Pyrus malus L. belongs to the family Rosaceae and its common name is apple tree.

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1 Apple Eco BOTANY Pyrus malus L. belongs to the family Rosaceae and its common name is apple tree. This three grows up to 10 m in height; tree top rounded, stem smooth, bark grayish-brown; the leaves are deciduous, alternate, acuminate, dark-green above and whitish below, with serrate margins and a stipule at the base; the flowers are hermaphrodite, pale pink, occasionally white, clustered in corymb inflorescences; the fruit, called apple, is a fleshy rounded pome that contains seeds. The flowering season is between December and May. Apple Eco extract is produced from the fruits of Pyrus malus, coming from organic crops. CHEMISTRY Table 1 shows the approximate chemical composition of apple (expressed as % of the fresh weight of the edible portion). % fresh weight of the edible portion Dry extract 16.0 Total sugar 11.1 Total acids a 0.6 Insoluble fraction 2.1 Pectin 0.6 Ash 0.3 Table 1. Approximate chemical composition of apple (expressed as % of the fresh weight of the edible portion) (Belitz, HD. & Grosch, W., 1997). a Expressed as malic acid. Table 1 shows that dry matter in apple is about 10-20%; main constituents are carbohydrates and organic acids; however, nitrogen compounds and lipids are scarce. Furthermore, certain secondary components are important because of their sensory value (pigments and aromatic compounds) and their nutritive value (vitamins and minerals) (Belitz, HD. & Grosch, W., 1997) V 01-01/

2 Carbohydrates Monosaccharides Besides glucose (1.8% edible portion) and fructose (5% edible portion), apples contain traces of other monosaccharides (Belitz, HD. & Grosch, W., 1997). Oligosaccharides Saccharose is the most abundant oligosaccharide in fruits. There are also minor amounts of other disaccharides (Belitz, HD. & Grosch, W., 1997). Alcohol sugars D-glucitol (sorbitol) is particularly abundant in all of the Rosaceae fruits (pomes, drupes); e.g. apple juice contains 300 to 800 mg/100 ml. Meso-inositol is widely distributed in all fruits (Belitz, HD. & Grosch, W., 1997). Polysaccharides All fruits contain cellulose, hemicellulose, pentosan and pectin. In apples it can also be found polysaccharides of fructose or fructans, like inuline or levan (Peschet, J.L. et al, 2001). Organic acids Malic acid is the most abundant organic acid in apples. Apples also contain low concentrations of a number of other acids, including the intermediates of the citric acid cycle, such as the cis-aconitic, succinic, pyruvic, citromalic, fumaric, glyceric, glycolic, glyoxylic, isocitric, lactic, oxalacetic, oxalic and 2-oxoglutaric acids. The most relevant phenol acids are the quinic, caffeic, chlorogenic and shikimic acids. Additionally, galacturonic and glucuronic acids can be found (Belitz, HD. & Grosch, W., 1997). Phenol compounds Phenol compounds are found in most of the fruits, where they often contribute to the fruit s flavor. Total phenolics content in apple (g/100 g fresh material) is between 0.1 and 1. Phenol compounds in fruits include: hydroxycinnamic acids, hydroxycoumarins, hydroxybenzoic acids, flavan-3-ols, flavan-3,4-diols, proanthocyanidins, anthocyanidins, flavanones, flavones and flavanols (Belitz, HD. & Grosch, W., 1997). Other minor compounds Apples also have nitrogenous compounds such as proteins or free amino acids (Asp being the main one), aromatic compounds such as 2-trans-hexenal (which gives apples their characteristic aroma), 3-cishexenal and its alcohols, and β-damascenone, in addition to vitamin C (2-10 mg/100g) and minerals such as potassium and phosphorus. TRADITIONAL USES Apple has always been a staple food for peoples living in cold climates. Apple trees are probably the earliest cultivated trees. Apples have the following properties: Laxative: contradictorily, apples can also be laxative because they regulate the intestinal flora and peristaltic movements. Eating apple first thing in the morning, helps resolving chronic constipation. Diuretic and depurative: because of its ability to absorb intestinal toxins, apples help in the treatment of skin chronic eczema, which is sometimes due to self-intoxication. Furthermore, they facilitate elimination of uric acid, urea and other metabolism residues, which is highly beneficial for people suffering from arthritis, rheumatism, gout and kidney stones. Because of their low sodium (salt) content, apple together with rice are recommended for people suffering from V 01-01/

