AC Dermal Respiratory Factor Advanced PF Efficacy Data · The results indicate that AC Dermal...

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Code: 20219PF INCI Name: Water & Saccharomyces Lysate Extract CAS #: 7732-18-5 & 8013-01-2 EINECS #: 231-791-2 & 232-387-9

Name of Study Results

Hyaluronic Acid Synthesis

The results indicate that AC Dermal Respiratory Factor Advanced PF is capable of increasing the percent concentration of hyaluronic acid by approximately 42% in comparison to the control. Therefore, we can assume that AC Dermal Respiratory Factor Advanced PF may be useful for retaining epidermal moisture.

ORAC Assay

AC Dermal Respiratory Factor Advanced PF exhibited antioxidant activity comparable to Trolox®. The antioxidant capacity of AC Dermal Respiratory Factor Advanced PF increased as the concentration increased, allowing us to deduce that its ability to minimize oxidative stress is dose dependent.

ProCollagen Assay

The ELISA indicates that AC Dermal Respiratory Factor Advanced PF is capable of increasing the expression of procollagen type 1-C peptide in the fibroblast cell culture model. AC Dermal Respiratory Factor was shown to be comparable to Magnesium Ascorbyl Phosphate in increasing the synthesis of Collagen Type I. These finding suggest that AC Dermal Respiratory Factor Advanced PF would be useful in cosmetic preparations to stimulate collagen type I production in-situ.

Reduction of Sunburn Pain

Topical application of AC Dermal Respiratory Factor Advanced PF is capable of producing consumer perceivable reductions in erythema and discomfort resulting from overexposure to light. It was also noted among those panelists complying with the one-week

AC Dermal Respiratory Factor Advanced PF Efficacy Data

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application period that there was a marked reduction in skin peeling to areas where lotion containing AC Dermal Respiratory Factor Advanced PF was applied.

Cellular Viability Assay

AC Dermal Respiratory Factor Advanced PF exhibited positive results by increasing cell metabolism. The increase in fluorescent signal indicates an increase in cellular metabolism and viability post AC Dermal Respiratory Factor Advanced PF treatment. For these reasons, we can assume AC Dermal Respiratory Factor Advanced PF is suitable for cosmetic applications designed to increase cell viability and metabolism.

High Resolution Ultrasound Skin Imaging Assay

As evidenced in a 4 week efficacy study of AC Dermal Respiratory Factor Advanced PF on skin, skin density was improved by 9.50% after one week and by 17.39% after 4 weeks when compared to the untreated control. When compared to the base cream AC Dermal Respiratory Factor Advanced PF improved skin density for the first two weeks of the trial, working 9.54% better than the base lotion after two weeks. Although still working better than the base lotion at the end of 4 weeks, AC Dermal Respiratory Factor Advanced PF improved density only 2.24% better than the base lotion. Results indicate that AC Dermal Respiratory Factor Advanced PF is capable of improving skin density when compared to both the untreated control as well as the base lotion. AC Dermal Respiratory Factor Advanced PF has a strong positive effect on skin’s density when used at recommended use levels.

Moisturizing Assay

As evidenced in a 4 week efficacy study of AC Dermal Respiratory Factor Advanced PF on skin, moisture levels were improved by 44.60% after 24 hours and by 95.0% after 4 weeks when compared to the untreated control. When compared to the base cream AC DRF Advanced PF improved moisturization by 12.14% and after 4 weeks AC DRF Advanced PF improved moisturization by 33.74%. Results indicate that AC Dermal Respiratory Factor Advaced PF is capable of increasing moisturization when compared to both the untreated control as well as the base lotion. The present study confirms that AC Dermal Respiratory Factor Advanced PF is not only capable of providing functional benefits but it is also capable of providing moisturizing and skin hydrating benefits when added to cosmetic applications.

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Pigmentation Assay

As evidenced in a 4 week efficacy study of AC Dermal Respiratory Factor Advanced PF on skin, erythema values were decreased 4.73% more than the untreated test after one week. After 4 weeks, erythema values were decreased by 15.16% more than the untreated site. When compared to the base cream AC Dermal Respiratory Factor Advanced PF decreased pigmentation 1.73% better after week one and after 4 weeks AC Dermal Respiratory Factor decreased pigmentation 13.11% more effectively than the base lotion alone. Results indicate that AC Dermal Respiratory Factor Advanced PF is capable of decreasing erythema when compared to both the untreated control as well as the base lotion. AC Dermal Respiratory Factor Advanced PF has a lightening effect on skin’s pigmentation when used at recommended use levels.

