INFLUENCE OF THE FE AND CU PRESENCE IN TEA EXTRACTS ON ANTIOXIDANT ACTIVITY

Anna Gramza-Michałowska¹, Rafał W. Wójciak², Józef Korczak³, Marzanna Hęś³, Julia Wiśniewska², Zbigniew Krejpcio^2
1 Department of Food Service and Catering, Faculty of Food Science and Nutrition, Poznań University of Life Sciences, Poland
² Department of Food Hygiene and Human Nutrition, The August Cieszkowski Agricultural University of Poznan, Poland
³ Department of Food Technology and Human Nutrition, The August Cieszkowski Agricultural University of Poznan, Poland

ABSTRACT

Tea leaves (Camelia sinensis L.) were extracted with two different solvents, namely water and ethanol. The absorptive atomic spectrometry technique – ASA was used to analyze the quantities of Fe and Cu in the tea extracts. Highest Cu level was marked in green tea ethanol extract. Fe content however was highest in black tea ethanol extract. Dependence of extracted Cu and Fe quantity from the solvent and tea leaf's genus was affirmed. Tea leaves can be the source of mineral components and trace elements, as well as the undesirable substances. Because both the iron and the copper can catalyze the oxidation processes, its presence is essential for the lipid stability. Metals content in lipids with addition of tea extracts shaped on level, which did not act prooxidatively. Cu content in lipid with green tea ethanol extract addition was five times higher; however Fe content in lipid with black tea ethanol extract addition was twice higher than in remaining samples. The result of tea extracts influence on oxidative stability of sunflower oil in Rancimat test showed best antioxidant activity in sample with green tea ethanol extract added.

Key words: tea extracts, Camelia sinensis, Cu, Fe, polyphenols, sunflower oil, Rancimat test.

INTRODUCTION

Tea leaves (Camelia sinensis L.) are source of such mineral elements as essential for health: zinc, manganese, iron, magnesium, copper, titanium, aluminum, strontium, bromine, sodium, potassium as well as nickel, chromium and also phosphorus, iodine and fluorine [5, 8, 9, 19]. It was confirmed that the content of metals might be an adequate discriminator of tea origin and that with growth of leaves the content of heavy metals such as lead, copper, cadmium lowers, what indicates that these metals are absorbed mainly during the early stages of plant growth [20]. Researches showed however that the metal content in tea leaves infusion depends on temperature and power of infusion as well as tea sort [16, 19]. Tea leaf can be the source of mineral components and trace elements indispensable for the man's health, as well as the undesirable substances. Because both the iron and the copper can catalyze the oxidation processes [1, 22, 24], its presence is essential in using the plant extracts for the lipid stabilization.

Objective of the research was to evaluate the Fe and Cu content in tea extracts and its influence on antioxidative activity in examined lipid systems.

MATERIALS AND METHODS

Tea extracts

Green and black Yunan tea aqueous and ethanol extracts were prepared according to method presented by Gramza et al. [10]. Tea samples were purchased from the market. Aqueous extracts were prepared by boiling grinded tea leaves, followed by stirring for 15 min at 80°C. Collected extracts were filtered, centrifuged and than lyophilized (HETO). Ethanol extracts were prepared after 24 h maceration of tea leaves in 95% ethanol, in ambient conditions. Collected extracts were filtered and centrifuged. Ethanol was evaporated on rotary evaporator (RVO 200A, INGOS). The powdered aqueous and ethanol extracts were kept frozen (-18°C) until further use. The extracts concentration was determined experimentally on level of 1000 ppm. The extraction procedures of tea leaves resulted in four extracts: green tea ethanol, green tea aqueous, back tea ethanol and black tea aqueous.

Tea extracts polyphenol content analysis

Total polyphenol content in tea extracts was measured with Folin-Ciocalteu Phenol Reagent [Fluka] using catechin [Sigma-Aldrich] as the reference standard – method described by Horwitz [12].

Tea extracts metals content analysis

Analysis of metals: Fe and Cu content in tea extracts were measured by flame atomic absorption spectrometry technique – ASA (FAAS-3, Zeiss) according to the method by Olejnik et al. [17]. Tea extracts samples were mineralized (450°C) to obtain carbon free white ash. Afterwards the ash was dissolved in 5 mL of nitric acid, filtered and analyzed. Results of six independent evaluations were expressed as ľg·g^-1 of the extract’s dry weight.

Lipid substrates

For the measurement of lipid stability of tea extracts sunflower oil (ZPT, Kruszwica) was purchased. Lipid samples with antioxidants added, and lipids with no additives (control sample) were incubated in Rancimat test conditions.

