Natural Preservatives

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1 Cosmetic Natural Preservatives 1. MSE-G 2. MSE-G3

2 MSE-G Natural preservatives that could apply to all cosmetic products With wide anti-microbiological activity Viscosity is not affected No reaction with macromolecules It is possible to apply it to any products MSE-G3 is a water phase product with anti-fungal activity

3 Characteristics Product MSE-G MSE-G3 INCI Name Physical test purpose Lactobacillus Ferment Scutellaria baicalensis Root Extract Glycyrrhiza glabra (Licorice) Root Extract Glycerin - Pale yellow liquid - ph(2% Solution) : 6.0±0.5 - Water soluble (slightly dissolved in water, but transparently dissolved with solubilizer 0.1%-0.2%) - dosage: 2.0% - can be added in water phase or after homogenization for cosmetics Camellia Sinensis Leaf Extract Allium sativum (Garlic) Bulb Extract Cocos nucifera (Coconut) Oil Lactobacillus Ferment Yeast Ferment Extract - Pale yellow liquid - ph(2% Solution) : 7.0±0.5 - Water soluble (slightly dissolved in water, but transparently dissolved with solubilizer 0.1%-0.2%) - dosage: 1.5%-2.0% - can be added in water phase or after homogenization for cosmetics & food improved anti fungal effect can be applied to Hydrogel&Sheet mask

4 Manufacturing 공정도 process MSE-G Scutellaria baicalensis Root Extract Glycyrrhiza glabra (Licorice) Root Extract Scutellaria baicalensis Root Glycyrrhiza glabra (Licorice) Root X10 hot water Extract Concentration Filtration Lactobacillus Ferment Lactobacillus Seed Modified MRS broth (Animal Origin Free) Filtration 5% w/w Fermentation (35, 48hrs) Precipitation Separation Filtration Dry Water Dispersion Formula (Glycerin) Q.C Packing

5 Manufacturing process MSE-G3 Garlic(Allium Sativum) Bulb Ext. Camellia Sinensis Leaf Ext. Garlic(Allium Sativum) Bulb Camellia Sinensis Leaf Coconut(Cocos Nucifera) Oil X10 hot water Extract Concentration Filtration Lactobacillus Ferment Lactobacillus Seed Modified MRS broth (Animal Origin Free) Filtration 5% w/w Fermentation (35, 48hrs) Precipitation Separation Filtration Dry Water Dispersion Formula (Glycerin, Yeast Ferment Ext.) Q.C Packing

6 MIC test MHB, incubation conc.(8.0x10 4 cfu/ml), ph7.0, Microdilution method, 35 /48hrs Strains MSE-G(%) MSE-G3(%) 1,2-Hexanediol(%) EHG (%) S.aureus E.coli P.aeruginosa C.albicans A.niger MSE-G3 is improved in anti fungal effect. natural preservatives need to be added by 10 fold of MIC, dissimilarly with synthetic preservatives, paraben. we assume that paraben is low MW, but natural preservatives is not.

7 Challenge test-formula Moisturizing Cream Content(%) Water to 100 Hexyl Laurate Glycerin Butyrospermum Parkii (Shea Butter) Betaine Beta-Glucan Sodium Hyaluronate Cetyl Alcohol Brassica Campestris (Rapeseed) Sterols Hydrolyzed Collagen Hydrogenated Lecithin Sorbitan Sesquioleate Stearyl Alcohol Ceramide Tocopheryl Acetate Sorbitan Stearate Sodium PCA BST-Gel L-arginine MSE-G / MSE-G1 / BHC-C 2.00

8 Challenge test_microbes Each preservative was added to cream and total cell number was observed after 7, 14, 21, and 28 days. Re-challenge test was performed if any microbe was not observed after 28 days. - Challenge test of microbes (inoculum cells : 1x10 6 cfu/g) Preservative Number of cells (CFU/g) 7days 14days 21days 28days Control >10 5 >10 5 >10 5 >10 5 MSE-G 2.0% ND ND ND MSE-G3 2.0% ND ND ND 1,2-Hexanediol 2% ND ND ND EHG 1% ND ND ND

9 Challenge MIC test test_fungi Each preservative was added to cream and total cell number was observed after 7, 14, 21, and 28 days. Re-challenge test was performed if any fungi was not observed after 28 days. - Challenge test of fungi (inoculum cells cfu/g) Preservative Number of cells (CFU/g) 7days 14days 21days 28days Control >10 5 >10 5 >10 5 >10 5 MSE-G 2.0% ND MSE-G3 2.0% ND ND 1,2-Hexanediol 2% ND EHG 1% ND

