ACB Yogurt Dermal Respiratory Factor PF Efficacy Data

Transcription

1 ACB Yogurt Dermal Respiratory Factor PF Efficacy Data Code: 20224PF INCI Name: Lactobacillus Ferment Lysate Filtrate CAS #: EINECS #: N/A Name of Study Results Oxygen Uptake Assay According to the results, ACB Yogurt Dermal Respiratory Factor PF may increase oxygen uptake by 115%, which is even more effective than Live Yeast Derivative. Therefore, we feel it is safe to determine that ACB Yogurt Dermal Respiratory Factor PF may be a useful anti-aging cosmetic ingredient as it shows that it can substantially increase oxygen uptake. Collagen Production Assay A series of test were conducted in-vitro within an amino acid deficient medium to determine the supplemental effects of ACB Yogurt Dermal Respiratory Factor PF on fibroblast cellular metabolism and Collagen I synthesis. Three dose levels were compared to a control, and the results were determined by an ELISA assay. These results showed that all dose levels are effective at increasing Collagen I Synthesis, though it is most effective at 4% because ACB Yogurt Dermal Respiratory Factor PF. ATP Synthesis Assay ACB Yogurt Dermal Respiratory Factor PF showed a significant increase in ATP levels in comparison with a control. After 24 hours there was a 42% increase in ATP levels from ACB Yogurt Dermal Respiratory Factor PF. Therefore we can assume that ACB Yogurt Dermal Respiratory Factor PF is able to increase ATP synthesis and the metabolic functions of the cells. Version#2/ info@activeconceptsllc.com +1 (704) Fax: +1 (704)

2 ORAC Assay ACB Yogurt Dermal Respiratory Factor PF exhibited potent antioxidant activity comparable to 100μM Trolox. The antioxidant capacity of ACB Yogurt Dermal Respiratory Factor PF increased as the concentration increased, as a result we can assure that its ability to minimize oxidative stress is dose dependent. Fibroblast Migration Assay According to the results, after 72 hours ACB Yogurt Dermal Respiratory Factor PF showed a significant increase in cellular proliferation in comparison to the control and LYCD. ACB Yogurt Dermal Respiratory Factor PF showed the highest level of absorbance at 0.96 nm. Therefore, ACB Yogurt Dermal Respiratory Factor PF may sufficient in replacing the nutritional value that was depleted from the medium and favors cell propagation. Anti-Irritation Assay Based on the subject evaluations we can determine that ACB Yogurt Dermal Respiratory Factor PF reduces irritation 71% better than the placebo. The before and after pictures of test subjects also support these evaluations. Cellular Viability Assay As shown in figure 1, ACB Yogurt Dermal Respiratory Factor PF did not significantly alter cell metabolism at any concentration. The increase in fluorescent signal indicates an increase in cellular metabolism and viability post ACB Yogurt Dermal Respiratory Factor PF treatment. For these reasons, we can assume ACB Yogurt Dermal Respiratory Factor PF is suitable for cosmetic applications designed to increase cell viability and metabolism Version#2/ info@activeconceptsllc.com +1 (704) Fax: +1 (704)

3 Oxygen Uptake Assay Phone: Fax: ACB Yogurt Dermal Respiratory Factor PF Code: 20224PF Abstract Our technical staff conducted a series of tests in vitro to determine the increased level of oxygen uptake, which indicates increased cellular respiration, due to the application of ACB Yogurt Dermal Respiratory Factor PF. Materials and Methods We used an in-vitro method correlating with a UV absorbance at 256nm. We used cultured human dermal fibroblasts maintained in culture flasks. The culture media was replaced with PBS supplemented with magnesium, calcium, and succinate. The cells were incubated for 4 hours and then harvested and resuspended in PBS with magnesium and calcium. The 2.0 x 10 6 Titers of cells were transferred to an oxygen electrode chamber where the basal oxygen consumption was measured by means of an oxygen electrode (Hansatech DW3). Untreated cells were used to obtain a baseline oxygen consumption measurement. The cell was then cleared and recharged with a fresh titer of cells to which was added 0.25% (w/w) of ACB Yogurt Dermal Respiratory Factor PF or a Live Yeast Cell Derivative (LYCD). Results are expressed relative to the number of viable cells recovered from the electrode chamber. Results 1 Page 1 of 2 Code 20224PF Figure 1. Oxygen Update of ACB Yogurt Dermal Respiratory Factor PF. Version#1/

4 Oxygen Uptake Assay Phone: Fax: Discussion According to the results, ACB Yogurt Dermal Respiratory Factor PF may increase oxygen uptake by 115%, which is even more effective than a Live Yeast Derivative. Therefore, we feel it is safe to say that ACB Yogurt Dermal Respiratory Factor PF may be a useful anti-aging cosmetic ingredient as it shows that it may substantially increase oxygen uptake. 2 Page 2 of 2 Code 20224PF Version#1/

