WO2007075016A1

WO2007075016A1 - Collagenase inhibitor containing poly-gamma-glutamic acid-vitamin c complex and use thereof

Info

Publication number: WO2007075016A1
Application number: PCT/KR2006/005684
Authority: WO
Inventors: Moon-Hee Sung; Chung Park; Jae-Chul Choi; Hiroshi Uyama; Haryoung Poo
Original assignee: Bioleaders Corporation
Priority date: 2005-12-29
Filing date: 2006-12-22
Publication date: 2007-07-05
Also published as: KR100628413B1

Abstract

The present invention relates to a collagenase inhibitor containing a poly-gamma-glutamic acid-vitamin C complex (PGA- VitC conjugate), and a composition for preventing skin wrinkles, which contains the PGA- VitC conjugate and can be used in drugs, cosmetics and foods. The PGA- VitC conjugate has not only the effect of inhibiting collagenase activity, but also antiaging effects, such as an antioxidant effect and a skin wrinkle-improving effect. Also, the collagenase inhibitor maintains the elasticity of the skin by keeping skin connective tissue taut and has high skin compatibility, excellent moisturization effect, moisture-absorbing effect and sustained-release effect. Thus, it is useful to provide cosmetic, pharmaceutical and food compositions for improving skin elasticity and reducing wrinkles.

Description

COLLAGENASE INHIBITOR CONTAINING POLY-GAMMA- GLUTAMIC ACID-VITAMIN C COMPLEX AND USE THEREOF

TECHNICAL FIELD

The present invention relates to an inhibitor of collagenase, an enzyme that degrades collagen present in the dermis, the collagenase inhibitor containing a poly- gamma-glutamic acid- vitamin C complex (PGA- VitC complex or PGA- VitC conjugate), and to a composition for preventing skin wrinkles, which contains the PGA- VitC complex and can be used in drugs, cosmetics, foods and the like.

BACKGROUND ART

Skin aging is classified into chronological aging which depends on the passage of time per se, and extrinsic aging which results from external factors (Claude, S. et al, Free Radical Biol. & Med., 26: 174, 1999). External factors that influence skin aging include wind, temperature, humidity, smoking, environmental pollution, ultraviolet light and the like, and particularly, aging caused by ultraviolet light is called photoaging. Ultraviolet light is divided, according to wavelength, into ultraviolet A (UVA, 320-400 nm), ultraviolet B (UVB, 290-320 nm), and ultraviolet C (UVC, 200-290 nm) (Fisher, G.J., New Engl. J. Med., 337: 1419, 1997), and among them, ultraviolet B was reported as the main cause of photoaging (Kang, S. et al, Arch. Dermatol, 133: 1208, 1997).

Generally, the dermal layer consists of a majority of type I collagen and a minor part of type III collagen, elastin, proteoglycan, fibronectin and the like (Bailly, C, J. Invest. Dermatol, 94:47, 1990). When skin is damaged by chronic sunlight exposure, the accumulation of abnormal elastotic material (i.e., solar elastosis) in the upper dermal collagen occurs (Yaar, M. et al, J. Invisting. Dermatol. Symp. Proc, 3:47, 1998), and proteoglycan increases, leading to a marked reduction in collagen, a major protein in dermis (Li, JJ. et al, Cancer Research, 56:483, 1996).

Collagen is a major structural protein produced in skin fibroblasts. It is an important protein present in the extracellular matrix and accounting for 30% of the total weight of body proteins and has a rigid triple-helical structure. The main functions of collagen include providing mechanical firmness of the skin, cushioning connective tissue, the binding of tissue, maintenance of cell adhesion, and inducing cell division and differentiation. Particularly, it functions to impart strength and tension to the skin to protect the skin from external stimuli or forces. The reduction of collagen that accounts for 90% of dermal volume was reported to have a very close connection with skin aging (Huang, C. et al, Proc. Natl. Acad. Sci. USA, 94:5826, 1997).

The synthesis and degradation of extracellular matrix, such as collagen, in the body, are suitably regulated. However, as aging progresses, the synthesis of the matrix is decreased, and the expression of matrix metalloproteinase (MMP), a collagen- degrading enzyme, is promoted to reduce skin elasticity and form wrinkles. Also, this collagen-degrading enzyme is activated by ultraviolet irradiation.

Recently, it has been found that, when collagen synthesis in vivo is activated, dermal matrix components will be increased, thus making it possible to expect effects, including wound healing, an increase in elasticity, and wrinkle improvement.

Thus, collagen has been used in cosmetics, foods, drugs and the like. Among less than 10 oligopeptides which is minimum active units of forming such collagen, peptides found to have a skin regeneration effect are known (US 4,665,054; WO 91/3488; WO 91/7431, etc.), but these peptides are known to have a problem in that a precipitate is formed, thus resulting in a great reduction in the stability of products. Myofibroblasts are also known as α- smooth muscle actin-positive fibroblasts. When fibroblasts are irradiated with UVA, single oxygen is generated. Reactive oxygen species derived from a singlet oxygen directly degrade or polymerize matrix components, which leads to the denaturation or fragmentation of the components, or cause a Maillard reaction, thus leading to the crosslinking of collagen fibers. Furthermore, reactive oxygen species also function to activate collagenase, a collagen-degrading enzyme which is generally present in an inactive form. Thus, the elimination of reactive oxygen species is effective for the prevention of skin aging through the inhibition of degradation or denaturation of matrix components or the inhibition of collagenase activity.