10 Affections on thickener with MSE-G 1 Thickener : Carbopol 940 (carbomer) Changes on ph Changes on Viscosity (RPM 10) Dosage : 2% Before After Before After MSE-G The changes in ph value are within 0.2 after applied with natural preservative, MSE-G Brookfield s DV-II is used as the model to detect the changes on viscosity (spindle N0. 3, 23 ) Changes on Viscosity of Carbopol 940 under different speed

11 Affections on thickener with MSE-G 2 Thickener : C2020 (carbomer) Changes on ph Changes on Viscosity (RPM 10) Dosage : 2% Before After Before After MSE-G The changes on ph value are ±0.02 with natural preservative, MSE-G Brookfield s DV-II is used as the model to detect the changes on viscosity (spindle N0. 3, 23 ) Changes on Viscosity of C2020 under different speed

12 Affections on thickener with MSE-G 3 Changes after the application to cosmetics (lotion) Changes on ph Changes on Viscosity (RPM 10) Dosage : 2% Before After Before After MSE-G The formula of thickener in this product C2020 : 0.2% Cosmedia SP : 0.5% Keltrol F(Xanthan Gum) : 0.2% Brookfield s DV-II is used as the model to detect the changes on viscosity (spindle N0. 3, 23 ) 1. The changes on ph value are within 0.1 with natural preservative, the viscosity is also not significantly changed 2. MSE-G would not induce huge changes on ph value and viscosity of cosmetics that contained carbopol or Gum thickener Changes on Viscosity of Carbopol under different speed

13 Affections on thickener with MSE-G3 1 Thickener : Carbopol 940 (INCI name : Carbomer) Dosage of thickener : Carbopol % Changes on ph Changes on Viscosity (RPM 10) Dosage : 2% Before After Before After MSE-G The ph value is increased within 0.08 with natural preservative, MSE-G3 Brookfield s DV-II is used as the model to detect the changes on viscosity (spindle N0. 3, 23 ) 2. The viscosity of Carbopol 940 is increased with natural preservative, MSE-G3 (MSE-G3 2%)

14 Affections on thickener with MSE-G3 2 Thickener : C2020 (INCI name : Acrylates / C10-30 Alkyl Acrylate Crosspolymer) Dosage of thickener : C % Changes on ph Changes on Viscosity (RPM 10) Dosage : 2% Before After Before After MSE-G The changes on ph value are within 0.05 with natural preservative, MSE-G3 2. The viscosity of C2020 is increased with natural preservative, MSE-G Brookfield s DV-II is used as the model to detect the changes on viscosity (spindle N0. 3, 23 ) cp C2020 C2020 (MSE-G3 2% 첨가 (MSE-G3 ) 2%) RPM

15 Affections on thickener with MSE-G&G3 Carbopol 940 C2020 Dosage 2% Changes on ph Changes on Viscosity (RPM 10) Before After Before After MSE-G MSE-G MSE-G MSE-G Brookfield s DV-II is used as the model to detect the changes on viscosity (spindle N0. 3, 23 )

16 Human Patch test Observation is done after 24hr of patch test Stability of natural preservatives in cosmetics is observed in this experiment Mark - ± Score Results of evaluation (number of persons) Test 1hr 24hrs Assessment - ± ± Vehicle Non irri. MSE-G 20% Non irri. MSE-G3 20% Non irri. 1,2-Hexanediol 20% Non irri. EHG 1% Non irri. * Vehicle : D.W. * Experiment was performed with 10 x recommend conc. * Volunteer : 10 No irritation is found with natural preservatives. # Evaluatuon : ± Doubtful reaction + Weak reaction (erythma, papule) ++ Strong reaction (erythma, papule, vesicle) - Negative reaction

17 Hydrogel Challenge

18 Test materials Gel base : Agar 1.5% + Carrageenan 0.5% + Xanthan Gum 0.3% Medication : 1 MSE-G 0.5% + Phenoxyethanol(PE) 0.2% 2 MSE-G 0.8% 3 MSE-G3 0.5% + PE 0.2% 4 MSE-G3 0.8% 5 PE 0.2% + M-paraben 0.1% Germ challenge : B + Y 10 5 cfu/ml 35 Mold 10 4 cfu/ml 28

19 Test Method 1. Heating the material of the Gel base and dissolved to prepare 100g. 2. Add the drug to each Erlenmeyer flask containing 100 g of Gel base. 3. Seed plate with 1 ml of B + Y (106 cfu/ml) and Mold (105 cfu/ml). 4. 9ml dispensing the plate when the Gel base is 45 ~ 48. [Final concentration of bacteria is B + Y (105 cfu / ml), Mold (104 cfu / ml)] 5. After sealing with Parafilm, B + Y was incubated at 35 and Mold was incubated at After 14 days, gel is divided into 8 portions on the plate and 4 pieces are placed in a falcon tube. 7. Add 10 ml of sterile saline solution to the falcon tube containing the gel. 8. After vortexing, agitate and spread 200 μl into new medium.

