The Influence of the Preparation of Tea Extracts on their Antioxidant Capacity and Reactivity

Transcription

1 The Influence of the Preparation of Tea Extracts on their Antioxidant Capacity and Reactivity K. Jung, M. Seifert, Th. Herrling Keywords: Tea, antioxidant activity, free radical, DPPH, ESR spectroscopy ABSTRACT Tea gives a powerful antioxidant beverage consumed in a lot of soft drinks today. The characterization of its antioxidative properties during and especially after its preparation procedure inclusive their long time stabilities is an important tool for the soft drink industry. The antioxidative capacity and reactivity are two parameters which gives a comprehensive description of the antioxidant properties. Both parameters are integrated in the Antioxidative Power AP, a 2D factor. The 2D volume and time-dependent approach of AP permits a higher differentiation between the antioxidative properties of the individual types of tea. The AP of the green tea beverage exceeds all other types of tea. Black tea has an AP which is six times lower than that of green tea. The reason for this lower antioxidative capacity and reactivity bases on the fermentation process of green tea. The fermentation process involves an enzymatic oxidation of green tea eliminating the antioxidative character of the phenols (oxidation of phenols). Green tea which has an excellent antioxidative capacity disposes also of a fast reactivity. Both parameters meet in an extraordinary high AP. Brew temperature and dwell time influence the AP in a different manner. Whereas brew temperatures between 25 C and 90 C have only a moderate influence (maximum 16%) on the AP, the increase of the dwell time from 1 min. to 30 min. enhances the AP by about 62%. Storing of the green tea beverage at 2-8 C (refrigerator) for 24 hours does not show any influence on the AP. Green tea is favoured as ingredient with excellent antioxidative properties ready for the application as antioxidant in various drinks. The AP method as a comprehensive tool select beverages with the most powerful antioxidant activity (in terms of capacity and reactivity) beginning from raw material up to the final product following all proceeding steps of green tea beverages. 70

2 1. Introduction Tea is one of the most widely consumed beverages. The tea plant (Camellia sinensis) has been used for over 5,000 years for its specific aroma, taste, and putative positive physiological functions. It has recently gained increasing attention because of its relevant content of antioxidant components. Tea is drunk to improve blood flow, eliminate toxins, and improve resistance to diseases. Like apples, grapes and cocoa, tea is a rich source of flavonoids and other polyphenols. The flavonoid content can be effected by different types of processing. Freshly harvested tea leaves are processed differently to produce specific types of tea (green, oolong, and black tea). Green tea is heated and dried to avoid enzymatic oxidation. Oolong tea is semifermented to permit a moderate level of enzymatic oxidation during processing and then dried. Black tea is the most thoroughly oxidized enzymatically. It is the degree of oxidation that affects the polyphenole profile of the tea. Many studies have shown that green and black teas can have a wide range of pharmaceutical properties including being antihypertensive [1], antioxidative [2, 3], antiarteriolemic [4, 5], anticarcinogenic [6, 7], and hypocholesterolemic [8, 9]. The effects of tea consumption on cancer and other diseases are conflicting, mainly because tea types greatly differ in their functionality depending on species, region of cultivation, manufacture. However, the concentration and activity of diverse antioxidants in the different teas should be first considered before drawing any conclusions about overall benefits of teas [10]. In fact, each tea differs both in terms of composition (antioxidant reactivity) and concentration (antioxidant capacity) of antioxidant compounds. Black teas [11] have low amounts (2-6% of extracted solids) of theaflavins (TF) and high thearubigen (20%) contents, whereas green teas have much higher catechin contents (30-42%), particularly EGCG (epigallocatechin gallate), which is the most abundant catechin. It is known that, due to the different structures, these substances show a different pattern of activity against the different free radical species. The dietary phytophenolics have been recognized largely as beneficial antioxidants that can scavenge harmful active oxygen species including O.- 2, H2 O 2, OH., and 1O. 2, but they can also act as prooxidant in some conditions. 71

