CN107531775A

CN107531775A - The purposes of collagen water decomposition thing and collagen water decomposition thing rich in collagen tripeptide

Info

Publication number: CN107531775A
Application number: CN201780001018.5A
Authority: CN
Inventors: 慎镛喆; 朴哲; 崔守林; 金利洙; 金贤雅; 赵允英; 郑惠珍
Original assignee: Amicogen Inc
Current assignee: Amicogen Inc
Priority date: 2016-02-05
Filing date: 2017-02-03
Publication date: 2018-01-02
Anticipated expiration: 2037-02-03
Also published as: KR101980361B1; KR20170093694A; CN107531775B; WO2017135754A1

Abstract

The present invention relates to the purposes of a kind of collagen water decomposition thing rich in collagen tripeptide and collagen water decomposition thing, and in particular to the collagen water decomposition thing that a kind of collagen water decomposition enzyme using the special abundant collagen tripeptide of generation manufactures and the composition for promoting hair tonic and improving skin condition including the active ingredient.The collagen water decomposition thing of the present invention is compared with existing collagen water decomposition thing, and the content showed increased of collagen tripeptide, human body is preferable to its absorptivity, can effectively prevent baldness, promotes hair tonic, improves wrinkle and skin moisture-keeping etc..

Description

Collagen water decomposition thing and collagen water decomposition rich in collagen tripeptide The purposes of thing

Technical field

This application claims in the excellent of the Korean Patent application submitted for 5 days 2 months the 10-2016-0014896th in 2016 First weigh, entire contents are incorporated herein by reference.

The present invention relates to a kind of collagen water decomposition thing rich in collagen tripeptide and collagen water decomposition thing Purposes, and in particular to the glue that a kind of collagen water decomposition enzyme of the collagen tripeptide abundant using special generation manufactures Former albumen water decomposition thing and the composition for promoting hair tonic and improving skin condition including the active ingredient.

Background technology

Collagen is a kind of by the dynamic of about 18 kinds of Amino acid profiles such as proline, hydroxy-proline, glycine, glutamic acid The fibroid protein of thing, the protein of 30% special tectonic is occupied in 5000 multiple proteins for forming human body.Especially It is that to have 70% of skin corium in substantial amounts of collagen, particularly skin in skin, bone, tendon be by collagen Form, be the composition principal component of skin.Collagen is to be formed by various amino acid with three distortions of polypeptide combination of shape and state Shape, molecular weight about 300000 or so, shape is very huge.

Low molecule collagen is the degraded production of the molecular weight being decomposed to form by Collagenase etc. about 5000 or so Thing, i.e. collagen peptide.The low molecule collagen is entered after human body decomposed again by the protein decomposition enzyme in human body after Amino acid form is ultimately formed to be absorbed by the body.Compared with the molecular weight 12000~70000 of most of protein, collagen egg White molecular weight about 300000 or so, volume is very huge, it is difficult to absorbs.In order to be easier to digest and absorb in vivo, low point Sub- collagen separates by extraction, is decomposed again after extractive process, be eventually converted into peptide shape of the molecular weight below 5000 State.

Collagen tripeptide is the small collagen type (molecular weight formed that is connected by 3 amino acid (glycine-X-Y) Typically 200~500), because molecular weight is small readily permeable in skin.If the amino acid of more than 4 is connected, due to molecule knot Structure is excessive and can not permeate in skin.Collagen tripeptide can combine immediately after passing through skin, significantly shorten and repair what is damaged The time of collagen tissue.And after human intake inside absorptivity it is higher than low molecule collagen, collagen can be maximized Live body effect of albumen.

Collagen is typically used as raw material with ox, pig, fish, squid etc., by obtaining collagen three after the water decomposition of enzyme Peptide combinations.But by using protein decomposition enzyme obtain composition three peptide contents it is very low, be rich in it is difficult to obtain The composition of tripeptides.Therefore the method for needing more effectively to refine the collagen water decomposition thing rich in tripeptides.

Hair has different growth cycles, by growth period (anagen), catagen (catagen), stand-down (telogen) grow up and come off.These be within 3~6 years a cycle repeatedly, per day normal 50~100 hairs that come off.One As baldness refer to that the hair in the growth stage in these cycles is less, catagen or hair in stand-down are more, make hair Send out improper and come off.

, there are a variety of sayings the reason for alopecia, and such as androgenic effect surplus, sebum secretion is superfluous, poor circulation, mistake Scalp hypofunction caused by oxide and bacterium etc., gene, aging, pressure etc..In alopecia reason, swash as hero A kind of cortisol (testosteron) of element is by the dihydrotestosterone of 5α-reductase enzymatic activity (dihydrotestosterone；DHT), the DHT is combined with specific hydrosol and is induced the protein for triggering alopecia to cause to take off Hair.Further, since these factors trigger sebum superfluous, cause the alopecia with scalp inflammation such as acne, seborrhea.

Baldness is generally acknowledged to be due to disease, subalimentation, aging, caused by hormone is unbalanced.This was done Many researchs, so far it has not been found that causing the basic reason of baldness.Various effort, but mesh are carried out also for hair growth disease The medicine (Finasteride (finasteride) and minoxidil (minoxidil)) of preceding only 2 kinds of hair growths obtains U.S. State FDA (Food and Drug Administration) official recognition.The Finasteride of 5α-reductase inhibitor is by conduct Widely used for the hair tonic accelerator of androgenetic alopecia (androgenetic alopecia).Hypotensive agent (anti- Hypertensive agent) minoxidil according to the opening of ATP- sensitiveness K- channels, can promote hair tonic.But the effect of medicine Being not can also having significant limitation and is temporary transient with perfect forecast.Therefore new more effective anticreep life is needed Hair treatment medicine.Even if to Anti-hair loss and hair tonic is promoted to have the minoxidil (minoxidil) of significant curative effect or glue at present The medicament of polysaccharide auxin (Trichosaccharide) etc., also there is failure and the unstability to human body, and initiation allows skin The side-effect problems such as skin allergy, therefore be badly in need of developing the composition for ensuring security and stable effect.

The content of the invention

【The technical issues that need to address】

During the related personnel of the present invention makes great efforts for collagen water decomposition thing of the manufacture rich in collagen tripeptide It was found that the new collagen water decomposition enzyme water decomposition collagen manufactured using bacillus subtilis, can be manufactured rich in collagen The collagen water decomposition thing of albumen tripeptides, the promotion hair tonic of the composition obtained by this water decomposition and improvement skin shape State compares than original collagen water decomposition thing significant effect.

Therefore, it is an object of the invention to provide a kind of collagen water decomposition thing rich in collagen tripeptide, including, (a) stage being heat-treated to the mixture of fish scale and water；(b) by centrifuging bacillus subtilis strain nutrient solution Afterwards, the method manufacture collagen catabolic enzyme that concentration is centrifuged the filtered fluid of gained and refined using ion-exchange chromatography Stage；(c) by the collagen catabolic enzyme input described in (a) stage mixture in after, in 25~45 DEG C of environment reclaimed water Decompose the stage of 5~15 hours；Wherein, collagen tripeptide accounts for 20~70wt% of overall moisture solution thing.

It is an object of the invention to provide a kind of Anti-hair loss of including collagen water decomposition thing active ingredient or promote life The food compositions of hair.

It is an object of the invention to provide a kind of Anti-hair loss of including collagen water decomposition thing active ingredient or promote life The Pharmaceutical composition of hair.

It is an object of the invention to provide a kind of Anti-hair loss of including collagen water decomposition thing active ingredient or promote life The cosmetic compositions of hair.

It is an object of the invention to provide a kind of the crease-resistant of including collagen water decomposition thing active ingredient, improves skin bullet Property, anti-aging or skin moisture-keeping cosmetic compositions.

It is an object of the invention to provide a kind of the crease-resistant of including collagen water decomposition thing active ingredient, improves skin bullet Property, anti-aging or skin moisture-keeping food compositions.

It is an object of the invention to provide a kind of collagen water for being used to manufacture Anti-hair loss or promote hair tonic therapeutic agent The purposes of analyte.

It is an object of the invention to provide a kind of put into collagen water decomposition thing effective dose to need the individual of the composition The method of Anti-hair loss or promotion hair tonic in body.

It is used to manufacture crease-resistant, improvement skin elasticity, anti-aging or skin moisture-keeping it is an object of the invention to provide one kind With the purposes of the collagen water decomposition thing of agent.

It is an object of the invention to provide a kind of put into collagen water decomposition thing effective dose to need the individual of the composition It is crease-resistant in body, improve skin elasticity, anti-aging or skin moisturization process.

【Solution】

To reach the purpose, the present invention provides a kind of collagen water decomposition thing rich in collagen tripeptide, including, (a) stage being heat-treated to the mixture of fish scale and water；(b) by centrifuging bacillus subtilis strain nutrient solution Afterwards, the method manufacture collagen catabolic enzyme that concentration is centrifuged the filtered fluid of gained and refined using ion-exchange chromatography Stage；(c) by the collagen catabolic enzyme input described in (a) stage mixture in after, in 25~45 DEG C of environment reclaimed water Decompose the stage of 5~15 hours；Wherein, collagen tripeptide accounts for 20~70wt% of overall moisture solution thing.

Present invention also offers a kind of Anti-hair loss of including collagen water decomposition thing active ingredient or promote hair tonic Food compositions.

Present invention also offers a kind of Anti-hair loss of including collagen water decomposition thing active ingredient or promote hair tonic Pharmaceutical composition.

Present invention also offers a kind of Anti-hair loss of including collagen water decomposition thing active ingredient or promote hair tonic Cosmetic compositions.

Present invention also offers a kind of the crease-resistant of including collagen water decomposition thing active ingredient, improves skin elasticity, resists Aging or the cosmetic compositions of skin moisture-keeping.

Present invention also offers a kind of the crease-resistant of including collagen water decomposition thing active ingredient, improves skin elasticity, resists Aging or the food compositions of skin moisture-keeping.

