J. Cosmet. Sci., 57, 57-64 (January/February 2006) Partially purified paeoniflorin exerts protective effects on UV-induced DNA damage and reduces facial wrinkles in human skin SANGHWA LEE, JUN MAN LIM, MU HYUN JIN, HYUNG KOOK PARK, EUI JUNG LEE, SANGJIN KANG, YOUN SOO KIM, and WAN GOO CHO, C. I. Center, Research Park, LG Household & Healthcare, Ltd. (S. L., J. M. L., M. H.J., H.K. P., E.j. L., S. K., W. G. C.), and CnU Skin Clinic & CnU Skin Laboratory, Ltd., (Y. S. K.), Taejeon, Korea. Accepted for publication October 3 , 200 5. Synopsis Partially purified paeoniflorin (PF), a new cosmetic ingredient from roots of Paeoniae lactiflora, has been developed. Its paeoniflorin content is about 64%, far higher than that of conventional, cosmetic-grade peony root extracts (-10% ). In this report, we studied the effects of PF on UV-induced DNA damage in both cultured human keratinocytes and hairless mouse skin. We also investigated the anti-wrinkle effects of PF-containing cosmetic preparations on human skin. From the in vitro and in vivo comet assay, it was revealed that PF protected cells from DNA damage induced by ultraviolet B (UVB) irradiation in both cultured normal human keratinocytes (19.4% decrease at 0.001 %) and hairless mouse skin keratinocytes (41 % decrease at 0.01 %). An eight-week clinical trial using 0.5% PF-containing formulation with 20 volunteers resulted in a statistically significant reduction in facial wrinkles (p 0.05 ). These results suggest that the partially purified paeoniflorin has potent anti-aging and anti-wrinkle activities and should be a useful ingredient for these purposes. INTRODUCTION Chronic exposure of human skin to solar ultraviolet radiation (UVR) is thought to be a major environmental factor that has serious adverse effects on the structure and function of the skin. UV irradiation may cause sunburn, immunosuppression, oxidative stress, and skin cancers, as well as premature skin aging, so-called "photoaging" (1). There is numerous evidence for the generation of reactive oxygen species (ROS) in skin upon UV exposure. Accumulation of molecular oxidative damage to cellular components such as lipid, protein, and DNA may lead to photoaging (2). There have been many articles concerning the protective effects of exogenous antioxidants on the harmful effects of UVR (3-5). In this context, we investigated by comet assay the protective effects of partially purified paeoniflorin (PF) against UV-induced DNA damage. PF came up as a 57

58 JOURNAL OF COSMETIC SCIENCE candidate because it was revealed that paeoniflorin, in our preliminary experiments, suppressed the expression of senescence-associated 13-galactosidase. Peony root (Paeoniae radix) is one of the most well-known herbs in China, Japan, and Korea, and has long been used as a component of traditional medicinal prescriptions. Several compounds including oxypaeoniflorin, albiflorin, benzoylpaeoniflorin, and pae- oniflorin are reported to be contained in peony root (6). Among them, paeoniflorin is known to be one of the principal bioactive components of peony root and has been reported to have immunoregulatory (7), anti-inflammatory, anti-coagulant (8), and seda- tive (9) effects. Recently, we developed a new cosmetic ingredient, partially purified paeoniflorin (PF), from the roots of P aeoniae lactiflora. It could be characterized by enriched paeoniflorin content (more than 60%), and Figure 1 shows the molecular structure of paeoniflorin. To verify the potential usefulness of PF as a cosmetic active ingredient, we investigated the effect of PF on the DNA damage caused by UVB irradiation. We also performed a clinical study with a cosmetic formulation containing 0.5% PF. In this report, we present the protective effects of PF on DVB-induced DNA damage as well as its anti-wrinkle activity in vivo. EXPERIMENT AL PREPARATION OF PARTIALLY PURIFIED PAEONIFLORIN (PF) PF was extracted and partially purified from the roots of Paeonia lactiflora. Briefly, peony root was ground and extracted with 7 5 % ethanol. The extract was concentrated by evaporation and resuspended in distilled water and fractionated with ethyl ether. The water-soluble part was loaded into a silica gel column, and the fraction containing peoniflorin was resolved with chloroform acetone (4: 1) as moving phase. The purity of paeoniflorin was determined to be about 64% by HPLC. CELL CULTURE Normal human keratinocytes (NHK) were cultured from adult foreskin. NHK cells were maintained in keratinocyte growth medium (KGM, Clonetics, USA) in a humidi- Figure 1. Molecular structure of paeoniflorin (C23H28O 11 M.W. 480.45), major constituent of PF.