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9 Oxygen Radical Absorbance Capacity (ORAC) Assay Phone: Fax: Tradename: ACB Yogurt Dermal Respiratory Factor PF Code: 20224PF CAS #: Test Request Form #: 936 Lot #: Sponsor: Active Concepts, LLC; 107 Technology Drive Lincolnton, NC Study Director: Erica Segura Principle Investigator: Meghan Darley Test Performed: Oxygen Radical Absorbance Capacity (ORAC) Introduction Reactive oxygen species (ROS) are generated by normal cellular processes, environmental stresses, and UV irradiation. ROS are detrimental to cellular structures and functional molecules (i.e DNA, proteins, lipids) as they act as strong oxidizing agents or free radicals. The oxygen radical absorbance capacity (ORAC) assay is a standard method used to assess antioxidant capacity of physiological fluids, foods, beverages, and natural products. The assay quantitatively measures a sample s ability to quench free radicals that have the potential to react with and damage cellular components. Oxygen Radical Absorbance Capacity (ORAC) assay was conducted to assess the antioxidant capacity of ACB Yogurt Dermal Respiratory Factor 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. involved 1in the use of its chemical products since the conditions of use are beyond our control. Statements concerning the possible use of our products are not intended as recommendations to Page 1 of 3 Version#1/ /Form#56

10 Oxygen Radical Absorbance Capacity (ORAC) Assay Phone: Fax: Materials A. Equipment: Synergy H1 Microplate reader (BioTek Instuments, Winooski, VT); Gen5 software (BioTek Instuments, Winooski, VT); Pipettes B. Buffers: 75mM Potassium Phosphate (ph 7.4); Deionized H 2 O C. Reagents: 2,2 -Azobis(2-methylpropionamidine) dihydrochloride (AAPH) (153mM); 6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox ); Fluorescein Sodium Salt (4nM) D. Preparation: Pre-heat (37 C) Synergy H1 Microplate reader; Prepare Trolox standards, sample dilutions, fluorescein solution, and AAPH. E. Microtitre Plates: Corning 96 Well Black Side/Clear Bottom Microplates Methods Solutions of ACB Yogurt Dermal Respiratory Factor PF and Trolox (positive control) were prepared in 75mM potassium phosphate buffer. Materials were prepared at three different concentrations/dilutions. Trolox was used as a reference for antioxidant capacity and prepared at a concentrations ranging from 12.5µM to 200µM in 75mM potassium phosphate buffer. For the ORAC assay, 25µL of test material and Trolox were combined with 150µL of fluorescein in 75mM potassium phosphate buffer and incubated in the Synergy HT Microplate reader at 37 C for 30 minutes. At the end of the incubation period, 25µL of AAPH were pipetted into each well. Fluorescent measurements were then taken every 2 minutes for approximately 2 hours at an excitation wavelength of 485nm and an emissions wavelength of 520nm. The AUC and Net AUC values of the standards and samples were determined using Gen5 2.0 Data Reduction Software using the below equations: AAA = R2 R1 + R3 R1 + R4 R1 + + RR Wheee R ii ffffffffffff rrrrrrr R1 NNN AAA = AAA ssssss AAA bbbbb The standard curve was obtained by plotting the Net AUC of different Trolox concentrations against their concentration. ORAC values of samples were then calculated automatically using the Gen5 software to interpolate the sample s Net AUC values against the Trolox standard curve. ORAC measurements for the test material were expressed in micro moles Trolox equivalents (µmte), where 1 ORAC unit is equal to 1 µmte. involved 2in the use of its chemical products since the conditions of use are beyond our control. Statements concerning the possible use of our products are not intended as recommendations to Page 2 of 3 Version#1/ /Form#56

11 Oxygen Radical Absorbance Capacity (ORAC) Assay Phone: Fax: Results ACB Yogurt Dermal Respiratory Factor PF exhibited potent antioxidant activity at a 4% concentration. Antioxidant Capacity (µmte) ORAC Assay µM Trolox 12.5µM Trolox 4% AC Yogurt DRF PF 53 2% ACB Yogurt DRF PF Figure 1: Antioxidant capacities Discussion As shown in figure 1, ACB Yogurt Dermal Respiratory Factor PF exhibited potent antioxidant activity comparable to 100µM Trolox. The antioxidant capacity of ACB Yogurt Dermal Respiratory Factor PF increased as the concentration increased, as a result we can assure that its ability to minimize oxidative stress is dose dependent. ACB Yogurt Dermal Respiratory Factor PF was designed to aid in cellular respiration, collagen production, and ATP synthesis and function as a probiotic. 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. involved 3in the use of its chemical products since the conditions of use are beyond our control. Statements concerning the possible use of our products are not intended as recommendations to Page 3 of 3 Version#1/ /Form#56