Scratch Assay

AC Dermal Respiratory Factor Advanced PF 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. AC Dermal Respiratory Factor Advanced PF was designed to increase cellular respiration, metabolism, and collagen synthesis. With the present study we can also be confident that this product has healing abilities and cell proliferation properties.

Reduction in Transepidermal Water Loss Assay

As evidenced in a four week efficacy study of AC Dermal Respiratory Factor Advanced PF on the skin, it can be used to effectively reduce transepidermal water loss with better results over time. When compared to the base cream AC Dermal Respiratory Factor Advanced PF was shown to decrease transepidermal water loss by 29.24% and by 50.00% when compared to the untreated control after four weeks. Results indicate that AC Dermal Respiratory Factor Advanced PF is capable of reducing TEWL, which allows for moisture retention. AC Dermal Respiratory Factor Advanced PF was designed to provide moisture retention benefits, 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 a decrease in transepidermal water loss therefore promoting moisture retention benefits when added to cosmetic applications.

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IL-6 ELISA

AC Dermal Respiratory Factor Advanced PET did not exhibit anti-inflammatory effects on LPS-treated fibroblasts. As expected, the changes in IL-6 production using AC Dermal Respiratory Factor Advanced PET appear to be dose dependent. Therefore, AC Dermal Respiratory Factor Advanced PET is more suitable for cosmetic applications other than anti-inflammation.

Collagen Type I ELISA

AC Dermal Respiratory Factor Advanced PET exhibited potent collagen synthesis activity. As expected, the rate of increase for collagen synthesis using AC Dermal Respiratory Factor Advanced PET appears to be dose dependent. The increase in collagen production may lead to improvement in the dermal-epidermal junction integrity as well as an improved scaffolding matrix. For these reasons, we can assume AC Dermal Respiratory Factor Advanced PET is suitable for cosmetic applications designed to boost collagen synthesis to aid in providing a younger and healthier complexion.

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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.

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

Sample: AC Dermal Respiratory Factor Advanced PF Code: 20219PF CAS #: 7732-18-5 & 8013-01-2 Test Request Form/Submission #: 535 Sponsor: Active Concepts, LLC; 107 Technology Drive Lincolnton, NC 28092 Study Director: Erica Segura Principle Investigator: Meghan Darley Test Performed: In-vitro Hyaluronic Acid Synthesis

SUMMARY

Hyaluronic acid is capable of improving epidermal elasticity by maintaining the water content of the intercellular matrix of epidermal tissue. Although most believe that the presence of hyaluronic acid is limited to the body’s joints, it is actually found throughout the body with a surprisingly high concentration in subcutaneous tissue. It is suspected that the viscous hyaluronic acid acts as a natural guard against subcutaneous desiccation. An ELISA assay was used to measure the synthesis of hyaluronic acid by cultures of human keratinocytes. The assay is based on the specificity of hyaluronic acid to a hyaluronic acid-binding protein. I. Introduction A. Purpose In vitro Hyaluronic Acid Synthesis was performed using human keratinocytes. II. Methods & Materials Human keratinocyes were cultured in KSFM medium and incubated for a period of 24 hours at a constant temperature of 37°C with the concentration of carbon dioxide in the incubator being limited to 5%. The keratinocytes were then treated with AC Dermal Respiratory Factor Advanced PF at 0.5% and 1% in diluted culture medium. The cells were then incubated under the same conditions as before for a period of 72 hours. The cultured supernatant was stored at -80°C before assays were performed. An ELISA kit purchased from Biogenic (Cat. No. O29001) was used for the assay. Results are relative to biological control which is untreated keratinocytes.

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

III. Results The results indicate that AC Dermal Respiratory Factor Advanced PF is capable of increasing the percent concentration of hyaluronic acid by approximately 42% in comparison to the control. Therefore, AC Dermal Respiratory Factor Advanced PF may be useful for retaining epidermal moisture.

Graph 1: Hyaluronic Acid Production comparing AC Dermal Respiratory Factor Advanced PF and Control.

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1 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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Oxygen Radical Absorbance Capacity (ORAC) Assay

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

Tradename: AC Dermal Respiratory Factor Advanced PF Code: 20219PF CAS #: 7732-18-5 & 8013-01-2 Test Request Form #: 198 Lot #: 26020 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)

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 AC Dermal Respiratory Factor Advanced PF. 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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Materials

A. Equipment: Synergy H1 Microplate reader (BioTek Instruments, Winooski, VT); Gen5 software (BioTek Instruments, 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 AC Dermal Respiratory Factor Advanced 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 (µMTE), where 1 ORAC unit is equal to 1 µMTE.

