J. Cosmet. Sci., 62, 49–56 (January/February 2011) 49 Inhibition of UV-induced ROS and collagen damage by Phyllanthus emblica extract in normal human dermal fi broblasts MUHAMMED MAJEED, BEENA BHAT, SUSMITHA ANAND, A. SIVAKUMAR, PRITEE PALIWAL, and K.G. GEETHA, Sami Labs Limited, Peenya Industrial Area, Bangalore 560058, India. Accepted for publication September 30, 2010. Synopsis As a part of ongoing research for novel natural cosmeceutical actives from plant extracts, this study demon- strates that Phyllanthus emblica fruit extract has shown its effi cacy in protection against ultraviolet-B (UVB) irradiation-induced reactive oxygen species (ROS) and collagen damage in normal human dermal fi broblasts. At a concentration of 0.5 mg/ml, emblica extract showed a signifi cant response of 9.5 ± 0.28-fold protection from UVB induced-collagen damage as compared to untreated cells. A known active, ascorbic acid, at a con- centration of 0.5 mg/ml, showed 3.7 ± 0.07-fold protection from UVB-induced collagen damage. While the untreated cells showed 84 ± 1.4% induction in ROS on UVB irradiation as compared to the non-irradiated cells, emblica extract treatment inhibited the induction of ROS to 15 ± 4% at a concentration of 0.5 mg/ml. Ascorbic acid inhibited the induction in ROS to 64 ± 2% at a concentration of 0.5 mg/ml. Emblica extract is a signifi cantly better natural active, with promising cosmeceutical benefi ts against photoaging. INTRODUCTION In the 21st century natural cosmetics are emerging as new skin care concepts with a perfect synergy between nature and technology. Phyllanthus emblica, commonly known in India as Amla (Sanskrit name Amalaki) is used in Ayurveda for its cosmetic applications due to its antioxidant properties. For several decades, the emblica fruit had been claimed to be a rich source of ascorbic acid, and its high antioxidant potential has been attributed to the presence of ascorbic acid (1). However, recent studies have confi rmed that only trace amounts of ascorbic acid are found in emblica extract and that the earlier reported anti- oxidant hydrolyzable tannins, emblicanins A and B, correspond to 1-O-galloy1-β-D-glucose (β-glucogallin) and mucic acid 1,4-lactone 5-O-gallate, respectively (2). The trace amount of free ascorbic acid in emblica extract suggests that the antioxidant effects exhibited by emblica fruits are due to gallic acid esters (2). Address all correspondence to Susmitha Anand: susmitha@samilabs.com.
JOURNAL OF COSMETIC SCIENCE 50 Ultraviolet-B (UVB) irradiation is a major environmental factor responsible for a high incidence of premature skin aging, referred to as photoaging, as well as skin cancer and melanoma. The majority of UV energy is absorbed by unidentifi ed photosensitizers in the cells that are postulated to generate reactive oxygen species (ROS). UV irradiation in- creases ROS production via PKCdelta signaling in primary murine fi broblasts (3). UVB irradiation has been demonstrated to produce ROS in the cells and skin, which induces the synthesis of matrix metalloproteinases (MMPs), causing skin photoaging (4). Photo- aging is a process involving alteration of type I collagen, the major component of dermis on exposure to UV radiation. Long-term exposure to UV radiation induces various cutaneous changes that differ because of physiological aging, including structural destruction of dermal collagen fi ber bundles, which comprise the major component of the dermis (5). Wrinkling, a representative change in skin surface associated with photoaging, is often seen at the corners of the eyes and in the space between the eyebrows (5). Type I collagen degra- dation is the major cause of wrinkle formation. Several factors promoting this degra dation process have been identifi ed, including UV radiation and ROS (6). Intracellular and extra- cellular oxidative stress initiated by ROS advance skin aging, which is characterized by wrinkles and atypical pigmentation. Because UV enhances ROS generation in cells, skin aging is usually discussed in relation to UV exposure. The use of antioxidants is an effec- tive approach to prevent symptoms related to photo-induced aging of the skin (7). Strat- egies to prevent or at least minimize ROS-induced photoaging and intrinsic aging of the skin necessarily include protection against UV irradiation and antioxidant homeostasis (8). The level of procollagen type I protein in photoaged skin is lower than that in naturally aged skin. The level of matrix metalloproteinase-1 protein and the activity of matrix metalloproteinase-2 were higher in the dermis of photoaged skin than in naturally aged skin (9). Emblica extract has type I pro-collagen promoting and anti-collagenase activity that is attributed to its anti-aging potential (10). Emblica fruit had been claimed to be a rich source of ascorbic acid, and its high antioxidant potential was earlier attributed to the presence of ascorbic acid. Also, it has become obvious that the high ascorbic acid content could contribute to the anti-aging and anti-photoaging properties of emblica extract. However, recent studies have shown only trace amounts of ascorbic acid in em- blica fruits (2). Therefore, we studied the comparative UVB protection effi cacy of ascorbic acid and emblica extract. To investigate the effect of emblica extract on UVB-induced collagen damage, we initially studied the cell viability response to emblica treatment in normal human dermal fi broblast cells. In continuation with earlier studies on emblica extract, the objective of the present study was to investigate the protective effect of em- blica extract in comparison to ascorbic acid against UVB-induced ROS and the collagen damage that results in photoaging. MATERIALS AND METHODS MATERIALS Normal human dermal fi broblasts (from adult donors) and its ready-to-use growth medium were obtained from PromoCell GmbH, Heidelberg, Germany. A neutral red stain (NR) was procured from Himedia Laboratories, Mumbai, India. A human collagen type I ELISA kit was purchased from Cosmo Bio (Carlsbad, USA). Procollagen type-I goat
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