Antioxidant activity of some foods containing phenolic compounds

International Journal of Food Sciences and Nutrition ( 2001 ) 52, 501– 508

Antioxidant activity of some foods containing phenolic compounds S. Karakaya, S.N. El and A.A. Taçcs Ege University, Engineering Faculty, Food Engineering Department, 35100, Bornova, Izmir, Turkey

This study was designed to determine the total phenols ( TP) and total antioxidant activity ( TAA) of some liquid and solid plant foods that are commonly consumed in Turkey. Total phenols were analysed according to the Folin– Ciocalteu method and antioxidant activities of these compounds in aqueous phase were assessed by measuring their direct ABTS– radical scavenging abilities. Total phenols varied from 68 to 4162 mg/l for liquid foods and from 735 to 3994 mg/kg for solid foods. TAA of liquid and solid foods ranged between 0.61– 6.78 mM and 0.63–8.62 mM, respectively. Total antioxidant activities of foods were well correlated with total phenols ( r 2 = 0.95). According to content of total phenols per serving, liquid foods were in the order of black tea > instant coffee > coke > red wine > violet carrot juice > apricot nectar > Turkish coffee > grape molasses > sage > white wine > linden flower, and solid foods were in the order of red grape > raisins > tarhana > dried black plum > dried apricot > grape > fresh paprika > fresh black plum > Urtica sp. > cherry > fresh apricot > paprika pickle > paprika paste.

Introduction Oxygen is essential to life; without it, living things cannot survive. Most of the potentially harmful effects of oxygen are believed to be due to the formation and activity of reactive oxygen species ( ROS) acting as oxidants, that is, compounds with a tendency to donate oxygen to other substances. Many reactive oxygen species are free radicals. Free radicals and other reactive oxygen species in the body are derived either from normal essential metabolic processes or from external sources. Their chemical reactivity can damage all types of cellular macromolecules including proteins, carbohydrates, lipids and nucleic acids. Several of these effects have been implicated in the causation of cataracts, atherosclerosis, some degenerative diseases and some type of cancer ( Kehrer & Correspondence to: S. Karakaya ISSN 0963-7486 printed/ISSN 1465-3478 online 01/060501-08 © 2001 Taylor & Francis Ltd DOI: 10.1080/09637480120057000

Smith, 1994; Langseth, 1995). The human body has several mechanisms of defence against free radicals and other reactive oxygen species. Important lines of defence are a system of enzymes, including glutathione peroxidase, superoxide dismutase and catalase and body fluids which act as antioxidants such as glutathione, ubiquinol and uric acid. Other components of antioxidants in the diet are vitamin E, vitamin C, carotenoids and phenolic compounds ( Shadidi & Naczk, 1995). Phenolic compounds are commonly found in both edible and non-edible plants. They are important in the plant for normal growth development and defence against infection and injury. Also, the presence of phenolic compounds in injured plants may have an important

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effect on the oxidative stability and microbial safety. Although phenolic compounds do not have any known nutritional function, they may be important to human health because of their antioxidant potency ( Hertog et al., 1995; Shadidi & Nazck, 1995; Hollman et al., 1996 ). The importance of the antioxidant constituents in the maintenance of health and protection from coronary heart disease and cancer is also raising interest among scientists, food manufacturers and consumers as the trend of the future is moving toward functional food with specific health effects (Velioglu et al., 1998; Kähkönen et al., 1999; Robards et al., 1999 ). The beneficial health-related effects of certain phenols or their potential antinutritional properties, especially when these compounds are present in large quantities in foods, are of importance to consumers. Therefore, it is of great interest to evaluate the antioxidant potential of foods. For this purpose, 13 liquid and 13 solid foods from the typical Turkish diet were selected and total phenols (TP) content and total antioxidant activities (TAA) were determined. Materials and methods Chemicals