20 Test Method Medication 45~48 9ml Gel Base 100g B+Y = 10 6 cfu/ml Mold = 10 5 cuf/ml 1ml Gel Base + Medication 14days incubating B+Y 35 M μl placing in 4 pieces Spreading Vortexing

21 Test Method Medication 45~48 9ml Gel base 100g Gel base + Medication 200 μl B+Y = 10 6 cfu/ml Mold = 10 5 cuf/ml 1ml placing in 4 pieces 14days incubating B+Y 35 M 28 B+Y = PCA Mold = PDA Spreading Vortexing

22 Test Result Challenge 14days Control MSE-G 0.5% + PE 0.2% MSE-G 0.8% MSE-G3 0.5% + PE 0.2% MSE-G3 0.8% PE 0.2% + M-p 0.1% ba cte ria 10 5 fun gu s 10 4 Recommend to prescribe MSE-G3 0.8% as a single prescription of natural preservatives, and MSE-G 0.5% + PE 0.2% as a combined prescription

23 Natural cosmetic Preservatives MSE-G & MSE-G3 Effect of Surfactant Check the effect of surfactant on antibacterial activity by paper disc diffusion assay Comparison of clear zone by using 7 surfactants, MSE-G and MSE-G3 alone and in combination 1 Amisoft CT-12S (TEA Cocoyl Glutamate) anion 2 Micolin S-430 (Sodium Lauryl Sulfate) anion 3 Corum 2C (Disodium Cocoamphodiacetate) anion 4 Mitaine CA (Cocamidopropyl Betaine) amphiprotic 5 Plantacare 2000up (Decyl Glucoside) nonionic 6 Plantacare 818up (Coco-Glucoside) nonionic 7 Plantacare 1200up (Lauryl Glucoside) nonionic 8 Miconium stac30 (steartrimonium chloride) cation 1 1+G 3 3+G 5 5+G G G MSE-G G G G 2 2+G 4 4+G 6 6+G 7 7+G 8 8+G 1 1+G3 3 3+G3 5 5+G3 C C MSE-G3 C C C 2 2+C 4 4+C 6 6+G3 7 7+G3 8 8+G3 8+C

24 2 2+C 4 4+C 6 6+G3 7 7+G3 8 8+G3 8+C Effect of Surfactant Natural cosmetic Preservatives MSE-G & MSE-G3 Check the effect of surfactant on antibacterial activity by paper disc diffusion assay Comparison of clear zone by using 7 surfactants, MSE-G and MSE-G3 alone and in combination Clear zone (mm) without surfactant anion MSE-G MSE-G3 BHC-C amphiprotic nonionic cation Amisoft CT-12S (TEA Cocoyl Glutamate) anion 2 Micolin S-430 (Sodium Lauryl Sulfate) anion 3 Corum 2C (Disodium Cocoamphodiacetate) anion 4 Mitaine CA (Cocamidopropyl Betaine) amphiprotic 5 Plantacare 2000up (Decyl Glucoside) nonionic 6 Plantacare 818up (Coco-Glucoside) nonionic 7 Plantacare 1200up (Lauryl Glucoside) nonionic 8 Miconium stac30 (steartrimonium chloride) cation 1. Both MSE-G and are all show no significant change when mixed with 4 (amphiprotic) 7, 8 (nonion, 1 cation) 1+G3 3 3+G3 5 5+G3 C C 2. Clear zone is bigger C when mixed C with preservative C in surfactant 1 (anion)

25 Effect of Surfactant Initial Toner Cleanser Natural cosmetic Preservatives MSE-G & MSE-G3 1 Control 2 MSE-G (2%) Serums MSE-G3 (2%) 4 BHC-C (2%) Lotion After 4weeks Light stability (Exposed to sunlight) Temperature stability (4 ) Temperature Stability (45 ) 1. For cleansers stored at 45 C, the yellow color becomes darker (seems due to the influence of the surfactant) No other abnormality

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