Cent. Eur. J. Biol. • 9(9) • 2014 • 888-900 DOI: 10.2478/s11535-014-0322-1 Central European Journal of Biology Phenolic production and antioxidant properties of some Macedonian medicinal plants Research Article Oliver Tusevski1, Aneta Kostovska2, Ana Iloska2, Ljubica Trajkovska2, Sonja Gadzovska Simic1* 1Department of Plant Physiology, Institute of Biology, Faculty of Natural Sciences and Mathematics, University “Ss. Cyril and Methodius”, P.O. Box 162, 1000 Skopje, Macedonia 2Biology Students Research Society (BSRS), Institute of Biology, Faculty of Natural Sciences and Mathematics, University “Ss. Cyril and Methodius”, P.O. Box 162, 1000 Skopje, Macedonia Received 24 September 2013; Accepted 26 December 2013 Abstract: Investigations have been made to study the production of phenolic compounds (total phenolics, flavonoids and phenylpropanoids) and total antioxidant capacity in 27 Macedonian traditional medicinal plants to improve its potential as a source of natural antioxidants. Antioxidant potential of plant extracts was analyzed by five different assays: cupric reducing antioxidant capacity (CUPRAC), phosphomolybdenum method (PM), reducing power (RP), 2,2-diphenyl-1-picrylhydrazyl (DPPH•) and 2,2’-azinobis(3- ethylbenzothiazoline-6-sulphonic acid (ABTS•+) radical scavenging activity. Origanum vulgare extract consistently exhibited the highest content of phenolic compounds and the strongest antioxidant capacity based on the tests performed, and can be proposed as a promising source of natural antioxidants. Melissa officinalis and Salvia ringens were also identified as valuable sources of antioxidant compounds. A positive linear correlation between antioxidant activity and total phenolics, flavonoids and phenylpropanoids indicates that these compounds are likely to be the main antioxidants contributing to the observed activities of evaluated plants. These findings suggest that the medicinal plants studied in this paper are good sources of bioactive compounds for the food and pharmaceutical industries. Keywords: Phenolics • Flavonoids • Phenylpropanoids • Antioxidant activity • Scavenging capacity • Medicinal plants © Versita Sp. z o.o. Abbreviations: 1. Introduction AAE - Ascorbic acid equivalents, Reactive oxygen species (ROS) are an integral part of ABTS - 2,2’-azinobis(3-ethylbenzothiazoline-6- normal physiological processes, continuously formed sulphonic acid, as a consequence of aerobic metabolism in eukaryotic CE - Catechin equivalents, cells. ROS at low-to-moderate concentrations play CUPRAC - Cupric reducing antioxidant capacity, important roles in cell physiology, such as regulation DPPH - 2,2-diphenyl-1-picrylhydrazyl, of cell growth, cellular signal transduction pathways, GAE - Gallic acid equivalents, and defence against pathogens [1,2]. In addition to PE - Pyrocatechol equivalents, their biological importance, overproduction of these PM - Phosphomolybdenum method, extremely reactive and unstable oxygen species RP - Reducing power, is considered to be the main contributor to various TE - Trolox equivalents, metabolic and cellular disturbances. These harmful by- TF - Total flavonoid content, products may also induce some oxidative damage to TP - Total phenolic content, functional biomolecules giving rise to oxidative stress. TPP - Total phenylpropanoid content Oxidative stress has been suggested to play a major * E-mail: [email protected] 888 O. Tusevski et al. role in the pathogenesis of many degenerative diseases acid, quercetin, catechin, α-tocopherol, BHT and BHA). in humans [3]. In modern medicine, maintaining the This study will be useful to determine the potential of balance between antioxidant defence system and ROS selected traditional medicinal plants as potential sources formation is believed to be a critical concept for healthy of natural antioxidants. biological systems [4]. Recently published data highlight the health benefits of medicinal herbs, fruit, vegetables, grains 2. Experimental Procedures and beverages as dietary antioxidants [5]. Natural plant products, which contribute health benefits to consumers, 2.1 Chemicals and apparatus had stronger antioxidant activity than that of synthetic All chemicals and reagents were of analytical grade antioxidants [6]. Synthetic antioxidants such as butylated and were purchased from Sigma-Aldrich (Germany). hydroxytoluene (BHT), butylated hydroxyanisole (BHA), All spectrophotometric measurements were performed tert-butylhydroquinone (TBHQ) and propyl gallate (PG) on UV–VIS spectrophotometer SpectraMAX 190 are widely used in the food industry to prevent oxidative (Molecular Devices, California, United States). deterioration [7]. However, it has been established that synthetic antioxidants appear to have carcinogenic and 2.2 Plant material