Phytofuse Renew Efficacy Data

Transcription

1 Tomorrow s Vision Today! Phytofuse Renew Efficacy Data Code: INCI Name: Selaginella Lepidophylla Extract CAS #: EINECS #: Type of Study Results Assessment of Hair Characteristics The results of the assessment indicate that when incorporated into a serum, Phytofuse Renew is capable of subjectively improving hair characteristics over all by 11% when compared to untreated hair and by 25% when being compared to the control serum. The only characteristic perceived to be positive when using the control serum was the increase in shine, however, it was rated 11% worse than completely untreated hair. Phytofuse Renew imparts benefits that tactilely and visually improve the sensorial assessment of typically curly, unruly or frizzy hair. This aqueous product is ideal for use in hair care formulations, especially as a replacement for silicones, to increase shine, smooth the cuticle, and moisturize the hair for a healthier appearance. Cellular Viability Assay Phytofuse Renew exhibited positive results by increasing cell metabolism. The increase in fluorescent signal indicates an increase in cellular metabolism and viability post Phytofuse Renew treatment. For these reasons, we can assume Phytofuse Renew is suitable for cosmetic applications designed to increase cell viability and metabolism. High Resolution Ultra Sound Skin Density Assay As evidenced in a 4 week efficacy study of Phytofuse Renew on skin, skin density was improved by 32.82% after 24 hours and by 40.10% after 4 weeks when compared to the untreated control. When compared to the base cream Phytofuse Renew improved skin density by 17.29% after 24 hours and after 4 weeks Phytofuse Renew improved density by 25.60%. Results indicate that Phytofuse Renew is capable of improving skin density when compared to both the untreated control as well as the base lotion. Phytofuse Renew has a positive effect on skin s density when used at recommended use levels. Version#1/ /Form#82 i n f a c t i v e c o n c e p t s l l c. c o m + 1 ( ) F a x : + 1 ( )

2 Tomorrow s Vision Today! IL-6 Elisa Assay Phytofuse Renew exhibited anti-inflammatory effects on LPS-treated fibroblasts. As expected, the changes in IL-6 production using Phytofuse Renew appear to be dose dependent. This decrease in IL-6 production indicates a reduced inflammatory environment which could decrease the signs of aging and reduce the formation of fine lines and wrinkles. For these reasons, we can assume Phytofuse Renew is suitable for cosmetic applications designed to provide soothing and anti-aging properties. Improvement in Moisturization Results indicated that after 24 hours, the solution containing 2.0% Phytofuse Renew moisturized the skin 17% more effectively than the base lotion alone, while enhancing hydration levels 26% better than the base lotion alone after three weeks of application. When comparing the emulsion containing 2.0% Phytofuse Renew to the untreated skin site, moisture levels increased by 71% after 24 hours and remained elevated by 95% at the end of the three week testing period. ORAC Assay As shown in figure 1, Phytofuse Renew exhibited antioxidant activity comparable to our known antioxidant, Trolox. The antioxidant capacity of Phytofuse Renew increased as the concentration increased, as a result we can assure that its ability to minimize oxidative stress is dose dependent. Phytofuse Renew was designed to provide moisturizing, antioxidant, and soothing properties. With the present study we can confirm that this unique ingredient is not only capable of providing functional benefits but it is also capable of providing antioxidant benefits when added to cosmetic applications. Transepidermal Water Loss Assay Results indicate continuous improvements in the barrier of the skin throughout the 3 week test period. After one week, the solution containing 2.0% Phytofuse Renew decreased TEWL 12% more effectively than the base lotion alone. After three weeks, the solution containing 2.0% Phytofuse Renew demonstrated even more effective barrier protection, decreasing TEWL 19% better than the base lotion alone. Scratch Assay Phytofuse Renew was able to increase cell migration and close the scratch at a rate comparable to the positive control. The mechanisms of the cells in the in vitro scratch assay mimic the mechanisms seen in in vivo wound healing therefore we can be assured that our results are translatable outside the laboratory. Phytofuse Renew was designed to be moisturizing and soothing, and provide enhanced aesthetics. With the present study we can also be confident that this product has healing abilities and cell proliferation properties. PM 2.5 Inhibition Phytofuse Renew was able to effectively prevent the accumulation/ deposition of PM 2.5um sized particles on the surface of the skin. Version#1/ /Form#82 i n f a c t i v e c o n c e p t s l l c. c o m + 1 ( ) F a x : + 1 ( )

3 Transepidermal Water Loss Study Tradename: Phytofuse Renew Code: CAS #: Test Request Form #: 300 Lot #: NC C Sponsor: Active Concepts, LLC; 107 Technology Drive Lincolnton, NC Study Director: Erica Segura Principle Investigator: Meghan Darley Test Performed: Transepidermal Water Loss Study Introduction An in-vivo study was conducted over a period of three weeks to evaluate the ability of Phytofuse Renew to enhance barrier function through reduction in Transepidermal Water Loss (TEWL). Results indicate that this material is capable of efficiently reducing TEWL which allows moisture retention. Materials A. Equipment: DermaLab Skin Combo Methods Ten volunteers M/F between the ages of 23 and 45 and who were known to be free of any skin pathologies participated in this study. A Dermalab Combo was used to measure TEWL on the subject s volar forearms. The instrument consists of a probe that is based upon the vapor gradient with an open chamber. This open chamber design maintains the free natural evaporation from the skin without interfering with the environment over the measurement area. This ensures unbiased and accurate readings. Operation of the water loss module is fully menu drive, allowing for pre-setting and standard deviation or measurement time. Baseline TEWL readings were taken on day one of the study. Following initial measurements, all subjects were asked to apply 5milligrams of each test material on their volar forearms. Measurements were taken immediately after application of the test materials and then weekly for three weeks. The test material consisted of 2% Phytofuse Renew in a base lotion. For added perspective, measurements of an untreated test site and a site treated with a base lotion (Cetaphil Moisturizing for All Skin Types) were recorded. involved 1in the use of its chemical products since the conditions of use are beyond our control. Statements concerning the possible use of our products are not intended as recommendations to Page 1 of 2 Version#4/