( + )-catechin hydrate (C-1251 ), Folin–Ciocalteu’s phenol reagent ( F-9252 ), myoglobin (from horse heart M-1882), sephadex G15 ( G15–120 )

were obtained from Sigma Chemical Company. Trolox ( 6-hydroxy-2, 5, 7, 8-tetramethylchroman-2-carboxylic acid) (catalogue no. 23, 881– 3) and ABTS ( 2– 29 -azinobis( 3-ethylben-

zothiazoline-6-sulphonic acid) diammonium salt) ( catalogue no. 27–72–1) were purchased from Aldrich Chemical Company. All other chemicals were supplied from Riedel-de-Haen Chemical Company and were of the highest quality available. Sources and preparation of samples Thirteen liquid foods (fruit juices, tea, coffee, etc.) and 13 solid foods (vegetables and fruits) were selected for sampling according to the data of the brand names of commonly consumed and/or purchased foods. These data were provided by the State Institute of Statistics of Turkey and from the records of the three hypermarkets in Izmir, Turkey. The 13 liquid foods were violet carrot juice, red wine, white wine, raki ( traditional alcoholic beverage consumed in Turkey), coke, Turkish coffee, instant coffee, black tea, sage, linden flower, juice of Urtica sp, apricot nectar and grape molasses. The 13 solid foods were fresh black plum, dried black plum, red grape, grape, raisins, fresh apricot, dried apricot, cherry, Urtica sp., fresh paprika, paprika paste, paprika pickle and tarhana ( traditional fermented food prepared by mixing yoghurt, wheat flour, baker’s yeast, onion, green pepper, fresh paprika, peppermint, tomato and salt). Different brand names of liquid foods were purchased from a hypermarket in Izmir, Turkey. Different brands of each liquid food were combined in equal servings to a composite ( Table 1). Composite of liquid foods with different production dates were used in triplicate analysis.

Table 1. Number of brands and preparation of liquid food samples

Liquid foods Black tea Turkish coffee Instant coffee Sage Linden flower Juice of Urtica sp. Coke Red wine White wine Raki

Number of brands

Amount of sample

Volume of added water ( 98°C)

( minute)

9 4 2 2 2 – 1 5 3 1

12 g 3.5 g 2g 5g 5g 5g 20 ml 20 ml 20 ml 20 ml

450 ml 75 ml 250 ml 200 ml 200 ml 200 ml – – – –

15 1 – 5 5 5 – – – –

Time

Treatment Stewing Stirring, boiling Stirring Stewing Stewing Stewing Evaporation of CO2 Evaporation of alcohol under vacuum to dryness then making up the volume to 20 ml with distilled water

Antioxidant activity with phenolic compounds 503

Grape molasses ( three brands), apricot nectar ( three brands) and violet carrot juice ( two brands ) were used directly after being diluted

with distilled water to appropriate concentrations. Other liquid foods were prepared as shown in Table 1. All liquid samples were stored at –40°C until analysed. Fresh black plums, fresh paprika, grape, red grape, fresh apricot and Urtica sp were purchased on the same day from an open air street market for 3 consecutive weeks. Foods purchased on different weeks were used for triplicate analysis. One brand name of dried black plum, paprika paste, paprika pickle, raisins and dried apricot were purchased from a hypermarket in Izmir, Turkey. Foods with different production dates were selected for triplicate analysis. Home-made tarhana samples were prepared at three different places using the same recipe. Fresh and dried solid foods were washed with distilled water and after their nonedible parts were removed they were placed in polyethylene bags which were mechanically sealed under nitrogen. All samples were then placed in a deep-freezer (–40°C). Just before the analysis, 100 g of sample was thawed and homogenized under nitrogen for 1 min by using a blender and an appropriate amount of sample was weighed for the analysis. Analysis of total phenols The extraction of total phenols was carried out according to the method of Vinson et al. ( 1995 ). A solution of 6 M HCl prepared in 75%

