CN103110534B - Combined essence of composite velvet antler polypeptide lyophilized powder and nano-scale buffering microemulsion for skin beauty and health care and preparation method of combined essence - Google Patents
Combined essence of composite velvet antler polypeptide lyophilized powder and nano-scale buffering microemulsion for skin beauty and health care and preparation method of combined essence Download PDFInfo
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Abstract
The invention relates to the fields of cosmetic for skin beauty, health care, skin aging delay and the like, and particularly relates to combined essence of composite velvet antler polypeptide lyophilized powder and nano-scale buffering microemulsion for skin beauty and health care and a preparation method of the combined essence. The preparation method comprises the following steps of: extracting separated composite velvet antler polypeptide lyophilized powder with high efficiency and natural biological activity from fresh velvet antler, and preparing the combined beauty essence by using the composite velvet antler polypeptide lyophilized powder and nano-scale buffering microemulsion capable of prompting transdermal absorption of functional components at a ratio. Therefore, the combined essence provided by the invention has the advantages of fast transdermal absorption speed of the functional components, long biological activity storage period, high biological use degree, good action effect and safe utilization. Furthermore, the combined essence has a terse preparation process and simple technical operation; not only can the energy and the cost be saved, but also the low-carbon environmental friendliness can be achieved, and an environment is prevented from being polluted; and the combined essence provided by the invention is applicable to industrialized mass production, and popularization and application.
Description
Technical field
The present invention relates to a kind of combination essence that can be used for skin beauty health-care and delay skin aging and preparation method thereof.It is use from fresh Cornu Cervi Pantotrichum extract compound antler polypeptide lyophilized powder that the antler polypeptide that separates, have natural bioactive and protective agent thereof are main composition, with there is the combination beauty treatment essence that promotes that the nanoscale buffering microemulsion of functional component Transdermal absorption is made into by a certain percentage, be specifically related to a kind of compound antler polypeptide lyophilized powder for skin beauty health-care and combine essence and preparation method thereof with nanoscale buffering microemulsion.
Background technology
One, the technical background of beauty treatment essence:
Essence is by extraction from some animal viscera or tissue, natural plants or Chinese herbal medicine, marine organisms, microorganism etc., separation, purification natural functional component or bioactive substance or skin-nourishing element etc. out, add in some high-quality liquid make-up and can be prepared into the beauty treatment essence with different beauty functions and skin anti-aging effect according to certain ratio, this is one of dosage form of current cosmetics and the favor of skin anti-aging external preparation Zhong Shou consumer and welcome.Be no matter at present which country essence product in the world, also no matter be the essence of which brand, no matter wherein contain what composition or there is the essence of which kind of function, have nothing but two kinds of dosage forms, i.e. two kinds of water aqua type essence and oil-type essences.In China, at present, mostly contain the needed nutrient of some skin histology and cells survival and growth thereof among the essence of beautifying face, cell growth factor, antioxidation biology enzyme, Moisture factor, biological active matter, natural plants and Chinese herbal medicine extract and plant volatile oil, and collagen hydrolysate liquor, tire Ox blood plasma or serum etc., therefore be born such as skin anti-aging essence, skin-nourishing essence, skin whitening essence, skin wrinkle resisting essence, skin moisture-keeping essence, skin speckle dispelling essence, breast enlargement body shaping essence, slimming essence, champignon essence etc.People think habitually, as long as often use at ordinary times that it just has that certain skin is anti-ageing, anti-old, crease-resistant, moisturizing, whitening, go the effects such as speckle.Occur when naturally aging of skin more wrinkle or because of seasonal variations skin become dry coarse, because of bad habit become face gloomy unglazed, during because of long-time illumination skin pigment intensification, all can the dissimilar essence of choice for use.
Two, the technical background of antler polypeptide
In recent years, the research worker of association area is in Cornu Cervi Pantotrichum, particularly fresh Cornu Cervi Pantotrichum, some have that highly biological active substance and functional component are studied, development and application, as antler polypeptide and the work of antler growth factor action oriented research are launched just successively.Due to Cornu Cervi Pantotrichum oneself growth and Regeneration and Repair speed very fast, therefore people infer in Cornu Cervi Pantotrichum and may have certain and generally organize pilose antler active polypeptide factors different or that kind is more, content enriches more (mainly comprising antler polypeptide and somatomedin), thereby control, effective stimulus the Fast Growth of Cornu Cervi Pantotrichum, and are promoted the rapid hypertrophy of Cornu Cervi Pantotrichum sclerotin and epidermal area in order.At present, antler polypeptide is studied and is applied by relevant medical domain: (1) is for wound healing: antler polypeptide can accelerate the formation of Experimental Fracture rat callus and the healing of fracture, improve the biomechanical property of fracture site, accumulation and calcium deposition to ossein in callus have facilitation; Antler polypeptide or a kind of short cell mitogen agent, can stimulate chondrocyte and Osteoblast like Cells; And antler polypeptide has repair to rat back skin injury, epidermis cell and fibroblast proliferation are also had to obvious facilitation, can accelerate skin wound healing.(2) for repairing of neural injury: total Velvet Antler Polypeptides can promote Differentiation of Neural Stem Cells in vitro.It can promote neuranagenesis after Rats'Sciatic Nerve Injury, accelerate its neural axon growth speed, contributes to the recovery of sciatic nerve function.Antler polypeptide can also promote peripheral nerve growth, is mainly by expanding neural periphery and internal blood vessel, improves the microenvironment of injured nerve, improves the effects such as arteriole blood flow and realizes; Meanwhile, also there is the removing of accelerating injured nerve degeneration necrosis material, increase the supply to neuranagenesis material, accelerate the recovery of axoplasm, and then prevent neuronal necrosis, promote the early stage regeneration function of nerve injury.(3) promote for immunologic function: murine interleukin and marrow nucleated cell decreasing that Cornu Cervi Pantotrichum mixed polypeptide has antagonism cyclophosphamide to induce, can improve total serum protein, albumin and globulin content and cytophagous phagocytic function; Hematid immunity function, special or non-specific immunity are had to certain castering action.(4) for antiinflammatory action: antler polypeptide can be brought into play obvious inhibitory action to various Acute and chronic inflammations, and can make adrenocortical irritability and stimulation strengthen.And antler polypeptide has an obvious antiinflammatory action.(5) improve antioxidation: antler polypeptide can improve the ability of mice removing free radical, reduces malonaldehyde (MDA) content in animal tissue's brain and hepatic tissue, suppresses cytolipin levels of peroxide and the biomembrane extent of damage; And can suppress in vitro rat brain, liver, the reaction of kidney foundation of microsomal Lipid Peroxidation and reduced form cytochrome C and form, improve the antioxidation of body.(6) for tumor suppression: to Clonal Rat Pheochromocytoma tumor, strain has significant short differentiation to antler polypeptide, and the propagation of energy inhibition tumor cell.Meanwhile, can make the life span significant prolongation of tumor model animal, antitumor action manifests.(7) sexual function is affected to: antler polypeptide increases lutropin content in adenopituicyte culture fluid, and testicle spermatogonia number increases, and the spermatogenic cell number of plies increases testosterone element secretion increasing.But, people's past is to traditional the decocting of the many employings of the extraction of fresh Cornu Cervi Pantotrichum, boil, endure or toast, air-dry, pulverize, the ancient modes such as wine bubble, destroy to a great extent thermo-labile, the polypeptide of changeableness, protein isoreactivity composition, the bioactive substance and the functional component thereof that wherein can be used for external preparation for skin aspect are few, mensuration and the analysis of adding over to fresh Cornu Cervi Pantotrichum functional component focus mostly at aspects such as general constituent and common biochemical components, therefore, Cornu Cervi Pantotrichum bioactive substance and functional component be fresh few application aspect skin, application aspect cosmetics aesthetic health care and skin anti-aging is more rare.