4 Transepidermal Water Loss Study Results TEWL Comparison Over Time 30% Percent (%) Decrease 25% 20% 15% 10% 5% 9% 3% 22% 12% 23% 24% 14% 19% Experimental vs. Untreated Control Experimental vs. Base Lotion 0% t = 24 1 week 2 week 3 weeks Figure 1: Improvements in barrier function following application of the test materials after a period of 3 weeks. Discussion As shown in Figure 2, results indicate continuous improvements in the barrier of the skin throughout the 3 week test period. After one week, the solution containing 2.0% Phytofuse Renew decreased TEWL 12% more effectively than the base lotion alone. After three weeks, the solution containing 2.0% Phytofuse Renew demonstrated even more effective barrier protection, decreasing TEWL 19% better than the base lotion alone. involved 2in the use of its chemical products since the conditions of use are beyond our control. Statements concerning the possible use of our products are not intended as recommendations to Page 2 of 2 Version#4/

5 Sensorial Assessment of Hair Characteristics Assay i n f a c t i v e c o n c e p t s l l c. c o m + 1 ( ) F a x : + 1 ( ) Tradename: Phytofuse Renew Code: Test Request Form #: 577 Sponsor: Active Concepts, LLC 107 Technology Drive, Lincolnton, North Carolina Study Director: Erica Segura Principle Investigator: Meghan Darley Abstract The condition of the cuticle (the outer most layer of the hair) significantly affects both, manageability and volume of our hair. Overtime as hair becomes damaged, the cuticle often lifts because of a variety of influences including environment and styling processes. This results in flat, dull hair that is difficult to manage. Improving the shine on the hair has been shown to instantly make it appear healthier and more youthful. Smoothing the cuticle not only eases manageability, but also helps to minimize physical damage that perpetuates the loss of body and difficulty in styling. Phyofuse Renew is an innovative, water-soluble active that was created to moisturize and protect the hair when incorporated into hair care products. When added to final formulations, this product is designed to increase the sheen of the hair while providing hydration and antioxidant properties for protection against stressors. The purpose of this study was to confirm whether or not Phytofuse Renew, incorporated into a hair serum, is capable of providing these benefits to the hair. A half head study was conducted to determine the comparison of using a serum manufactured using Phytofuse Renew vs. a control serum. The volunteer s hair was photographed. The images of the half head study were used in conjunction with a sensory study to assess the shine, volume, dry and wet combability, thickness, smoothness, hydration, softness and manageability. Based on the results obtained, Phytofuse Renew is capable of enhancing the shine and overall appearance of the hair, as well as increasing frizz control for a smooth style. Materials and Methods The study was conducted using a female participant with long, naturally curly hair. The subject was asked to wash her hair and blow dry, as normal. A half head study was then conducted. After shampooing and conditioning, the hair was parted segmenting the hair in half, one half of the participant s wet hair was then treated with the Phytofuse Renew Serum. The hair on the other half of the head was treated with a control serum not containing any test product, following the wash. Following the application of the control serum and the Phytofuse Renew Serum, the participant was asked to comb out wet hair for a sensory assessment of wet combability, and then the participant s hair was blown dry using a flat paddle brush on both sides of the head. Once the hair was completely dry, the participant was asked to assess the volume, shine, dry combability, thickness, smoothness, hydration, softness and manageability of both halves of her hair. Assessments were made using a rubric from 1 to 10, with 1 being the lowest and 10 being the highest. See attachment 1. 1 Page 1 of 5 Version#2/ /Form#76

6 Sensorial Assessment of Hair Characteristics Assay i n f a c t i v e c o n c e p t s l l c. c o m + 1 ( ) F a x : + 1 ( ) Results Baseline Assessment of Untreated Hair Half Head Assessment of Control Serum Half Head Assessment of Phytofuse Renew Serum Experimental Serum vs. Untreated Control Experimental Serum vs. Control Serum Shine % 14% Volume % 50% Dry Combability % 0% Wet Combability % -14% Thickness % 33.00% Smoothness % 28% Hydration % 50% Softness % 20% Manageability % 43% Mean % 25% Table 1. Results of Sensory Assessment of Hair Rating from 1 (worst) to 10 (best) Graph 1. Rating of hair characteristics following sensory assessment 2 Page 2 of 5 Version#2/ /Form#76

7 Sensorial Assessment of Hair Characteristics Assay i n f a c t i v e c o n c e p t s l l c. c o m + 1 ( ) F a x : + 1 ( ) Figure 1. Full head Baseline, Untreated Hair Half-Head Treated with Phytofuse Renew Serum Half-Head Control Serum Figure 2. Half-Head Zoomed Photo of Hair Treated with Test Serum vs. Control Serum 3 Page 3 of 5 Version#2/ /Form#76

