CN101947312B - Modified polyethylene glycol superoxide dismutase (mPEG-SOD) nanoemulsion capable of being directly externally used for skin and preparation method thereof - Google Patents

Abstract

The invention discloses modified mPEG-SOD nanoemulsion capable of being directly externally used for skin and a preparation method thereof. The mPEG-SOD nanoemulsion is prepared from the following raw materials in part by weight: 7 parts of isopropyl myristate, 30 parts of caprylocaproyl macrogolglycerides, 10 parts of polyglyceryl-3-dioleate, 50 parts of mPEG-SOD aqueous solution and 3 parts of vitamin E. The mPEG-SOD nanoemulsion solves the problem that the SOD has low stability, is easy to deactivate, and has poor penetrability for external application to the skin. The modified polyethylene glycol superoxide dismutase (mPEG-SOD) nanoemulsion capable of being directly externally used for the skin is a preparation with good stability and capacity of persistently protecting the bioactivity of the SOD, and can quickly penetrate the skin so as to greatly improve the biological effect of the SOD. The preparation method has the advantages of simple preparation process, easy actual operation, no need of special instrument and equipment, and high-efficiency and quick preparation.

Description

But mPEG-SOD nano-emulsion of a kind of direct cutaneous external and preparation method thereof

Technical field

The invention belongs to medical technical field, but relate to Pegylation superoxide dismutase (mPEG-SOD) nano-emulsion of a kind of direct cutaneous external and preparation method thereof.

Background technology

(1) general introduction of SOD

Since people such as nineteen sixty-eight American scholar McCord and Fridovich at first find and successfully from ORBC, extract superoxide dismutase (Superoxide Dismutase; Be called for short SOD) since, the research of SOD and application receive the common concern of different in the world specialties, different subject research worker.People from different fields, different degree, different scopes; With diverse ways and means; The chemical nature of SOD, biological characteristics, molecular structure, pharmacology, medicineization and clinical, medical, chemical industry etc. are studied and are inquired into, and successfully set up a whole set of fast and effectively SOD separate and analytical method.Particularly people such as McMord in 1969 since having delivered about [biological significance of ultra-oxygen anion free radical and superoxide dismutase] on the biochemical magazine of the U.S.; People are to free radical and biology; The relation of free radical and medical science; Understanding is significantly improved, and research has also had breakthrough, and makes the old and feeble free radical theory of professor's Harman proposition in 1956 be able to checking, replenish and develop.Teach as Michelson and write in the symposium collected works preface of the French Banyuls that organized by European Molecular Bioglogy Organization in June, 1976 (class's Reno this) first superoxides and superoxide dismutase: McMord in 1969 and Fridovich have at first found the SOD vigor and have delivered the article about the biological significance of superoxide radical from ORBC; A series of and the creationary research work in henceforth following one by one molecular biology aspect about oxygen metabolism almost can be compared with caused result after nineteen fifty-three Watson and Crick deliver famous dna double helical structure theory mutually.Comment that this section is penetrating is summarized and has been affirmed that SOD finds promoting important function and the value that the scientific research development is risen.

(2) SOD source, distribution and kind and basic characteristics thereof

SOD is a metalloid enzyme, and it extensively is present in all aerobic and aerotolerant each histiocytes of organism of nature.Because Cu.Zn-SOD; Mn-SOD;

that produces in the biological oxidation process made in these three kinds of SOD isozyme debridements specifically of Fe-SOD; In fact the cell that produces SOD is had protection and protective effect to active oxygen, therefore, American scholar Keele imagination; All aerobic metabolism cells; Comprise that the simplest microorganism all possibly contain SOD, really, people such as Yost successfully separated from E.Coli.B and have obtained Mn-SOD in 1973; And McCord in 1971 before this once carried out assay determination to more than 20 kind in nature to the SOD content in the different aerobe of oxygen demand, found that the aerobic metabolism of existence and cell of SOD is closely related.SOD content in the anaerobe that usually in air, can survive is lower than aerobic SOD slightly, and this maybe be through different relevant with contained oxygen demand and the generation active oxygen ability of different biologies.Up to the present; The specialty research worker has been separated from antibacterial, fungus, protozoacide, algae, insecticide, Fish, vegetable, beans, wild plant, crops (wheat, straw) and each internal organs of animal and histiocyte and has been obtained SOD; And these SOD that obtain are carried out further separation and purification confirm with analysis and research backs; These SOD mainly are divided into three types of Cu.Zn-SOD, Mn-SOD and Fe-SOD.

1.Cu.Zn-SOD: be the pale blue green, mainly be present in the eukaryotic cytoplasm, generally speaking, all have Cu.Zn-SOD in animal, plant, algae, microorganism and some the prokaryote body.Specifically, the main source of these enzymes has: people's (lung, brain, sperm, erythrocyte), horse (liver, erythrocyte), cattle (heart, liver, sperm, erythrocyte), sheep (liver, erythrocyte), pig (field, liver, kidney, erythrocyte), Canis familiaris L. (erythrocyte), chicken (liver, erythrocyte), duck (erythrocyte), goose (erythrocyte), arrow fish liver, the leaf of spinach, Plantula Brassicae chinensis leaf, Semen Phaseoli Vulgaris, Semen phaseoli radiati, Semen phaseoli radiati, straw, wheat, Mai Ye, Fructus Rosae Normalis, rose hip, Lentinus Edodes fungus, bakery yeast, beer yeast, slightly stick with paste neurospora etc.From these separate sources things, extract the Cu.Zn-SOD of purification; Except that indivedual character differed greatly, other all had similar character, but the Cu.Zn-SOD of these separate sources; Its pheron part is different with the kind source; Amino acid whose arrangement preface along or form some differences still arranged, and this species diversity has just caused the non-identity on its enzyme biology and the physicochemical property again.

The basic characteristics of Cu.Zn-SOD show on its molecular weight and molecular weight subunit, molecular structure, aminoacid composition, electrophoretic property, spectral characteristic and electron paramagnetic resonance spectrum (EPR) etc.This enzyme molecular weight; Except the enzyme molecular weight 135000 of from lung, purifying, and have outside 4 subunits, the molecular weight of other sources Cu .Zn-SOD is all about 32000; The dimer that each is made up of two equal subunits, and all contain atom Cu and an atom Zn on each subunit.Behind polypropylene vinegar amine gel electrophoresis, protein staining and SOD vital staining clearly show the active region band of three enzymes, also present three enzymatic activity district bands after the isoelectrofocusing; The isoelectric point, IP of these three kinds of components is respectively 5.2; 5.8,6.2, this enzyme has been located maximum light absorption at visible region 670nm (660-680nm); SOD concentrates in the absorption Spectrum characteristic of visible region, has reflected the optical characteristics of two copper ions in this enzyme prothetic group; (250～270nm) have located maximum light absorption, and at 280～320nm place certain light absorption are arranged, and the absorption Spectrum characteristic of its ultraviolet region is decided by certain content of amino acids in this enzymatic structure albumen at this enzyme of ultraviolet region 265nm.Moreover, this enzyme also has key property, i.e. a paramagnetism; Research and analysis prove that cupric has paramagnetism in the Cu.Zn-SOD molecule, just because of this reason; The Cu.Zn-SOD of oxidation state demonstrates distinctive electron paramagnetic wave spectrum, when the active center of Cu.Zn-SOD enzyme is researched and analysed, proves that the Cu.Zn-SOD activity not only is decided by pheron; But also be decided by prothetic group-Cu and the Zn in this enzymatic structure; Further research discloses this enzyme amino acid composition, all contains a color amino, a methionine and three tyrosine residues in each Cu.Zn-SOD molecule, and other is the highest with glycine content.This different aminoacids kind and content also are another important levy of this enzyme on molecular structure.