In recent years, Chinese scholars finds and has reported in fresh Cornu Cervi Pantotrichum, not only to have the antler polypeptide that can promote cytoribosome and protein synthesis in succession, but also extract and separated the cell growth factor with polypeptide active substance from fresh Cornu Cervi Pantotrichum, and prove that this pilose antler active polypeptide and cytokine thereof are approximately made up of 68 aminoacid, its molecular weight moons 7200 dalton, the character of sterling antler polypeptide is pale yellow powder, belongs to water-soluble substances, soluble in water.Also confirm simultaneously, in the antler polypeptide separating in extraction, also contain the various kinds of cell somatomedin that other can promote cell division and propagation, as the keratinocyte factor (KGF), epidermal growth factor (EGF), fibroblast growth factor (FGF), insulin like growth factor (IGF), platelet derived growth factor (PDGF), vascular endothelial cell growth factor (VEGF), nerve growth factor (NGF), transforming growth factor (TGF) etc., be really an antler polypeptide and cell growth factor word bank.And these have antler polypeptide and the cell growth factor of natural bioactive, be important functional component and the bioactive substance that at present generally acknowledged skin beauty health-care, delay skin aging and skin barrier function rebuild.Therefore, the discovery of antler polypeptide and cell growth factor thereof separates with extracting, for its application among skin beauty health-care is laid a good foundation.
Three, the technical background of microemulsion
Microemulsion (microemulsion, ME) be a kind of outward appearance clear being formed by water, oil phase, surfactant and cosurfactant, size droplet diameter is less than 100nm's and all very stable liquid systems of thermodynamics and kinetics.Microemulsion is famous with minimum microgranule, its emulsion droplet can freely be dispersed among another kind of liquid and form dispersion system of colloid, its emulsion droplet shape mostly is spherical, and the basic uniformity of size, good fluidity, even can not make it layering through long-time placement, heating or autoclaving or centrifugalize under uniform temperature yet.Even in phase composition critical point, destroyed because temperature changes, but in the time returning to normal temperature, this system can independently return to again microemulsion state.Microemulsion and common emulsion have three basic points: the first, and the formation of ordinary emulsion generally needs the external world that energy is provided, and microemulsion is spontaneous formation; The second, ordinary emulsion is thermodynamic unstable system, deposit in process generation is coalescent and be finally divided into oil, water is biphase.Microemulsion is the equal stabilising system of thermodynamics and kinetics, can not occur coalescent.The 3rd, the particle diameter of ordinary emulsion is conventionally at micron order, and the particle diameter basic controlling of microemulsion is at 10-100nm, and can be used as carrier fat-soluble and water-soluble nutrient substance and functional component.Nutrient substance in microemulsion and functional component, according to its lipotropy and hydrophilic difference, may mainly be present in oil phase, water, oil-water interfacial film phase, or distribute between each phase.The character of dissociating, the lipid of nutrient substance and functional component may change to some extent, and like this, nutrient substance just may present different stability and distribution characteristics etc. thereof in microemulsion system from functional component.Microemulsion is as functional component carrier and promote Transdermal absorption penetrating agent, compared with emulsion, microemulsion particle diameter is small to nanoscale and distribute very evenly, therefore, more be conducive to merge mutually and be uniformly distributed with bioactive substance and functional component, make its transdermal effect better, bioavailability is higher; And compared with other, microemulsion character is more stable, and the easier industrialization of preparation or industrialization with liposome etc.
the weak point of current similar technology or product
The at present weak point of beauty treatment essence application: the production of beauty treatment essence is easy, processing ease, easy to use and truly have lot of advantages and advantage in practical application and action and efficacy thereof, but its obvious defect and deficiency also have much.The weak point of beauty treatment essence application mainly contains following aspect at present: first, the ethanol that great majority contain variable concentrations for the essence of beautifying skin, when use, there is certain olfactory stimulation and skin irritation, particularly some is easy to cause skin allergy and xerosis cutis to the skin quality of alcohol sensible, and eyes are also had to zest in various degree, second, the beauty treatment effect of essence and effect mainly depend on wherein contained or the functional component and the biological activity thereof that add, but a lot of functional components and biological activity thereof are easy to degraded or hydrolysis among liquid or solvent, to such an extent as to much the molecular structure destruction of functional components, loss of bioactivity, physiological function reduce, the 3rd, easily volatilization and lost part solvent of the frequent unlatching of beauty treatment essence, wherein dissolubility and the stability of functional component are reduced, not only affect sense organ and the quality of product, and have influence on stability and the actual effect of product, therefore, essence be conventionally difficult to ensure deposit and storage period not oversize, the 4th, beauty treatment essence is in frequent use procedure as do not note health, is easy to make it to pollute, grow a large amount of harmful or invasive organisms, thereby affects skin health, even cause cross infection, the 5th, for promoting functional component and bioactive substance Transdermal absorption, some beauty treatment has been added certain density short transdermal penetration agent in essence, and some short transdermal penetration agent more easily causes that skin allergy or stimulation, excessive use even have certain bio-toxicity (as DMSO etc.), the 6th, even great majority are that water-soluble substances has wherein added some oil soluble material in beauty treatment essence, need to add some emulsifier or co-emulsifier and just can make that it is dispersed, dissolve completely, even but add some emulsifier or co-emulsifier and be also difficult to maintain the permanent stability of essence and some emulsifying agent or co-emulsifier and use and once had the report that causes skin allergy, the 7th, the more important thing is, solvent in essence itself does not have the effect that promotes functional component transdermal penetration and absorption, even people's artificial some so-called short skin penetrants that add in essence at present, but its actual short transdermal penetration effect is very limited again, particularly due to the histological characteristic of skin barrier structure, some exogenous materials are strictly limited, comprise protein, water, sugar, lipid, vitamin, the materials such as inorganic ions or cosmetic industry composition pass through, most functional components all cannot or be difficult to bring into play due biological action by complete keratodermatitis barrier, therefore, the bioavailability of adding functional component in essence and bioactive substance to is less, biological action and result of use are limited.
The weak point of antler polypeptide application at present: antler polypeptide has biological action and application widely as having bioactive functional component, but antler polypeptide is mainly applied at present in medical research and part clinical field, there is not yet the formal product of applying in skin beauty health-care and delay skin aging.And, if adopt antler polypeptide prepared by existing method to apply and still have certain limitation and weak point in skin beauty health-care and delay skin aging product.Be mainly manifested in: (1) is though adopt traditional heating, strong acid or pheron to be hydrolyzed that antler polypeptide content of peptides that fresh Cornu Cervi Pantotrichum obtains is higher and yield is higher, but heating, strong acid or pheron method for hydrolysis be more wasting energy and contaminated environment not only, and gained antler polypeptide has lost natural bioactive to a great extent, when practical application, its biological action and physiologic function are limited, are also difficult to bring into play to greatest extent and reach the effect that antler polypeptide should have; (2) adopt conventional at present acid alcohol method to extract separating obtained antler polypeptide, its molecular conformation, natural characteristic etc. also increase compared with the more reservation of traditional method, biologic activity, but acid alcohol method gained antler polypeptide is comparatively coarse, purity is not high and contain more insoluble impurities and corticosteroid hormone and gonadal hormone, and these are directly used in skin beauty health-care and delay skin aging aspect may exist skin irritation and irritated risk and side effect etc.; (3) adopt column chromatography or high performance liquid chromatogram to be further purified, though can obtain molecular weight distribution homogeneity better, higher, the good antler polypeptide of biological activity of purity, but adopt this method gained antler polypeptide not only yield be very low, complex operation, need that special instruments and equipment, application cost are high, price etc., and this method is difficult to industrialization and expands examination and large-scale production, also cannot popularization and application; Add that this method rejects other composition completely, comprises the functional component with Synergistic, therefore practical application effect is also very limited; (4) antler polypeptide is difficult for preserving and is easy to degraded, biological activity and also greatly reduces in liquid, and adopts lyophilized powder and be aided with protective agent and synergist is only preferred plan.
