ACB Kale Protein Blend Efficacy Data - Active...

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Tomorrow’s Vision… Today! ® [email protected] +1 (704)-276-7100 Fax: +1 (704)-276-7101 Version#2/12-9-15/ Form#82 Code: 20036 INCI Name: Hydrolyzed Kale Protein & Hydrolyzed Carrot Protein & Hydrolyzed Lemon Protein CAS #: 100209-45-8 & 100209-45-8 & 100209-45-8 EINECS #: 309-353-8 & 309-353-8 & 309-353-8 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 anti- aging hair care that yields voluminous, soft and shiny hair aesthetics. ACB Kale Protein Blend Efficacy Data

Transcript of ACB Kale Protein Blend Efficacy Data - Active...

Page 1: ACB Kale Protein Blend Efficacy Data - Active Conceptsactiveconceptsllc.com/wp...ACB-Kale-Protein-Blend-Efficacy-Data... · incorporated into a shampoo, ACB Kale Protein Blend . is

Tomorrow’s Vision… Today!®

i n f o @ a c t i v e c o n c e p t s l l c . c o m • + 1 ( 7 0 4 ) - 2 7 6 - 7 1 0 0 • F a x : + 1 ( 7 0 4 ) - 2 7 6 - 7 1 0 1

Version#2/12-9-15/ Form#82

Code: 20036 INCI Name: Hydrolyzed Kale Protein & Hydrolyzed Carrot Protein & Hydrolyzed Lemon Protein CAS #: 100209-45-8 & 100209-45-8 & 100209-45-8 EINECS #: 309-353-8 & 309-353-8 & 309-353-8

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 anti-aging hair care that yields voluminous, soft and shiny hair aesthetics.

ACB Kale Protein Blend Efficacy Data

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Tomorrow’s Vision… Today!®

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Version#2/12-9-15/ Form#82

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

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Tomorrow’s Vision… Today!®

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Version#2/12-9-15/ Form#82

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.

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Version#1/02-27-2015/Form#76

Active Concepts Salon Half-Head Study

Information contained in this technical literature is believed to be accurate and is offered in good faith for the benefit of the customer. The company, however, cannot assume any liability or risk 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 use our products in the infringement of any patent. We make no warranty of any kind, expressed or implied, other than that the material conforms to the applicable standard specification.

i n f o @ a c t i v e c o n c e p t s l l c . c o m • + 1 ( 7 0 4 ) - 2 7 6 - 7 1 0 0 • F a x : + 1 ( 7 0 4 ) - 2 7 6 -71

Tradename: ACB Kale Protein Blend Code: 20036 CAS #: Lot #: 150106C & 150106D Test Request Form #: 787 Sponsor: Active Concepts, LLC – 107 Technology Drive, Lincolnton, North Carolina 28092 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

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Page 2 of 7

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Version#1/02-27-2015/Form#76

Active Concepts Salon Half-Head Study

Information contained in this technical literature is believed to be accurate and is offered in good faith for the benefit of the customer. The company, however, cannot assume any liability or risk 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 use our products in the infringement of any patent. We make no warranty of any kind, expressed or implied, other than that the material conforms to the applicable standard specification.

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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 5.00 4.00 7.00 7.00 10.00 Volume 4.00 4.00 7.00 3.00 8.00 Dry Combability 2.00 2.00 6.00 6.00 10.00 Wet Combability 1.00 2.00 5.00 6.00 10.00 Thickness 6.00 8.00 7.00 6.00 7.00 Smoothness 5.00 5.00 9.00 8.00 10.00 Hydration 7.00 6.00 10.00 7.00 10.00 Softness 7.00 3.00 10.00 6.00 10.00 Managability 3.00 4.00 7.00 6.00 9.00 Mean 4.44 4.22 7.56 6.11 9.33

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 1. Average Results of Participant’s Assessment

Table 2. Average Change Experienced for Participant’s Sensory Assessment

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Page 3 of 7

3

Version#1/02-27-2015/Form#76

Active Concepts Salon Half-Head Study

Information contained in this technical literature is believed to be accurate and is offered in good faith for the benefit of the customer. The company, however, cannot assume any liability or risk 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 use our products in the infringement of any patent. We make no warranty of any kind, expressed or implied, other than that the material conforms to the applicable standard specification.

i n f o @ a c t i v e c o n c e p t s l l c . c o m • + 1 ( 7 0 4 ) - 2 7 6 - 7 1 0 0 • F a x : + 1 ( 7 0 4 ) - 2 7 6 -71