MMP (matrix metalloproteinase) is a family of various enzymes involved in degradation of ECM (extracellular matrix) and BM (basement membrane) and is divided, according to structural and functional properties, into four subfamilies: interstitial collagenase, stromelysin, gelatinase, and membrane-type MMP (MT- MMP) (Fisher, G.L. et al, J. Clin. Invest., 101 : 1432, 1998).

Also, MMP is a metalloproteinase having zinc in the active center thereof and is secreted in the form of zymogen from most cells, including skin keratinocyte and fibroblasts. For enzymatic activity, MMP is structurally modified, so that the amino-terminal end thereof is truncated and activated. The activity of the activated MMP is regulated by inhibitors, such as α2-macroglobulin or TIMPs (tissue inhibitors of metalloproteinase). Fisher et al. reported that even one-time UV irradiation increases the NMP activity of the skin markedly and degrades collagen in the skin, thus suggesting that MMPs influence degradation of collagen in the dermal layer and play a very import role in photoaging (Fisher, GJ. et al, Photochem. Photobiol, 69: 154, 1999; Lee, K.S. et al, J. Dermatol ScL, 17: 182). With respect to the most major cause of skin aging, collagenase, a collagen- degrading enzyme, is secreted directly or indirectly by the molecular synthesis of epidermal cells (cells of tthe upper skin layer) to react with reactive oxygen species, so that a structure that maintains the skin is slowly degraded to show visible changes such as wrinkles. Herein, in young dermis, collagen fibers form a cubic structure, and elastin, an elastic fiber present between the collagen fibers, acts like a spring to maintain the tightness and firmness of the skin. On the other hand, in old skin, collagen is degraded by collagenase, and elastin is also crosslinked between chains to reduce skin elasticity, and thus the skin loses tightness and firmness and becomes wrinkled (FIG. 1).

Based on the skin moisturizing effect of collagen, cosmetics containing collagen have been marketed. However, these cosmetics are for application to a skin surface, and the percutaneous absorption of polymer, collagen is difficult, thus making it impossible to expect the moisturizing effect of the cosmetics. Thus, it is not considered that these cosmetics essentially improve functions of the skin.

Also, the following substances are known in the art as collagen synthesis-promoting agents: retinoic acid (Fisher, GJ. et al., Photochem. Photobiol, 69:154), TGF (trans-forming growth factor) (Cardinale, G. et al, Adv. Enzymol, 41 :425, 1974), a protein derived from animal placenta (JP 8-231370), betulinic acid (JP 8-208424), chlorella extracts (JP 9-40523; JP 10-36283), and the like. However, the retinoic acid has limitations on its use, because it is unstable, causes severe irritation upon application to the skin and shows side effects, such as erythema, and has a stability problem. Also, the chlorella extracts and the like have an insignificant effect, and thus it is considered that these substances cannot provide the effect of improving skin functions by substantially promoting collagen synthesis of skin.

In more direct attempts to overcome the above-described problems, studies and patent applications have been made, which relate to functional cosmetic compositions having the effects of preventing and reducing wrinkles by promoting the proliferation of fibroblasts and the expression of collagen, a dermal protein, and inhibiting the expression of collagenase, an enzyme that degrades the dermal protein. Examples thereof include: N-butyric acid (Korean Patent Registration No. 10-0519890); 3-aminopropane phosphoric acid (Korean Patent Registration No. 10- 0174165); a complex comprising vitamin C (L-ascorbic acid) and Terminalia chebula Rets, extract, microencapsulated in 2-hydroxypropylated-β-cyclodextrin (HPBCD), and phytosphingosine (Korean Patent Registration No. 10-0424726); an epidermal growth factor (EGF) with VitC (Korean Patent Registration No. 10- 0364288); peptide-bonded VitC derivatives (Korean Patent Registration No. 10- 0459679); a procyanidin oligomer (Korean Patent Registration No. 10-0439627); a human epidermal growth factor (Korean Patent Registration No. 10-0433373); retinol and an epidermal growth factor (EGF) (Korean Patent Registration No. 10- 0377397).

Furthermore, studies and patent applications relating to functional cosmetic compositions for wrinkle prevention, based on natural substances, have recently been made. Examples thereof include: extracts from Patrinia scabiosaefolia, Polygoni Cuspidati Radix, Psoraleae Semen and Sophora flavescens (Korean Patent Registration No. 10-0515418); chestnut bark extract (Korean Patent Registration No.