3 The combined effects of the different antioxidants contained must be taken into account when considering the health benefit of food. The total Antioxidative Power AP [12] of tea is not related to a particular kind of polyphenol but to the combined activity of diverse antioxidants, including phenolic acids and polyphenols. In this regard, we have measured the antioxidative capacity and activity as an integrated value the Antioxidative Power AP. The molecular combination of synergistic antioxidants exert much higher activity, thus demonstrating that antioxidants work within a complex pathway which involves concerted activity of synergistic antioxidants in counteracting the negative effects of free radical species [13,14]. The AP of tea mainly relies on its water-soluble components, because these represent the principal antioxidant molecules in a tea. The lipid soluble antioxidants are represented by carotenoids which are present in very limited concentration. In this study we have mainly compared the AP of green tea beverages undergo preparation procedures with different parameters like variation in brew temperature and dwell time. In order to gain better insight into the antioxidant and radical scavenging properties of green tea, we examined their AP, indeed as their ability to counteract the reactive free radical DPPH [15,16] by means of Electron Spin Resonance (ESR) spectroscopy [17, 18]. The AP method allows an accurate quantification and qualification of the free radical scavenging properties of the antioxidant compounds. The measuring unit of the AP is [(number of scavenged free radicals) / (mg input of antioxidant substance * reaction time in minutes)]. The aim was to better understand the complex pattern of activities of green tea by determining if differences exist by using different preparation procedures. For ranking the AP of green tea in relation to other types of tea corresponding experiments were performed at the beginning of this study. 2. Materials and Methods 2.1 Chemicals and probes The test-radical reagent 2,2- diphenyl-1-picryl-hydrazil (DPPH) was purchased from Sigma (München, Germany). Other reagents were of the highest grade of purity commercially available. Different tea types commercially available were measured for 72

4 their scavenging activity against the test radical DPPH. The different tea types measured for their antioxidant capacity and reactivity were: chamomile; green tea; black tea; herbal tea; rose hip/hibiscus. 50 mg of tea was reduced to small pieces by a mortar and was brewed with 90 C hot water. After dwelling and shaking at different periods of the tea beverage and a spin down the supernatant was dissolved in a ratio of 1:10 in EtOH (50%). Different net weights of the tea beverages were mixed with the DPPH-ethanol solution (100%) and were prepared for the ESR measurement. 2.2 Experimental instrumentation The measurements of the antioxidant capacity and reactivity of the different tea types and the influence of different preparation procedures were performed with the test radical DPPH. The DPPH concentration which varies in dependence of the amount and reactivity of the antioxidants containing in the probe is measured by Electron Spin Resonance (ESR). Opposite to optical methods which measure the only change of the intensity of the probe solution colored by DPPH the ESR has two distinct advantages: 1. Measurement of turbid solutions. 2. Measurement of colored solutions caused by the probe especially in the optical range of the DPPH solution (magenta, violet). The measurements discussed in this article were performed with the X-band ESR spectrometer Miniscope (Magnettech, Germany) and the following technical parameters: 100 G sweep width, 100 Gain, 1 G modulation amplitude, 7 mw attenuation, 3365 G central field. 2.3 Method for the determination of the Antioxidative Power (AP) The properties of the antioxidant products are characterized by the quantitative and qualitative ability to remove their antagonists the dangerous oxygen free radicals and their secondary products like lipid peroxides. The reactive oxygen species (ROS) are characterized by a high reactivity resulting in a short life time of ms ns. Aggressive and reactive free radicals need also reactive antioxidants for their elimination. That s why the de- 73

5 termination and at last the declaration of the reactivity as a quality parameter of an antioxidant product is absolutely necessary. A new parameter describing the properties of an antioxidant for removing ROS in humans should comprised information of its antioxidant capacity (quantitative factor) and reactivity (qualitative factor). This new 2D parameter called Antioxidative Power AP is characterized by the capacity to remove a certain number of free radicals in a certain time interval depending naturally from the quantity and reactivity of the applied antioxidant and resulting in the following relation: Quantity N of reduced free radicals characterized by free electrons (spins) or quantity of applied concentration c of DPPHspins times their reduction amplitude RA divided by the characteristic net weight w c of the antioxidant product times the corresponding reaction time t r. RA *_N(DPPH-spin) RA * c(dpph) AP(t,r) = = (1) t r w c t r w c with the measuring units of the AP in DPPH-spins / mg min or mol/l DPPH / mg min. The con- versation between the different units is carried out by the Avogrado number. Whereas the characteristic net weight w c describes the capacity or potential of the product with the incorporated antioxidant to remove free radicals the reaction time t r describes its reactivity determined by the oneelectron reduction potential E 0 [19]. All methods [20] known up to now measure only the antioxidant capacity as a stationary parameter without the dependence on reaction time between the antioxidant and the free radical. The AP considers this time by the measurement and calculation of the reaction time t r in relation to its corresponding characteristic net weight w c. The test substance is solved in one milliliter of a EtOH 50% solution. 200 µl of these antioxidative solutions are mixed with 200 µl of the test radical solution (0.2 mm DPPH in EtOH 95%) resulting in a final concentration of 0.1 mm DPPH in a water/ethanol solution (25/75). Immediately after mixing of the test radical with the antioxidative solution the ESR measurement is started. The measurement and calculation of the AP were performed with differ- 74

