This study was aimed to evaluation of the antioxidant activity of Thymus tosevii extracts in inhibition of thermal autooxidation of lard. Ether and ethyl acetate extracts from overgrown herbal plants were prepared and added to the antioxidant and peroxide free lard. Wheeler method was used as an accelerated method for monitoring the oxidative stability of lipids. The progress of the oxidation was observed by measuring the peroxide value. It was found that all of the investigated representatives of genus Thymus showed certain antioxidant activity.

Lipid peroxidation is a well-established mechanism of cellular injury in both plants and animals, and is used as an indicator of oxidative stress in cells and tissues. Cellular damage due to lipid peroxidation cause derangement, such as ischemia-reperfuzion injury [Armstrong et al., 1975], coronary arteriosclerosis [Steinberg et al., 1989], and diabetes mellitus [Sundaram et al., 1996], as well as associating with aging and carcinogenesis [Ames, 1983]. It is well established that lipid peroxidation is one of the reactions set into motion as a consequence of the formation of free radicals in cells and tissues. The one-electron reduction products of O₂, superoxide anion (O₂^-), hydrogen peroxide (H₂O₂) and hydroxyl radical (OH^.) actively participate in the initiation of lipid peroxidation [Frankel, 1985].

Synthetic antioxidants are usually added to foods to preserve fats (BHT, BHA, etc). Recently, there is a considerable interest in the food industry and in preventive medicine in the development of "natural antioxidants" from plant material [Loliger, 1991]. Plant tissues are the main biological systems that synthesize a-tocopherol, ascorbic acid, and carotenoids, but in addition they are also rich in a wide variety of phenolic compounds [Cuvelier et al., 1996]. Flavonoids and other plant phenolics have been reported to have multiple biological effects such as antioxidant [Kim et al., 1994; Yanishlieva et al., 1995], anti-inflammatory [Haraguchi et al., 1996] and antimicrobial activities [Taylor et al., 1996].

The objective of this study was investigation of the inhibitory activities of Thymus tosevii extracts on the thermal autooxidation of lard.

Plant material

Speciments of air dried herbal parts of Thymus species included in the investigations were from: Thymus tosevii var. tosevii (collected near Kičevo, in 1997); T. tosevii var. longifrons (collected on mountain Mavrovo in 1997); T. tosevii var. degenii (collected from locality Vrben in 1997) and T. tosevii var. degenii (from locality Lazaropole, 1997).

Extraction procedure

Milled plant material (1.0 g) was extracted by continual mixing with 100 ml mixture of ethanol:water (7:3, v/v), 24h at room temperature. After filtration the solution was evaporated until only water remained. Water phase was extracted first, with diethylether to obtained ether extract, and secondly, with ethyl acetate to obtained ethyl acetate extracts. Both extracts were dried over anhydrous sodium sulphate. Filtrated and concentrated under vacuum up to the concentration of 1 g per 10 ml of extract (10%). Primary prepared extracts were further diluted with ethyl acetate to obtained 0.5, 1.0 and 5.0% working concentrations of the extracts.

Preparation of lard

Standard substances of BHA (Merck, Germany), caffeic acid and luteolin (Extrasintese, France) dissolved in ethyl acetate were added to lard in amount of 0.02% final concentration. Each of the ether and the ethyl acetate extracts were added to the lard in final concentration of 0.04%. The blank sample (control) contained ethyl acetate in concentration of 0.04%.

Determination of total flavonoides

Total flavonoides content was determined by spectrometry, using AlCl₃ as complex forming reagent. 1 ml of extract was hydrolyzed with concentrated HCl (25 %) in acetone media under reflux in duration of 30 min. After cooling and filtration, flavone aglycones were taken into ethylacetate media in separating funnel, dried over anhydrous sodium sulphate, filtrated in 50 ml volumetric flask and filled up with ethyl acetate. Two 10 ml portions of last solution were placed in two 25 ml volumetric flasks. 1 ml 2% solution of AlCl₃ in 5.0% acetic acid in methanol was added into one flask and than both was filled up with acetic acid in methanol. After 30 min absorbance of the solution with AlCl₃ was measured at l=390 nm against the solution without AlCl₃ (blank solution).

Calibration curve

Standard substance of luteolin was used for preparation of 100 mg^.ml^-1 stock solution in methanol. Five dissolution were made in concentration range from 1.0 to 10.0 mg^.ml^-1. Absorbance of the each solution was measured at 390 nm, 30 minutes after adding AlCl₃ against the respective solutions without AlCl₃.

