Arch Personal Care Products L.P.
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1 Arch Personal Care s L.P. NAB GrassRoots Extract SAP Code# Proposed INCI Designation: Collinsonia canadensis (stoneroot) & Lophophyrum elongatum (wheatgrass) & Oryzopisis miliciea (ricegrass) Extract Background Grasses in general, especially in their early stages of growth, are excellent sources of antioxidants, photoprotective and genoprotective materials. In the grass stages of growth, rapid metabolism and high exposure to UV is a consistent theme. Thus, such plants must have a high level of nutrients and a very high level of anti-oxidants and photoprotectants. The combination of rapid growth and UV exposure necessitates the growing plants to develop unique strategies to off set the extreme oxidative environment and protect its genetic material while undergoing rapid growth. One of the most familiar harvested young grasses used mostly in nutritional areas is wheatgrass. Wheatgrass is the green growing, early form of the wheat grain. Wheatgrass is a completely different entity from the mature wheat stalk filled with grain. Fully mature wheat is classified as a grain, while young wheatgrass is classified as a vegetable. The ingestion of wheatgrass juice has been claimed to be the new Fountain of Youth, just as green tea was proclaimed to be about a decade ago. 1 While some of the claimed benefits are fanciful, research does bear out a multitude of benefits derived from using the material. Recent information from nutritional research indicates that wheatgrass is a whole food that has been harvested at the absolute peak of its nutritional value. 2 Think of it as concentrated solar energy, or simply as an off-the-shelf source of the very best that green plants have to offer. As one of nature s richest foods, it provides a natural source of the vitamins, minerals, amino acids, fatty acids and enzymes (over 80 have been identified) 2 our bodies need to nourish cells and help maintain a healthy lifestyle. Because of its inherent composition, wheatgrass juice is considered a natural cleanser. Based upon this research, another material of interest is the rice plant. Like wheat, rice, in its early grass life form also has one of the highest rates of metabolism and is subject to high doses of UV. Rice plants cultivated under high UV, nutrient rich conditions, produce higher than normal levels of anti-oxidants. 06/02/05 v1.4 Key Attributes DNA Protection Anti-Oxidant Anti-Irritant
2 Arch Personal Care s L.P. Information Arch Personal Care s has discovered that both of these green grasses provide not only useful nutrients to the body, but also to the skin. From this knowledge, we are pleased to offer NAB GrassRoots Extract, an extract derived from wheatgrass (Lophophyrum elongatum) and ricegrass (Oryzopisis miliciea), in which all phases of growth and extraction have been controlled to maximize bioactivity. These grass extracts are further combined with stoneroot (Collinsonia canadensis) extract, an accepted skin soothing, anti-itch agent. Stoneroot is a member of the mint family and has long been used by the American Indians as a poultice for bruises, cuts and sores. Its leaves and roots were brewed to make medicinal teas and washes, or lotions, for cuts and wounds by generations of American Indians and pioneering settlers in the mountains of Virginia, the Carolinas, Kentucky, and Tennessee. The name stoneroot refers either to the plant s knobby, stone-hard root or to the mountaineers use of a tea brewed from the root as a diuretic in the treatment of kidney or bladder stones. Stoneroot contains volatile oils, tannins, and saponins. 