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Results AC Dermal Respiratory Factor Advanced PF began exhibiting antioxidant activity at a 0.5% concentration.

Figure 1: Antioxidant capacities

Discussion As shown in figure 1, AC Dermal Respiratory Factor Advanced PF exhibited antioxidant activity comparable to 100µM Trolox®. The antioxidant capacity of AC Dermal Respiratory Factor Advanced PF increased as the concentration increased, as a result we can assure that its ability to minimize oxidative stress is dose dependent. AC Dermal Respiratory Factor Advanced PF was designed to aid in cellular respiration and metabolism, and function to increase collagen synthesis. 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 applications.

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AC Dermal Respiratory Factor Advanced PF

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

Sample: AC Dermal Respiratory Factor Advanced PF Code: 20219PF CAS #: 7732-18-5 & 8013-01-2 Test Request Form/Submission #: 536 Sponsor: Active Concepts, LLC; 107 Technology Drive Lincolnton, NC 28092 Study Director: Erica Segura Principle Investigator: Meghan Darley Test Performed: In-vitro Procollagen Assay

SUMMARY

Fibroblasts are the main source of the extracelluar matrix peptides including the structural proteins, collagen and elastin. Procollagen is a large peptide synthesized by fibroblasts in the dermal layer of the skin and is the precursor for collagen. As the peptide is processed to form a mature collagen protein, the propeptide portion is cleaved off (type I C-peptide). Both the mature collagen protein and the type I C-peptide fragment are then released into the extracellular environment. As collagen is synthesized, the type I C-peptide fragment accumulates into the tissue culture medium. Since there is a 1:1 stoichiometric ratio between the two parts of the procollagen peptide, assaying for type I C-peptide reflects the amount of collagen synthesized. Type 1 C-peptide was assayed via an ELISA based method. I. Introduction A. Purpose A fibroblast cell culture model was used to assess the ability of AC Dermal Respiratory Factor Advanced PF to exert an effect on collagen synthesis. II. Materials A series of type I C-peptide standards were prepared ranging from 0 ng/ml to 640 ng/ml. Next, an ELISA microplate was prepared by removing any unneeded strips from the plate frame followed by the addition of 100 µl of peroxidase-labeled anti procollagen type I-C peptide to each well used in the assay. Twenty (20) µl of either sample (collected tissue culture media) or standard were then added to appropriate wells and the microplate was covered and allowed to incubate for 3 ± 0.25 hours at 37°C. After the incubation, the wells were aspirated and washed three times with 400 µl of wash buffer. After the last wash was removed 100 µl of peroxidase substrate solution (hydrogen peroxide + tetramethylbenzidine as a chromagen) was added to each well and the plate was incubated for 15 ± 5 minutes at room temperature. After the incubation, 100 µl of stop solution (1 N sulfuric acid) was added to each well and the plate was read using a microplate reader at 450 nm.

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III. Method Procollagen Concentration To quantify the amount of procollagen present, a standard curve was generated using known concentrations of procollagen type 1-C peptide. A linear regression was then performed to establish the line that best fits these data points. Mean absorbance values for the test materials and untreated samples were then used to estimate the amount of procollagen type 1-C peptide present in each sample. IV. Results The data obtained from this study met criteria for a valid assay and the controls performed as anticipated. AC Dermal Respiratory Factor Advanced PF was able to procollagen production 54.48% at the highest concentration tested.

1%Magnesiu

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AdvancedPF

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ng/mL 2575 2421 2810 2727 2568 2194% Increase 50.33% 47.17% 54.48% 53.10% 50.19% 41.70%

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Figure 1: Procollagen Assay Results

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V. Conclusion The ELISA indicates that AC Dermal Respiratory Factor Advanced PF is capable of increasing the expression of procollagen type 1-C peptide in the fibroblast cell culture model. AC Dermal Respiratory Factor Advanced PF was shown to be comparable to Magnesium Ascorbyl Phosphate, Sodium Ascorbate, and Biodynes TRF Improved 25 in increasing the synthesis of procollagen. When used at 0.5% AC Dermal Respiratory Factor Advanced PF yielded 2727 ng/ml and at 1.0% yielded 2810 ng/ml Type 1 C-Peptide and was able to increase procollagen synthesis 54.48% and 53.10%, respectively, over the untreated control. These findings suggest that AC Dermal Respiratory Factor Advanced PF is useful in cosmetic preparations to stimulate collagen type 1 production in situ. .