3 high blood pressure; inclusion of both foods in the diet, effectively lowers blood pressure and reduces edema. Lipid-lowering: apple s pectin absorbs bile salts in the intestine, which reduces the amount of row material for the organism to produce cholesterol. Strengthens and balances the nervous system: probably due to its phosphorus content. Apple is recommended in cases of nervousness, stress and mental fatigue. Apple is also highly recommendable to delay aging processes as it has powerful antioxidant effects and stimulates cellular regeneration. COSMETIC PROPERTIES Trans-epidermal water loss (TEWL) regulatory activity This activity is due to the carbohydrates and α-hydroxyacids (AHA) in apple. Carbohydrates are active principles extensively used in cosmetics. These compounds are hygroscopic, namely they adsorb water thus contributing to keep a healthy moisture level in the horny layer. These active compounds build hydrogen bonds, thus preventing massive water loss and reducing dehydration. Additionally, some of these compounds make a protective coat on the skin, thus preventing and slowing down transepidermal water loss. Most AHAs are natural active compounds that promote normal keratinization and desquamation. Those with multiple hydroxyl groups are moisturizing antioxidants, and are especially gentle for sensitive skin (Yu, RJ. & Van Scott, EJ., 2002). Thus, Apple Eco is highly recommendable to formulate cosmetic products with moisturizing activity on skin and hair. Antioxidant activity Because of its high content in phenol compounds and vitamin C, apple has antioxidant properties, scavenges free radicals and protects the organism from cell deterioration. Phenol compounds The antioxidant activity of phenol compounds results from the combination of their iron-chelating activity and their scavenging activity on aging-causing free radicals (Pérez Trueba, G., 2003). In a subsequent study (Chaudhary, P. et al, 2006), the photo-protective capacity of total polyphenols found in the epicarp and mesocarp of apples was assessed. The previous administration of these polyphenols in animal cells dose-dependently decreased DNA damage and cell death induced by solar radiation. Polyphenols inhibited the Fenton reaction mediated by 2-desoxyribose (2-DR) and by lipids, they captured DPPH radicals and superoxide anions, and they reduced Fe 3+ to Fe 2+. It was also noted that apple polyphenols inhibited DNA single and double strand breakage. All these results show that these compounds have a powerful protective action against cell damage caused by sun exposure. Their anti-free radical and antioxidant properties were also demonstrated. Vitamin C is a powerful antioxidant very useful in the treatment of photo-aging, similar to vitamin E. Several animal studies have revealed a reduction of UVB-induced erythema and its burns by pretreatment with vitamin C at 10% or with psoralen. Other models carried out on human skin showed similar effects, either using only ascorbate or combining it with vitamin E (Chiu, A. & Kimball, AB., 2003). V 01-01/

4 Thus, Apple Eco is useful to formulate cosmetic products for the protection of skin and hair against oxidative processes. Cell regeneration stimulating activity This activity is due to the α-hydroxyacids (AHA) content of apple. AHA act at the stratum corneum level. They act on the cohesion between corneocytes in the deepest layers, so that they make the stratum corneum thinner and improve skin flexibility. Their action mechanism is not fully understood. Different theories have been proposed to explain their action. The earliest theory proposes that AHAs impairing effects on corneocyte cohesion is mainly due to their action on intercellular ionic bonds (Berardesca, E. & Maibach, H., 1995). Corneocytes are bound together through covalent (disulfide bonds) and noncovalent bonds; the later ones include ionic bonds, established between negatively charged groups (such as phosphate, sulphate, carboxyl) and positively charged groups (amine groups). AHAs effects would thus be based on their ability to inhibit certain enzymes involved in the formation of phosphate and sulphate groups in the cellular walls. AHAs might compete with sulphate and phosphate for the sulphotransferase and phosphotransferase enzymes, which are involved in the formation of mucopolysaccharides, phospholipids, sterols and glycoproteins. Some AHAs inhibit certain phosphotransferases and specific kinases. For example, citric acid inhibits the glucose-6-phosphotransferase and the phosphofructokinase enzymes. Additionally, AHAs may also act as substrates that accept phosphate groups, yielding phosphorilated alphahydroxyacids in certain metabolic pathways. More recently, Wang, X. (1999) proposed another model to explain their action mechanism. AHAs have been observed to act on calcium, which is essential to build most of the intercellular joints and to maintain their structural integrity. It has been found that AHAs reduce the calcium concentration in the epidermis and that calcium gets eliminated from the intercellular joints by chelation. The resulting drop in calcium concentration brings about cellular decohesion and desquamation. This effect is reinforced by the action of epidermal proteolytic enzymes. Thus, Apple Eco is recommendable to formulate cosmetic products with exfoliating and anti-aging activity. BIBLIOGRAPHIC EFFICACY STUDY Among other components, apples have a composition rich in polysaccharides, oligosaccharides and monosaccharides. Fructose and its derivatives are present in significant amounts, and they have moisturizing and emollient properties, as evidenced in the following study. 1. Experimental method In 2005, Kim, K.H., et al, conducted a study to evaluate the moisturizing action and emollient properties of levan, a polysaccharide composed of fructose (with β-2,6 bonds) which can be produced by plants and also by certain microorganisms, such as Zymomonas mobilis. The species of microorganisms that generate fructan usually grow in cold and dry areas, and therefore this compound is known to act as an osmoprotectant avoiding excessive water loss. V 01-01/