8 Sensorial Assessment of Hair Characteristics Assay i n f a c t i v e c o n c e p t s l l c. c o m + 1 ( ) F a x : + 1 ( ) Half-Head Treated with Phytofuse Renew Serum Half-Head Control Serum Figure 3. Half-Head Zoomed-Out Photo of Hair Treated with Test Serum vs. Control Serum Percent (%) Difference Graph 2. Hair Assessment results for sensory characteristics 4 Page 4 of 5 Version#2/ /Form#76

9 Sensorial Assessment of Hair Characteristics Assay i n f a c t i v e c o n c e p t s l l c. c o m + 1 ( ) F a x : + 1 ( ) Discussion The results of the assessment indicate that when incorporated into a serum, Phytofuse Renew is capable of subjectively improving hair characteristics over all by 11% when compared to untreated hair and by 25% when being compared to the control serum. The only characteristic perceived to be positive when using the control serum was the increase in shine, however, it was rated 11% worse than completely untreated hair. When used in a post-wash serum, Phytofuse Renew improved shine, smoothness, hydration and manageability by 60%, 29%, 50% and 25% respectively, in comparison to untreated hair. The control serum improved shine 40% when compared to untreated hair, but significantly decreased hair volume, combability, thickness, softness and overall manageability. These comparative results demonstrate the half head study results, shown in figure 2 and figure 3, to further visually support that the Phytofuse Renew Serum creates hair that is shinier and smoother, with the appearance of manageability, verse the control serum. Phytofuse Renew imparts benefits that tactilely and visually improve the sensorial assessment of typically curly, unruly or frizzy hair. This aqueous product is ideal for use in hair care formulations, especially as a replacement for silicones, to increase shine, smooth the cuticle, and moisturize the hair for a healthier appearance. 5 Page 5 of 5 Version#2/ /Form#76

10 Cellular Viability Assay Analysis Tradename: Phytofuse Renew Code: CAS #: Test Request Form #: 162 Sponsor: Active Concepts, LLC; 107 Technology Drive Lincolnton, NC Study Director: Erica Segura Principle Investigator: Meghan Darley Test Performed: Cellular Viability Assay Introduction The cellular viability assay is useful for quantitatively measuring cell-mediated cytotoxicity, cell proliferation and mitochondrial metabolic activity. Increased metabolism in a cell indicates ample cellular respiration and adenosine triphosphate (ATP) production. ATP is the molecular energy of cells and is required in basic cell function and signal transduction. A decrease is ATP levels indicates cytotoxicity and decreased cell function while an increase in ATP levels indicates healthy cells. The cellular viability assay was conducted to assess the ability of Phytofuse Renew to increase cellular metabolic activity in cultured dermal fibroblasts. Assay Principle The assay utilizes a nonfluorescent dye, resazurin, which is converted to a fluorescent dye, resorufin, in response to chemical reduction of growth medium from cell growth and by respiring mitochondria. Healthy cells that are in a proliferative state will be able to easily convert resazurin into resorufin without harming the cells. This method is a more sensitive assay than other commonly used mitochondrial reductase dyes such as MTT. An increase in the signal generated by resazurin-conversion is indicative of a proliferative cellular state. This information is presented in good faith but is not warranted as to accuracy of results. Also, freedom from patent infringement is not implied. This information is offered solely for your investigation, verification, and consideration. Version#5/ /Form#64

11 Cellular Viability Assay Analysis Materials A. Kit: PrestoBlue Cell Viability Reagent (Invitrogen, A13261) B. Incubation Conditions: 37 C at 5% CO 2 and 95% relative humidity (RH) C. Equipment: D. Cell Line: E. Media/Buffers: F. Culture Plate: G. Reagents: H. Other: Forma humidified incubator; ESCO biosafety laminar flow hood; Light microscope; Pipettes Normal Human Dermal Fibroblasts (NHDF) (Lonza; CC-2511) Dulbecco's Modified Eagle Medium (DMEM); Penicillin-Streptomycin (50U- 50mg/mL); Fetal Bovine Serum (FBS); Phosphate Buffered Saline (PBS) Falcon flat bottom 96-well tissue culture treated plates PrestoBlue reagent (10X) Sterile disposable pipette tips Methods Human dermal fibroblasts were seeded into 96-well tissue culture plates and allowed to grow to confluency in complete DMEM. A 10-fold serial dilution was performed resulting in Phytofuse Renew concentrations on 1%, 0.1%, and 0.01% in complete DMEM and incubated with fibroblasts for 24 hours. Ten microliters of viability reagent was added to 90µL of cell culture media in culture wells. This information is presented in good faith but is not warranted as to accuracy of results. Also, freedom from patent infringement is not implied. This information is offered solely for your investigation, verification, and consideration. Version#5/ /Form#64

12 Cellular Viability Assay Analysis Results The data obtained from this study met criteria for a valid assay and the controls performed as anticipated. Phytofuse Renew at all concentrations is able to increase cellular metabolism compared to the control. Cellular metabolism results are expressed as a percentage of the control Phytofuse Renew Viability (% of control) 150% 130% 110% 90% 70% 50% % % % 1% 0.10% 0.01% Concentration of Sample Figure 1: Cellular Metabolism of Phytofuse Renew treated fibroblasts expressed in terms of percent of control. Discussion As shown in figure 1, Phytofuse Renew exhibited positive results by increasing cell metabolism. The increase in fluorescent signal indicates an increase in cellular metabolism and viability post Phytofuse Renew treatment. For these reasons, we can assume Phytofuse Renew is suitable for cosmetic applications designed to increase cell viability and metabolism. This information is presented in good faith but is not warranted as to accuracy of results. Also, freedom from patent infringement is not implied. This information is offered solely for your investigation, verification, and consideration. Version#5/ /Form#64