The weak point of microemulsion application at present: although have at present microemulsion product, some perhaps its particle diameter also can reach nanoscale, but the microemulsion of preparation at present, although its prescription and ratio thereof are not quite similar, particularly its water is all with deionized water or distilled water, though this is most important to whole formation of microemulsion, but adopt distilled water as water preparation microemulsion to its pH value without buffering and Stabilization, more particularly for beautifying skin, defying age, Regeneration and Repair, promote functional cosmetics such as skin cell proliferation to there are requirements at the higher level to the stability of pH value, especially for the active polypeptide and the cytokine class cosmetics that promote epidermis and hypodermal cell propagation, not only need best pH value environment, and, also need stable pH condition could more be conducive to Skin Cell growth and propagation, moreover, Skin Cell existence, growth and division, propagation also need certain ion concentration and osmotic pressure, and these, only adopt original water (distilled water) used be cannot possess, be also defect and the deficiency of its application.Water in nanoscale microemulsion formula of the present invention abandons distilled water and the current retrieval of 0.01mol/L pH7.2 phosphate buffered saline that adopts not yet finds that there is people time prepared by microemulsion, uses, utilize this microemulsion formula and preparation method not only for Skin Cell is grown and propagation provides good microenvironment, and, also for it provides comparatively stable and suitable pH condition and superior ion concentration and osmotic pressure; Simultaneously, also available buffer or neutralize this product after being applied to skin of face because the pH value of skin of face is compared with low and occur fluctuation or the decline of whole microemulsion system pH value, thus be more conducive to the growth of skin tissue cell and giving full play to of propagation and regenerative cell physiological function.
Summary of the invention
The present invention is directed to some defect and the deficiency of current correlation technique and preparation thereof, invent a kind of compound antler polypeptide lyophilized powder for skin beauty health-care and nanoscale buffering microemulsion combination essence.Antler polypeptide in compound antler polypeptide lyophilized powder in this combination essence not only has the features such as good native conformation, biological activity and molecular weight are little, and in compound antler polypeptide lyophilized powder, also has antler polypeptide active protection and the synergetic effect additive of coupling completely; Nanoscale in this combination essence buffering microemulsion not only have particle diameter little, be evenly distributed, instant compound antler polypeptide lyophilized powder, promote functional component and bioactive substance infiltration and the advantage such as Transdermal absorption is good, also there is skin tissue cell and grow and breed the microenvironments such as the most favourable and suitable pH value, ion concentration and osmotic pressure.And, compound antler polypeptide lyophilized powder and nanoscale buffering microemulsion combination essence, its percutaneous absorbability is better, stability is better, sense organ is good, health giving quality is better, it is safer to use and convenience.And, the present invention also aims to provide a kind of compound antler polypeptide lyophilized powder for skin beauty health-care and nanoscale buffering microemulsion to combine the preparation method of essence.Meanwhile, for the problem such as some defect and deficiency of above-mentioned correlation technique and preparation thereof, the scheme that the invention provides these defect problems of solution is as follows.
Design principle of the present invention
General principle of design of the present invention is: utilize antler polypeptide efficient natural biological activity and the mutual aid synergism after compound has with protective agent and synergist unique skin anti-aging, epidermis reparation and skin barrier function are rebuild with aesthetic health care biological effect and physiology's effect and antler polypeptide biological activity and be difficult for preserving and be prepared into lyophilized powder etc. for a long time in liquid; Simultaneously; utilize nanoscale buffering microemulsion to there is good solubility and pardon and the activating effect to antler polypeptide and protective agent and synergist; particularly nanoscale buffering microemulsion transmits the promotion antler polypeptide of carrier and the Transdermal absorption effect of protective agent and synergist thereof etc. as good percutaneous, invents a kind of compound antler polypeptide lyophilized powder for skin beauty health-care and nanoscale buffering microemulsion combination essence.Specific design principle of the present invention is: first design a kind of can success from fresh Cornu Cervi Pantotrichum, extract separate there is natural bioactive, Technology and the operational approach of the antler polypeptide of efficient physiological role obtain colory micromolecule antler polypeptide; There is natural bioactive, efficient physiological role and promote skin tissue cell propagation and associated protein is combined into, the micromolecule antler polypeptide of expression and secretion carries out compound preparation and prepares compound antler polypeptide lyophilized powder according to suitable ratio with different types of antler polypeptide freeze drying protectant and synergetic effect additive this again, make it to have longer action effect, better synergy and storage life more of a specified duration etc.; Then, design and prepare and can mate completely, mix with this compound antler polypeptide lyophilized powder and there is the nanoscale buffering microemulsion that promotes antler polypeptide and protection synergist Transdermal absorption thereof, and solvent and percutaneous transmission and Transdermal absorption carrier using it as compound antler polypeptide lyophilized powder; Finally, manufacture experimently and determine compound antler polypeptide lyophilized powder and nanoscale buffering microemulsion optimal proportion and make the combination essence with skin anti-aging and beauty and health care functions.Make this compound antler polypeptide lyophilized powder for skin beauty health-care of the present invention and nanoscale buffering microemulsion combination essence, before unmixed, can retain for a long time native conformation and the molecular structure of antler polypeptide, can continue again to bring into play height biological activity and the physiological function of antler polypeptide, also solve antler polypeptide long-term preservation in liquid and be easy to decompose or degrade and constantly lose the problems such as its biological activity; Simultaneously, the characteristics and advantages such as easy to use and comfortable that makes that nanoscale buffering microemulsion both retained that beauty treatment essence has, there is again the function that efficient percutaneous transmits carrier and promotes Transdermal absorption, also build and provide the cell micro-environment that is conducive to skin tissue cell growth, growth, propagation and metabolism the best, be suitable nutritional condition, somatomedin, pH value, ion concentration, osmotic pressure etc., make this compound antler polypeptide lyophilized powder and nanoscale buffering microemulsion combination essence there is in actual applications better skin anti-aging and aesthetic health care effect.
Technical scheme of the present invention
(1) the present invention combines prescription and the ratio thereof of essence:
1. the compound antler polypeptide lyophilized powder for skin beauty health-care and nanoscale buffering microemulsion combination essence, it is characterized in that it is made up of compound antler polypeptide lyophilized powder and two parts of nanoscale buffering microemulsion with skin anti-aging and beauty and health care functions, its percentage ratio that raw material forms is separately as follows respectively:
(1) compound antler polypeptide lyophilized powder raw material composition percentage by weight is: antler polypeptide: 0.01～0.50; Mannitol: 0.10～1.50; Glycine: 0.10～1.50; Proline: 0.10～1.50; Hydroxyproline: 0.10～1.50; Albumin: 0.20～2.50.
(2) nanoscale buffering microemulsion raw material composition percentage by weight is: pH7.2 phosphate buffered saline (0.01mol/L): 20.0～60.0; Isopropyl myristate: 5.0～25.0; Labraso: 15.0～55.0; Polyglyceryl fatty acid ester: 5.0～30.0.
