CN108084253A - Rich hydroxyproline albumen and the medicinal and cosmetic composition containing this albumen - Google Patents

Rich hydroxyproline albumen and the medicinal and cosmetic composition containing this albumen Download PDF

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Publication number
CN108084253A
CN108084253A CN201611017917.8A CN201611017917A CN108084253A CN 108084253 A CN108084253 A CN 108084253A CN 201611017917 A CN201611017917 A CN 201611017917A CN 108084253 A CN108084253 A CN 108084253A
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China
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glycoprotein
albumen
ginkgo
molecular weight
hydroxyproline
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CN201611017917.8A
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Chinese (zh)
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石佳媛
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Individual
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/02—Cosmetics or similar toiletry preparations characterised by special physical form
    • A61K8/11—Encapsulated compositions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
    • A61K8/645—Proteins of vegetable origin; Derivatives or degradation products thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/70—Web, sheet or filament bases ; Films; Fibres of the matrix type containing drug
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
    • A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
    • A61L15/22—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons containing macromolecular materials
    • A61L15/32—Proteins, polypeptides; Degradation products or derivatives thereof, e.g. albumin, collagen, fibrin, gelatin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
    • A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
    • A61L15/42—Use of materials characterised by their function or physical properties
    • A61L15/44—Medicaments
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/10—General cosmetic use
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
    • A61L2300/252—Polypeptides, proteins, e.g. glycoproteins, lipoproteins, cytokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
    • A61L2300/412—Tissue-regenerating or healing or proliferative agents

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Chemical & Material Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • Epidemiology (AREA)
  • Engineering & Computer Science (AREA)
  • Medicinal Chemistry (AREA)
  • Hematology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Organic Chemistry (AREA)
  • Materials Engineering (AREA)
  • Birds (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Botany (AREA)
  • Dermatology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Cosmetics (AREA)

Abstract

The invention discloses a kind of hydroxyproline-rich glycoproteins, this glycoprotein can be obtained from Chinese yew, ginkgo, tomato and carrot cell culture by acid alcohol extraction, with following characteristics：Average molecular weight is 20,000Da, fluctuation range 10,000~38,000, it is to be measured by gel infiltration and electric arteries and veins；High water solubility.

