Antioxidant Activity In Vitro of Selected Peruvian Medicinal Plants

Biomolecules can be oxidized by free radicals. This oxidative damage has an important etiological role in aging and the development of diseases like cancer, atherosclerosis, and other inflammatory disorders. Synthetic antioxidants, like butylated hydroxyanisole, are good free radical scavengers, however, the synthetic antioxidants can be carcinogenic. Therefore, there is an increasing interest in searching for antioxidants of natural origin. We report here the results of a screening for antioxidant activity of 53 ethanolic extracts from 40 Peruvian plants used in traditional medicine for the treatment of several infectious and inflammatory diseases. The antioxidant activity in vitro was measured by means of the 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging assay. Rutin, a natural antioxidant, was used as a control. Twenty one (39.6%) of the plant extracts showed an EC50 lower than 50 μg/ml. The plants with the highest activity were Gentianella nitida, Iryanthera lancifolia, Lepechinia meyenii, Oenothera multicaulis, Philodendron solimoesense and Tetracera volubilis, which showed an EC50 of 13.70, 14.08, 16.65, 16.89, 8.80, and 5.29 μg/ml, respectively. The crude ethanolic extract of Tetracera volubilis has better antioxidant activity in vitro than the pure natural antioxidant rutin (EC50 = 7.16 μg/ml). INTRODUCTION The oxidative damage caused by free radicals is considered to be related to the development of diseases like atheroesclerosis, cancer, brain dysfunction, arthritis and other inflammatory disorders (Halliwell, 1991; Finkel and Holbrook, 2000). Several synthetic antioxidants, like butylated hydroxyanisole or butylated hydroxytoluene, are effective free radical scavengers, however, they are being restricted because they can be carcinogenic (Ito et al., 1983; Safer and Al-Nughamish, 1999). Thus, there is a growing interest in searching for antioxidants of natural origin, especially those present in medicinal plants. The purpose of this study is to evaluate the in vital antioxidant activity of 40 plants used in Peruvian traditional medicine for the treatment of several infectious and inflammatory disorders. Plants that have been used extensively for the treatment of inflammatory or infectious diseases may contain radical scavengers compounds that could be used as natural antioxidants. MATERIAL AND METHODS Plant Material Plants were collected between May and July 2001 from five different regions of Peru (Table 1). Plants were collected and identified by botanists Irma Fernández (IF), Joaquina Albán (JA), Alfredo Tupayachi (HV), Abundio Sagástegui (AS) and Genaro Yarupaitán (GY). The respective voucher specimens are deposited at the Department of Chemistry of the Pontificia Universidad Católica del Perú, in Lima. Air-dried powdered plant material was extracted by percolation at room temperature with 95% ethanol. The solvent was then evaporated to dryness under reduced Proc. WOCMAP III, Vol. 1: Bioprospecting & Ethnopharmacology Eds. J. Bernáth, É. Németh, L.E. Craker and Z.E.Gardner Acta Hort 675, ISHS 2005 104 pressure at a temperature lower than 40°C. Yield of extracts in terms of dry starting materials are listed in Table 1. For the antioxidant assay, the extracts were resuspended in ethanol. Antioxidant Assay The antioxidant activity in vital was measured by means of the 1,1-diphenyl-2picrylhydrazyl (DPPH) free radical scavenging assay. When DPPH is dissolved in ethanol at room temperature, DPPH produces a violet solution containing the stable DPPH radical. In the presence of an antioxidant compound, the DPPH radical is reduced, producing decoloration of the solution. The DPPH free radical assay was carried out according to the method of Mensor et al. (2001), with some modifications. Briefly, one ml of a 0.3 mM DPPH ethanol solution was added to 2.5 ml of sample solutions (final concentrations of 10 and 50 μg/ml) and the mixtures were incubated at room temperature for 30 minutes. The absorbances were then measured at 517 nm using a spectrophotometer (Perkin Elmer, Lambda 2). The percentage antioxidant activity (%AA) was calculated according to the following formula: %AA = Abscontrol (Abssample Absblank) x 100% Abscontrol Abssample= 1 ml 0.3 mM DPPH + 2.5 ml plant extract solution Absblank = 1 ml ethanol + 2.5 ml plant extract solution Abscontrol = 1 ml DPPH 0.3 mM in ethanol + 2.5 ml ethanol Rutin (Sigma, St. Louis, MO) was used as a positive control. Those extracts with the highest antioxidant activity (more than 25% AA at a plant extract concentration of 10 μg/ml) were diluted at 6 different concentrations between 1 and 30 μg/ml in order to assess their EC50. The EC50 values were obtained by extrapolation from linear regression analysis and denote the concentration of plant extract required to scavenge 50% of the DPPH free radicals. The results shown in Table 1 are mean values of at least three independent determinations. RESULTS AND DISCUSSION Table 1 summarizes the antioxidant activities in vital of 53 ethanolic extracts from 40 Peruvian medicinal plants belonging to 21 different families. Twenty one (39.6%) of the plant extracts have an EC50 lower than 50 μg/ml. The plants with the highest antioxidant activities were Gentianella nitida, Iryanthera lancifolia, Lepechinia meyenii, Oenothera multicaulis, Philodendron solimoesense and Tetracera volubilis. An important hypoglycemic activity was demonstrated for the G. nitida water extract in two rat models, which is attributed to the presence of several xanthones (Lacaille-Dubois et al., 1996; Callo et al., 2001). No antioxidant activities have been reported previously for this plant. The I. lancifolia stems and bark ethanol extracts showed a strong DPPH radical scavenging activity. Silva et al. (1999) demonstrated that an extract from the pericarps of I. lancifolia inhibits the lipid peroxidation of rat brain homogenates. This activity is due to the presence of two flavonolignans and the compounds iryantherin K and L. It is not known yet if these compounds are also able to scavenge DPPH radicals. L. meyenii is popularly used for the treatment of cough and bronchitis. Several terpenes have been isolated from this plant (Mango et al., 1990; Bruno et al., 1991), however, none of those compounds have been investigated for antioxidant actity. No previous phytochemical or pharmacological investigations have been done on O. multicaulis, P. solimoesense and T. volubilis. It is noteworthy that the crude extracts of the stems of P. solimoesense and the