(3) compound antler polypeptide lyophilized powder and nanoscale buffering microemulsion combination essence composition percentage by weight are: compound antler polypeptide lyophilized powder: 0.01-10.0; Nanoscale buffering microemulsion: 90.0-99.99.
2. for the compound antler polypeptide lyophilized powder of skin beauty health-care and a nanoscale buffering microemulsion combination essence, it is characterized in that preferred percent that it be made up of the each raw material of compound antler polypeptide lyophilized powder with skin anti-aging and beauty and health care functions and nanoscale cushion the preferred percent that the each raw material of microemulsion forms and distinguish as follows:
(1) preferred weight percent of compound antler polypeptide lyophilized powder raw material composition is: antler polypeptide: 0.02; Mannitol: 0.50; Glycine: 0.50; Proline: 0.50; Hydroxyproline: 0.50; Albumin: 0.98.
(2) preferred weight percent of nanoscale buffering microemulsion raw material composition: pH7.2 phosphate buffered saline (0.01mol/L): 36.0; Isopropyl myristate: 14.0; Labraso: 37.5; Polyglyceryl fatty acid ester: 12.5.
(3) percentage by weight of compound antler polypeptide lyophilized powder and nanoscale buffering microemulsion combination essence composition: compound antler polypeptide lyophilized powder: 3.0; Nanoscale buffering microemulsion: 97.0.
3. for the compound antler polypeptide lyophilized powder of skin beauty health-care and a nanoscale buffering microemulsion combination essence, it is characterized in that they are made up of 3% compound antler polypeptide lyophilized powder and 97% nanoscale buffering microemulsion.3% compound antler polypeptide lyophilized powder is made up of antler polypeptide, mannitol, glycine, proline, hydroxyproline and albumin; 97% compound antler polypeptide lyophilized powder solvent-nanoscale buffering microemulsion by: water (0.01mol/LpH7.2 phosphate buffered saline), oil phase (isopropyl myristate), surfactant (Labraso) and cosurfactant (polyglyceryl fatty acid ester) are formed.
(2) the present invention combines essence technology of preparing and operational approach:
1, the technology of preparing of compound antler polypeptide lyophilized powder and operational approach:
(1) accurately take just from male sika deer set up a separatist regime by force of arms get off containing the fresh Cornu Cervi Pantotrichum of blood, and be cut into after fritter, directly put among the container of high speed electric homogenizer.
(2) preparation pH4.0 acetic acid-sodium acetate (HAc-NaAc) buffer solution is as fresh Cornu Cervi Pantotrichum acidolysis homogenate; Then, according to the ratio of 10 times, fresh Cornu Cervi Pantotrichum, this acidolysis homogenate of prior pre-cooling being added to, have among the high speed electric homogenizer of fresh Cornu Cervi Pantotrichum fritter, and carry out immediately acidolysis homogenate.
(3) collect and merge above-mentioned fresh Cornu Cervi Pantotrichum acidolysis homogenate, being positioned over immediately High speed refrigerated centrifuge high speed centrifugal, leaving and taking the supernatant (supernatant) after high speed centrifugation, and measuring its volume with graduated cylinder.
(4) after this supernatant is filtered with coarse filter, then be that 100,000 daltonian polysulfone hollow fibre ultrafilter membranes carry out ultrafiltration with ultrafilter and molecular weight; The careful collection daltonian whole permeate in molecular weight≤100,000 (ultrafiltration permeate first), and metering volume (ml).
(5) be that 10,000 daltonian polysulfone hollow fibre ultrafilter membranes carry out ultrafiltration again by daltonian molecular weight≤100,000 whole permeate (ultrafiltration permeate first) with ultrafilter and molecular weight; The careful collection daltonian whole permeate in molecular weight≤10,000 (second ultrafiltration permeate), and metering volume (ml).
(6) be that 300 daltonian NF membrane concentrate the excessive permeate of above-mentioned volume by daltonian molecular weight≤10,000 whole permeate (second ultrafiltration permeate) with nanofiltration device and molecular weight, the concentrated solution (three nanofiltration trapped fluids) that can obtain being held back by NF membrane, wherein contains molecular weight antler polypeptide and cytokine between 300-10000 dalton, relevant to beautifying skin and defying age.And after this NF membrane, can remove gonadal hormone and the corticosteroid hormone of beautifying skin forbidding, as testosterone element (molecular weight is 288.42), hydrocortisone (molecular weight is 362.46), female alcohol (molecular weight is 270), estradiol (molecular weight is 272.38), estriol (molecular weight is 288), Progesterone (molecular weight is 314.47) etc.
(7) detect and adjust antler polypeptide content in NF membrane trapped fluid, it is reached after 2mg/ml, get again this adjustment liquid 100ml (antler polypeptide total content 0.20g), add respectively mannitol 5.0g, glycine 5.0g, proline 5.0g, hydroxyproline 5.0g, albumin 9.8g, fully mix homogeneously.
(8) the above-mentioned antler polypeptide complex liquid containing antler polypeptide concentrated solution and protective agent and synergist is divided and is filled in the cillin bottle that specification is 10ml after 0.45 μ m bacteria filter ultrafiltration, in every cillin bottle, add this complex liquid 1.0ml, so can 100 cillin bottles of subpackage in; Then, according to cryodesiccated normalization procedure, be placed into and in freezer dryer, carry out lyophilization, can obtain the compound antler polypeptide lyophilized powder containing antler polypeptide 2mg, mannitol 50mg, glycine 50mg, proline 50mg, hydroxyproline 50mg, albumin 98mg in each cillin bottle, after adding a cover, carry out immediately gland seal, and save backup in-20 DEG C of cryogenic refrigerators.
2, the preparation of nanoscale buffering microemulsion:
(1) at ambient temperature, first according to Labraso: polyglyceryl fatty acid ester=3: 1 ratio, accurately draw Labraso 375ml and polyglyceryl fatty acid ester 125ml, and be placed among the 1000ml container of same cleaning; Then, more fully stir with JB-3A type time constant-temperature magnetic stirring apparatus, rotating speed 200rpm/min, 10min, makes it to mix completely the mixed liquor 500ml into Labraso and polyglyceryl fatty acid ester continuously.
(2) measure respectively 360ml pH7.2 phosphate buffered saline (0.01mol/L) and 140ml isopropyl myristate, add to respectively in 500ml Labraso and polyglyceryl fatty acid ester mixed liquor; Fully stir with time constant-temperature magnetic stirring apparatus, rotating speed 200rpm/min, 30min continuously, can obtain total amount of liquid is the water white nanoscale buffering microemulsion of 1000ml.
(3) this nanoscale buffering microemulsion is divided respectively and is filled in the water white cillin bottle that specification is 10ml, in each cillin bottle, add this liquid 10ml, so can divide and install in 100 cillin bottles; After adding a cover, carry out immediately gland seal, and be placed in cool place, dry, ventilation saves backup.Like this, the cillin bottle quantity of nanoscale buffering microemulsion is housed identical with the quantity of the cillin bottle that contains compound antler polypeptide lyophilized powder.
(4) now, the preferred weight percent of this nanoscale buffering microemulsion raw material composition just in time: pH7.2 phosphate buffered saline (0.01mol/L) 36.0, isopropyl myristate 14.0; Labraso 37.5, polyglyceryl fatty acid ester 12.5.
