CN102743300B - Sea cucumber glycopeptide, preparation method and application in preparation of cosmetics - Google Patents

Abstract

The invention discloses a sea cucumber glycopeptide, which is characterized in that the molecular weight is less than 3000Da, a sugar chain is connected to serine or threonine through GalNAc, and the sea cucumber glycopeptide has a structure of-Gal beta 1-3GalNAc alpha-O-Ser/Thr. The sea cucumber feed is prepared from sea cucumber serving as a raw material through processes such as enzymolysis and the like, and has the advantages of wide raw material source, safety, reliability, simple preparation process, low cost and convenience in application. The prepared sea cucumber glycopeptide has obvious functions of promoting the growth of fibroblasts and protecting the fibroblasts from ultraviolet radiation damage, so that the sea cucumber glycopeptide can be applied to the preparation of cosmetics, can effectively prevent the ultraviolet radiation damage and avoid skin aging caused by ultraviolet radiation and the like.

Description

Apostichopus glycopeptide, preparation method and preparing the application in cosmetics

Technical field

The present invention relates to a kind of Stichopus japonicus zymolyte, especially a kind of preparation technology is simple, cost is low, there is significant promotion fibroblastic growth and to fibroblast ultraviolet radiation damage, there is Apostichopus glycopeptide, the preparation method of protective effect and preparing the application in cosmetics.

Background technology

At present, sun care preparations of a great variety, its sun-proof principle is different.If Chinese invention patent number is ZL02817885.8, name is called that the patent of invention of " silica-coated mixed crystal oxide particle, its preparation method and the cosmetic material prepared with it " discloses the cosmetics that one has sun-proof (pre-anti-ultraviolet causes skin injury) function, add there is the silica-coated mixed crystal oxide particle that blocks UV resistance and make, namely by physical method prevention of uv damages in cosmetics base stock.A kind of sun care preparations is also had to be add fibroblast growth factor in base material of cosmetics, by short epidermal cell repairing propagation prevention of uv damages, but because of existing fibroblast growth factor complex manufacturing, expensive, for which limit its extensive use.

At present, the small-molecule active substances such as the equal mucopolysaccharide of the active substance with Stichopus japonicus prepared by raw material, peptide and saponin.Up to now, also not about taking Stichopus japonicus as raw material, enzyme-squash techniqued Apostichopus glycopeptide and its relevant report applied in cosmetics.

Summary of the invention

The present invention is the above-mentioned technical problem in order to solve existing for prior art, provides that a kind of preparation technology is simple, cost is low, has and promote fibroblastic growth and have Apostichopus glycopeptide, the preparation method of protective effect to fibroblast ultraviolet radiation damage and preparing the application in cosmetics significantly.

Technical solution of the present invention is: a kind of Apostichopus glycopeptide, it is characterized in that molecular weight is less than 3000Da, and sugar chain is connected on serine or threonine by GalNAc, has-Gal β 1-3 GalNAc α-O-Ser/Thr structure.

Amino acid contained molar content is: aspartic acid and agedoite 11.41 %, threonine 4.93 %, serine 5.23%, glutamic acid and glutamine 14.68%, proline 6.28 %, glycine 12.33%, alanine 13.05%, valine 3.27%, isoleucine 2.61%, leucine 3.9%, tyrosine 1.61%, phenylalanine 2.18%, histidine 1.22%, lysine 3.8%, arginine 5.78%, methionine 1.75%, cysteine 1.48%; Sugar content is 10 ~ 20% of gross mass, and relative humidity is not more than 10%, and dissolubility is 1 gram/30 ml waters.

