Isolation, identification and evaluation of natural antioxidants from aromatic herbs cultivated in Lithuania

Abstract

Oxidative spoilage of lipid-rich foods decreases their shelf-life and leads to undesirable chemical and physical changes. Nowadays natural antioxidants are generally preferred. The major part of industrially used antioxidants consists of radical scavengers, which inhibit the oxidative chain reaction by inactivating free radicals formed during peroxidation of lipids. Aromatic and medicinal herbs are rich sources of natural radical scavenging compounds. The research described in this thesis focuses on the evaluation of several aromatic plants grown in Lithuania as a possible source of food antioxidants. Various aspects of lipid oxidation, of antioxidative mechanisms and of natural sources of radical scavenging antioxidants are outlined in Chapter 1.Employment of model systems for antioxidant studies is often preferred over lipid stability tests due to their speed and the simplified comparison and interpretation of the obtained results. Preliminary screening of extracts from aromatic plants was carried out using theβ-carotene-linoleic acid model system (β-CLAMS). The method is based on a competition between the plant antioxidant and an oxidisable indicator (β-carotene) for free radicals, which are generated during temperature-accelerated oxidation of linoleic acid. The agar-diffusion and spectrophotometricβ-CLAMS tests singled out thyme ( Thymus vulgaris L.) and sage ( Salvia officinalis L.) as the most promising sources of natural antioxidant compounds (Chapter 2). The screening results also revealed that supercritical fluid extraction (SFE) as well as solvent extraction with acetone and methanol-water were the two most efficient techniques for isolation of antioxidant constituents from sage and thyme (Chapter 2). In further antioxidant activity tests, the spectrophotometricβ-CLAMS technique was adapted to microtiter plates, which gave a better reproducibility and a shorter analysis time. The modified and improved spectrophotometricβ-CLAMS confirmed the preliminary screening results. Sage and thyme acetone extracts again demonstrated the highest radical scavenging activity with relative antioxidant activities similar to those of 2,6-di- tert -butyl-4-methylphenol (BHT) (Chapter 3).The activity of primary antioxidants can be evaluated directly by monitoring the reduction of free radicals. Such experiments can be carried out off-line, i.e. by the introduction of previously isolated antioxidant compound(s) into an equilibrated free radical containing model system, or on-line, by adding a solution of a free radical to the eluate of an HPLC column. The on-line techniques allow for a rapid and selective detection of radical scavenging substances in the presence of many inactive constituents with a minimum of preparatory manipulations. Two on-line model systems based on the reduction of radical intermediates of the luminol chemiluminescence (CL) reaction and on the reduction of the stable 2,2'-diphenyl-1-picrylhydrazyl radical (DPPH·) were used for the detection of antioxidant compounds in thyme and sage isolates. Ten compounds in the thyme acetone oleoresin and six in sage gave an inhibition signal on the CL detector (Chapter 4).An adequate HPLC separation and a sensitive detection of radical scavenging are two key-elements that determine the success of on-line experiments. Improved separation efficiency especially for polar analytes was observed after an acetonitrile/water gradient program was introduced and after lowering of the pH of the HPLC solvents (Chapter 5). A stable, pulse-free flow, appropriate instrumental set-up and optimised compositions of the CL and the DPPH·reagents improved the sensitivity of the on-line detection. The minimum detectable amounts registered in this study varied from 0.06 ng for rosmarinic acid to 2400 ng for carvacrol (HPLC-CL detection) and from 0.2 ng for eugenol to 19 ng for BHT (HPLC-DPPH·detection) (Chapter 5). It was found that the HPLC-CL system was on average more sensitive, however the HPLC-DPPH·method was more robust.The on-line HPLC-DPPH·method as well as TLC plates sprayed with DPPH·solution were used for the activity-guided isolation of radical scavenging compounds from a leaf extract of thyme. Nine active compounds were isolated from the methanolic and the acidic aqueous alcoholic extract by means of solvent partitioning, normal phase chromatography, size exclusion chromatography and reversed phase MPLC or HPLC (Chapter 6). Rosmarinic acid, eriodictyol, taxifolin, luteolin 7-glucuronide, p -cymene 2,3-diol and p -cymene 2,3-diol 6-6´-dimer were identified by a combination of UV, CD, mass spectrometry and 1H and 13C NMR. Two weakly active, volatile compounds, thymol and carvacrol, were identified by GC-MS and their structures confirmed by GC and TLC analysis with reference compounds. The structure of a highly active new phenylpropanoid trimer 3'-O-(8''-Z-caffeoyl)-rosmarinic acid was elucidated from its UV and CD spectra, FD mass spectrum, and 1H-, 13C-, COSY and direct and long range 1H-13C NMR spectra (Chapter 6). In off-line Trolox Equivalent Antioxidant Capacity (TEAC) and DPPH·assays, this compound was a weaker and stronger radical scavenger than rosmarinic acid, respectively.The presence in T. vulgaris of radical scavenging compounds of different polarity and with distinctive reactivity to different radicals offers the possibility to use extracts from this plant as natural antioxidants for various lipid-containing food systems.</p