KR100628413B1 - Collagenase inhibitor containing poly-gamma-glutamic acid-vitamin c complex and use thereof - Google Patents

Abstract

The present invention relates to a collagenase inhibitor containing a poly-gamma-glutamic acid (PGA) -vitamin C complex and a composition for preventing skin wrinkles that can be used as a drug, a cosmetic, a food, and the like.

The PGA-vitamin C complex according to the present invention not only inhibits collagenase that breaks down collagen, but also has anti-aging effects such as antioxidant effects and skin wrinkle improvement, thereby preventing skin connective tissues from loosening and maintaining skin elasticity. It has a high skin compatibility, moisturizing, hygroscopicity and sustained release effect, so it is useful to provide a medicine, cosmetics and food composition useful for skin elasticity and wrinkle improvement.

Poly gamma glutamic acid-vitamin C complex, collagenase, anti-wrinkle composition

Description

Collagenase inhibitor containing polygamma glutamic acid-vitamin C complex and use thereof {Collagenase Inhibitor Containing Poly-gamma-glutamic Acid-Vitamin C Complex and Use Thereof}

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

Figure 2 is a graph showing the collagenase inhibition of the PGA-vitamin C complex according to the present invention. Here, the PGA-VC conjugate is a PGA-vitamin C complex, VC is a vitamin C, PGA, VC mixture is a PGA, vitamin C mixture, respectively.

Figure 3 is a photograph confirming the degree of MMP-1 inhibition by the PGA-vitamin C complex according to the present invention using a cDNA made through reverse transcription PCR (reverse transcription-PCR, RT-PCR).

Figure 4 is a bar graph showing the degree of MMP-1 inhibition by the PGA-vitamin C complex according to the present invention through an Active MMP-1 ELISA experiment.

The present invention is an inhibitor and agent of collagenase (collagenase) which is an enzyme that breaks down the collagen present in skin dermis containing poly-gamma-glutamic acid (PGA) -vitamin C complex, cosmetics, It relates to a composition for preventing skin wrinkles that can be used as food.

Skin aging is divided into chronological aging process over time and extrinsic aging process by external factors (Claude, S., et al., Free radical Biol . & Med ., 26: 174, 1999). External factors affecting skin aging include wind, temperature, humidity, tobacco smoke, pollution, ultraviolet rays, etc. In particular, aging by ultraviolet rays is called photoaging. Ultraviolet rays are divided into ultraviolet A (UVA, 320-400 nm), ultraviolet B (UVB, 290-320 nm) and ultraviolet C (UVC, 200-290 nm) depending on the wavelength (Fisher, GJ, New). Engl . J. Med ., 337: 1419, 1997), among others, ultraviolet B has been reported as a major factor of photoaging (Kang, S., et. al ., Arch . Dermatol ., 133: 1208, 1997).

Generally, the dermal layer is composed of the majority of type I collagen and some type III collagen, elastin, proteoglycan, fibronectin, etc. (Bailly, C., J. Invest . Dermatol ., 94:47, 1990), in case of chronic sun damage, abnormal elastotic material is deposited in the upper collagen of the dermis (Yaar, M., et. al ., J. Invisting . Dermatol . Symp . Proc ., 3:47, 1998), proteoglycans are increased and collagen, the main protein of the dermis, is significantly reduced (Li, JJ, et. al ., Cancer Research , 56: 483, 1996).

Collagen is a major matrix protein produced in skin fibroblasts and is present in extracellular epilepsy and is an important protein that accounts for 30% of the total weight of biological proteins. Its main functions are mechanical firmness of skin, resistance of connective tissue and binding of tissue, support of cell adhesion, induction of cell division and differentiation, and especially to give skin strength and tension to protect skin from external stimulation or force. Play a role. It is reported that the reduction of collagen, which accounts for 90% of the dermal layer, is closely related to skin aging (Huang, C., et. al ., Proc . Natl . Acad . Sci . USA , 94: 5826, 1997).

In vivo, the synthesis and degradation of extracellular matrix such as collagen is properly regulated, but the synthesis decreases as aging progresses, and the expression of matrix metalloproteinase (MMP), an enzyme that degrades collagen, is promoted. The elasticity of the skin is reduced and wrinkles are formed. Ultraviolet irradiation also activates these degrading enzymes.

Recently, the synthesis of collagen in vivo has been used to cosmetics, foods, medicines and the like as it can be expected to increase the dermal matrix components can be expected to the effect of wound healing, increased elasticity, wrinkles and the like. Peptides of which skin regeneration effect has been found among 10 or less oligopeptides, which are the smallest active units forming such collagen, are known (US 4,665,054, WO 91/3488, WO 91/7431, etc.), but precipitation of these peptides forms. It is known that there is a problem that the stability of the product is greatly reduced.