13 High Resolution Ultrasound Skin Imaging Assay Tradename: Phytofuse Renew Code: CAS #: Test Request Form #: 534 Lot #: NC C Sponsor: Active Concepts, LLC; 107 Technology Drive Lincolnton, NC Study Director: Erica Segura Principle Investigator: Meghan Darley Test Performed: High Resolution Ultrasound Skin-Imaging Assay Introduction An in-vivo study was conducted over a period of four weeks to evaluate the effect on skin density of Phytofuse Renew. 10 M/F subjects between the ages of participated in the study. Data gathered from the high resolution ultrasound imaging yielded results that indicate that this material is capable of significantly improving skin density compared to the control. Materials A. Equipment: DermaLab Skin Combo (Ultrasound Probe) Methods High Resolution Ultrasound Skin imaging is based on measuring the acoustic response after an acoustic pulse is sent into the skin. The energy of the acoustic pulse is low and will not affect the skin in any way. When the acoustic pulse is emitted and hits different areas of the skin, part of the pulse will be reflected and part will be transmitted further into the skin. The reflected signal travels back and is picked up by the ultrasound transducer. After processing the signal, a cross-sectional image appears on the screen. This image represents an intensity, or amplitude, analysis of the signals. The intensity of the signals that are received refer to a color scale. Dark colors represent areas of the skin with low reflection. This means that there are no changes or very small changes in density between the structures in the skin. Bright colors represent areas with strong reflections, signifying substantial changes in density between structures. 1 Page 1 of 4 Version#3/ /Form#75

14 High Resolution Ultrasound Skin Imaging Assay 10 volunteers M/F between the ages of 23 and 45 and who were known to be free of any skin pathologies participated in this study. The DermaLab ultrasound probe was used to determine the skin density of the subject s volar forearms. Following initial measurements, all subjects were asked to apply 2 mg of each test material on their volar forearms. Measurements were taken 24 hours after application of test materials and then weekly for 4 weeks. The test material consisted of 2.0% Phytofuse Renew in a base lotion. For added perspective, measurements of an untreated test site and a site treated with a base lotion (Cetaphil Moisturizing for All Skin Types) were recorded. Results Phytofuse Renew showed improvements in skin density at a 2.0% concentration. Please note, each value is an average of three consecutive readings per test site. Individual Raw Data: t = 24 1 week 2 weeks 3 weeks 4 weeks Subject 1-Test Untreated Control Base Lotion Control Subject 2-Test Untreated Control Base Lotion Control Subject 3-Test Untreated Control Base Lotion Control Subject 4-Test Untreated Control Base Lotion Control Subject 6-Test Untreated Control Base Lotion Control Page 2 of 4 Version#3/ /Form#75

15 High Resolution Ultrasound Skin Imaging Assay T = 24 1 week 2 weeks 3 weeks 4 weeks Subject 7-Test Untreated Control Base Lotion Control Subject 8-Test Untreated Control Base Lotion Control Subject 9-Test Untreated Control Base Lotion Control Subject 10-Test Untreated Control Base Lotion Control Raw Data: t=24 1 week 2 week 3 week 4 week Experimental (2.0% Phytofuse Renew in Base Lotion) Untreated Control Base Lotion Control Experimental vs. Untreated Base Lotion vs. Untreated Control Experimental vs. Base Lotion t=24 1 week 2 week 3 week 4 week 32.82% 44.88% 46.76% 43.28% 40.10% 13.24% 17.44% 19.82% 13.57% 11.54% 17.29% 23.36% 22.49% 26.16% 25.60% 3 Page 3 of 4 Version#3/ /Form#75

16 High Resolution Ultrasound Skin Imaging Assay Percent (%) Difference Between Test Sites 50.00% 45.00% 40.00% 35.00% 30.00% 25.00% 20.00% 15.00% 10.00% 5.00% 0.00% Comparative Analysis of Skin Density t=24 1 week 2 week 3 week 4 week Experimental vs. Untreated Base Lotion vs. Untreated Control Experimental vs. Base Lotion Figure 1: Percent difference in skin density recordings between test materials Discussion As evidenced in a 4 week efficacy study of Phytofuse Renew on skin, skin density was improved by 32.82% after 24 hours and by 40.10% after 4 weeks when compared to the untreated control. When compared to the base cream Phytofuse Renew improved skin density by 17.29% after 24 hours and after 4 weeks Phytofuse Renew improved density by 25.60%. Results indicate that Phytofuse Renew is capable of improving skin density when compared to both the untreated control as well as the base lotion. Phytofuse Renew has a positive effect on skin s density when used at recommended use levels. 4 Page 4 of 4 Version#3/ /Form#75

