دانلود رایگان ترجمه مقاله آزمایش فعالیت آنتی اکسیدانی روغن اسانس مرزنجوش – الزویر 2004

استفاده از متدهای مختلف برای آزمایش فعالیت آنتی اکسیدانی روغن اسانس مرزنجوش

Use of different methods for testing antioxidative activity of oregano essential oil

Abstract

The antioxidant properties of the essential oil from oregano in relation to its chemical composition were examined. The antioxidant activity was investigated with three different methods: the β-carotene bleaching (BCB) test, the 2,2′-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging method and the thiobarbituric acid reactive species (TBARS) assay. It was found that the total essential oil, its fraction as well as its pure constituents have a significant antioxidant effect when tested by each method, respectively. Generally the antioxidant activity of the oregano essential oil is less effective than the ascorbic acid, but comparable with the α-tocopherol and the synthetic antioxidant butylated hydroxytoluene (BHT). The synergy among minor oxygen containing compounds was suggested as possible factor, which influenced the antioxidant power of the oregano essential oil. The antioxidant concentrations influenced its antioxidant power, too.

1. Introduction

The most widely used synthetic antioxidants in food (butylated hydroxytoluene BHT, butylated hydroxyanisole BHA, propyl galate PG and tertiary butyl hydroquinone TBHQ) have been suspected to cause or promote negative health effects (Barlow, 1990; Branen, 1975; Chan, 1987; Namiki, 1990; Pokorny, 1991). For this reason there is a growing interest in studies of natural additives as potential antioxidants. Many sources of antioxidants of plant origin have been studied in recent years. Among these the antioxidant properties of many aromatic plants and spices have shown to be effective in retarding the process of lipid peroxidation in oils and fatty foods and have gained the interest of many research groups. Numerous types of antioxidants with varied activities were identified. Their antioxidant effect was due to the presence of hydroxyl groups in their chemical structure (Shahidi, 2000; Shahidi, Janitha, & Wanasundara, 1992; Vekiari, Oreopoulou, Tzia, & Thomopoulos, 1993). Several non-volatile compounds such as carnosol, quercetine, caffeic acid and rosmarinic acid are well known to be good scavengers of free radicals, but some volatile compounds from essential oils possess also the potential as natural agents for food preservation. A number of studies on antioxidant activities of essential oils from various aromatic plants reported that the oregano essential oil, rich in thymol and carvacrol, has a considerable antioxidant effect on the process of the lard oxidation (Lagouri, Blekas, Tsimidou, Kokkini, & Boskou, 1993; Tsimidou & Boskou, 1994). Yanishlieva and Marinova (1995) examined the antioxidant activity of hexane extracts of oregano grown in Bulgaria, as well as the mechanism of action of pure thymol and carvacrol (Yanishlieva, Marinova, Gordon, & Raneva, 1999). In zour previous work (Milos, Mastelic, Jerkovic, & Katalinic, 2000) we presented the chemical composition and the antioxidant effect of oregano glycosidically bound volatiles on the lard oxidation process. Recent publications (Abdalla & Roozen, 1999, 2001; Bendini, Gallina Toschi, & Lercker, 2002; Cervato, Carabelli, Gervasio, Cittera, Cazzola, & Cestaro, 2000; Damechki, Sotiropoulou, & Tsimidou, 2001; Martinez-Tome, Jimenez, Ruggieri, Frega, Strabbioli, & Murcia, 2001; Vichi, Zitterl-Eglseer, Jugi, & Fraz, 2001) showed antioxidative activities of oregano. The aim of the present study was to examine the antioxidant properties of oregano essential oil by using three different methods, namely, the b-carotene bleaching (BCB) test, the 2,20 -diphenyl-1-picrylhydrazyl (DPPH) radical scavenging method and the thiobarbituric acid reactive species (TBARS) assay.

2. Experimental

2.1. Materials

Oregano (Origanum vulgare L., ssp. hirtum) was collected in central Dalmatia in October 2001. The plant material consisted of flowered tops and stalks (15–20 cm). Air-drying of oregano was performed in a shady place at room temperature for 10 days. The plant material was used for the isolation of the essential oil, immediately after drying. A hundred grams of the dried plant material was subjected to a 3-h hydrodistillation using a modified Clevenger-type apparatus. The body of the modified Clevenger-type apparatus consists of two concentric tubes. The inner tube is graduated and filled with water and a known amount of n-pentane, whose role is to retain the essential oil and to separate it from the water. Also, instead of a condenser of the ‘‘cold-finger’’ type an Allihn type condenser is used. The obtained essential oil was dried over anhydrous sodium sulfate and stored under nitrogen in sealed vial at 20 C until required. The voucher specimens of oregano plant material and essential oil are deposited in the Department of Biochemistry and Food Chemistry, Faculty of Chemical Technology, Split, Croatia. The oregano essential oil (0.5 g) was fractionated on a silica gel (30–60 mm, Mallinckrodt Baker B.V., Deventer, The Netherlands) column (length 20 cm; i.d. 2 cm). Pentane (50 ml) was used to obtain a fraction, which contained only nonpolar hydrocarbons (CH fraction), and diethyl ether (50 ml) was used to obtain a fraction of polar (oxygen containing, CHO fraction) compounds. These fractions were concentrated to 0.5 ml and subjected to thin layer chromatography (TLC) on silica gel plates in order to check results of the column chromatography separation. Different solvents were used as a mobile phase: n-hexane for CH fraction and n-hexane:ethyl acetate 85:15 (v/v) for CHO fraction. Two percent vanillin-sulphuric acid was used as a detection reagent. The fractions obtained by column chromatography were also subjected to GC/MS analysis (GC/MS conditions as described in Section 2.2) and good separation results were confirmed. In order to obtain a fraction of phenolic compounds, 1 g of the essential oil was dissolved in 5 ml pentane and extracted with sodium hydroxide solution (20%) in water. In this manner, phenolic compounds were removed from the pentane layer. The aqueous phase, containing dissolved phenolic compounds sodium salts, was neutralized with hydrochloric acid solution (10%) in water. Finally, isolation of the phenolic compounds was performed by extraction with pentane (5  5ml). The effectiveness of this separation method was tested by TLC on silica gel plates (mobile phase: n-hexane:ethyl acetate 85:15 v/v) and a purity of the phenolic compounds fraction was confirmed. These separation results were confirmed by GC/MS analysis too (GC/MS conditions as described in Section 2.2). Butylated hydroxytoluene, a-tocopherol, 2,20 -diphenyl-1-picrylhydrazyl, thiobarbituric acid, b-carotene, ascorbic acid and all of the applied solvents were of pro analysis purity and were purchased from Fluka Chemie, Buchs, Switzerland. Anhydrous sodium sulfate was obtained from Merck, Darmstadt, Germany.