Myofibroblasts are also known as α-smooth muscle actin positive fibroblasts. When UVA is irradiated on fibroblasts, singlet oxygen is generated, and reactive oxygen species derived from singlet oxygen can directly decompose or polymerize matrix components to cause denaturation or fragmentation, or maillard reaction. ) To crosslink the collagen fibers. In addition, active oxygen also has a function of converting collagenase, a collagen degrading enzyme present in an inactive form, into an active form. Therefore, scavenging free radicals is effective in preventing aging of the skin in connection with degradation of matrix components, inhibition of degeneration, and inhibition of collagenase activity.

Matrix metalloproteinases (MMPs) are a family of enzymes involved in the degradation of ECM (extracellular matrix) and BM (basement membrane), depending on their structure and functional properties. ), Four subfamily of stromelysin, gelatinase and membrane-type MMP (MT-MMP) (Fisher, GL, et. al ., J. Clin . Invest ., 101: 1432, 1998).

In addition, MMP is a metalloproteinase with zinc in its active center, which is secreted in the form of a zymogen in most cells, including keratinocytes and fibroblasts of the skin in vivo. . In order to have enzymatic activity, MMPs undergo structural modifications resulting in cleavage and activation of amino terminal sites, and activated MMPs are controlled by inhibitors such as α2-macroglobulin or TIMPs (tissue inhibitors of metalloproteinase). Fisher et al. Reported that MMP activity of the skin is increased in a single UV irradiation and that the collagen in the skin is significantly disrupted, thereby affecting collagen breakdown of the dermal layer and playing a very important role in photoaging (Fisher). , GJ, et al ., Photochem . Photobiol ., 69: 154, 1999, Lee, KS, et al ., J. Dermatol . Sci ., 17: 182).

The main cause of skin aging is collagenase, a collagenase that is directly or indirectly secreted by molecular synthesis between epidermal cells, the upper layer of skin, and the structure that supports skin by reacting with free radicals slowly collapses to the eyes such as wrinkles. In the young dermis, collagen, a collagen fiber, forms a three-dimensional structure, and elastic elastic fiber, elastin, acts like a spring to maintain the skin's firmness and elasticity. Destroyed by the agent, elastin also loses its elasticity and elasticity of the skin because the cross-linking is formed between the chains and lose elasticity (Fig. 1).

Conventionally, cosmetics containing collagen have been released by using collagen's skin moisturizing effect, but these cosmetics are applied to the surface of collagen, and it is essentially skin function because it is difficult to absorb the percutaneous absorption of collagen as a polymer. This is not to be improved.

In addition, retinoic acid (Fisher, GJ, et al ., Photochem . Photobiol ., 69: 154) as a collagen synthesis promoter, TGF (trans-forming growth factor) as a carcinogenesis factor (Cardinale G., et al ., Adv . Enzymol ., 41: 425, 1974), proteins from animal placenta (JP 8-231370), betulinic acid (JP 8-208424), chlorella extracts (JP 9-40523, JP 10-36283) and the like are known. However, in case of retinoic acid, it is unstable, severely irritated when applying the skin, and the amount of use is limited due to side effects such as thorax and stability problems. Chlorella extract, etc., is insignificant so that it is not possible to expect the effect of improving skin function by substantially promoting skin collagen synthesis. There was this.

In order to improve this, a functional cosmetic composition which promotes the proliferation of fibroblasts and the expression of collagen, a dermal constituent protein, and inhibits the expression of collagenase, an enzyme that breaks down the dermal constituent protein, thereby preventing and improving skin wrinkles. The research and the patent application which improved it are being made.

This includes N-butyric acid (N-butyric acid, Republic of Korea Patent No. 10-0519890), 3-aminopropane phosphoric acid (Korea Registration No. 10-0174165), 2-hydroxypropylated-β-cyclo Vitamin C (L-ascorbic acid) and Gaza in Terminaline (HPBCD) chebula Rets . ) Epidermal growth factor (EGF) (PET) and peptides are combined with a complex of microencapsulation extracts and Phytosphingosine (Republic of Korea Patent Registration 10-0424726), Vitamin C Vitamin C derivatives (Republic of Korea Patent Registration 10-0459679), procyanidin oligomer (Republic of Korea Patent Registration 10-0439627), human epidermal growth factor (Republic of Korea Patent Registration 10-0433373), retinol and epidermal growth factor : EGF) (Republic of Korea Patent Registration 10-0377397) and the like.

In addition, the recent research and patent applications for the functional cosmetic composition for preventing wrinkles made of natural materials.

