ACB Yogurt Dermal Respiratory Factor PF Efficacy Data

Transcription

1 Tomorrow s Vision Today! ACB Yogurt Dermal Respiratory Factor PF Efficacy Data Code: 20224PF INCI Name: Lactobacillus Ferment Lysate Filtrate CAS #: EINECS #: N/A Name of Study Type of Study Results Oxygen Uptake Assay In-vitro 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 in-vitro 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 In-vitro 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. ORAC Assay In-vitro 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. Version#1/ /Form#82 i n f a c t i v e c o n c e p t s l l c. c o m + 1 ( ) F a x : + 1 ( )

2 Tomorrow s Vision Today! Fibroblast Migration Assay In-vitro 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 In-vivo 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 In-vitro 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#1/ /Form#82 i n f a c t i v e c o n c e p t s l l c. c o m + 1 ( ) F a x : + 1 ( )

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5 Tomorrow s Vision Today! ACB Yogurt Dermal Respiratory Factor PF Redness Reduction Study Code: 20224PF INCI Name: Lactobacillus Ferment Lysate Filtrate Suggested Use Levels: % Abstract An in-vivo tape-stripping study was conducted to evaluate the benefits of ACB Yogurt DRF PF as an antiirritant using visible redness as a marker. Materials and Methods The study was conducted with 5 M/F subjects between the ages of 24 and 45 years old. The subjects were asked to evaluate the reduction in irritation using ACB Yogurt DRF PF compared to the unloaded control lotion. Volunteers applied Lubriderm Lotion, which has been mixed with 5% ACB Yogurt DRF PF to an irritated patch of skin on their right or left volar forearm. As a control, the subjects will also apply an unloaded lotion to an equally irritated patch of skin on another secion of the same volar forearm. Using surgical tape, the skin on the volar forearm of each subject will be tape-stripped eight times or until visible redness is produced, followed by immediate application of the test product and the placebo. Volunteers will evaluate anti-irritation visually (not sensorial). Results Subject #1 The control lotion is identical to the variable lotion with the exception of the ACB Yogurt DRF PF ingredient. Irritation will be induced via tape-stripping using surgical tape. Subjectss will apply the test product and the placebo then evaluated anti-irritation visually. After the subject s arm has been exposed to each treatment for fifteen minutes, each will then be asked to evaluated the visual reduction in redness of both lotions and self-grade the effectiveness of each lotion on a scale of Before and after pictures will also be taken of all volunteers. Control ACB Yogurt DRF PF Table 1. Subject 1 Test for the ACB Yogurt DRF PF before and after in-vivo redness reduction study. Page 1 of 3 Version 1/ / Form 2 info@activeconceptsllc.com +1 (704) Fax: +1 (704)

6 Tomorrow s Vision Today! Subject #2 Subject #4 Control ACB Yogurt DRF PF Control ACB Yogurt DRF PF Figure 2. Subject 2 test for the ACB Yogurt DRF PF before and after in-vivo redness reduction study. Figure 4. Subject 4 test for the ACB Yogurt DRF PF before and after in-vivo redness reduction study. Subject #3 Subject #5 Control ACB Yogurt DRF PF Control ACB Yogurt DRF PF Figure 3. Subject 3 test for the ACB Yogurt DRF PF before and after in-vivo redness reduction study. Figure 5. Subject 5 test for the ACB Yogurt DRF PF before and after in-vivo redness reduction study. Page 2 of 3 Version 1/ / Form 2 info@activeconceptsllc.com +1 (704) Fax: +1 (704)

7 Tomorrow s Vision Today! ACB Yogurt DRF PF Unloaded Control Subject Subject Subject Subject Subject Figure 6. Table of over-all reduction scores and averages by subject self-grading process. 8 Visual Reduction in Irritation (1=Lowest/10=Highest) ACB Yogurt DRF Control Lotion Figure 7. The ACB Yogurt DRF PF was self-graded by subjects showing a 47% reduction in redness on a scale of Discussion The results indicate that ACB Yogurt DRF PF is an effective ingredient for alleviating redness caused by physical irritation of the epidermis. Page 3 of 3 Version 1/ Form 2 info@activeconceptsllc.com +1 (704) Fax: +1 (704)

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10 Oxygen Radical Absorbance Capacity (ORAC) Assay i n f a c t i v e c o n c e p t s l l c. c o m + 1 ( ) F a x : + 1 ( ) Tradename: 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 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. 1 Page 1 of 3 Version#1/ /Form#56

11 Oxygen Radical Absorbance Capacity (ORAC) Assay i n f a c t i v e c o n c e p t s l l c. c o m + 1 ( ) F a x : + 1 ( ) Materials A. Equipment: Synergy H1 Microplate reader (BioTek Instuments, Winooski, VT); Gen5 software (BioTek Instuments, Winooski, VT); Pipettes B. Buffers: 75mM Potassium Phosphate (ph 7.4); Deionized 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 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: AAUUCC = RR2 RR3 RR RR WWh RR ii ll aa ii RR1 RR1 RR1 RR1 NN AAUUCC = AAUUCC ss pp ee AAUUCC The standard curve was obtained by plotting the Net AUC of different Trolox concentrations against their concentration. ORAC values of samples were then calculated automatically using the Gen5 software to interpolate the sample s Net AUC values against the Trolox standard curve. ORAC measurements for the test material were expressed in micro moles Trolox equivalents (µmte), where 1 ORAC unit is equal to 1 µmte. 2 Page 2 of 3 Version#1/ /Form#56

12 Oxygen Radical Absorbance Capacity (ORAC) Assay i n f a c t i v e c o n c e p t s l l c. c o m + 1 ( ) F a x : + 1 ( ) 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 2% ACB Yogurt PF DRF PF 53 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. 3 Page 3 of 3 Version#1/ /Form#56

13 Phone: Fax: Results 1 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 speci fication. Page 1 of 2 Code 20224PF Version#1/

14 Phone: Fax: 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 speci fication. Page 2 of 2 Code 20224PF Version#1/

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17 Cellular Viability Assay Analysis i n f a c t i v e c o n c e p t s l l c. c o m + 1 ( ) F a x : + 1 ( ) Tradename: ACB Yogurt Dermal Respiratory Factor PF Code: 20224PF CAS #: Test Request Form #: 937 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. Version#1/ /Form#64

18 Cellular Viability Assay Analysis i n f a c t i v e c o n c e p t s l l c. c o m + 1 ( ) F a x : + 1 ( ) 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. This information is presented in good faith but is not warranted as to accuracy of results. Also, freedom from patent infringement is not implied. This information is offered solely for your investigation, verification, and consideration. Version#1/ /Form#64

19 Cellular Viability Assay Analysis i n f a c t i v e c o n c e p t s l l c. c o m + 1 ( ) F a x : + 1 ( ) 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 significantly alter cellular metabolism compared to the control. Cellular metabolism results are expressed as a percentage of the control. Viability (% of control) 20224PF ACB Yogurt DRF PF 150% 130% 110% 98.08% % 92.38% 90% 70% 50% 1% 0.10% Concentration of Sample 0.01% Figure 1: Cellular Metabolism of ACB Yogurt Dermal Respiratory Factor PF-treated fibroblasts expressed in terms of percent of control. Discussion 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. This information is presented in good faith but is not warranted as to accuracy of results. Also, freedom from patent infringement is not implied. This information is offered solely for your investigation, verification, and consideration. Version#1/ /Form#64

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21 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

22 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