methanol/water solution and 1.2 M HCl prepared in 50% methanol/water solution were used as solvents for liquid and solid foods, respectively. The mixtures in screw-capped tubes were placed in a 90°C oven and shaken at 15 min intervals for a period of 2 h. After the 2-h period, the tubes were allowed to cool to room temperature and then diluted with distilled water to an appropriate volume. Total phenols were analysed according to Folin–Ciocalteu method (Singleton & Rossi, 1965 ) by using ( + )-catechin hydrate as the standard and the results were given as catechin equivalents ( CE). Measurement of total antioxidant activity Total antioxidant activity ( TAA) was determined by the method of Miller et al. ( 1993 ). This spectrophotometric technique measures the relative abilities of antioxidants to scavenge the 2– 29 -azinobis ( 3-ethylbenzothiazoline6-sulphonate ) radical cation ( ABTS + ) in comparison with the antioxidant potency of standard amounts of Trolox. The radical cation ABTS + , which is produced by the ferrylmyoglobin radical generated from metmyoglobin and H2 O2 in the presence of peroxidase, is a blue/green chromogen with a characteristic absorption at 734 nm. Results and discussion Total phenols Total phenols of samples varied from 68 to 4162 mg/l CE for the liquid foods and from 735

Table 2. Total phenols ( TP) of liquid and solid foodsa ,b Liquid foods Violet carrot juice Red wine White wine Coke Turkish coffee Instant coffee Black tea Sage Linden flower Apricot nectar Grape molasses

a b

Total phenols ( mg/l) 772 2003 280 834 2389 1242 1492 291 68 736 4162

± ± ± ± ± ± ± ± ± ± ±

119 66 69 178 350 148 191 69 19 151 490

Mean ± standard deviation. Total phenols were calculated as catechin equivalents ( CE).

Solid foods Fresh black plum Dried black plum Red grape Grape Raisins Fresh apricot Dried apricot Cherry Urtica sp. Fresh paprika Paprika paste Paprika pickle Tarhana

Total phenols ( mg/kg) 1435 3679 2206 1580 3994 735 3075 1054 1254 2039 1476 901 3717

± ± ± ± ± ± ± ± ± ± ± ± ±

406 566 612 371 576 224 345 270 221 332 408 243 328

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Table 3. Serving sizes of liquid and solid foods Liquid foods Violet carrot juice Red wine White wine Coke Turkish coffee Instant coffee Black tea Sage Linden flower Apricot nectar Grape molasses

Serving size ( ml) 240 100 100 330 70 240 240 240 240 240 30

to 3994 mg/kg CE for the solid foods ( Table. 2). Total phenols of juice of Urtica sp. and raki were determined below 5 m M which was the least detectable concentration in the method. For this reason, further analysis were not applied to these samples. The Food and Drug Administration ( FDA) established the lists of ‘Reference Amounts Customarily Consumed Per Eating Occasion’. The FDA defines the term ‘serving’ or ‘serving size’ as an amount of food customarily con-

Figure 1. Total phenols per serving for liquid foods.

Solid foods Fresh black plum Dried black plum Red grape Grape Raisins Fresh apricot Dried apricot Cherry Urtica sp. Fresh paprika Paprika paste Paprika pickle Tarhana

Serving size ( g) 65 40 75 75 40 65 40 65 60 50 30 50 40

sumed per eating occasion by persons 4 years of age or older expressed in a common household measure that is appropriate to other food ( Federal Register, 1993). In this respect, total phenols of all samples were calculated on the basis of their serving size. Serving size of all samples are represented in Table 3. As shown in Figure 1, total phenols (mg/ serving ) of liquid samples were found to be in the following order: black tea > instant coffee > coke > red wine > violet carrot juice > apricot nectar >


Figure 2. Total phenols per serving for solid foods.