For example, paddle, Ho Jang-geun, Pagoji, ginseng extract (Korea Patent Registration 10-0515418), Yulpi extract (Korea Patent Registration 10-0308178), Gwakhyang ( Agastache rugosa), cheomho (Lagenaria siceraria var. clavata Ser . ), Aromas ( Aquilaria agallocha Roxb . ) Extract (Korean Registered Patent 10-0309071), Soybean, Malt, Wheat Extract (Korea Registered Patent 10-0337113), Ginseng ( Panax ginseng CA Meyer) extract (Republic of Korea Patent Registration 10-0361433), soybean, malt, yeast extract is encapsulated in hydrogenetirescichin (Republic of Korea Patent Registration 10-0471372), Compound K is a major metabolite of ginseng using nanoemulsification technology (20-O-β-D- glucopyranosyl -20 (S) - Prototype wave incident diol) a fine emulsion particles and liposomes inclusion therein (Republic of Korea Patent registration 10-0465977), skirt mushroom (Schizophyllum commune Fr.) Derived β-1,6-branched-β-1,3-glucan (Korean Patent No. 10-0295623), ginseng aglycone (Korean Patent No. 10-0365071), containing exedroids and iridoids Plant extract (Korea Patent 10-0373928), Pueraria Myripica ( Pueraria mirifica ) Extract, Ginseng Extract Polysaccharides from fructofuranos, a major component of fructofuranose (PG-fractane), combined with beta (2 → 6) Extract (Korea Patent Registration 10-0474110), Jeonho ( Anthriscus sylvestris Hoffmann Extract or Parsley ( Petroselinum sativum Miller ) extract (Korea Patent Registration 10-0507292), Cordyceps sinensis extract (Korea Patent Registration 10-0514315) and the like.

Looking at the above patents related to anti-aging of the skin, it is a cosmetic composition that promotes collagen synthesis or contains a peptide or plant extract as a collagenase inhibitor, such a prior art to promote collagen synthesis or to prevent collagen synthesis It is based on inhibiting activity. In particular, indole-3-acetic acid is a plant growth hormone that produces tree fruits and flowers. It regenerates the skin and inhibits collagenase, which reduces skin elasticity. Although it has been spotlighted as a raw material, it is known to be difficult to stabilize due to its weak light and heat. In addition, doxycycline, cartilage-derived molecule, Rb (retinoblastoma) among tetracycline antibiotics (Chun SJ, et al ., Arthritis & Rheumatism , 50:78, 2004), triterpenoid family, oleanolic acid, corsepin and its derivatives (Korea Patent 10-0514315), many collagenase activity inhibitors, ursol Collagenase expression inhibitors (Korean Patent No. 10-0155613) containing linic acid are known, but there are problems except side effects or instability in vivo.

Therefore, while having an inhibitory effect against collagenase, a collagen degrading enzyme, one of the main causes of skin aging, it is possible to maintain a stable activity in vivo, and to develop a composition useful for improving skin elasticity and preventing wrinkles without side effects. Needed.

On the other hand, the present inventors developed the use of poly-gamma-glutamic acid (poly-gamma-glutamic acid, PGA, WO 04/007593), a natural material having a wide variety of functions, as a sticky mucus component of the Korean traditional soybean fermented food, Cheonggukjang. While it was found that the PGA-vitamin C complex may improve the stability of the vitamin and increase the moisturizing properties of the skin (Korea Patent Registration 10-0485727), but in the technical field of the present invention belongs to the PGA- vitamin C complex Collagenase inhibitory effect was not known

 Accordingly, the present inventors have made diligent efforts to improve the problems of the prior art that polygamma glutamic acid-vitamin C complex is a substance capable of maintaining the elasticity of the skin by inhibiting collagenase that breaks down collagen which is a component of skin connective tissue. The present invention was completed by developing a collagenase inhibitor having excellent hygroscopicity, moisturizing property, skin suitability and sustained release effect, and having an active oxygen scavenging effect, and a composition for preventing skin wrinkles that can be used for various purposes. .

After all, the main object of the present invention is to provide a collagenase inhibitor containing polygammaglutamic acid-vitamin C complex.

Another object of the present invention is to provide a skin wrinkle preventing agent, cosmetic, and food composition containing the PGA-vitamin C complex as an active ingredient.

In order to achieve the above object, the present invention provides a collagenase inhibitor containing PGA-vitamin C complex.

The present invention also provides a composition for preventing skin wrinkles containing PGA-vitamin C complex as an active ingredient.

In the present invention, the molecular weight of the PGA may be characterized by 1 to 15,000 kDa. In addition, the collagenase may be characterized in that MMP-1, but is not limited thereto.

In the present invention, the anti-wrinkle composition may be characterized in that the cosmetic, pharmaceutical, food composition, PGA-vitamin C complex is 0.01 to 60% by weight, preferably 0.1 to 50% by weight relative to the total weight of the composition It may contain, but is not limited thereto.

The anti-wrinkle composition for inhibiting collagenase of the present invention may be used in the form of general external medicines or functional cosmetic preparations, and when formulated, commonly used fillers, extenders, binders, wetting agents, disintegrants, surfactants, etc. It can be prepared by using a diluent or excipient of, or by adding to the base of various kinds of cosmetics. Liquid preparations for skin administration include suspensions, solutions, emulsions, and the like, and various excipients such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water, liquid, and paraffin, In addition, various kinds of cosmetic bases may be included.

By using the composition for preventing skin wrinkles containing the PGA-vitamin C complex of the present invention as an active ingredient, cosmetics such as external skin ointment, astringent cosmetics, softening cosmetics, nourishing cosmetics such as cosmetic lotion, moisturizing cosmetics, face wash, body cosmetics, Cosmetics such as lotions, creams, essences and packs can be prepared, and when used as foods, they can be prepared as functional drinks, capsules or powders such as drinks, but they are effective in improving skin elasticity and preventing wrinkles by inhibiting collagenase. If it can be obtained is not limited thereto.