3 NAB GrassRoots Extract incorporates the protective nutrients of wheatgrass and ricegrass along with the soothing repair properties of stoneroot in this botanical blend. Manufacturing Process The growth and extraction methods used to produce the grasses in NAB GrassRoots Extract focus on concentrating the nutrient content of each plant in their early growth stage. During the first three weeks of growth, with optimal UV exposure, increased levels of free radical scavengers are created in the sprouting plants due to the vulnerability of the young plants. It is at this stage that the wheatgrass and ricegrass are harvested. To achieve optimal growth conditions and ensure highly reproducible levels of key components, our source material, a strain of wheatgrass called Lophophyrum elongatum, is grown in nutrient rich soil in the southwestern United States. The ricegrass, Oryzopisis miliciea common white rice, is grown in southern India. The grasses are planted in the early spring and harvested every three weeks throughout the summer. Freshly harvested plants are extracted in water and purified. Next, the extract is mixed 50/50 with stoneroot which has been found to have a mineral and organic content that offers a soothing effect on the skin. The combination of wheatgrass, ricegrass and stoneroot extracts that makes up NAB GrassRoots Extract has been tested extensively for in vitro cellular effects and demonstrable effects in vivo in a controlled clinical test. Efficacy In Vitro Anti-Oxidant Assay Cytochrome C Oxidation An in vitro cytochrome C oxidation/reduction assay in the presence of isolated neutrophils was used to assess the anti-oxidant activity of the NAB GrassRoots Extract. This is an important assay as singlet oxygen causes extensive damage to skin cells through everyday metabolism as well as UV exposure. Reduction of cytochrome C via free radical activity causes an increase in its absorbance at 550 nm. Thus, cytochrome C was used as a marker to assess the
3 Arch Personal Care s L.P. inhibitory activity of NAB GrassRoots Extract against singlet oxygen generated via chemical means. Very low active concentrations were evaluated as this chemical assay is extremely sensitive. At a concentration of 10 ug/ml NAB GrassRoots Extract showed a 85% inhibition of cytochrome C oxidation. Inhibitory activity was also observed at higher and lower concentrations. Comparatively, NAB GrassRoots Extract was more effective than propyl gallate and Vitamin E and as effective as BHT in this assay (see Table 1). This study gives an indication of the inherent antioxidant efficacy of NAB GrassRoot Extract. The recommended use level of this extract in cosmetic products is between 2 and 5%. Table 1. Inhibition of Cytochrome C Oxidation Test Material 0.1 ug/ml 1.0 ug/ml 10 ug/ml 25 ug/ml NAB GrassRoots Extract 64% 78% 85% 94% Tocopherol acetate 17% 38% 33% 25% Propyl gallate 0% 11% 24% 33% BHT 71% 75% 91% 94% Protection of UV-Induced Nucleic Acid Damage While UV damage to cellular integrity and membrane peroxidation often results in immediate visible damage such as inflammation and sun burn, damage to subcellular components such as DNA often results in no obvious immediate effects, however, extended damage over time causes problems. Unchecked oxidation, whether through UV exposure or metabolic processes results in damage to DNA building blocks. This can ultimately alter the function of critical cellular proteins resulting in cell dysfunction, death or uncontrolled growth. While any anti-oxidant would seem to provide protection, this is not the case, since critical solubility characteristics are required to allow for penetration into the viable cell s nucleus. An in vitro cell culture assay was utilized to determine the effectiveness of NAB GrassRoots Extract at reducing the oxidation of the DNA base guanine or guanosine to an oxidized form when cells are exposed to varying UV doses. Fibroblasts actively growing in culture are exposed to an extreme dose of UVA radiation (about 30 minutes exposure to 15 J/cm2) with and without the inclusion of protective ingredients. Cells are washed free of additives, re-suspended in fresh serum, and incubated with a fluorescent FITC conjugated probe specific for modified guanosine (OxyDNA Kit, Bio81DNA, available from Biotrim, Inc.) The degree of DNA modification was measured via quantitative fluorescent microscopy using image analysis. The data is shown in Figure 1. Figure 1. Protection of UV-Induced Nucleic Acid Damage - In Vitro Relative Relative Amount Amount of Guanosine of Oxidized Dimers Guanosine (pixels) Placebo NAB GrassRoots 1% NAB GrassRoots 3% Positive Control % Protection No UV Exposure Low UV Exposure % Protection ** Fluorescence is comparatively quantified by pixel analysis within a defined microscopic field