6 ent tea sorts. Their antioxidant reactivity is characterized by the reaction time t r and their antioxidant capacity is determined by the corresponding characteristic net weight w c. Both data results in the 2D factor AP. 2.4 Standardization of the AP For better handling of the evaluated antioxidant activity of different products the AP has to be standardized to the activity of the reproducible antioxidant substance ascorbic acid supplied by Sigma Germany. The AP is based on the antioxidant activity of one milliliter of a solution with a concentration of 1ppm vitamin C (ascorbic acid). This basic antioxidant activity is defined as an antioxidative unit (AU).The AP of a solution of a concentration of 1 ppm vitamin C corresponds to 1 AU by the following relation: 3. Results and Discussion 3.1 Measurement of the Antioxidative Power AP of different tea types Five different tea types were tested: (1) Chamomile, (2) Green tea, (3) Black tea, (4) Herbal tea, (5) Rose hip/hibiscus. The different teas were tested corresponding to the operation sequence described above. The calculated AP s of the tea types are represented in Table 1 and Figure 1. Table 1 shows the calculated AP values represented as reduced spins pro mg antioxidant product times the reducing time of the test radical DPPH caused by the antioxidant product. The standardization of the AP with a uniform measuring unit the AU is seen in a further column. The comparison of the measured AP with the corresponding reaction time t r of the tea sorts is seen in Figure 1a and with the corresponding net weight w c it is seen in Figure 1b. An enormous difference is obviously seen between the AP= AU of green tea and an AP < AU of the other tea products. The high AP of green tea bases on both on its high reactivity (small t r ) and high AP (1 ppm vitamin C) = 1 AU = spins/mg min. (2) According to this the antioxidative power AP of vitamin C corresponds to an AP(vitamin C) = 10 6 AU = spins/mg min. In comparison the antioxidative power of the used green tea was determined to AP (green tea ) = AU = spins/mg min. 75

7 Table 1: Measured Reaction Time t r, Corresponding Net Characteristic Weight w c and Calculated Antioxidative Power AP of Different Tea Types Brewed at 50 C and a Dwell Time of 3 Minutes. AP AP t r w c (spins/mg min) (AU) (min) (mg) Chamomile 7.296* Green tea 2.376* Black tea 3.617* Herbal tea 2.484* Rose hip/hibiscus 4.356* Figure 1a: Antioxidative Power AP (AU) of different tea types (bar plot) and line diagram of their corresponding reaction times t r (min). 76

8 Figure 1b: Antioxidative Power AP (AU) of the different tea types (bar plot) and line diagram of their corresponding characteristic net weights w c (mg). capacity (small w c ). Whereas the relative low AP = 1746 AU of rose hip is determined by a moderate antioxidant capacity (w c = mg) and a very low antioxdant reactivity (t r = min.) the AP = 2923 AU of chamomile is determined by a very low antioxidant capacity (w c = 0.178mg) and a moderate antioxidant reactivity (t r = min). Figures 1a and 1b show distinctly the effect of the parameters t r and w c on the AP of a product. While green tea consists of antioxidants with high reactivity demonstrated by the short reaction time of t r (green tea) = min. rose hip tea consists of antioxidants with a long reaction time of t r (rose hip) = min. which is 6.44 times longer. A net weight which is 8.42 times higher than the w c (green tea) = mg of green tea is necessary to reach the same radical reduction. On the other hand black tea and herbal tea consist of antioxi- 77

9 dants with reaction times of t r (black tea) = min. and t r (herbal tea) = min. resulting in smaller differences between the characteristic net weights of w c (black tea) = mg and w c (herbal tea) = mg. Their AP is definitely determined by the reactivity of their individual antioxidants and resulting in AP(black tea) = AU and AP (herbal tea) = 9955 AU. While the AP of nearly all tea types other than chamomile are determined by the reaction time t r and the corresponding characteristic net weights w c chamomile consists of antioxidants with a moderate reactivity of t r (chamomile) = min which is higher than that of other tea sorts except for green tea. But the high characteristic net weight of chamomile with w c (chamomile) = mg leads to the conclusion that the concentration of antioxidants is much lower than in the other tea types. So the AP can give information about the quality (parameter t r ) and quantity (parameter w c ) of the antioxidants contained in the product. The fact, that a clear inverse correlation between the AP and t r can not be seen in Figures 1a and 1b, shows, that different tea types contain different antioxidants with different radical scavenging activities. The different reaction times t r show that there are different antioxidants in the individual tea types beside their individual concentrations which both are responsible for the AP. The long reaction time t r of rose hip/hibiscus shows that here works other antioxidants than in green tea. 3.2 Influence of brew temperature and dwell time on the AP of green tea beverages The 2D AP method enables a better comprehensive description of the antioxidative character of a probe and the external influences affect on it. The determination of the AP of a commercial product is one thing. In a first step we have shown the big variance between the AP of beverages made of four different tea types namely green tea, mint tea, dandelion and red clover. The first characterisation of the tea beverages was performed by the AP determination using a brew temperature of 90 C and a dwell time of 10 minutes. During the dwell time the tea beverages were continuously shaken. After centrifugation the supernatant was removed and measured in an ESR Spectrometer. The measured and calculated AP values are listed in Table 2 which range from AU for 78