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AC Dermal Respiratory Factor Advanced PF Reduction of Sunburn Pain and Erythema

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.


Abstract: A protocol was developed to evaluate the ability of AC Dermal Respiratory Factor Advanced PF to reduce the discomfort produced by over-exposure to UV radiation. Materials and Methods: Twenty panelists (m/f 27-45) were asked to evaluate two lotions containing either AC Dermal Respiratory Factor Advanced PF (2.8% w/w) or Benzocaine (0.5% w/w). The panelists were asked to apply the products immediately following overexposure to the sun and to record their initial perception as well as their perception after two hours. The product was evaluated for reduction in erythema and reduction in sunburn pain. The results were graded on a scale of 1 to 5 with 5 being complete reduction. Application continued for 1 week, with the product being applied twice per day and initial perception recordings being taken and perceptions after two hours. The average perceived reduction was determined at the end of the study to report the soothing benefits of AC Dermal Respiratory Factor Advanced PF. Results:

Graph 1. Comparison in the reduction of erythema and sunburn pain between Benzocain and AC Dermal Respiratory Factor Advanced PF

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AC Dermal Respiratory Factor Advanced PF Reduction of Sunburn Pain and Erythema



Table of Results:

Reduction of Erythema

Reduction of Sunburn Pain

Benzocaine AC DRF Advanced PF Benzocaine AC DRF Advanced PF Panelist t = 0 Hr t = 2 Hr t = 0 Hr t = 2 Hr t = 0 Hr t = 2 Hr t = 0 t = 2 Hr 1 0 0 0 2 1 1 3 4 2 0 1 0 1 1 1 4 5 3 0 0 0 0 2 2 3 4 4 0 0 0 3 0 1 2 3 5 1 1 0 2 1 1 4 4 6 0 0 1 1 1 1 5 5 7 0 0 0 2 2 2 3 3 8 0 0 1 1 0 1 3 3 9 0 0 0 2 2 2 4 4 10 0 0 0 1 1 1 3 3 11 0 0 0 3 1 1 2 3 12 1 0 0 0 2 2 5 5 13 0 0 0 1 1 2 4 6 14 0 0 0 2 2 2 4 4 15 0 1 1 1 1 1 5 5 16 0 0 0 1 1 1 4 4 17 0 0 0 1 1 1 4 4 18 0 1 0 2 0 1 3 3 19 0 0 0 3 1 1 2 2 20 0 0 0 0 2 1 5 5 Mean 0.1 0.2 0.15 1.45 1.15 1.3 3.6 3.95

Table 1. Results for panelist comparison in the reduction of erythema and sunburn pain between Benzocaine and AC Dermal Respiratory Factor Advanced. Discussion: Topical application of AC Dermal Respiratory Factor Advanced PF is capable of producing consumer perceivable reductions in erythema and discomfort resulting from overexposure to light. It was also noted among those panelists complying with the one-week application period that there was a marked reduction in skin peeling to areas where lotion containing AC Dermal Respiratory Factor Advanced PF was applied.

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Cellular Viability Assay Analysis


Tradename: AC Dermal Respiratory Factor Advanced PF Code: 20219PF CAS #: 7732-18-5 & 8013-01-2 Test Request Form #: 902 Lot #: 36082

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 AC Dermal Respiratory Factor Advanced PF 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.





Materials

A. Kit: PrestoBlue™ Cell Viability Reagent (Invitrogen, A13261) 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 96-well tissue culture treated plates G. Reagents: PrestoBlue™ reagent (10X) H. Other: 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 AC Dermal Respiratory Factor Advanced PF concentrations of 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 and a fluorometric measurement was taken at 560nm for excitation and 590nm for emission. Results The data obtained from this study met criteria for a valid assay and the controls performed as anticipated. AC Dermal Respiratory Factor Advanced PF did not exhibit significant effects on the cellular metabolism. Cellular metabolism results are shown as mean fluorescence units (MFU) and expressed as percentage change, calculated by the below equation:

𝑃𝑃𝑃𝑃𝑃𝑃𝑃 (%)𝐶ℎ𝑎𝑃𝑎𝑃 = 𝑀𝑀𝑀𝐶𝐶𝐶𝐶𝐶𝐶𝐶 −𝑀𝑀𝑀𝑆𝑆𝑆𝑆𝐶𝑆

𝑀𝑀𝑀𝐶𝐶𝐶𝐶𝐶𝐶𝐶 × 100





Figure 1: Cellular Metabolism of AC Dermal Respiratory Factor Advanced PF-treated fibroblasts expressed in terms of percent of control.