4 Arch Personal Care s L.P. As the data in Figure 2 indicates, at both a 1% and 3% concentration, NAB GrassRoots Extract significantly inhibited the amount of DNA damage. Damage reduction was over 80% at a concentration of 3% NAB GrassRoots Extract. The positive controls were samples covered with a physical UVA filter reducing incident irradiation by about 98%. In Vivo Irritation Study The effect of 3% NAB GrassRoots Extract to reduce irritation of hands exposed to hot water (110 F) and 3% Sodium lauryl sulfate for 15 minute repeat immersion periods was evaluated. Test subjects applied the test material to one hand only and immersed both hands into hot, soapy water for 15-minute periods. Subjects removed their hands from the water; dried them and evaluations were made. Subjects repeated this procedure five times. Skin irritation was assessed by increases in skin blood flow with Laser Doppler. As the results in Table 2 indicate, while petrolatum had protective effects, due to its barrier function, as shown by almost completely eliminating increased blood flow, the NAB GrassRoots Extract at 3% had protective effects against skin irritation. With multiple immersions, blood flow increased to only 80.7 units compared to This represents a protective effect of about 50%. Table 2. Skin Irritation (Blood Flow Permid Units) Test Materials Single Repeated Immersion Immersions (5) Baseline (No Immersion) No Application % NAB GrassRoots Extract Petrolatum presented is the increase in skin blood flow units compared to the same site pretreatment values. Increased values were measured one minute after removal of hands from the hot, soapy test solution. The results presented are the averages of at least 10 observations. In Vivo UV-Induced Lipid Peroxidation The method of Smith et al. was used to assess the ability to prevent lipid and free fatty acid peroxidation in vivo. 4 Forearms of 5 test subjects were irradiated with approximately 0.5 MED for seven consecutive days with product treatment twice a day. At the start of the study and after completion, lipids were extracted from the skin surface with sequential extraction with 2% SLS and hexane. The extracts were pooled and assessed for the amount of lipid peroxides via HPLC. The amount of peroxide was expressed as a ratio with respect to extracted sterol esters to correct for efficiency of extraction at each time point. As the following data indicates, while application of sunscreen completely eliminated UV generated peroxidation, 3% NAB Grass Roots Extract was also very effective at reducing lipid damage in vivo by more than 40%.
5 Arch Personal Care s L.P. Figure 2. In-Vivo, UV Induced Lipid Peroxidation. The lower the ratio of peroxides:sterol esters X 10 6, the greater the protection Ratio of Peroxides:Sterol Esters X Pre-Treatment Post UV & Treatment Control SPF 15 3% NAB GrassRoots Clinical Study A 4 week study was conducted to evaluate a 3% NAB GrassRoots Extract aqueous gel formulation for its soothing effect. The study was done on 8 subjects, and was run in the fall in Florida; most subjects were experiencing skin irritation and redness at the start of the study. Moisturization, clinical assessment and photographs were taken at the start and completion of the study. The testing protocols included evaluation of the following parameters: Skin Hydration measured via Nova Meter. Prior to test probe reading, subjects equilibrated for 39 minutes in an isolated room to a relative humidity between 30%-40% and temperature of C. Firmness measured via Ballistometry to evaluate both superficial (epidermal) and integral (dermal) skin firmness. The ratio of the height of the first rebound peak to the second rebound peak was analyzed as the measurement point. Superficial Facial Lines (SFL) measured via the Packman method as a clinical assessment for the visible appearance of lines and wrinkles. The face was divided into 6 sectors for analysis. Assessments were done blinded by two investigators. Global Grading Clinical visual assessment of global appearance from 1 to 10 where 10 is the best appearance. Redness Grading Clinical visual assessment of skin redness from 0 to 10 where 0 is devoid of redness and 10 is very red.
