Summary of the invention
The purpose of this invention is to provide a kind of cream with effects of beautification and nourishing face, the mechanism that it takes place according to skin aging, from each different side, scientific composition, develop a kind of cream that in cosmetics, adds biological preparation and Chinese medicine ingredients with effects of beautification and nourishing face, the antagonism and the process of delaying decrepitude of skin, finally reach skin-nourishing, preserve moisture, crease-resistant, whiten, the cosmetics of effects such as speckle removing and skin care.
Technical scheme of the present invention is: design a kind of cream with effects of beautification and nourishing face, it comprises the major ingredient and the attached material of makeup of known cosmetics, it is characterized in that: also comprise α-mannatide 10%-20%, Margarita powder 80%-90% in its main component.
Described composition includes α-mannatide 10%-20%, Margarita powder 10%-70%, tea polyphenols 10-80%.
Described composition includes α-mannatide 10%-30%, Margarita powder 10%-30%, tea polyphenols 10%-40%, hyaluronic acid sodium 10%-30%.
Described composition includes the Chinese medicine 10%-30% of α-mannatide 10%-20%, Margarita powder 10%-30%, tea polyphenols 10%-20%, hyaluronic acid sodium 10%-20%, oxidation and removing free radicals.
Described composition includes α-mannatide 10%-20%, Margarita powder 5%-20%, tea polyphenols 5%-10%, the Chinese medicine 10%-30% of hyaluronic acid sodium 10%-20%, oxidation and removing free radicals, the Chinese medicine 10%-40% of ultra-violet radiation resisting.
Described composition includes the antianaphylactic Chinese medicine 15%-30% of Chinese medicine 10%-20%, the Chinese medicine 15%-30% of ultra-violet radiation resisting, antiinflammatory of α-mannatide 10%-20%, Margarita powder 5%-10%, tea polyphenols 10%-20%, hyaluronic acid sodium 5%-20%, oxidation and removing free radicals.
The Chinese medicine of described oxidation and removing free radicals has: Fructus Schisandrae Chinensis, Radix Angelicae Sinensis, Cordyceps, green tea, Semen Ginkgo, Radix Salviae Miltiorrhizae, Cortex Magnoliae Officinalis, Fructus Jujubae, Radix Pseudostellariae, Semen Ziziphi Spinosae, Radix Rehmanniae; The Chinese medicine of described ultra-violet radiation resisting has: Radix Bupleuri, Radix Scutellariae, Rhizoma Chuanxiong, Radix Ginseng, Ganoderma, Radix Notoginseng, Rohdea japonica Roth; The antianaphylactic Chinese medicine of described antiinflammatory has: Radix Puerariae, Radix Scutellariae, Fructus Aurantii Immaturus, Radix Glycyrrhizae, Fructus Mume, Pheretima, Cortex Moutan, Placenta Hominis, the Radix Astragali, Radix Stephaniae Tetrandrae, Ramulus Luffae, Folium Pyrrosiae, Herba Ephedrae, Rhizoma Atractylodis, Herba Spirodelae, Caulis Polygoni Multiflori, Fructus Atriplicis Sibiricae.
The pastille composition is to contain α-mannatide 200g, Margarita powder 300g, hyaluronic acid sodium 200g, Fructus Schisandrae Chinensis 30g, Radix Angelicae Sinensis 30g, Cordyceps 30g, green tea 25g, Semen Ginkgo 40g, Radix Salviae Miltiorrhizae 20g, Cortex Magnoliae Officinalis 20g, Fructus Jujubae 35g, Radix Pseudostellariae 30g, Semen Ziziphi Spinosae 20g, Radix Rehmanniae 20g in every 1000g medicine in the described cream.
The pastille composition is to contain α-mannatide 200g, Margarita powder 130g, tea polyphenols 100g, hyaluronic acid sodium 135g, Fructus Schisandrae Chinensis 20g, Radix Angelicae Sinensis 10g, Cordyceps 10g, green tea 25g, Semen Ginkgo 30g, Radix Salviae Miltiorrhizae 20g, Cortex Magnoliae Officinalis 20g, Fructus Jujubae 35g, Radix Pseudostellariae 30g, Semen Ziziphi Spinosae 20g, Radix Rehmanniae 20g, Radix Bupleuri 35g, Radix Scutellariae 20g, Rhizoma Chuanxiong 30g, Radix Ginseng 20g, Ganoderma 25g, Radix Notoginseng 25g, Rohdea japonica Roth 40g in every 1000g medicine in the described cream.
