ACB Kale Protein Blend Efficacy Data

Transcription

1 Tomorrow s Vision Today! ACB Kale Protein Blend Efficacy Data Code: INCI Name: Hydrolyzed Kale Protein & Hydrolyzed Carrot Protein & Hydrolyzed Lemon Protein CAS #: & & EINECS #: & & Type of Study Results Hair Testing The results of the assessment indicate that when incorporated into a shampoo, ACB Kale Protein Blend is capable of improving hair characteristic 44% more than a control shampoo and 36% more than the control conditioner. When used in both the shampoo and conditioner at 2%, ACB Kale Protein Blend improved shine, volume, dry and wet combability, smoothness, hydration, softness and manageability by 37%, 59%, 54%, 50%, 32%, 35%, 55% and 38% respectively, better than the control shampoo. These results can be further supported by figures 1 through 10, where clearly the half of the subject's head treated with 2.0% ACB Kale Protein Blend appears more voluminous, shiny, soft and healthy. When comparing hair characteristics of the baseline assessments to the post style assessments, the benefits of including 2% ACB Kale Protein Blend in a shampoo and conditioner are even more apparent. In relation to the baseline readings, the test-half of the head improved all subjective parameters, improving shine, volume, dry and wet combability, thickness, smoothness, hydration, softness and manageability by 41%, 47%, 75%, 87%, 14%, 47%, 30%, 30%, and 63% respectively, with an the test product improving hair conditions on average by 48% when compared to the untreated baseline. For this reason, ACB Kale Protein Blend would be an ideal product to use in cosmetic applications designed for antiaging hair care that yields voluminous, soft and shiny hair aesthetics. Version#2/ / Form#82 info@activeconceptsllc.com +1 (704) Fax: +1 (704)

2 Tomorrow s Vision Today! Cellular Viability Assay As shown in figure 1, ACB Kale Protein Blend exhibited comparable results by increasing cell metabolism. An increase in fluorescent signal indicates an increase in cellular metabolism and viability. ACB Kale Protein Blend does not appear to have negative effects on cellular metabolism and can safely be used in cosmetic materials. Hydration Assay Both the innovative hydrolysis method used in the production of ACB Kale Protein Blend and the traditional method of hydrolysis induced when creating Wheat Hydrolysate yeild a protein hydrolysate capable of producing hair hydrating benefits at virtually identical levels. This demonstrates a novel approach in hydrolysis, such as the one used in the manufacturing of ACB Kale Protein Blend provides just as much moisture as the Wheat Hydrolysate with proven hydration benefits. ORAC Assay As shown in figure 1, ACB Kale Protein Blend exhibited similar strong antioxidant properties similar to 200μM concentration of Trolox, our highest standard used. The antioxidant capacity of ACB Kale Protein Blend increased as the concentration increased, as a result we can assure that its ability to minimize oxidative stress is dose dependant. ACB Kale Protein Blend was designed to provide hair and skin benefits such as conditioning and moisturization and, in addition, act as a film former. However 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 potent antioxidant benefits when added to cosmetic and personal skin and hair care applications. TEWL As shown in Figure 1, results indicate continuous improvements in the barrier of the skin throughout the 3 week test period. After one week, the solution containing 2.0% ACB Kale Protein Blend decreased TEWL 8% more effectively than the base lotion alone. After three weeks, the solution containing 2.0% ACB Kale Protein Blend demonstrated even more effective barrier protection, decreasing TEWL 21% better than the base lotion alone. When compared to the untreated control, the solution containing 2.0% ACB Kale Protein Blend decreased transepidermal water loss by 19% after one week and by 25% after three weeks. Typical Amino Acid Composition See attached Version#2/ / Form#82 info@activeconceptsllc.com +1 (704) Fax: +1 (704)

3 Tomorrow s Vision Today! Volumizing Assay Immediate results showed and average increase in hair diameter of 13.8% with an average increase of 13.4% four hours following the initial application. After placing individual hair strands under a microscope, Microscopy Imaging of the individual strands were taken to visually demonstrate the increase in hair diameter achieved when using ACB Kale Protein Blend at 2.0% in a solution compared to the use of Wheat Hydrolysate at 2.0% in a solution. ACB Kale Protein Blend is able to effectively volumize the hair for thicker and younger looking hair, a revolutionary step for anti-aging hair care products. Version#2/ / Form#82 info@activeconceptsllc.com +1 (704) Fax: +1 (704)