Hereinafter, the present invention will be described in more detail.

PGA used in the present invention is a mucus material of a polymer in which D, L-glutamic acid is combined with gamma-glutamyl (γ-glutamyl), and is a Korean traditional soybean fermented food made of rice straw. Produced from Bacillus sp. Strains isolated from traditional soybean fermented foods, Kinema, Nepal, PGA-Vitamin C complex of the present invention is an edible, water-soluble, anionic, biodegradable polymer material to be used as a raw material for absorbents, moisturizers and cosmetics Could be.

Recently, development and industrialization of hydrogel, which is an eco-friendly material having absorbent, biodegradable and plasticity, have been promoted using PGA as a raw material. Irradiating the aqueous solution with a chemical crosslinking agent treatment method results in crosslinking reaction between molecules of PGA, and thus obtains a PGA resin characterized by absorbency, biodegradability, and plasticity. The PGA-vitamin C complex of the present invention is used as a diaper. It can be applied to hygiene products such as food, horticulture industry.

In addition, studies on the composition of PGA, the effect of manganese ions on PGA production, the use of water-soluble polymers by ultrasonic decomposition, and the development of low water-soluble plastics by synthesis of ester derivatives (Ito, Y., et al ., Biosci , Biotechnol , Biochem , 60: 1239, 1996), PGA production by Bacillus subtilis and utilization as health foods with osteoporosis treatment effect as calcium dissolving agent (JP 6-32742), Effect of reducing water pollution by reducing (EP 838160), use as solidified biodegradable fiber, film and film molded product by dissolution, precipitation and drying of PGA (JP 7-138364, JP 5-117388), for drug carrier There are also reports on polymers (JP 6-92870, JP 6-256220), etc. Therefore, using the PGA-vitamin C complex of the present invention as described above, low water-soluble plastics, health foods having a therapeutic effect on osteoporosis, water pollution It may be used as an improving agent, biodegradable fiber or film and film molded body, drug carrier polymer, and the like.

The present inventors, as specifically described in Example 1, Bacillus subtilis Soybean (Bacillus subtilis var chungkookjang , KCTC 0697BP), and PGA-vitamin C complex in water-soluble powder was obtained using a gel in which vitamin C was dissolved.

As a result of confirming the inhibition of collagenase by the PGA-vitamin C complex, the collagenase inhibition effect by the PGA-vitamin C complex is the best compared to the case where PGA, vitamin C and PGA and vitamin C mixed solution, In addition, it was confirmed that the antioxidant, sustained release effect and skin compatibility effect by the PGA-vitamin C complex.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

In the following examples, only the collagenase inhibitor containing the PGA-vitamin C complex is described, but the composition that inhibits the activity of collagenase is useful for improving the elasticity of the skin and preventing wrinkles, as an anti-wrinkle agent, cosmetic and food. It will be apparent to those skilled in the art that it can be used.

Example 1: Preparation of poly-gamma-glutamic acid (PGA) and PGA-vitamin C complex

PGA was prepared according to the method described in the prior patent (WO 04/007593) by the inventors.

That is, inoculated with a culture solution of Bacillus subtilis var chungkookjang (KCTC 0697BP) strain in a fermentation broth containing a basic medium to obtain a sample solution containing PGA and then left to stand to remove the polysaccharides in the fermentation broth, the PGA precipitate Distilled water was added to dissolve the solution, and proteolytic enzyme was added thereto, followed by standing reaction in a thermostat to decompose the extracellular protein present in the PGA sample. The average molecular weight of the PGA thus obtained was 13,000 kDa, and it was confirmed that 95% or more of the molecules were in the range of the molecular weight of 3,000 to 15,000 kDa.

PGA-Vitamin C complex was prepared according to the method described in the prior patent (Republic of Korea Patent Registration 10-0498812) by the inventors.

That is, the PGA was put into a dry test tube, dissolved in DMSO (dimethyl sufoxide), and stirred to obtain a transparent liquid. 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide (N-Hydroxysuccinimide) were obtained in the obtained clear liquid. ) Was added and stirred at room temperature, followed by addition of DMSO solution in which vitamin C was dissolved to form a gel. The formed gel was immersed in a large amount of water, water was exchanged several times, and then lyophilized to obtain a water-soluble powdery PGA-vitamin C complex.

Example 2: Inhibition of collagenase of PGA-vitamin C complex

After dissolving the PGA-vitamin C complex synthesized in Example 1 to 1% concentration, the collagenase of 400 unit / mL so that the dissolved PGA-vitamin C complex solution is 0.002, 0.003, 0.004, 0.006, 0.008% concentration The mixture was mixed and reacted at 37 ° C. for 20 minutes. To 25 mg of bovine collagen, 0.5 mL of 0.05M TES (N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid) buffer (pH 7.0) was added, followed by reaction at 37 ° C. for 15 minutes and the enzyme reactant. After mixing, the mixture was reacted at 37 ° C. for 5 hours.