Turkish coffee > grape molasses > sage > white wine > linden flower. Total phenols ( mg/serving ) of solid foods were in the order of red grape > raisins > tarhana > dried black plum > dried apricot > grape > fresh paprika > fresh black plum > Urtica sp. > cherry > fresh apricot > paprika pickle > paprika paste ( Figure. 2). In general, phenolic compounds of plants may vary with maturity period, variety and climatic conditions (Prior et al., 1998 ). Phenolic compounds contribute to the resistance of plants to physical stress which is caused by the injuries that take place during mechanized harvesting or due to insect bites and biological stress resulting from being infected by fungi, bacteria and viruses. Immediately after injury, there is an oxidation of pre-existing phenols and after their subsequent degradation, phenol content decreases. At the later stage, the usual response of a plant to stress is an increase in the total phenols. This response was shown in a study conducted on a kind of orange. After 48 h of healing, over a twofold increase in total phenols was found in the peel of wounded Valencia orange (Shadidi & Nazck, 1995). The content of foods can also be markedly affected during food processing. Phenolic compounds of processed foods were found to be approximately 50% lower than those of fresh products ( Hertog et al., 1992; Shadidi & Nazck, 1995).

Phenolic compounds constitute up to 35% of the dry weight of tea. The composition of phenolic compounds of tea may vary depending on variety, geographical origin, environmental conditions and uncontrolled fermentation conditions. Control of fermentation has important effects on the flavour and colour of tea and these depend on the degree of oxidation of tea phenols. Oolong tea and Pounchong tea are semi-fermented products with red and yellow colour, respectively, and have some of the characteristics of green and black tea. Phenolic compounds of Oolong tea are 70% oxidized while phenols of Pounchong tea are 30% oxidized due to enzymatic and non-enzymatic reactions that take place during fermentation ( Xie et al., 1993, Vinson et al., 1995 ). Phenolic compounds of coffee are produced from thermal degradation of carbohydrates, chlorogenic acid and lignins. Both roasting time and temperature were reported to affect the phenolic content and composition (Shadidi & Nazck, 1995). Phenolic compounds are important components of wine and they contribute to sensory characteristics such as colour, flavour, astringency and hardness of it. The composition of phenols in wine depends on the type of fruits used for vinification, extraction procedures of phenolic compounds, procedures employed for

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wine making and chemical reactions that occur during the ageing of wine. The content of phenolic compounds of wine has probably been more extensively studied than any other beverage. According to the results of the studies, the content of total phenolics of white wine changed from 50 mg/l to 2000 mg/l in gallic acid equivalents ( GAE). However, the content of total phenolics of a typical white wine was found as 250 mg/l. Total phenolics of a typical red wine ranged between 1000 and 4000 mg/l in GAE although some certain types of red wine contained as high as 6500 mg/l ( Shadidi & Nazck, 1995; Soleas et al., 1997 ). As wines, contents and relative proportions of phenols in juices may change as a result of enzymatic browning reactions and the formation of haze ( Shadidi & Nazck, 1995). The content of total phenols in foods may vary depending on these mentioned factors. From this point of view, significant differences could be noted among the results of researches on the content of total phenols in foods. Studies usually reporting a large range of total phenols make the comparison difficult. Discrepancies may be due to differences in cultivars or varieties ( Hertog et al., 1992). There is no recommended daily intake for phenolic compounds. Accurate data on population-wide intakes of phenolic compounds are not available ( Robards et al., 1999 ). In our recent study, we estimated the daily consumption of black tea, linden flower, sage, rosehip, violet carrot juice, grape molasses, tarhana, honey and Urtica sp. with 100 healthy adult volunteers using a food frequency questionnaire. In that study, the intakes of black tea, linden flower, sage, grape molasses, violet carrot juice and tarhana were reported to be ranging between 38–360 ml, 4–360 ml, 4–360 ml, 0.3–10 ml, 8–143 ml, 1.3– 40 g per day, respectively. According to the consumption profiles, intakes of phenolic compounds from black tea, linden flower, sage, grape molasses, violet carrot juice and tarhana were calculated as 57–357 mg, 0.27–24 mg, 1–105 mg, 1– 42 mg, 6–110 mg and 5–149 mg per day, respectively ( Karakaya & El, 1999). Total antioxidant activity ( TAA) Total antioxidant activities ( TAA) of liquid and solid samples are given in Table 4 Total antioxidant activity of white wine, coke, sage