The pastille composition is to contain α-mannatide 200g in every 1000g medicine in the described cream, Margarita powder 100g, tea polyphenols 100g, hyaluronic acid sodium 85g, Fructus Schisandrae Chinensis 15g, Radix Angelicae Sinensis 10g, Cordyceps 10g, green tea 20g, Semen Ginkgo 20g, Radix Salviae Miltiorrhizae 10g, Cortex Magnoliae Officinalis 10g, Fructus Jujubae 15g, Radix Pseudostellariae 20g, Semen Ziziphi Spinosae 20g, Radix Rehmanniae 20g, Radix Bupleuri 20g, Rhizoma Chuanxiong 15g, Radix Ginseng 30g, Ganoderma 25g, Radix Notoginseng 30g, Rohdea japonica Roth 45g, Radix Puerariae 5g, Radix Scutellariae 10g, Fructus Aurantii Immaturus 5g, Radix Glycyrrhizae 20g, Fructus Mume 15g, Pheretima 10g, Cortex Moutan 20g, Placenta Hominis 10g, Radix Astragali 10g, Radix Stephaniae Tetrandrae 10g, Ramulus Luffae 15g, Folium Pyrrosiae 5g, Herba Ephedrae 5g, Rhizoma Atractylodis 5g, Herba Spirodelae 15g, Caulis Polygoni Multiflori 10g, Fructus Atriplicis Sibiricae 10g.
Characteristics of the present invention are: α-mannatide is a kind of biological response modifier, can infiltrate skin inside fast, local microenvironment is regulated, make the cell internal and external environment be in poised state, keep normal metabolism of cell and function, strengthen skin infection, antiallergic and stress function etc.; Margarita powder contains big angulation glutelin, trace element and several amino acids, can improve the activity of SOD, GSP-PX, quicken the free radical that scavenger cell produces because of peroxidating, (the free radical energy loss white matter of mourning or grieve over the deceased mainly is by the modified amino acid residue to the biomembrane lipid peroxidization that inhibition is caused by free radical, causes the change of protein structure and conformation, cause peptide chain interruption, polymerization and crosslinked, make collagen protein stiff, follow the string and dilatancy, make skin wrinkle occur.)。In addition, Margarita powder can also promote the processes of wound repair of skin, strengthens the permeability of Skin Cell to nutrient substance, improves the water storage function of skin layer, makes moisture be weighing apparatus attitude distribution etc. inside and outside Skin Cell; Tea polyphenols has and protein, polysaccharide, the bonded ability of alkaloid, make us producing the astringent sensation, skin is had good adhesive ability, thick pore is shunk, make lax skin-tightening face reduce wrinkle, also can reduce the excessive secretion of greasy skin sebum.Tea polyphenols also has very strong oxidation resistance, and UVA and UVB are had very strong filtration, reduces the damage of ultraviolet to skin, the activity of energy restraint of tyrosinase, reduce the metabolism intensity of melanocyte, reduce melanic formation, have the skin-whitening effect, to multiple microorganisms such as virus, antibacterial, funguses, particularly the skin pathogenic bacterium there is the inhibition ability, the effect of antiinflammatory is arranged, and can promote cell metabolism, cultivate skin vitality, make it keep young fine and smooth; Hyaluronic acid sodium has water conservation, slows down functions such as horn cell breaks up, the effect of removing free radical; Radix Ginseng, Poria energy SOD activity improving are removed free radical, with the same effect that ultra-violet radiation resisting is arranged of Radix Bupleuri, to stop skin aging, help skin regeneration and reparation; Radix Puerariae, Radix Scutellariae be anti-inflammatory, antiallergic, raising immunologic function in various degree all, to reduce free-radical generating, removes the risk factor of skin aging or black speck.
The specific embodiment
Because the present invention is carried recoverable space craft (spacecraft or retrievable satellite) with the Alpha-hemolytic streptococcus strain, utilize the comprehensive function of factors such as little in the space (zero) gravity, cosmic ray, alternating magnetic field, fine vacuum, hyperpyrexia deep cooling, make the dna double chain structure fracture of bacterial strain, genome is reset, thereby produces new bacterial strain.After returning ground, bacterial strain through space treatment is cultivated and screened, by studying contents such as its morphological characteristic, cultural characteristic, biochemical reactions, metabolite, hereditary character and protein expression, hereditary stability, pilot scale production target, optimize the stable strain of plus variant, inherited character wherein, the medicine of the ulcerative colitis that after cultivation and fermentation, obtains medical treatment.This strain in China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation, is numbered CGMCCNo.1082.
Particularly Alpha-hemolytic streptococcus D33 bacterial strain behind ground screening, separation and the purification, selects higher, the secreted mannan peptide content of fermentation unit higher high yield, quality strains T33 strain through space treatment mutation.By Institute of Microorganism, Academia Sinica whole-cell protein SDS-polyacrylamide gel (SDS-PAGE) analysis of T33 bacterial strain and D33 bacterial strain and the genomic DNA restricted enzyme cutting analysis (RFLP/PFGE) of bacterial strain are compared, can prove that its gene of T33 bacterial strain that space mutagenesis and ground filter out has variation.This mutant strain is stable in heredity, stronger production adaptability is arranged, the mannan peptide content that is produced by its fermentation improves, content of peptides improves 3 to 5 times than national standard (80%), institute for drug control, Xi'an sample presentation is measured and is reached 271.6%, and fermentation content on average exceeds more than three times than matched group product content.This strain is through the spaceship-carried space flight of Shenzhou series of spacecraft, and further screening and culturing breeding forms stable hereditary property, the tangible high-yield highly-effective strain of variation features to strain by BeiJing ZhongKe Institute of Micro-biology of institute and biology department of Northwest University.The mannan peptide content of α in the every fermentation unit of this strain-space flight strain production has improved more than several times.(the material 1 of seeing Appendix: science and technology bureau Shaanxi Province, Shaanxi Province scientific and technological achievement assay certificate material
Adnexa material 2: the mannatide that the space flight strain is produced is produced bacterial strain and is carried notarization through " No. three, divine boat " airship.