tumour-promoting action [8]. Therefore, it is of great Twenty-seven medicinal plant species from twelve importance to find new sources of safe and inexpensive families (Table 1) were collected from various locations antioxidants of natural origin in order to use them in at Galichitsa Mountain, Republic of Macedonia. foods and pharmaceutical formulations. The plant species used in this study belong to the Medicinal plants synthesize antioxidant compounds family Lamiaceae (nine tested species), Asteracae as secondary products. These compounds are mainly (four tested spices), Fabaceae, Plantaginaceae (three phenolics that serve as plant defence mechanisms tested spices), Rosaceae, Malvaceae, Gentianaceae, to counteract ROS and avoid oxidative damage. Scrophulariaceae, Linaceae, Caprifoliaceae, Polyphenols possess ideal structural chemistry for Caryophyllaceae and Verbenaceae (one tested species). free radical scavenging activity, and they have been Voucher specimens of collected plants are deposited in shown to be more effective antioxidants in vitro than the Herbarium Collection of Biology Students Research tocopherols and ascorbate. The antioxidant activities of Society (BSRS), Institute of Biology, Faculty of Natural phenolics are due to a number of different mechanisms, Sciences and Mathematics, University “Ss. Cyril and such as free-radical scavenging, hydrogen or electron Methodius”, Skopje. donation, singlet oxygen quenching, metal ion chelation, and acting as scavengers of superoxide, peroxide and 2.3 Extraction procedure hydroxyl radicals [9]. In addition, it has been reported Plant material was air-dried, lyophilized and then that the antioxidant potential of medicinal plants grounded into a fine powder by laboratory mill. correlates with their phenolic compound content [10]. Antioxidant compounds were extracted from There are approximately 3500 vascular plant powdered plant material (0.02 g) with 80% methanol species in Macedonia, of which 700 have medicinal in an ultrasonic bath for 30 min. The extracts were properties, however only 120 species are generally centrifuged (15 min at 12000 rpm) and the supernatant utilised in folk and official medicine. To our knowledge, was used for quantification of total phenolics, flavonoids there is no available information or systematic survey of and phenylpropanoids, as well for antioxidant activity antioxidant activity in wild-growing Macedonian plants. using different assays. Ascorbic acid (AA), quercetin The aim of this study is to perform a preliminary screen (Q), catechin (C), α-tocopherol (α-T), butylated of antioxidant activities from 27 traditional medicinal hydroxytoluene (BHT) and butylated hydroxyanisole plant species collected on Galichitsa Mountain, Republic (BHA) were used as reference compounds. of Macedonia. Therefore, the main objectives of this Standard chemicals were dissolved in pure methanol study were: 1) to determine total content of phenolics, at a concentration of 0.5 mg mL-1 and prepared prior to flavonoids and phenylpropanoids in methanolic extracts each assay. from selected medicinal plants; 2) to assess antioxidant activity by using a number of chemical in vitro assays; 2.4 Quantification of phenolic compounds and 3) to explore correlation between phenolic contents 2.4.1 Total phenolic content (TP) and antioxidant activity in tested samples. The results Total phenolic (TP) content in methanolic extracts was for antioxidant activity were compared with those determined according to the Folin-Ciocalteu colorimetric obtained with different reference compounds (ascorbic method [11] with the following modifications. An aliquot 889 Antioxidant properties of Macedonian medicinal plants TP TF TPP Plant species (Familly) Plant part Common name (mg GAE/g DW)b (mg CE/g DW)c (mg PE/g DW)d Achillea holosericea Sibth. & Sm. (Asteraceae) Aerial parts / 24.52 ± 1.50 11.11 ± 0.96 9.84 ± 0.31 Agrimonia eupatoria L. Aerial parts Common agrimony 59.57 ± 0.08 20.14 ± 0.20 18.34 ± 1.05 (Rosaceae) Alcea pallida Waldst. et Kit. Flowers Hollyhock 10.90 ± 0.36 1.42 ± 0.19 1.13 ± 0.12 (Malvaceae) Anthyllis vulneraria L. Aerial parts Woundwort 12.02 ± 0.42 2.22 ± 0.15 1.81 ± 0.19 (Fabaceae) Astragalus glycyphyllos L. Aerial parts Wild liquorice 15.93 ± 0.47 1.62 ± 0.40 0.94 ± 0.08 (Fabaceae) Centaurium erythraea Rafn. Aerial parts Common centaury 22.28 ± 1.07 4.48 ± 0.13 3.56 ± 0.38 (Gentianaceae) Cichorium intybus L. Aerial parts Chicory 33.36 ± 0.14 8.06 ± 0.24 10.25 ± 0.79 (Asteraceae) Clinopodium vulgare L. Aerial parts Wild basil 57.07 ± 4.15 46.33 ± 2.22 35.13 ± 2.75 (Lamiaceae) Digitalis ferruginea L. Aerial parts Rusty foxglove 13.24 ± 1.28 2.86 ± 0.54 1.81 ± 0.18 (Plantaginaceae) Digitalis lanata Ehrh. Aerial parts Woolly Foxglove 22.16 ± 1.02 11.19 ± 1.38 6.66