0

2

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6

8

10

12

Ratin

g fr

om 1

(wor

st) t

o 10

(bes

t)

Assessment of Hair Characteristics Baseline Assessment of UntreatedHair

Assessment of Half Head shampooedwith Untreated Control

Assessment of Half Head Shampooedwith ACB Kale Protein Blend

Assessment of Half HeadConditioned with Untreated Control

Assessment of Half HeadConditioned with ACB Kale ProteinBlend

Graph 1. Rating of hair characteristics following sensory assessment

Half-Head Treated with Positive Control Shampoo and Conditioner

Figure 1. Full head Baseline, Untreated Hair Figure 2. Half-Head Treated

Half-Head Treated with

2.0% ACB Kale Protein Blend

+ Base Shampoo & Conditioner

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Page 4 of 7

4

Version#1/02-27-2015/Form#76

Active Concepts Salon Half-Head Study

Information contained in this technical literature is believed to be accurate and is offered in good faith for the benefit of the customer. The company, however, cannot assume any liability or risk 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 use our products in the infringement of any patent. We make no warranty of any kind, expressed or implied, other than that the material conforms to the applicable standard specification.

i n f o @ a c t i v e c o n c e p t s l l c . c o m • + 1 ( 7 0 4 ) - 2 7 6 - 7 1 0 0 • F a x : + 1 ( 7 0 4 ) - 2 7 6 -71

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

Figure 5. Full head Baseline, Untreated Hair Figure 6. Half-Head Treated

Half-Head Treated with

2.0% ACB Kale Protein Blend

+ Base Shampoo & Conditioner

Half-Head Treated with Positive Control Shampoo and Conditioner

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Page 5 of 7

5

Version#1/02-27-2015/Form#76

Active Concepts Salon Half-Head Study

Information contained in this technical literature is believed to be accurate and is offered in good faith for the benefit of the customer. The company, however, cannot assume any liability or risk 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 use our products in the infringement of any patent. We make no warranty of any kind, expressed or implied, other than that the material conforms to the applicable standard specification.

i n f o @ a c t i v e c o n c e p t s l l c . c o m • + 1 ( 7 0 4 ) - 2 7 6 - 7 1 0 0 • F a x : + 1 ( 7 0 4 ) - 2 7 6 -71

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

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Page 6 of 7

6

Version#1/02-27-2015/Form#76

Active Concepts Salon Half-Head Study

Information contained in this technical literature is believed to be accurate and is offered in good faith for the benefit of the customer. The company, however, cannot assume any liability or risk 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 use our products in the infringement of any patent. We make no warranty of any kind, expressed or implied, other than that the material conforms to the applicable standard specification.

i n f o @ a c t i v e c o n c e p t s l l c . c o m • + 1 ( 7 0 4 ) - 2 7 6 - 7 1 0 0 • F a x : + 1 ( 7 0 4 ) - 2 7 6 -71

Graph 3. Hair Assessment results for sensory characteristics observed between the positive control and test products

0%10%20%30%40%50%60%70%80%90%

Shine

Volume

Dry Combability

Wet Combability

ThicknessSmoothness

Hydration

Softness

Managability

Changes in Hair Characteristics from Baseline to Post Style

0%

10%

20%

30%

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Volume

Dry Combability

Wet Combability

ThicknessSmoothness

Hydration

Softness

Managability

Treated Half vs. Control Half After Application & Style

Graph 2. Hair Assessment results for sensory characteristics

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Page 7 of 7

7

Version#1/02-27-2015/Form#76

Active Concepts Salon Half-Head Study

Information contained in this technical literature is believed to be accurate and is offered in good faith for the benefit of the customer. The company, however, cannot assume any liability or risk 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 use our products in the infringement of any patent. We make no warranty of any kind, expressed or implied, other than that the material conforms to the applicable standard specification.

i n f o @ a c t i v e c o n c e p t s l l c . c o m • + 1 ( 7 0 4 ) - 2 7 6 - 7 1 0 0 • F a x : + 1 ( 7 0 4 ) - 2 7 6 -71

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 anti-aging hair care that yields voluminous, soft and shiny hair aesthetics.

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Page 1 of 3 Version#1/05-22-15/Form#64

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.