Discussion

In this study, AC Dermal Respiratory Factor Advanced PF (code 20219PF) was tested to evaluate its effects on the viability of normal human dermal fibroblasts (NDHF). At concentrations of both 0.1% and 0.01% AC Dermal Respiratory Factor Advanced PF (code 20219PF), nor the preservatives contained therein exhibited any inhibition of cell viability. It can therefore be concluded that at normal use concentrations AC Dermal Respiratory Factor Advanced PF (code 20219PF) is not cytotoxic.

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High Resolution Ultrasound Skin Imaging Assay [email protected] • Phone: +1-704-276-7100 • Fax: +1-704-276-7101

Tradename: AC Dermal Respiratory Factor PF Code: 20219PF CAS #: 7732-18-5 & 8013-01-2 Test Request Form #: 811

Lot #: NC140218-A Sponsor: Active Concepts, LLC; 107 Technology Drive Lincolnton, NC 28092 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 AC Dermal Respiratory Factor (DRF) Advanced PF. 10 M/F subjects between the ages of 23-45 participated in the study. Results indicate that this material is capable of significantly improving skin density compared to the control. Materials A. Equipment: DermaLab Skin Combo (Ultrasound Probe)

Methods 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. 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. Baseline elasticity readings were taken on day one of the study.

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Following initial measurements, all subjects were asked to apply 2 mg of each test material on their volar forearms. Measurements were taken immediately after application of test materials and then weekly for 4 weeks. The test material consisted of 2% AC Dermal Respiratory Factor Advanced PF 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 AC Dermal Respiratory Factor Advanced PF showed improvements in skin density at a 2.0% concentration. Please note, each value is an average of three consecutive readings per test site.

Averages T = 0 T = 1 Week

T = 2 Weeks

T = 3 Weeks

T = 4 Weeks

Experimental (2.0% AC DRF Advanced PF + Base Lotion)

68.40 76.38 84.22 82.86 81.00

Base Lotion 77.90 76.38 76.89 79.71 79.22 Untreated 71.10 69.75 73.89 68.29 69.00

Chart 1. Average Increase in Skin Density per Individual Test Site

Percent (%) Change T = 0 T = 1 Week

T = 2 Weeks

T = 3 Weeks

T = 4 Weeks

Base Lotion vs. Untreated 9.56% 9.50% 4.06% 16.74% 14.81%

Experimental (2.0% AC DRF Advanced PF + Base Lotion) vs. Untreated

-3.80% 9.50% 13.98% 21.34% 17.39%

Experimental (2.0% AC DRF Advanced PF + Base Lotion) vs. Base Lotion

-12.20% 0.00% 9.54% 3.94% 2.24%

Chart 2. Comparison of Skin Density Changes between Two Test Sites

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Figure 1: Ultrasound Results Comparing Test Sites to Untreated Site

Figure 2: Ultrasound Results Comparing the Difference between the Test Site and the

Control Site

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Collagen Ultrasound Comparison of Experimental + Control to Untreated Site

Base Lotion vs. Untreated

Experimental (2.0% AC DRFAdvanced PF + Base Lotion)vs. Untreated

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0.00%

5.00%

10.00%

15.00%

T = 0 T = 1 Week T = 2 Weeks T = 3 Weeks T = 4 Weeks

Perc

ent (

%) D

iffer

ence

Collagen Ultrasound Experimental vs. Base Lotion Treatment

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Discussion As evidenced in a 4 week efficacy study of AC Dermal Respiratory Factor Advanced PF on skin, skin density was improved by 9.50% after one week and by 17.39% after 4 weeks when compared to the untreated control. When compared to the base cream AC Dermal Respiratory Factor Advanced PF improved skin density for the first two weeks of the trial, working 9.54% better than the base lotion after two weeks. Although still working better than the base lotion at the end of 4 weeks, AC Dermal Respiratory Factor Advanced PF improved density only 2.24% better than the base lotion. Results indicate that AC Dermal Respiratory Factor Advanced PF is capable of improving skin density when compared to both the untreated control as well as the base lotion. AC Dermal Respiratory Factor Advanced PF has a strong positive effect on skin’s density when used at recommended use levels.