5 In order to study the moisturizing action of levan, two parameters were measured: transepidermal water loss (TEWL) and the level of skin moisturization. Transepidermal water loss was assessed in a group of volunteers aged between 22 and 37. The measurements were conducted in an acclimatized room at 20-25ºC, with relative humidity of 50±5%. The volunteers spent 20 minutes there to acclimatize before the measurements to be used as pre-treatment baseline values were taken using a Vapometer. Subsequently, a 20 µl sample (solution with ethanol and levan at 5%) was applied in a 4 cm 2 area of the forearm (the same one that had been previously quantified). The measurements were taken at regular intervals and were compared with values obtained for samples containing hyaluronic acid (1%) instead of levan, and also with distilled water alone. The water content of skin was extrapolated according to its electrical conductivity, since the greater the amount of this molecule, the greater the ability of skin to conduct electricity. This property was assessed with a Corneometer (CM825) in an area 40 mm in diameter of the forearms of 10 volunteers aged between 22 and 37. Panelists were kept in an acclimatized room at 25ºC with a relative humidity of 45-55%. Afterwards, 10 µl of 5 different formulas (0.2% of levan) were applied on the volunteers. The values from these formulas were then compared with the results obtained with other formulas containing hyaluronic acid (0.2%) and with other formulas simply containing distilled water. 2. Results The results show that levan clearly decreases transepidermal water loss from the skin after its application, as opposed to the result obtained with distilled water. Its levels are similar to the values obtained with hyaluronic acid. Corneometer measurements also show that the water content that skin keeps with the application of levan is higher than the level that is maintained with only distilled water, and similar to the level achieved by hyaluronic acid. 3. Conclusions According to the results obtained, levan and by extension fructo-polysaccharides are excellent moisturizing agents to keep skin hydrated and reduce transepidermal water loss. Therefore, due to its content in polysaccharides and sugars in general, Apple Eco is highly recommended for cosmetic products with moisturizing, emollient and conditioning activity, as it helps keep skin hydrated and in good condition. COSMETIC APPLICATIONS Action Active Cosmetic Applications TEWL regulation Antioxidant Cell regeneration stimulating activity Carbohydrates AHAs Phenol compounds AHAs -Moisturizing -Emollient -Conditioner -Anti-aging -Photo-protection -Hair color protection -Exfoliating -Anti-aging V 01-01/

6 RECOMMENDED DOSE The recommended dose is between 0.5% and 5.0%. BIBLIOGRAPHY Belitz, HD. & Grosch, W. Química de los Alimentos. Zaragoza: Ed. Acribia S.A., 1997; p: (613 BEL). Berardesca, E. & Maibach, H. AHA mechanisms of action. Cosmetics &Toiletries, 1995; 110 (6): (ref. 1327). Chaudhary, P., Shukla, S.K., Kumar, I.P., Namita, I., Afrin, F., Sharma, R.K. Radioprotective properties of apple polyphenols: An in vitro study. Molecular and Cellular Biochemistry, 2006; 288 (1-2): Chiu, A. & Kimball, AB. Topical vitamins, minerals and botanical ingredients as modulators of environmental and chronological skin damage. Br J Dermatol., 2003; 149: Kim, K.H., Chung, C.B., Kim, Y.H., Kim, K.S., Han, C.S., Kim, C.H., R&D Center Bioland Ltd. (Korea) and Metabolic Engineering Research Laboratory KRIBB (Korea). Cosmeceutical properties of levan produced by Zymomonas mobilis. J. Cosmet. Sci., Nov-Des 2005; 56: Pérez-Trueba, G. Los flavonoides: antioxidantes o prooxidantes. Rev. Cubana Invest. Biomed., 2003; 22 (1): Peschet, J.L., Puigserver, A. and Biagini, A. Evaluation of fructans in various fresh and stewed fruits by high-performance anion-exchange chromatography with pulsed amperometric detection. Journal of Chromatography A., Jun 2001; vol 920 (1-2): Wang, X. A theory for the mechanism of action of the α-hydroxy acids applied to the skin. Medica Hypotheses, 1999; 53 (5): (ref. 4261). Yu, RJ. & Van Scott, EJ. Hydroxycarboxylic acids, N-acetylamino sugars, and N-acetylamino acids. Skinmed., 2002; 1 (2): Webs: Panel.Pubmed_RVDocSum&ordinalpos= V 01-01/