10-0308178); extracts from Agastache rugosa, Lagenaria siceraria var. clavata Ser. and Aquilaήa agallocha Roxb. (Korean Patent Registration No. 10-0309071); extracts from soybean, malt and wheat (Korean Patent Registration No. 10- 0337113); a Panax ginseng CA Meyer extract (Korean Patent Registration No. 10- 0361433); encapsulation of soybean, malt and yeast extracts in hydrogenated lecithin (Korean Patent Registration No. 10-0471372); encapsulation of compound K (20-O-β-D-glucopyranosil-20(S)-protopanaxadiol), the main metabolites of ginseng, in fine emulsion particles and liposome, using a nanoemulsifying technique (Korean Patent Registration No. 10-0465977); Schizophyllum commune Fr. -derived β-l,6-branched-β-l,3-glucan (Korean Patent Registration No. 10- 0295623); ginseng aglycone (Korean Patent Registration No. 10-0365071); a plant extract containing ecdysteroids and iridoides (Korean Patent Registration No. 10- 0373928); a Pueraria mirifica extract, polysaccharide ginsan extracted from ginseng, a fructan polysaccharide (PG-fructan) having beta (2→6) linked fructofuranose, and extracts from Pueraria mirifica, Butea superba, and Mucuna collettii (Korean Patent Registration No. 10-0474110); an Anthriscus sylvestris Hoffmann extract or a Petroselinum sativum Miller extract (Korean Patent Registratioon No. 10-0507292); and a Cordyceps militaris extract (Korean Patent Registration No. 10-0514315).

The above patents relating to skin aging prevention disclose cosmetic compositions containing peptides or plant extracts as agents for promoting collagen synthesis or inhibiting collagenase, and aim to promote collagen synthesis or inhibit collagenase activity in order to prevent skin aging. Particularly, indole-3 -acetic acid is a plant growth hormone causing a tree to bear fruit or to bloom and is a substance, which regenerates the skin and inhibits collagenase that reduces the elasticity of the skin. It has received much attention as a raw material for functional cosmetics, but is known as a substance which is weak against light and heat and thus difficult to stabilize. Also, among tetracycline-based antibiotics, a large number of collagenase activity inhibitors, including doxycycline, cartilage-derived molecules, Rb (retinoblastoma) (Chun, SJ. et al, Arthritis & Rheumatism, 50:78, 2004), triterpenoid-based substances, oleanolic acid, corsepin and its derivatives (Korean Patent Registration No. 10-0514315), and collagenase inhibitor containing ursolinic acid (Korean Patent Registration No. 10-0155613), are known, but mostly have problems in that they show side effects or are unstable in vivo.

Accordingly, there is an urgent need to develop a composition useful for skin elasticity improvement and wrinkle prevention, which has an inhibitory effect against collagenase, a collagen-degrading enzyme which is one major cause of skin aging, and, at the same time, can maintain stable activity in vivo and has no side effects.

Meanwhile, the present inventors conducted studies to develop the use of natural material, poly-gamma-glutamic acid (PGA; WO 04/007593), which is a sticky viscous component of chungkookjang, Korean traditional fermented soybean food, and has various functions, and, as a result, found that PGA- VitC conjugate can improve the stability of vitamin and increase skin moisturizing ability (Korean

Patent Registration No. 10-0485727). However, the collagenase inhibitory effect of the PGA- VitC conjugate is not yet known.

Accordingly, the present inventors have made extensive efforts to solve the above- described problems occurring in the prior art and, as a result, found that the poly- gamma-glutamic acid- VitC conjugate (complex) can maintain the elasticity of the skin by inhibiting collagenase degrading collagen, the component of skin connective tissue, and has high skin compatibility and an excellent moisture- absorbing effect, a moisturizing effect and a sustained-release effect, thereby completing the present invention.

SUMMARY OF THE INVENTION

Therefore, it is a major object of the present invention to provide a collagenase inhibitor containing PGA- VitC conjugate.

Another object of the present invention is to provide a composition for preventing skin wrinkles, which contains the PGA- VitC conjugate as an active ingredient.

To achieve the above objects, the present invention provides a collagenase inhibitor containing the PGA- VitC conjugate. Also, the present invention provides a composition for preventing skin wrinkles, which contains the PGA- VitC conjugate as an active ingredient.

The other features and embodiments of the present invention will be more clearly understood from the following detailed description and the accompanying claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram showing that collagen in the dermis of young skin is degraded by collagenase to become the dermis of old skin.

FIG. 2 is a graphic diagram showing the collagenase inhibitory effect of the PGA- VitC conjugate according to the present invention. In FIG. 2, PGA-VC conjugate:

PGA- VitC conjugate, VC: VitC, and PGA,VC mixture: a mixture of PGA and VitC.

FIG. 3 is a photograph showing the results of agarose gel analysis conducted to examine inhibition of MMP-I by PGA- VitC conjugate according to the present invention using cDNA prepared through reverse transcription-PCR (RT-PCR).

FIG. 4 is a bar graph showing the results of active MMP-I ELISA analysis conducted to examine inhibition of MMP-I by PGA- VitC conjugate according to the present invention.