50% of 8% ninhydrin solution dissolved in methyl cellosolve in 50% solution of 1.52 mM stannous chloride (SnCl ₂ ) and 0.4 M citric acid 1 mL was added to the reaction solution and then boiled for 20 minutes. After cooling, 5 mL of 50% n-propanol was added, and the mixture was left at room temperature for 15 minutes, and the absorbance was measured at 600 nm.

Collagenase activity was calculated as the concentration of L-leucine released from collagen, and collagenase inhibition by PGA was associated with PGA against collagenase apoptosis activity values that did not react with PGA. Calculated as a relative ratio to the value of the genease enzyme activity (Mandl, I. et. al ., Isolation and characterization of proteinase and collagenase from Cl . Histolyticum, J. Clin . Invest 32: 1323, 1953).

As shown in FIG. 2, collagenase inhibition resulted from PGA-vitamin C complex was stronger than that of PGA, vitamin C, and PGA-vitamin C mixed solution. It was confirmed that the inhibition, especially when the concentration of PGA-vitamin C complex concentration of 0.5 mM was confirmed that the degree of inhibition of collagenase reached almost 90%.

Example 3: Inhibition measurement of collagenase of PGA-vitamin C complex by cell assay

The degree of collagen degradation by collagenase of each sample of PGA, vitamin C, PGA and vitamin C complex, and PGA-vitamin C complex was analyzed by reverse dermal transcription using human dermal fibroblasts. PCR, RT-PCR)), Western blot analysis, and active MMP-1 ELISA method was performed in the following manner.

Human skin fibroblasts were cultured in a 37 ° C. incubator fed 5% CO ₂ to a culture solution in which 10% FBS (fetal bovine serum) was added to DMEM (dulbecco's modified eagle's medium). The cells used in the experiment were 2-8 passageages (1 passage was a period in which 1 × 10 ⁵ cells grew to 1 × 10 ⁶ cells), and 1 × 10 ⁶ cells in a 60 mm culture dish for each condition. Was inoculated and incubated for 18 hours before being used for the experiment.

After removing the cultured cell medium, 0.05% PGA (poly-gamma-glutamic acid), 0.05% vitamin C, 0.05% PGA + 0.05% vitamin C, 0.05% PGA-vitamin C in 2 mL PBS (phosphate buffered saline) buffer The complex was placed in each culture dish and incubated in a 37 ° C. incubator fed with 5% CO ₂ for 1 hour. After 1 hour, UV A (UVA: 360 nm) was irradiated to each culture dish at 5 J / cm ² , then replaced with serum-free DMED medium (serum free DMEM media) and fed with 5% CO ₂ for 24 hours. Incubation was incubated in an incubator. After incubation, the medium was collected and tested for active MMP-1 ELISA, the cells were collected, mRNA was extracted, RT-PCR was performed, and protein lysates were collected for western blot analysis.

One) RT - PCR through MMP -1 inhibition measurement

After RT-PCR was removed from the cells inoculated in a 60 mm petri dish, 1 mL of TRIZOL (Invitrogen Corp.) solution was added and pipetting was repeated. Collect the solution from the petri dish and collect them in a 1.5 mL tube. The reaction was carried out at room temperature for minutes. 0.2 mL chloroform was added to the reaction sample, followed by strong vortexing and reaction at room temperature for 5 minutes. After the reaction was centrifuged for 15 minutes at 4 ℃, 13,000 rpm and the supernatant was separated. The separated supernatant was collected in a new 1.5 mL tube, 0.5 mL isopropyl alcohol was added, mixed well, and reacted at room temperature for 10 minutes. After the reaction was again centrifuged for 10 minutes at 4 ℃, 13,000 rpm, the supernatant was removed, 1 mL of 75% ethanol (EtOH) was added and vortexed, and centrifuged at 4 ℃, 13,000 rpm for 20 minutes. The supernatant was removed, and the precipitate was dried at room temperature for 10 minutes, and then diethyl pyrocarbonate (DEPC) distilled water was dropped onto the RNA precipitate, and RNA was quantified by spectrophotometry.

2 μg of the quantified RNA was put in a PCR tube, 1 μL of oligo-dT random primer was added, and the volume was filled with DEPC distilled water so that the final volume was 15 μL. The RT-PCR reaction was performed for 5 minutes at 70 ° C., and then 10 μL of a buffer mixture (M-MLV (moloney murine leukemia virus) 5 × reaction buffer 2 μL, 10 mM dNTP 1.25). Add μL, RNaseOUT (Invitrogen Corp.) 10 units, DEPC distilled water 5.5μL, M-MLV reverse transcriptase (M-MLV RTase, Invitrogen Corp.) 200 units) and react at 60 ° C for 60 minutes, followed by 10 minutes at 80 ° C. Reacted to produce cDNA. 1 μL of the cDNA was added to a new PCR tube, and 10 pmol of substrate metalloproteinase (MMP) -1 primer (forward: 5'-TGACTTTTAAAACATAGTCTATGTTCA-3 ', reverse: 5'-TCTTGGATTGATTTGAGATAAGTCATAGC-3') was added to each 1 μL and then premixed. 10 μL (premix, Bioneer Corp.) was added, and the final volume was filled with DEPC distilled water so that the final volume was 20 μL, followed by PCR. PCR reactions were pre-denaturated at 95 ° C. for 5 minutes, followed by 30 cycles (each cycle at 40 ° C. at 95 ° C., 40 seconds at 55 ° C., 80 seconds at 72 ° C.) and elongation ( extension) was carried out at 72 ° C. for 5 minutes.