Description

Rich hydroxyproline albumen and the medicinal and cosmetic composition containing this albumen
Technical field
Invention is related to hydroxyproline-rich glycoprotein, and this albumen can be obtained from vegetable material, further relate to the medicinal of it With cosmetic purposes.
Background technology
The known some glycoprotein for coming from animal, if collagen and proteoglycans are after as former state or in the suitable composition of incorporation It carries out that during the coating of surface beneficial effect can be generated.
Collagen is the glycoprotein of a kind of rich proline and hydroxyproline, can be directly used in or be mixed with other polypeptide matrices For treatment and undesirable skin hydration and elastic relevant wrinkle and other the defects of damaging appearance after conjunction.But, come from dynamic The application of the collagen of object is restricted, because wherein there is the danger by virus and endotoxin contamination.Although come from the change of plant Object is closed there is no this risk, but its application in cosmetics is extremely limited so far：For example, as it is known that cosmetic combines Object contains the full plants of the chopping of the crude extract or such as avocado etc of the plant of such as aloe etc.
Have been recognized that, the Plant glycoprotein for being referred to as extensin be as caused by the plant cell for being in proliferation period, And with the structure similar to animal collagen.Average molecular weight is disclosed in EP-A-05334078 more than 100,000Da's Application of the extensin in cosmetics.However, disclosed so far all includes using brine for extracting the method for extensin Solution extracts the vegetable material in various sources, is then purified with the strong acid of such as trichloroacetic acid etc, these methods cannot obtain For the suitable product of cosmetics, due to there is asking for solubility, stability, reproducibility and its chemical-physical feature consistency Topic.
It has been found that by following methods can obtain similar in construction to above-mentioned extensin but molecular weight it is relatively low, Soluble hydroxyproline-rich glycoprotein higher in acidic aqueous solution, this method include the cell of the plant of in vitro culture selection, And extract the above-mentioned cell being grown in suitable culture medium with acidity alcohol solution.
The content of the invention
The aqueous solution of glycoprotein of the present invention keeps stablizing, and any sugared egg that insoluble product can be caused to be formed will not occur White polymerization.In addition, the viscosity of this solution is especially high, and it is not dependent on its concentration；It is especially surprising that its 0.1% Concentration i.e. have the film forming and aquation power identical with 1% collagen or 5% phytalbumin solution.
Vegetable material to be extracted is obtained from the fermentation culture medium of Chinese yew, ginkgo, tomato and carrot cell.It is preferred that it uses Cell obtained from Chinese yew, ginkgo and tomato.Cell culture technology be it is common, including with from such as leaf, skin, root, stem or The callus culture of the plant parts of seed etc carries out suspension culture, and this method is as described in Dobbs and Roberts (Experiments inPlant Tissne Culture, 2nd ed., Cambridge University Press, New York, 1985).
Above-mentioned plant callus is often used as suitable liquid training after sterilizing and selectively adding antibacterial agent The inoculum of base is supported, as described in the handbook in above-mentioned Dobbs and Roberts.It is especially suitable for culture medium of the invention Murashige and Skoog culture mediums.Add such as proline, reducing agent, ethylene or such as Ethephon or L- amino ring The additive that can discharge the compound of ethylene etc of propanecarboxylic acid etc is suitable for improving the yield of purpose glycoprotein.
It is preferred that using methyl α-naphthyl acetate as auximone, with 6- (γ, γ-dimethylamino) purine for cytokinin, with dimension Raw element and 3% sugar are carbon source.Can appropriate vitamin C be added according to selected material, to prevent final product brown stain.
Fermentation time is between 3-12 days, preferably between 5-6 days.Culture medium is centrifuged once finding to terminate, It is preferably ethyl alcohol extraction cell material with alcohol and under conditions of there are diluted inorganic acid, preferably hydrochloric acid or sulfuric acid. This method can make some enzyme inactivations that may destroy glycoprotein stability of the present invention, particularly make polyphenol oxidase and tyrosine oxygen Change enzyme inactivation, these enzymes can polymerize glycoprotein, so as to form insoluble product.
Alcohol extraction is carried out under conditions of having inorganic acid, can thoroughly extract alkaline glycoprotein, and it is right to have proven to its This purposes has high selectivity.Than ethanol, also can be used such as methanol or isopropanol etc can the miscible alcohol of water. Obtained water-alcohol extraction is neutralized, then concentrate and is heated to 70-100 DEG C of temperature, is preferably 80 DEG C or so, approaches Precipitation albuminate completely.Classification ultrafiltration is carried out by concentrating clarifying suspension, and to the liquid, to remove high and low molecular weight Substance.Ultrafiltration be with block molecular weight be 10,000-40,000 Da polysulfonate sorrel, such as CentriconROr RomiconRInto Row, fiber can be hollow or curling.Electrodialysis is carried out to obtained filtration product, to remove unwanted object Matter, such as salt and low molecular weight sugar.Obtained solution after filtered and dialysis, can be directly used in cosmetics or drug or by its Smaller size smaller is concentrated into, then freezes or is atomized.
Gel infiltration is carried out by high pressure liquid chromatography (HPLC) (HPLC), the product of the present invention is analyzed and identified, it is described HPLC is made of a Waters pump installation, and there are one Ultrahydrogel Linear Waters for toolRColumn group 30cm × 0.5cm and Waters UV absorption detectors, 484 types.0.067M potassium dihydrogen phosphates, 0.1M NaCl and 6 × 10 will be contained- 4MNaN3Aqueous solution be used as eluant, eluent.The glycoprotein sample being analysed to is dissolved in same elution solution (3mg/10ml), is measured Purpose thing and it is selected as object of reference of the molecular weight between cromoci (12,400Da) and glucan blue (2,000,000 Da) Target amount；Can by 12.5% polyacrylamide gel and 4% stacking gel electrophoresis separately or concurrently measure the production Object or in-between product.The sample being analysed to is dissolved in the buffer solution containing SDS and 0.1% mercaptoethanol, makes the precipitation capacity be 100~300mg.Carried out under the stabling current of 20mA migration 4 it is small when.With 5 kinds of standard molecular weights (7kD, 14kD, 24kD, 54kD And 66kD) draw metering curve.The molecular weight of two master tapes is calculated as 22.5kD and 25kD by the metering curve, and two compared with The molecular weight of weak band is 31kD and 34kD.It can be from the various cell explants from different plants with methods disclosed herein Middle acquisition molecular weight is quite and amino acid forms comparable product mixtures.It gives to come from Ginkgo Cells by way of example below and carry The amino acid analysis result of the glycoprotein taken.
Amino acid peak area %
Asp 4.399
Glu 4.328
Hyp 17.505
Ser 7.065
Gly 6.056
Hys 1.782
Arg 2.471
Thr 4.739
Pro 10.036
Ala 8.2
Tyr 2.388
Val 6.162
Met 1.154
Ile 2.479
Leu 5.525
Phe 1.862
Lys 14.254
Above-mentioned data are equivalent to the percentage of total amino acid content in glycoprotein mixture.Sugar in the mixture for arabinose and Galactolipin.The average value of the ratio between amino acid and sugar is 2: 1 in various products.
As described above, the product of the present invention not only can be applied to medicine field but also can be applied to cosmetic field.For drug For field, which can be mixed in gel or ointment or with being used to burn on hospital gauze or the special treatment of wound. It usually to be sterilized in this occasion to the product or sterilising filtration and lyophilized.
The cosmetic and dermatologic formulations of the present invention can be prepared with conventional method.The example of type of service include water into Spray, washing lotion, solution, lotion, gel, ointment and missible oil.
The cosmetic and dermatologic formulations of the present invention can be about 0.01~about 50% rich hydroxyl containing weight percent Proline glycoprotein is preferably 0.05~5%, also containing common excipient.It, can due to the high stability of glycoprotein of the present invention To obtain the medicinal and Toiletry preparation containing more than 50% soluble hydroxyproline-rich glycoprotein.
The glycoprotein of the present invention can be directly added into in medicinal and Toiletry preparation or carried out microencapsulation, it is long-acting to generate Hydration.This microcapsules can be hydrophilic or lipophilic.The preparation of the present invention can contain to be had mutually for anticipation target Other active ingredients of benefit or useful activity.
The following examples illustrate the present invention further.
Example 1
Prepare the gingkgo callus and liquid culture for glycoprotein production
In 0.1%Tween 80(R)Ginkgo tender leaf is washed in solution, prepares the explant of this tender leaf.Blade is cut into about 0.5cm sizes, and pre-sterilized 1 minute with 75% ethyl alcohol.Then sterilized with 2% liquor natrii hypochloritis, and 3 are washed in sterile water It is secondary.Gained explant is transferred in the Murashige ＆ Skoog culture mediums in culture dish, is contained in the culture medium 3% sucrose is simultaneously with the addition of swashing for Lynsmeyer ＆ Skoog vitamins and such as 2,4- dichlorphenoxyacetic acids and methyl α-naphthyl acetate etc Element.Above-mentioned product is cultivated 20 days in the dark, at 23 DEG C.At the end of this period, frangible callus is obtained, it Be easy to growth and can in the mode continuous passage of squamous subculture under identical conditions because can use it for preparation Liquid Culture Base.The callus is filled to the Erlenmeyer flasks of 200ml Murashige ＆ Skoog culture mediums, the training for inoculation It supports and methyl α-naphthyl acetate and 6 (γ, γ-dimethylamino) purine is with the addition of in base, the 3% sucrose conduct of raw peacekeeping of Lynsmeyer＆Skoog dimensions Carbon source.By the flask culture 4 days under the conditions of stirring and continuous illumination.Then the biomaterial of cell is harvested for extracting sugar Albumen.
Example 2
Glycoprotein is prepared by Ginkgo Cells
Low-speed centrifugal is carried out to the culture obtained by 1 method of example, and with 1 liter of 70% ethyl alcohol containing 1% sulfuric acid to harvest Cell (fresh weight 1.5kg) is extracted.Extraction 2 times is repeated, so as to quantitative recovery alkaline glycoprotein.Neutralize after to extract into Row filtering, to remove dregs, concentrates at 50 DEG C, in a vacuum until all ethyl alcohol is all completely removed.This is aqueous Concentrate heats 30 minutes at 85 DEG C, and centrifuges again, to remove precipitation, will precipitate reject.It is blocked with 40,000Da The Centricon of the limitRUltrafiltration is carried out to the settled solution obtained, to exclude higher molecular weight.
Again with hollow-fiber film of the molecular weight for 10,000Da is blocked to the progress ultrafiltration of above-mentioned filtration product, to remove non-saccharide Albumen low molecular weight substance.Then dialyse to filtrate, and be condensed into 1% solid residue.Obtain 1.5 liters it is slightly sticky Product, this product can be directly used in cosmetic composition.In electrophoretic analysis, which contains 6 bands, wherein 4 Molecular weight be 16,000,22,000,33,000 and 36,000Da.