DETAILED DESCRIPTION OF THE INVENTION, AND PREFERRED EMBODIMENTS The present invention relates to a collagenase inhibitor containing a PGA- VitC conjugate, and a composition for preventing skin wrinkles, containing the collagenase inhibitor.

In the present invention, the PGA preferably has a molecular weight of 1-15,000 kDa. Also, collagenase is preferably MMP-I, but the scope of the present invention is not limited thereto.

In the present invention, the composition for preventing skin wrinkles is preferably a cosmetic, pharmaceutical or food composition, and the PGA- VitC conjugate is preferably contained in an amount of 0.01-60 wt%, and more preferably 0.1-50 wt%, based on the total weight of the composition, but the scope of the present invention is not limited thereto.

The inventive composition for preventing skin wrinkles, which inhibits collagenase, can be used in the form of general external drug or functional cosmetic formulations. The inventive composition can be formulated using general diluents or excipients, such as fillers, extenders, binders, wetting agents, disintegrants and surfactants, or formulated by adding it to various cosmetic bases. Liquid formulation for skin application include suspensions, internal solutions, and emulsions, and contain general simple diluents, including water and liquid paraffin, and various excipients, for example, wetting agents, flavoring agents, aromatics and preservatives, as well as various cosmetic bases.

The inventive composition for preventing skin wrinkles, which contains the PGA-

VitC conjugate as an active ingredient, can be used to prepare drugs, such as ointments for external skin application, lotions, such as astringent lotion, milk lotion and nourishing milk lotion, cosmetics, such as moisturizing cosmetics, cleansing foam, body cosmetics, lotions, creams, essence, and packs, and foods, including functional beverages, such as drinks, capsules or powder. However, the scope of the present invention is not limited thereto, and the inventive composition can be prepared in any formulation, as long as it inhibits collagenase to provide an advantageous effect on skin elasticity improvement and wrinkle prevention.

The PGA used in the present invention is a viscous polymer consisting of D,L- glutamic acid bound to γ-glutamyl, and is produced from Bacillus sp. strains isolated from, for example, Chungkookjang (Korean traditional fermented soybean food prepared using ricestraw), Natto (Japanese traditional fermented soybean food) and Kinema (Nepalese traditional fermented soybean food). The PGA-VitC conjugate is an eatable, aqueous, anionic, biodegradable polymer material, which can be used as a moisture absorber, a moisturizer and a raw material for cosmetics.

Recently, studies focused on developing and industrializing eco-friendly material, hydrogel having water absorptivity, biodegradability and plasticity, using PGA as a raw material, have been conducted. In such studies, when an aqueous solution containing PGA is treated with a chemical crosslinking agent and irradiated with radiation, the crosslinking reaction between the molecules of PGA can occur to obtain PGA resin having water absorptivity, biodegradability and plasticity. The inventive PGA- VitC conjugate obtained according to this principle can be applied in hygienic products such as diapers, food and horticultural industries.

Moreover, the following studies and patents, for example, have been reported: the effects of manganese ions on the composition and production of PGA, the use of PGA as water-soluble polymers by untrasonic decomposition, and the development of low- water-soluble plastics by the synthesis of ester derivatives (Ito, Y. et ah, Biosci. Biotechnol. Biochem., 60: 1239, 1996); the production of PGA by Bacillus subtillis, and the use of PGA as a calcium dissolving agent in healthy foods having an osteoporosis therapeutic effect (JP 6-32742); effects of reducing the content of phosphorus in water systems to reduce water contamination (EP 838160); the use of PGA as biodegradable solid fibers or films and molded films by the dissolution, precipitation and drying of PGA (JP 7-138364 and JP 5-117388); and polymers for drug carriers (JP 6-92870 and JP 6-256220). Thus, the inventive PGA- VitC conjugate is a highly useful natural polymer, which can be used as low-water- soluble plastics, healthy foods having osteoporosis therapeutic effects, agents for reducing water contamination, biodegradable fibers or films and molded films, polymers for drug carriers, and the like.

In the present invention, as described in Example 1 below in detail, a water-soluble powdery PGA- VitC conjugate was prepared by isolating PGA from Bacillus subtilis var chungkookjang (KCTC 0697BP) and treating the PGA with a gel containing VitC dissolved therein.

The effect of the PGA- VitC conjugate on collagenase inhibition was analyzed and, as a result, it could be seen that the effect of the PGA- VitC conjugate on collagenase inhibition was excellent compared to the cases where each of PGA, VitC and a mixture of PGA and VitC was added. Furthermore, the PGA- VitC conjugate had an excellent antioxidant effect, a sustained-release effect and a skin compatibility effect.

Example

The present invention will hereinafter be described in further detail by examples. However, it is to be understood that these examples can be modified into other various forms, and the scope of the present invention is not intended to be limited to such examples. Such examples are given to more fully describe the present invention for a person skilled in the art.