Present invention also offers a kind of collagen water decomposition thing for being used to manufacture Anti-hair loss or promote hair tonic therapeutic agent Purposes.

Collagen water decomposition thing effective dose is put into the individual for needing the composition present invention also offers a kind of Anti-hair loss or the method for promoting hair tonic.

It is used to manufacture crease-resistant, improvement skin elasticity present invention also offers one kind, it is anti-aging or skin moisture-keeping agent The purposes of collagen water decomposition thing.

Collagen water decomposition thing effective dose is put into the individual for needing the composition present invention also offers a kind of It is crease-resistant, improve skin elasticity, anti-aging or skin moisturization process.

The present invention described further below.

The invention provides a kind of collagen water decomposition thing, including,

(a) stage being heat-treated to the mixture of fish scale and water；

(b) after by centrifuging bacillus subtilis strain nutrient solution, concentration centrifuges the filtered fluid of gained and profit The method refined with ion-exchange chromatography manufactures the stage of collagen catabolic enzyme；

(c) by the collagen catabolic enzyme input described in (a) stage mixture in after, in 25~45 DEG C of environment The stage of 5~15 hours of water decomposition；Wherein, collagen tripeptide accounts for 20~70wt% of overall moisture solution thing.

(a) stage is, it would be desirable to extract collagen fish scale mixed with water after high-temperature process process, be one The collagen that fish scale is included extracts the stage in water.The fish scale and the ratio of water that (a) stage adds are without spy Different to require, the weight ratio of preferable fish scale and water is 2 to 8.

In (a) stage from fish scale extract collagen temperature conditionss be 80~120 DEG C, preferably 85~ 110 DEG C, but this temperature is not limited to, optimal temperature conditionss are according to the extraction time of collagen and the ratio of fish scale and water Produce change.Heat treatment time in (a) stage produced change according to temperature conditionss, for 1~10 hour, preferably 2~9 Hour, it is within 3~8 hours optimal, but not limited to this.

(b) stage is the stage for preparing the water decomposition enzyme needed for water decomposition collagen, with water of the prior art Decompose the enzyme used during collagen to compare, the especially high water decomposition thing of the content of collagen tripeptide can be manufactured, it is enzyme Preparatory phase.The manufacture method of the present invention staff's collagen water decomposition enzyme in (b) stage by described in, is speciallyyed permit to Republic of Korea The Room, which was submitted, applies for a patent application (application numbers：No. KR10-2015-0124453), entire contents are incorporated by reference into this Text.

The bacterial strain that the bacillus subtilis in the present invention is deposit number KCTC12866BP, but not limited to this.

According to an embodiment of the invention, the collagen that the present invention staff includes for separation bacillus subtilis strain nutrient solution Protease, ion-exchange chromatography is carried out under different conditions.The ion-exchange chromatography in (b) stage is available Cation-exchange chromatography, when the concentration of sodium chloride now is 0.1~0.3M, the rate of recovery of target enzyme is optimal.Therefore, institute When the ion-exchange chromatography for stating (b) stage is cation-exchange chromatography, preferable concentration is 0.1~0.3M chlorination Sodium.

According to an embodiment of the invention, the ion-exchange chromatography in (b) stage can utilize anion-exchange chromatography, When the concentration of sodium chloride now is 0.09~0.155M, the rate of recovery of target enzyme is optimal.Therefore, (b) stage from When sub- exchange chromatography is anion-exchange chromatography, preferable concentration is 0.09~0.155M sodium chloride.

(c) stage in the present invention is the glue that the collagen extract that mixing (a) stage prepares manufactures with (b) stage Former protease, carry out the stage of collagen water decomposition.

In embodiments of the invention, in the collagen catabolic enzyme that (b) stage manufactures under 25~45 DEG C of temperature environment Keep the enzymatic activity of maximum duration.When temperature conditionss are more than 45 DEG C, the enzymatic activity at initial stage can with temporary increase, but with when Between elapse, active loss is increasing, can not fully demonstrate the effect of collagen water decomposition, opposite, and temperature is less than 25 DEG C when, enzymatic activity is too low, exist can not water decomposition collagen the problems such as.

Therefore, the water decomposition temperature conditionss in (c) stage are preferably 25~45 DEG C, more preferably 27~42 DEG C, or 30 ~40 DEG C are optimal.

The present invention may include that the collagen water decomposition thing obtained after (c) stage by described in, which passes through, to be centrifuged, ion Exchange chromatography, the stage concentrated after foreign matter is removed the methods of filtering.The stage for removing foreign matter and concentrating can be led to using this area The method often used, is not particularly limited.

The technical characteristic of the collagen water decomposition thing manufactured by the manufacture method in the present invention is, with using it He compares at the collagen water decomposition thing of water decomposition enzyme manufacture, collagen tripeptide content showed increased.

Collagen tripeptide is that (molecular weight is general for the small collagen type that is connected by 3 amino acid (glycine-X-Y) 200~500), because molecular weight is small readily permeable in skin.If the amino acid of more than 4 is connected, due to molecular structure mistake It can not permeate in skin greatly.Collagen tripeptide can combine immediately after passing through skin, significantly shorten the collagen repaired and damaged The time of albumen tissue.And after intake inside absorptivity it is higher than low molecule collagen, the live body of maximizing collagen Effect.Therefore, the content of the collagen tripeptide in collagen water decomposition thing is higher, and live body utilization rate is higher, maximizing The physiological activity of collagen.

According to an embodiment of the invention it has been confirmed that the collagen three of the collagen water decomposition thing of the present invention The content of peptide accounts for the about 53wt% of total moisture solution thing.Also, in the collagen water decomposition thing manufactured using other water decomposition enzymes Substantially not including collagen tripeptides (with reference to embodiment 4 and comparative example).Collagen is difficult to be absorbed by the body in itself, therefore Water decomposition is that small-sized tripeptides form is optimal.The collagen tripeptide being easily absorbed by the body contained in the water decomposition thing of the present invention Content it is more apparent very high compared with the collagen water decomposition thing reported in the past the advantages of.

The collagen tripeptide specifically, the collagen water decomposition thing and whole water decomposition things in the present invention compare Content be 20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42, 43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67, 68,69,70wt%.Moreover, with whole water decomposition things compare collagen tripeptide content in the range of 20~70wt% when, It is not limited to above-mentioned numerical value.Preferably 30~70wt%, 40~70wt%, 50~70wt%, 30~60wt%, 40~ 60wt%.50~60wt% is optimal, but not limited to this.

The present invention provides a kind of Anti-hair loss of including collagen water decomposition thing active ingredient or promotes the medicinal of hair tonic Composition.

In this specification【Alopecia】For the state that the phenomenon or hypotrichosis that hair comes off from scalp attenuate；【Anticreep Hair】To prevent and suppressing the depilation phenomenon；【Promote hair tonic】To promote the new life of hair and protecting original hair to be good for Health.

Generally【Hair tonic】The growth stage complete, and from stand-down be converted to growth period and delay from growth period to Catagen.The cycle of hair is broadly divided into growth stage, catagen, 3 stages of stand-down.In the growth stage with the quick increasing of cell Grow, hair follicle grows to deep skin and forms hair.Obvious transitional period, hair follicle during this are interrupted in cell division in catagen Gradually regression and hair tonic is stopped.It it is finally stand-down, the hair follicle of regression specifically includes dense papilla (dermal Papilla) the plumule (germ) of cell.It is initially with the cell in the plumule in the new round growth stage phenomenon of stand-down Fast breeding, the expansion of papilla and the synthesis of basilar memebrane key element guiding.

Therefore, it is necessary to by promoting or extending the growth stage, the loss of hair is prevented, i.e. anti-alopecia-stopping or guiding hair Regeneration, i.e. promote hair tonic.Composition made according to the present invention includes the effect of the existing hair of solidification and anti-alopecia-stopping, adds Thick one or more had in the improvements, and hair-growing effects such as hair.

Including compared with original water decomposition thing, hence it is evident that the collagen water of the collagen tripeptide containing very high content The present composition of analyte active ingredient, the human body body absorption and sclerderm absorptivity of collagen can be effectively improved, And infiltration rate is fast, to Anti-hair loss and promote hair-growing effects notable.

Specifically according to an embodiment of the invention, to the collagen water decomposition thing in the present invention and utilization this area water Decompose what trap inside the collagen water decomposition thing of the water decomposition enzyme manufacture typically used during collagen was evaluated As a result, the AUC of the collagen water decomposition thing in the present invention_last,C_maxObvious very high, T_maxIt is shorter, show absorption in vivo It is very outstanding.It may determine that the content of the collagen tripeptide of the collagen water decomposition thing in the present invention is non-according to the above Chang Gao, therefore the very fast absorptivity of body absorption is very outstanding.

According to another embodiment of the invention, the combination containing the collagen water decomposition thing active ingredient in the present invention Thing effectively facilitates the propagation of the hair follicle cell of the shedder sample of catagen, suppresses the progress of catagen, and has maintenance The effect in growth period, the growth to hair have remarkable result.In addition, according to the trichogenous of the composition of the present invention If effect and the rice ground your (Minoxidil) used as positive control group are equally very outstanding.

Collagen tripeptide content in the collagen water decomposition thing of the present invention is very high, the collagen egg absorbed in vivo The amount of white derivative tripeptides is relatively more, and effect is also relatively good.

The present invention provides a kind of Anti-hair loss of including collagen water decomposition thing active ingredient or promotes the food of hair tonic Composition.

These food compositions are except including the collagen water decomposition beyond the region of objective existence as active ingredient, with general food group Compound identical includes other supplementary elements such as various flavouring agents or natural carbohydrate.The natural carbohydrate bag Include monosaccharide, such as glucose, fructose etc.；Disaccharides, such as maltose, sucrose etc.；And polysaccharide, such as dextrin, ring paste The glycitols such as the conventional carbohydrate and xylitol of essence etc., sorbierite, antierythrite.The flavouring agent can effectively utilize natural flavor Agent (thaumatin), qualities of stevia extract (such as rebaudioside A, glycyrrhizin etc.) and synthesis flavouring agent (saccharin, Aspartame Deng).

