Purpose for Men Skin Care

Transcription

1 Purpose for Men Skin Care 31, Geodudanji 1gil, Dongnae-myeon, Chuncheon, Gangwon, Republic of Korea Page1 1

2 GUARDIANT is a good agent for Men Skin Care These days, not only women men are interested in beauty and health. Men highly interested in cosmetic and they are spending their time and money for their skin care. Normally people think it s easy to care men s skin but this is not true. Even they have thick epidermal and abundant sebum and collagen, it is decreased with ages and shaving every in the morning, rough cleansing, smoking, drinking affects to skin. These are damages to skin cell and it makes sensitive skin condition which causes skin trouble. GUARDIANT was made for the purpose of the men skin care and excellent effect to protect skin. GUARDIANT strengthens skin barrier and increasing skin recovery in epidermal by promoting Involucrin, TGase-1, Cornified envelope and provides deep moisturizing effect. Page2 2

3 PART Ⅰ General Information 1. About Salicornia Herbacea Research of Salicornia Herbacea Men s Skin 4. What is Skin barrier? PART Ⅱ Technical Data 1. Explain of GUARDIANT Stability study Safety study 14 PART Ⅲ Efficacy Data 1. Cell proliferation Effect Anti-wrinkle Effect 3. Moisturizing Effect Enhancement effect of Skin barrier 26 PART Ⅳ Conclusion 1. Summary Application 31 Page3 3

4 1. About Salicornia Herbacea Common name Latin name INCI name Main Ingredient Biological Effect Marshfire Glasswort, Samphire Salicornia Herbacea Salicornia Herbacea Extract Betaine, Mineral, Amino acid, Moisturizing, Anti-wrinkle, strengthen skin barrier Samphire is known as glasswort, sea asparagus and sea pickle and generally grows along the coast. It tolerates immersion in salt water. Because it grows near the coast and absorbs useful ingredients in nourished sea water samphire contains a lot of vitamins, minerals and other healthy nutrients of the sea. It highly includes many calcium, iron, sodium, etc. It has abundant amino acid and minerals. Calcium in samphire is 6 times more than milk called king of calcium, Iron in samphire is 40 times more than seaweed. Samphire has good effect for acne, freckles and other skin trouble and has detox effect. Minerals and amino acids are helps skin recovery and provide moisture to skin. As a representative plant growing in salty land, it has useful ingredients than any other plants living in land or under the water. It is referred to as a one of the most valuable gift that mud flats and sea gives us as well as natural resources. It easily can be found in Shinan-gun, the no.1 culture place for samphire. In Shinan-gun, there are total 1004 island and is the largest tideland salt farm in Korea. Page4 4

5 Betaine in Samphire Betaine is one of botanical ingredient and a natural moisturizing ingredient. Betaine having detox effect and helps cell recovery so that relaxes organs in the body and get refreshment. Also, it stimulates blood circulation so that helps skin recovery and acts as an anti-cancer agent. Chemical composition of Salicornia herbacea L Items Contents (%) Items Contents (mg/100g) Dietary fiver Total phenolics 430 Total nitrogen 1.33 Total chlorophyii Total sugar 15.2 Choline 29 Uronic acid 2.6 Betaine 888 Ref) Chemical Components of Native Plant, Salicornia herbacea L, 2002 Betaine modulates NF-kB signaling pathway through enhancement of thiols Department of Pharmacy, Graduate School, Pusan National University, 2005 Abstract : Betaine is an important human nutrient that is obtained from a variety of foods. Betaine as a methyl donor is known to convert homocysteine to methionine in the methionine metabolism. Results showed that betaine enhanced thiols level and BHMT gene expression. Betaine decreased homocysteine level causing age-related increases. And then betaine suppressed reactive species, NF-κB activation and its related gene expressions of cyclooxygenase, inducible NO synthase, vascular cell adhesion molecule-1, and intracellular cell adhesion molecule-1 during the aging process. Furthermore, betaine attenuated oxidative-induced NF-κB signaling pathways via enhancement of thiols in the cell culture system. Based on these results, it is concluded that betaine enhances thiols through homocysteine metabolism and contributes cellular redox-status. Thus, betaine might be used as a useful agent to attenuate vascular aging and further development of age-related diseases. Page5 5