Adnexa material 3: the mannatide that the space flight strain is produced is produced bacterial strain and is returned ground screening report through the lift-launch of " No. three, divine boat " airship
Adnexa material 4: Microbe Inst., Chinese Academy of Sciences produces the form Physiology and biochemistry probation report book of bacterial strain and ground contrast bacterium about the mannatide that " No. three, divine boat " airship carries the production of space flight strain
Adnexa material 5: Microbe Inst., Chinese Academy of Sciences produces SDS-PAGE and the RFLP/PFGE Analysis and Identification statement of bacterial strain and ground contrast bacterium about the mannatide that " No. three, divine boat " airship carries the production of space flight strain
Adnexa material 6: the mannatide oral liquid examining report that institute for drug control, Shaanxi Province " No. three, divine boat " space flight strain is produced
Adnexa material 7: scientific and technological novelty assessment report copy
Adnexa material 8: notice is accepted in China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation culture presevation)
Strain improvement of the present invention: on the basis of Alpha-hemolytic streptococcus growth rhythm, selected than the proper growth strain in period, adopt the plate growth bacterium colony, immerse methods such as the bacteria suspension in other material, germ-carrying sand, carry " No. three, divine boat " spacecraft on March 25th, 2002.Carried out 6 days 0 18 hours flying at space track (200 kilometers of perigee altitudes, 350 kilometers of altitude of the apogees) at rail.The specific condition of space mainly is presented as special environments such as microgravity, fine vacuum, high radiation, alternating magnetic field.The strain of outer-space flight be placed in one with the diverse environment of tellurian ecological environment in, the electronics, proton and the mental retardation heavy particle that mainly comprise coming self-magnetic field to capture; Cosmic ray such as proton, particle and heavier high energy heavy particle from the milky way galaxy; From the proton of sun magnetic storm and heavy particle etc.These particles act on the dna double chain structure in the microbial cell nuclear, can cause double-strand break.Microgravity, high vacuum environment can make the base sequence of DNA recombinate, promptly genomic reorganization and variation.The new bacterial strain that the variation back produces, variation in various degree all can take place in its morphological characteristic, cultural characteristic, biochemical reactions, metabolite, hereditary character and protein expression, pilot scale production target etc.After ground is returned in spacecraft, through further screening, only optimize wherein 0.2% plus variant and the stable bacterial strain of inherited character, make the production strain through cultivation.This strain that after spaceship-carried mutation, selects December in 2003 29 days by China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation, be numbered CGMCCNo.1082.
Experiment and middle trial production result show, stable on inherited character through the novel space strain behind the space mutagenesis, stronger production adaptability is arranged, the mannan peptide content that is produced by its fermentation improves, content of peptides improves 3 to 5 times than national standard (80%), institute for drug control, Xi'an sample presentation is measured and is reached 271.6%, and fermentation content on average exceeds more than three times than matched group product content, and fermentation period shortens greatly.
Preparation method:
The space flight strain is produced the preparation process of mannatide:
The mannatide of space flight strain of the present invention production is by following method preparation: space strain preparation-one grade fermemtation-second order fermentation-fermentation liquid is purified, and final production goes out mannatide.
1. space strain preparation:
With through the Alpha-hemolytic streptococcus of space mutagenesis breeding meticulous selection-breeding as producing strain.
A. inclined-plane seed culture medium: Carnis Bovis seu Bubali cream 0.5%, yeast extract 0.6%, peptone 0.5%, glucose 0.4%, sodium chloride 05%, agar 3%, sheep blood 8%; PH7.4.
121 ℃ of inclined-plane seed culture medium sterilising temps, 30 minutes time, steam pressure 0.12Mpa.
Unpacking strain cryovial with aseptic broth bouillon dilution, inserts the blood inclined-plane under aseptic condition, the rearmounted 38 ℃ of constant temperature culture of inoculation 30 hours.
B. meat soup seed culture medium: Carnis Bovis seu Bubali cream 0.5%, yeast extract 0.6%, peptone 0.5%, glucose 0.4%, sodium chloride 0.5%; PH7.4.
121 ℃ of meat soup seed culture medium sterilising temps, 30 minutes time, steam pressure 0.12Mpa.
With cultured slant strains under aseptic condition by 9% inoculum concentration, insert in the meat soup seed culture medium 38 ℃ of constant temperature culture 30 hours.