Need to prove that to the encode gene of Cu.Zn-SOD of the mankind, research worker is also furtherd investigate, the Cu.Zn-SOD that contains in verified higher mammal and the human Cytoplasm, its gene of encoding is positioned on the 21st pair of chromosome.

2.Mn-SOD: the Mn-SOD of separation and purification, be rose pink, be present in and remove in exo-erythrocytic all mitochondrions.Generally speaking, all have Mn-SOD in zooblast mitochondrion and the prokaryote body, specifically, the main source of these enzymes has: (1) eucaryotic organism comprises red algae, beer yeast, bakery yeast, Semen phaseoli radiati, Hepar Gallus domesticus, Hepar Mus, people liver, Cor Bovis seu Bubali etc.; (2) procaryote comprises escherichia coli (E.Coli.B), mycobacteria (mycobacterium lepraemurim, Mycobacterium phlei), bacillus stearothermophilus, spherical Rhodopseudomonas, the withered hot bacterium of streptococcus (fecal bacteria) (aquatic withered hot bacterium, have a liking for the withered hot bacterium of temperature), actinomycetes etc.; Though its source of these Mn-SOD is different, very similarly biological characteristics and physicochemical property are arranged, the different Mn-SOD in some source also exists some qualitative difference certainly.

The basic characteristics of Mn-SOD also mainly show on its molecular weight and molecular weight subunit, aminoacid composition, absorption spectrum, paramagnetic resonance spectrum method property and enzymatic activity.Result of study shows, derives from the Mn-SOD in the prokaryotic cell, and its molecular weight is mostly about 10000; Except the Mn-SOD from three kinds of cells purifications such as Mycobacterium phlei, withered hot bacterium, actinomyces is that the tetramer and mycobacteria are the trimer, other is by the dimer that two subunits are formed, and each subunit contains a manganese atom respectively; And derive from the Mn-SOD in the eukaryotic cell mitochondrion; Except the Mn-SOD that from red algae, extracts was an aggressiveness of two subunits compositions, all the other all were the tetramers that four subunits are formed, and its molecular weight is about 80000; See that from molecular weight subunit the Mn-SOD polypeptide chain is greater than Cu.Zn-SOD.The Mn-SOD of grain line body and algae or mushroom is in amino acid whose arrangement; Though not exclusively roughly the same, on the whole, similar; They and Cu.Zn-SOD are relatively; The most outstanding difference is that Cu.Zn-SOD only contains a tryptophan, and each subunit only has a tyrosine, and Mn-SOD contains more tyrosine and tryptophan.(473～480nm) have located maximum light absorption to this enzyme at visible region 475nm; But slightly variant with kind source difference, possibly be to decide, therefore by the state of charge of Mn-SOD is incomplete same; Its oxidation or reduction all can influence visible absorption character; Just for this reason, its extinction coefficient are different, and the optical absorption characteristics of performance is also different.Located similar general proteinic maximum light absorption peak at ultraviolet region 280nm (280-283nm); This ultra-violet absorption spectrum characteristic possibly be because this enzyme contains the cause of the tyrosine and the tryptophan of a great deal of, its paramagnetic resonance characteristic; Mn-SOD and Cu.Zn-SOD are different; Natural Mn-SOD does not have paramagnetic resonance spectrum, just shows its paramagnetic resonance spectrum characteristic after having only the Mn-SOD degeneration, and identical by line with the resonance wave spectrum of MnCl2; Mn is relevant in the activity of this enzyme and the prothetic group, but its stable pheron that depends on.

The genetics research result confirms, contained Mn-SOD in the mitochondrion in higher mammal and the people's cell, and its gene of encoding it is positioned on the 6th pair of chromosome.

3.Fe-SOD: this enzyme and Mn-SOD, Cu.Zn-SOD are the same, all are that SOD has isozyme, its common effect characteristics, but all catalysis

Disproportionation is H ₂O ₂And O ₂, but on many character and biological property, Fe-SOD is similar to Mn-SOD, and be not similar to Cu.Zn-SOD, and this maybe be different relevant with the source kind with the structure of three kinds of SOD isozymes.

It is faint yellow that Fe-SOD is, and such SOD exists only in the prokaryote.Specifically, these enzymes be mainly derived from prokaryote, as have a liking for the green bacterium of thiosulfate, wine and women-sensual pursuits chomophoric bacterium, desulfurization vibrio, huge tooth born of the same parents bacillus, escherichia coli B, mycobacterium tuberculosis, Paracoccus denitrificans, luminous bacillus (comprising luminous bacillus at interval), knitline algae, ovum shape pseudomonas, cloud tongue etc.; Its molecular weight size is between Cu.Zn-SOD and Mn-SOD, and is about about 38000, and most sources are that Fe-SOD is two dimers that equal subunit is formed; Its Fe content is relevant with the source of this enzyme, and the subunit that has has only the 1/2Fe atom, and the subunit that has but has 1 Fe; Because of it derives from some prokaryotic cells; The special more high animal and human's class of its race differs greatly, and is therefore, comparatively rare on Fe-SOD basis and clinic study center; Practical application is rare especially, and this paper does not give unnecessary details.But what particularly point out is; Fe-SOD catalysis

reaction is secondary response; Its rate constant value increases with pH and descends; And under physics and chemical factor influence, the active normal bigger variation that takes place of Fe-SOD should be noted in the application especially.

(3) biological effect of SOD

SOD is one type and has the active high-molecular biologic enzyme of heigh antioxidation; Its chemical nature remains protein; It has all characteristics of general enzyme, be equally living cells composition, produce the reactive protein that has the efficient catalytic function in vivo and in vitro by living cells.

In fact; Why the global SOD that becomes famous receives much attention and favors; Not merely be because it is the achievement that U.S. scientist finds and succeeds in developing; Also be not only because it is the composition of living cells; But dismutation reaction can take place through catalysis ultra-oxygen anion free radical in it, alleviate or eliminates ultra-oxygen anion free radical

and metabolite or derivant inductive oxidation or peroxidation tackle the damage and the harm of body.Particularly; SOD is as the unique enzyme-specific that can effectively remove ultra-oxygen anion free radical

in the body, and the cofactor of the nonprotein part in its structure or prothetic group are metal ion and exist with the metallo-chelate form.This particular structural of SOD just; Make it to because the inductive aging of ultra-oxygen anion free radical , inflammation, tumor, Radiation sickness, autoimmune disease, cardiac-cerebral ischemia are poured into relevant diseases such as syndrome, cataract again and had good prevention effect, and SOD all has certain auxiliary curative effect to some sub-health status, cancer chemotherapy and radiotherapy, digestive system disease (chronic gastritis, atrophic gastritis etc.), respiratory system disease (asthma, bronchitis etc.), dermatosis (contact dermatitis, solar dermatitis, chloasma etc.), burn and reparation etc.Do simple an introduction in the face of the biological effectiveness of SOD down.

1, the relation of SOD and inflammatory reaction: inflammation is a kind of protective response after body receives the external microbe invasion.Phagocyte plays an important role in inflammatory reaction; They produce respiratory burst in phagocytosis or irriate; Consume oxygen; Activation pbosphohexose branch road; Discharge a large amount of

and OH isoreactivity oxygen, thus coup injury endotheliocyte, erythrocyte, fibroblast, platelet and sperm, and leukocyte itself also can be by the oxygen free radical injury of it oneself generation.