6 2. Research of Salicornia Herbacea Analysis of Mineral composition Mineral Contents (mg/g) Sodium(Na) Calcium(Ca) Potassium(K) Magnesium(Mg) 54 Zinc(Zn) 29.6 Iron(Fe) 66.2 As a result of analysis of mineral composition, we can check lots of useful ingredient in samphire. Mineral balances between acid and alkali and improves enzyme function. In samphire, there are lots of useful ingredients including sodium, calcium, and potassium. Analysis of Amino acid composition Amino acid Contents (mg/g) Taurine 21.4 Aspartic acid Glutamic acid Glycine 80.4 Alanine 88.7 Methionine 52.2 Lysine Histidine Amino acids supply abundant nutrient to skin. Samphire shows many useful amino acids. It contains valine, leucine and proline and Taurin which is rare ingredient in land plants. These ingredients strengthen connective tissue and keep skin smooth and elastic. Page6 6

7 3. Men s Skin Man s skin has smaller sebaceous or oil glands. This is opposite to what most people believe to be true. Absorption of active ingredients in skin care product is primarily through these glands, and therefore this is a consideration in the formulation of products for proper men s skin care. Rough and Dried skin Man s skin is thicker 6 times more than woman s skin and slowly aged and has high skin elasticity than woman because they have thick epidermis, abundant lipids production and collagens. But later, when they are old, its skin has deep and more wrinkles. Testosterone improves lipid production so that their skin easily oil resulting skin trouble. Men have rough skin. Men have less moisture and stratum corneum than women therefore they need to provide abundant moisture and nutrients to skin. By provide moisture keep skin hold moisture for vital and glossy skin. Importance of Skin barrier Each corneocyte is surrounded by a protein shell called a cell envelope. The cell envelope is composed primarily of two proteins, loricirn and involucrin. These proteins contain extensive links between each other making the cell envelope the most insoluble structure of the corneocyte. The two subtypes of cell envelopes are described as "rigid" and "fragile" based on the interaction of lamellar bilayer with the cell envelope. Page7 7

8 4. What is Skin barrier? Skin is always exposed to outside environment and acts a protective barrier and prevents loss of moisture in skin. Stratum corneum plays role as a skin barrier and has crosslinked structure connecting with Keratin, Ceramides, Cholesterol, Collagen, fatty acids, etc. It provides structural stability and elasticity and acts a skin barrier. In addition to the structural role of supporting elasticity protect inner skin cells. Finally it has Cornified envelope (CE), an outer skin layer and protect skin from the outer environment. It is mainly consisted of involucrin and loricrin and other components like small proline-rich proteins, cystain A, envoplakin. Amont them, Involucrin is a major component for forming cornified envelope and acts as a biomarker promoting thick and strong Cornified envelope. TGase-1 is an enzyme which combine Involucrins and helps elastic and strong CE. This is a picture of keratinocyte can be found in healthy skin. It has hexagon shape in normal condition but when our skin is damaged, it loses its polygonal shape. Involucrin helps it gets back hexagon shape, a healthy condition by improving and stimulating normalization of keratinocyte. And TGase-1 accelerates recovery by combining Involucrins activity. GUARDIANT strengthen skin barrier and protects our skin as well as keep moisture. GUARDIANT improves production of Involucrin and TGase-1 so that makes strong and thick Cornified envelope. Strong CE protects skin from harmful environmental factor and inhibits water evaporation. Not even damaged skin, it can effective to normal skin to strengthen skin barrier. By treating fundamental problems in the skin with 2 major factors, it protects men s skin more effectively and makes vital and healthy skin condition. Page8 8

9 1. Explain of GUARDIANT Overall Procedure for the preparation of GUARDIANT Material purchase and also certificate of origin & quality Grinding whole dried Salicornia Herbacea a) Extraction and then 1st Filtration Centrifugation (4500rpm, 30min) and then 2nd Filtration Measurement of dry content on 110 Produce of final product and sterilization Yes No Quality control Yes Product packaging ready for market a ; Extraction with 70% EtOH - Extraction time ; 12hr - Extraction temperature ; 20~25 - Filtration: paper filtration (pore size: 1 μm ) for remove precipitation and suspended particles Page9 9