2. sweat:
Fermentation medium: Carnis Bovis seu Bubali cream 0.4%, yeast extract 0.5%, peptone 0.5%, glucose 0.4%, sodium chloride 0.5%.
121 ℃ of fermentation medium sterilising temps, 30 minutes time, steam pressure 0.12Mpa.
A. one grade fermemtation jar: with good meat soup strain under aseptic condition by 10% inoculum concentration, insert first class seed pot, 29 ℃ of constant temperature culture 30 hours, tank pressure was no more than 0.02Mpa, ventilation is advisable can stir culture fluid, fully stirs continuously.
B. second order fermentation jar: with the one grade fermemtation culture fluid under aseptic condition by 20% inoculum concentration, insert fermentation tank, 29 ℃ of constant temperature culture 70 hours, tank pressure is 0.02Mpa not, jar is put in deactivation.Deactivation is adopted to heat and is made the fermentation liquid temperature reach 100 ℃, is incubated 60 minutes, and cooling is left standstill.
3. leaching process:
A, fermentation liquid concentrate, and make concentrated solution volume and fermentating liquid volume ratio be controlled at 1: 15-1: 20 or be determined by circumstances.
The concentrated solution of b, fermentation liquid adds 80-99% ethanol, and the volume ratio is controlled at 1: 1.5-1: 5.5 or be determined by circumstances.Fully stir leave standstill after, centrifugal removal supernatant, the precipitation dissolved in distilled water, accent pH5.0 obtains lysate and lysate is left standstill.
C, the more centrifugal removal impurity of lysate is obtained supernatant, accurately measure the clear liquid volume, calculate required amount of alcohol by the supernatant stereometer, adjust pH slowly joins ethanol in the supernatant, and fully stirs, and leaves standstill the centrifugal removal supernatant in back and obtains precipitate.
D, precipitate reuse dissolved in distilled water are transferred pH, and centrifugal, supernatant adds ethanol precipitation again.Such technology repeatable operation to intermediate detection qualified till, the precipitate that obtains is the mannatide crude product that the space flight strain is produced.Vacuum drying 3-8 hour, promptly obtain the mannan peptide product that the space flight strain is produced.
The check of the product that said method obtains:
[character] this product is white or little yellow amorphous powder; Odorless, tasteless.
This product is easily molten in water, and is insoluble in ethanol, chloroform and acetone.
Specific optical rotation: get this product, accurate claim surely, be dissolved in water and be diluted to the solution that contains lOmg among every lml approximately, measure (two appendix vI of Chinese Pharmacopoeia version in 2000 E) in accordance with the law, specific optical rotation is+70 ℃ to+80 ℃.
[discriminating] 1, get this product l0mg, add water 0.5ml and make dissolving, add a-naphthols alcoholic solution (5-100) lml, shake up, slowly add sulphuric acid 0.5ml along tube wall, after several minutes, the interface is aubergine.
2, get this product, add water and make the solution that contains lmg among every lml, get about lOul point on filter paper, dry, fix, put high salpeter solution and (get periodic acid 1.2g with dehydrated alcohol, after adding water 3Oml dissolving. add O.2mol/L sodium acetate solution 1.5ml and ethanol lOOml, mixing is promptly.Put the dark place and preserve, can use the several months) the middle immersion 5 minutes, take out, wash with 70% alcoholic solution, (get potassium iodide 5g, sodium thiosulfate 5g adds water lOOml and makes dissolving to put reducing solution, add ethanol 150m1,2mol/L hydrochloric acid solution 2.5ml again, with adding, face the time spent and join with stirring) the middle immersion 5 minutes, take out, wash with 70% alcoholic solution, put in the pinkish red industry sulphuric acid test solution and soaked about 30 minutes, take out, (get sodium pyrosulfite 0.4g with sodium metabisulfite solution, add hydrochloric acid lml, being dissolved in water makes into lOOml, promptly), and flushing, dry, the place should be aubergine at the filter paper point sample.
3, get test sample and reference substance is an amount of, add respectively the chlorination sodium injection make contain lmg among every lml solution as need testing solution and reference substance solution, press the test of mannatide immunogenicity determining method, test sample and contrast QC should all not have haemolysis to be taken place.
[inspection] 1, acidity: get this product, add water and make the solution that contains IOmg among every 1ml, measure (two appendix VI of Chinese Pharmacopoeia version in 2000 H) in accordance with the law, pH value should be 3.0-5.0.
2, trap: get this product, add water and make the solution that contains 0.4mg among every lml, according to spectrophotography (two appendix VI of Chinese Pharmacopoeia version in 2000 A), wavelength place at 260nm, its trap must not be greater than 0.25, and at the wavelength place of 280nm, its trap must not be greater than 0.20.
3, total ammonia amount: get this product, measure according to N2 method (two appendix VII of Chinese Pharmacopoeia version in 2000 D, second method). press dry product and calculate, contain total nitrogen and should be 0.8-2.0%.