A preparation method for above-mentioned Apostichopus glycopeptide, is characterized in that carrying out as follows:

A. wall of sea cucumber Stichopus japonicus is carried out homogenized 0 ~ 10 DEG C time;

B. in gained homogenate, add 0.01 ~ 0.1M phosphate buffer of 1 ~ 3 times of volume pH 7 ~ 8 and be the compound enzyme of 0.1 ~ 1% with homogenate mass ratio, enzymolysis 12 ~ 48 h; Described compound enzyme is the mixture that alkaline protease, papain and trypsin are 3:3:4 by unit of activity ratio;

C. centrifuging and taking supernatant, employing molecular cut off is that the Ultra filtration membrane device of 3000 below Da is separated, and gets the liquid through ultrafilter membrane;

D. by Sephadex LH-20 molecular sieve chromatography, OD is collected _280nmabsworption peak;

E. collected liquid is passed through macroporous adsorbent resin and active carbon postlyophilization, obtained Apostichopus glycopeptide.

A kind of above-mentioned Apostichopus glycopeptide is preparing the application in cosmetics.

Preparing the application in sun care preparations.

The Apostichopus glycopeptide of gross mass 0.1 ~ 1 % is mixed in described base material of cosmetics.

The present invention is raw material with Stichopus japonicus, and be prepared from through techniques such as enzymolysis, raw material sources are extensive and safe and reliable, and preparation technology is simple, cost is low, be convenient to application.Prepared Apostichopus glycopeptide has significant promotion fibroblastic growth and has protective effect to fibroblast ultraviolet radiation damage; therefore can apply preparing in cosmetics; can effective prevention of uv damages, avoid the skin aging because ultraviolet radiation etc. causes.

Accompanying drawing explanation

Fig. 1 is the ultraviolet spectrogram of the embodiment of the present invention 1,2,3.

Detailed description of the invention

Embodiment 1:

Preparation method is carried out as follows:

A. wall of sea cucumber Stichopus japonicus is carried out homogenized 10 DEG C time;

B. in gained homogenate, add 0.01 ~ 0.1M phosphate buffer of 3 times of volume pH 7 ~ 8 and be the compound enzyme of 0.1% with homogenate mass ratio, enzymolysis 48 h; Described compound enzyme is the mixture that alkaline protease, papain and trypsin are 3:3:4 by unit of activity ratio;

C. centrifuging and taking supernatant, employing molecular cut off is that the Ultra filtration membrane device of 3000 below Da is separated, and gets the liquid through ultrafilter membrane;

D. by Sephadex LH-20 molecular sieve chromatography (2.5 × 60 ~ 100cm), OD is collected _280nmabsworption peak;

E. collected liquid is passed through macroporous adsorbent resin (HPD system row) desalination and active carbon postlyophilization, obtained Apostichopus glycopeptide.

Alkaline protease used is obtained (CAS numbering: 9014-01-1A0271), the raw materials such as alkaline protease, papain and trypsin are commercially available prod.

Embodiment 2:

Preparation method is carried out as follows:

A. wall of sea cucumber Stichopus japonicus is carried out homogenized 0 DEG C time;

B. in gained homogenate, add 0.01 ~ 0.1M phosphate buffer of 1 times of volume pH 7 ~ 8 and be the compound enzyme of 1% with homogenate mass ratio, enzymolysis 12 h; Described compound enzyme is the mixture that alkaline protease, papain and trypsin are 3:3:4 by unit of activity ratio;

C. centrifuging and taking supernatant, employing molecular cut off is that the Ultra filtration membrane device of 3000 below Da is separated, and gets the liquid through ultrafilter membrane;

D. by Sephadex LH-20 molecular sieve chromatography (2.5 × 60 ~ 100cm), OD is collected _280nmabsworption peak;

E. collected liquid is passed through macroporous adsorbent resin (HPD system row) desalination and active carbon postlyophilization, obtained Apostichopus glycopeptide.

Raw material is with embodiment 1.

Embodiment 3:

Preparation method is carried out as follows:

A. wall of sea cucumber Stichopus japonicus is carried out homogenized 5 DEG C time;

B. in gained homogenate, add 0.01 ~ 0.1M phosphate buffer of 1 times of volume pH 7 ~ 8 and be the compound enzyme of 0.5% with homogenate mass ratio, enzymolysis 30 h; Described compound enzyme is the mixture that alkaline protease, papain and trypsin are 3:3:4 by unit of activity ratio;

C. centrifuging and taking supernatant, employing molecular cut off is that the Ultra filtration membrane device of 3000 below Da is separated, and gets the liquid through ultrafilter membrane;

D. by Sephadex LH-20 molecular sieve chromatography (2.5 × 60 ~ 100cm), OD is collected _280nmabsworption peak;

E. collected liquid is passed through macroporous adsorbent resin (HPD system row) desalination and active carbon postlyophilization, obtained Apostichopus glycopeptide.

