of its SOD sample.We find by animal acute toxicity experiment recently, glycocoll-copper, cupric glutamate and acidum clofenamicum copper and diclofenac copper complex, the disposable oral Lethal Dose 50 (LD of its small white mouse
50) all less than 500mg/Kg, belong to low toxicity and moderate toxicity material.Amino acid is non-toxic substance, introduce cupric ion and obviously show toxicity later on, and acidum clofenamicum and diclofenac also shows the toxicity with the similar size of amino acid copper complex after to be anti-inflammation analgesis medicament and cupric ion cooperate.Although the nontoxic or little poison of this explanation part, the toxicity behind the introducing cupric ion is very important.This shows that though copper is one of bioelement of people, excessive absorption cupric ion also can cause damage to body.The SOD stand-in that with copper are central ion have certain application value, if but the medicinal enzyme of or external application for oral administration as human body must be subjected to the restriction of toxicity size.
Iron, manganese, zinc and amino acid belong to nontoxic after forming title complex substantially, show its LD by animal acute toxicity experiment
50All bigger, for example the numerical value of halfcystine iron, tyrosine iron complex is all more than 5000mg/Kg.Chronic animal toxicity experiment shows that also the amino acid iron complex of low dosage is to not damage of animal body.Can also see that by the experiment of animal pharmaceuticals dynamic metabolism this class material can be absorbed by the animal body, and can increase its intravital SOD contents level, so this class iron amino acid title complex has using value preferably.Yet the SOD stand-in by these part preparations also have obviously water-soluble relatively poor weak point, the material that has even fat-soluble also bad, and this has just caused the limitation of practical application.If adopt organic solvent such as ethanol to increase as secondary solvent that it is water-soluble, can overcome the deficiency of this respect, but ethanol, acetone and other organic solvent all there is the active effect of its SOD sample of obvious reduction, make it remove O
2 -Ability reduce, and be proportionate with its usage quantity.Chinese patent CN1138587A, CN1225631A are the SOD stand-in of model with the small molecules, its structure and natural SOD differ and far are starting point, the copper, iron, the manganese compound that form with coordination of rich imidazoles nitrogen and nitrogenous macrocyclic ligands have been announced, these title complexs have certain SOD sample activity, but also have above-mentioned weak point.Therefore screen nontoxic, high reactivity, good water solubility and with the approaching stand-in of natural SOD structure are best selections.
Summary of the invention
The invention is characterized in that the polycondensation aspartic acid metal SOD stand-in of preparation have good water solubility, nontoxic, characteristics such as molecular weight is moderate, have broad application prospects.
The polycondensation aspartic acid is nontoxic, biodegradable, can select in different scopes by its molecular-weight average of control of polymerization process.The polycondensation aspartic acid is novel water-soluble polymer, at present can mass production, be applied among the different fields as flocculation agent etc.
As part, water insoluble and other organic solvents commonly used of the aspartic acid polycondensate that we use dissolve in the alkaline aqueous solution (pH 〉=8.5).Cause trouble for separation purification afterwards for avoiding in preparation process, introducing external metallic cation, we adopt the ammoniacal liquor of 1mol/L as solvent, hydrolysis takes place in aspartic acid polycondensate dissolving back under alkaline condition, the ring-type chain link of hydrolysis aftercondensated thing is opened into the chain chain link, solution is scarlet, and pH is 9.5.There is when avoiding it to cooperate oxyhydroxide to generate with metal ferrous ion or mn ion, transfer to earlier and add mn ion solution back flow reaction again after adding ferrous ions soln again after the acidity or transferring to neutrality with acid solution with acid solution, 4～8 hours reaction times, 60～100 ℃ of temperature of reaction, the mol ratio of ferrous ion or mn ion and polycondensation aspartic acid chain link is 1: 2～1.5: 2.The alkaline hydrolysis process of aspartic acid condenses can be expressed as:
We use molecular-weight average be 3000 polycondensation aspartic acid as part and metal ion reaction, under the situation of not considering polycondensate polymerization degree size, think a divalent-metal ion can with the chain link combination of two polycondensates.The chain link mol ratio of choosing divalent metal and polycondensate when feeding intake is 1: 2～1.5: 2, and the ratio that increases metal-salt can increase productive rate and also help reacted separation purification simultaneously.