Rancimat test

Rancimat test (Metrohm, Switzerland) based on conductometric measure of volatile acids dissociation products formed during lipids oxidation process. Lipid sample (2.5 g) was oxidized in a reaction vessel at 110°C; air flow (20 l·h^-1). The end of induction period was characterized by the sudden increase of water conductivity, due to dissociation of volatile carboxylic acids [15]. On the ground of received induction periods (Ip) reprints antioxidant effectivity of applied extracts were marked. Protection coefficient (Pc) was determined as the relation of induction period of sample with antioxidant to induction period of control sample. Antioxidant: tea extracts addition was on 1000 ppm, α-tocopherol and BHT addition was on 200 ppm level. All antioxidants were put in to lipid systems after earlier dissolution in 80% ethanol and than evaporated. Pc values > 1 confirm antioxidant properties; Pc < 1 confirms prooxidant activity of examined additives.

Statistical analysis

The results were obtained from a minimum of six independent experiments and averaged. Data were analyzed by the analysis of variance (p £ 0.05) to estimate the differences between values of compounds tested. Results were processed by the computer program Statistica 6.0.

RESULTS AND DISCUSSION

Metal content analysis conducted with ASA showed different concentrations of Fe and Cu as it is shown on Figure 1 and 2. There was no significant differences in Fe content in green tea extracts, although it was noted in black tea extracts (p < 0.01), higher in ethanol than aqueous extract. The significant differences were found in extracts differing type of solvent and leaves used for the extraction (p < 0.01). No presence of metals was found in pure solvents used for the extraction.

Fig. 1. Fe content in Yunan tea extracts

Cu level was significantly differing among the extracts (Fig. 2). Highest content of Cu was found in green tea ethanol extract – 56.91 [ľg·g ^-1 dry weight].; significantly lower contents were found in green tea and black tea aqueous extracts respectively 6.91 and 10.73 ľg·g^-1 dry weight. Statistical analysis showed the dependency of extracted Cu content from the solvent (p < 0.05) and kind of tea (p < 0.05).

Fig. 2. Cu content in Yunan tea extracts

Substances mainly responsible for chelating activity of plant extracts were determined. Results of total polyphenol content expressed as catechin equivalent varied in green and black tea leaves extracts. Highest polyphenol content was observed in ethanol extracts. Total polyphenol content was as followed: green tea ethanol extract – 798.21 mg·g^-1; green tea aqueous extract – 287.36 mg·g^-1; black tea ethanol extract – 605.11 mg·g^-1 and black tea aqueous extract – 198.52 mg·g^-1 extracts dry weight. Statistical analysis allowed to submit significant differences in polyphenols content in all samples (p < 0.001).

Main factors influencing the metal content are horticulture conditions and plant species [7, 20]. Green tea leaves are rich of minerals and trace elements, essential for human body. Drinking tea infusions could be a source of mineral substances [23]. Tea leaves also contain undesirable substances like excess of copper (plant protection agent residue) and atmospheric contaminations [16, 20].

Fernandez-Caceres et al. [8] analyzed the metals content and showed that Fe content in green tea leaves oscillate on levels 108.5-321.8 ľg·g^-1 of dry weight and black tea 176.0-946.2 ľg·g^-1 dry weight. Copper content was – 37.0 ľg/1g for green tea and 22.5 – 37.0 ľg/1g black tea leaves respectively. According to Chu and Juneja [5] green tea leaves contain 100 – 200 ľg·g^-1 dry weight Fe and 15-20 ľg·g^-1 dry weight Cu. In tea leaves infusion it was evaluated 1ľg Fe /1g of leaves, no copper was found. Infusion temperature is very important factor influencing the metals content. Tascioglu and Kok [19] found that the increase of brewing temperature from 18-80°C influences the metal content. Higher temperature than 60°C resulted in decrease of Fe and Cu in the infusion of black tea, which might be caused by the instability of soluble complexes, which might precipitate, or dissolute. In green tea infusions temperature increase did not influenced Fe content, although it significantly affected Cu content. Additionally it was stated that the level metal extraction in different temperatures depends also from tea kind and infusion strength.

Knowledge of metal content is very important in use of extracts as antioxidants in lipid systems, because Fe and Cu are known as oxidation catalysts [2, 6, 14]. Fe and Cu contents in lipid systems were also evaluated (Table 1). It was stated that metals content is not a prooxidative level. Cu content in green tea ethanol extract was five times higher and Fe in black tea ethanol extract twice higher than in remaining samples.

