CN100534410C - Application of hirudinoid protease chalone incosmetic - Google Patents
Application of hirudinoid protease chalone incosmetic Download PDFInfo
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- CN100534410C CN100534410C CNB2004100047095A CN200410004709A CN100534410C CN 100534410 C CN100534410 C CN 100534410C CN B2004100047095 A CNB2004100047095 A CN B2004100047095A CN 200410004709 A CN200410004709 A CN 200410004709A CN 100534410 C CN100534410 C CN 100534410C
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- protease
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Abstract
An application of leech's proteinase depressant in cosmetics for increasing the content of elastin in skin tissue to eliminate wrinkles on skin and beautify the skin is disclosed. Said proteinase depressant is extracted from leech.
Description
Technical field
The present invention relates to a kind of new purposes of Hirudo protease chalone, particularly the application in crease-resistant beauty and make-up articles for use.
Background technology
The human body skin structure is divided into three layers usually, is followed successively by subcutaneous tissue, corium and epidermis from deep to shallow.Epidermis is most active one deck in the skin, is made up of basal layer, granular layer and horny layer, gives skin with protective effect by continuous keratinization.Subcutaneous tissue is made up of connective tissue, nerve fiber, lymphatic vessel and little blood vessel etc., and its major function is to alleviate bump, heat insulation and store heat.The cell of skin corium inside seldom mainly is made of fibrous connective tissue.Collagen fiber, elastic fiber and reticular fiber etc. are wherein arranged, and they have very important relation to elasticity, gloss and the tension force etc. of skin.The lax, wrinkling etc. aging of skin all is to occur among the corium.Elastoser is a kind of enzyme that extensively is present in the degraded elastin laminin (elastic fiber) in the human tissue cell, and content is abundant in epidermis and hypodermal cell.People this enzyme active relatively low in juvenile years skin, and the metabolism of elastin laminin is active, is keeping the skin better elastic.When human senility, the activity of this enzyme strengthens gradually in the skin, the elastin laminin in the hydrolysis skin corium, and the anabolism of elastin laminin slows down simultaneously, and the elasticity of skin is reduced gradually, causes cutis laxa, wrinkling etc. senile change.Therefore, suppress the activity of elastoser in the skin effectively, reduce the hydrolysis rate of elastin laminin, improve the content of the elastin laminin in the skin corium, can play effect crease-resistant, beauty treatment.
People such as Jung isolate a kind of elastase inhibitor (TheJournal of Biological Chemistry in nineteen ninety-five in the Hirudo body, 1995,270:13879-13884), called after Hirudo protease chalone can be efficiently and suppress elastase activity specifically, and is all very stable under acid-base status, adopted genetic engineering means, realized external the efficiently expressing of Hirudo protease chalone, therefore sought the application of this albumen aspect crease-resistant cosmetics, had great importance.
Summary of the invention
The new purposes that the purpose of this invention is to provide a kind of Hirudo protease chalone is in the cosmetic product preparation, in the hope of reaching the effect of crease-resistant beauty treatment.
For achieving the above object, the present invention has adopted following technical scheme:
Hirudo protease chalone is to be present in the intravital a kind of small protein of Hirudo, Hirudo protease chalone is the protein with sequence 2 amino acid residue sequences in the appended sequence table, or the amino acid residue sequence of described sequence is passed through replacement, disappearance or the interpolation of one or several amino acid residue and has identical active by the deutero-protein of described sequence with the amino acid residue sequence of described sequence.
Described Hirudo protease chalone can pass through biological extraction method (Jung et al, The Journal ofBiological Chemistry, 1995,270:13879-13884) or genetic engineering vivoexpression method (Chinese patent that awaits the reply, application number: 02146788.9) obtain.This protein is to thermally-stabilised, and through 80 ℃ of heating 30 minutes, activity did not weaken, at room temperature long preservation.Experimental result shows that Hirudo protease chalone is the efficient and specific inhibitor of elastoser.It also has more weak inhibitory action to trypsin and Chymotrypsin, but its effect is all not and to 10% of elastoser, to other enzyme, does not then have obviously as papain, pepsin, thrombin etc. and to act on.Our experiment proves simultaneously Hirudo protease chalone is applied to animal skin surfaces, after handling after a while, the content of elastin laminin in the skin histology is increased, and do not have tangible untoward reaction.Illustrate that Hirudo protease chalone can be used as the functional component of crease-resistant cosmetics, is used for cosmetic formulations.