As with the inductive pleuritis of SOD treatment carrageenin, can reduce hydrothorax and leukocytic accumulation.SOD can also suppress arthritis, reduces the deposition of IgG at glomerule, disturbs the accumulation of leukocyte at glomerule inflammation position.SOD can suppress the change of the organ permeability that acute tracheitis that xanthine/xanthine oxidase causes causes, and reduces the accumulation of polymorphonuclear leukocyte in pulmonary, and SOD is used to treat inflammation and has good application prospects.

LPS is the main stimulus object of bacterial inflammation, can induce the expression of many inflammatory factors (TNF-α, IL-1 etc.).Lu Andesi people such as (Loenders) utilizes the lps injection rat to cause pneumonia, finds that SOD combines with the epithelial cell of macrophage, neutrophilic granulocyte and trachea associated lymphoid tissue.At this moment, the SOD in the blood utilizes neutrophilic granulocyte as carrier, and it is transported to inflammation part, and SOD has the effect that phorbol ester is induced neutrophilic granulocyte and macrophage release free radical that suppresses.This shows, in conjunction with neutrophilic granulocyte and the macrophage of SOD have the antagonism active oxygen, remove the autoprotection function of free radical.

Similar result of study also shows; SOD also can discharge cytokine through suppressing macrophage; Weaken the neutrophilic granulocyte inflammation of LPS mediation; Through weakening the migration of Langerhans cell to lymph node, significantly suppressed the contact hypersensitivity of scytitis, demonstrate anti-inflammatory, anti-infectious function that SOD has.And SOD also can weaken the acute inflammation that causes because of hyperoxia.

2, the relation of SOD and nervous system disease: FR induces oxidative stress and damage thereof in many nervous system disease, to play a crucial role, and the normal antioxidation mechanism (comprising the effect of SOD) of brain maybe be very important to preventing these diseases.Zooscopy confirms that SOD can regulate the concentration of outer free radical of brain cell and peroxide, improves the cognitive function of brain.

For the brain atrophy patient, free radical (FR) or reactive oxygen species (ROS) can directly damage macromolecular substances in the brain cell, thereby cause cerebral tissue cell death widely.And at cerebral tissue gradually in the process of atrophy, outside antioxidation mechanism (comprising the effect of SOD) can make the effect of free radicals damage of continuous generation and blocked.And SOD can play a significant role on the neural degree of necrosis after the minimizing brain atrophy.

There are dependency in SOD mutant and familial amyotrophic lateral sclerosis (AIS), and the SOD of sudden change can form nitric acid albumen with the pernitric acid reaction, finally causes the selectivity degeneration of motor neuron in the lateral sclerosis of spinal cord (AIS).Treatment to lateral sclerosis of spinal cord (AIS) can be adopted immunosuppressant, neurotrophic factor etc., but antioxidant SOD should be the most potential a kind of medicine.

To parkinson disease (PD) early stage with patients with terminal blood in active detection of SOD find that parkinson disease (PD) SOD in late period is active significantly to descend, the damage of free-radical oxidation mechanism possibly be parkinson disease (PD) formation reason.And parkinson disease (PD) show that iron content changes in the brain, and mitochondrial function is impaired, and the anti-oxidation protection system weakens (minimizing that mainly is SOD and glutathion), so early stage SOD uses, should be a kind of very potential ancillary drug.

3, the relation of SOD and cardiovascular system diseases: SOD is expression in blood vessel wall, and mainly by the VSMC secretion, it is the topmost existence form of SOD in the human aorta.SOD in the blood vessel mainly is present in the extracellular matrix, and other has sub-fraction in cell surface and blood plasma.

The whole process, particularly oxygen-derived free radicals that the inductive oxidative stress of superoxide radical plays an important role in the generation of atherosclerosis (AS), evolution and superoxide radical

has also been participated in cardiovascular disease generation, development is in the generation of the oxidative modification thing of low density lipoprotein, LDL (LDL) and play a part very crucially for the formation of atherosclerosis (AS).SOD can remove superoxide radical

and suppress or reduce MDA to form; Alleviate peroxide injury to vascular endothelial cell; Thereby the protection vascular system is avoided the damage of oxygen-derived free radicals, prevents the formation of atherosclerosis (AS).

Research also shows, the expression decreased of SOD during coronary heart disease, and active reduction of SOD can cause the patients with coronary heart disease blood vessel inner skin cell function disorderly.And; When atherosclerosis (AS); The generation of superoxide radical in the blood vessel

is showed increased also, and the superoxide radical that increases has simultaneously also disturbed neural adjusting to vascular tone.Therefore, improve SOD in the blood vessel expression can, to preventing the highest cardiovascular disease of human M & M significant.

In addition; SOD also can suppress the surplus balance in vivo of

and nitric oxide (NO), stops the generation and the development of atherosclerosis (AS).Simultaneously, adjusting SOD and and the active balance of NO are also significant to preventing and treating atherosclerosis (AS).

4, the relation of SOD and ischemical reperfusion injury:, when restoration of blood flow is logical again, cause the state of an illness further to worsen on the contrary in remissions such as bypass surgery coronarius, thrombolytic art, variant angina pectoris, coronary vasospasms.This be because, behind the myocardial ischemia-reperfusion

Producing increases, and passes through the lipid peroxidation injury of myocardium cell membrane of film, thereby causes Ca ²⁺Get in a large number and produce arrhythmia in the cell. also energy loss blood trouble endothelial cell and myocardial cell inner mitochondria film, endoplasmic reticulum and lysosome membrane simultaneously; Thereby destroyed the homergy of body, formed the myocardial ischemia-reperfusion syndrome.At this moment, if just during this time inject the symptom that an amount of SOD can effectively alleviate " myocardial ischemia-reperfusion syndrome ", and if before recovering blood reperfusion, use SOD, even might prevent the appearance of " myocardial ischemia-reperfusion syndrome ".

Moreover; When myocardial ischemia-reperfusion; Because producing,

increase; Approach through the mediation of oxidative stress damaging action causes cardiac function to worsen; The existence and the exogenous SOD that gives of inherent antioxygen material possibly reduce the myocardial necrosis degree, reduce myocardial infarction area, and the reparation behind cardiac muscle and the blood vessel injury and function kept have positive effect.SOD except that being applied to the myocardial ischemia-reperfusion syndrome, also can be used for before the tremulous pulse ischemia-reperfusions such as brain, kidney, liver, small intestinal or refilling process among.

5, the relation of SOD and organ transplantation: organ transplantations such as liver, kidney, heart, skin; Also exist the situation of recovering hemoperfusion; Therefore; SOD also can be applicable to the syndrome that occurs in the operation processs such as preservation and transplanting, the replantation of amputated limb, shaping, the beauty treatment of organs such as liver, kidney, heart, skin, can alleviate the damage that free radical causes like this, guarantees the success of transplant operation.

Through nanotechnology; Sod gene is imported in the law during ischemia damage model mice body; Make the interior SOD of liver increase nearly 10 times;

eased down to normal level; The alanine aminotransferase level reduces, and hepatocellular ischemical reperfusion injury significantly reduces, this to liver transplantation after the protectiveness treatment significant.

6, the relation of SOD and pulmonary disease: SOD is free radical scavenger and a polyphenoils main in the lung tissue.Human SOD mainly is present in extracellular matrix, trachea and the alveolar gap of blood vessel.And too much superoxide radical

generation of pulmonary reaches and SOD balance destruction, can cause the generation of many pulmonary disease.Therefore, the adjusting of antioxygen material such as SOD and replenish disease in control pulmonary and the many tracheal diseases very important.