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Moisturization/Hydration Assay [email protected] • Phone: +1-704-276-7100 • Fax: +1-704-276-7101


Lot #: NC140218-A Sponsor: Active Concepts, LLC; 107 Technology Drive Lincolnton, NC 28092 Study Director: Erica Segura Principle Investigator: Meghan Darley Test Performed: Moisturization/Hydration Assay

Introduction

An in-vivo study was conducted over a period of three weeks to evaluate the moisturization benefits of AC Dermal Respiratory Factor (DRF) Advanced PF. 10 M/F subjects between the ages of 23-45 participated in the study. Results indicate that this material is capable of significantly increasing moisturization compared to the control. The moisturization assay was conducted to assess the moisturizing ability of AC Dermal Respiratory Factor (DRF) PF. Materials A. Equipment: DermaLab Skin Combo (Hydration/ Moisture Pin Probe)

Methods The moisture module provides information about the skin’s hydration by measuring the conducting properties of the upper skin layers when subjected to an alternating voltage. The method is referred to as a conductance measurement and the output is presented in the unit of uSiemens (uS). A moisture pin probe is the tool used to gather hydration values. 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. A Dermalab Corneometer was used to measure the moisture levels on the subject’s volar forearms. The Corneometer is an instrument that measures the amount of water within the skin. The presence of moisture in the skin improves conductance therefore results in higher readings than dry skin. Therefore the higher the levels of moisture, the higher the readings from the Corneometer will be. Baseline moisturization readings were taken on day one of the study.

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Following initial measurements, all subjects were asked to apply 2 mg of each test material on their volar forearms. Measurements were taken immediately after application of test materials and then weekly for 4 weeks. The test material consisted of 2% AC Dermal Respiratory Factor Advaned PF 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 AC Dermal Respiratory Factor Advanced PF showed very high moisturizing capabilities at a 2.0% concentration. Please note, each value is an average of three consecutive readings per test site.

Averages T = 0 T = 24 Hours

T = 1 Week

T = 2 Weeks

T = 3 Weeks

T = 4 Weeks

T = -24 Hours

T = -1 Week


100.10 157.90 164.00 187.56 223.71 229.44 141.44 105.88

Base Lotion 94.20 140.80 145.00 151.89 175.00 171.56 118.11 97.13 Untreated 92.00 109.20 107.50 110.22 124.43 117.67 103.33 91.25

Chart 1. Average Moisture Increase and Regression Scores of Individual Test Sites

Percent (%) Change T = 0 T = 24 Hours

T = 1 Week

T = 2 Weeks

T = 3 Weeks

T = 4 Weeks

T = -24 Hours

T = -1 Week

Base Lotion vs. Untreated 2.39 28.94 34.88 37.80 40.64 45.80 14.30 6.44


8.80 44.60 52.56 70.16 79.79 95.00 36.88 16.03


6.26 12.14 13.10 23.48 27.84 33.74 19.76 9.01

Chart 2. Comparative Moisture Increase and Regression Scores Between Individual Test Sites

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Figure 1. Average increase in moisturization per test site

Figure 2. Percent difference in moisturization between two test sites over four weeks

0.00

50.00

100.00

150.00

200.00

250.00

T = 0 T = 24Hours

T = 1Week

T = 2Weeks

T = 3Weeks

T = 4Weeks

T = -24Hours

T = -1Week

Moi

stur

izat

ion

(μSi

emen

s)

Moisturization Average Moisture Readings

Experimental (2.0% AC DRFAdvanced PF + Base Lotion)

Base Lotion

Untreated

0.00

10.00

20.00

30.00

40.00

50.00

60.00

70.00

80.00

90.00

100.00

T = 0 T = 24Hours

T = 1Week

T = 2Weeks

T = 3Weeks

T = 4Weeks

T = -24Hours

T = -1Week

Perc

ent (

%) D

iffer

ence

Bet

wee

n Te

st S

ites

Moisturization Percent (%) Difference


Experimental (2.0% AC DRFAdvanced PF + Base Lotion) vs.Untreated

Experimental (2.0% AC DRFAdvanced PF + Base Lotion) vs.Base Lotion

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Figure 3. Regression in skin moisturization after application of experimental material ceased