After completion of the PCR reaction, the prepared cDNA was electrophoresed on 1.0% agarose gel containing ethidium bromide (EtBr), and the density of each band was measured by using densitometry. (FIG. 3). Here, C1 means a control group not irradiated with UVA, C2 means a control group irradiated with UVA, and was verified using a β-actin antibody to confirm that the same amount of protein was added to each well. As shown in Figure 3, PGA, VitC, PGA + VitC mixture, PGA-VitC complex, respectively, all put into the production of β-actin, but for MMP-1 PGA-vitamin C complex according to the present invention It was confirmed that the amount of production of MMP-1 is significantly reduced.

2) Active MMP -One ELISA  Through experiment MMP -1 inhibition measurement

The active MMP-1 ELISA experiment was performed using the human active MMP-1 fluorescence assay kit of the R & D system (F1M00) using the prepared culture solution. A working standard solution and a sample solution were prepared in advance, and 100 μL of assay diluent RD1-64 was added to each well. 150 μL of standard solution and sample solution were added to each well, and the reaction solution was removed for 3 hours at room temperature. Then, the reaction solution was removed, and 400 μL of washing buffer was added to each well using a multi-channel pipette. Put and wash 4 times. 200 μL of APMA (p-aminophenylmercuric acetate) was added to the standard solution and the sample solution, and the resultant was covered with an adhesive strip to block light, and then reacted at 37 ° C. for 2 hours under appropriate humidity. The reacted solution was removed, and 400 µL of washing buffer was added to each well using a multi-channel pipette and washed four times. 200 μL of substrate was added to each well and covered with an adhesive cloth, followed by reaction at 37 ° C. for 17-20 hours. After the reaction, relative fluorescence units (RFU) of each well were measured at 320 nm and 405 nm, C1 is a control group not irradiated with UVA, and C2 is a control group irradiated with UVA (FIG. 4).

As shown in Figure 4, when the PGA, PGA + vitamin C mixture was added to the active MMP-1 concentration was not significantly changed, when the vitamin C is added, the concentration of MMP-1 is relatively low, according to the present invention When the PGA-vitamin C complex was added, the concentration of active MMP-1 was significantly reduced.

Example 4: Antioxidant Effect of PGA-Vitamin C Complex

The degree of free radical removal by the PGA-vitamin C complex prepared in Example 1 was confirmed. DPPH (1,1-diphenyl-2-picrylhydrazyl) used is a radical colored according to the oxidation / reduction environment, and the radical removal ability of PGA-vitamin C in the present invention was confirmed by measuring the O.D. value.

The PGA-Vitamin C complex was dissolved in distilled water or a solvent and then mixed with 100 μL of 200 μM DPPH in a 100 μL sample in a liquid state. After the mixture was left at room temperature for 10 minutes, absorbance was measured at A518 nm with distilled water or a mixture of solvent (C), PGA, vitamin C, PGA and vitamin C as a control, and EDA (Electron Donating Ability) was measured. Absorbance-experimental group absorbance / control absorbance × 100 (Table 1).

Experimental group PGA VitC PGA-VitC Complex EDA (%) 0 90 86

As shown in Table 1, the PGA-vitamin C complex showed free radical scavenging ability at 100 μM or more at about 86%, similar to vitamin C. However, PGA-vitamin C complex did not show free radical scavenging activity. From the results, it was confirmed that the PGA-vitamin C complex has a similar antioxidant effect as vitamin C.

Example 5: Sustained-release effect of PGA-vitamin C complex

Whether the PGA-Vitamin C complex prepared in Example 1 has sustained release in the intestinal absorption was performed as follows according to the method described in the prior patent by the inventors (Republic of Korea Patent Registration 10-0498812).

That is, 30 four-week-old male Balb / c mice were used to raise basic feed and distilled water in a mouse cage controlled to a proper temperature and a 12-hour contrast cycle. After 1 hour, 1.5 hours, and 2 hours after oral administration of the PGA-vitamin C complex, the mice were anesthetized with ether, and the entire small intestine from the duodenum to the ileum was detached from the abdomen of the mouse, followed by The contents were washed with cold saline in two parts. Next, the small intestine tissue was homogenized with a homogenizer with the addition of appropriate cold saline. The homogenized small intestine tissue was centrifuged at 8,000 × g for 20 minutes at 4 ° C., and the soluble portion of each fraction and the insoluble precipitate were separated and stored at −20 ° C., and the contained vitamin C was analyzed by HPLC.

As a result, the intestinal absorption rate of vitamin C increased with time, from which it was confirmed that the PGA-vitamin C complex of the present invention has a significant sustained release.