The important medium of superoxide radical

when being acute lung injury, therefore polyphenoils such as increase or the raising SOD of pulmonary can play effective protective effect to

inductive acute lung injury.SOD also has the certain protection effect to some other diseases of pulmonary, can significantly protect it to avoid the pulmonary fibrosis damage that bleomycin causes like people SOD at the orientation expression of Mus pulmonary.

The scale-model investigation of adult's respiration inhibition syndrome (ARDS) confirms; Excretory too much

the isoreactivity oxygen species of neutrophilic granulocyte has caused injury of lung, and makes that sb.'s illness took a turn for the worse.And in time, reasonable supplement SOD can effectively alleviate or reduce this type of injury of lung.Emphysema patient also can use SOD, but should when the pathological changes initial stage, the elastance of lung fiber suffered damage as yet, not use, and curative effect is better.

7, SOD and autoimmune disease (auto immune disease; AID) relation: the pathogenesis of various dissimilar autoimmune diseasees respectively has its different features, but

prostaglandin and the hydrolase that generated by macrophage, monocyte and neutrophilic leukocyte play an important role on pathological changes.Moreover; The Oxidation and the peroxide injury of superoxide radical

; Can cause protein denaturation and protein synthesis mistake, thereby form autoimmune disease.Zoopery confirms; The scavenger of SOD and other oxygen-derived free radicals can suppress the chronic disease process of autoimmune disease; SOD can prevent and treat all kinds of autoimmune diseasees through to scavenging action.Can be used for treating lupus erythematosus, scleroderma, dermatomyositis etc. like SOD, demonstrate certain curative effect.Particularly for rheumatoid arthritis patients, as acute stage pathological changes do not form preceding use, its curative effect effect will be better.And; Have among the human lupus erythematosus patients serum and a kind ofly can make destructive Jie of chromosome split the factor; The existence of this factor occurs being proportionate with

, so better with the SOD therapeutic effect.

8, SOD and Radiation sickness and radiation proof relation: radiation can cause the interior hydrone generation of body too much

and the chain reaction of these

that too much generate initiation free radicals; Cause body that the reaction of

is increased the weight of, even form Radiation sickness.SOD can be used to prevent or treat the diseases such as cystitis, dermatomyositis, lupus erythematosus and leukopenia that cause owing to radiotherapy, and to receiving the personnel of ionizing radiation, also injectable SOD is as preventive measure.

9, senile cataract: the formation of senile cataract also mainly is the result of superoxide radical to the crystallin oxidation.Before senile cataract forms, perhaps in that to suffer from senile cataract early stage, even before getting into the geratic period, promptly begin often to take some antioxidants, suitably replenish in other words and inject a certain amount of SOD, have certain prevention and initial stage therapeutic effect.If in case form cataract, then need surgical removal, fail to respond to any medical treatment with SOD.

(4) structural modification of SOD

SOD is a kind of special metalloenzyme; The substrate of its effect is the formation of ultra-oxygen anion free radical

ultra-oxygen anion free radical and a lot of diseases and substantial connection is arranged; Its special role mechanism receives the common concern of medical circle.As medicinal SOD, though still exclude pharmacopeia at present, existing abroad commodity selling, its commodity are Orgotein, Ormetein, Onotsein, Palosein, Paroxinorn.The development of the medicinal SOD preparation of compatriots and production develop rapidly, and some SOD preparations occur in succession, and through numerous scientists' cooperation, its basic medical research and clinical trial also get into certain phase.But,, poor with biomembranous affinity because free SOD half-life in blood is short; Problems such as certain antigenicity are arranged, to a great extent limit its extensive use clinically, for this reason; Research worker is stepped up research again both at home and abroad, and its molecular structure is transformed and modified.

1, Polyethylene Glycol-SOD (Polyethylene glycol-SOD, PEG-SOD)

Because free SOD half-life in blood is short, metabolic rate is soon in order to improve its clinical efficacy; People such as Mc.Cord developed the PEG-SOD derivant in 1979; They make PEG combine with free SOD with chemical method; Process the PEG-SOD derivant,, confirm that PEG-SOD extended to 24-35 hour than the half-life in the F-SOD blood by original 5-6 minute through assay determination.Although PEG-SOD has strided forward major step aspect the half-life in prolonging blood, find that the holdup time of SOD and curative effect are not in full accord in the blood in research and the clinical practice, one of reason is the affinity problem of SOD and target cell.Because the affinity of PEG-SOD and target cell is low, curative effect is undesirable, so PEG-SOD fails to be promoted the use of clinically.But PEG-SOD is indubitable for the raising of SOD stability.

2, liposome-SOD (Liposome-SOD, L-SOD)

It is in the liposome of main component that people such as nineteen eighty-two Michelsion imbed SOD with phospholipid, processes liposome-SOD derivant.Through analyzing and detecting and find that L-SOD can operate in the blood circulation well attached on the erythrocyte membrane; Particularly SOD is to after going in the liposome to form L-SOD; It is not easy to decompose in blood, and molecular weight brings up to 9.6 ten thousand from 3.3 ten thousand, and the half-life extends to 7～24 hours.The author points out, the composition of the liposome through changing L-SOD can also obtain different organs is had the different SDO derivants of obvious tropism.For example, cationic liposome-SOD tends to lung more, and after anionic property liposome-SOD tended to liver, free SOD intravenous injection more, major part concentrated on kidney, L-SOD each internal organs that then can extensively distribute.The application of L-SOD on the animal pathological model is more, and like fibrosis cicatrix due to radiation, intramuscular injection just is clearly better for twice weekly.Usefulness low temperature such as Chan (50 ℃) cause rat brain infringement (edema; Vascular permeability increase etc.); If before K cryogenic treatment, inject free SOD and L-SOD respectively to rat, the result finds, the rat of injection L-SOD; Brain damage and edema and vascular permeability increase obviously alleviate, and free SOD is then invalid in injection.All obtain satisfactory effect in European and Japan with some hepatopathy of L-SOD treatment, autoimmune disease, rheumatism, kawasaki disease, Behcet disease and Crohn disease etc.In addition, L-SOD and some anticarcinogen and usefulness can reduce its side effect, guarantee normal tissue cell.Bleomycin A5 for example, amycin, anticarcinogens such as daunomycin have and in cancerous cell, produce the mechanism that active oxygen destroys the DNA chain.In normal cell, the enzyme that decomposes bleomycin A5 is arranged, thereby it is less to Normocellular murder by poisoning, but myocardial cell lacks the enzyme that decomposes amycin and daunomycin, thereby they are big to the toxicity of cardiac muscle.Owing to getting into the normal cell that comprises myocardial cell, L-SOD is difficult for getting into cancerous cell, if with L-SOD and amycin, and daunomycin, bleomycin A5 merges use, both can bring into play its antitumaous effect, can reduce again cardiac muscle and Normocellular serious side effects.

Vein injects when dissociating SOD, and SOD is many attached to the cell outside, is difficult to get in the cell.And during intravenous injection L-SOD, L-SOD can penetrate in the cell, on the sidewall attached to cell.The free SOD in the attached cell outside; Can not bring into play scavenging action and Detoxication to

in the cell; Can not bring into play Normocellular protective effect; On the contrary, also can influence and weaken cancer therapy drug destroying of cancerous cell subtracted effect; And in the entering cell,, not only can reach free radical elimination effect in the born of the same parents, and can not disturb cancer therapy drug that cancerous cell subtracted effect extremely attached to the L-SOD of cell inwall.Thereby, normal cell is protected, make cancerous cell be able to subtract extremely.Also have the author to propose, vein injects when dissociating SOD, and blood level possibly of short durationly raise significantly; Make free radical and free radical resisting concentration unbalance, both be unfavorable for the body homergy, also be unfavorable for the anti-mechanism of unloading of body; And behind the L-SOD entering cell; Discharge free SOD on a small quantity lentamente, can not make concentration rising suddenly in the blood, the defense mechanism of body is not had influence.