Determination of peroxide value

Thermal autooxidation of lard was carried out in a thermostat chamber at 60°C. At definite time intervals samples were removed and the peroxide value (Pb) was determined by the Wheeler method [Trajković et al., 1983]. The experiment was carried out in triplicate.

Total flavonoid contents in wild Thymus extracts were determined by spectrometry, using AlCl₃ as complex forming reagent. In the concentration range of 1.0-10.0 mg^.ml^-1, luteolin obeys Lambert-Beer's low with the following regression equation: A=-0.003 + 0.18g (r=0.999). The average contents of total flavonoids in the extracts, expressed as luteolin, are summarized in Table 1.

The content of total flavonoids in the Thymus tosevii species ranged from 71.8 to 225.0 mg^.ml^-1 in ether extracts and from 32.0 to 130.0 mg^.ml^-1 in ethyl acetate extracts. Ether extracts of T. tosevii var. tosevii, T. tosevii var. degenii (Vrben) and T. tosevii var. degenii (Lazaropole) contained more total flavonoids (225.0, 71.8 and 72.5 mg^.ml^-1, respectively) than their ethyl acetate extracts (130.0, 52.5 and 32.0 mg^.ml^-1, respectively). An exception was the ether extract of T. tosevii var. longifrons, which contained less total flavonoids (98.0 mg^.ml^-1) than its ethyl acetate extract (101.0 mg^.ml^-1). Both ether and ethyl acetate extracts of T. tosevii var. tosevii were significantly richer in flavonoids than the other samples.

Structure dependent antioxidant activity of flavonoids is a well-documented phenomenon [Huang et al., 1992]. There is a general agreement that ortho-dihydroxylation of the B ring contributes markedly to the antioxidant activity of flavonoids, and all compounds with the 3',4'-dihydroxy configuration studied thus far possessed antioxidant activity. On the contrary, meta-5,7-hydroxilation of A ring apparently had little, if any, effect on antioxidant activity [Pratt, 1992]. Flavonoids from Macedonian Thymus sp. posses various hydroxyl patterns, ortho-dihydroxyl in ring B as well as 1,3-dihidroxyl systems on ring A, in combination with 2,3-double bond [Kulevanova, 1997]. Testing their antioxidative activity was carried out using extracts in which flavonoids were identified and determined.

The inhibitory activities of T. tosevii var. tosevii extracts on the thermal autooxidation of lard are presented at Graph 1. Considering the significantly lower peroxide value of extracts compared to the control, it is obvious that the extracts in all concentrations have shown inhibitory activity. Within 17 days, BHA and caffeic acid have shown very strong antioxidant activity. All the extracts have demonstrated great inhibition for 5 days, keeping the peroxide value below 8 mmol O₂^.kg^-1. Inhibitory effect raises according to the concentration. Thus, the largest inhibition was obtained with 5 % extracts. Regarding the type of the extracts, 5% ether extracts from T. tosevii var degenii (Lazropole) and T. tosevii var. longifrons (Graph 2 and Graph 3, respectively) showed greater inhibiting action. High inhibition also showed 5% ethyl acetate extracts from T. tosevii var. degenii (Vrben) and T. tosevii var. tosevii (Graph 1 and Graph 4, respectively). Moreover, 5% ethyl acetate extract from T. tosevii var. tosevii demonstrated stronger activity than lyteolin (Graph 1). The inhibitory activity on autooxidation of lard was not in linear correlation to the flavonoids content in the extracts. Probably a possible synergism of flavonoids with other components present in the wild Thymus extracts may be responsible for this effect.
 

Graph 1. Antioxidative activity of ether and ethyl acetate extracts of T. tosevii var. tosevii	Graph 2. Antioxidatite activity of ether and ethyl acetate extracts of T. tosevii var. degenii
Graph 3. Antioxidatite activity of ether and EtOAc extracts of T. tosevii var. longifrons	Graph 4. Antioxidatite activity of ether and ethyl acetate extracts of T. tosevii var. degenii

Ether and ethyl acetate extracts of four Thymus species from Macedonian flora possessed certain antioxidant activity, showed by the inhibition of thermal autooxidation of lard termostating on higher temperature (60°C). All investigated extracts contained various total flavonoids content. The correlation between the antioxidant activity and flavonoids content was low, meaning that besides flavonoids, there were probably other components responsible for the antioxidative activity.

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