In particular, the following examples describe only a collagenase inhibitor containing the PGA- VitC conjugate. However, it will be obvious to those skilled in the art that a composition for inhibiting collagenase activity, which contains the PGA- VitC conjugate, is useful for skin elasticity improvement and wrinkle prevention, and thus can be used in wrinkle-preventing drugs, cosmetics and foods.

Example 1; Preparation of PGA and PGA- VitC conjugate

PGA was prepared according to the method described in the previous patent (WO 04/007593) filed in the name of the present inventors. That is, a culture broth of Bacillus subtilis var chungkookjang (KCTC 0697BP) was inoculated into a fermentor containing a basal medium to provide a PGA-containing sample solution. Then, the sample solution was left to stand to remove polysaccharides from the fermented solution, and the PGA precipitate was added with distilled water to dissolve. Next, protease was added to the solution and left to stand in incubator to degrade extracellular proteins present in the PGA sample, followed by dialyzing in a sufficient amount of distilled water to remove free glutamic acid, and concentrating to provide pure PGA. From which it could be seen that the PGA thus obtained had an average molecular weight of 13,000 kDa, more than 95% of the PGA molecules had a molecular weight ranging from 3,000 to 15,000 kDa, and the PGA had a molecular weight distribution ranging from 1 kDa to 15,000 kDa.

A PGA- VitC conjugate (complex) was prepared according to the method descried in the previous patent (Korean Patent Registration No. 10-0498812) owned by the present inventors. That is, the PGA was placed in a dry test tube and dissolved in DMSO (dimethyl sufoxide), followed by stirring, thus obtaining transparent liquid. To the obtained transparent liquid, l-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride) and N-hydroxysuccinimide were added, and stirred at room temperature, and then a DMSO solution containing VitC dissolved therein was added thereto, thus forming a gel. The formed gel was immersed in a large amount of water, which was then replaced several times with fresh water. Then, the resulting material was freeze-dried, thus affording a water-soluble powdery PGA- VitC conjugate. Example 2; Analysis on collagenase inhibition by PGA- VitC conjugate

The PGA- VitC conjugate synthesized in Example 1 was dissolved at a concentration of 1%, and mixed with 400 unit/mL collagenase at collagenase concentrations of 0.002%, 0.003%, 0.004%, 0.006% and 0.008%, followed by reaction at 37 "C for 20 minutes. Meanwhile, 25 mg of bovine collagen was added to 0.5 mL of 0.05M TES (N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid) buffer (pH 7.0) and then allowed to react at 37 ^°C for 15 minutes. The collagen reaction solution was mixed with the collagenase reaction solution, and then allowed to react at 37 °C for 5 hours.

1.52 mM stannous chloride (SnCl₂) and 1 mL of a solution mixture of 50% solution of 0.4 M citric acid and 50% solution of 8% ninhydrin in methyl cellosolve were added to the reaction solution and then boiled for 20 minutes. The reaction solution was cooled, 5 ml of 50% n-propanol was added thereto, and the mixture was left to stand at room temperature for 15 minutes and measured for absorbance at 600 nm.

Collagenase activity was calculated as the concentration of L-leucine liberated from collagen, and collagenase inhibition caused by PGA was calculated as the relative ratio of the enzymatic activity of collagenase, which reacted with PGA, to the enzymatic activity of collagenase, which did not react with PGA (Mandl, I. et ah, J. Clin. Invest, 32:1323, 1953).

The results of measurement of collagenase inhibition caused by the PGA- VitC conjugate are shown in FIG. 2. As shown in FIG. 2, collagenase was strongly inhibited when the PGA- VitC conjugate was used, compared to when each of PGA, VitC and a mixture of PGA and VitC was used. Particularly, when the PGA-VitC conjugate was used at a concentration of 0.5 mM, collagenase inhibition reached about 90%.

Example 3: Cell assay for collagenase inhibition by PGA- VitC conjugate

A test on the degradation of collagen by each sample of PGA sample, VitC, a mixture of PGA and VitC and PGA- VitC conjugate was conducted in the following manner using human dermal fibroblasts by reverse transcription-PCR (RT-PCR), western blot analysis, and active MMP-I ELISA analysis.

Human dermal fibroblasts were cultured in a DMEM (dulbecco's modified eagle's medium) containing 10% FBS (fetal bovine serum), in a 5% CO₂ incubator at 37 ^°C . The cells used in the test were for 2-8 passages (1 passage is a period during which lxlθ⁵ cells grow into 1x10⁶ cells). 1 x 10⁶ cells were inoculated into a 60 mm culture dish in the respective conditions and cultured for 18 hours before use in the test.

After the medium was removed from the cultured cells, 0.05% PGA (poly-gamma- glutamic acid), 0.05% VitC, 0.05% PGA + 0.05% VitC and 0.05% PGA- VitC conjugate, each contained in 2 niL of PBS (phosphate buffered saline) buffer, were placed in the respective culture dishes and incubated in a 5% CO₂ incubator at 37 ^°C for 1 hour. After 1 hour, each culture dish was irradiated with UVA (360 nm) at 5 J/cm², and replaced with serum free DMEM media, and then incubated in a 5% CO₂ incubator at 37 ^°C for 24 hours. After the incubation, the media were collected and subjected to active MMP-I ELISA, and the cells were collected, from which mRNA was isolated and subjected to RT-PCR. Also, protein lysates were collected and subjected to western blot.