The present invention can the biological extraction method or the Hirudo protease chalone that makes of genetic engineering vivoexpression method as raw material, the preparation cosmetic formulations can be multiple dosage forms such as cream, unguentum, water preparation, powder, oil preparation.The cosmetic formulations of above-mentioned various dosage forms all can be according to the conventional method preparation in cosmetic formulations field.
The present invention creatively is used for cosmetic formulations with Hirudo protease chalone, by its inhibitory action to elastoser, improves the content of elastin laminin in skin histology, and then brings into play the effect of its crease-resistant beauty treatment, has a good application prospect.
The specific embodiment:
Synthesizing of embodiment 1, Hirudo protease chalone expressing gene
1, the synthetic of small fragment: sequence 1 Hirudo protease chalone gene is divided into two-stage nitration, and first section sequence is cDNA normal chain, and second section sequence is the cDNA complementary strand.3 ' end at first section adds 8 and second section 3 ' complementary base, and 105 bases of length overall are the sequence in the sequence table 2, adds 8 and first section 3 ' at second section 3 ' end and holds complementary base, and 111 bases of length overall are the sequence in the sequence table 3.Add NdeI and NotI restriction enzyme site respectively at two fragments, 5 ' end, synthetic with dna synthesizer.
With the synthetic fragment of dna synthesizer, the complete sequence Hirudo protease chalone gene with single circulation PCR reaction connection composition sequence 1 as shown in Figure 1, proves that the sequence that obtains is correct.
1, cuts this PCR fragment, in the pGEM-T-EASY plasmid of packing into, make up the pGEM-GUM cloning vehicle.
2, the structure of pGEM-GUM expression plasmid
Employing is available from the efficient expression plasmid pPIC9K of American I nvitrogen company, and with NdeI and NotI enzyme action pGEM-GUM plasmid and pPIC9K plasmid respectively, gel electrophoresis is collected target fragment, with the connection of spending the night of 16 ℃ of T4 ligases.Through the conversion of conventional method, choose bacterium, amplification obtains expression plasmid pGEM-GUM, and its structure collection of illustrative plates is as shown in Figure 2.Enzyme action is identified, is proved that the structure of expression plasmid is correct.
3, with plasmid pGEM-GUM SacI linearisation. adopt Zhejiang Xin Zhi company electricity gene introducing apparatus, linearizing pGEM-GUM is imported in the GS115 Pichia yeast (available from Invitrogen company),, select through cultivating, processes such as screening obtain the monoclonal bacterium of anti-G418mg/ml.
4, select the monoclonal bacterium, be inoculated in the 5ml culture medium, 30 ℃ are spent the night, and change over to then in the 250ml culture medium, continue to be cultured to OD
600=10-20 collects thalline, is diluted to OD with no carbon source culture medium
600=50, continue to cultivate 24 hours, adding methanol to final concentration is 2% abduction delivering, adds methanol once every 24 hours, to 96 hours.The centrifuging and taking supernatant carries out gel electrophoresis analysis and functional examination.Get the 10ml supernatant, use the 12%SDS-PAGE electrophoresis, and dye through Coomassie brilliant blue.As seen the result in the position of 6KD, has inductive protein expression, and according to the BSA contrast, the estimation expression is 103mg/L.
In the present embodiment, induce meaning very big with methanol when cultivating the monoclonal thalline, before not inducing, basic driftlessness albumen in the culture fluid, as shown in Figure 3, significant target protein band is arranged in the culture fluid after inducing, and size is identical with the albumen of sequence 2, and does not have significant target protein band in the culture fluid before inducing.