4, immunogenicity: get test sample and reference substance is an amount of, add the chlorination sodium injection respectively and make the solution that contains lOmg among every lml, make 1: 2, l with phosphate buffer respectively again: 4,1: 8,1: 16,1: 32, l: 64,1: 128,1: 256 diluent is as need testing solution and reference substance solution, check (attached mannatide immunogenicity determining method) in accordance with the law, the least concentration of the insoluble blood vessel of test sample should be higher than the reference substance respective concentration-doubly more than.
5, loss on drying is got this product, is dried to constant weight at 105 ℃, subtracts weight loss and must not cross 5.0% (two appendix VIII of Chinese Pharmacopoeia version in 2000 L).
6, heavy metal is got this product, at 1.0g, checks that in accordance with the law (Chinese Pharmacopoeia version VIII in 2000 H) contains heavy metal and must not cross 20/1000000ths.
7, the undue toxicity gets this product, adds the chlorination sodium injection and makes the solution that contains 0.5mg among every 1ml, checks (Chinese Pharmacopoeia version appendix in 2000 XI C) in accordance with the law. press the intravenous injection administration, and should (injection) up to specification.
[assay]
1. the preparation of reference substance solution
Precision takes by weighing through 105 ℃ of D-mannose reference substance 0.1g that are dried to constant weight and puts in the lOOml measuring bottle, is dissolved in water and is diluted to scale. shake up; Precision is measured 5ml, puts in the measuring bottle of lOOml, adds water to scale, shakes up. contain mannose 50ug among every 1ml.
2. need testing solution is equipped with
It is an amount of to get this product, and accurate the title decides, and is dissolved in water and makes the solution that contains 40ug among every 1ml.
3. the preparation of standard curve
Precision takes by weighing reference substance solution 0,0.2,0.4,0.6,0.8,1.Oml, put respectively in the tool plug test tube, respectively add water to 1.0ml, add 3% phenol solution 1.Oml again, shake up, pour sulphuric acid 4.5ml, shake up, being positioned over room temperature, is blank with 0 pipe. measure trap according to spectrophotography (two appendix IV of Chinese Pharmacopoeia version in 2000 A) at the wavelength place of 490nm.To corresponding trap, calculate regression equation with mannose ug number.
4. algoscopy
Precision is measured need testing solution 1.Oml, operates from " adding 3% phenol solution 1.Oml again " under the sighting target directrix curve preparation in accordance with the law, measures trap, by the regression equation calculation mannose content.
Mannatide immunogenicity determining method (complement combined techniques);
1, test solution
A. phthalate buffer (pH7.2)
Get sodium chloride 8.5g, sodium hydrogen phosphate 0.565g and potassium dihydrogen phosphate 0.135g, add water to 1000ml and make its dissolving, add 10% Adlerika 1ml, shake up.
The B.l% sheep erythrocyte suspension
The preparation of sheeps blood erythrocyte: (get glucose 20.5g, sodium citrate 8.0g, sodium chloride 4.2g in filling equivalent A Shi liquid by sheep jugular vein sterile blood sampling, citric acid 5.5g, add water to 1000mI and make dissolving, 100 ℃ the sterilization 30 minutes) sterile chamber in, 4 ℃ of preservations.
The preparation of 1% sheeps blood erythrocyte suspension: it is an amount of to get above-mentioned sheeps blood erythrocyte, and with sodium chloride injection washing three times, each centrifugal 5 minutes (2000 rev/mins), sheeps blood erythrocyte is amassed in pressure, makes l% sheeps blood erythrocyte suspension with sodium chloride injection.Get suspension, make need testing solution for 20 times with the sodium chloride injection dilution, other gets 20 times of equivalent suspension thin ups as blank solution, according to spectrophotography (two appendix IV of Chinese Pharmacopoeia version in 2000 A), wavelength place at 541nm measures, its trap should be 0.65-0.70, should regulate the concentration of 1% sheep erythrocyte suspension as overrun.
C. hemolysin and sensitization sheeps blood erythrocyte
The preparation of hemolysin: get above-mentioned hematocrit sheeps blood erythrocyte, make 25% sheeps blood erythrocyte suspension with sodium chloride injection. get 1 of rabbit, the above-mentioned sheeps blood erythrocyte suspension of intravenous injection, once a day, totally seven times, first three time 3ml, three 5ml in back, last is injected blood sampling in back seven days.Separation of serum, 56 ℃ of deactivations in .30 minute, packing is preserved below O ℃.
The mensuration of amboceptor unit: it is an amount of to get hemolysin, add phosphate buffer and make 1 respectively: l000,1: 2000,1: 3000, l: 4000,1: 5000,1: 6000,1: 7000,1: 8000,1: 9000, l: 10000 diluent, respectively getting 0.lml puts in the test tube, add 1% Sanguis caprae seu ovis cell suspension 0.1ml, shake up, adding dilution factor is l: 30 guinea pig serum (complement) 0.2ml and phosphate buffer 0.2ml, shake up, 37 ℃ of insulations 30 minutes, the high dilution of complete hemolysis pipe is 1 unit hemolysin.