(1) RT-PCR analysis of MMP-I inhibition For RT-PCR, the culture broth was removed from the cells inoculated into the 60 mm culture dish, and the cells were repeatedly pipetted with 1 mL of TRIZOL (Invitrogen Corp.) solution. Then, the solution in the culture dish was collected into a 1.5 mL tube and allowed to react at room temperature for 5 minutes. 0.2 mL of chloroform was added to the reaction sample, and the mixture was strongly vortexed and then allowed to react at room temperature for 5 minutes. After the reaction, the reaction solution was centrifuged at 4^°C and 13,000 rpm for 15 minutes, and the supernatant was isolated. The isolated supernatant was collected into a 1.5 mL fresh tube, 0.5 mL of isopropyl alcohol was added thereto to mix, and the mixture was allowed to react at room temperature for 10 minutes. After the reaction, the reaction solution was centrifuged again at 4°C and 13,000 rpm for 10 minutes, and the supernatant was removed. 1 mL of 75% ethanol (EtOH) was added thereto and vortexed, and the solution was centrifuged at 4 ^°C and 13,000 rpm for 20 minutes. The supernatant was removed, and the precipitate was dried at room temperature for 10 minutes. Then, DEPC (diethyl pyrocarbonate) solution water was dropped onto the RNA precipitate, and the RNA was quantified using spectrophotometry.

2 μg of the quantified RNA was placed in a PCR tube, 1 μL of an oligo-dT random primer was added into the tube, and DEPC distilled water was added thereto to a final volume of 15 μL. In the RT-PCR reaction, the above-prepared mixture was allowed to react at 70 °C for 5 minutes, and then 10 μL of a buffer mixture, 2 μL of M-MLV (moloney murine leukemia virus) 5 x reaction buffer, 1.25 μL of 10 mM dNTP, 10 units of RNaseOUT (Invitrogen Corp.). ⁵-⁵ μ^L of DEPC distilled water, and 200 units of M-MLV RTase (Invitrogen Corp.), were added thereto. The resulting mixture was allowed to react at 42 ^°C for 60 minutes, and then at 80 ^°C for 10 minutes, thus preparing cDNA. 1 μL of the cDNA was placed in a fresh PCR tube, and 1 μL of each of 10 pmol matrix metalloproteinase (MMP)-I primers (forward: 5'-TGACTTTT A AAAC ATAGTCT ATGTTC A-3' (SEQ ID NO: 1), and reverse: S'-TCTTGGATTGATTTGAGATAAGTCATAGC-S' (SEQ ID NO: 2)) was added into the tube. Then, 10 μL of a premix (Bioneer Corp.) was added into the tube, and DEPC distilled water was added thereto to a final volume of 20 μL. Then, the mixture was subjected to a PCR reaction. The PCR reaction was performed in the following conditions: pre-denaturation at 95 °C for 5 min, and then 30 cycles, each consisting of 40 sec at 95 ^°C , 40 sec at 55 °C and 80 sec at 72 ^°C , followed by extension at 72 ^°C for 5 min.

After completion of the PCR reaction, the constructed cDNA was electrophoresed on 1.0% agarose gel containing ethidium bromide (EtBr), and the density of each band was measured using a densitometry (see FIG. 3). In FIG. 3, Cl represents a control group non-irradiated with UVA, and C2 represents a control group irradiated with UVA. In order to confirm whether the protein was added in the same amount for each well, verification was performed using a β — actin antibody. As shown in FIG. 3, in the cases where each of PGA, VitC, the PGA + VitC mixture and the PGA- VitC conjugate (PGA conj VitC) was added, there was no change in the production of β-actin, but in the case where the PGA- VitC conjugate according to the present invention was added, the production of MMP-I was markedly reduced.

(2) Analysis of MMP-I inhibition by active MMP-I ELISA test

An active MMP-I ELISA test was carried out using the prepared culture broth and human active MMP-I fluorescent assay kit of R&D system (FlMOO). A working standard solution and a sample solution were previously prepared and 100 μL of assay diluent RD1-64 was added into each well. 150 μL of each of the working standard solution and the sample solution was added into each well, and the mixture was allowed to react at room temperature for 3 hours. Then, the reaction solution was removed, and each well was washed four times with 400 μL of wash buffer using a multi-channel pipette. 200 μL of APMA (p-aminophenylmercuric acetate) was added to each of the standard solution and the sample solution, and the mixture was covered with an adhesive strip in order to block light. Then, the mixture was allowed to react at 37°C for 2 hours at a suitable humidity. The reaction solution was removed, and each well was washed with 400 μL of wash buffer using a multichannel pipette. 200 μL of substrate was added into each well, and each well was covered with an adhesive strip and allowed to react at 37°C for 17-20 hours. After the reaction, the relative fluorescence units (RFU) of each well at 320 nm and 405 nm were measured, and the measurement results are shown in FIG. 4. In FIG. 4, Cl represents a control group non-irradiated with UVA, and C2 represents a control group irradiated with UVA.