3, preparation and the usage of compound antler polypeptide lyophilized powder and nanoscale buffering microemulsion combination essence:
(1) preparation of compound antler polypeptide lyophilized powder and nanoscale buffering microemulsion combination essence: respectively get 1 cillin bottle open bottle cover that compound antler polypeptide lyophilized powder and nanoscale buffering microemulsion are housed.Immediately nanoscale is cushioned to microemulsion and be poured in compound antler polypeptide lyophilized powder, it is repeatedly put upside down and mixes gently 3min, until compound antler polypeptide lyophilized powder is completely dissolved; With the dropper on water white transparency drop bottle, this complete lysate is all transferred in the water white transparency drop bottle with dropper and mixed, adds a cover again, become the compound antler polypeptide lyophilized powder of 10ml and nanoscale buffering microemulsion combination essence, can directly be carried out skin or facial external.Due in every cillin bottle that has a compound antler polypeptide lyophilized powder containing antler polypeptide 2mg, mannitol 50mg, glycine 50mg, proline 50mg, hydroxyproline 50mg, albumin 98mg, and every lyophilized powder just can preparation cost invention combine essence after need to adding 10ml nanoscale buffering microemulsion, that is contain antler polypeptide 2mg, mannitol 50mg, glycine 50mg, proline 50mg, hydroxyproline 50mg, albumin 98mg in every 10ml combination essence.As calculated with preparation 100ml combination essence, by 10 times of the corresponding expansions of each composition in 10ml combination essence, in every 100ml combination essence, in compound antler polypeptide lyophilized powder, the degree of each material is just in time: antler polypeptide: 0.02; Mannitol: 0.50; Glycine: 0.50; Proline: 0.50; Hydroxyproline: 0.50; Albumin: 0.98.And the percentage by weight of compound antler polypeptide lyophilized powder and nanoscale buffering microemulsion combination essence composition also just in time: compound antler polypeptide lyophilized powder 3.0, nanoscale buffering microemulsion 97.0.
(2) using method of compound antler polypeptide lyophilized powder and nanoscale buffering microemulsion combination essence: before formal use, first remove the aging cutin (only in the time using first) of skin of face according to medical cosmetology Standardization Requirement; While use, according to the clean skin of face of clean method of routine later; After skin bone dry, i.e. desirable the present invention combines essence 10-20 and drips among palm and make it dispersed; Then, embrocate gently or pat face with this, until it is substantially dried and absorbs cleanly; As still dry in skin, can repeat again once by this method; Used 2-3 every day, and using continuously 30 days is a life cycle.As some aging face comparatively obviously or severe patient, can use 3 cycles continuously until obtain positive effect according to above-mentioned usage.
innovative point of the present invention and beneficial effect
1, antler polypeptide is used for oral to improve the purposes such as body's immunity as main composition at present, also have antler polypeptide as injection for bone injury reparation and promote the purposes such as osteoblastic proliferation, there is not yet at present report both at home and abroad but become to be used for preparing skin protection cosmetics and cosmetic health product and skin anti-aging product using antler polypeptide as main effect.The present invention adopts compound antler polypeptide lyophilized powder and the rationally application of nanoscale buffering microemulsion and mutual supplement with each other's advantages binding pattern matching while using combination essence, and be directly used in nutrition skin-protection, epidermal cell proliferation and activation, and crease-resistant, speckle dispelling, laser, epidermis injury and keratodermatitis that grinding or fruit acid Exfoliating Scrub (the aging cutin layer of no cell structure) excessively etc. cause strip off rear reparation, this is first for the skin beauty health-cares such as epidermal barrier function reconstruction and skin anti-aging field, and at its compositional system and prescription element and Proportionality design, with and preparation method thereof there is novelty with aspects such as application models.In other words, there is not yet at present the product of the preparation using antler polypeptide as main composition both at home and abroad for all essences of skin anti-aging and aesthetic health care, the more compound antler polypeptide lyophilized powder of no-trump and nanoscale buffering microemulsion are according to the combination essence of specific prescription and ratio matching while using.
2, prepare at present with commercially available antler polypeptide is many and Related product is mainly derived from traditional handicraft and acid alcohol method is extracted the crude product antler polypeptide separating from Cornu Cervi Pantotrichum, wherein not only contain steroid 17-hydroxy-11-dehydrocorticosterone and the gonadal hormone of in more insoluble impurities and cosmetics product, prohibiting interpolation and (comprise estradiol, estriol, Progesterone, testosterone element, dehydroepiandrosterone, hydrocortisone etc.), and, the native conformation of these antler polypeptides is all affected in the time that extraction separates in various degree with molecular structure and biological activity etc., even occur obviously changing, restriction and affected its range of application and result of use.But, not only antler polypeptide content is high for the compound antler polypeptide that adopts the present invention to prepare, molecular weight, and, contain the multiple cell growth factor relevant to skin anti-aging and aesthetic health care (as epithelical cell growth factor, fibroblast growth factor, transforming factor, insulin like growth factor) etc. there is the collaborative composition of potentiation of natural bioactive, especially adopting in preparation method of the present invention adopts the daltonian NF membrane of molecular weight 300 to carry out after ultrafiltration, not only play the regulating and controlling effect to antler polypeptide concentration, and steroid 17-hydroxy-11-dehydrocorticosterone and gonadal hormone intrinsic in fresh Cornu Cervi Pantotrichum are removed.
3, to extract the raw material that antler polypeptide adopts be containing the fresh Cornu Cervi Pantotrichum of blood for preparation method of the present invention, traditional method and acid alcohol method extract antler polypeptide be adopt through a large amount of tap waters rinse and distilled water rinsing do not contain XUELURONG.Sanguis Cornu Cervi Pantotrichum just source itself is rare, very rare and precious and contain the bioactive substances relevant to skin beauty health-care and delay skin aging that enrich in a large number.The present invention is used not only can obtain in Cornu Cervi Pantotrichum the antler polypeptide of high-quality more containing XUELURONG, and, also can obtain Sanguis Cornu Cervi Pantotrichum and comprise that antler polypeptide is at interior bioactive substance simultaneously; The more important thing is, directly adopt just and set up a separatist regime by force of arms from male sika deer the fresh Cornu Cervi Pantotrichum extraction antler polypeptide getting off, also can greatly simplify extraction process, increase work efficiency, save preparation time, improve the polypeptide utilization of resources, alleviate environmental pollution, reduce water waste, reduce production cost etc.
4, the scope of the more traditional acid alcohol method of the pH value pH3.0-3.5 of preparation method extraction antler polypeptide of the present invention acetic acid-sodium acetate buffer used system is brought up to pH3.8-4.0 scope, make polypeptide formation condition gentleer, macromole hydrolysis or degradation reaction are more reasonable, thereby, both can improve antler polypeptide yield, can obtain again more high-quality antler polypeptides, can also better keep its native conformation and biological activity, and, can also retain wherein more many cells somatomedin etc. and skin beauty health-care and delay skin aging associated biomolecule active substance.
5, when preparation method of the present invention is extracted antler polypeptide, thoroughly abandoned and in traditional acid alcohol method, added the contaminated environment such as high-purity and high concentration ethanol, dangerous, the chemical extraction partition method that also can make part of polypeptide degeneration again, and change into by physical partition methods such as different molecular weight ultrafilter membranes, these innovations not only can obtain the antler polypeptide of higher yield, and can better keep its native conformation and biological activity, and can also retain wherein more many cells somatomedin etc. and skin beauty health-care and delay skin aging associated biomolecule active substance.