10 S p e c i f i c a t i o n INCI Name CHARACTERISTICS Appearance Odor GUARDIANT Water Butylene Glycol 1,2-Hexanediol Ethylhexylglycerin Salicornia Herbacea Extract SPECIFICATIONS Light yellow Liquid Typical ph Specific Gravity at 20 C Identification test 1) Identification test 2) Heavy Metal Arsenic (As) Preservative Total Aerobic Count Dark green Red-Purple Ring 20ppm 2ppm Non-added Less than 100 cfu/g E. coli Not detected Salmonella Not detected Storage condition Sealed containers should be Stored at a temperature of 10~30 (50~86 ), Quality might be affected after opening packing, please refer to MSDS for more informations. After opening the drums, sterilization is no more guaranteed Expiration Date 2 years in sealed original packing, stored in due conditions Page10 10

11 2. Stability study Stability to ph We have evaluated the stability of GUARDIANT at different ph, temperature, ethanol, at 1% to 5% in final products. ph of GUARDIANT solution was measured using the potentiometric method. Measurements were made at room temperature within a ph range of 2 to 10.. ph V/V 1% % % ; stable, ±; slightly unstable GUARDIANT shows generally stability between 2 to 10 tested ph. Accordingly it allows the use of various formulations in final products. Page11 11

12 Stability to temperature This study was made at the ph of the solution (ph close to 5.0) at temperatures ranging from -20 to 65, for 6 days. Test sample Temp 1day 2day 3day 4day 5day 6day GUARDIANT ; stable, ±; slightly unstable Temp V/V 30min 60min 120min 30min 60min 120min 30min 60min 120min 1% % % ; stable, ±; slightly unstable GUARDIANT shows stability during 6 days under -20, 60 condition and within tested range of temperature 40 ~80 during 2 hours. Accordingly, it allows the use of any formulations in final products. Page12 12

13 Stability in the presence of ethanol The study of solubility in various water/ethanol mixtures was made at room temperature at the ph of the solution (ph close to 5.0) (V/V) Ethanol / H2O (V/V) 10/90 30/70 50/50 70/30 90/10 1% % % ; stable, ±; slightly unstable GUARDIANT is good formulation in under 30% of Ethanol contents. Page13 13

14 3. Safety study Repeated insult patch test (RIPT) OBJECTIVE To determine the irritation and/or sensitization potential of a test material after repeated application under occlusive, semi-occlusive or open patches to the skin of human subjects. TEST MATERIAL GUARDIANT 10% solution in distilled water STUDY DATES This study was initiated on Sep 8 th, 2013 and was completed on Oct 11 th, PANEL SELECTION Panels of human subjects, male and female, randomly selected. No individuals were empanelled if they exhibited or had a history of acute or chronic dermatologic, medical, or physical conditions that could interfere with dermal scoring. TEST METHOD Patches were applied to the same site on Monday, Wednesday, and Friday for a total of 9 applications during the Induction period. The subjects remove the patches 24hours after each application. 24hour rest periods follow each removal. Prior to each reapplication, site(s) were graded for dermal irritation and sensitization Dermal scores 0 No visible skin reaction ± Barely perceptible erythema (minimal) 1+ Mild erythema (diffuse) 2+ Well defined erythema 3+ Erythema and oedema 4+ Erythema and oedema with vesiculation Page14 14

15 Ten to 21 days after application of the final induction patch, challenge patch(es) are applied to previously unpatched sites, adjacent to the original induction patch sites. The challenge sites 24~72hours after application. REFERENCE Standard Operating Procedures, Clinical Trials , Repeat Insult Patch Test (RIPT) RESULT Subject Number Subject Initials Induction Scores Challenge Scores hr 48hr 72hr 1 KJW KYI KMH LJH JJS LBR KHS JIJ KHM KNI CONCLUSION Based on the test population of 10 subjects and under the conditions of this study, the GUARDIANT 10% solution identified did not demonstrate a potential for eliciting dermal irritation on sensitization. Page15 15