Claims (4)

1. hydroxyproline-rich glycoprotein can use acid alcohol extraction from Chinese yew, ginkgo, tomato and carrot cell culture Middle acquisition, with following characteristics：- average molecular weight is 20,000Da, fluctuation range 10,000~38,000, pass through gel Infiltration and electrophoresis measure；- high water solubility.
2. a kind of method for the glycoprotein for being used to prepare claim 1, this method include：
A) Chinese yew, ginkgo, tomato and carrot cell 3-15 days are cultivated in a kind of fluid nutrient medium；
B) the alcohol extracting cell material mixed under conditions of having diluted mineral acid with water soluble；
C) neutralize, concentrate and the extract is heated at a temperature of 70~100 DEG C；
D) centrifuge, be classified ultrafiltration and dialyse.
3. the cosmetic as active component of the glycoprotein containing claim 1 and pharmaceutical formulation have aquation, film forming, toning With epulosis characteristic.
It is water into spray, washing lotion, solution, lotion, gel, ointment, missible oil and medical yarn 4. preparation as claimed in claim 3 Cloth form.
CN201611017917.8A 2016-11-20 2016-11-20 Rich hydroxyproline albumen and the medicinal and cosmetic composition containing this albumen Pending CN108084253A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201611017917.8A CN108084253A (en) 2016-11-20 2016-11-20 Rich hydroxyproline albumen and the medicinal and cosmetic composition containing this albumen

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201611017917.8A CN108084253A (en) 2016-11-20 2016-11-20 Rich hydroxyproline albumen and the medicinal and cosmetic composition containing this albumen

Publications (1)

Publication Number Publication Date
CN108084253A true CN108084253A (en) 2018-05-29

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Application publication date: 20180529