6, while application among skin anti-aging and aesthetic health care as natural biological preparation; main composition antler polypeptide not only needs the long period to retain its original molecular structure and natural bioactive thereof; and; also need composite other protective agent and synergist and mutual aid synergist thereof, to strengthen and to improve the recent and long-term effect of its biological action and physiologic function.Composition is worked in coordination with in compound antler polypeptide lyophilized powder of the present invention has been pressed skin anti-aging and beauty and health care functions effect appropriate design just corresponding protective agent and synergist and mutual aid thereof, and integral formula in compound antler polypeptide lyophilized powder is also different with current Related product from combination and ratio thereof.
7, in the present invention, compound antler polypeptide lyophilized powder and nanoscale buffering microemulsion is all suitable for industrialization, is also easy to large-scale mass production.According to the invention process, those skilled in the relevant art are easy to realize.Therefore, be conducive to the universal of this invention and apply.
8, compound antler polypeptide lyophilized powder is the important functional component of the present invention and bioactive substance, be designed to solid-state form, not only be easy to packaging and transport, be also not easy fragmentation and leakage, be also not easy to be subject to pollute, pathogenic microbes is also difficult to grow and breed; And, the very easily preservation of this solid-state form of compound antler polypeptide lyophilized powder, low temperature and room temperature all can, greatly extend storage life and the operating period of this product.Meanwhile, the functional component such as antler polypeptide and bioactive substance are also difficult for decomposing or degraded when solid-state.Therefore, this solid-state form of compound antler polypeptide lyophilized powder can either retain the biological activity of its functional component, can keep again its biological action and physiologic function, and makes that its effect is better, effect is more obvious.
9, in compound antler polypeptide lyophilized powder except antler polypeptide; also has various kinds of cell somatomedin etc.; and protection, activation, collaborative and its biological action of potentiation and physiologic function assist a ruler in governing a country composition; make that biological activity is higher, constituent is more complete, bioavailability is better, biological effect is stronger; thereby; more be conducive to the performance of the collaborative and synergy effect of mutual aid, be also more conducive to performance skin anti-aging and beauty and health care functions multi-faceted, multi-level, many target spots.
10, compound antler polypeptide lyophilized powder as the most important functional component of the present invention, with and corresponding protective agent and synergist and the collaborative composition of mutual aid thereof as contained and activation without coupling, suitable, stable solvent system, will be difficult to reach the object of expection, also be difficult to bring into play to greatest extent its high-performance bio effect and best physiological role.Nanoscale buffering microemulsion of the present invention not only has feature and the actual effect that can dissolve fast with good protection antler polypeptide natural bioactive, and, this nanoscale buffering microemulsion can be the existence of original position skin tissue cell and regenerative cell, growth and proliferation and differentiation provide stable and good biochemical microenvironment, and, can also promote to greatest extent compound antler polypeptide and protective agent thereof, constantly infiltration and Transdermal absorption of the collaborative composition of synergist and mutual aid, make the compound antler polypeptide of the most important functional component of the present invention with and corresponding protective agent, the collaborative composition of synergist and mutual aid is brought into play to greatest extent its high-performance bio and is learned effect and best physiological role.
11, compound antler polypeptide lyophilized powder and nanoscale buffering microemulsion combination essence adopt the mode of matching while using, are also the differences that is different from conventional beauty treatment essence.According to the invention process, can either keep combining the quality of essence, can prevent that again the bioactive ingredients such as antler polypeptide from losing its biological activity because of the hydrolysis in essence substrate and solvent thereof or degraded.Particularly, adopt nanoscale buffering microemulsion as essence substrate and solvent, can make each composition in compound antler polypeptide lyophilized powder more easily dissolve and disperse; And, the combination essence that adopts nanoscale buffering microemulsion to prepare as essence substrate and solvent, granule is small, it is more comfortable and pleasant to be evenly distributed, to use; Simultaneously, the combination essence that adopts nanoscale buffering microemulsion to prepare as essence substrate and solvent, can carry more bioactive substances in compound antler polypeptide lyophilized powder, and effectively promote these bioactive substances and functional component Transdermal absorption, make its bioavailability better, make antler polypeptide more stable, biological action and with better function.
12, the water in nanoscale microemulsion formula of the present invention is abandoned distilled water and the current retrieval of 0.01mol/LpH7.2 phosphate buffered saline that adopts not yet finds that there is people and uses time prepared by microemulsion, also has no directly application in beauty treatment essence.Utilize this phosphate buffered saline to carry out microemulsion formula and preparation method, not only for Skin Cell is grown and propagation provides good microenvironment, and, also for it provides comparatively stable and suitable pH condition and superior ion concentration and osmotic pressure; Simultaneously, also available buffer or neutralize this product after being applied to skin of face because the pH value of skin of face is compared with low and occur fluctuation or the decline of whole microemulsion system pH value, thus be more conducive to the growth of skin tissue cell and giving full play to of propagation and regenerative cell physiological function.
Brief description of the drawings
Fig. 1. preparation technology's flow process and the Technology Roadmap of compound antler polypeptide lyophilized powder.
Fig. 2. preparation technology's flow process and the Technology Roadmap of nanoscale buffering microemulsion.
Fig. 3. preparation technology's flow process and the Technology Roadmap of combination essence.
Fig. 4. the contrast schematic diagram of administration side and control sides water content in experimental example 2.
Fig. 5. in experimental example 3, volunteer self-appraisal and researcher contrast schematic diagram to different time moisture of skin situation scoring after using product of the present invention.
Fig. 6. in experimental example 3, volunteer self-appraisal and researcher contrast schematic diagram to different time skin elasticity situation scoring after using product of the present invention.
Fig. 7. in experimental example 3, volunteer self-appraisal and researcher contrast schematic diagram to different time skin complexion situation scoring after using product of the present invention.
Fig. 8. in experimental example 3, volunteer self-appraisal and researcher contrast schematic diagram to different time skin microgroove situation scoring after using product of the present invention.
Detailed description of the invention
Implementation example 1: the preparation of compound antler polypeptide lyophilized powder:
(1) accurately take just from live body Cervus nippon Temminck set up a separatist regime by force of arms get off containing the fresh Cornu Cervi Pantotrichum 100g of blood, use sharp kitchen knife or hacksaw to be cut into size and be about 0.5cm
3the fritter of left and right, is placed in 4 DEG C of refrigerators and saves backup.
(2) use respectively clean dried and the volumetric flask preparation sodium acetate solution of 0.2mol/L and the acetum of 0.2mol/L of sterilizing; Then, accurately measure respectively sodium acetate solution 80ml and acetum 920ml, and be placed in same container and mix, adjust this pH to 4.0, be settled to 1000ml with distilled water again, make it to become acetic acid-sodium acetate (HAc-NaAc) buffer solution of pH4.0, build immediately capacity bottle stopper, and carry out pre-cold standby among being positioned over 4 DEG C of refrigerators at once.
(3) immediately the fresh Cornu Cervi Pantotrichum fritter of blood that contains of above-mentioned pre-cooling is all positioned among the container of FSH-II type high speed electric homogenizer, and using acetic acid-sodium acetate (HAc-NaAc) buffer solution of preparing the pH4.0 that also pre-cooling is good as fresh Cornu Cervi Pantotrichum acidolysis homogenate; Then in above-mentioned 1000ml pre-cooling acetic acid-sodium acetate (HAc-NaAc) buffer solution being added at twice in the ratio of 10 times, fresh Cornu Cervi Pantotrichum, have among the container containing the high speed electric homogenizer of the fresh Cornu Cervi Pantotrichum fritter of blood, start high speed electric homogenizer 20min at every turn and carry out acidolysis homogenate.
(3) gradation is collected and is merged Cornu Cervi Pantotrichum acidolysis homogenate, and it is centrifugal to be placed in immediately High speed refrigerated centrifuge high speed.The centrifuging temperature of High speed refrigerated centrifuge is 4 DEG C, and rotating speed is 8500rpm/min, centrifugation time 20min.