16 Ames test for mutagenicity OBJECTIVE To screen for mutagens through the simple and inexpensive procedure that uses a bacterial test organism. It is a biological assay used in genetics, generally genetic toxicology, to test for mutagenic properties of a chemical compound. STUDY DATES This study was initiated on Sep, 9th, 2013 and was completed on Sep 17th, TEST ORGANISMS The test organism is a histidine-negative (his - ) auxotrophic strain of salmonella typhimurium that will not grow on a medium deficient in histidine unless a back mutation to his + (histidine-positive) has occurred. PRINCIPLE OF TEST METHOD It is recognized that the mutagenic effect of a product is frequently influenced by the enzymatic pathway of an organism, whereby non-mutagens are transformed into mutagens and vice versa when introduced into human system. REFERENCE K. Mortelmans, E. Zeiger., Mutation Research 455 (2000) 29-60, The Ames Salmonella. Microsome mutagenicity assay. RESULT Test samples TA98 His+ revertants/plate TA100 Spontaneous test NQO % GUARDIANT CONCLUSION Based on the test procedure and under the conditions of this study, the GUARDIANT 10% solution identified did not demonstrate a potential for mutagenicity. Page16 16

17 UV absorbance measurement OBJECTIVE UV absorbance measurement test is to determine of phototoxicity of ingredient in vitro condition. The test can result of safety about phototoxicity by evaluating UV absorbance. PRINCIPLE OF TEST METHOD UVA(320~400nm), UVB(290~320nm), UVC(200~290nm), visible rays (400~700nm). The shortest wavelengths of UVC (and those even shorter; nm) are blocked by ordinary air. MATERIAL Sample Concentration GUARDIANT 0.1% GUARDIANT 0.5% GUARDIANT 1% GUARDIANT 2% RESULT CONCLUSION GUARDIANT has no absorbance of UV and has no risk of phototoxicity. Page17 17

18 MATERIAL SAFETY DATA SHEET DATE : February, 19, 2014 WRITTEN BY : Sung Yong, Lee SIGNATURE : 1. PRODUCT AND COMPANY IDENTIFICATION Product name : Product code : Use : Manufacturer : GUARDIANT PE1389 Raw material for cosmetic RADIANT. 31 Geodudanji 1-Gil Dongnae-myeon Chuncheon Gangwondo Republic of Korea. Tel : Fax : E mail Emergency call radiantcmo@chol.com G2. COMPOSITION AND IMFORMATION OF INGREDIENTS Chemical Name : Molecular weight CAS Number (Butylene Glycol) EINECS (Butylene Glycol) INCI Name : None N.A Water Butylene Glycol 1,2-Hexanediol Ethylhexylglycerin Salicornia Herbacea Extract Hazardous ingredients: None 3. HAZARDS Information provided on the health effects of this product is based on individual components. All ingredients are bound and potential for hazardous exposure as shipped is minimal. However, some vapours may be released upon heating and the end-user (fabricator) must take the necessary precautions (mechanical ventilation, respiratory protection, etc) to protect employees from exposure. Main hazards Health risks Environment risks Routes of exposure 4. FIRST AID Skin : Eyes: Ingestion : No Known Health hazards Experience shows no acute irritancy or toxic effects. Handle the product with good working practice avoiding dispersion into the environment. Inhalation, Ingestion, Skin/Eye contact. Wash off with soap and plenty of water. Immediately irrigate with water. Do not induce vomiting without medical advice. Inhalation : Move to fresh air in case of accidental inhalation of fumes from overheating or combustion. When symptoms persist or in all cases of doubt seek medical advice. Page18 18