The present invention food compositions manufacture it is identical with the Pharmaceutical composition, can be used as health food or Person is added in various food.Can add the present invention composition food such as beverage class, meat, chocolate, foodstuff, Biscuits, Pizza, steep face, other noodles, chewing gum class, carbohydrate, ice cream, alcoholic beverage class, mix vitamin agent and Health food etc..

And the food compositions can include various battalion except the collagen water decomposition beyond the region of objective existence as active ingredient Support agent, vitamin, mineral matter (electrolyte) synthesis flavouring agent and natural flavours, colouring agent and sweetening agent (cheese, chalk Power etc.), pectic acid, pectate, alginic acid, alginate organic acid, protective solid sweetening agent, PH conditioning agents, stabilize Agent, preservative, glycerine, alcohol, soda carbonating agent etc..In addition, the composition in the present invention can include manufacture day Right fruit juice and the pulp of fruit drink and vegetable beverage etc..

Collagen water decomposition thing as the active ingredient of the present invention is a kind of natural materials, and nontoxicity has no side effect, Can be with Anti-hair loss, or promote the long-term use for the purpose of hair tonic.

The present invention the food compositions can be including collagen water decomposition thing active ingredient Anti-hair loss or Promote the health food of hair tonic.

Health food in the present invention can manufacture and be processed as lozenge for the purpose of Anti-hair loss or promotion hair tonic, The forms such as capsule, powder, granule, liquid, pill.

In the present invention【Health food】It is according to health food relevant law regulations the 6727th, is that use has to human body The food that the functional raw material or composition of benefit are manufactured or processed, purpose are the construction and function or physiology of regulation human body Effect etc. as health purpose acquisition beneficial effect and intake.

The health food of the present invention can include conventional food additives, be not specified by clear and definite assigned food additive Suitability in the case of, the general provisions that are published according to the food additives of food pharmaceuticals state security department's certification and general Experimental method etc. deserves the related specification of composition and benchmark as basis for estimation.

It is described【Food additives publish】The composition of loading is for example, ketone, glycine, calcium citrate, nicotinic acid, Chinese cassia tree The syntheticses such as acid；The natural addition such as Perimmon element, licorice, cellulose crystallite, kaoliang color, guar gum The mix preparation classes such as agent, Pidolidone salt pref, noodles alkaline agent, preservative, tar colorant preparation.Such as the health food of ingot shape By admixed excipients, bonding agent, disintegrating agent and other additives in the active ingredient collagen water decomposition thing of the present invention, and Lubricant compression forming is added after being granulated by common method or is directly compressible into mixture described in type.And according to demand in institute State in the health food of ingot shape and add flavouring agent.

Hard rubber wafer in the health food of capsule shape is using conventional hard capsules, and by the active ingredient of the present invention The mixture filling manufacture of the additive such as admixed excipients in collagen water decomposition thing.Soft capsule is by collagen water decomposition The mixture of the additive such as admixed excipients is manufactured with being filled into the capsule preparations such as gel in thing.It is described soft according to demand Matter capsule can include the additives such as the pigment agents such as glycerine or sorbierite, colouring agent, preservative.

The health food of pellet is by published manufacture method, by the active ingredient collagen water decomposition thing of the present invention The mixture modulation shaping of middle admixed excipients, bonding agent, disintegrating agent and other additives, and according to demand using white sugar or Other sugar-coat agent manufacture sugar-coat, or cover surface using materials such as starch, talcums.

Granular health food is by published manufacture method, by the active ingredient collagen water decomposition of the present invention The mixture modulation of admixed excipients in thing, bonding agent, disintegrating agent and other additives granulates, and according to demand, can be with Including additives such as flavouring agent, flavor enhancements.

The health food can be beverage class, meat, chocolate, foodstuff, Biscuits, Pizza, steep face, other faces Class, chewing gum class, carbohydrate, ice cream, alcoholic beverage class, mix vitamin agent and health food etc..

According to the present invention Pharmaceutical composition, can individually include the present invention in collagen water decomposition thing or can be with volume Include more than one pharmaceutical carrier, excipient or diluent outside.

Pharmaceutical carrier can comprise additionally in oral carrier or parenteral carrier.Oral carrier can include lactose, and starch is fine Tie up plain conductor, magnesium stearate, stearic acid etc..And parenteral carrier can include water, appropriate oil, normal saline solution, water solubility Glucose and glycol etc., and can additionally include stabilizer and preservative.Suitable stabilizer has sodium hydrogensulfite, sulphur The antioxidants such as sour sodium or ascorbic acid.Suitable preservative has chlorination benzalkonium chloride, methyl or P-hydroxybenzoic acid third Ester and methaform etc..In addition documents below contents (Remington's can be referred to as the medicinal carrier used PHarmaceutical Sciences,19th ed.,Mack Publishing Company,Easton,PA,1995)。

Any input method can be used to put into the mammal including the mankind for the Pharmaceutical composition of the present invention.It is such as oral Or parenteral input.Parenteral investing method has intravenously, intramuscular, intra-arterial, in marrow, in pleura, and sclerderm in heart, Subcutaneously, intraperitoneal, in nasal cavity, intestinal tube is local, is put into sublingual or rectum, but not limited to this.Preferably orally put into this hair Bright Pharmaceutical composition.For example, the Pharmaceutical composition of the present invention is modulated into injection-type preparation, and use the injection needle of 30 gauges Gently puncture the method for skin or be applied directly to the method for skin surface and put into.

The Pharmaceutical composition of the present invention such as content, for oral use or parenteral system can be divided according to input approach Agent is molded.

Composition of the invention for preparations for oral administration can be powder, granule, lozenge, pill, sugar-coat lozenge, capsule Agent, liquid agent, gel, syrup, agent, suspension etc. is starched, pass through manufacture method modulation shaping disclosed in this area.For example, For oral use dose active component and solid excipient are allocated after crush, then add appropriate adjuvant post-processing as particle mixing Thing, obtain lozenge or sugar-coat lozenge.Suitable excipient has including lactose, glucose, sucrose, sorbierite, mannitol, xylose The carbohydrate of alcohol, antierythrite, maltitol etc.；Include the starch of cornstarch, wheaten starch, rice starch, potato starch etc. Class；Include the cellulose family of cellulose, methylcellulose, sodium carboxymethylcellulose, hydroxypropyl methyl cellulose etc.；Including solidifying The filler of glue, polyvinylpyrrolidone etc..The Pharmaceutical composition of the further present invention, which adds, includes anti-coagulants, lubricant, Spices, emulsifying agent and preservative etc..

For preparations for oral administration, with injection, creams agent, emulsion agent, externally-applied soft ointment, essential oil agent, NMF, gel The forms such as agent, spray and snuff, pass through manufacture method modulation shaping disclosed in this area.These forms are all remembered Be loaded in prescription document disclosed in pharmaceutical chemistry (Remington's PHarmaceutical Science, 15th Edition, 1975.Mack Publishing Company,Easton,Pennsylvania 18042,Chapter 87:Blaug, Seymour in).

The Pharmaceutical composition of the present invention as external preparation for skin Pharmaceutical composition in use, as Anti-hair loss, promotion hair tonic with And improve the skin preparations for extenal use of scalp state, with creams, gel, sticking patch, spray, ointment, emplastrum, emulsion agent, smear The Pharmaceutical composition modulation shaping of the skin preparations for extenal use form of agent, paste or opoultice, but it is not limited to this.

Total effective dose of the Pharmaceutical composition of the present invention puts into patient as single input amount (single dose), also may be used Classification therapeutic scheme (fractionated to put into patient for a long time as multiple input amount (multiple dose) treatment protocol).The Pharmaceutical composition of the present invention according to the weight of the state of an illness, can adjust the content of active ingredient. Preferably, divided according to the glycoprotein of the present invention, the every overall dosage not daily 1kg of body weight of daily patient is about 0.01ug ~1,000mg, or 0.1 μ g~100mg are optimal.But the dosage of the therapeutic agent candidate substances of the invention is, it is necessary to examine Consider the input approach and treatment number of times of Pharmaceutical composition, and be also contemplated that the age of patient, body weight, health status, sex, The many factors such as the weight of symptom, eating habit and drainage status determine effective input amount to patient.For this area skill For art personnel, the specific use using the Pharmaceutical composition of the invention as Anti-hair loss and the pharmaceutical formulation for promoting hair tonic On the way, appropriate adjustment is made to effective input amount.For the shape of the Pharmaceutical composition of the present invention, approach and input side are put into Method can be adjusted flexibly.

The present invention provides a kind of Anti-hair loss of including collagen water decomposition thing active ingredient or promotes the beauty of hair tonic Use composition.

According to the cosmetic compositions of the present invention, the active ingredient except including the collagen water decomposition thing in the present invention Outside, in addition to it is usually used in the compositions of cosmetic compositions.Such as including antioxidant, stabilizer, solubilizer, vitamin, pigment And the usual auxiliaries and carrier of dyestuff etc..

Moreover, do not damaging collagen water in addition to the collagen water decomposition thing in the composition of the present invention In the case of the Anti-hair loss of analyte and the effect of promotion hair tonic, can be mixed with existing Anti-hair loss agent and preparation for baldness makes With.