(4), after centrifugal completing, leave and take the supernatant (supernatant) after centrifugal, and with its volume of graduated cylinder accurate-metering.Then after this supernatant is first filtered with the coarse filter connecting in ultrafiltration apparatus, then be, that 100,000 daltonian polysulfone hollow fibre ultrafilter membranes carry out ultrafiltration first through molecular weight in this ultrafiltration apparatus; The careful collection daltonian whole permeate in molecular weight≤100,000 (ultrafiltration permeate first), and metering volume (ml).Meanwhile, the daltonian trapped fluid in reject molecular weight >=100,000 is for other purposes outside the present invention.
(5) be that 10,000 daltonian polysulfone hollow fibre ultrafilter membranes carry out ultrafiltration for the second time with ultrafilter and molecular weight again by daltonian above-mentioned molecular weight≤100,000 whole permeate (ultrafiltration permeate first); The careful collection daltonian whole permeate in molecular weight≤10,000 (second ultrafiltration permeate), and metering volume (ml).Meanwhile, reject molecular weight≤100,000 dalton and >=1 ten thousand daltonian trapped fluid are for other purposes outside the present invention.
(6) be that 300 daltonian NF membrane concentrate the excessive permeate of above-mentioned volume with nanofiltration device and molecular weight again by collecting the daltonian whole permeate in molecular weight≤10,000 (second ultrafiltration permeate), the concentrated solution (three nanofiltration trapped fluids) that can obtain being held back by NF membrane, wherein contains molecular weight antler polypeptide and cytokine between 300-10000 dalton, relevant to beautifying skin and defying age.Research is found, the molecular weight that separation and Extraction obtains from fresh Cornu Cervi Pantotrichum contains various kinds of cell somatomedin in 10000 dalton and following protein peptide matters thereof, as insulin like growth factor, epidermal growth factor, fibroblast growth factor, nerve growth factor subclass material, transforming growth factor etc.; And after this NF membrane, can remove gonadal hormone and the corticosteroid hormone of beautifying skin forbidding, as testosterone element (molecular weight is 288.42), hydrocortisone (molecular weight is 362.46), female alcohol (molecular weight is 270), estradiol (molecular weight is 272.38), estriol (molecular weight is 288), Progesterone (molecular weight is 314.47) etc.
(7) detect through antler polypeptide content in nanofiltration device and 300 dalton's NF membrane trapped fluids, then, adjust antler polypeptide content in NF membrane trapped fluid by mode concentrated or dilution, make it reach 2mg/ml.
(8) accurately draw the antler polypeptide liquid 100ml (antler polypeptide total content 0.20g) through being adjusted into 2mg/ml; Then, each mannitol 5.0g, glycine 5.0g, proline 5.0g, hydroxyproline 5.0g, albumin 9.8g of accurately taking, respectively they are joined among 100ml (antler polypeptide total content 0.20g) antler polypeptide liquid, finally fully mix with JB-3A type time constant-temperature magnetic stirring apparatus, 200 revs/min of rotating speeds, 10-20min continuously, until solid content is wherein completely dissolved.
(9) by the above-mentioned complex liquid that contains antler polypeptide concentrated solution and protective agent and synergist; be that 0.45 μ m bacteriological filtration film carries out can obtaining aseptic antler polypeptide complex liquid after heat sterilization through specification, require in superclean bench, it to be divided respectively immediately that to be filled to the specification of having sterilized be among 10ml cillin bottle according to sterile working.In every cillin bottle, add 1.0ml, so can 100 cillin bottles of subpackage in; Then, then according to cryodesiccated normalization procedure, be placed into and in freezer dryer, carry out lyophilization; Add a cover rapidly, and carry out gland seal immediately, can obtain the compound antler polypeptide lyophilized powder containing antler polypeptide 2mg, mannitol 50mg, glycine 50mg, proline 50mg, hydroxyproline 50mg, albumin 98mg in each cillin bottle; Then, it is placed in rapidly to freezing saving backup among-20 DEG C of refrigerators.
Implementation example 2: the preparation of nanoscale buffering microemulsion:
(1) using isopropyl myristate (IPM) as oil phase, Labraso (S) is as surfactant, polyglyceryl fatty acid ester (C) is cosurfactant, and pH7.2 phosphate buffered saline (0.01mol/L) is as water.Under room temperature by IPM with water respectively with 9.8: 0.2,9: 1,8: 2,7: 3.....2: the ratio of 8,1: 9,0.2: 9.8 is mixed, the mass ratio (S/C) of setting surfactant and cosurfactant is that Km value is respectively 1,2,3,4 and prepare S/C volumetric solution, the volumetric solution of preparation is used for to titration IPM and water and forms Binary Liquid.In titration process, in the time that Binary Liquid becomes clarification, have or not opalescence produce and judge whether to form microemulsion in conjunction with Zeta particle size determination by perusal, record the consumption of S/C volumetric solution while forming microemulsion and calculate oil phase, water, the percentage ratio of the each composition of surfactant and cosurfactant in microemulsion system, the ratio of each composition can know formation microemulsion by computed in software analysis time.When result shows Km=3, microemulsion region maximum, simultaneously for the consumption etc. that reduces surfactant considers, therefore in the time of Km=3 we to select the each component ratio of nanoscale buffering microemulsion be pH7.2 phosphate buffered saline (0.01mol/L): isopropyl myristate (IPM): (S/C) be 36%: 14%: 50%.
(2) among aseptic, first according to Labraso: polyglyceryl fatty acid ester=3: 1 ratio, accurately measures Labraso 375ml and polyglyceryl fatty acid ester 125ml with graduated cylinder clean, dry, that sterilized respectively; Afterwards, at room temperature, they are poured among same cleaning, dry, the 1000ml container of sterilizing again; Then, more fully stir with JB-3A type time constant-temperature magnetic stirring apparatus, 200 revs/min of rotating speeds, 10-20min continuously, makes it abundant stirring, mixes the mixed liquor 500ml into Labraso and polyglyceryl fatty acid ester completely.
(3) measure respectively 360ml pH7.2 phosphate buffered saline (0.01mol/L) and 140ml isopropyl myristate, and they are added to respectively in the mixed liquor of above-mentioned 500ml Labraso and polyglyceryl fatty acid ester; Then fully stir with JB-3A type time constant-temperature magnetic stirring apparatus, 200 revs/min of rotating speeds, 30min continuously, can obtain total amount of liquid is the water white nanoscale buffering of 1000ml microemulsion.
(4) be that 0.45 μ m bacteriological filtration film carries out after heat sterilization by this nanoscale buffering microemulsion through specification, require in superclean bench, it to be divided respectively immediately that to be filled to the specification of having sterilized be among the water white cillin bottle of 10ml according to sterile working.In each cillin bottle, add this liquid 10ml, so subpackage can obtain 100 aseptic nanoscale buffering microemulsion that XiLin is bottled; Add a cover immediately, carry out rapidly gland seal, and be placed in cool place, dry, ventilation, lucifuge place and save backup.Like this, the cillin bottle quantity of nanoscale buffering microemulsion is housed identical with the quantity of the cillin bottle that contains compound antler polypeptide lyophilized powder.
(5) and, the preferred weight percent of this nanoscale buffering microemulsion raw material composition just in time: pH7.2 phosphate buffered saline (0.01mol/L) 36.0, isopropyl myristate 14.0; Labraso 37.5, polyglyceryl fatty acid ester 12.5.