Example 6: Skin Safety Test of PGA-Vitamin C Complex

Skin safety of the cosmetic composition containing the PGA-vitamin C complex was measured. Specifically, 30 subjects (average age 25 years, age distribution 19-40 years) were divided into two groups A and B, the cosmetic composition of the present invention in group A, and the cosmetic composition of the comparative formulation example in the group B should be chest chambers. The patch test was performed using Haye's Test Chamber. At this time, the holder of skin lesions such as psoriasis and eczema, or those who are pregnant, lactating or taking antihistamines were excluded from the experiment. The test site was washed with 70% ethanol, dried, and then, 15 μg of the sample was dropped into the chamber in groups A and B, respectively, and then fixed on the upper arm, which is the test site.

The patch was applied for 24 hours, and after removing the patch, the test site was marked with a marking pen, and the test site was observed after 24 hours, 48 hours, and 72 hours, respectively, and the judgment was made by the International Contact Dermatitis Research Group; ICDRG) (Table 2 and Table 3).

International Contact Dermatitis Association Decision sign Criteria evaluation Average ± Suspicious or micro reactions and erythema Smile stimulation 0-0.9 + Erythema + cirrhosis Light stimulation 1.0-2.9 ++ Erythema + cirrhosis + vesicle Heavy stimulation 3.0-4.9 +++ Erythema + cirrhosis + blisters Strong stimulation 5.0 or more - No response Non-irritating 0

Skin Safety Results According to the Jurisdiction Regulations time Cosmetics Containing PGA-Vitamin C Complex Cosmetics without PGA-vitamin C complex 24 hours 0 0.5 48 hours 0 0.5 72 hours 0 0

As shown in Table 3, the composition containing the PGA-vitamin C complex of the present invention was found to be a safe cosmetics without irritation to the skin with an average degree of stimulation of 0, from which the PGA-vitamin C complex of the present invention is highly It was confirmed that the skin suitability of (Table 3).

Hereinafter, a softening lotion (skin), a lotion, an essence, a face wash (cleansing foam) and a shampoo are illustrated as examples of the formulation of the present invention, but the formulation including the cosmetic composition of the present invention is not limited thereto.

Formulation Example 1: Flexible Lotion (Skin)

Butylene glycol, glycerin, polyoxyethylene (60) hardened castor oil, betaine, citric acid, sodium citrate, and preservatives were added to the purified water, followed by stirring and dissolving. The PGA-vitamin C complex prepared in Example 1 was added thereto, stirred well, and aged to prepare a flexible lotion including the PGA-vitamin C complex, and the content of each component is shown in Table 4.

Raw material Formulation Example 1 Content (w / w%) Comparative Formulation Example 1 Content (w / w%) PGA-Vitamin C Complex 50.0 - Butylene Glycol 7.0 7.0 glycerin 5.0 5.0 Polyoxyethylene (60) hardening castor oil 0.2 0.2 ethanol 5.0 5.0 Betaine 2.0 2.0 Citric acid 0.02 0.02 Sodium citrate 0.06 0.06 antiseptic a very small amount a very small amount Spices a very small amount a very small amount Purified water Remaining amount Remaining amount

Formulation Example 2: Lotion (emulsion)

The PGA-vitamin C complex, butylene glycol, glycerin, carboxyvinyl polymer, arginine, preservative and purified water obtained in Example 1 were heated with stirring at 70 to 75 ° C. Squalane, butylene glycol dicaprylate / dicaprate, sorbitan stearate, polysorbate 60, glyceryl stearate and stearyl glycerate mixture, which were heated by stirring at 75 to 80 ° C. were added thereto, followed by emulsification. When the mixture was cooled to about 45 ° C. while stirring, the fragrance was added to the mixture, followed by cooling to 30 ° C., followed by aging, to prepare a lotion including the PGA-vitamin C complex.

Raw material Formulation Example 2 Content (w / w%) Comparative Formulation Example 2 Content (w / w%) PGA-Vitamin C Complex 40.0 - Butylene glycol 8.0 8.0 glycerin 5.0 5.0 Squalane 10.0 10.0 Butylene Glycol Dicaprylate / Dicaprate 5.0 5.0 Sorbitan stearate 1.5 1.5 Polysorbate 60 1.0 1.0 Glyceryl Stearate 0.5 0.5 Stearyl glycyrrhetinate 0.2 0.2 Carboxy Vinyl Polymer 0.1 0.1 Arginine 0.1 0.1 antiseptic a very small amount a very small amount Spices a very small amount a very small amount Purified water Remaining amount Remaining amount

Formulation Example 3: essence

After stirring and mixing the cytocyterol, polyglyceryl 2-oleate, ceramide, ceteareth-4 and cholesterol, and then emulsified by adding a mixed solution of PGA-vitamin C complex, dicetyl phosphate, concentrated glycerin and purified water, When the mixture was cooled to 45 ° C. while stirring, the fragrance was added to the mixture, stirred, and cooled to 30 ° C. to mature. Carboxyvinyl polymer, xanthan gum and preservatives were added thereto, stabilized, and aged to prepare an essence including the PGA-vitamin C complex. The contents of each component are shown in Table 6.