Table 1. Fe and Cu content added with tea extracts to lipid systems (mg·kg-1 extract’s dry weight)

EXTRACT		Cu [mg·kg-1 d.w.]	Fe [mg·kg-1 d.w.]
GREEN	ETHANOL	0.059	0.019
	AQUEOUS	0.007	0.016
BLACK	ETHANOL	0.012	0.046
	AQUEOUS	0.011	0.022
Metal content inducing prooxidation process in lipid systems1		0.05 – 0.07	0.3 – 1.0

1-Anderson & Lingnert 1998, Evans et al 1951

Lipid stability analysis in Rancimat test allowed evaluating the induction periods of examined lipid samples. As a lipid substrate the sunflower oil was used. Lipids with antioxidants added induction periods are shown on Figure 3. Higher induction period in comparison to control sample without antioxidant added confirms better antioxidant activity of the extract. Analysis of the sunflower oil stability in Rancimat test showed, that tea extract had significant influence. Longest induction period was found in lipid with green tea ethanol extract added (6.45 h).

The protection coefficiency in sample with green tea ethanol extract was similar to α-tocopherol, where protection coefficiency was 1.62, but higher than in BHT (1.11). The statistical analysis did not found significant influence of Cu and Fe presence in tea extracts on lipid stability.

Fig. 3. Tea extracts influence on oxidative stability of sunflower oil in Rancimat test conditions (1000 ppm)

Ho et al. [11] studied green, black and oolong tea extracts activity in Rancimat test. The highest activity was found in lipid with addition of green tea extract, lowest in oolong tea extract. Results of present research do correlate with results of Chen et al. [3, 4]. Green tea extract in rapeseed oil, incubated in 100°C possessed stronger antioxidant activity than BHT and rosemary extract in concentration of 0.02%.

Von Gadow et al. [21] showed that one of tea component – catechin, possessing strong antioxidative activity, was also very active in lard in Rancimat test conditions (90ºC). Its activity was stronger than BHT and α-tocopherol. Protection coefficiency was as follows: α-tocopherol 5.01; BHT 6.47; catechin 43.22; quercitin 42.7; rutin 4.79.

The results of above analyses confirm probable metal ions chelating properties of tea extracts. Confirmation of those assumptions was present in other investigations of the authors [10]. It was stated that increase of antioxidant concentration resulted the increase of chelating Fe²⁺ ions ability and was highest in addition of 1000 ppm to analyzed system. Other research showed that metal ions chelating ability of flavonoids was due to the presence of ortho-dihydroxyl group in B-ring and catechol structure in C-ring [18], and affirmed that flavonoids were able to bind with metal ions across 3- or 5- hydroxyl and 4– keto- substituents or hydroxyl groups in –ortho position of B-ring [13]. It was stated that other tea components like theaflavins also showed high copper and iron ions chelating ability.

CONCLUSIONS

Tea leaves can be the source of mineral components and trace elements, as well as the undesirable substances. Because both the iron and the copper can catalyze the oxidation processes, its presence is essential in using the plant extracts for the lipid stabilization. The tea extracts metals content analysis by the absorptive atomic spectrometry technique showed considerable differentiation of Cu and Fe concentrations. The highest Cu level was marked in green tea ethanol extract. Fe content however was highest in black tea ethanol extract. Dependence of extracted Cu and Fe quantity from the solvent and tea leaf's genus was affirmed. Presence of these metals was not found in applied solvents.

Metals content was also estimated in lipids with addition of extracts as antioxidants. It shaped on level, which how literature passes, did not act prooxidatively. Cu content in lipid with green tea ethanol extract addition was five times higher; however Fe content in lipid with black tea ethanol extract addition was twice higher than in remaining samples. The results of tea extracts influence on oxidative stability of sunflower oil showed best antioxidant activity in sample with green tea ethanol extract added.

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Anna Gramza-Michałowska
Department of Food Service and Catering, Faculty of Food Science and Nutrition, Poznań University of Life Sciences, Poland
phone: +4861 848 7331
fax: +4861 848 7430
Wojska Polskiego 31
60-624 Poznań
Poland
email: angramza@up.poznan.pl

Rafał W. Wójciak
Department of Food Hygiene and Human Nutrition,
The August Cieszkowski Agricultural University of Poznan, Poland
Wojska Polskiego St. 28, 60-624 Poznan, Poland

Józef Korczak
Department of Food Technology and Human Nutrition,
The August Cieszkowski Agricultural University of Poznan, Poland
Wojska Polskiego St. 28, 60-624 Poznan, Poland

Marzanna Hęś
Department of Food Technology and Human Nutrition,
The August Cieszkowski Agricultural University of Poznan, Poland
Wojska Polskiego St. 28, 60-624 Poznan, Poland

Julia Wiśniewska
Department of Food Hygiene and Human Nutrition,
The August Cieszkowski Agricultural University of Poznan, Poland
Wojska Polskiego St. 28, 60-624 Poznan, Poland

Zbigniew Krejpcio
Department of Food Hygiene and Human Nutrition,
The August Cieszkowski Agricultural University of Poznan, Poland
Wojska Polskiego St. 28, 60-624 Poznan, Poland

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