Implementation example 3: preparation and the usage of combination essence:
(1) preparation of compound antler polypeptide lyophilized powder and nanoscale buffering microemulsion combination essence: respectively get 1 cillin bottle open bottle cover that compound antler polypeptide lyophilized powder and nanoscale buffering microemulsion are housed.Directly nanoscale is cushioned to microemulsion and be poured in compound antler polypeptide lyophilized powder, add a cover repeatedly and put upside down and mix, until compound antler polypeptide lyophilized powder dissolves completely; With the dropper on water white transparency drop bottle, this lysate is all transferred in the water white transparency drop bottle with dropper and mixed, adds a cover again, become 10 milliliters of compound antler polypeptide lyophilized powders and nanoscale buffering microemulsion combination essence.Due in every cillin bottle that has a compound antler polypeptide lyophilized powder containing antler polypeptide 2mg, mannitol 50mg, glycine 50mg, proline 50mg, hydroxyproline 50mg, albumin 98mg, and every lyophilized powder just can preparation cost invention combine essence after need to adding 10ml nanoscale buffering microemulsion, that is contain antler polypeptide 2mg, mannitol 50mg, glycine 50mg, proline 50mg, hydroxyproline 50mg, albumin 98mg in every 10 milliliters of combination essences.As calculated with preparation 100ml combination essence, by 10 times of the corresponding expansions of each composition in 10ml combination essence, in every 100ml combination essence, in compound antler polypeptide lyophilized powder, the degree of each material is just in time: antler polypeptide: 0.02; Mannitol: 0.50; Glycine: 0.50; Proline: 0.50; Hydroxyproline: 0.50; Albumin: 0.98.And the percentage ratio (W/V) of compound antler polypeptide lyophilized powder and nanoscale buffering microemulsion combination essence composition also just in time: compound antler polypeptide lyophilized powder 3.0, nanoscale buffering microemulsion 97.0.
(2) using method of compound antler polypeptide lyophilized powder and nanoscale buffering microemulsion combination essence: before formal use, first remove the aging cutin (only in the time using first) of skin of face according to medical cosmetology Standardization Requirement; While use, according to the clean skin of face of clean method of routine later; After skin bone dry, i.e. desirable the present invention combines essence 10-20 and drips among palm and make it dispersed; Then, embrocate gently or pat face with this, until it is substantially dried and absorbs cleanly; As still dry in skin, can repeat again once by this method; Used 2-3 every day, and using continuously 30 days is a life cycle.As some aging face comparatively obviously or severe patient, can use 3 cycles continuously until obtain positive effect according to above-mentioned usage.
The present invention who further illustrates above-mentioned preparation below by experimental example combine essence beneficial effect.
Experimental example 1: the transdermal penetration rate experiment of antler polypeptide combinations of factors essence.
The main research of this experiment antler polypeptide combinations of factors essence (compound antler polypeptide lyophilized powder and nanoscale buffering microemulsion combination essence) compares the transdermal filter experiment of these two objects by Franz In vitro penetration instrument with simple compound antler polypeptide lyophilized powder (with pH7.2 phosphate buffered saline (0.01mol/L) dilution).Result shows that antler polypeptide combinations of factors essence is higher than the compound antler polypeptide lyophilized powder unit are accumulation Transdermal absorption amount of dissolving with phosphate buffered saline merely, the combination essence that adopts nanoscale buffering microemulsion to prepare as essence substrate and solvent is described, can have more effectively promoted these bioactive substances and functional component Transdermal absorption.
Experimental example 2: the skin repair effect of antler polypeptide combinations of factors essence:
6 of healthy rats, male and female half and half, after rat back skin preserved skin 24h, smear 10 minutes in depilation district 2cm × 2cm scope gently repeatedly with 50% glycolic acid, absorb unnecessary liquid with cotton balls, are skin keratin and strip off damage model.Then smear antler polypeptide combinations of factors essence in Skin Wounds place one side, opposite side is smeared normal saline, smear every day twice, continuous 15 days, in experimentation, observe animal skin and integral animal, after use, whether there will be zest and untoward reaction, and in the time of 15 days, detect the water content of injured skin.
Experimental result shows that smearing antler polypeptide after skin injury can obviously improve rat and strip off the skin of damage, this experiment was smeared after 15 days, administration side scar is obviously less than control sides, and detect administration side water content apparently higher than control sides (as Fig. 4), two groups of differences have statistical significance (P < 0.05).
Experimental example 3: the clinical trial of antler polypeptide combinations of factors essence:
Random recruitment meets the routine healthy person in middle and old age volunteer of healthy volunteer 30 of inclusion criteria and exclusion standard, be women, the range of age is 40～57 years old, and in year mean age (48.12 ± 5.45), occupation is medical personnel 26 people, common laborer 4 people.According to aesthetic medicine test and clinical observation design, face was coated with and put on the skin twice every day, 10 of each uses (approximately 0.5ml left and right), the combination essence of the natural pilose antler active polypeptide factor lyophilized powder of uninterrupted use and activator (liquid) preparation continuously, life cycle is 4 weeks.And, in 4 weeks before aesthetic medicine and clinical trial, in use procedure, after using, follow up a case by regular visits to weekly and adopt digital photographing to take pictures, then, volunteer oneself and researcher apply relevant evaluation scale again and photoshop cs5 software carries out clinical observation on the therapeutic effect and objective evaluation.Scale is marked by 1～4 point.Wherein, 1 is best for skin after using, and 4 is that after using, skin is the poorest.
Result: as scheme natural pilose antler active polypeptide factor lyophilized powder combination essence to have clear improvement (as Fig. 5～8) such as healthy elder trial volunteer skin of face moisture, elasticity, the colour of skin, microgrooves.In Fig. 5～8, * represents compared with before test, and scoring average difference has statistical significance (P < 0.05), and * * represents that scoring average has significant difference (P < 0.01) compared with before test.
Implementation example of the present invention is only the specific descriptions that the preferred embodiment of the present invention is carried out; not design of the present invention and scope are limited; do not departing under the prerequisite of design philosophy of the present invention; various modification and improvement that those skilled in the art make technical scheme of the present invention; all should fall into protection scope of the present invention; the technology contents of request protection of the present invention, has all been documented in claims.
Claims (1)
1. the compound antler polypeptide lyophilized powder for skin beauty health-care and nanoscale buffering microemulsion combination essence, it is characterized in that described combination essence is made up of two part institutes of compound antler polypeptide lyophilized powder and nanoscale buffering microemulsion with skin anti-aging and beauty and health care functions, the weight percent of the each raw material composition of the described compound antler polypeptide lyophilized powder when percentage by weight difference of the described each raw material composition of nanoscale buffering microemulsion is as follows:
(1) compound antler polypeptide lyophilized powder raw material forms the percentage by weight in described combination essence:
(2) in nanoscale buffering microemulsion, each raw material forms the percentage by weight in described microemulsion:
(3) compound antler polypeptide lyophilized powder and the percentage by weight of nanoscale buffering microemulsion in described combination essence:
Compound antler polypeptide lyophilized powder: 3.0%
Nanoscale buffering microemulsion: 97.0%.
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| CN106420564A (en) * | 2016-10-31 | 2017-02-22 | 江苏煌上田投资有限公司 | Cornu cervi pantotrichum external restoration composition |
| CN108721137A (en) * | 2018-07-05 | 2018-11-02 | 广州慧雪生物科技有限公司 | A kind of freeze-dried powder for repairing skin |
| CN112891243A (en) * | 2021-04-25 | 2021-06-04 | 吉林省东北亚生物科技有限公司 | Cosmetic containing anti-wrinkle composition and preparation method thereof |
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