Table 4. Total antioxidant activity ( TAA; mM) and ratio of TAA total phenols( TP) of samplesa Total antioxidant activity( TAA) mM

TAA/TP ´ 10 3

Liquid foods Violet carrot juice Red wine Turkish coffee Instant coffee Black tea Apricot nectar Grape molasses

2.67 5.30 4.88 3.31 4.44 0.61 6.78

3.5 2.6 2.0 2.7 3.0 0.8 1.6

Solid foods Fresh black plum Dried black plum Red grape Grape Raisins Fresh apricot Dried apricot Cherry Fresh paprika Paprika paste Paprika pickle Tarhana

2.67 8.08 6.84 2.75 8.62 0.63 6.85 1.65 3.67 2.19 0.63 6.60

1.9 2.2 3.1 1.7 2.2 0.9 2.2 1.6 1.8 1.5 0.7 1.8

a

Total phenols were calculated as catechin equivalents

( CE).

and linden flower were not detected. The ratios of TAA to total phenols for all samples are also shown in Table 4. Total antioxidant activity of the liquid foods varied from 0.61 mM for apricot nectar to 6.78 mM for grape molasses and for solid foods it varied from 0.63 mM for fresh apricot and paprika pickle to 8.62 mM for raisins. The ratios of total antioxidant activities to total phenols of all samples were in the order of violet carrot juice > red grape > black tea > instant coffee > red wine > dried apricot = dried black plum = raisins > Turkish coffee > fresh black plum > tarhana = fresh paprika > grape > grape molasses = cherry > paprika paste > fresh apricot > apricot nectar > paprika pickle. There was a relationship ( r 2 = 0.95 ) between total phenols and TAA for all samples. However, it is clear that this relationship is relevant to in this test system. Meyer et al. ( 1997 ) reported that the antioxidant activity of polyphenols were strongly dependent on the test system and substrate in order to be protected by the antioxidant. Sol-


eas et al. ( 1997) assayed TAA of 32 Ontario wines from various producers by using myoglobin/ABTS method. They reported that TAA of the samples were within the range of 6.6– 28.6 mmol/l. Also, Walters et al. (1997) tested red wines and found that the TAA of the samples ranged between 12.61– 22.24 mmol/l. In some studies, TAA of several juices and fruits have been reported as automated oxygen radical absorbance capacity ( ORAC). Results showed significant variation in the TAA among fruits with strawberry having the highest ORAC activity ( Cao et al., 1996; Wang et al., 1996 ). Velioglu et al. ( 1998 ) determined antioxidant activity and total phenolic in selected fruits, vegetable and grain products using b -carotene bleaching method. Their results indicated that there was a positive and highly significant relationship between total phenolics and antioxidant activity, when all plant materials were included in the statistical analysis. Kähkönen et al. ( 1999 ) studied antioxidant activity of plant extracts

containing phenolic compounds using inhibition of methyl linoleate oxidation method and no significant correlation was found between the total phenolic content and antioxidant activity. It could be clearly seen that the relationship between the total phenols and antioxidant activity is strongly related to the test system and the substrate in order to be protected by the antioxidant. In conclusion, our data suggest that studied samples can supply phenolic compounds with potential antioxidant activity. Phenolic compounds are considered to be non-essential nonnutrient dietary components. In view of their potentially health-promoting activities, this opinion might need to be modified in the future. Acknowledgement s—This work was funded by Scientific and Technical Research Council of Turkey ( TUBITAK). We thank Professor Nicholas J. Miller ( International Antioxidant Research Centre, UMDS-Guy’s Hospital, London, UK) for valuable comments on this study.

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