J. Cosmet. Sci., 60, 395–403 (July/August 2009) 395 Type I pro-collagen promoting and anti-collagenase activities of Phyllanthus emblica extract in mouse fi broblasts PITHI CHANVORACHOTE, VARISA PONGRAKHANANON, SUDJIT LUANPITPONG, BOONTARIKA CHANVORACHOTE, SUMALEE WANNACHAIYASIT, and UBONTHIP NIMMANNIT, Department of Physiology (P.C.) and Pharmaceutical Technology (International) Program (V.P., S.L., B.C., S.W., U.N), Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok, Thailand. Accepted for publication February 11, 2009. Synopsis As part of an ongoing search for the novel pharmacological activities of Phyllanthus emblica, the present study has shown its type I collagen promoting and anti-collagenase effects on primary mouse fi broblast cells. At a concentration of 0.1 mg/ml, emblica extract signifi cantly increased the type I pro-collagen level up to 1.65- fold, and 6.78-fold greater than that of an untreated control, determined by immunocytochemistry and Western blot analysis, respectively. Emblica extract caused an approximately 7.75-fold greater type I pro- collagen induction compared to the known herbal collagen enhancer asiaticoside at the same treatment con- centration (0.1 mg/ml). Moreover, emblica extract inhibited collagenase activity in a dose-dependent manner. Maximal inhibition was observed (78.67 ± 3.51%) at a concentration of 1 mg/ml. In summary, emblica ex- tract has a promising pharmacological effect that benefi ts collagen synthesis and protects against its degrada- tion and could be used as a natural anti-aging ingredient. INTRODUCTION Anti-aging active compounds from herbal extracts are currently in demand in the cosmetics industry. They not only help to prevent skin damage, but also to regenerate the new skin components. Among a great number of potent biologically active herbal extracts, Phyllanthus emblica or emblica fruit extracts represent several pharmacologi- cal properties attributable to their high vitamin C content (1). In addition, emblica fruit contains several biologically active tannins proposed to have a potent antioxidant activity (2). Address all correspondence to Ubonthip Nimmannit and Pithi Chanvorachote.

JOURNAL OF COSMETIC SCIENCE 396 Skin aging is a process involving an alteration of type I collagen, the major component of dermis. An increase in type I collagen degradation and a decrease in its regeneration are considered major causes of wrinkle formation (3). Type I collagen is originally synthe- sized from intracellular type I pro-collagen containing propeptide extensions at both ends of the molecule (4,5). The pro-collagen is secreted to the extracellular matrix where the propeptides are removed and the collagen molecules aggregate to form the fi bril (5,6). The matrix metalloproteinases (MMPs) are a large family of zinc-dependent endopro- teases degrading all extracellular matrix proteins (ECMs) including collagen. MMP-1, interstitial collagenase, mediates type I and type III collagen degradation (7). Several fac- tors promoting this degradation process have been identifi ed, including ultraviolet (UV) radiation and reactive oxygen species (ROS) (8). Due to their potent antioxidant activity, emblica extracts appear to have promise as effectors for anti-aging actives. The objective of the present study is to investigate the novel pharmacological activities of emblica extract, namely type I collagen promoting and anti-collagenase activities. MATERIALS AND METHODS MATERIALS Dulbecco’s Modifi ed Eagle’s medium (DMEM), L-glutamine, fetal bovine serum (FBS), penicillin/streptomycin, and phosphate-buffered saline were obtained from Gibco-BRL (Gaithersburg, MD). Formaldehyde, triton X-100, glycerol, sodium formate, 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and dimethyl sulfoside (DMSO) were obtained from Sigma-Aldrich (St. Louis, MO). Pro-collagen type I rabbit polyclonal antibody, horseradish peroxidase-coupled isotype-specifi c secondary antibod- ies, and FITC-coupled secondary antibodies were obtained from Santa Cruz Biotechnol- ogy (Santa Cruz, CA). A protease inhibitior mixture was obtained from Roche Molecular Biochemicals (Switzerland). A chemiluminescence detection system was obtained from Amersham Biosciences (Piscataway, NJ), and a Western blot system was obtained from Bio-Rad (Hercules, CA). An EnzChek® gelatinase/collagenase assay kit was purchased from Molecular Probes®, Invitrogen (Carlsbad, CA). ISOLATION OF MOUSE EMBRYONIC FIBROBLASTS Isolation of fi broblast cells from mouse embryo was carried out according to a previously described method (Bradley, Baylor College of Medicine, Waco, TX). Briefl y, mouse uterus was dissected from 13- or 14-post coitum pregnancy female mice. Each embryo was separated and washed with phosphate buffer solution. Then the embryo was forced through a syringe that was fi tted with an 18G 11/2" needle into Dulbecco’s Modifi ed Eagle’s medium (DMEM). Primary fi broblasts were the only cells that attached and proliferated after fi ve days incubation at 37°C. Cells were cultured in DMEM containing 10% fetal bovine serum, 2 mmol/l L-glutamine, and 100 units/ml of penicillin/strepto- mycin in a 5% CO2 environment at 37°C. The cells from passages 2–5 were used for the experiments.