Raw material is with embodiment 1.

Test one:

Take sample (Apostichopus glycopeptide) 5mg of the embodiment of the present invention 1 or 2 or 3, be dissolved in 3ml distilled water, then sample solution be divided into two equal portions.A copy of it adds 1.5ml distilled water; Another part adds 1.5m 0.4mol/L NaOH solution; After reaction 2h, two parts of solution carry out uv scan respectively within the scope of 200 ~ 400nm, result is as shown in Figure 1: the uv absorption of the Apostichopus glycopeptide after 240 nm place NaOH process significantly strengthens, and illustrates in Apostichopus glycopeptide to deposit to have-Gal β 1-3 GalNAc α-O-Ser/Thr structure.

Embodiment 1 or 2 or 3 gained Apostichopus glycopeptide, time-of-flight mass spectrometry result shows it m/zfor being less than 3000Da, sugar chain is connected on serine or threonine by GalNAc, has-Gal β 1-3 GalNAc α-O-Ser/Thr structure.

Molar content amino acid contained is after testing: aspartic acid and agedoite 11.41 %, threonine 4.93 %, serine 5.23%, glutamic acid and glutamine 14.68%, proline 6.28 %, glycine 12.33%, alanine 13.05%, valine 3.27%, isoleucine 2.61%, leucine 3.9%, tyrosine 1.61%, phenylalanine 2.18%, histidine 1.22%, lysine 3.8%, arginine 5.78%, methionine 1.75%, cysteine 1.48%; Sugar content is 10 ~ 20% of gross mass, and relative humidity is not more than 10%, and dissolubility is 1 gram/30 ml waters.

Test two:

Apostichopus glycopeptide obtained for embodiment 1 or 2 or 3 is applied in cosmetics, can is the commodity dosage forms such as powder, tablet and injection, mixes with existing cosmetics during use.Also in the base material of cosmetics, can make cosmetics by the Apostichopus glycopeptide mixed containing gross weight 0.1 ~ 1%, base material of cosmetics can be existing ordinary cosmetics.

Protective skin cream containing Apostichopus glycopeptide measures fibroblast growth promoting activity:

1. sample preparation

Protective skin cream is formula shown in table 1:

Table 1

Composition	Mass percent	Composition	Mass percent
				Glyceryl monostearate	8	Propylparaben	0.5
Stearic acid	8	Glycerol	10
				Hexadecanol	1	Triethanolamine	1
Propyl parabene	0.1	Distilled water	69.9
				Dimethicone	1	Essence	0.5

(1) containing the protective skin cream sample preparation of Apostichopus glycopeptide: namely press table 1 by oil phase raw material and oil soluble emulsifying agent, 80 DEG C are mixed and heated under constantly stirring, maintain 20 min sterilizings, Aqueous Phase Raw Material and water soluble emulsifier are added in distilled water, 80 DEG C are heated under stirring, maintain 20min sterilizing, then cool, stirring and emulsifying in aqueous phase will be slowly added to for millesimal Apostichopus glycopeptide in mass ratio with protective skin cream, again aqueous phase is joined in oil phase, add essence when temperature is down to 45 DEG C, continue to stir cooling, obtain the protective skin cream containing Apostichopus glycopeptide.Getting 0.5g is dissolved in 2.5ml distilled water containing the protective skin cream of Apostichopus glycopeptide, and 10000 rpm are centrifugal, and 20 min get supernatant, through 0.2 μm of membrane filtration, and 4 DEG C of preservations.

(2) preparation of Apostichopus glycopeptide sample: the Apostichopus glycopeptide solution preparing 2.4 μ g/ml with water, 10000 rpm are centrifugal, and 20 min get supernatant, through 0.2 μm of membrane filtration, 4 DEG C of preservations.