Discussion As evidenced in a 4 week efficacy study of AC Dermal Respiratory Factor Advanced PF on skin, moisture levels were improved by 44.60% after 24 hours and by 95.0% after 4 weeks when compared to the untreated control. When compared to the base cream AC DRF Advanced PF improved moisturization by 12.14% and after 4 weeks AC DRF Advanced PF improved moisturization by 33.74%. Results indicate that AC Dermal Respiratory Factor Advaced PF is capable of increasing moisturization when compared to both the untreated control as well as the base lotion. Furthermore, when examining the moisture levels on the skin after application of test materials stopped, it was determined that AC Dermal Respiratory Factor Advanced PF is capable of sustaining increased skin moisturization when compared to the skin site that remained untreated through the duration of the study. After 24 hours, the site testing 2.0% AC DRF Advanced PF + Base Lotion was approximately 36.88% more moisturized than the site which received no treatment. After one week, the experimental test site was still yielding moisturization results that were 16.03% higher than the untreated site. Additionally, in comparison to the site tested with the base lotion alone, the site treated with 2.0% AC DRF Advanced PF + Base Lotion moisturized the skin 19.76% better after 24 hours and was still 9.01% more effective in moisturizing the skin when reading were taken one week after the applications of both test materials ceased. The present study confirms that AC Dermal Respiratory Factor Advanced PF is not only capable of providing functional benefits but it is also capable of providing moisturizing and skin hydrating benefits when added to cosmetic applications.

0.0010.0020.0030.0040.0050.0060.0070.0080.0090.00

100.00

T = 0 T = 24Hours

T = 1Week

T = 2Weeks

T = 3Weeks

T = 4Weeks

T = -24Hours

T = -1Week

Perc

ent (

%) D

iffer

ence

Moisture Regression Experimental Treatment vs. Untreated

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


Lot #: NC140218-A Sponsor: Active Concepts, LLC; 107 Technology Drive Lincolnton, NC 28092 Study Director: Erica Segura Principle Investigator: Meghan Darley Test Performed: Pigmentation Assay Introduction

An in-vivo study was conducted over a period of four weeks to evaluate the effect on pigmentation of AC Dermal Respiratory Factor (DRF) Advanced PF. 10 M/F subjects between the ages of 23-45 participated in the study. Results indicate that this material is capable of reducing erythema. The pigmentation assay was conducted to assess the effects of AC Dermal Respiratory Factor Advanced PF. Materials

A. Equipment: DermaLab Skin Combo (Pigmentation Probe)

Methods The pigmentation measurement of the DermaLab Combo is performed using a handheld probe. This probe accommodates the color sensor, filters, optics, and light source. The light source is composed of two high intensity white LEDs, as well as a guiding light, which illuminates the target during positioning of the probe. Once the probe is in place, the LEDs flash at full power to illuminate the target area. Erythema levels are measured and recorded. 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 pigmentation probe was used to measure the erythema levels of the subject’s volar forearms. Baseline pigmentation readings were taken on day one of the study.

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Following initial measurements, all subjects were asked to apply 2 mg of each test material on their volar forearms. Measurements were taken immediately after application of test materials and then weekly for 4 weeks. The test material consisted of 2% AC Dermal Respiratory Factor Advanced PF 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 AC Dermal Respiratory Factor Advance PF showed reductions in erythema at a 2.0% concentration. Please note, each value is an average of three consecutive readings per test site.

Averages T = 0 T = 1 Week

T = 2 Weeks

T = 3 Weeks

T = 4 Weeks


12.65 12.09 11.59 11.29 11.19

Base Lotion 12.63 12.30 12.82 12.90 12.88 Untreated 11.96 11.96 12.61 12.46 13.19

Chart 1: Average Pigmentation Results per Individual Test Site

Percent (%) Change T = 0 T = 1 Week

T = 2 Weeks

T = 3 Weeks

T = 4 Weeks

Base Lotion vs. Untreated 5.60% 2.82% 1.67% 3.56% -2.36%


5.77% 1.04% -8.11% -9.40% -15.16%


0.16% -1.73% -9.62% -12.51% -13.11%

Chart 2: Difference between Two Test Sites

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Figure 1: Pigmentation Results for Individual Test Sites

Figure 2: Pigmentation Results Compared in Percent Difference between Test Sites

10.00

10.50

11.00

11.50

12.00

12.50

13.00

13.50


Erythema Average Results for Individual Test Sites

Experimental (2.0% AC DRFAdvanced PF + Base Lotion)