5, protein purification: experiment is with containing 50mM Tris-HCl (pH8.0), 1M (NH
4)
2SO
4Balance phenylsepharose chromatographic column was gone up sample with culture supernatant with 1ml/ minute, after cleaning, reuse 50mMTris-HCl (pH 8.0) eluting, collect eluent, obtain the Hirudo protease chalone of purity about 95%, measure protein content with the Lowry method.
The functional examination of embodiment 3, Hirudo protease chalone of the present invention
Get 5 test tubes, be denoted as normal saline, induce preceding supernatant, induce the back supernatant, add 40ul 0.2M Tris-HCl successively respectively, pH 8.0,20ul 0.37mg/ml Pancreas Sus domestica gland elastoser (SIGMA, E-1250), the normal saline of 40ul, induce before supernatant or induce the back supernatant refined solution (1umol/L), mixing adds 400ul 0.1mM reaction substrate SucA3PNA (SIGMA) again, and room temperature reaction is after 10 minutes, in 410nm place colorimetric, make comparisons with normal saline, calculate enzymatic activity and suppress percentage rate, computing formula is OD
410Measure sample/OD
410Normal saline.The result induces preceding supernatant and normal saline group no significant difference as shown in Figure 4, is 98% and induce the enzymatic activity inhibition percentage rate of back supernatant.Show that expression product is Hirudo protease chalone, and better elastic protease inhibitory action is arranged.
The preparation of embodiment 4, Hirudo protease chalone cream of the present invention:
Get adjuvant protective skin cream substrate 99 grams, add totally 1.0 milliliters of spissated Hirudo protease chalone refined solution and distilled water, mixing and stirring, the ultimate density that makes Hirudo protease chalone is 1umol/L, packing is standby, is Hirudo protease chalone protective skin cream.Get adjuvant protective skin cream substrate 99 grams again, add 1.0 milliliters of normal saline, mixing and stirring, packing is normal saline contrast protective skin cream.
The protective skin cream matrix formulations be (W/W, %): A, oil phase: cocos nucifera oil (caprylic/capric) ester 10.00; 16, octadecanol and 16, the poly-glucose 7.50 of octadecyl; Acid esters (PL1618) is a small amount of; Different hexadecanol 5.00; Polydimethylsiloxane 350csks 15.00; 2-iso-octyl-2 cyano group-3,3-diphenylacrylate ester 5.00; Vitamin E 3.00; Glyceryl stearate 2.00.B water: deionized water 43.50; Glycerol 5.00.C additive: hydrolysis almond protein 3.00; Sunscreen 1.00.Transfer PH to 5.5 with 12.5% citric acid.
Protective skin cream substrate preparation method: oil phase A component is poured in the container, be stirred and heated to 75-80 ℃, in another container, mix the aqueous phase B component, be stirred and heated to 75-80 ℃.Add B in A under stirring, insulation mixed 15 minutes.Be cooled to 50 ℃, doping C.Stir cool to room temperature.
The Hirudo protease chalone protective skin cream (1umol/L) of above-mentioned preparation is adopted in experiment.Get by Military Medical Science Institute's animal center 20 of Kunming mouses are provided, male female half and half, body weight 18-22 gram, be divided into two groups at random, be Hirudo protease chalone group and normal saline group, back unhairing, coating Hirudo protease chalone protective skin cream and normal saline contrast protective skin cream respectively, every day three times, coating is 30 days continuously.Put to death mice next day after stopping using, and peels off skin, removes fat, weighs, and adopts Sandberg et al (Connective TissueResearch.25; 139-48,1990) method is measured the elastin laminin content in the skin histology.Result such as Fig. 5, Hirudo protease chalone protective skin cream can obviously improve the proteic content of elasticity in the skin histology.