The preparation of sensitization sheeps blood erythrocyte: before facing usefulness, the sheeps blood erythrocyte suspension with 2% mixes with 2 unit haemolysis rope equal-volumes, and 37 ℃ are incubated 15 minutes promptly.
Complement: get the Cavia porcellus more than three, the heart blood sampling, centrifugalize serum is preserved below 0 ℃.
The mensuration of complement unit: it is an amount of to get complement, add phosphate buffer, make 1: 40, l respectively: 60, l: 80, l: 100, l: 120,1: 140,1: 160,1: 180 diluent, respectively get and O.2ml put in the test tube, add the 0.2ml phosphate buffer, shake up, 37 ℃ of insulations are after 30 minutes, add sensitization sheeps blood erythrocyte 0.2ml respectively, shake up, 37 ℃ are incubated 30 minutes again. and the high dilution of complete hemolysis pipe is the complement of 1 unit.
Antibody: it is an amount of to get the mannatide reference substance, add the chlorination sodium injection and make the reference substance solution immunizing rabbit that contains lOmg among every lml, the next day adopt ear vein injection reference substance solution once.Inject for the first time 0.2ml, for the second time respectively inject 0.5ml to the 5th time, respectively inject 1.Oml the 6th time to the tenth time, the tenth once respectively injects 2.Oml to the 15 time, and last is injected blood sampling in back three days, centrifugalize serum, 56 ℃ of deactivations in following 30 minutes, (before facing usefulness, needing 56 ℃ of deactivations once more in 30 minutes) preserved in packing below 0 ℃.
The mensuration of antibody unit: it is an amount of to get antibody, add phosphate buffer and make 1: 2,1: 4,1: 8,1: 16,1: 32.1: 64, l respectively: 128 diluent, respectively getting 0.lml puts in the test tube, add mannatide reference substance solution 0.lml and 2 unit complements O.2ml, shake up, place more than 4 hours in 4-8 ℃, put 37 ℃ of insulations 30 minutes, the high dilution of insoluble blood vessel is 1 unit antibody.Establish antigen simultaneously and (do not add antibody, replace with the 0.1ml phosphate buffer), antibody (do not add antigen, replace with the 0.lml phosphate buffer), complement (do not add antigen, antibody, replace with the 0.2ml phosphate buffer) control tube, more than three kinds of control tube haemolysis entirely; The sensitization sheeps blood erythrocyte that other establishes (do not add antigen, antibody and complement, replace with the 0.4ml phosphate buffer) control tube is haemolysis not.
2, immunogenicity determining method
It is an amount of to get rapid glycopeptide reference substance of manna and test sample, make the reference substance solution and the need testing solution of variable concentrations according to the regulation under the medicine item, respectively getting 0.1ml puts in the test tube, add the 2 antibody 0.1ml of unit and the 2 complement 0.2m1. of unit shake up, more than 4 hours, put 37 ℃ of insulations 30 minutes in 4-8 ℃ of placement, add sensitization sheeps blood erythrocyte 0.2ml, shake up, 37 ℃ are incubated 30 minutes again.Observe the haemolysis situation of each pipe, the least concentration of insoluble blood vessel is represented the immunogenicity of mannatide.Establish antigen simultaneously and (do not add antibody, replace with the 0.1ml phosphate buffer), antibody (do not add antigen, replace with the 0.1ml phosphate buffer), complement (do not add antigen, antibody, replace) control tube with the 0.2ml phosphate buffer, more than three kinds of control tube haemolysis entirely: the sensitization sheeps blood erythrocyte that other establishes (do not add antigen, antibody and complement, replace with the 0.4ml phosphate buffer) control tube is haemolysis not.
Embodiment 1: get said medicine α-mannatide 200g, Margarita powder 300g, hyaluronic acid sodium 200g, Fructus Schisandrae Chinensis 30g, Radix Angelicae Sinensis 30g, Cordyceps 30g, green tea 25g, Semen Ginkgo 40g, Radix Salviae Miltiorrhizae 20g, Cortex Magnoliae Officinalis 20g, Fructus Jujubae 35g, Radix Pseudostellariae 30g, Semen Ziziphi Spinosae 20g, Radix Rehmanniae 20g.During preparation with above Chinese medicine dense fry in shallow oil twice → merge medicine juice → filtration → with the suitable adjuvant of α-mannatide, Margarita powder, tea polyphenols, hyaluronic acid sodium mixing → add → stir make become the cream → quality inspection of O/W type qualified → packing, finished product.
Concrete production process and adjunct ingredient ratio are carried out according to the preparation technology's (see Chinese Pharmacopoeia Commission's volume, Chemical Industry Press publishes) and the cosmetic standard of 2000 editions cream of Pharmacopoeia of People's Republic of China.
Other embodiment and embodiment 1 production process are identical, and adjuvant adds also identical, just the medicament composing prescription difference.