Moreover, the cosmetic compositions of the present invention can be modulated into the arbitrary shape for deserving industry universal.For example, hair tonic Oil, hair conditioner, hair-care essence liquid, hair care breast, nutritional breast of defending the doctrine, shampoo, irrigation , treatment waxes of taking care of hair, Hair-conditioning essential oil, hair care Frost, nourishing cream of taking care of hair, moisturizing breast of defending the doctrine, scalp massage frost, pomade, hair sprays, send out film, nutrient hair mask, hair soap, hair washing Breast, foam of having one's hair wash, hair oil, hair drier, agent for treatment of hair, hair dye, agent for permanent hair waving, decolorising agent, hair styling gel, hair elite The form that breast, hair cosmetic agent, hair care spraying, hair moisturiser, hair cream, hair jelly etc. all aoxidize, but not limited to this.

According to an embodiment of the invention, collagen water decomposition thing of the invention improves skin to the crease-resistant of skin, moisturizing etc. Skin state aspect has significant effect.The collagen water decomposition thing manufactured by the collagen water decomposition enzyme of the present invention The excellent for the collagen water decomposition thing that skin condition improvement substantially manufactures than existing collagen water decomposition enzyme. Judge that the collagen water decomposition thing in the present invention is rich in collagen tripeptide with this, it is higher to human body utilization rate, and collagen The physiologically active of albumen plays more preferable.

It can clearly confirm the collagen water decomposition thing in the present invention to skin condition by crossing specific embodiment Improvement.

The present invention provides a kind of anti-including the crease-resistant of the collagen water decomposition thing active ingredient, improvement skin elasticity Aging or the cosmetic compositions of skin moisture-keeping.

In addition the present invention provides a kind of including the crease-resistant of the collagen water decomposition thing active ingredient, improves skin bullet Property, anti-aging or skin moisture-keeping food compositions.

Foregoing content can be all suitable for the cosmetic compositions and food compositions.

The present invention provides a kind of collagen water decomposition thing for being used to modulate Anti-hair loss or promote hair tonic therapeutic agent Purposes.

The present invention provides a kind of anticreep for putting into collagen water decomposition thing effective dose in the individual for needing the composition Hair or the method for promoting hair tonic.

Present invention offer is a kind of crease-resistant for modulating, improvement skin elasticity, described in anti-aging or skin moisture-keeping agent The purposes of collagen water decomposition thing.

The present invention provide it is a kind of collagen water decomposition thing effective dose is put into it is crease-resistant in the individual for needing the composition, Improve skin elasticity, anti-aging or skin moisturization process.

It is described in the present invention【Effective dose】When putting into individual, to promote hair tonic and improvement, treatment, pre- anticreep The amount of hair and other effects, and improve, treatment, prevention wrinkle of skin, skin elasticity, the amount of skin aging and skin moisturization； It is described【Individual】Extracted for animal, preferably mammal, the particularly animal including the mankind or from animal thin Born of the same parents, tissue, organ etc..The individual can also be the patient for needing the effect.

【The technique effect of invention】

The present invention relates to a kind of collagen water decomposition thing rich in collagen tripeptide and collagen water decomposition thing Purposes, collagen water decomposition thing of the invention compared with existing collagen water decomposition thing, collagen tripeptide Content showed increased, human body are preferable to its absorptivity, can effectively prevent baldness, promote hair tonic, improve wrinkle and skin is protected It is wet etc..

Brief description of the drawings

Fig. 1 is to utilize point of the cationic ion resin of the collagen catabolic enzyme manufactured by embodiments of the invention From the figure for refining result.A is the SDS-PAGE analysis results (M of the collagen catabolic enzyme of refinement:The big tick marks of protein, conc:Concentrating sample, wash:Clean, E1~E5:Elution samples 1~5).B is, according to the buffer concentration in AKTA primer The collagen catabolic enzyme extractive process not changed is identified as maximum (arrow is respectively the mark of elution samples).

Fig. 2 is to utilize point of the anionic ion resin of the collagen catabolic enzyme manufactured by embodiments of the invention From the figure for refining result.A is the SDS-PAGE analysis results (M of the collagen catabolic enzyme of refinement:The big tick marks of protein, P1:Represent peak 1).B is the collagen catabolic enzyme extractive process mark not changed according to buffer concentration in AKTA primer Know for maximum (arrow is respectively the mark of elution samples).

Fig. 3 is temperature and the enzymatic activity result schematic diagram of the collagen catabolic enzyme manufactured by embodiments of the invention Table.

Fig. 4 is the enzyme elapsed according to time and temperature of the collagen catabolic enzyme manufactured by embodiments of the invention Activity change result schematic diagram table.

Fig. 5 is PH and the enzymatic activity result schematic diagram table of the collagen catabolic enzyme manufactured by embodiments of the invention.

Fig. 6 is to pass through the collagen of the collagen catabolic enzyme that embodiments of the invention manufacture and general protein decomposition enzyme HPLC data (the A of albumen tripeptides productivity contrast:CTP standard substances, B:Collagen catabolic enzyme BP, C:Alkali protease Alcalase 2.4L FG,D:Flavor protease Flavourzyme 1000L, E:Papain Collupulin MG).

Fig. 7 is that, using the collagen catabolic enzyme manufactured by embodiments of the invention, production is rich in collagen tripeptide Collagen water decomposition thing manufacturing process's schematic diagram.

Fig. 8 is the glue in the water decomposition thing produced using the collagen catabolic enzyme manufactured by embodiments of the invention HPLC data (the A of former albumen tripeptides (CTP) content analysis:CTP standard substances, B:Collagen catabolic enzyme BP, C:Basic protein Enzyme Alcalase 2.4L FG).

Fig. 9 is to put into experiment to the animal experiment body that catagen depilation is induced using dexamethasone (Dexamethasone) During material, the growth period of hair follicle and effect assessment results icon ((A) defeathering the 9th day, (B) defeathering of catagen ratio are influenceed 11st day, (C) defeathering the 14th day, (D) defeathering the 17th day, anagen：Growth stage, catagen：Catagen, Nor：It is not used, ground Sai meter Song processing, the normal group of a defeathering, Control：Handled using dexamethasone and the solvent control of solvent is put into after defeathering Group, BP-CH：Handled using dexamethasone and the group of the collagen water decomposition thing of the present invention, Minox are put into after defeathering：Use The positive control group of minoxidil is put into after dexamethasone processing and defeathering).

Figure 10 is, dynamic to testing after the animal experiment body input substances lost hair or feathers using Induced By Dexamethasone catagen The influence result schematic diagram that the body hair growth of thing is brought.

Figure 11 is to be observed by tissue staining body, the skin of the animal experiment body to be lost hair or feathers using Induced By Dexamethasone catagen Result schematic diagram (the Dex of skin tissue hair follicle growth：Dexamethasone).

Figure 12 is to be irradiated by UV after inducing wrinkle and putting into each material, use silica gel and the woods mould of epoxy resin Result figure (the N of plate observation：Normally, C：Control, P：Positive control, COL17, COL34：Pass through in general collagen catabolic enzyme Collagen water decomposition the thing 17mg/kg or 34mg/kg of production input group, CTP17, CTP34：The collagen of the present invention Water decomposition thing 17mg/kg or 34mg/kg input group).

Figure 13 is, after experiment just terminates, gathers the skin of the hairless mouse of SKH-1, carries out the result of horse pine trichrome staining Schematic diagram (N：Normally, C：Control, P：Positive control, COL17, COL34：The glue produced by general collagen catabolic enzyme Former albumen water decomposition thing 17mg/kg or 34mg/kg input group, CTP17, CTP34：The collagen water decomposition thing of the present invention 17mg/kg or 34mg/kg input group).

Figure 14 is, after experiment just terminates, gathers the skin of the hairless mouse of SKH-1, passes through immune-blotting method method confirmation group Knit the quantized result schematic diagram (N of interior collagen 1A (A) and MMP-1 (B) protein discovery amount：Normally, C：Control, P： Positive control, COL17, COL34：By in general collagen catabolic enzyme produce collagen water decomposition thing 17mg/kg or Person 34mg/kg input group, CTP17, CTP34：Collagen water decomposition the thing 17mg/kg or 34mg/kg of the present invention throwing Enter group).

Figure 15 is, when the 5th week interstage of experiment and the 10th week, confirms the grease (A) of mouse skin, moisture (B) Result schematic diagram (the N of state：Normally, C：Control, P：Positive control, l, COL17, COL34：Pass through in general collagen point Solve collagen water decomposition the thing 17mg/kg or 34mg/kg of enzyme production input group, CTP17, CTP34：The collagen of the present invention Albumen water decomposition thing 17mg/kg or 34mg/kg input group).

Figure 16 is, after putting into each material to the Balb-c mouse for inducing dry skin, evaluates the knot of skin moisture content Fruit schemes (N：Normally, C：Control, P：Positive control, COL17：Collagen water decomposition the thing 17mg/kg, COL34 of the present invention：It is logical Cross the collagen water decomposition thing 34mg/kg of in general collagen catabolic enzyme production input group).

Figure 17 is, after putting into each material to the Balb-c mouse for inducing dry skin, what is tickled by pruitus is secondary Number measurement result schematic diagram (N：Normally, C：Control, P：Positive control, COL17：The collagen water decomposition thing 17mg/ of the present invention Kg, COL34：The collagen water decomposition thing 34mg/kg produced by general collagen catabolic enzyme input group).

Figure 18 is, each material is put into the Balb-c mouse for inducing dry skin, and gathers after off-test each old Mouse skin histology, the quantized result schematic diagram (A of skin moisture-keeping factor protein matter discovery amount is confirmed by immune-blotting method method： Collagen 1A, B：AQP3, C：HAS2, N：Normally, C：Control, P：Positive control, control), COL17：The glue of the present invention Former albumen water decomposition thing 17mg/kg, COL34：The collagen water decomposition thing produced by general collagen catabolic enzyme 34mg/kg input group).

Embodiment

Embodiments of the invention described further below.

Following examples are only the example of the present invention, and present disclosure is not limited to following examples.

The manufacture of collagen water decomposition enzyme

The manufacture method of collagen water decomposition thing in the present invention and the collagen water manufactured by methods described Analyte related content is with reference to korean patent application the 10-2014-0158019th.

