j. Cosmet. Sci., 53, 321-335 (November/December 2002) In vitro antioxidant and in vivo photoprotective effects of a lyophilized extract of Capparis spinosa L. buds F. BONINA, C. PUGLIA, D. VENTURA, R. AQUINO, S. TORTORA, A. SACCHI, A. SAIJA, A. TOMAINO, M. L. PELLEGRINO, and P. de CAPRARIIS, Department of Pharmaceutical Sciences, School of Pharmacy, University of Catania, Catania (F. B., C.P., D.V.), Department of Pharmaceutical Sciences, University of Salerno, Salerno (R.A., S.T., P.d.C.), Department of Pharmaceutical Chemistry, University Federico II, Naples (A.Sac.), Department of Pharmacology of Natural Substances & General Physiology, University of Rome "La Sapienza," Rome (A.Sai.), and Department Farmaco-Biologico, University of Messina, Messina (A. T., M.L.P.), Italy. Accepted for publication April 29, 2002. Synopsis The aim of the present study was to evaluate the in vitro antioxidant and in vivo photoprotective activities of a lyophilized extract of Capparis spinosa L. (LECS) obtained by methanolic extraction from the flowering buds of this plant. For the in vitro experiments, LECS was tested employing three different models: (a) bleaching of the stable 1,1-diphenyl-2-picrylhydrazyl radical (DPPH test) (b) peroxidation, induced by the water-soluble radical initiator 2,2'-azobis(2-amidinopropane) hydrochloride, of mixed dipalmitoylphospha- tidylcholine/linoleic acid unilamellar vesicles (LUVs) (LP-LUV test) and (c) UV-induced peroxidation of phosphatidylcholine multilamellar vesicles (UV-IP test). The in vivo antioxidant/radical scavenger activity was assessed by determining the ability of topically applied LECS to reduce UVB-induced skin erythema in healthy human volunteers. From the results obtained in in vitro and in vivo tests, LECS showed a significant antioxidant effect. Furthermore, by chromatographic fractionation and spectroscopic methods, we identified the major constituents of LECS, and particularly some flavonols (kaempferol and quercetin derivatives) and hydroxycinnamic acids (caffeic acid, ferulic acid, p-cumaric acid, and cinnamic acid). INTRODUCTION Inflammatory and degenerative processes following acute and chronic UV-light skin irradiation are known to be mediated by overproduction of reactive oxygen species (ROS) and free radicals and by impairment of antioxidant systems (1-3). Thus it is today largely recognized that, under environmentally challenged conditions, skin topical ap- Address all correspondence to F. Bonina. 321

322 JOURNAL OF COSMETIC SCIENCE plication and systemic administration of antioxidants could support physiological mechanisms to maintain or restore a healthy skin barrier (4). Clear experimental evidence supports the development of new and effective pharmaceutical and cosmetic strategies involving antioxidant ingredients to prevent UV radiation-induced skin pathological conditions and photoaging (5-8). Particularly, a number of recent studies are focused on the effect of several natural antioxidant compounds (lipoic acid, fiavonoids, vitamin E, silymarin, pycnogenol, etc.) in protecting human skin against solar UV-simulated light- induced damage (6,7,9,10). Plant polyphenolic compounds are endowed with a broad biological profile, which is related to their antioxidant/free radical scavenging capability and thus to their effica- cious protective effect in oxidative stress conditions. In fact, biophenols have been shown to interfere not only with the propagation reaction in the lipoperoxidative chain, but also with the formation of free radicals, either by chelating the transition metal or by inhibiting the enzymes involved in the initiation reaction (11). Capparis spinosa L. (family Capparidaceae) is a plant originating in dry regions in west or central Asia and diffused particularly in the Mediterranean basin. The floral buttons of C. spinosa are employed as a flavoring in cooking (12) and, as well as those of other Capparis species, are also used in traditional medicine for their diuretic, antihypertensive, poultice and tonic effects (13), and in certain pathological conditions are related to uncontrolled lipid peroxidation (14-16). Furthermore, whole extracts of the floral but- tons, applied topically in cosmetic bases, are reported to possess stimulant, bioactivating, hydrating properties on dry, aged, and undernourished skin (17). Previous studies (18,19) have shown the presence in C. spinosa floral buttons of various poliphenolic compounds and in particular in fiavonoids such as quercitin and kaempferol glycosides. In the present study we have employed a lyophilized extract of Capparis spinosa (LECS) obtained by methanolic extraction from the flowering buds of this plant. The first part of our research was focused on the qualitative and quantitative character- ization of the LECS phenolic profile (mainly represented by fiavonols and hydroxycin- namic acids) to better understand its chemical composition. Then we evaluated the LECS for its in vitro antioxidant/radical scavenging activity and in vivo UVB-induced skin erythema-inhibiting ability. To evaluate the in vitro antioxidant and radical scavenger activities of LECS, we em- ployed three experimental models: (a) bleaching of the stable 1,1-diphenyl-2-picrylhy- drazyl radical (DPPH test) (b) peroxidation, induced by the water-soluble radical ini- tiator 2,2'-azobis(2-amidinopropane) hydrochloride, of mixed dipalmitoylphosphatidyl choline/linoleic acid unilamellar vesicles (LP-LUV test) and (c) the protective effect against the UV light-induced peroxidation on phosphatidylcholine multilamellar vesicles, as a model membrane (UV-IP test). Since the interaction with the microenvi- ronment of the lipid cell bilayers plays a key role in the membrane-dependent biological activity (such as the antioxidant activity) of a drug, the concomitant employment of different tests, in a homogenous solution (DPPH test) and in membranous systems (LP-LUV and UV-IP tests), may give useful indications for determining the actual effectiveness and suitability of a potential antioxidant (20). Furthermore, LECS was tested in vivo to assess its ability to reduce UVB-induced skin erythema (monitored by reflectance spectrophotometry) when topically applied in healthy human volunteers. This test is regarded as one of the most suitable models for