19 5. FIRE FIGHTING NFPA: Health: 1 Flammability: 1 Reactivity: Unsuitable extinguishing media Flammable properties Flash point (test method): Flammable limits in air, % by volume: Autoignition temperature: Products of combustion: Extinguishing Media: Fire Fighting Environmental Concerns: Fire Fighting Instructions: 109 (228 F) (Closed Cup) Upper: No Information Lower: No Information 394 (741 F) Carbon monoxide and butadiene. Use alcohol type aqueous film forming foam for large fires. Use CO2 or dry chemical for small fires. Thoroughly decontaminate bunker gear and other fire-fighting equipment before re-use. Water spray should be used to cool fire-exposed structures and vessels. Water or foam may cause frothing. Water spray can be used to reduce the intensity of flames and to dilute spills to a non-flammable mixture. Keep personnel removed from and upwind of fire. If potential for exposure to vapors or products of combustion exists, wear full fire fighting turnout gear and NIOSH approved self-contained breathing apparatus. Oxidizing chemicals may accelerate the burning rate in a fire situation. 6. ACCIDENTAL RELEASE Remove heat and sources of ignition. Drums and packing in danger should be cooled by pulverized water, as heating could provoke a rise in pressure with explosion or deflagration risks. Prevent entry into watercourses and pipeworks. Immediately mop up with suitable absorbant equipment for subsequent correct disposal according to current legislation. `7. HANDLING AND STORAGE Store in a dry place and away from light to insure the quality. Keep the drums well closed in a well aired place. Keep away from heat and sources of ignition. Do not smoke. In case of important heating of the liquid, there is a risk of formation of explosive mixtures with air. Risk of fire in case of contact with hot area, sparks or flames. 8. EXPOSURE CONTROL AND PERSONAL PROTECTION Respiratory protection Hand Protection : Eye Protection : Skin and body protection : Engineering measures Facial mask Protective gloves Glasses with air-tight protection Long sleeved clothing and safety shoes Heat only in areas with appropriate exhaust ventilation. Provide 9. PHYSICAL AND CHEMICAL PROPERTIES appropriate exhaust ventilation at machinery. Appearance: Odour: Liquid Yellow Liquid Typical Specific Gravity at 20 : Solubility in water: ph : Soluble Page19 19

20 Identification Test 1 ): Dark green Identification Test 2 ): Red-Purple Ring Vapour pressure : N.A. Density : N.A Preservative : Non-added 10. STABILITY AND REACTIVITY Thermal decomposition: Distillation without decomposition at normal pressure, No thermal decomposition in case of correct storage and handling. Dangerous decomposition products: No dangerous decomposition products if storage and handling conditions are respected. In case of fire or thermal decomposition, release of carbon monoxide and carbonic anhydride. Dangerous reactions : Reacts violently with powerful oxidising agents. Hazardous decomposition products :May emit irritant/toxic vapour/fumes under fire conditions. 11. TOXICOLOGY Acute toxicity : No toxical effect known Sensitisation : Not sensitizing Inhalation : Inhalation is possible only under aerosol conditions, and vapour/fumes might be irritant/toxic. 12. ECOLOGICAL INFORMATION No ecologic effect known 13. DISPOSAL Recommended method to dispose the product without danger: Dispose in accordance with the current legislation preferably using high temperature incineration or In a biological purification station in accordance with the current legislation. 14. TRANSPORT INFORMATION Not dangerous for transport. (ROAD-RAIL, SEA, AIR) 15. REGULATORY INFORMATION Labelling according E.E.C. directives : Not submitted to labelling 16. OTHER INFORMATION This information is furnished without warranty, except that it is accurate to the best knowledge of RADIANT INC. The data on this sheet relates only to the specific material designated herein. Page20 20

21 Cell proliferation effect OBJECTIVE The accurate assessment of cell number and cell proliferation is useful in many high content assays and is a key read out in cell growth applications. In order to investigate the possibility of GUARDIANT as a cosmetic ingredient, we measured its proliferation effect by MTT assay. STUDY DATES This study was initiated on August 26 th, 2013 and was completed on September 6 th, 2013 TESTING MATERIALS Test concentration 1%, 2% GUARDIANT TESTING METHOD Cell culture CCD-986sk fibroblast and HaCaT keratinocyte cells were cultured at 37 C (5% CO2) in DMEM supplemented with 5% FBS, 50 units/ml penicillin, 50 μg/ml streptomycin, and 2 mm glutamine. MTT assay Cells were plated at cells/100 ul into 96-well plates and then incubated. After 24 h, 100 ml of MTT stock solution (5 mg/ml) was added to the plate, followed by incubation for additional 4 h. The medium was then removed, and any formazan dye was extracted with 100 ul of 0.04 N HCl in isopropanol, and the absorbance was measured in a microplate reader at 570 nm with a background correction at 650 nm. Page21 21