That is, mTB medium (the yeast extract2.4%, tryptone1.2%, glycerol 1% of 100ml KH₂PO₄2.31%, H₂HPO₄12.54%) in after culture bacillus subtilis strain (deposit number KCTC12866BP) Centrifuged 15 minutes in 6500rpm.Filtered fluid is transferred to after new flexible pipe and is concentrated into 5 times using 30kDa filter.Make With AKTA prime equipment, by ion-exchange chromatography, the filtered fluid after concentration is refined.

Refinement condition now is that A is buffered into (50mM Tris-HCl (pH7.5)) buffers use as binding, and (50mM Tris-HCl (pH7.5), 0.5M NaCl) is buffered using B to tilt, and flow velocity is 5ml/min.Refinement uses cation Exchange chromatography and anion-exchange chromatography, ion exchange resin use resin cation SP sepHarose resin respectively (GE Healthcare, new-Jersey, USA) and resin anion (R.A.) Q sepHarosse resin (GE healthcar, New-Jersey, USA).

0.5M NaCl inclinations are set in cation-exchange chromatography first, and protein is refined according to each division.Such as figure Shown in 1B, confirm in SDS-PAGE, refine the protein refinement degree that protein maximum division (E1~E5) is shown in chart, hair Protein containing prediction target enzyme in existing E1, E2, particularly can confirm that in E1 and is decomposed containing substantial amounts of collagen Enzyme (Figure 1A arrow represents).

E1~E5 NaCl concentration is E1：0.1~0.2M, E2：0.2~0.3M, E3：0.3~0.4M, E4：0.4~ 0.5M, E5：0.5M.It is possible thereby to learn, by cation-exchange chromatography separate the NaCl concentration of target enzyme for 0.1~ 0.3M.Preferably, the rate of recovery of target enzyme is optimal in the case of the 0.1~0.2M of NaCl concentration of E1 parts.Then, by the moon from Sub- exchange chromatography refines E1.

The buffering now used is 0.25M NaCl, and sets identical gradient with cation-exchange chromatography.Such as figure Shown in 2B, when the anion exchange resin refined in chart is 0.09~0.115M NaCl, there is the maximum (figure of protein 2B arrow represents).

The refinement figure that should be divided is confirmed from SDS-PAGE, can confirm that most of target enzyme has refined clean (figure 2A P1).It is possible thereby to learn, NaCl concentration is target in the case of 0.09~0.155M when passing through anion-exchange chromatography The rate of recovery of enzyme is optimal.The new collagen protease extracted is named as BP below.

BP enzyme reaction condition test

Tested as optimum temperature, carry out enzyme reaction in 20~70 DEG C of temperature, and measure potency.As shown in figure 3, temperature For 0~55 DEG C when, produce enzymatic activity, the specific activity (specific of peak particularly shown in the environment of 50 DEG C Activity) and relative activity (relative activity), activity drastically declines since 60 DEG C of temperature.

Suitable for a large amount of productions, to be tested the stabilisation of temperature.Temperature is adjusted to 30 DEG C, 35 DEG C, 40 DEG C with And 50 DEG C, and the other sample of each temperature was not sampled from the time of 0~12 hour and confirms respective residual activity.

As shown in figure 4, the lower enzyme of temperature shows more continual and steady state, it is small by 12 between 35 DEG C~40 DEG C When after remain about 59.3% activity.As can be seen here, it is optimal the production process temperature of collagen tripeptide to be adjusted into 35 DEG C.

Next optimal pH value is confirmed.By pigskin, gel, matrix is not respectively put into buffering (50mM according to pH value citrate-Na₂HPO₄(pH5.0~6.0), 50mM Tris-HCl (pH6.0~9.0), 50mM Na₂CO₃-Na₂HCO₃(pH9.0 ~10.0) activity of enzyme is determined after melting in) and confirms optimal pH.Highest activity is presented in BP in the environment of pH 7.4, Higher activity is presented under pH 6~10 differential gap environment.But as shown in figure 5, it can confirm that slant acidity or meta-alkalescence In the case of enzyme reduced activity.

The process using the collagen catabolic enzyme (BP) manufacture collagen tripeptide is illustrated below.

Confirm BP collagen tripeptide prouctiveness

The fish scale of pretreatment is put into the reactor set in mixer.According to 20：80 weight ratio equably mixes Fish scale and water, and about 90 DEG C of temperature be heat-treated 5 hours after, prepare 4 samples.4 samples use collagen respectively Catabolic enzyme (BP) (manufacturer：Amicogen), alkali protease 2.4L FG (Alcalase 2.4L FG) (manufacturer： Novozyme, dealer：Viosys), flavor protease 1 (Flavourzyme 1), 000L (manufacturer：Novozyme, sell Business：Viosys), papain MG (Collupulin) (manufacturer：Novozyme, dealer：Great bell business) enzyme, compare The prouctiveness of collagen tripeptide.

The reaction condition of each enzyme is following (temperature, PH, enzyme usage amount, reaction time).

-BP：35 DEG C, pH 7.4,30unit/g (fish scale), 12 hours

-Alcalase 2.4L FG：60 DEG C, pH 7.0,30unit/g (fish scale), 12 hours

- Flavourzyme 1,000L：55 DEG C, pH 6.0,30unit/g (fish scale), 12 hours

-Collupulin MG：60 DEG C, pH 7.0,30unit/g (fish scale), 12 hours

After ferment treatment, the heat treatment carried out 30 minutes under 80 DEG C of temperature environment is deactivated, also, 4 kinds of enzymes are carried out Comparative analysis, confirm the line balancing rate of BP collagen tripeptide.

The collagen water decomposition thing to manufacture is analyzed using HPLC (gilson, Gilson Inc).In SuperdexTM Peptide10/300GL posts, mobile phase (10mM Tris-Cl (pH7.4), 0.15M NaCl, 5mM CaCl₂), flow velocity： 0.5ml/min condition is in analysis collagen tripeptide (collagen tripeptide, CTP).CTP standard substance uses By the Gly-Pro-Hyp (glycine-proline-hydroxyproline, GPH) of 3 kinds of Amino acid profiles.

As shown in fig. 6, CTP standard substances shown in analysis result formed maximum at about 55 minutes or so.Compare 4 kinds of enzymes Degrading activity is it is known that, collagen later using the ratio of the maximum formation of the collagen water decomposition thing of BP progress water decompositions Albumen is degraded.And standard substance shows larger maximum in the same period, CTP content can be learnt very It is high.Using the collagen water decomposition thing and other of Alcalase 2.4L FG water decompositions using Flavourzyme 1000L or The collagen water decomposition thing of the person's collupuin MG water decompositions low molecule content that compares is very high, but CTP contents almost do not have Have.It is possible thereby to learn that the production effects of BP to collagen tripeptide are notable.

Use the manufacture of BP collagen water decomposition thing

In order to manufacture the collagen water decomposition thing containing collagen tripeptide, the manufacture method using BP is established.

The fish scale of pretreatment is put into the reactor set in mixer.According to 20：80 weight ratio equably mixes Fish scale and water, and be heat-treated 5 hours in about 90 DEG C of temperature.

Under 35 DEG C of temperature environment, the liquid after heat treatment is put into 10%NaOH, adjusts the pH of solution Maintain 7.4.The BP of about 30unit/g (fish scale) left and right is put into the liquid after adjustment, and under 35 DEG C of temperature environment Reaction 12 hours.After ferment treatment, the heat treatment carried out 30 minutes under 80 DEG C of temperature environment is deactivated.Use ultracentrifugation After the foreign matter in collagen tripeptide liquid after separator removal ferment treatment, pass through ion column and remove the foreign matters such as metal ion.

Using vacuum pump-down thickener, after the liquid concentration to Brix 35%, by active carbon purifying, taken off Color removes peculiar smell.By the secondary filter of active carbon purifying liquid (membrane filtration, membrane filtration), it is carried out it is degerming after, make With spray dryer powdered, and Quality Detection is carried out, by packing stage, produce the collagen water decomposition rich in tripeptides Thing (Fig. 7)

Use the manufacture of the collagen water decomposition thing of general water decomposition enzyme

In order to compared with the manufacturing process using new BP, using being used in general low molecule collagen decomposition process Alcalase 2.4L FG (manufacturers：Novozyme, dealer：Viosys the line balancing rate of collagen tripeptide) is compared.

The fish scale of pretreatment is put into the reactor set in mixer.According to 20：80 weight ratio equably mixes Fish scale and water, and about 90 DEG C of temperature be heat-treated 5 hours.

Under 35 DEG C of temperature environment, the liquid after heat treatment is put into 10%NaOH, adjusts the pH of solution Maintain 7.5.The Alcalase enzymes of about 30unit/g (fish scale) left and right are put into the liquid after adjustment, and in 35 DEG C of temperature Reacted 12 hours under degree environment.After ferment treatment, the heat treatment carried out 30 minutes under 80 DEG C of temperature environment is deactivated.

After the foreign matter in the collagen tripeptide liquid after ferment treatment is removed using ultracentrifugation separator, pass through ion column Remove the foreign matters such as metal ion.Using vacuum pump-down thickener, after the liquid concentration to Brix 35%, pass through activated carbon Purification, carries out colour fading and removes peculiar smell.Active carbon purifying liquid is passed through into filter press filtering or secondary filter (membrane filtration, membrane Filtration), it is carried out it is degerming after, using spray dryer powdered, and after carrying out Quality Detection, collagen is carried out to it Albumen tripeptides (CTP) content analysis.

CTP content analysis is carried out with the identical method of embodiment 2.The final tune manufactured using BP and Alcalase Collagen water decomposition thing with previous stage is analyzed.

As shown in figure 8, on CTP contents it is known that using the collagen water decomposition thing that BP is manufactured and CTP reference materials The maximum of matter shows that position is identical, but is not shown on same position using the Alcalase collagen water decomposition things manufactured Maximum.