3、SM-SOD

SM-SOD is meant the derivant (Stylenemleimide-SOD or Polytyrene-Comaleic acid-SOD write a Chinese character in simplified form SM-SOD) that styrene maleic amide and SDO form.During making, with maleic acid and styrene reaction, generate polystyrene maleic acid (SM) earlier, the latter is connected with SOD again, can form SM-SOD.Generally speaking, the Lys residue on the 1 molecule SOD molecule is connected with 2 molecule SM and amido link, forms SM-SOD.SOD molecular weight 3.3 ten thousand is not if be made into SM-SOD, after the intravenous injection; This hydrophilic protease is removed by kidney very soon, because, the behavior of undergraduate course class medicine in blood; The molecular size, form, charge property, the hydrophobicity that depend on them, the fine structure on surface and kidney are removed.Generally speaking, diameter is difficult for being removed by kidney the above person of 8nm.Albumin diameter 7nm, molecular weight (MW) about 6.6 sub-very much ovalizes are electronegative, with the protein of kidney basement membrane solution acid the electrostatic repulsion effect arranged, and renal function is just often difficultly removed by kidney.Yet bilirubin, fatty acid, carbamide equimolecular quantity are little more than the SOD molecular weight, but are difficult for being filtered by kidney; Its reason is that these hydrophobic type aniones mainly become complex with albumin bound in blood, has the scholar that this principle amplification is enlarged and is used for hydrophilic enzyme drug SOD, and they select SM conduct and albumin and the bonded part of SOD; SM is combined to form the SM-SOD derivant earlier with SOD; In the quiet injection body of SM-SOD, owing to SM can combine with albumin rapidly in vivo, therefore; After SM-SOD is injected into blood, the rapid and albumin bound of SM-SOD in blood.Generally whether molecule SM-SOD and molecule albumin bound, the SOD derivant of this albumin parcel can be by protease hydrolysis, and antibody contacts with it with leukocyte also difficulty, and this has not only protected the SOD enzyme not to be hydrolyzed destruction, and has reduced its antigenicity.SM-SOD-albumin complex is difficult for being removed by kidney, and the half-life extends to 6 hours in the blood.In addition, SM itself is protonated or become non-charged species, and fat-soluble raising is easy to combine with biomembrane.Molecular surface has the SOD (SM-SOD) of hydrophobic organic anion in microcirculation, can assemble to the low diseased region of pH effectively; It is the focus tropism; This gathering is reversible, and after pathological changes changed, SM-SOD returned in the blood and albumin bound again; Form the white egg complex of SOD-SM-again, continue performance useful physiological function.The Japan scholar crosses limit letter and waits the people to report; The rat of myocardial ischemia-reperfusion; Ventricular premature beat type arrhythmia phenomenon appearred after several seconds, if before ischemia-reperfusion respectively to free SOD of rat intravenous injection and SM-SOD, the result finds; Arrhythmia does not appear in the rat of intravenous injection SM-SOD, the free then nonreactive rhythm of the heart effect of SOD of injection.This explains that also there are notable difference in SM-SOD and F-SOD on action effect.

4, enzymology-SOD (Acyl-SOD, simple Ac-SOD)

The SM-SOD half-life in vivo obviously prolongs, and certain focus tropism is arranged, and this is the strong point that SM-SOD uses in vivo.But the blood vessel in the tissues such as cornea, binding film, skin seldom or do not have, and provides the action effect of SOD little through intravasation and blood circulation to them, and SOD is electronegative lyoenzyme, with its eye dripping or be coated in skin, is difficult to be retained in diseased region.For this reason, Japanese scholar An Dong unit is just waiting the people to research and develop enzymology-SOD, hydrophobic fat acid is attached on the Lys residue of SOD molecule, makes it to form and the high Ac-SOD derivant of the double-deck affinity of cell membrane lipid.The SOD activity that Ac-SOD is active and environmental pollution is treated is roughly the same, but the affinity of Ac-SOD and film is strong, thereby can gather close the combination with cornea binding film, skin etc.Rabbit cornea inflammation or ulcer with Ac-SOD treatment endotoxin induced have obviously suppressed leukocyte infiltration, have delayed the development of pathological changes.And under similarity condition, adopt free SOD then not have this effect.

5, other modification SODs

Ctelan combines cattle Cu.Zn-SOD with glutaraldehyde, in blood again with albumin bound, forming molecular weight is 200000 dalton left and right sides derivants, the half-life extends to 6-8 hour in the blood.Gong Tian etc. combine the Mn-SOD of Serraria (Serratieae) with the protease of this bacterium, and then form α M-SOD derivant with people's α-macroglobulin, and the antigenicity of Serraria Mn-SOD is descended, and can keep its activity for a long time in the blood.

(5) SOD external preparation for skin situation

Descend since the good equality people in the little storehouse of Japanese scholar confirms in the tissue of dermatosis affected part that SOD is active, after LPO content raise, in recent years, department of dermatologry was specially learned this was carried out a large amount of research and discussion, existing at present great mass of data report, and various skin is sick relevant with the SOD activity.

The research worker of Ningxia Medicine College, the whole blood SOD that has measured 4 patients and 21 members of family in two kinds of xeroderma pigmentosums (XP) family is active.The result shows: (1) XP patient's whole blood SOD activity is 1114.3 ± 80.0U/gHb; (2) heterozygous is 1224.9 ± 63.6U/gHb; (3) normal control group SOD activity of the same age is 1532.9 ± 31.00U/gHb and 1341.7 ± 78.7U/gHb.Patient's whole blood SOD activity is compared with matched group of the same age, and significance difference (P＜0.05) is arranged.Heterozygous is compared with matched group of the same age, and difference does not have significance meaning (P＞0.05).But see from numerical value; Its SOD activity really is lower than matched group of the same age; This group results suggest: SOD is active low in the morbidity of (1) XP and the patient's body;

is through suppressing DNA repairase and dna polymerase activity; Make DNA duplicate and repair process produces mistake, thereby and cause a series of clinical symptoms with DNA excision repair-deficiency person combined effect.(2) SOD-1 is active obviously low, and heterozygous SOD-1 activity is on the low side, show the DNA repair enzyme genetic relevant with primary disease maybe with SOD-1 gene linkage, be positioned on No. 21 chromosomes relevant.Therefore the author thinks, explore and use SOD preparation for treating XP patient, and possibly be a kind of comparatively effective method.

The author studies different SOD compound enzyme content to antihistaminic experiment, and selects chlorphenamine as the positive control medicine.The result finds, finite concentration and active SOD composite enzyme solution and chlorphenamine all can be to antihistaminics, and with similar to antihistaminic circular ten minutes, this shows that the application of SOD in the external preparation for skin medicine has certain theory and experiment basis.

About the diadermic problem of SOD; The author once selected three kinds of experimental animals (white mice, Cavia porcellus, rabbit) to make an experiment specially; One side is a control sides, smears not contain the SOD preparation: the same position of opposite side is a control sides, smears the same substrate that contains the SOD preparation; After one week, bark fetching undertissue measures the SOD activity respectively.The result shows that the active highly significant of the test side subcutaneous tissue SOD ground that smears the SOD preparation is higher than matched group skin (P＜0.01), and a kind of result of experimental animal shows same rule.