22 RESULT CONCLUSION GUARDIANT was treated to human fibroblast cells (CCD-986sk) to analyze effects on cell proliferation. As a result of study, GUARDIANT has excellent cell proliferation compared with control and we can estimate that it is a potential agent for improving skin barrier. Page22 22

23 Anti-wrinkle effect OBJECTIVE Matrix metalloproteinases (MMPs) play an important role in physiological and pathological processes. MMP-1 plays a significant role in the degradation of fibrillar collagens in extracellular matrix remodeling. To investigate the effect of GUARDIANT on anti-wrinkle effect, we measured the expression level of MMP-1 STUDY DATES This study was initiated on August 26 th, 2013 and was completed on September 6 th, 2013 TESTING MATERIALS - 1%, 2% GUARDIANT TESTING METHOD MMP-1 assay Add 100 µl of Assay Diluent to each well. Add 100 µl of Standard, control, or sample to each well. Cover with a plate sealer, and incubate at room temperature for 2 hours. Aspirate and wash 4 times. Add 200 µl of Conjugate to each well. Cover with a new plate sealer, and incubate at room temperature for 2 hours on the shaker. Aspirate and wash 4 times. Add 200 µl Substrate Solution to each well. Add 50 µl of Stop Solution to each well. Read at 450 nm within 30 minutes. RESULT CONCLUSION From this data, we found that GUARDIANT inhibits MMP-1 production in human fibroblasts and 2% of GUARDIANT has similar MMP-1 inhibition compared with positive control, TGF-β. Therefore GUARDIANT might be improving collagen metabolism in dermal fibroblasts and has anti-wrinkle effect. Page23 23

24 Moisturizing effect OBJECTIVE Hyaluronan (also called hyaluronic acid or hyaluronate or HA) is an anionic, nonsulfated glycosaminoglycan distributed widely throughout connective, epithelial, and neural tissues. HA is produced mainly by fibroblasts. And also it is powerful moisturizing agent. By checking hyaluronate, we evaluate moisturizing effect of GUARDIANT STUDY DATES This study was initiated on August 26 th, 2013 and was completed on September 6 th, 2013 TESTING MATERIALS Test concentration GUARDIANT 1%, 2% TESTING METHOD Hyaluronic acid assay - Add 100 µl of Standards and samples into corresponding wells. Add 150 µl of Diluent to the Blank control wells and 100 µl of Diluent to the Zero HA control wells. Add 50 µl of working HA Detector to all wells except the blank control wells. The plate incubate for one hour at 37 ºC. Transfer 100 µl to the detection plate. Incubate at 4 ºC for 30 minutes. Wash the plate 3 times with 200 µl of 1X Wash Buffer. Add 100 µl of working Enzyme to each well. Incubate at 37 C for 30 minutes. Add 100 µl working Substrate Solution to each well. Measure the absorbance of each well at 405 nm beginning at T = 15 min. Page24 24

25 RESULT CONCLUSION To investigate moisturizing effect, we did hyaluronic acid assay. This result showed that GUARDIANT significantly increases hyaluronic acid as dose dependent manner and has strong moisturizing effect compared with positive control, EGF. Page25 25

26 Enhancement effect of skin barrier OBJECTIVE The top layer of skin cells is the most important function in maintaining the effectiveness of the barrier. Individual cells overlie each other and are tightly packed, preventing bacteria from entering and maintaining the water-holding properties of the skin. To investigate the effect of GUARDIANT on skin barrier, we measured the expression level of Transglutaminase-1, Cornified envelope and involucrin. STUDY DATES This study was initiated on August 26 th, 2013 and was completed on September 6 th, 2013 TESTING MATERIALS Test concentration 1%, 2% GUARDIANT TESTING METHOD Transglutaminase-1 assay ml samples were added to each well and were allowed to incubate at 37 for 1 h. The plates were incubated with 100 ml of TGase-1 antibody diluted 1:1000. The plates were washed three times in PBS and then incubated for 1 h with 100 ml peroxidase conjugated goat anti-mouse antibody diluted 1:2500. The microplates were washed three times. The buffer (100 ml) containing the O- phenylenediamine dihydrochloride and the urea hydrogen peroxide tablet. The amount of TG-I was measured by quantifying the O-phenylenediamine dihydrochloride reaction with an ELISA reader at 490 nm and subtracting the background at 650 nm. Cornified Envelope - Cornified envelopes (CE) were extracted from HaCaT cells (8x10 5 /test) by exhaustive boiling and sonication in 2% sodium dodecyl sulfate, 20 mm dithiotreitol, 0.1 M Tris-HCl (ph 8.0), and 0.5 mm ethylendiamine tetraacetate. CE formation was quantified by spectrophotometry at 600 nm and was normalized to the protein content. Reverse transcription-polymerase chain reaction (PCR) - Two micrograms of total RNA was reverse transcribed using oligo(dt) primers and AMV reverse transcriptase. PCR amplification of the complementary DNA was carried out for 35 consecutive cycles in 50 ml of amplification buffer containing 2.5 U per reaction Tag polymerase and 50 and 30 primers each in concentration of 0.5 mm. Page26 26