17 IL-6 ELISA Analysis Tradename: Phytofuse Renew Code: CAS #: Test Request Form #: 204 Sponsor: Active Concepts, LLC; 107 Technology Drive Lincolnton, NC Study Director: Erica Segura Principle Investigator: Meghan Darley Test Performed: Interleukin (IL)-6 Enzyme-Linked Immunosorbent Assay (ELISA) Introduction Interleukin-6 is a proinflammatory cytokine known to play an active role in inflammation, immunology, bone metabolism, reproduction, arthritis, neoplasia, and aging. IL-6 signals through the nuclear factor-kappa B (NF-κB) pathway that results in the transcription of inflammatory mediators, including matrix metalloproteinase-1 (MMP-1). MMP s are responsible for breaking down the extracellular matrix and collagen in the skin leading to wrinkles, fine lines, and loss of skin elasticity. Reducing the level of IL-6 and other inflammatory mediators is believed to slow down degradation of the skin matrix and, possibly, stimulate its replenishment. Interleukin-6 ELISA was conducted to assess the changes in IL-6 levels in Phytofuse Renew -treated in vitro cultured human dermal fibroblasts. Assay Principle This ELISA utilizes a colorimetric reaction employing antibodies with antigen specificity to human IL-6. Monoclonal antibodies specific for IL-6 epitopes are coated on a microtiter plate. In positive samples, IL-6 will bind to these antibodies and are tagged a second time with another IL-6-specific antibody labeled with horseradish peroxidase (HRP). The addition of the chromagen solution, containing 3,3,5,5 -tetramethylbenzidine, provides the colorimetric reaction with HRP that is quantitated through optical density (OD) readings on a microplate spectrometer. The standard curve provides a reference from the OD readings for the amount of collagen in each sample. This information is presented in good faith but is not warranted as to accuracy of results. Also, freedom from patent infringement is not implied. This information is offered solely for your investigation, verification, and consideration. Version#3/ /Form#63

18 IL-6 ELISA Analysis Materials A. Kit: IL-6 ELISA Kit (Biosource; KAC1261) B. Incubation Conditions: 37 C at 5% CO 2 and 95% relative humidity (RH) C. Equipment: Forma humidified incubator; ESCO biosafety laminar flow hood; Microplate Reader; Pipettes D. Cell Line: Normal Human Dermal Fibroblasts (NHDF) (Lonza; CC-2511) E. Media/Buffers: Dulbecco's Modified Eagle Medium (DMEM); Penicillin- Streptomycin (50U-50mg/mL); Fetal Bovine Serum (FBS); Phosphate Buffered Saline (PBS); Amphotericin (45pg/mL) F. Culture Plate: Falcon flat bottom 12-well tissue culture treated plates G. Reagents: Lipopolysaccharide (LPS) (1µg/mL) H. Other: Sterile disposable pipette tips; wash bottles Methods Human dermal fibroblasts were seeded into 12-well tissue culture plates and allowed to grow to confluency in complete DMEM. 1%, 0.1%, 0.01% concentrations of Phytofuse Renew were added to complete DMEM containing 1µg/mL LPS and incubated with fibroblasts for 24 hours. Complete media containing 1µg/mL LPS was used as the positive controls and complete DMEM was used a negative control. Standards were prepared in concentrations ranging from 2476pg/mL to 0pg/mL. 50µL of Solution B was added to wells for standards and assay controls and 50µL of Solution A was added to experiment wells. 100µL of standards, controls, and samples were added to appropriate wells. After a one hour incubation at room temperature and washing, 50µL Solution A and 100µL anti-il-6 conjugate was added to all wells. Following a one hour incubation and washing, 100 µl chromagen solution was added for the colorimetric reaction. One-hundred µl stop solution was added to stop the reaction after 15 minutes. The optical density was read at 450nm on the Synergy HT Microplate Reader. A standard curve was created by reducing the data and generating a linear curve fit. The IL-6 concentration of Phytofuse Renew treated-fibroblasts was determined by extrapolation from the standard curve and expressed in pg/ml. This information is presented in good faith but is not warranted as to accuracy of results. Also, freedom from patent infringement is not implied. This information is offered solely for your investigation, verification, and consideration. Version#3/ /Form#63

19 IL-6 ELISA Analysis Results The data obtained from this study met criteria for a valid assay and the positive and negative controls performed as anticipated. Phytofuse Renew, at concentrations of 1%, 0.1%, and 0.01%, was able to decrease IL-6 production compared to our positive control. IL-6 levels are expressed by the following formula: = Average IL 6 Concentrations Dilution Factor IL-6 Concentration (pg/ml) Phytofuse Renew IL-6 ELISA % Phytofuse Renew % Phytofuse Renew 0.01% Phytofuse Renew Positive Control Negative Control Figure 1: Phytofuse Renew -treated fibroblasts IL-6 concentrations This information is presented in good faith but is not warranted as to accuracy of results. Also, freedom from patent infringement is not implied. This information is offered solely for your investigation, verification, and consideration. Version#3/ /Form#63

20 IL-6 ELISA Analysis IL-6 production percent change is calculated by the following formula: = Positive Control Avg.Concentration Sample Avg.Concentration Positive Control Avg.Concentration Percent Dercrease in IL-6 Concentration Percent Decrease (%) % Phytofuse Renew 0.1% Phytofuse Renew 0.01% Phytofuse Renew Series Figure 2: Percent change in IL-6 production compared to positive control Discussion As shown in figure 1, Phytofuse Renew -exhibited anti-inflammatory effects on LPS-treated fibroblasts. As expected, the changes in IL-6 production using Phytofuse Renew appear to be dose dependent. This decrease in IL-6 production indicates a reduced inflammatory environment which could decrease the signs of aging and reduce the formation of fine lines and wrinkles. For these reasons, we can assume Phytofuse Renew is suitable for cosmetic applications designed to provide soothing and anti-aging properties. This information is presented in good faith but is not warranted as to accuracy of results. Also, freedom from patent infringement is not implied. This information is offered solely for your investigation, verification, and consideration. Version#3/ /Form#63