The research worker of The 2nd Army Medical College basis Isotope Lab is also carried out corresponding transdermal test in order to observe the Transdermal absorption situation of SOD-I, and they smear for mouse back skin with 125I labelling human SOD (125I-Hsod-1) with the preparation of the aqueous solution of phosphate buffer; The result finds; 1h behind the skin, blood can be measured radioactivity 125I, and every ml blood counting reaches 500～600cpm; Except that individual point, the radioactivity of blood cell all is higher than the radioactivity of blood plasma; The radioactive substance of each histoorgan also can be measured after skin is coated with 1h.So inference, SOD-1 can partly get into body through skin.

In sum, SOD has medical value widely, but because natural SOD less stable, inactivation is difficult for preserving easily, and has limited the application surface of SOD.In recent years, more and more widely, its main cause comprises two aspects to SOD in the application on the skin care item, the one, and cosmetics additive can prevent skin aging, plays skin effect; The 2nd, the prevention and treat relevant dermatosis.These have at home and abroad obtained to use widely, and many superior cosmetics have all added SOD, and it is processed facial film, milk, cream.Domestic have a not product such as old frost of big precious SOD frost, violet SOD frost and SOD.But the stability of SOD still becomes a big bottleneck of application; In some cosmetics for external use, mainly be through drug administration carrier, optimize the stability that preservation condition is come enhancement of SOD at present, avoid its inactivation; As use SOD external-use liposome cream treatment mottle clinically, obtain very big success.Zhang Huailiang adopts lecithin, cholesterol to prepare the carrier of liposome as SOD, and research shows that the SOD activity can preserve more than 6 months in cream.

Carry out the research work of the chemical modification of a large amount of SOD in China, and obtained good result.Chemical modification be exactly on molecular level the group to SOD be connected with some biocompatibility macromole through artificial method, thereby reach the character that changes SOD, improve its half-life, reduce immunogenicity, weaken the hydrolysis of protease.Such as SOD with monomethyl Polyethylene Glycol (mPEG) modified; Have less, the less enzyme degradation of stronger biological activity, loss of activity still less, longer half-life, blood concentration fluctuation, less immunogenicity and antigenicity, less toxicity, better dissolubility, etc.

Though to a certain extent, improved the stability of SOD, remedied a lot of defectives in the SOD application through chemical modification.But in the application of SOD percutaneous drug delivery, the skin permeation rate of SOD is too low, can only partly get into body through skin, is difficult to reach the effect of treatment.So on using, the SOD permeability remains the problem of a key on the external preparation for skin.

Summary of the invention

The objective of the invention is, propose a kind of have stable good, but can protect the SOD biological activity lastingly and improve Pegylation superoxide dismutase (mPEG-SOD) nano-emulsion and preparation method thereof of a kind of direct cutaneous external of SOD external preparation for skin permeability.

Because the nano-emulsion carrier belongs to the nanoscale emulsion, the first, can be wrapped in the SOD of modified in the nano-emulsion, further improve the stability of SOD composition; The second, the nano-emulsion carrier improved functional component dispersion, make functional component be present in the system with nanoscale, improved the transdermal penetration property of SOD at the external preparation for skin product, maximization improves the utilization rate of functional component.The 3rd, the SOD that uses Pegylation is as the external functional component, and combining nano breast carrier formation mPEG-SOD nano-emulsion, has very much the external advantage.

But the purpose that realizes foregoing invention provides a kind of Pegylation superoxide dismutase (mPEG-SOD) nano-emulsion of direct cutaneous external.

1, technical scheme of the present invention

(1) but Pegylation superoxide dismutase (mPEG-SOD) nano-emulsion of a kind of direct cutaneous external of the present invention form by the following weight parts proportion raw material:

(2) but Pegylation superoxide dismutase (mPEG-SOD) nano-emulsion of a kind of direct cutaneous external of the present invention form by the following weight parts proportion raw material:

(3) preferred weight part proportioning of the present invention is:

(4) but Pegylation superoxide dismutase (mPEG-SOD) nano-emulsion of a kind of direct cutaneous external of the present invention, the mass concentration of SOD aqueous solution is 0.03% in its prescription.

(5) but another object of the present invention provides a kind of method for preparing of Pegylation superoxide dismutase (mPEG-SOD) nano-emulsion of direct cutaneous external, may further comprise the steps:

1) mPEG-SOD with recipe quantity is dissolved in distilled water, and this solution is as the mPEG-SOD aqueous solution, subsequent use;

2) take by weighing consumption according to each component ratio in the prescription, subsequent use, the weight vitamin E of recipe quantity is dissolved in the isopropyl myristate, subsequent use;

3) with the sad capric acid polyethyleneglycol glyceride of recipe quantity, polyglycereol-3-dioleate, the mPEG-SOD aqueous solution of fully mix homogeneously, and adding recipe quantity adds the liquid for preparing in (2) then;

And with whole system under 25 ℃ of conditions of room temperature or in the natural room temperature, start the time constant-temperature magnetic stirring apparatus, with 200rpmmin ^-1The rotating speed magnetic agitation, fully mixing is transparence liquid until whole system, but is a kind of Pegylation superoxide dismutase (mPEG-SOD) nano-emulsion of direct cutaneous external;

4) but Pegylation superoxide dismutase (mPEG-SOD) nano-emulsion of this a kind of direct cutaneous external is sub-packed in rapidly among the lucifuge glass container of different size, add a cover packing, and place 4 ℃ of airtight preservations of refrigerator to get final product rapidly.

2, beneficial effect of the present invention

But Pegylation superoxide dismutase (mPEG-SOD) nano-emulsion of a kind of direct cutaneous external of the present invention in sum is compared with prior art, has following beneficial effect:

(1) SOD is after mPEG modifies, and promptly after the PEGization, its main biological function remains unchanged; Also has following advantage: prolonged the half-life of SOD, increased the water solublity of SOD, reduced malicious pair and make sheet; Reduce immunogenicity, improve medicine stability in vivo and in vitro.On this basis, but the present invention uses Pegylation superoxide dismutase (mPEG-SOD) nano-emulsion of a kind of direct cutaneous external of nano-emulsion technology preparation.Strengthened the advantage of mPEG-SOD further.

(2) but Pegylation superoxide dismutase (mPEG-SOD) nano-emulsion of a kind of direct cutaneous external of the present invention; The nano-emulsion particle diameter is little; Superoxide dismutase (SOD) dispersion that is contained improves; In external preparation for skin, can promote the Transdermal absorption of superoxide dismutase (SOD), solved the low problem of superoxide dismutase (SOD) preparation capable of permeating skin absorbance.

(3) but the present invention prepares a kind of Pegylation superoxide dismutase (mPEG-SOD) nano-emulsion of direct cutaneous external; MPEG itself modifies the SOD structure; Improved the stability of SOD; The special construction of adding nano-emulsion is wrapped in mPEG-SOD albumen in the nanometer nodule in the breast, does not contact with external environment.Nano-emulsion carrier itself has necessarily antibiotic, bacteriostasis in addition, just can not prevent product rotten so need not add chemical preservative, and this has further improved the stability of mPEG-SOD.

(4) but Pegylation superoxide dismutase (mPEG-SOD) nano-emulsion of a kind of direct cutaneous external of the present invention preparation is preparation under temperature conditions Celsius about 25 ℃, therefore have important value for activity and the content of protecting the SOD functional component.The present invention can keep its biological activity and biological action to greatest extent.