Raw material Formulation Example 3 Content (w / w%) Comparative Formulation Example 3 Content (w / w%) PGA-Vitamin C Complex 10.0 - Cytostolol 1.70 1.70 Polyglyceryl 2-oleate 1.50 1.50 Ceramide 0.7 0.7 Ceteares-4 1.2 1.2 cholesterol 1.5 1.5 Dicetylphosphate 0.4 0.4 Concentrated glycerin 5.0 5.0 Carboxy Vinyl Polymer 0.2 0.2 Xanthan Gum 0.2 0.2 antiseptic a very small amount a very small amount Spices a very small amount a very small amount Purified water Remaining amount Remaining amount

Formulation Example 4: Facial cleanser (cleansing foam)

Sodium N-acyl glutamate, glycerin, PEG-400, and propylene glycol were added to the purified water, followed by mixing. Then, a small amount of PGA-vitamin C complex was added thereto, and EDTA-4Na was added thereto, followed by heating and stirring at 80 ° C. for stirring. POE (15) oleyl alcohol ether, laurin derivative and methyl paraben mixed solution heated at 80 ° C. were added to the mixture, followed by stirring. Then, the mixture was gradually cooled to prepare a cleanser including a PGA-vitamin C complex. The content of each component is shown in Table 7.

Raw material Formulation Example 4 Content (w / w%) Comparative Formulation Example 4 Content (w / w%) PGA-Vitamin C Complex 20.0 - N-acyl glutamate 20.0 20.0 glycerin 10.0 10.0 PEG-400 15.0 15.0 Propylene glycol 10.0 10.0 POE (15) oleyl alcohol ether 3.0 3.0 Lauric derivatives 2.0 2.0 Methyl paraben 0.2 0.2 EDTA-4Na 0.03 0.03 Spices 0.2 0.2 Purified water Remaining amount Remaining amount

Formulation Example 5: shampoo

Glycerine and EDTA-4Na were added to purified water, and the resulting mixture was heated and dissolved at 80 DEG C, followed by stirring by adding TEA lauryl sulfate, sodium lauryl ether sulfate, lauryl amidopropyl betaine and lauryl acid diethanol amide. Citric acid was added thereto and neutralized at 50 ° C., followed by adding and stirring the PGA-vitamin C complex and Zinc pyrithione at 45 ° C. to prepare a shampoo including the PGA-vitamin C complex. 8 is shown.

Raw material Formulation Example 5 Content (w / w%) Comparative Formulation Example 5 Content (w / w%) PGA-Vitamin C Complex 20.0 - TEA Lauryl Sulfate 20.0 20.0 Sodium Lauryl Ether Sulfate 30.0 30.0 Lauryl Amidopropyl Betaine 2.0 2.0 Lauryl acid diethanol amide 3.0 3.0 glycerin 3.0 52.0 EDTA-2Na 0.05 0.05 Methyl paraben 0.2 0.2 Citric acid 0.03 0.03 Zinc pyrithione 0.02 0.02 Spices 0.2 0.2 Purified water Remaining amount Remaining amount

As described above in detail specific parts of the present invention, it is apparent to those skilled in the art that such specific descriptions are merely preferred embodiments, and thus the scope of the present invention is not limited thereto. something to do. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

The present invention is effective to provide a collagenase activity inhibitor and a composition for preventing skin wrinkles containing poly gamma glutamic acid (PGA) -vitamin C complex. The collagenase inhibitor according to the present invention not only has an effect of inhibiting the activity of collagenase, but also has an anti-aging effect such as an antioxidant effect and an effect of improving skin wrinkles by acting on the matrix metalloproteinase, and the skin connective tissue is loose. It prevents losing and maintains the elasticity of the skin, and has a high skin compatibility, moisturizing and hygroscopic properties, it will be useful in providing cosmetics, pharmaceuticals, food compositions useful for skin elasticity and wrinkle improvement.

Claims (8)

A cosmetic composition for preventing skin wrinkles containing polygamma glutamic acid (PGA) -vitamin C complex having collagenase inhibitory activity as an active ingredient. The cosmetic composition for preventing skin wrinkles according to claim 1, wherein the polygamma glutamic acid (PGA) has a molecular weight of 1 to 15,000 kDa. delete A food composition for preventing skin wrinkles containing polygamma glutamic acid (PGA) -vitamin C complex having collagenase inhibitory activity as an active ingredient. The food composition for preventing skin wrinkles according to claim 4, wherein the polygamma glutamic acid (PGA) has a molecular weight of 1 to 15,000 kDa. A pharmaceutical composition for preventing skin wrinkles containing polygamma glutamic acid (PGA) -vitamin C complex having collagenase inhibitory activity as an active ingredient. According to claim 6, wherein the polygamma glutamic acid (PGA) has a molecular weight of 1 ~ 15,000 kDa, characterized in that the anti-wrinkle pharmaceutical composition. delete

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