4 Active Concepts Salon Half-Head Study Tradename: ACB Kale Protein Blend Code: CAS #: Lot #: C & D Test Request Form #: 787 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 as a result of a variety of influences including environment and styling processes. This results in flat, dull hair that is difficult to manage. Improving the body of the hair has been shown to instantly make it appear healthier and more youthful. Increasing combability not only eases manageability, but also helps to minimize physical damage that perpetuates the loss of body and difficulty in styling. ACB Kale Protein Blend is a product designed to provide volume, hydration and conditioning properties to the hair. However, this unique ingredient also enhances shine, dry and wet combability, manageability and the smoothness of the hair. The purpose of this study was to confirm whether or not ACB Kale Protein Blend is capable of providing these additional benefits. A half head study was conducted to determine the comparison of using a shampoo incorporating ACB Kale Protein Blend vs. a control shampoo, in addition to the comparison of using a conditioner incorporating ACB Kale Protein Blend vs. a control conditioner. Each volunteer s hair was photographed. The images of the half head study were used in conjunction with a sensory assessment subjectively rating shine, volume, dry and wet combability, thickness, smoothness, hydration, softness and manageability. This assessment was conducted both before and after treatment. Based on the results obtained, ACB Kale Protein Blend is capable of enhancing the volume and overall health of the hair perfect for use in anti-aging hair care product lines. Materials and Methods The study was conducted using five participants with a variety of hair types. Each subject had their baseline photo taken prior to having their hair washed. The participant was also asked to complete a survey rating their hair prior to treatment on a scale of 1 to 10, with 1 being the lowest and 10 being the highest, using the following parameters 1 involved in 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 7 Version#1/ /Form#76

5 Active Concepts Salon Half-Head Study shine, volume, dry and wet combability, thickness, smoothness, hydration, softness and manageability. Half of the head was shampooed and conditioned with a base shampoo/ base conditioner + 2.0% ACB Kale Protein Blend. The hair on the other half of the head was treated with the base shampoo and conditioner alone. After the application and rinse of the test and positive control products, each participant s hair was blown dry using a round brush on both sides of the head. Once the hair was completely dry, the participant was asked to again 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. Results Baseline Assessment of Untreated Hair Assessment of Half Head shampooed with Untreated Control Assessment of Half Head Shampooed with Kale Protein Blend Assessment of Half Head Conditioned with Untreated Control Assessment of Half Head Conditioned with Kale Protein Blend Shine Volume Dry Combability Wet Combability Thickness Smoothness Hydration Softness Managability Mean Table 1. Average Results of Participant s Assessment Percent Difference - Comparison of Control Shampoo vs. 2.0% ACB Kale Protein Blend + Shampoo Percent Difference - Comparison of Control Conditioner and 2.0% ACB Kale Protein Blend + Conditioner Percent Difference Baseline Assessment vs. Half-Head Kale Protein Blend Shine 43% 30% 41% Volume 43% 75% 47% Dry Combability 67% 40% 75% Wet Combability 60% 40% 87% Thickness -14% 14% 14% Smoothness 44% 20% 47% Hydration 40% 30% 30% Softness 70% 40% 30% Managability 43% 33% 63% Mean 44% 36% 48% Table 2. Average Change Experienced for Participant s Sensory Assessment 2 involved in 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 7 Version#1/ /Form#76

6 Active Concepts Salon Half-Head Study Assessment of Hair Characteristics Rating from 1 (worst) to 10 (best) Baseline Assessment of Untreated Hair Assessment of Half Head shampooed with Untreated Control Assessment of Half Head Shampooed with ACB Kale Protein Blend Assessment of Half Head Conditioned with Untreated Control Assessment of Half Head Conditioned with ACB Kale Protein Blend Graph 1. Rating of hair characteristics following sensory assessment Half-Head Treated with Positive Control Shampoo and Conditioner Half-Head Treated with 2.0% ACB Kale Protein Blend + Base Shampoo & Conditioner Figure 1. Full head Baseline, Untreated Hair Figure 2. Half-Head Treated 3 involved in 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 3 of 7 Version#1/ /Form#76

7 Active Concepts Salon Half-Head Study Half-Head Treated with Positive Control Shampoo and Conditioner Half-Head Treated with 2.0% ACB Kale Protein Blend + Base Shampoo & Conditioner Figure 3. Full head Baseline, Untreated Hair Figure 4. Half-Head Treated Half-Head Treated with Positive Control Shampoo and Conditioner Half-Head Treated with 2.0% ACB Kale Protein Blend + Base Shampoo & Conditioner 4 Figure 5. Full head Baseline, Untreated Hair involved in 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 4 of 7 Figure 6. Half-Head Treated Version#1/ /Form#76