(5) this product preparation process is simple, and practical operation is easy, preparation efficiently fast, does not need special instruments and equipment, and energy savings and cost are free from environmental pollution again, are fit to industrialization production.

3, preparation points for attention of the present invention

(1) product used container of when preparation must strictly according to the rules clean and sterilization, and preferably adopt strong acid to soak back water and dash, clear water washes clean again, dry with distilled water immersion the back, places after the baking oven high-temperature sterilization drying subsequent use again; In addition, when carrying out the product preparation, used container all will be paid special attention to prevent to contain impurity or pollutions such as metal ion and microorganism, in order to avoid influence product quality and stability.

(2) SOD is the private present of the auspicious professor in Dinke, therefore, modifies and should in time link up with the fourth professor during preparation and get in touch in early stage, and is synthetic and prepare so that this seminar can accomplish the SOD of q.s.

(3) prepare in strict accordance with above-mentioned dosage and method.When carrying out this product preparation; Should add according to above-mentioned flow sequence, its additive capacity must require accurate, and; In the interpolation of other raw material; Could add a kind of raw material down after must waiting a kind of raw material of adding fully to dissolve again,, influence the preparation of whole nano-emulsion system in order to avoid occur muddyly.

(4) when preparation external mPEG-SOD nano-emulsion, need select and definite S/C mass ratio (km value); And definite S/C mass ratio (km value) 1,2,3,4; Be to confirm at last after carrying out the biphase binary liquid test of titration water oil and pass through pseudo-ternary phase diagram analyzing; Only, be only comparatively ideal km value confirming and calculating the km value that obtains maximum nano-emulsion zone.Above-mentioned method for preparing confirms that through testing Km value has been 3, but actual when carrying out the product preparation, surfactant (S) and cosurfactant in this ratio still must be after mixing also will be more abundant mixing, prepare effect and quality in order to avoid influence.

(5) because the proportion or the density different relationships of liquid solution; When carrying out the product preparation, the capacity of selected preparation container must be a bit larger tham the product volume of actual preparation, like preparation 100g product; Need to select the preparation container of 150-200mL; So both be convenient to each composition and fully mixed and dissolve, and also be convenient to ultrasonic concussion or stirring and evenly mixing, and be unlikely to overflow outward.

(6) though blank nano-emulsion itself has the characteristics of certain bacteria growing inhibiting; Other interpolation antiseptic is not arranged, so when carrying out the product preparation, except that the preparation container must carry out strictness cleaning and sterilization in this formula for a product; Also should be during concrete the preparation in strict accordance with rule of operation; Under aseptic condition and according to the sterile working, require to operate as far as possible, avoid this nano-emulsion sample to be polluted, influence product quality.

(7) sample of preparation completion must be packed with lucifuge, and places shady and cool dry ventilation lucifugal place to preserve as far as possible, keep fire away or thermal source.In addition, after product uses at ordinary times, need not place low temperature or refrigerator to preserve, but must seal tightly, be positioned over natural room temperature and keep in Dark Place and get final product.

(8) these article possibly have zest to skin, please suspend use immediately as using the position significant reaction to occur; In actual use, be sure not these article are done ophthalmic; Skin is injured, have wound or redness must not use, use the back red and swollen if any continuing, and should stop using; Baby and child must not use; The skin sensitivity person should do the skin sensitivity property testing before using, and tests completely without irritative response and can use.

(9) SOD is a peptide matters, so-20 ℃ of left and right sides cold preservations of these solid feed needs are comparatively suitable, face with facing and join, and each amount of preparation should not be too big, behind the preparation SOD aqueous solution, joins existing usefulness, as too many or too much for use at present, should place 2-8 ℃ of refrigerator short-term to preserve as far as possible.

(10) the mPEG-SOD nano-emulsion muddiness can occur after putting into refrigerator, but recovers can form nano-emulsion voluntarily after the room temperature, does not influence use.

Description of drawings

But Fig. 1. a kind of preparation technology's flow process and technology path of Pegylation superoxide dismutase (mPEG-SOD) nano-emulsion of direct cutaneous external.

The specific embodiment

Further specify product of the present invention and method for preparing below in conjunction with the practical implementation example

Embodiment 1: but a kind of method for preparing of Pegylation superoxide dismutase (mPEG-SOD) nano-emulsion of direct cutaneous external

But a kind of preferred weight part proportioning of each raw material of Pegylation superoxide dismutase (mPEG-SOD) nano-emulsion of direct cutaneous external is:

But a kind of concrete method for preparing of Pegylation superoxide dismutase (mPEG-SOD) nano-emulsion of direct cutaneous external (100g is an example with preparation):

1. mPEG-SOD aqueous solution preparation: 0.015g mPEG-SOD is dissolved in the 49.985g distilled water.With this solution as water (I) 50g;

2. accurately take by weighing the isopropyl myristate of 7g with analytical balance, place another, and the vitamin E that takes by weighing 3g is dissolved in wherein, with clear oil phase (II) 10g that is labeled as of marking pen through among 50mL conical flask of decontamination;

3. according to the sad capric acid polyethyleneglycol glyceride (S) that has designed in advance: the ratio of polyglycereol-3-dioleate (C)=3: 1; Accurately take by weighing the sad capric acid polyethyleneglycol glyceride of 30g and 10g polyglycereol-3-dioleate respectively, and the two raw material is placed among the clean conical flask of 100mL of the 3rd process decontamination; Add a cover, and this conical flask is placed rapidly on the liquid flash mixer, open the liquid flash mixer; And it is transferred to the II shelves, fully mix homogeneously makes it to form emulsifying agent/co-emulsifier (S/C) mixture; Behind the 3-5min; The closing liquid flash mixer takes off conical flask, and with clear S/C mixture (III) 40g that is labeled as of marking pen;

4. according to oil phase (II): S/C mixture (III): the ratio of water (I)=10%: 40%: 50%, phase (I) 50g that fetches water respectively earlier, S/C mixture (III) 40g place among the 4th the clean conical flask of the 200mL through decontamination;

5. after above-mentioned water (I), S/C mixture (III) two liquid phases fully being mixed; Again under 25 ℃ of conditions of room temperature or in the natural room temperature; Put it into and shake in the ultrasonator also about ultrasonic 2min; Perhaps under 25 ℃ of conditions of room temperature or in the natural room temperature, start the time constant-temperature magnetic stirring apparatus, with 200rpmmin ^-1Rotating speed magnetic agitation 5min, then, in this container, directly add oil phase (II) 10g again; And with whole system in ultrasonator about ultrasonic 5min; Perhaps under 25 ℃ of conditions of room temperature or in the natural room temperature, start the time constant-temperature magnetic stirring apparatus, with 200rpmmin ^-1Rotating speed magnetic agitation 20min;

6. close ultrasonator or time constant-temperature magnetic stirring apparatus; Take off the 200mL conical flask; Observe its outward appearance limpid in this way colourless, fluidity and good dispersion, obviously visible opalescence person is arranged, but be Pegylation superoxide dismutase (mPEG-SOD) nano-emulsion of a 100g direct cutaneous external;

7. but Pegylation superoxide dismutase (mPEG-SOD) nano-emulsion of this 100g direct cutaneous external is sub-packed in rapidly among the lucifuge glass container of different size, adds a cover rapidly, packing, and place 4 ℃ of airtight preservations of refrigerator to get final product;

8. according to International or National or enterprise's relevant criterion and requirement; Stochastic sampling from above-mentioned packing product; At first adopt the distribution of particles and the size of this nano-emulsion sample of Ma Erwen instrument detecting; After meeting the quality standard of nano-emulsion, again according to the specific requirement of national departments concerned, the system that carries out index of correlation detects; After waiting to judge or estimating it and meet national relevant regulations and standard and authentication code fully, can be used as promptly that launch is sold or special-purpose in the beauty parlor.