For preventing the ferrous ion oxidation, can under the condition of nitrogen protection, react.Use nitrogen to be protective material, in actual production process, produce trouble sometimes.We take to add under acidic conditions and the equimolar oxammonium hydrochloride of ferrous sulfate is a protective material, have also obtained effect preferably.
The rising temperature of reaction can increase speed of response, and we take to react and can carry out under 60～100 ℃ temperature.The increase reaction times can be improved productive rate, considers that from each factor of economic dispatch the reaction times is chosen 6～8 hours and is advisable.
Along with the carrying out of reaction, product increases gradually, but polycondensation aspartic acid metal complexes has good solvability in the aqueous solution, and system does not have tangible phase change in the entire reaction course.Temperature of reaction system drops to room temperature and does not find that also the solubleness of reactant and product has bigger difference later.After overnight under 4 ℃ the condition, there is precipitation to separate out reaction system, is different from reactant through the differential thermal analysis throw out.
Be under 7.8 the condition, to have measured SOD stand-in catalysis O of the present invention at pH with improved nitroblue tetrazolium(NBT) (NBT) luminescence method
2 -The activity of disproportionation reaction.The natural SOD of different concns and polycondensation aspartic acid metal complexes are to O
2 -Inhibiting rate sees Table 1.Natural SOD or stand-in are to O
2Inhibiting rate is big more, illustrates that then its SOD sample activity is high more.As can be seen from Table 1, natural SOD and stand-in are with the concentration measurement of g/L the time, and the natural SOD's of specific activity of polycondensation Ferrous diaspartate is active lower slightly, the activity of polycondensation AspMn and natural quite, and all be to reduce with its concentration to reduce, this illustrates that it has similar SOD sample activity.
Natural SOD of table 1. different concns and poly aspartic acid metal complexes are to O
2 -Inhibiting rate
| Concentration (g/mL) | Natural SOD | The polycondensation Ferrous diaspartate | The polycondensation AspMn |
| 10 -5 10 -6 10 -7 10 -8 10 -9 | 84.5 76.8 48.9 32.2 16.0 | 74.4 53.8 35.4 12.1 7.2 | 84.3 71.7 58.7 36.3 14.5 |
With the PE-240C elemental analyser carbon, hydrogen, the nitrogen element content of polycondensation aspartic acid metal complexes are analyzed, the result shows that the measurement result of two kinds of title complexs conforms to substantially with its theoretical value (the results are shown in Table 2), and this shows the basically identical that its molecular composition and expection are imagined.
Table 2. polycondensation asparagus fern ammonia ferrous results of elemental analyses
| The polycondensation Ferrous diaspartate | The polycondensation AspMn | |||
| Molecular formula (C 8H 8O 6N 2Fe)n | Chain link amount 284 | Molecular formula (C 8H 8O 6N 2Me)n | Chain link amount 283 | |
| Measured value | Theoretical value | Measured value | Theoretical value | |
| C/％ | 33.10 | 33.80 | 33.25 | 33.92 |
| H/％ | 3.04 | 2.83 | 2.97 | 2.83 |
| N/％ | 10.15 | 9.86 | 9.45 | 9.90 |
Through infrared measurement, aspartic acid polymkeric substance ferrous infrared absorption is: 3344cm
-1Strong absorption (NH stretching vibration); 1665cm
-1(-COO-); 1544cm
-1(NH flexural vibration); 1414cm
-1586cm
-1The infrared absorption of polycondensation AspMn is: 3278cm
-1(NH stretching vibration); 1667cm
-1(-COO-); 1443cm
-11140cm
-11082cm
-1
Compare original 3300cm with polycondensation Sodium L-aspartate salt
-1Near-the NH stretching vibration peak and-position at COO-peak not have variation substantially, poly aspartic acid manganese-disappearance of NH flexural vibration peak, during poly aspartic acid is ferrous-and NH flexural vibration peak also weakens to some extent, and this explanation metal ion (M) has coordination with N on the-NH.In conjunction with ultimate analysis, infer that tentatively these two kinds of materials may be the inner complex salt structure again:
Polycondensation amino acid coordination compound involved in the present invention is expected to further the detection by a series of pharmacology, toxicity and three genicity tests, thereby replaces natural SOD in many-sided Application of Additives such as medicine, makeup, food, healthcare products and feeds.