(3) control sample: get 0.5g protective skin cream and be dissolved in 2.5ml distilled water, 10000 rpm are centrifugal, and 20 min get supernatant, through 0.2 μm of membrane filtration, 4 DEG C of preservations.

2. sample is to the mensuration of fibroblastic growth facilitation:

Fibroblast be the 10th generation In vitro culture human fibroblasts.

Be 4.5 × 10 by quantity ⁴individual/ml is in the human fibroblasts 100 μ l of exponential phase, different samples (protective skin cream sample, Apostichopus glycopeptide sample, control sample containing Apostichopus glycopeptide) each 2.5ml of solution, and the RPMI-1640 liquid medium extremely every hole 200 μ l added containing 10% hyclone, at 5%CO ₂37 ^oafter cultivating 24h in C incubator, add 20 μ L MTT(5g/L), then after cultivating 4h, discard culture medium, add 180 μ l DMSO, after 60s that constant temperature oscillator vibrates, detect absorbance by microplate reader at 570 nm/630 nm wavelength places.

Each sample solution establishes 3 Duplicate Samples.

Growth promotion rate is calculated: growth promotion rate=［ (experimental group inhales shading value – matched group absorbance)/matched group absorbance ］ × 100% according to the absorbance recorded.

Result is as table 2.

Table 2

Sample	Matched group	Containing the protective skin cream of Apostichopus glycopeptide	Apostichopus glycopeptide
				Fibroblastic growth promotion rate (%)	0	47.4	58.1

Result shows: the protective skin cream containing Apostichopus glycopeptide and Apostichopus glycopeptide have growth promoting function to human body fibroblast.

Test three:

Because fibroblast has the effect of ultraviolet injury protection; for this reason can by Apostichopus glycopeptide being that millesimal amount is added in existing cosmetics with cosmetics base stock mass ratio; make protective skin cream, eye cream, sun screen, hand cream and massage cream etc., skin can be coated on and cause skin injury for pre-anti-ultraviolet.

Get Apostichopus glycopeptide 0.1g, 0.2g of the embodiment of the present invention 1 or 2 or 3 respectively, respectively add 100ml distilled water, be diluted to 0.1% and 0.2%, be stirred to evenly.Then respectively get respectively in 3ml sample liquid and 3mlTCA holding test tubes.Then each sample liquid is got 1.5ml and to be placed in centrifuge tube at-4 DEG C with the centrifugal 20min of rotating speed 10000r/min.Get supernatant ultraviolet-uisible spectrophotometer and survey anti-uv-ray.Result shows that high peaks appears in absorption curve in 260-315nm ultra-violet (UV) band, and show that Apostichopus glycopeptide is comparatively large at the rate of absorbing UV of this wave-length coverage, therefore Apostichopus glycopeptide has certain uvioresistant ability.

Claims (1)

1. a preparation method for Apostichopus glycopeptide, described Apostichopus glycopeptide molecular weight is less than 3000Da, and sugar chain is connected on serine or threonine by GalNAc, has-Gal β 1-3 GalNAc α-O-Ser/Thr structure, it is characterized in that carrying out as follows:

A. wall of sea cucumber Stichopus japonicus is carried out homogenized 0 ~ 10 DEG C time;

B. in gained homogenate, add 0.01 ~ 0.1M phosphate buffer of 1 ~ 3 times of volume pH 7 ~ 8 and be the compound enzyme of 0.1 ~ 1% with homogenate mass ratio, enzymolysis 12 ~ 48 h; Described compound enzyme is the mixture that alkaline protease, papain and trypsin are 3:3:4 by unit of activity ratio;

C. centrifuging and taking supernatant, employing molecular cut off is that the Ultra filtration membrane device of 3000 below Da is separated, and gets the liquid through ultrafilter membrane;

D. by Sephadex LH-20 molecular sieve chromatography, OD is collected _280nmabsworption peak;

E. collected liquid is passed through macroporous adsorbent resin and active carbon postlyophilization, obtained Apostichopus glycopeptide.