21 Improvements in Moisturization Tradename: Phytofuse Renew Code: CAS #: Test Request Form #: 300 Lot #: NC C Sponsor: Active Concepts, LLC; 107 Technology Drive Lincolnton, NC Study Director: Erica Segura Principle Investigator: Meghan Darley Test Performed: Improvements in Moisturization Introduction An in-vivo study was conducted over a period of three weeks to evaluate the moisturization benefits of Phytofuse Renew. 10 M/F subjects between the ages of participated in the study. Results indicate that this material is capable of significantly increasing moisturization compared to the control. Materials A. Equipment: DermaLab Skin Combo Methods 10 volunteers M/F between the ages of 27 and 56 and who were known to be free of any skin pathologies participated in this study. Ten volunteers (M/F) between the ages of 27 to 56 and who were known to be free of any skin pathologies participated in these studies. Volunteers were asked to apply an O/W emulsion containing 2% Phytofuse Renew to their volar forearms twice a day for three weeks. Measurements were taken using the DermaLab Combo. For added perspective measurements of an untreated test site and a site treated with the O/W emulsion with no additives were also recorded. The first study was conducted to measure the moisturizing ability of the product. Measurements were taken at T=0, week 1, week 2 and week 3 using an Impedance Meter. This piece of equipment employs an impedancebased electronic sensing system to evaluate conductance. For added perspective, measurements of an untreated test site and a site treated with a base lotion (Cetaphil Moisturizing for All Skin Types) were recorded. 1 Page 1 of 2 Version#4/

22 Improvements in Moisturization Results Moisturization Comparison Over Time Percent (%) Difference 120% 100% 80% 60% 40% 20% 0% -20% 110% 108% 95% 71% 26% 27% 26% -3% t = 24 1 week 2 week 3 weeks Experimental vs. Untreated Control Experimental vs. Base Lotion Figure 1: Improvements in moisturization following application of the test materials after a period of 3 weeks. Discussion Results indicated that after 24 hours, the solution containing 2.0% Phytofuse Renew moisturized the skin 17% more effectively than the base lotion alone, while enhancing hydration levels 26% better than the base lotion alone after three weeks of application. When comparing the emulsion containing 2.0% Phytofuse Renew to the untreated skin site, moisture levels increased by 71% after 24 hours and remained elevated by 95% at the end of the three week testing period. 2 Page 2 of 2 Version#4/

23 Oxygen Radical Absorbance Capacity (ORAC) Assay i n f a c t i v e c o n c e p t s l l c. c o m + 1 ( ) F a x : + 1 ( ) Tradename: Phytofuse Renew Code: CAS #: Test Request Form #: 36 Lot #: NC A Sponsor: Active Concepts, LLC; 107 Technology Drive Lincolnton, NC Study Director: Erica Segura Principle Investigator: Meghan Darley Test Performed: Oxygen Radical Absorbance Capacity (ORAC) Introduction Reactive oxygen species (ROS) are generated by normal cellular processes, environmental stresses, and UV irradiation. ROS are detrimental to cellular structures and functional molecules (i.e DNA, proteins, lipids) as they act as strong oxidizing agents or free radicals. The oxygen radical absorbance capacity (ORAC) assay is a standard method used to assess antioxidant capacity of physiological fluids, foods, beverages, and natural products. The assay quantitatively measures a sample s ability to quench free radicals that have the potential to react with and damage cellular components. Oxygen Radical Absorbance Capacity (ORAC) assay was conducted to assess the antioxidant capacity of Phytofuse Renew. Assay Principle This assay is based upon the effect of peroxyl radicals generated from the thermal decomposition of 2, 2 -azobis- 2-methyl-propanimidamide dihydrochloride (AAPH) on the signal intensity from the fluorescent probe, fluorescein, in the presence of an oxygen radical absorbing substance. The degree of change is indicative of the amount of radical damage and the presence of antioxidants results in an inhibition in the free radical damage to the fluorescein. The antioxidant protection of the sample can be calculated by comparing it to a set of known standards. Trolox, a water soluble vitamin E analog, with known antioxidant capabilities is used in this ORAC assay as the standard for measuring the antioxidant capacity of unknown substances. ORAC values, expressed in µm of Trolox equivalents (TE), are calculated using the area under the curves (AUC) of the test product, Trolox, and the control materials. Trolox equivalency is used as the benchmark for antioxidant capacity of mixtures since it is difficult to measure individual components. 1 Page 1 of 3 Version#3/ /Form#56