Embodiment
Embodiment 1:
Take by weighing molecular-weight average and be 3000 aspartic acid polycondensate 2.0g and be dissolved in the ammoniacal liquor of 15mL 1mol/L, solution is the scarlet clear solution, and regulating the pH value with the hydrochloric acid soln of 10mol/L is 4.Other takes by weighing ferrous sulfate 1.5g and equimolar oxammonium hydrochloride is dissolved in the 15mL distilled water.Two kinds of solution are mixed in the 250mL ground triangular flask, and mixing the back system is the scarlet clear solution still, and reaction system is placed thermostat water bath, be warming up to 80 ℃ gradually, magnetic agitation was taken out after 6 hours, and system is the scarlet clear solution still, is cooled to room temperature.Have cotton-shaped suspended substance to occur after 4 ℃ of conditions are spent the night, the throw out behind the suction filtration gets light orange powder, productive rate about 50% with distillation washing twice behind the natural air drying.
Embodiment 2:
Take by weighing aspartic acid polymkeric substance 2.0g and be dissolved in the ammoniacal liquor of 15mL 1mol/L, regulating the pH value with the hydrochloric acid soln of 10mol/L is 7.Other takes by weighing manganous sulfate 1.7g and is dissolved in the 15mL distilled water.Two kinds of solution are mixed in the 250mL ground triangular flask, and mixing the back system is the scarlet clear solution, takes out after magnetic agitation refluxed 6 hours, and system is the scarlet clear solution still, is cooled to room temperature.Have cotton-shaped suspended substance to occur after 4 ℃ of conditions are spent the night, the throw out behind the suction filtration washes twice with distillation, and natural air drying gets white powder.Productive rate about 60%.
Claims (4)
1. the SOD stand-in of a class polycondensation amino acid coordination, it is characterized in that with the polycondensation aspartic acid being that part gets with ferrous ion or mn ion complex reaction in the aqueous solution: at first with alkaline aqueous solution dissolving polycondensation aspartic acid, transfer to acid solution and to add mn ion solution back flow reaction again after adding ferrous ions soln again after the acidity or transferring to neutrality with acid solution, 4～8 hours reaction times, 60～100 ℃ of temperature of reaction, the mol ratio of ferrous ion or mn ion and polycondensation aspartic acid chain link is 1: 2～1.5: 2.
2. the SOD stand-in of a class polycondensation amino acid coordination according to claim 1 is characterized in that described alkaline aqueous solution is the alkaline aqueous solution of ammonia.
3. the SOD stand-in of a class polycondensation amino acid coordination according to claim 1 and 2 is characterized in that ferrous ion and mn ion adopt the soluble salt solution of ferrous ion, bivalent manganese.
4. the SOD stand-in of a class polycondensation amino acid coordination according to claim 1 and 2 is characterized in that adopting under the acidic conditions oxammonium hydrochloride protection ferrous ion not oxidized.
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| CN 02124983 CN1281742C (en) | 2002-06-29 | 2002-06-29 | Polycondensed amino-acid coordinated SOD simulatives |
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| CN 02124983 CN1281742C (en) | 2002-06-29 | 2002-06-29 | Polycondensed amino-acid coordinated SOD simulatives |
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| CN100347180C (en) * | 2005-12-26 | 2007-11-07 | 北京华美天意科技开发有限公司 | SOD simulated compound with short peptide as ligand and preparation method thereof |
| CN105565910B (en) * | 2015-12-16 | 2017-03-08 | 山东远联化工有限公司 | The preparation method of biologically active polypeptide calcareous fertilisers |
| CN105949793B (en) * | 2016-05-31 | 2018-04-06 | 西北师范大学 | The preparation of soybean protein chitosan microball combination amino acids metal complex and the application as antioxidant |
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