6 Arch Personal Care s L.P. As the data in Table 3 outlines, after 4 weeks of treatment, improvement was seen in skin redness, irritation, hydration and lines and wrinkles. Table 3. Panel Study Efficacy of 3% NAB GrassRoots Extract in relation to skin redness, irritation, lines and wrinkles, and hydration. Parameter Baseline 1 month % change Skin Hydration (Nova) Firmness Superficial Facial Lines Global Grading Redness Grading Summary Arch Personal Care s has demonstrated through in vitro studies that NAB GrassRoots Extract can protect against UV irradiation at the skin surface, reducing lipid peroxide formation, and protect cellular nucleic acids and enzymes such as cytochrome C from UV damage. These results, combined with the in vivo testing, show that NAB GrassRoots Extract is a highly effective extract blend distributing protective agents throughout the cell, thus providing extensive protection to the skin. Typical Properties Appearance Amber liquid Non-volatile matter (1g/16h/@ 105 C) 3.0% minimum ph 6.0 to 8.0 Ash (800 C) 1.5% maximum Microbial Content 500 opg maximum Recommended Use Levels 2-5% Toxicological Summary Epi-Derm (tested 100% concentration) Epi-Ocular (tested 100% concentration) RIPT (tested 100% concentration) Non-Irritating Minimal Non-sensitizing References 1 Pelle, E., Ames, D., Paddle, G. and Smith, W.P. In Vitro model to test alpha tocopherol efficacy: protection against UV induced lipid peroxidation. Annual of the NY Academy of Sciences, 570, 491, (1989) 2 Wheat Grass Nature s Finest Medicine, by Steve Meyerowitz, Sproutman Publications, 1999, p Website: 4 Pelle, E., Maes, D. Padulo, G. and Smith, W.P. In Vitro model to test anti-oxidant potential. Arch. Biochem., Biophys., 283, (1990)
7 Arch Personal Care s L.P. Appendix Additional supporting studies completed on NAB GrassRoots Extract are as follows: In-Vitro Protection of Liposome Oxidation Assessment of lipid and free fatty acid damage via peroxidation was assessed as described by Smith et al 1,3 UV dosage was a low dose 1-2 mj/cm2 of irritation for 30 minutes and 5-8 mj/ cm2 for 1 hour for a high UV dose A Kratos solar simulator was used to deliver a combined UVA-UVB dose As the results in Figure 1 show, NAB GrassRoots Extract was very effective in reducing the peroxidation of lipids via both low and high UV doses 0 5% NAB GrassRoots Extract was more effective than BHT UV Induced Lipid Peroxidation of Liposomes 120 % Inhibition No Active.01% GrassRoots.032% GrassRoots.05% GrassRoots.1% GrassRoots.5% GrassRoots.1% BHT 0 Low UV High UV Figure 1 UV Induced Lipid Peroxidation of Liposomes In Vivo Cell Renewal Activity - Prevention of UV induced hyper-proliferation UV radiation causes free radical damage in the skin which results in a hyper-proliferative response This increase in cellular metabolism results in immediate cosmetic damage to the skin and longer-term consequences The use of sunscreens and photoprotectants can reduce this hyperproliferative response To assess such changes, the Dansyl Chloride technique was used to measure rates of skin cell turnover in a control, unexposed skin, and skin exposed to UV with and without photoprotection The test was conducted as follows: Two sites of the stratum corneum on the forearm of each of 10 subjects was irradiated with 1 MED of UV then stained with fluorescent dansyl chloride by applying semi-occlusive patches of 5% dansyl chloride milled in petrolatum for 24 hours After assuring that the stain was completely taken up by the stratum corneum layers by viewing under a quartz mineral lamp, subjects were instructed to apply water or the test product (untreated, SPF treated or 3% NAB GrassRoots Extract) to the appropriate site twice per day Visual inspection of the sites under the quartz lamp was made until the stain disappeared, noting the time required for complete turnover of the full thickness stratum corneum Tel Fax archpc@archchemicals.com
8 Arch Personal Care s L.P. It was found that cell renewal rates on UV exposed skin were dramatically increased, indicating a hyper-proliferative response This effect can be eliminated by the use of sunscreens As Table 1 shows, irradiation of the skin with 2 UV doses of approximately 2 MEDs resulted in a hyperproliferative response of 66% Pre-application of an SPF 15 sunscreen almost completely eliminated this effect as expected NAB Grass Roots Extract at 3% reduced hyper-proliferation to only 23% 7DEOH&KDQJHVLQ6NLQ&HOO5HQHZDO5DWHV 7HVW0DWHULDO 3UH6WXG\ $IWHU89 &KDQJH 7UHDWPHQW &RQWURO GD\V GD\V 63) GD\V GD\V 1$%Š *UDVV5RRWV([WUDFW GD\V GD\V 9DOXHVSUHVHQWHGDUHDYHUDJHVRIVXEMHFWV Tel Fax archpc@archchemicals.com
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