As shown in FIG. 4, in the cases where each of PGA and the PGA + vitamin mixture was added, there was no significant change in the concentration of active MMP-I, and in the case where VitC was added, the concentration of MMP-I was relatively reduced. However, it was confirmed that in the case where the PGA- VitC conjugate was added, the concentration of active MMP-I was significantly reduced.

Example 4; Antioxidant effect of PGA-VitC conjugate

Free radical removing ability of the PGA- VitC conjugate prepared in Example 1 was examined. The used DPPH (l,l-diphenyl-2-picrylhydrazyl) is a radical showing a color depending on a change in an oxidation/reduction environment. In the present invention, the radical removing ability of the PGA- VitC conjugate was examined by measuring O.D. value.

The PGA-VitC conjugate was dissolved in distilled water or solvent, and 100 μL of the liquid sample was mixed with 100 μL of 200μM DPPH. The mixture solution was left to stand in a dark chamber at room temperature for 10 minutes and measured for absorbance at A518 nm in comparison with PGA, VitC and the PG A/ VitC mixture as control groups. Also, EDA (electron donating ability) was calculated from an equation of (absorbance in control group - absorbance in test group)/absorbance in control group x 100 (see Table 1).

Table 1

As a result, as shown in Table 1, the PGA- VitC conjugate showed free radical removing ability of more than 86%, which is similar to VitC, but PGA did not show free radical removing ability. These results suggest that the PGA- VitC conjugate had an antioxidant effect similar to that of VitC.

Example 5: Sustained-release effect of PGA- VitC conjugate

A test on whether the PGA- VitC conjugate has sustained-release properties in intestinal absorption was carried out in the following manner according to the method described in the previous patent (Korean Patent Registration No. 10- 0498812) owned by the present inventors.

Specifically, thirty 4-week-old Balb/c mice were bred in a mouse cage with a 12-hr light/ 12-hr dark cycle while the animals were given free access to feed and distilled water. At 1 hr, 1.5 hr and 2 hr after orally administering the PGA- VitC conjugate, the mice were anesthetized with ether, the entire small intestine region from the duodenum to the ileum was removed from the abdomens of the mice, and was divided into the upper and lower parts, the contents of which were washed out with cold physiological saline. Then, the small intestine tissue was homogenized with a homogenizer while suitable cold physiological saline was added thereto. The homogenized small intestine tissue was centrifuged at 4^°C and 8,000 x g for 20 minutes, and the soluble part and non-soluble part of each fraction were separated from each other and stored at -20 ^°C , while VitC contained therein was analyzed by HPLC.

From the test results, it could be seen that the intestinal absorption rate of VitC was increased with the passage of time, suggesting that the PGA- VitC conjugate according to the present invention had significant sustained-release properties.

Example 6; Test of skin safety of PG A- VitC conjugate

The skin safety of a cosmetic composition containing the PGA- VitC conjugate was measured. Specifically, 30 volunteers (average age: 25 years old; age distribution: 19-40 years old) were divided into two groups A and B. The groups A and B were subjected to a skin patch test with the inventive cosmetic composition and a comparative cosmetic composition, respectively, using the Haye's Test Chamber. Herein, persons who had skin lesions, such as psoriasis or eczema, pregnant women and nursing mothers, or persons who were being administered with antihistamine agents, were excluded from the test. The test site was washed with 70% ethanol and dried. Next, for each person of the groups A and B, 15 μg of each of the samples was dropped into the chamber, and then fixed onto the upper arm site as the test site.

The patch was applied for 24 hours, and after the patch was removed, the test site was marked with a marking pen. At 24 hours, 48 hours and 72 hours, the test site was observed, and evaluated according to the criteria of International Contact Dermatitis Research Group (ICDRG) (see Table 2 and Table 3).

As a result, as shown in Table 3, the inventive composition containing the PGA- VitC conjugate had 0 degree of skin irritation, suggesting that the composition is a safe composition having no skin irritation. This indicates that the inventive PGA- VitC conjugate had a high degree of skin compatibility (see Table 3). Table 2: Evaluation criteria of International Contact Dermatitis Research Group

Table 3: Results of skin safety according to judgment criteria of International Contact Dermatitis Research Group

Hereinafter, skin lotion, milk lotion, essence, cleaning foam and shampoo will be illustrated as formulation examples of the present invention, but formulations containing the inventive cosmetic composition are not limited thereto.