Sequence table
(1) general information:
(ii) denomination of invention: the application of Hirudo protease chalone in cosmetics
(iii) sequence number: 2
(2) sequence 1:
(i) sequence signature:
(A) length: 174bp
(B) type: nucleic acid
(C) chain: two strands
(D) topological structure: linearity
(ii) molecule type: cDNA
(xi) sequence description: sequence 1
gttgacgaaa?acgctgaaga?cacccatggt?ttgtgcggtg?aaaaaacctg?ctctccagct 60
caagtctgtc?taaacaacga?atgcgcttgc?actgcaatca?gatgcatgat?cttctgtcct 120
aacggtttca?aagttgatga?aaacggttgc?gaatacccat?gtacctgcgc?ttga 174
(3) sequence 2:
(i) sequence signature:
(A) length: 57 aminoacid
(B) type: aminoacid
(D) topological structure: linearity
(ii) molecule type: polypeptide
(xi) sequence description: sequence 2
Val?Asp?Glu?Asn?Ala?Glu?Asp?Thr?His?Gly?Leu?Cys?Gly?Glu?Lys
1 5 10 15
Thr?Cys?Ser?Pro?Ala?Gln?Val?Cys?Leu?Asn?Asn?Glu?Cys?Ala?Cys
20 25 30
Thr?Ala?Ile?Arg?Cys?Met?Ile?Phe?Cys?Pro?Asn?Gly?Phe?Lys?Val
35 40 45
Asp?Glu?Asn?Gly?Cys?Glu?Tyr?Pro?Cys?Thr?Cys?Ala
50 55 57
Claims (3)
1, a kind of have the inhibiting Hirudo protease of elastoser chalone in the application of preparation in the crease-resistant beauty and make-up articles for use, and wherein Hirudo protease chalone is the protein with sequence 2 amino acid residue sequences in the sequence table.
2, application according to claim 1 is characterized in that: described Hirudo protease chalone can obtain by biological extraction method or genetic engineering vivoexpression method.
3, application according to claim 1 is characterized in that: its beauty and make-up articles for use dosage form is cream, unguentum, water preparation, powder, oil preparation.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2004100047095A CN100534410C (en) | 2004-02-24 | 2004-02-24 | Application of hirudinoid protease chalone incosmetic |
| PCT/CN2005/000214 WO2005084612A1 (en) | 2004-02-24 | 2005-02-23 | The new usage of elastase inhibitors |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2004100047095A CN100534410C (en) | 2004-02-24 | 2004-02-24 | Application of hirudinoid protease chalone incosmetic |
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| Publication Number | Publication Date |
|---|---|
| CN1660041A CN1660041A (en) | 2005-08-31 |
| CN100534410C true CN100534410C (en) | 2009-09-02 |
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| CNB2004100047095A Expired - Fee Related CN100534410C (en) | 2004-02-24 | 2004-02-24 | Application of hirudinoid protease chalone incosmetic |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107519036A (en) * | 2017-09-06 | 2017-12-29 | 深圳肽之妃科技有限公司 | A kind of striae of pregnancy spray containing polypeptide and preparation method thereof |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103006512B (en) * | 2012-12-21 | 2014-06-11 | 山东侨牌集团有限公司 | Hirudin anti-winkle face-softening conditioning cream and production process thereof |
| CN103006511B (en) * | 2012-12-21 | 2015-04-22 | 山东侨牌集团有限公司 | Hirudin skin-whitening moisture-keeping conditioning cream and production process thereof |
-
2004
- 2004-02-24 CN CNB2004100047095A patent/CN100534410C/en not_active Expired - Fee Related
Non-Patent Citations (4)
| Title |
|---|
| Isolation and characterization of guamerin, a new humanleukocyte elastase inhibitor from Hirudo nipponia. Hyo Il Jung.The Journal Biological Chemistry,Vol.270 No.23. 1995 |
| Isolation and characterization of guamerin, a new humanleukocyte elastase inhibitor from Hirudo nipponia. Hyo Il Jung.The Journal Biological Chemistry,Vol.270 No.23. 1995 * |
| 弹性蛋白降解与皮肤衰老及其护肤品开发. 魏少敏.日用化学工业，第5期. 1997 |
| 弹性蛋白降解与皮肤衰老及其护肤品开发. 魏少敏.日用化学工业，第5期. 1997 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107519036A (en) * | 2017-09-06 | 2017-12-29 | 深圳肽之妃科技有限公司 | A kind of striae of pregnancy spray containing polypeptide and preparation method thereof |
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| CN1660041A (en) | 2005-08-31 |
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Granted publication date: 20090902 Termination date: 20130224 |