Cellular Viability Assay Analysis

i n f o @ a c t i v e c o n c e p t s l l c . c o m • + 1 ( 7 0 4 ) - 2 7 6 - 7 1 0 0 • F a x : + 1 ( 7 0 4 ) - 2 7 6 -71

Tradename: ACB Kale Protein Blend Code: 20036 CAS #: 100209-45-8 & 100209-45-8 & 100209-45-8 Test Request Form #: 1268 Lot Number: NC-150401

Sponsor: Active Concepts, LLC; 107 Technology Drive Lincolnton, NC 28092 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.

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Page 2 of 3 Version#1/05-22-15/Form#64

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.

Cellular Viability Assay Analysis

i n f o @ a c t i v e c o n c e p t s l l c . c o m • + 1 ( 7 0 4 ) - 2 7 6 - 7 1 0 0 • F a x : + 1 ( 7 0 4 ) - 2 7 6 -71

Materials

A. Kit: Cellular Senescence Assay Kit (Chemicon® International; KAA002) B. Incubation Conditions: 37°C at 5% CO2 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.

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Page 3 of 3 Version#1/05-22-15/Form#64

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.

Cellular Viability Assay Analysis

i n f o @ a c t i v e c o n c e p t s l l c . c o m • + 1 ( 7 0 4 ) - 2 7 6 - 7 1 0 0 • F a x : + 1 ( 7 0 4 ) - 2 7 6 -71

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.

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.

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Tomorrow’s Vision… Today!®

[email protected] • +1 (704)-276-7100 • Fax: +1 (704)-276-7101

ACB Kale Protein Blend Hydrolyzed Protein Hydration Comparison Assay

Version 1/ 05.22.15/ Form 2Page 1 of 1

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.

Abstract

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.

Materials and Methods

Results

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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.

Code: 20036INCI Name: Hydrolyzed Kale Protein & Hydrolyzed Carrot Protein & Hydrolyzed Lemon Protein Suggested Use Levels: 1.0 - 10.0%TRF#: 787

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Page 1 of 3

1

Oxygen Radical Absorbance Capacity (ORAC) Assay

i n f o @ a c t i v e c o n c e p t s l l c . c o m • + 1 ( 7 0 4 ) - 2 7 6 - 7 1 0 0 • F a x : + 1 ( 7 0 4 ) - 2 7 6 -71

Information contained in this technical literature is believed to be accurate and is offered in good faith for the benefit of the customer. The company, however, cannot assume any liability or risk 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 use our products in the infringement of any patent. We make no warranty of any kind, expressed or implied, other than that the material conforms to the applicable standard specification.

Version#1/05-22-15/Form#56

Tradename: ACB Kale Protein Blend Code: 20036 CAS #: 100209-45-8 & 100209-45-8 & 100209-45-8 Test Request Form #: 1267 Lot Number: NC-150401

Sponsor: Active Concepts, LLC; 107 Technology Drive Lincolnton, NC 28092 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.

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Page 2 of 3

2

Oxygen Radical Absorbance Capacity (ORAC) Assay

i n f o @ a c t i v e c o n c e p t s l l c . c o m • + 1 ( 7 0 4 ) - 2 7 6 - 7 1 0 0 • F a x : + 1 ( 7 0 4 ) - 2 7 6 -71

Information contained in this technical literature is believed to be accurate and is offered in good faith for the benefit of the customer. The company, however, cannot assume any liability or risk 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 use our products in the infringement of any patent. We make no warranty of any kind, expressed or implied, other than that the material conforms to the applicable standard specification.

Version#1/05-22-15/Form#56

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 H2O 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 = 0.5 + 𝑅2𝑅1

+𝑅3𝑅1

+𝑅4𝑅1

+ ⋯+𝑅𝑅𝑅1

→ 𝑊ℎ𝑒𝑒𝑒 𝑅 𝑖𝑖 𝑓𝑓𝑓𝑓𝑒𝑒𝑖𝑓𝑒𝑅𝑓𝑒 𝑒𝑒𝑟𝑟𝑖𝑅𝑟

𝑁𝑒𝑁 𝐴𝐴𝐴 = 𝐴𝐴𝐴𝑠𝑠𝑠𝑠𝑠𝑠 − 𝐴𝐴𝐴𝑏𝑠𝑠𝑏𝑏 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.