27 Tape stripping - Tape-stripped stratum corneum was cut into small pieces and immersed in 1 ml of dissociation buffer, followed by boiling at 100 for 10 min. Disaggregated corneocytes, which had been detached from the tape, were collected as a precipitate by centrifugation at 4000 g for 10 min. Extraction with dissociation buffer was repeated three times to remove soluble materials extensively, and the remaining insoluble materials were used as CEs. Staining of CEs Primary antibodies against CE components used in this study were monoclonal anti-human involucrin, monoclonal anti-isopeptidebond, monoclonal anti-human filaggrin, rabbit anti-mouse loricrin, rabbit anti-human envoplakin. RESULT [Skin barrier enhancement effect by improvement of TGase-1] [Skin barrier enhancement effect by improvement of Cornified envelope] Page27 27

28 [Skin barrier enhancement effect by improvement of Involucrin] [Skin barrier enhancement effect by Corneocyte maturation] Page28 28

29 CONCLUSION [Skin barrier enhancement effect by improvement of TGase-1] Transglutaminase (TGase) is a enzyme catalyzing an intermolecular bond between proteins. TGases-1 is known to participate in outer skin layer, Cornified envelop(ce). From the result, GUARDIANT showed increase of TGases-1 activity. Therefore GUARDIANT has enhancement effect of skin barrier by increasing Tgase-1 activity. [Skin barrier enhancement effect by improvement of Cornified envelope] GUARDIANT enhances the activity of cornified envelope (CE) and in case of 2% of GUARDIANT, it showed excellent cornified envelope activity compared with control. By promoting maturation of the cornified envelope, strengthen skin barrier and protect skin from external environment. [Skin barrier enhancement effect by improvement of Involucrin] Involucrine is well known as a differentiation biomarker in stratum corneum. From the result of involucrine acivity of GUARDIANT, it showed similar activity with positive control. Therefore, we can estimated that GUARDIANT increases involucrin production leading enhancement of skin barrier [Skin barrier enhancement effect by Cornecyte maturation] After shaving, stratum corneum in skin is damaged so couldn t have hexagon shape and leads skin trouble. When treated with 2% of GUARDIANT, it improves cell recovery with hexagon shaped skin cell as you can see above picture. GUARIDANT, promoting corneocyte maturation and helps normalize damaged startum corneum effectively. Page29 29

30 SUMMARY Men s skin has thicker skin layer and lipids than women. Though they have abundant collagens, shave every morning and rough cleansing or skin care leads to dehydration and sensitive skin condition. Therefore men need cosmetic specially concentrated on men s skin GUARDIANT increases Involucrin production which is a component of the keratinocyte crosslinked envelope in skin layer. GUARDIANT enhance the production of cornified envelope (CE), which is an insoluble structure surrounding the corneocytes, and maintain the barrier function in the skin by increasing Involucrin combination. GUARDIANT protects skin from dryness or roughness by accelerating skin barrier mechanism and keep moisture condition for vital and glossy skin. < Proposed mechanism for GUARDIANT protecting skin in Human skin> Page30 30

31 APPLICATION [Cleansing] -Shaving cream -Facial wash [Skin care] - Toner - Lotion - Cream Page31 31