8 Active Concepts Salon Half-Head Study Half-Head Treated with Positive Control Shampoo and Conditioner Half-Head Treated with 2.0% ACB Kale Protein Blend + Base Shampoo & Conditioner Figure 7. Full head Baseline, Untreated Hair Figure 8. Half-Head Treated Half-Head Treated with Positive Control Shampoo and Conditioner Half-Head Treated with 2.0% ACB Kale Protein Blend + Base Shampoo & Conditioner Figure 9. Full head Baseline, Untreated Hair Figure 10. Half-Head Treated 5 involved in 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 5 of 7 Version#1/ /Form#76

9 Active Concepts Salon Half-Head Study Changes in Hair Characteristics from Baseline to Post Style Softness Managability Shine 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% Volume Dry Combability Hydration Wet Combability Smoothness Thickness Graph 2. Hair Assessment results for sensory characteristics Treated Half vs. Control Half After Application & Style Shine 60% Managability 50% 40% 30% Volume Softness 20% 10% 0% Dry Combability Hydration Wet Combability Smoothness Thickness 6 involved in 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 6 of 7 Graph 3. Hair Assessment results for sensory characteristics observed between the positive control and test products Version#1/ /Form#76

10 Active Concepts Salon Half-Head Study Discussion The results of the assessment indicate that when incorporated into a shampoo, ACB Kale Protein Blend is capable of improving hair characteristic 44% more than a control shampoo and 36% more than the control conditioner. When used in both the shampoo and conditioner at 2%, ACB Kale Protein Blend improved shine, volume, dry and wet combability, smoothness, hydration, softness and manageability by 37%, 59%, 54%, 50%, 32%, 35%, 55% and 38% respectively, better than the control shampoo. These results can be further supported by figures 1 through 10, where clearly the half of the subject's head treated with 2.0% ACB Kale Protein Blend appears more voluminous, shiny, soft and healthy. When comparing hair characteristics of the baseline assessments to the post style assessments, the benefits of including 2% ACB Kale Protein Blend in a shampoo and conditioner are even more apparent. In relation to the baseline readings, the test-half of the head improved all subjective parameters, improving shine, volume, dry and wet combability, thickness, smoothness, hydration, softness and manageability by 41%, 47%, 75%, 87%, 14%, 47%, 30%, 30%, and 63% respectively, with an the test product improving hair conditions on average by 48% when compared to the untreated baseline. For this reason, ACB Kale Protein Blend would be an ideal product to use in cosmetic applications designed for antiaging hair care that yields voluminous, soft and shiny hair aesthetics. 7 involved in 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 7 of 7 Version#1/ /Form#76

11 Cellular Viability Assay Analysis Tradename: ACB Kale Protein Blend Code: CAS #: & & Test Request Form #: 1268 Lot Number: NC 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 ACB Kale Protein Blend 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. Page 1 of 3 Version#1/ /Form#64

12 Cellular Viability Assay Analysis Materials A. Kit: Cellular Senescence Assay Kit (Chemicon International; KAA002) B. Incubation Conditions: 37 C at 5% CO 2 and 95% relative humidity (RH) C. Equipment: Forma humidified incubator; ESCO biosafety laminar flow hood; Light microscope; 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) F. Culture Plate: Falcon flat bottom 6-well tissue culture treated plates G. Reagents: 5-bromo-4-chloro-indolyl-β-D-galactopyranoside (X-gal) H. Other: Sterile disposable pipette tips; wash bottles; 15mL conical tubes, 1.5mL microcentrifuge tubes 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 ACB Kale Protein Blend 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. Page 2 of 3 Version#1/ /Form#64

13 Cellular Viability Assay Analysis Results The data obtained from this study met criteria for a valid assay and the controls performed as anticipated. ACB Kale Protein Blend exhibited significant effects on cellular metabolism compared to the control. Cellular metabolism results are expressed as a percentage of the control. Cellular Viability Viability (% of Control) % ACB Kale Protein Blend 0.10% ACB Kale Protein 0.01% ACB Kale Protein Blend Blend Figure 1: Cellular Metabolism of ACB Kale Protein Blend-treated fibroblasts expressed in terms of percent of control. Discussion As shown in figure 1, ACB Kale Protein Blend exhibited comparable results by increasing cell metabolism. An increase in fluorescent signal indicates an increase in cellular metabolism and viability. ACB Kale Protein Blend does not appear to have negative effects on cellular metabolism and can safely be used in cosmetic materials. 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. Page 3 of 3 Version#1/ /Form#64