Formulation Example 1: Skin lotion

Butylene glycol, glycerin, polyoxyethylene (60) hydrogenated caster oil, betain, citric acid, sodium citrate and a preservative were added to purified water, stirred and then dissolved. Then, a solution of fragrance in ethanol was added thereto. To the mixture, the PGA- VitC conjugate prepared in Example 1 was added, sufficiently stirred, and aged, thus preparing a skin lotion containing the PGA- VitC conjugate. The content of each component in the skin lotion is shown in Table 4 below. Table 4

Formulation Example 2; Milk lotion (emulsion)

The PGA- VitC conjugate prepared in Example 1, butylene glycol, glycerin, a carboxyvinyl polymer, arginine, a preservative and purified water were mixed with each other and heated at 70-75 ^°C with stirring. Meanwhile, squalane, butylene glycol dicaprylate/dicaprate, sorbitan stearate, polysorbate 60, glyceryl stearate and stearyl glycyrrhetinate were mixed with each other and heated at 75-80 °C with stirring, and the mixture was added to the above mixture containing the PGA- VitC conjugate, to form an emulsion. The emulsion was cooled to about 45 ^°C with stirring, a fragrance was then added thereto. The resulting mixture was stirred, cooled to 30 ^°C and then aged, thus preparing a milk lotion containing the PGA- VitC conjugate. The content of each component in the milk lotion is shown in Table 5 below. Table 5

Formulation Example 3; Essence

Sitosterol, polyglyceryl 2-oleate, ceramide, ceteares-4 and cholesterol were mixed with each other with stirring. Then, to the mixture, a mixture solution of the PGA- VitC conjugate, dicetyl phosphate, concentrated glycerin and purified water was added to form an emulsion. The emulsion was cooled to 45 ^°C with stirring, and a fragrance was then added thereto. Then, the mixture was stirred, cooled to 30 ^°C and aged. Then, the mixture was stabilized by adding a carboxyvinyl polymer, xantan gum and a preservative thereto, and then aged, thus preparing an essence containing the PGA- VitC conjugate. The content of each component in the essence is shown in Table 6 below. Table 6

Formulation Example 4: Cleansing foam

N-acylglutamic acid sodium salt, glycerin, PEG-400 and propylene glycol were mixed with each other in purified water, and the PGA- VitC conjugate was added thereto in small portions. Then, EDTA-4Na was added thereto, and the mixture was heated at 80 ^°C with stirring to dissolve. Meanwhile, POE(15) oleylalcohol ether, lauryl derivatives and methyl paraben were mixed and heated at 80 ^°C , and the mixture was added to the mixture containing the PGA- VitC conjugate. The resulting mixture was stirred, and a fragrance was added thereto. Then, the mixture was slowly cooled, thus affording a cleansing foam containing the PGA- VitConjugate. The content of each component in the cleansing foam is shown in Table 7 below. Table 7

Formulation Example 5; Shampoo

To purified water, glycerin and EDTA-4Na were added, and the mixture was dissolved by heating it at 80 ^°C . Then, TEA lauryl sulfate, sodium lauryl sulfate, lauryl amidopropyl betain and laurylic acid diethanolamide were added thereto and stirred. The mixture was neutralized by addition of citric acid at 50 °C , and the PGA- VitC conjugate and zinc pyrithione were added thereto at 45 °C . The mixture was stirred, thus obtaining a shampoo containing the PGA- VitC conjugate. The content of each component in the shampoo is shown in Table 8 below.

Table 8

INDUSTRIAL APPLICABILITY

As described and proven above in detail, the present invention provides the collagenase activity inhibitor containing the poly-gamma-glutamic acid (PGA)-VitC conjugate, and the composition for preventing skin wrinkles, which contains the PGA- VitC conjugate. The collagenase inhibitor according to the present invention has not only the effect of inhibiting collagenase activity, but also antiaging effects, such as the effect of inhibiting matrix metalloproteinase to provide an antioxidant effect and reduce skin wrinkles. Also, the collagenase inhibitor maintains the elasticity of the skin by keeping skin connective tissue taut and has high skin compatibility, moisturizing ability and moisture-absorbing ability. Thus, it is useful to provide cosmetic, pharmaceutical and food compositions for improving skin elasticity and reducing wrinkles.

While the present invention has been described with reference to the particular illustrative embodiment, it is not to be restricted by the embodiment but only by the appended claims. It is to be appreciated that those skilled in the art can change or modify the embodiment without departing from the scope and spirit of the present invention.

Claims

THE CLAIMS What is claimed is:

1. A cosmetic composition for preventing skin wrinkles, which contains the PGA- VitC conjugate having collagenase inhibiting activity, as an active ingredient.

2. The cosmetic composition according to claim 1, wherein the molecular weight of said PGA is 1-15,000 kDa.

3. A food composition for preventing skin wrinkles, which contains the PGA- VitC conjugate having collagenase inhibiting activity, as an active ingredient.

4. The food composition according to claim 3, wherein the molecular weight of said PGA is 1-15,000 kDa.

5. A pharmaceutical composition for preventing skin wrinkles, which contains the PGA- VitC conjugate having collagenase inhibiting activity, as an active ingredient.

6. The pharmaceutical composition according to claim 5, wherein the molecular weight of said PGA is 1 - 15 ,000 kDa.