24 Oxygen Radical Absorbance Capacity (ORAC) Assay i n f a c t i v e c o n c e p t s l l c. c o m + 1 ( ) F a x : + 1 ( ) Materials A. Equipment: Synergy H1 Microplate reader (BioTek Instuments, Winooski, VT); Gen5 software (BioTek Instuments, Winooski, VT); Pipettes B. Buffers: 75mM Potassium Phosphate (ph 7.4); Deionized H 2 O C. Reagents: 2,2 -Azobis(2-methylpropionamidine) dihydrochloride (AAPH) (153mM); 6- Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox ); Fluorescein Sodium Salt (4nM) D. Preparation: Pre-heat (37 C) Synergy H1 Microplate reader; Prepare Trolox standards, sample dilutions, fluorescein solution, and AAPH. E. Microtitre Plates: Corning 96 Well Black Side/Clear Bottom Microplates Methods Solutions of Phytofuse Renew and Trolox (positive control) were prepared in 75mM potassium phosphate buffer. Materials were prepared at 10%. Trolox was used as a reference for antioxidant capacity and prepared at a concentrations ranging from 12.5µM to 200µM in 75mM potassium phosphate buffer. For the ORAC assay, 25µL of test material and Trolox were combined with 150µL of fluorescein in 75mM potassium phosphate buffer and incubated in the Synergy HT Microplate reader at 37 C for 30 minutes. At the end of the incubation period, 25µL of AAPH were pipetted into each well. Fluorescent measurements were then taken every 2 minutes for approximately 2 hours at an excitation wavelength of 485nm and an emissions wavelength of 520nm. The AUC and Net AUC values of the standards and samples were determined using Gen5 2.0 Data Reduction Software using the below equations: The standard curve was obtained by plotting the Net AUC of different Trolox concentrations against their concentration. ORAC values of samples were then calculated automatically using the Gen5 software to interpolate the sample s Net AUC values against the Trolox standard curve. ORAC measurements for the test material were expressed in micro moles Trolox equivalents (µmte), where 1 ORAC unit is equal to 1 µmte. 2 Page 2 of 3 Version#3/ /Form#56

25 Oxygen Radical Absorbance Capacity (ORAC) Assay Results i n f a c t i v e c o n c e p t s l l c. c o m + 1 ( ) F a x : + 1 ( ) Phytofuse Renew began exhibiting antioxidant activity at a 10% concentration Phytofuse Renew ORAC Assay Antioxidant Capacity (µmte) µM Trolox 12.5µM Trolox 10% Phytofuse Renew Figure 1: Antioxidant capacities Discussion As shown in figure 1, Phytofuse Renew exhibited antioxidant activity comparable to our known antioxidant, Trolox. The antioxidant capacity of Phytofuse Renew increased as the concentration increased, as a result we can assure that its ability to minimize oxidative stress is dose dependent. Phytofuse Renew was designed to provide moisturizing, antioxidant, and soothing properties. With the present study we can confirm that this unique ingredient is not only capable of providing functional benefits but it is also capable of providing antioxidant benefits when added to cosmetic applications. 3 Page 3 of 3 Version#3/ /Form#56

26 Scratch Assay Analysis i n f a c t i v e c o n c e p t s l l c. c o m + 1 ( ) F a x : + 1 ( ) Tradename: Phytofuse Renew Code: CAS #: Test Request Form #: 141 Sponsor: Active Concepts, LLC; 107 Technology Drive Lincolnton, NC Study Director: Erica Segura Principle Investigator: Meghan Darley Test Performed: Scratch Assay Introduction Wounded tissue begins a complex and structured series of events in order to repair the damaged region. Some of these events include upregulation of angiogenic factors causing increased vascularization, increased deposition of extracellular matrix, and increased cell proliferation. The wound healing process begins as cells polarize toward the wound, initiate protrusion, migrate, and close the wound area. These processes reflect the behavior of individual cells as well as the entire tissue complex. The scratch assay was conducted to assess the wound healing properties of Phytofuse Renew treated in-vitro cultured human dermal fibroblasts. Assay Principle The in-vitro scratch assay is a well-known and widely used method to study cell migration and proliferation. This assay is based on the observation that when an artificial gap or scratch is made on a confluent cell monolayer, the cells will migrate towards the opening and close the scratch. The basic steps involve creating a scratch in a cell monolayer and capturing images throughout the healing or cell migration process. Through these images we can quantify the rate of cell migration. This information is presented in good faith but is not warranted as to accuracy of results. Also, freedom from patent infringement is not implied. This information is offered solely for your investigation, verification, and consideration. Version#2/

27 Scratch Assay Analysis Materials i n f a c t i v e c o n c e p t s l l c. c o m + 1 ( ) F a x : + 1 ( ) A. Incubation Conditions: 37 C at 5% CO 2 and 95% Relative Humidity (RH) B. Equipment: Forma Humidified Incubator, ESCO Biosafety Laminar Flow Hood, Inverted Microscope; Camera; Pipettes C. Cell Line: Normal Human Dermal Fibroblasts (NHDF) (Lonza; CC-2511) D. Media/Buffers: Complete and Serum-Free Dulbecco's Modified Eagle Medium (DMEM); Fetal Bovine Serum (FBS) E. Reagents: Paraformaldehyde (3.7%); Crystal Violet Stain F. Culture Plate: Falcon Flat Bottom 6-Well Tissue Culture Treated Plates G. Other: Sterile Disposable Pipette Tips; Wash Bottles; 15mL Conical Tubes Methods Human dermal fibroblasts were seeded into 6-well tissue culture plates and allowed to grow to confluency in complete DMEM. A 0.5% concentration of Phytofuse Renew was added to the serum-free DMEM and incubated with fibroblasts for the extent of the experiment. Complete DMEM containing 10% FBS was used a positive control and serum-free DMEM was used a negative control. When cell growth reached confluency scratches were made across the well in a cross or X pattern. The wells were washed with sterile PBS and fresh media containing Phytofuse Renew, and the positive and negative controls were added. Initial images were captured immediately after the scratch took place and every 24-hours afterwards, up to 96-hours. Cells were fixed with 3.7% paraformaldehyde and stained with crystal violet for enhanced microscopy. ImageJ software was used to analyze the images and calculate the area of the scratch and the closure rate. Results The data obtained from this study met criteria for a valid assay and the positive and negative controls performed as anticipated. Phytofuse Renew at a 0.5% concentration was able to increase cell migration and wound healing compared to our negative control. Percent scratch closure and migration rate are expressed by the following formula: This information is presented in good faith but is not warranted as to accuracy of results. Also, freedom from patent infringement is not implied. This information is offered solely for your investigation, verification, and consideration. Version#2/