Test Example 1, evaluation of the present invention and study on the stability

The present invention detects through ultramicroscope, but a kind of Pegylation superoxide dismutase (mPEG-SOD) nano-emulsion outward appearance clear of direct cutaneous external, visible opalescence, has certain fluidity, and droplet diameter distribution is between 10～100nm.Thermodynamic stability of the present invention is good, and stable storage is good, through room temperature keep sample investigation, strong illumination experiment, hot test and low-temperature test, finds that the present invention is not stratified, even also not stratified in the high speed centrifugation environment.

1) the naked eyes outward appearance is observed: but a kind of Pegylation superoxide dismutase (mPEG-SOD) nano-emulsion outward appearance clear of direct cutaneous external, visible opalescence, have certain fluidity.Detect through transmission electron microscope, droplet diameter distribution is between 10～100nm.

2) temperature stability experiment:

But Pegylation superoxide dismutase (mPEG-SOD) nano-emulsion of a kind of direct cutaneous external of the present invention is packed in the hermetic container; Place under 4 ℃, 25 ℃, 40 ℃, the 60 ℃ conditions and investigate; Press certain hour (0,1,3,6,12,24 month) at interval, measure study on the stability project (having or not layering).The result shows that mPEG-SOD nano-emulsion outward appearance of the present invention is still transparent, does not find muddiness or deposited phenomenon.

3) light stability experiment: but Pegylation superoxide dismutase (mPEG-SOD) nano-emulsion of a kind of direct cutaneous external of the present invention is packed in the hermetic container; Place under the daylight; Light at room temperature is according to measuring in the 5th day, the tenth day, and the result does not relatively have difference before placing.The result finds that outward appearance of the present invention is still transparent, does not find muddy or deposition, and good stability is described.

4) accelerated stability experiment: but get Pegylation superoxide dismutase (mPEG-SOD) nano-emulsion of a kind of direct cutaneous external of the present invention, place high speed centrifuge supporting centrifuge tube, with 13000rpmmin ^-1The centrifugal 30min of speed, do not have layering, still transparent, explain that this product stability is good.

But the bioactive test of SOD in Pegylation superoxide dismutase (mPEG-SOD) nano-emulsion of Test Example 2, a kind of direct cutaneous external of the present invention

But prepare a kind of Pegylation superoxide dismutase (mPEG-SOD) nano-emulsion of direct cutaneous external and traditional mPEG-SOD cream frost method for preparing according to the present invention; Under identical condition, prescription effective component of the present invention is mixed with mPEG-SOD nano-emulsion and mPEG-SOD cream frost respectively; Under identical storage condition, measure the SOD activity respectively respectively at placing 30 days front and back; The result finds, but the SOD activity in a kind of Pegylation superoxide dismutase (mPEG-SOD) nano-emulsion of direct cutaneous external is active obviously high than the SOD in the SOD cream frost.

But Pegylation superoxide dismutase (mPEG-SOD) nano-emulsion of Test Example 3, a kind of direct cutaneous external, cream frost, emulsion preparations transdermal test in vitro infiltration comparative experiments

But carry out a kind of Pegylation superoxide dismutase (mPEG-SOD) nano-emulsion, cream frost, emulsion preparations transdermal test in vitro infiltration comparative experiments of direct cutaneous external with the Franz diffusion cell.The Franz diffusion cell keeps 37 ℃ of water-baths, and with rat skin fixed supply pond with accept between the pond, corium is towards accepting the pond, and effectively infiltrating area is 5cm ², accept to fill with acceptable solution in the pond, prevent to produce bubble; Accepting pool volume is 10mL; To prepare mPEG-SOD nano-emulsion, mPEG-SOD cream frost respectively, mPEG-SOD Emulsion 0.5g is uniformly coated on the skin for each three parts, places coverslip on the Supply House, prevents water evaporates.Open magnetic stirrer with 300r.min ^-1Rotating speed stirs, and respectively at 1,2,4,6,9h takes out acceptable solution 1mL (the sampling back replenishes the equal-volume acceptable solution), with sample solution 15000r.min ^-1Get supernatant behind the high speed centrifugation and carry out the HPLC analysis.Try to achieve each dosage form of SOD accumulative total Transdermal absorption amount by accumulation Transdermal absorption amount Q (formula 1), but calculate again the direct cutaneous external Pegylation superoxide dismutase (mPEG-SOD) nano-emulsion, cream frost,

The transdermal test in vitro permeability such as the following table of emulsion preparations.

The dermal osmosis speed that can know the mPEG-SOD nano-emulsion is respectively mPEG-SOD Emulsion and mPEG-SOD cream frost 1.36 and 1.65 times.The Franz diffusion cell is widely used in the transdermal test in vitro permeability test of medicine at present, and it can comparison image simulation medicine release conditions in vivo, has great importance for Study of Clinical Application.But medicine actual release conditions in vivo still is that some is different in the Franz diffusion cell with it.The result of study of Franz diffusion cell has important directive significance for people, but can not ignore the difference of it and truth.

Claims (5)

1. but the mPEG-SOD nano-emulsion of a direct cutaneous external is characterized in that it is made up of the following weight parts proportion raw material:

Wherein the mass concentration of mPEG-SOD is 0.03% in the mPEG-SOD aqueous solution.

2. but the mPEG-SOD nano-emulsion of a kind of direct cutaneous external according to claim 1 is characterized in that it is made up of following preferred weight part proportion raw material:

Wherein the mass concentration of mPEG-SOD is 0.03% in the mPEG-SOD aqueous solution.

But 3. the mPEG-SOD nano-emulsion of a kind of direct cutaneous external according to claim 1, wherein preferred weight part proportioning of each raw material is:

Wherein the mass concentration of mPEG-SOD is 0.03% in the mPEG-SOD aqueous solution.

But 4. the mPEG-SOD nano-emulsion of a kind of direct cutaneous external according to claim 3, but it is characterized in that the method for preparing of the mPEG-SOD nano-emulsion of described a kind of direct cutaneous external may further comprise the steps:

(1) mPEG-SOD with recipe quantity is dissolved in distilled water, and this solution is as the mPEG-SOD aqueous solution, subsequent use;

(2) take by weighing consumption according to each component ratio in the prescription, subsequent use, the vitamin E of recipe quantity is dissolved in the isopropyl myristate, subsequent use;

(3) with sad capric acid polyethyleneglycol glyceride, the polyglycereol-3-dioleate of recipe quantity, the mPEG-SOD aqueous solution of fully mix homogeneously, and adding recipe quantity adds the liquid for preparing in (2) then; And with whole system in natural room temperature, start the time constant-temperature magnetic stirring apparatus, with 200rmin ^-1The rotating speed magnetic agitation, fully mixing is transparence liquid until whole system, but is a kind of mPEG-SOD nano-emulsion of direct cutaneous external;

(4) but the mPEG-SOD nano-emulsion of this a kind of direct cutaneous external is sub-packed in rapidly among the lucifuge glass container of different size, add a cover packing, and place 4 ℃ of airtight preservations of refrigerator to get final product rapidly.

5. but the mPEG-SOD nano-emulsion of a kind of direct cutaneous external according to claim 4, but the average droplet size that it is characterized in that the mPEG-SOD nano-emulsion through the prepared a kind of direct cutaneous external of claim 4 method is in the scope of 10～100nm.

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