The medicament composing prescription of embodiment 2 is: the pastille composition is to contain α-mannatide 200g, Margarita powder 130g, tea polyphenols 100g, hyaluronic acid sodium 135g, Fructus Schisandrae Chinensis 20g, Radix Angelicae Sinensis 10g, Cordyceps 10g, green tea 25g, Semen Ginkgo 30g, Radix Salviae Miltiorrhizae 20g, Cortex Magnoliae Officinalis 20g, Fructus Jujubae 35g, Radix Pseudostellariae 30g, Semen Ziziphi Spinosae 20g, Radix Rehmanniae 20g, Radix Bupleuri 35g, Radix Scutellariae 20g, Rhizoma Chuanxiong 30g, Radix Ginseng 20g, Ganoderma 25g, Radix Notoginseng 25g, Rohdea japonica Roth 40g in every 1000g medicine in the described cream.
The medicament composing prescription of embodiment 3 is: the pastille composition is to contain α-mannatide 200g in every 1000g medicine in the described cream, Margarita powder 100g, tea polyphenols 100g, hyaluronic acid sodium 85g, Fructus Schisandrae Chinensis 15g, Radix Angelicae Sinensis 10g, Cordyceps 10g, green tea 20g, Semen Ginkgo 20g, Radix Salviae Miltiorrhizae 10g, Cortex Magnoliae Officinalis 10g, Fructus Jujubae 15g, Radix Pseudostellariae 20g, Semen Ziziphi Spinosae 20g, Radix Rehmanniae 20g, Radix Bupleuri 20g, Rhizoma Chuanxiong 15g, Radix Ginseng 30g, Ganoderma 25g, Radix Notoginseng 30g, Rohdea japonica Roth 45g, Radix Puerariae 5g, Radix Scutellariae 10g, Fructus Aurantii Immaturus 5g, Radix Glycyrrhizae 20g, Fructus Mume 15g, Pheretima 10g, Cortex Moutan 20g, Placenta Hominis 10g, Radix Astragali 10g, Radix Stephaniae Tetrandrae 10g, Ramulus Luffae 15g, Folium Pyrrosiae 5g, Herba Ephedrae 5g, Rhizoma Atractylodis 5g, Herba Spirodelae 15g, Caulis Polygoni Multiflori 10g, Fructus Atriplicis Sibiricae 10g.
This skin irritation test with cream of effects of beautification and nourishing face of the present invention: select 6 of 2.5-3.0kg healthy rabbits, every rabbit back left-right symmetric selects 3 places to shave a mao 2.5cm
2, carry out with the own control test method(s), be coated with 0.5g cream or blank substrate, every day twice, the administration at random of left side or right side, the reacting phenomenon of skin behind observation and record 24h, the 72h at every turn.The result shows: do not see local excitations such as skin rubefaction, dermexanthesis, vesicle reaction behind coating 24h, the 72h, illustrate that looks improving and the skin nourishing cream of the present invention is to be perfectly safe, do not have any untoward reaction.
The animal pharmacodynamic study
Wrinkle of skin is one of common sign of human senility.The main component of skin histology is a collagen protein, and in the collagen protein in what and the skin of distinctive hydroxyproline content some other amino acid whose content what are closely related with skin aging.
1 experiment material
1.1 reagent
Six kinds of cream, α-mannatides of the present invention are provided by our company; Olay nutrition essence frost is the commercial goods; Ethylene glycol: Shandong Chemical Inst; Acetone: Changzhou Wanda chemical reagent work produces; Benzyl alcohol: reagent one factory in Shanghai produces; Sodium hydroxide: Chinese chemical reagent three factories; Citric acid: Chinese chemical industry station pilot plant; Sodium chloride: Qingdao Sifang District chemical reagent factory produces; Sodium citrate: Shenyang reagent one factory; Caprylic acid: Beijing Chemical Plant; The tri-chlorination peptide: Japan produces; Thiodiglycol: Japan produces; TGA: Shanghai pharmaceutical factory of The 2nd Army Medical College; The about 9cm of quantitative filter paper: Hangzhou Xinhua Paper Making Mill.
1.2 instrument
40 type stepless speed regulation electric blender: Jiangyin, Jiangsu scientific research apparatus factory produces; B70-30 type electric suction apparatus: hospital equipment factory in Shanghai produces; 202-2 type electrically heated drying cabinet: Shanghai City Shanghai County experimental apparatus factory produces; 835 type automatic amino acid analyzers: Japan produces.
1.3 animal: Kunming mouse is provided by The Fourth Military Medical University's Experimental Animal Center.