To be using the CTP content quantitative result of the comparison of the collagen water decomposition thing of BP and Alcalase manufactures, BP 53.1%, but Alcalase is 0%.It is possible thereby to learn, the collagen rich in collagen tripeptide can be manufactured using BP Water decomposition thing.

Collagen water decomposition thing using BP is compared with the trap of the collagen water decomposition thing using Alcalase

<5-1>Animal test method

Ether anesthesia male mouse, after being inserted at its right carotid artery with polyethylene pipe, using transfusion back strap by sleeve pipe from Extracted out by place, and adjust its position, allow suture mouth after the free activity of rat.Mouse fasting 12 hours to intubation with On, it is only supplied drinking water.6 are selected in each group respectively, according to the collagen water decomposition of the embodiment 4 and comparative example 1 The collagen water decomposition thing (BP-CH) using BP and the collagen moisture using Alcalase of thing manufacture method manufacture Solution thing (Alcalase-CH) is dissolved in distilled water and put into 500mg/kg (5mL/kg) capacity respectively.After input on a time period (15,30,45,60,90,120,180,240 and 360 points) use centrifugal separation technology separation bleeding after arteria carotis blood sampling Slurry.The blood plasma isolated freezen protective under -20 DEG C of temperature environment, while analyze the hydroxyproline in blood plasma with LC-MS/MS The concentration of (Hydroxyproline, Hyp) is simultaneously quantitative.

<5-2>LC-MS/MS analytic approach

Sample pretreatment：After internal standard liquid (the μ g/mL of Gabapentin 0.1) mixing is added in 50 μ L blood plasma, then add After adding the μ L of trichloroacetic acid 50 of 5% concentration to mix, filtered fluid is obtained after 13000rpm is centrifuged 10 minutes.Filtered fluid is dilute After releasing 2 times, take out in 2 μ L injections LC-MS/MS and analyzed

Analytical equipment：Agilent1260HPLC system and Agilent6460triple-quadrupole mass spectrometer

Post：Zorbax SB-Aq, particle size 1.8 μ, 2.0mm, i.d. × 100mm, l.

Column temperature：30℃

Mobile phase：0.1% formic acid (A) acetonitrile (B) gradient elution

Flow velocity：0.25mL/min

Injection rate：2μL

<5-3>Trap evaluation method

After formulating the plasma concentration v. time curve for each target component of each group, plasma concentration v. time curve is sought The gross area (AUC；Area under the plasma concentration-time curve), with this comparison trap.AUC Pass through linear trapezoid method (linear trapezoidal method) value of quadraturing.Extrapolation is relatively difficult in terminal phase, therefore not Need the unlimited AUC for seeking total AUC, asking untill 6 hours of final blood sampling point_last.Maximum plasma concentration (C_max) and maximum plasma Concentration arrival time (T_max) directly can read in the graph.AUC, C between each group is analyzed by one-way ANOVA_max, T_maxNumerical value, and pass through Duncan conspicuousnesses on proof method Research statistics afterwards.

<5-4>The mounting test result of collagen water decomposition thing

The ACU values and cmax value of free hydroxyproline, the conspicuousness between BP-CH and Alcalase-CH groups are poor in blood It is different.This is to illustrate that the hydroxyproline absorptivity in blood is higher.Moreover, the Tmax values and Alcalase- of input BP-CP group CH faciations are than very fast.It is possible thereby to learn, hydroxyproline is absorbed rapidly after BP-CH is absorbed, and stay in blood Time is shorter, may infer that from the soak time from blood to tissue and accelerates (table 1).

It is known that can be true in change passage in the blood of the special acid hydroxyproline of collagen by this experiment Recognize, the sink effect using BP collagen water decomposition thing is substantially higher than using Alcalase collagen water decomposition thing.

【Table 1】

The hydroxyproline of collagen water decomposition thing using BP and the collagen water decomposition thing using Alcalase Trap results contrast

Abbreviation：

AUC_last, from the gross area that the time is zero plasma concentration time curve of time of measuring to the end, 6 hours；C_max, Peak plasma concentration；T_max, reach Cmax time；

^aSD：Standard deviation；

^b：CTP groups and the significant difference (p of Alcalase-CP groups<0.05).

^c：T_maxIntermediate value (scope)

Use the Anti-hair loss and promotion hair-growing effects of BP collagen water decomposition thing

<6-1>Experimental animal food manufactures

Collagen water decomposition thing, BP enzymes are manufactured with the collagen water decomposition manufacture method identical method of embodiment 4 Treating capacity with 17unit/g processing, produce collagen water decomposition thing of the CTP contents 28% or so, used for edible On the way.

<6-2>The establishment of experimental animal and catagen shedder sample

Experimental animal is C57BL/6 mouse.Due to melanin in the hair follicle of the species be present, and according to the amount skin of melanin The colour of skin can produce change, therefore (growth period hair cycle can be judged according to the colour of skin：Black, catagen and stand-down：It is pink Color).Initially enter stand-down within 6~7 weeks after life.Because of this characteristic, the animal is selected as test body, and 35 are introduced from DBL (loyal north) The C57BL/6 Female Rats (17g-20g) of 6 weeks after only giving birth to.

Trial flock be respectively set as using Induced By Dexamethasone catagen dexamethasone processing group, dexamethasone processing with And verification albumen water decomposition thing (BP-CH) the input group using BP manufactures, solvent control group (control) and positive control group (3% minoxidil) processing group etc..Each group has 8 mouse to form.When putting into solvent control group and collagen water decomposition thing Body weight is measured, and is put into 2,000mg/10ml/kg input amounts continuous oral one time a day, positive control group also smears rice daily Promise ground you.

Using animal with mao device or hair remover is pushed away, will be used rapidly after the back hair removing of the experimental animal of 7 weeks after life Water cleans.Test sample of input and minoxidil are continuously put into 10 days defeathering daily from 7th day.Defeathering connects from the 9th day Continuous 5 days 0.1% dexamethasone for smearing 1ml at the back of experimental animal daily.Experimental animal is in final sample test and rice Promise you input after ether anesthesia death in the 2nd day.

<6-3>Suppress depilation catagen and maintain growth period effect to confirm

Daily clinical symptoms for confirming experimental animal and whether dead.In order to visually confirm the guiding effect in growth period Fruit, a film,fault is clapped weekly, in order to observe the effect for suppressing catagen, clap a film,fault within every 3 days.Photo after shooting utilizes Color in the overall defeathering position of ImageJ determination test animals is by the changing ratio of ash to black, long-term and regression of seeking survival The ratio of phase

The result of experiment is illustrated in table 2 below, Fig. 9 and Figure 10 above.

【Table 2】

Suppress the catagen ratio (%) in the defeathering position of catagen test body

Above-mentioned numerical value is average value ± SE.

The significant difference of the control group of Dunnet's tests：* p ＜ 0.01.

The numerical value of the table is the catagen progress ratio at the position that defeathering induces catagen.That is, the higher catagen of ratio Progression rates are high, and ratio is lower, just represent and successfully suppress catagen.The BP-CH marked in the table is the collagen for absorbing the present invention The fauna of albumen water decomposition thing.

Shown in the table 2, Figure 10 and Figure 11, the ratio of the defeathering growth period of the 9th day and catagen is shown, normal right According to group and substances input group without too big difference, but minoxidil smears group and only makees the solvent of dexamethasone processing Control group is compared, and statistically shows that growth stage ratio is higher, and catagen ratio is reduced.±

Defeathering the 11st day, 14 days, the back pinkiness for starting solvent control group on the 17th, untill off-test always Keep catagen.The defeathering growth stage ratio that substances input group and minoxidil smear group from the 11st day statistically has Obvious increase.

<6-3>Tissue detection

According to the embodiment<5-1>After being tested, the defeathering position of experimental animal is gathered, observes hair follicle state.

That is, experimental animal is excessively anaesthetized it is lethal after, dermal tissue harvesting keeping 10% concentration neutral formalin In solution, after FFPE, histotomy is made with the section of 5 μ m thicks, and use hematoxylin eosin staining.

It is as shown in figure 11 to deserve result.

As shown in figure 11, it is thinning to carry out the epidermis for the solvent control group that dexamethasone processing induces catagen, is not formed Hair follicle.The epidermis for the group (BP-CH) for absorbing collagen water decomposition thing after dexamethasone processing is thickening, and hair follicle is flourishing and is molded. It is also that can confirm that the thickening hair follicle of epidermis is flourishing that minoxidil, which smears group,.

That is, collagen water decomposition thing of the invention promotes propagation and the raising of hair follicle cell under catagen depilation state The growth result of hair is notable.

Use the improvement wrinkle effect of BP collagen water decomposition thing

After the life for the SKH-1 hairless mouses system that this experiment uses 6 weeks sample (female) be from SAMTAKO BIO KOREA After (Ebony Hill city of Gyeonggi, South Korea) adopts, in southeast medical chemistry research institute animal house (animal facility registration card：No. 412) enter The one-week quarantine of row and the healthy animal of domestication raising, design temperature (22 ± 3) DEG C, relative humidity (50 ± 10) %, lighting hours 12 hours (07：00~19：00) feeding environment performs.

As shown in table 3, each group other point 10,7 groups is always divided into and are tested.In addition to normal group, other wrinkles induce Group's equal graded irradiation UVB10 weeks.Irradiation group divides control group and positive control group (retinoic acid 2mg/kg, i.p.), other point of dosage Do not come into operation the collagen water decomposition thing (COL) of general collagen catabolic enzyme (Alcalase) and the collagen of the present invention Albumen water decomposition thing (CTP) 17mg/kg, 34mg/kg, oral input 14 weeks.Feed is experimental animal solid-state feed (SAMTAKO BIO KOREA, South Korea), drinking water does not limit.

【Table 3】

The composition of experimental animal

Positive control group with reference to Belinda M methods (The journal of investigative dermatology, 112(3)；271-278,1999.), with the vitamin A acid of abdominal cavity investing method input 0.05%.