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15 Phone: Fax: Figure 2. Irritated patch on subject 7 before treating with variable. Figure 3. Irritated patch on subject 7 fifteen minutes after treating with variable. 2 Page 2 of 3 Version1/ /Form#2

16 Phone: Fax: Figure 4. Irritated patch on subject 2 prior to treating with placebo. Figure 5. Irritated patch on subject 2 fifteen minutes after treating with placebo. Discussion: Based on the subject evaluations we can determine that ACB Yogurt Dermal Respiratory Factor PF reduces irritation 71% better than the placebo. The before and after pictures of test subjects also support the evaluations as one can clearly see that figure 2 (treated with variable) is less red and has a reduced area of irritation compared to figure 4 (treated with placebo). 3 Page 3 of 3 Version1/ /Form#2

17 Cellular Viability Assay Analysis Phone: Fax: Tradename: ACB Yogurt Dermal Respiratory Factor PF Code: 20224PF CAS #: Test Request Form #: 937 Lot #: Sponsor: Active Concepts, LLC; 107 Technology Drive Lincolnton, NC Study Director: Erica Segura Principle Investigator: Meghan Darley Test Performed: Cellular Viability Assay Introduction The cellular viability assay is useful for quantitatively measuring cell-mediated cytotoxicity, cell proliferation and mitochondrial metabolic activity. Increased metabolism in a cell indicates ample cellular respiration and adenosine triphosphate (ATP) production. ATP is the molecular energy of cells and is required in basic cell function and signal transduction. A decrease is ATP levels indicates cytotoxicity and decreased cell function while an increase in ATP levels indicates healthy cells. The cellular viability assay was conducted to assess the ability of ACB Yogurt Dermal Respiratory Factor 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. This information is presented in good faith but is not warranted as to accuracy of results. Also, freedom from patent infringement is not implied. This information is offered solely for your investigation, verification, and consideration. Page 1 of 3 Version#2/ /Form#64

18 Cellular Viability Assay Analysis Phone: Fax: Materials A. Kit: PrestoBlue Cell Viability Reagent (Invitrogen, A13261) B. Incubation Conditions: 37 C at 5% CO 2 and 95% relative humidity (RH) C. Equipment: 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 ACB Yogurt Dermal Respiratory Factor PF 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 and a fluorometric measurement was taken at 560nm for excitation and 590nm for emission. This information is presented in good faith but is not warranted as to accuracy of results. Also, freedom from patent infringement is not implied. This information is offered solely for your investigation, verification, and consideration. Page 2 of 3 Version#2/ /Form#64

19 Cellular Viability Assay Analysis Phone: Fax: Results The data obtained from this study met criteria for a valid assay and the controls performed as anticipated. ACB Yogurt Dermal Respiratory Factor PF did not have negative effects on cell metabolism. Cellular metabolism results are shown as mean fluorescence units (MFU) and expressed as percentage change, calculated by the below equation: PPPPPPP (%)Chaaaa = MMM CCCCCCC MMM SSSpll MMM CCCCCCC 100 Relative Fluorescence Units (RFU) Viability Assay 20224PF ACB Yogurt Dermal Respiratory Factor PF Complete Media 1% 20224PF ACB Yogurt Dermal Respiratory Factor PF 0.1% 20224PF ACB Yogurt Dermal Respiratory Factor PF 0.01% 20224PF ACB Yogurt Dermal Respiratory Factor PF RFU % Change 0.00% % -9.77% -6.61% 100% 80% 60% 40% 20% 0% -20% -40% -60% -80% -100% Percent (%) Change Figure 1: Cellular Metabolism of ACB Yogurt Dermal Respiratory Factor PF-treated fibroblasts expressed in terms of percent of control. Discussion In this study, ACB Yogurt Dermal Respiratory Factor PF (code 20224PF) was tested to evaluate its effects on the viability of normal human dermal fibroblasts (NDHF). At concentrations of 1%, 0.1%, and 0.01% ACB Yogurt Dermal Respiratory Factor PF (code 20224PF), nor the preservatives contained therein exhibited any inhibition of cell viability. It can therefore be concluded that at normal use concentrations ACB Yogurt Dermal Respiratory Factor PF (code 20224PF) is not cytotoxic. This information is presented in good faith but is not warranted as to accuracy of results. Also, freedom from patent infringement is not implied. This information is offered solely for your investigation, verification, and consideration. Page 3 of 3 Version#2/ /Form#64