14 Tomorrow s Vision Today! ACB Kale Protein Blend Hydrolyzed Protein Hydration Comparison Assay Code: INCI Name: Hydrolyzed Kale Protein & Hydrolyzed Carrot Protein & Hydrolyzed Lemon Protein Suggested Use Levels: % TRF#: 787 Abstract Results Hydrolyzed proteins, such as Oat, Soy and Wheat have been used in hair care as a traditional means to hydrate the hair and provide strengthening properties. Until recently, hyrolysis was induced using acid, water, or fermentation. Active Concepts has impelemented an innovative hydrolysis approach to the newest and most bio-available vegetable protein on the market, ACB Kale Protein Blend. This microorganism prompted hydrolysis creates the by product, lactic acid, as a secretion which provides comparable hydrating benefits to the leading hydrolyzed proteins on the market. Percent (%) Increase Increase in Hair Hydration Untreated Control 2.0% ACB Kale Protein Blend 2.0% Wheat Hydrolysate Materials and Methods The hair samples used in this study were tested using identical intervals and percentages of two protein hydrolysates, ACB Kale Protein Blend and Wheat Hydrolysate. The materials used in the procedure to determine the diameter of each strand were an untreated control hair sample, the control hair sample (2.0% Wheat Hydrolysate in an Aqueous Solution), and the sample treated with the test material (2.0% ACB Kale Protein Blend in an Aqueous Solution). Using a 9003 DPM Nova Impedance Meter, hydration levels of each strand of hair were measured. Both the 2.0% Wheat Hydrolysate Aqueous Solution and the 2.0% ACB Kale Protein Blend Aqueous Solution were shown to increase moisture levels by comparable amounts in the respective hair strands. Figure 1.Percent increase in hair hydration using ACB Kale Protein Blend and Wheat Hydrolysate Discussion Both the innovative hydrolysis method used in the production of ACB Kale Protein Blend and the traditional method of hydrolysis induced when creating Wheat Hydrolysate yeild a protein hydrolysate capable of producing hair hydrating benefits at virtually identical levels. This demonstrates a novel approach in hydrolysis, such as the one used in the manufacturing of ACB Kale Protein Blend provides just as much moisture as the Wheat Hydrolysate with proven hydration benefits. Page 1 of 1 Version 1/ / Form 2 info@activeconceptsllc.com +1 (704) Fax: +1 (704)

15 Oxygen Radical Absorbance Capacity (ORAC) Assay Tradename: ACB Kale Protein Blend Code: CAS #: & & Test Request Form #: 1267 Lot Number: NC Sponsor: Active Concepts, LLC; 107 Technology Drive Lincolnton, NC Study Director: Erica Segura Principle Investigator: Meghan Darley Test Performed: Oxygen Radical Absorbance Capacity (ORAC) Assay Introduction Reactive oxygen species (ROS) are generated by normal cellular processes, environmental stresses, and UV irradiation. ROS are dangerous 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 ACB Kale Protein Blend. 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 involved in 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 3 Version#1/ /Form#56

16 Oxygen Radical Absorbance Capacity (ORAC) Assay 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 ACB Kale Protein Blend and Trolox (positive control) were prepared in 75mM potassium phosphate buffer. Materials were prepared at three different concentrations/dilutions. 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: AAA = R2 R1 + R3 R1 + R4 R1 + + RR Wheee R ii ffffffffffff rrrrrrr R1 NNN AAA = AAA ssssss AAA bbbbb 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 per ml (µmte), where 1 ORAC unit is equal to 1 µmte. 2 involved in 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 3 Version#1/ /Form#56

17 Oxygen Radical Absorbance Capacity (ORAC) Assay Results ACB Kale Protein Blend exhibited very potent antioxidant activity at a 0.5% concentration. ORAC Assay Antioxidant Capacity (nmte) nM Trolox nM Trolox % ACB Kale Protein Blend % ACB Kale Protein Blend % ACB Kale Protein Blend Figure 1: Antioxidant capacities Discussion As shown in figure 1, ACB Kale Protein Blend exhibited similar strong antioxidant properties similar to 200µM concentration of Trolox, our highest standard used. The antioxidant capacity of ACB Kale Protein Blend increased as the concentration increased, as a result we can assure that its ability to minimize oxidative stress is dose dependant. ACB Kale Protein Blend was designed to provide hair and skin benefits such as conditioning and moisturization and, in addition, act as a film former. However 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 potent antioxidant benefits when added to cosmetic and personal skin and hair care applications. 3 involved in 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 3 of 3 Version#1/ /Form#56