2 methods and result
Get 90 of Kunming mouses, male and female half and half, body weight are 18-22g, be divided into 9 groups at random by sex and body weight, be respectively: blank group, first kind of cream group of the present invention, second kind of cream group of the present invention, the third cream group of the present invention, the 4th kind of cream group of the present invention, the 5th kind of cream group of the present invention, the 6th kind of cream group of the present invention, α-mannatide powder group, Olay nutrition essence frost group.Each test sample 2g with the water mixing batter, is evenly spread upon respectively and respectively organizes mouse back, application area 4cm * 3cm, once a day, continuous 35 days.Smeared back 24 hours in last, the hair that each group mouse back is tried to distinguish shaves off (in order to avoid the interference of the protein in the hair) and the skin that will distinguish is cut, behind the tissues such as removing skin fat, distilled water flushing filter paper is wiped away dried, put in 37 ℃ of baking ovens dry 24 hours, and took out and shred and, break into homogenate with high speed homogenizer then with acetone defat 48 hours, put again in 37 ℃ of baking ovens dry 24 hours, for the sample of measuring usefulness.Sample thief 40mg at 110 ℃ of following hydrochloric acid hydrolysiss, evaporates deacidification in 80 ℃ of water-baths, add the 0.02mol/L dissolving with hydrochloric acid again after, produce 835-50 type automatic amino acid analyzer with Japan and carry out several amino acids Determination on content such as hydroxyproline.The results are shown in Table 1,2.
The various cream of table 1 are to the influence of mouse skin hydroxyproline content
Annotate: compare with the blank group
*P<0.01,
* *P<0.001.
The various cream of table 2 are sweet to mouse skin, the influence of paddy, proline content (g/100g)
Annotate: compare with the blank group
*P<0.05,
*P<0.01,
* *P<0.001.
Table 1,2 experimental data show: six kinds of cream of the present invention can make the content of hydroxyproline in the mouse skin, glycine, glutamic acid and proline significantly raise, and along with the reinforcement of prescription, above-mentioned amino acid whose content also raises thereupon gradually.Use the mice of α-mannatide and Olay nutrition essence frost, above-mentioned amino acid whose content only has trickle rising in its skin, compares no significant difference with the blank group.Above-mentioned experimental result shows: six kinds of cream of the present invention reduce wrinkle formation, delaying decrepitude of skin so that reach significantly be better than on the effect of looks improving and the skin nourishing single with α-mannatide and Olay nutrition essence frost.
The checking of the curative effect of cream of the present invention: Liu so-and-so, woman, 38 years old, cadre.Tell annual spring face and occur redly through regular meeting, the small pox that the grain of rice is big is difficult to disappear, and consideration may be relevant with anaphylactogen such as pollen.In this year, skin of face is dark and gloomy, and the yellowish-brown speckle also appears in corse sweat pore, and mottle obviously increases the weight of after the Exposure to Sunlight.Once used several freckle removing and whitening products, but poor effect.With looks improving and the skin nourishing cream of the present invention, every day twice, in sooner or later being applied to face after clean.After 20 days, conscious skin is tight flexible, the soft and smooth exquisiteness of touching, and the colour of skin is glossy than before, and the mottle color is thin out, and area has been desalinated about 4cm before than medication
2, continuing 2 months (2 courses of treatment) of medication, skin is ruddy delicate, is rich in gloss, soft and smooth exquisiteness, mottle disappears.Following up a case by regular visits to 1 year mottle does not have recurrence, and spring, skin of face was not met quick reaction appearance.
The curative effect statistics of cream of the present invention: trial volunteer 50 examples, male 17 examples, women 33 examples, age reckling 17 years old, the maximum 67 years old, 34.76 years old mean age.Dry skin 41 examples, ichthyosis 4 examples wherein, neutral skin 6 example and oily skin 3 examples.Every routine experimenter uses looks improving and the skin nourishing cream of the present invention, and sooner or later each softened gently and put on the skin with being applied to face about 0.5g after clean every day, used one month (course of treatment) continuously.Effect criterion: excellent: conscious comfortable, objective sign (drying, squama, erythema etc.) disappearance; Very: the conscious objective sign (drying, squama, erythema etc.) that do not accommodate disappears 〉=2/3; Difference: do not see obvious change before and after using.The result: in 50 examples, effect is judged the superior's 46 examples, accounts for 92%; Ichthyosis experimenter 4 examples use the back skin symptom to alleviate, and effect is judged good person's 4 examples, accounts for 8%, and total effective rate is 100%.Whole experimenters do not have 1 example and untoward reaction occurs.
In addition the treatment data of 268 routine patient with chloasma is added up as follows: 268 routine experimenters are the women, 20 years old of age minimum, maximum 48 years old, 36.5 years old mean age.The course of disease is the shortest 6 months, and is the longest 16 years.Gestation back onset 138 examples, cosmetics cause 62 examples, companion's hepatopathy 16 examples, surplus is agnogenio.Wherein some cases is with menoxenia.Every routine experimenter uses looks improving and the skin nourishing cream of the present invention, and sooner or later each softened gently and put on the skin with being applied to face about 0.5g after clean every day, used continuously 2 months.Effect criterion: cure: the chloasma complete obiteration, skin color is normal; Produce effects: chloasma disappears more than 50%; Effectively: chloasma reduces less than 50%, and the speckle color shoals; Invalid: mottle disappears not as good as 20% or changes not obvious person.Result: cure 104 examples, account for 38.88%; Produce effects 82 examples account for 30.55%; Effective 52 examples account for 19.44%; Invalid 30 examples account for 11.2%, and the statistics total effective rate is 88.8%.