28 Scratch Assay Analysis i n f a c t i v e c o n c e p t s l l c. c o m + 1 ( ) F a x : + 1 ( ) Area Enclosure Scratch Area (mm 2 ) Positive Control Negative Contol Phytofuse Renew Time Post Scratch (Hours) Figure 1: Area of scratch Percent Scratch Closure % Closure Positive Control Negative Contol Phytofuse Renew Time Post Scratch (Hours) Figure 2: Percent scratch closure Migration Rate (nm 2 /hour) Migration Rate Time Post Scratch (Hours) Positive Control Negative Contol Phytofuse Renew Figure 3: Cell migration rate This information is presented in good faith but is not warranted as to accuracy of results. Also, freedom from patent infringement is not implied. This information is offered solely for your investigation, verification, and consideration. Version#2/

29 Scratch Assay Analysis i n f a c t i v e c o n c e p t s l l c. c o m + 1 ( ) F a x : + 1 ( ) A t=0 t=96 t=96 B C Positive Control D E F Phytofuse Renew Negative Control G H I Figure 4: Images at t=0 hours (A, D, G) and t=96 hours (B, E, H) for Phytofuse Renew, positive control, and negative control. At experiment completion (t=96 hours), cells were fixed in paraformaldehyde and stained with crystal violet (C, F, I). Discussion Phytofuse Renew was able to increase cell migration and close the scratch at a rate comparable to the positive control. The mechanisms of the cells in the in vitro scratch assay mimic the mechanisms seen in in-vivo wound healing therefore we can be assured that our results are translatable outside the laboratory. Phytofuse Renew was designed to be moisturizing and soothing, and provide enhanced aesthetics. With the present study we can also be confident that this product has healing abilities and cell proliferation properties. This information is presented in good faith but is not warranted as to accuracy of results. Also, freedom from patent infringement is not implied. This information is offered solely for your investigation, verification, and consideration. Version#2/

30 Anti-Pollution Assay Analysis Tradename: Phytofuse Renew Code: CAS #: Test Request Form #: 1689 Lot #: Sponsor: Active Concepts, LLC; 107 Technology Drive Lincolnton, NC Study Director: Erica Segura Principle Investigator: Maureen Danaher Test Performed: Pollution Protection Assay Introduction The role of pollution in the appearance of the premature wrinkles and age spots has because a new frontier in antiaging active ingredients. While we have known about the harmful effects of pollution on our health for years, new research indicates air pollution plays a detrimental role in extrinsic aging. Carbon and metal micro particles found in polluted air embed in the dermis causing oxidative stress, initiating the inflammatory cascade leading to the breakdown on the collagen, elastin, and other structural components in the skin. Additionally, polyaromatic hydrocarbons overstimulate the aryl hydrocarbon receptors on keratinocytes and melanocytes resulting in the hyperpigmentation and the appearance of age spots. Providing a physical barrier will prevent embedment of carbon particles, thus reducing the signs extrinsic aging. Our pollution protection assay was conducted to assess the ability of Phytofuse Renew to provide immediate protection from carbon air pollution. Page 1 of 4 Version#2/

31 Anti-Pollution Assay Analysis Materials A. Equipment: Dissecting microscope; Digital camera; Pipettes B. Reagents: Micronized activated charcoal; Cetaphil Moisturizing for All Skin Types C. Other: Disposable pipette tips; wash bottles Methods Volunteers, male and female, between the ages of 23 and 45 and who were known to be free of any skin pathologies participated in this study. All subjects were asked to apply 2 mg of each test material, experimental, control, and untreated on their volar forearms. Lotions were allowed to dry completely before the addition of 5 mg of micronized charcoal. The micronized charcoal used has a particle size of 2.5 microns (PM 2.5) or less that mimics the small particulates found in polluted air. Each treatment area was washed five times using deionized water. Images were taken pre- and post-wash using a dissecting microscope. The test material consisted of 2% Phytofuse Renew in a Cetaphil Moisturizing for All Skin Types. For added perspective, images of an untreated test site and a site treated with Cetaphil Moisturizing for All Skin Types were recorded. Color analysis was conducted on the images and results depicted in optical density values and pigmentation histograms. Images were inverted and standard coloration values recorded and assigned absorbance units. The lower the mean optical density value the better protection against carbon particle embedment or PM 2.5 inhibition. Page 2 of 4 Version#2/

32 Anti-Pollution Assay Analysis Results The data obtained from this study met criteria for a valid assay and the controls performed as anticipated. Phytofuse Renew at a concentration of 2% was able to provide protection from carbon pollution. Figure 1: Phytofuse Renew Histogram Images - Inhibition on PM 2.5 Figure 2: Phytofuse Renew Images Page 3 of 4 Version#2/

33 Anti-Pollution Assay Analysis Figure 3: Untreated Histogram Images - Inhibition on PM 2.5 Figure 4: Untreated Images Discussion As shown in figure 1, Phytofuse Renew (code 16586) was able to provide pollution protection as specified by micronized carbon residue. The small amount of carbon that remains compared to the untreated control indicates the ability of Phytofuse Renew to provide barrier protection against everyday air pollution and slow the extrinsic aging process. It can therefore be concluded that at normal use concentrations Phytofuse Renew can be used as a skin pollution protection active ingredient. Page 4 of 4 Version#2/