18 Transepidermal Water Loss Study Tradename: ACB Kale Protein Blend Code: CAS #: & & Test Request Form #: 1269 Lot Number: NC 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 ACB Kale Protein Blend 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% ACB Kale Protein Blend 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#1/

19 Transepidermal Water Loss Study Results Percent (%) Decrease 30% 25% 20% 15% 10% 5% 0% TEWL Comparison Over Time 25% 22% 21% 19% 14% 8% 8% 3% t=24 1 week 2 weeks 3 weeks Experimental vs. Untreated Control Experimental vs. Base Lotion Figure 1: Improvements in barrier function following application of the test materials after a period of 3 weeks. Discussion As shown in Figure 1, results indicate continuous improvements in the barrier of the skin throughout the 3 week test period. After one week, the solution containing 2.0% ACB Kale Protein Blend decreased TEWL 8% more effectively than the base lotion alone. After three weeks, the solution containing 2.0% ACB Kale Protein Blend demonstrated even more effective barrier protection, decreasing TEWL 21% better than the base lotion alone. When compared to the untreated control, the solution containing 2.0% ACB Kale Protein Blend decreased transepidermal water loss by 19% after one week and by 25% after three weeks. 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#1/

20 Phone: Fax: 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. Version1/

21 Tomorrow s Vision Today! ACB Kale Protein Blend Hair Volumizing Assay Hydrolyzed Protein Comparison Code: INCI Name: Hydrolyzed Kale Protein & Hydrolyzed Carrot Protein & Hydrolyzed Lemon Protein Suggested Use Levels: % TRF#: 787 Abstract Results Hydrolyzed proteins, such as Oat, Soy and Wheat have been used in hair care as a traditional means to hydrate the hair and provide strengthening properties. Until recently, hydrolysis was induced using acid, water, or fermentation. Active Concepts has implemented an innovative hydrolysis approach to the newest and most bio-available vegetable protein on the market, ACB Kale Protein Blend. This microorganism prompted hydrolysis creates the by product, lactic acid, as a secretion which provides volumizing and anti-aging benefits measured. Percent (%) Increase Effects on Hair Diameter Immediately Following Application 4 Hours After Application Materials and Methods The hair samples used in this study were tested using identical intervals and percentages of two protein hydrolysates, ACB Kale Protein Blend and Wheat Hydrolysate. The materials used in the procedure to determine the diameter of each strand were an untreated control hair sample, the control hair sample (treated with 2.0% Wheat Hydrolysate Solution), and the sample treated with the test material (2.0% ACB Kale Protein Blend). Each hair was imaged and measured before a solution was applied. The hairs were then removed from the slide and either placed in the 2.0% solution of the Wheat Hydrolysate or the 2.0% solution of ACB Kale Protein Blend. Each hair was removed, measured and imaged then placed aside. After four hours, each hair was reimaged and measured to demonstrate the sustained volume potential of each respective hydrolyzed protein. ACB Kale Protein Blend Wheat Hydrolysate Figure 1.Percent increase in hair diameter over time after treatment with ACB Kale Protein Blend and Wheat Hydrolysate Microscopy Imaging of Individual Hair Strands Figure 2. Individual strand immediately following treatment with Wheat Hydrolysate (note beading) Figure 3. Individual strand immediately following treatment with ACB Kale Protein Blend Page 1 of 2 Version 1/ / Form 2 info@activeconceptsllc.com +1 (704) Fax: +1 (704)

22 Tomorrow s Vision Today! ACB Kale Protein Blend Hair Volumizing Assay Hydrolyzed Protein Comparison Code: INCI Name: Hydrolyzed Kale Protein & Hydrolyzed Carrot Protein & Hydrolyzed Lemon Protein Suggested Use Levels: % TRF#: 787 Results (Cont.) Figure 4. Individual strand four hours after treatment with Wheat Hydrolysate Figure 5. Individual strand four hours after treatment with ACB Kale Protein Blend Discussion Immediate results showed and average increase in hair diameter of 13.8% with an average increase of 13.4% four hours following the initial application. After placing individual hair strands under a microscope, Microscopy Imaging of the individual strands were taken to visually demonstrate the increase in hair diameter achieved when using ACB Kale Protein Blend at 2.0% in a solution compared to the use of Wheat Hydrolysate at 2.0% in a solution. ACB Kale Protein Blend is able to effectively volumize the hair for thicker and younger looking hair, a revolutionary step for anti-aging hair care products. Page 2 of 2 Version 1/ / Form 2 info@activeconceptsllc.com +1 (704) Fax: +1 (704)