Ultraviolet lamp is irradiated using the luminous sunlamps of UVB in cage, and light source 302nm, UVB intensity is 0.3mW/cm²Height be irradiated.

Ultraviolet irradiation amount is determined using UV-radiometer.It is every with day interval after mouse is closed in irradiation cage Week irradiation 3 times, concurrent irradiation [0 week 10 weeks：60mJ/m²(1M.E.D), 1 week：120mJ/m²(2M.E.D), 2~3 weeks：180mJ/m² (3M.E.D), 4~5 weeks：240mJ/m²(4M.E.D) 5~10 weeks：240mJ/m²(4M.E.D)]

<7-1>Observe the form of wrinkle of skin

Wrinkle of skin state observation is at 5~14 weeks of experimental animal, after carrying out light anaesthesia, is clapped using digital camera After taking the photograph the photo of rear back surface, with SILFLO (Flexico developments LTD.Tokyo, Japan) silicone rubber impression material Material makes skin of back wrinkle copy, and uses the state of the other comparative observation wrinkle of trial flock after microscope photographing.

The form for visually observing wrinkle of skin is as shown in figure 12.Observation uses the SILFLO SKH-1 hairless mouses made The result of skin Plate for coping model, it is thicker to irradiate the wrinkle thickness ratio of UVB control group (C group), and interval is wider, and wrinkle is deep, Put into P group and the COL 17 of vitamin A acid, 34mg/kg input groups, wrinkle is less compared with C faciations or wrinkle thickness of thin, line Thinner (Figure 12).

As can be seen here, even if the UVB of irradiation 10 weeks, collagen water decomposition thing of the invention is to SKH-1 hairless mouses Epidermal keratinocytes play effective effect, are believed to be effectively improved the formation and prevention of wrinkle of skin.

<7-2>Observe skin histology

The skin histology cut is placed on after fixing 24 hours in 10% neutral formalin solution of room temperature, using routine Method washed, be dehydrated, transparence, infiltration etc. is after process, is cut into slices using FFPE and being made with 4 μ m thicks, and soak Steep in one evening of Bouin solution, and after use hematoxylin eosin staining, with the collagen in observation by light microscope skin corium Fiber (collagen fiber).

Moreover, by the skin histology cut be placed in 10% neutral formalin solution it is fixed after, washing, and with 60%~ 100% alcohol carries out dewater treatment successively, and blocking using being made after FFPE.Using rotary microtom by it with 5 μ M thickness is cut into slices, and using after hematoxylin eosin staining, is observed with light microscope (Nikon Co., Tokyo, Japan).

During excessive exposure UVB, epidermis becomes coarse, and normal collagen construction is destroyed, causes the arrangement of collagen not Rule, elastic fibers are reduced.

Change and the result of loss amount and form of the elastic fibers in dermal layer of the skin are observed, can be in control group (C Group) in confirm the elastic fibers substantially deformed, but positive control group and COP group, the elastic fibers of CTP group pass through 14 weeks Sample process after with C faciations are obvious becomes dense (Figure 13).

Moreover, the result of the collagen amount of observation intradermal, the collagenous fibres for only irradiating UVB C group are destroyed, arrange Irregular, collagen amount also compared with normal group (N group), is inclined in reducing.But carry out the P of vitamin A acid processing Relatively dense and rule is more presented with C faciations in group, COL group and CTP group.The glue of CTP group particularly compared with COL faciations Former protein content is more good (Figure 13) with dense degree.

<7-3>Confirm skin histology collagen, collagen catabolic enzyme protein generation (Collagen 1A, MMP-1)

Skin is excessive to be exposed under UV environment, and MMP-1 can be caused excessively to generate, and active oxygen excess generation, is resisted Oxidation is destroyed.And the secretion of inflammatory cytokine increases in the presence of keratinocyte, hinders intradermal Collagen synthesis in fiber subcellular fraction, and promote the activity of catabolic enzyme, form excessive wrinkle.For this relevant people of the present invention Member puts into general collagen catabolic enzyme (COL group) and the collagen moisture of the present invention to irradiation UVB SKH-1 hairless mouses After solving thing (CTP group), collagen synthesis factor Collagen 1A and collagen catabolic enzyme are confirmed by Western blotting The generation of MMP-1 protein.

For analysing protein, after off-test, it is every that the mice skin tissue of collection is put into cell lysis buffer solution After the other 200 μ L of 20mg, tissue is crushed using homogenizer, and in 800rpm, the centrifugation of progress 10 seconds, only using filtering Liquid.After the ice hatching that 24 disappear, with 14000rpm, the centrifugation of 20 minutes is carried out under 4 DEG C of environment.Utilize Bradford Assay2) after quantitative protein, the SDS-PAGE (polyacrylamide gel electrophoresis) of protein is recycled according to size classes.Profit PVDF (polyvinylidene chloride Polyvinylidene fluoride) is moved to half dry type transmission system (Bio-Rad, USA) After film, a hour is handled using the confining liquid (5% skim milk, 1 × TBST buffer) containing 5% skim milk.Use 1 × TBST buffer are cleaned 3 times after 10 minutes, after the Collagen 1A, MMP-1, MMP-13 that handle primary antibodies, 4 An evening is reacted under DEG C environment, and is cleaned 3 times after 10 minutes using 1 × TBST buffer herein.Examined using western blot After test agent box (Abfrontier, WEST SAVE GOLD) produces reaction with film, Chemi-Doc (BIORAD XRS are used System) equipment observation generation degree, and be compared after being adjusted with GAPDH.

Figure 14 is its result schematic diagram.

Irradiate the collagen synthesis factor Collagen 1A of the UVB C group obvious (p of growing amount<0.05) reduce, but The Collagen 1A growing amounts put into the group of sample have been lifted.Particularly put into the P group and COL of vitamin A acid The p of 34, CTP 34 masses<0.05 significant increase (Figure 14 A).

Confirm collagen catabolic enzyme MMP-1 protein generation, it is found that the MMP-1 growing amounts in irradiation UVB C group increase Add.Opposite, the MMP-1 growing amounts (p in CTP is 17,34 crowds<0.05) significantly reduce, CTP 17,34 can be learnt with this Group is significantly more outstanding (Figure 14 B) than positive control group and COL group.

Based on the above results, the irradiation of excessive ultraviolet causes the increase of collagen catabolic enzyme, collagen synthesis enzyme Generation is reduced, and after being handled 14 weeks using the collagen water decomposition thing of the present invention, is compared than general collagen water decomposition thing, Increase Collagen 1A growing amount, reduce MMP-1 generation, there is significantly anti-wrinkle effect.

<7-4>Confirm the moisturizing of skin histology

When causing dermal tissue insult because of ultraviolet irradiation, the grease of skin and the content of moisture are reduced therewith.This reality Applying a main purpose is, confirms to irradiate the grease in the skin histology of the mouse of the ultraviolet of 10 weeks and whether moisture subtracts It is few, and, whether collagen water decomposition thing of the invention can repair the reduction of these greases and moisture.

Put into general collagen water decomposition thing (COL group) and the collagen water decomposition thing (CTP group) 5 of the present invention In week, after 10 weeks, use the grease and the (Courage- of moisture measurement apparatus Corneometer CM 820 of skin Hazake, Koln, Germany) carry out the measurement of grease and moisture.In 21.5 ± 2 DEG C of constant temperature and humidity, relative humidity 40 Certain limit measurement moisture is identified in the environment of ± 5%, after stablizing 30 minutes.

Figure 15 is its result schematic diagram.

As shown in figure 15, because between 10 weeks ultraviolet irradiation, can confirm that control (C) group grease and moisture and The normal control group (N) of ultraviolet is not irradiated compared to significantly reduction.In addition, carry out at collagen water decomposition thing of the present invention For the fauna of reason compared with normal control faciation, irradiation 5 weeks and 10 weeks is presented higher grease and moisture, and And such effect increases with the content of collagen water decomposition thing, grease and moisture also together increase.Particularly originally The skin moisturization of input is than general collagen water decomposition thing (COL in the collagen water decomposition thing (CTP group) of invention Group) more outstanding (Figure 15 A, B).

Utilize the improvement skin moisturization of BP collagen water decomposition thing

Guide (health food test method(s) instructs guide, and beauty related functionality is tested, 2004.) is instructed with reference to the food medicine Room, Such as the content of table 4, each group does not select 10, is always divided into 5 groups and is tested.Normal group (N group) uses Balb-C mouse, skin Dry the NC/Nga Tnd mouse for the increase transepidermal water discharge commonly used in sample selection moisturizing experiment.To induce dry skin shape State, with reference to Okawa et al. methods (Dermatol Sci.66 (2)：136-43,2012.), 1 week before dissection starts, and will make up Cotton (2 × 2cm) is soaked in 1：After being placed on back 15 seconds after the acetone and diethyl ether solution of the mixing of 1 ratio, then wiped with distilled water Same position 30 seconds, prepared in advance for dry skin.

For do not induce dry skin normal control group (Normal group, N group) and, induce and dry skin and only throw Enter the negative control group (Control group, C group) of solvent, by oral 4 weeks of 0.9% salt solution, positive control group (P group) used Certification at food and drug safety is obtained, improves dry skin and improves NAG (the N- acetamido glucoses of skin moisture content Sugar, N-acetyl glucosamine).It is that N-acetyl glucosamine 17mg/kg are orally put into absorb dosage.

It is better than COL34 moistening effect in addition, though the CTP17 in the fact Example 7-4 is low.Therefore, according to reality The collagen water decomposition thing 17mg/kg (CLT17 group) of the invention of the manufacture of example 4 is applied, with the glue manufactured according to the embodiment 1 Former albumen water decomposition thing 34mg/kg (COL34 group) is oral respectively to be put into 4 weeks.Feed is experimental animal solid-state feed (SAMTAKO BIO KOREA, South Korea), drinking water does not limit.