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3

Oxygen Radical Absorbance Capacity (ORAC) Assay

i n f o @ a c t i v e c o n c e p t s l l c . c o m • + 1 ( 7 0 4 ) - 2 7 6 - 7 1 0 0 • F a x : + 1 ( 7 0 4 ) - 2 7 6 -71

Information contained in this technical literature is believed to be accurate and is offered in good faith for the benefit of the customer. The company, however, cannot assume any liability or risk 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 use our products in the infringement of any patent. We make no warranty of any kind, expressed or implied, other than that the material conforms to the applicable standard specification.

Version#1/05-22-15/Form#56

Results ACB Kale Protein Blend exhibited very potent antioxidant activity at a 0.5% concentration.

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.

199

13

206

100 83

0

50

100

150

200

250

200nM Trolox 12.5nMTrolox

0.5% ACBKale Protein

Blend

0.1% ACBKale Protein

Blend

0.05% ACBKale Protein

Blend

Ant

ioxi

dant

Cap

acit

y (n

MTE

)

ORAC Assay

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Transepidermal Water Loss Study

i n f o @ a c t i v e c o n c e p t s l l c . c o m • + 1 ( 7 0 4 ) - 2 7 6 - 7 1 0 0 • F a x : + 1 ( 7 0 4 ) - 2 7 6 -71

Version#1/05-22-15

Information contained in this technical literature is believed to be accurate and is offered in good faith for the benefit of the customer. The company, however, cannot assume any liability or risk 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 use our products in the infringement of any patent. We make no warranty of any kind, expressed or implied, other than that the material conforms to the applicable standard specification.

Tradename: ACB Kale Protein Blend Code: 20036 CAS #: 100209-45-8 & 100209-45-8 & 100209-45-8 Test Request Form #: 1269 Lot Number: NC-150401 Sponsor: Active Concepts, LLC; 107 Technology Drive Lincolnton, NC 28092 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.

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Transepidermal Water Loss Study

i n f o @ a c t i v e c o n c e p t s l l c . c o m • + 1 ( 7 0 4 ) - 2 7 6 - 7 1 0 0 • F a x : + 1 ( 7 0 4 ) - 2 7 6 -71

Version#1/05-22-15

Information contained in this technical literature is believed to be accurate and is offered in good faith for the benefit of the customer. The company, however, cannot assume any liability or risk 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 use our products in the infringement of any patent. We make no warranty of any kind, expressed or implied, other than that the material conforms to the applicable standard specification.

Results

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.

8%

19%

22% 25%

3%

8%

14%

21%

0%

5%

10%

15%

20%

25%

30%

t=24 1 week 2 weeks 3 weeks

Perc

ent (

%) D

ecre

ase

TEWL Comparison Over Time

Experimental vs. Untreated Control Experimental vs. Base Lotion

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Version1/02-25-15

[email protected] • Phone: +1-704-276-7100 • Fax: +1-704-276-7101

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.

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Tomorrow’s Vision… Today!®

[email protected] • +1 (704)-276-7100 • Fax: +1 (704)-276-7101

ACB Kale Protein Blend Hair Volumizing AssayHydrolyzed Protein Comparison

Version 1/ 05.22.15/ Form 2Page 1 of 2

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.

Abstract

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.

Materials and Methods

Results

13.8 13.4

0.492.2

0

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6

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Immediately Following Application 4 Hours After Application

Per

cen

t (%

) In

crea

se

Effects on Hair Diameter

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

Figure 2. Individual strand immediately following treatment with Wheat Hydrolysate (note beading)

Figure 3. Individual strand immediately following treatment with ACB Kale Protein Blend

Microscopy Imaging of Individual Hair Strands

Code: 20036INCI Name: Hydrolyzed Kale Protein & Hydrolyzed Carrot Protein & Hydrolyzed Lemon Protein Suggested Use Levels: 1.0 - 10.0%TRF#: 787

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Tomorrow’s Vision… Today!®

[email protected] • +1 (704)-276-7100 • Fax: +1 (704)-276-7101

ACB Kale Protein Blend Hair Volumizing AssayHydrolyzed Protein Comparison

Version 1/05.22.15/ Form 2Page 2 of 2

Results (Cont.)

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.

Discussion

Figure 4. Individual strand four hours after treatment with Wheat Hydrolysate

Figure 5. Individual strand four hours after treatment with ACB Kale Protein Blend

Code: 20036INCI Name: Hydrolyzed Kale Protein & Hydrolyzed Carrot Protein & Hydrolyzed Lemon Protein Suggested Use Levels: 1.0 - 10.0%TRF#: 787