Base Lotion

Untreated

-20.00%

-15.00%

-10.00%

-5.00%

0.00%

5.00%

10.00%


Perc

ent (

%) D

iffer

ence

Erythema Comparative Data Between Two Sites


Experimental (2.0% AC DRFAdvanced PF + Base Lotion) vs.Untreated

Experimental (2.0% AC DRFAdvanced PF + Base Lotion) vs.Base Lotion

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Discussion As evidenced in a 4 week efficacy study of AC Dermal Respiratory Factor Advanced PF on skin, erythema values were decreased 4.73% more than the untreated test after one week. After 4 weeks, erythema values were decreased by 15.16% more than the untreated site. When compared to the base cream AC Dermal Respiratory Factor Advanced PF decreased pigmentation 1.73% better after week one and after 4 weeks AC Dermal Respiratory Factor decreased pigmentation 13.11% more effectively than the base lotion alone. Results indicate that AC Dermal Respiratory Factor Advanced PF is capable of decreasing erythema when compared to both the untreated control as well as the base lotion. AC Dermal Respiratory Factor Advanced PF has a lightening effect on skin’s pigmentation when used at recommended use levels.

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

Tradename: AC Dermal Respiratory Factor Advanced PF Code: 20219PF CAS #: 7732-18-5 & 8013-01-2 Test Request Form #: 901 Lot Number: 36082

Sponsor: Active Concepts, LLC; 107 Technology Drive Lincolnton, NC 28092 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 AC Dermal Respiratory Factor Advanced PF-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.

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Materials

A. Incubation Conditions: 37°C at 5% CO2 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 5% concentration of AC Dermal Respiratory Factor Advanced PF 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 AC Dermal Respiratory Factor Advanced PF, 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. AC Dermal Respiratory Factor Advanced PF at a 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:

𝑆𝑆𝑆𝑆𝑆𝑆ℎ 𝐴𝑆𝐴𝑆 𝑡=𝑥 − 𝑆𝑆𝑆𝑆𝑆𝑆ℎ 𝐴𝑆𝐴𝑆 𝑡=0𝑆𝑆𝑆𝑆𝑆𝑆ℎ 𝐴𝑆𝐴𝑆 𝑡=0

× 100 = % 𝑆𝑆𝑆𝑆𝑆𝑆ℎ 𝐶𝐶𝐶𝐶𝐶𝑆𝐴

𝐶ℎ𝑆𝑎𝑎𝐴 𝑖𝑎 𝐴𝑆𝐴𝑆 𝐶𝑜 𝑆𝑆𝑆𝑆𝑆𝑆ℎ (𝑎𝑛2)𝑀𝑖𝑎𝑆𝑆𝑆𝑖𝐶𝑎 𝑇𝑖𝑛𝐴 𝑡=𝑥

= 𝑀𝑖𝑎𝑆𝑆𝑆𝑖𝐶𝑎 𝑅𝑆𝑆𝐴

𝑊ℎ𝐴𝑆𝐴 𝑥 = 𝑆𝑖𝑛𝐴 (ℎ𝐶𝐶𝑆𝐶) 𝑝𝐶𝐶𝑆 𝐶𝑆𝑆𝑆𝑆𝑆ℎ

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Figure 1: Area of scratch

Figure 2: Percent scratch closure

0

0.5

1

1.5

2

2.5

3

3.5

0 24 48 72 96

Scra

tch

Area

(mm

2 )

Time Pose Scratch (Hours)

Area Enclosure

Positive Control Negative Control AC Dermal Respiratory Factor Advanced PF

0

20

40

60

80

100

120

24 48 72 96

Perc

ent (

%) C

losu

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Time Post Scratch (Hours)

Percent Scratch Closure


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Figure 3: Cell migration rate

Figure 4: Images at t=0 hours (A, D, G) and t=96 hours (B, E, H) for AC Dermal Respiratory Factor Advanced PF, positive control, and negative control. At experiment completion (t=96 hours), cells were fixed in paraformaldehyde and stained with crystal violet (C, F, I).

0

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40000

60000

80000

100000

120000

24 48 72 96

Mig

ratio

n Ra

te (n

m2 /

hou

r)

Time Post Scratch (Hours)

Migration Rate


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Discussion AC Dermal Respiratory Factor Advanced PF 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. AC Dermal Respiratory Factor Advanced PF was designed to increase cellular respiration, metabolism, and collagen synthesis. With the present study we can also be confident that this product has healing abilities and cell proliferation properties.

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