【Table 4】

Experimental animal group

<8-1>Measure skin moisture content

The cuticula of the epidermis of health contains 15~20% moisture, and skin is presented when the moisture of epidermis is less than 10% Dry, it is lackluster and elastic, cause wrinkle to increase.The water that the sense of touch of the soft water profit of skin will be leaned in horny layer of epidermis Divide and maintain.

The moisture of cuticula is the lipid by being generated on skin, as existing in the sebum film and cuticula of adsorbent Water soluble ingredient natural moisturizing factor (NMF：Natural Moisture factor) determine, the moisture evaporation of epidermis with The changes of contents of ceramide and natural moisturizing factor has substantial connection.

To confirm the collagen water decomposition thing 17mg/kg (CTP 17) or disclosed collagen of the present invention After collagen water decomposition thing 34mg/kg (COL 34) that catabolic enzyme is manufactured is put into 4 weeks, the influence that is brought to skin moisture-keeping And characteristic, before experiment one week at that time, use (the Courage- of analyzer Corneometer CM 820 of skin moisture content Hazake, Koln, Germany) position for inducing dry skin is measured.In 21.5 ± 2 DEG C of constant temperature and humidity, relative humidity Certain limit measurement moisture is identified in the environment of 40 ± 5%, after stablizing 30 minutes.

Figure 16 is its result schematic diagram.

Though maintaining the moisture of the epidermis of the Balb-C mouse of normal group (N group), dry skin is induced Control group (C group) moisture compared with normal control faciation significantly reduces.In addition, positive control group, CTP 17, COL 34 crowds There are the effect of increase skin moisture content, wherein CTP 17 best results.

There is more excellent skin moisturization than COL 34mg/kg in the case of CTP inputs 17mg/kg, it can be seen that, The moistening effect of the higher collagen water decomposition thing of the invention of collagen tripeptide content discloseder collagen egg than Plain boiled water analyte significant effect.

<8-2>The effect for the pruritus that dry skin triggers

Skin is the carrier for carrying various immune substances, and the part reflected at first to exterior materials.Skin Wall is injured, reduces water combining power while causing the water loss of hard epidermis to increase, ultimately results in dry skin, skin occurs Itch.Therefore, after putting into various materials to animal specimen, the effect for relaxing pruitus is evaluated.

Scratch the state that behavior (Scratching behavior) viewing test animal scratched in 10 minutes.Same cluster Test body be placed in same cage, confirmation scratches number.The continuous action for continuing more than 2 seconds continues the number meter during n seconds Number is that the of short duration movement counting within n times, 2 seconds is 1 time.

Figure 17 is its result schematic diagram.

After inducing dry skin, the result for the number that scratched within confirming 10 minutes is shown, triggers the control l group (C of xerosis Group) in p<0.05 attention rank scratch number increase while, respectively put into sample group in the number that scratches substantially subtract Few (p<0.05).

On the one hand, the alleviation effects of pruritus caused by dry skin are fitst water in CTP 17, in addition to COL 34, than sun Property control group is more outstanding.

<8-3>The protein recovery effects (Collagen 1A, AQP3, HAS2) of the skin moisture-keeping factor

For analysing protein, after adding cell pyrolysis liquid in skin histology, tissue is destroyed using homogenizer, and use Filtered fluid is extracted after centrifuge filtering.By Bradford assay to quantification of protein after, perform polyacrylamide gel electricity Swim SDS-PAGE (Poly acrylamide Gel Electrophoresis).Using half dry type transmission system (Bio-Rad, USA after) being moved to PVDF (Polyvinylidene fluoride) membrane, Chemi-Doc (BIORAD XRS are used System) equipment observation generation degree, and be compared after being adjusted with GAPDH.

(i) collagen is the shape for participating in cell, the function point analysis such as differentiation movement and the synthesis of protein, and wound Vital constituent during mouth recovers.Trigger xerodermia, cause the collagen content in skin to reduce, finally lead Formation of wrinkle etc. is caused to produce deleterious effect to skin.

(ii) aquaporin -3 (AQP3) plays critically important effect to the aquation (hydration) of skin.Skin is done Dry, AQP3 generation will be reduced, and had influence on transepidermal water and be lost in (TEWL).Aquaporin present in cell membrane (AQP3) Increase, will preserve the moisture in skin, eventually become anti-ageing factor (J.Lipid Res.46：2706,2005.).Therefore One of an important factor for aquaporin -3 (AQP3) present in confirmation cell membrane is the moisture level of confirmation skin (Biology of the cell.97(7)：479-486 2005.).

(iii) glucan (hyaluronic acid, HA) is that macromolecule osamine present in extracellular connection tissue gathers Sugar, play the protection wall effect for stopping moisture evaporation.With advancing age, the beta-dextran content for making the effect of moisture protective bulkhead is anxious Play declines.Outside environmental elements middle-ultraviolet lamp maintains glucan to play minus effect skin.Because of this inherent or transient cause Portugal The reason for reduction of glycan content can cause skin to reduce santal, pachylosis, wrinkle etc..Therefore it is to protect to maintain beta-dextran content Hold moisture of skin and maintain the secret formula of ruddy complexion skin.Glucan is to continue through hyaluronic acid synthetase (HAS) synthesis.Therefore HAS gene increases, and the content of the glucan in skin can also rise, the moisturizing to skin, skin elasticity, and wrinkle etc. is asked Topic has energetically effect.

Therefore, the related personnel of the present invention is by detected by Western blot, to the collagen water decomposition thing of the present invention because Dry skin and cause the influence of skin moisture-keeping factor reduction to be evaluated.

Figure 18 is its result schematic diagram.

As shown in figure 18, with dry skin is induced, Collagen 1A, AQP3 and HAS content ratio can confirm that just Normal control group significantly reduces.On the other hand, positive control group, CTP 17 and COL 34 crowds are compared with control group (C group), respectively The major part of individual albumen quality has been recovered.Wherein CTP 17 has significant skin moisture-keeping factor recovery effects, and input amount is only COL 34 half, but it is more outstanding than COL 34 effect.

That is, absorptivity is very good inside the collagen water decomposition thing of the invention rich in collagen, even if throwing It is few to enter amount, can also reach remarkable result.

Collagen water decomposition thing of the invention has outstanding anti-wrinkle effect and skin moisturization as can be seen here, can Reach effective resisting age of skin and improve the effect of skin elasticity.

【Utilization possibility in industry】

The present invention relates to a kind of collagen water decomposition thing rich in collagen tripeptide and collagen water decomposition thing Purposes, collagen water decomposition thing of the invention compared with existing collagen water decomposition thing, collagen tripeptide Content showed increased, human body are preferable to its absorptivity, can effectively prevent baldness, promote hair tonic, improve wrinkle and skin is protected It is wet etc., there is higher industry to utilize possibility.

Claims

A kind of 1. collagen water decomposition thing rich in collagen tripeptide, it is characterised in that the collagen water decomposition thing It is to be prepared by following phases,

(a) stage being heat-treated to the mixture of fish scale and water；

(b) after by centrifuging bacillus subtilis strain nutrient solution, concentration centrifuge gained filtered fluid and using from The stage of the method manufacture collagen catabolic enzyme of sub- exchange chromatography purification；

(c) by the collagen catabolic enzyme input described in (a) stage mixture in after, in 25~45 DEG C of Environmental Water Solve the stage of 5~15 hours；

Wherein, collagen tripeptide accounts for 20~70wt% of overall moisture solution thing.
2. collagen water decomposition thing as claimed in claim 1, it is characterised in that the bacillus subtilis is deposit number KCTC12866BP bacterial strain.
3. collagen water decomposition thing as claimed in claim 1, it is characterised in that the ion-exchange chromatography in (b) stage Method uses cation-exchange chromatography, and concentration is 0.1~0.3M sodium chloride (NaCl).
4. collagen water decomposition thing as claimed in claim 1, it is characterised in that the ion-exchange chromatography in (b) stage Method uses anion-exchange chromatography, and concentration is 0.09~0.155M sodium chloride (NaCl).
5. a kind of Anti-hair loss or the food compositions for promoting hair tonic, it is characterised in that including any described in claim 1-4 Collagen water decomposition thing and active ingredient is used as using the collagen water decomposition thing.
6. a kind of Anti-hair loss or the pharmaceutical composition for promoting hair tonic, it is characterised in that including any described in claim 1-4 Collagen water decomposition thing and active ingredient is used as using the collagen water decomposition thing.
7. a kind of cosmetic compositions, it is characterised in that including any described collagen water decomposition thing in claim 1-4 And active ingredient is used as using the collagen water decomposition thing.
It is 8. a kind of for cosmetic compositions that are crease-resistant, improving skin elasticity, prevent skin aging or skin moisture-keeping, its feature Be, including in claim 1-4 any described collagen water decomposition thing and using the collagen water decomposition thing as Active ingredient.
9. a kind of exist for food compositions that are crease-resistant, improving skin elasticity, prevent skin aging or skin moisture-keeping, its feature In, including any described collagen water decomposition thing and using the collagen water decomposition thing as having in claim 1-4 Imitate composition.
10. the collagen water decomposition thing as described in any in a kind of 1-4 such as claim in manufacture Anti-hair loss or promotes hair tonic to use Purposes in therapeutic agent.
11. a kind of Anti-hair loss or the method for promoting hair tonic, it is characterised in that including any in the claim 1-4 by effective dose Described collagen water decomposition thing is put into the individual for needing the composition.
12. the collagen water decomposition thing as described in any in a kind of 1-4 such as claim is manufacturing crease-resistant, improvement skin elasticity, Purposes in anti-aging or skin moisture-keeping agent.
13. it is a kind of crease-resistant, improve skin elasticity, the method for preventing skin aging or skin moisture-keeping, it is characterised in that including that will have Any described collagen water decomposition thing is put into the individual for needing the composition in the claim 1-4 of effect amount.