10 Table 2: Measured Reaction Time t r and Calculated Antioxidative Power AP of Different Teas Brewed at 90 C and a Dwell Time of 10 Minutes. AP (AU) t r (min) Green tea Mint tea Dandelion Red clover dandelion to AP = AU for green tea. Their reactivity ranges from t r = min for dandelion to green tea t r = min. The highest AP value and the highest reactivity against free radicals were measured for green tea. Green tea was used for further experiments. To test the influence of different brew temperatures this parameter was changed in a further experiment. The brew temperature was varied between 25 C, 50 C, 60 C, 70 C, 80 C and 90 C. A constant dwell time of 10 minutes was used for all these measurements. The measured AP values inclusive the reaction time t r are represented in Table 3 and Figure 2. A maximum AP inclusive the shortest reaction Table 3: Measured Reaction Time tr and Calculated Antioxidative Power AP of Green Tea Brewed at Different Temperatures (25-90 C) and a Dwell Time of 10 Minutes. Green Tea AP (AU) t r (min) Brew temperature 25 C C C C C C

11 Figure 2: Antioxidative Power AP (bar plot) and reaction time t r (line diagram) of green tea beverages brewed at different temperatures for a dwell time of 10 minutes. time t r were measured for a brew temperature of 60 C. For brew temperatures over 60 C the AP decreases again. The reason seems to be an increase of the reaction time t r for brew temperatures over 80 C. This longer reaction time is correlated with lower antioxidative reactivity of the beverage resulting in the insight that an other antioxidant composition is active. Obviously some reactive antioxidants/polyphenols are removed and some other antioxidants/polyphenols are extracted at higher temperatures from the green tea leaf. Varying the brew temperature the composition and reactivity of the antioxidants within the beverage can be influenced and definitely described by the AP. The influences of different dwell times from 1min., 5 min., 10 min., 20 min. to 30 min. were tested in a further experiment where the brew temperature kept constant at 50 C. The AP results are listed in Table 4 and represented in Figure 3. It is clearly seen that the AP increases with longer dwell time by 64%. In contrast there is a decrease of reaction time t r by about 14%. It is seen that the 80

12 Table 4: Measured Reaction Time t r and Calculated Antioxidative Power of Green Tea Brewed at 50 C and Different Dwell Periods from 1 Minute to 30 Minutes. Green tea 50 C AP (AU) t r (min) Dwell time 1 min min min min min Figure 3: Antioxidative Power AP (bar plot) and reaction time t r (line diagram) of green tea beverages brewed at 50 C as a function of different dwell periods between 1 minute and 30 minutes. increase of the AP is not directly correlated with the decrease of the reaction time. This fact is seen up to dwell times of 30 81

13 Table 5: Measured Reaction Time t r and Calculated Antioxidative Power of Green Tea Brewed at 50 C and Dwell Time of 10 Minutes Followed by Different Storage at 4-8 C for a Period of 10 Minutes to 24 hour. Green tea 50 C AP (AU) t r (min) Storage time 10 min h h minutes where a minimum of the reaction time t r is seen at 20 minutes followed by a small increase up to a dwell time of 30 minutes. Responsible for the increase of the AP is mainly the number of extracted antioxidants. Of course, the amount of extracted antioxidants/polyphenols from the tea leaf increase with longer dwell time. Whereas an increase of the brew temperature from 25 to 60 C (dwell time of 10 minutes) results in an increase of the AP by 3.4% the extension of the dwell time from 1 to 30 minutes (brew temperature of 50 C) results in an increase of the AP by about 62%. This fact shows that variations of the brew temperature (temperatures over 25 C presumed) have only a minimum influence on the AP whereas an elongation of the dwell time can considerably increase the AP. A brew temperature of about 60 C and a dwell times of about 20 to 30 minutes seems to result in a green tea beverage with an optimum AP. An interesting question for the preparation of tea beverages is their handling over long times, especially the stability of the AP over long storage periods. Storage periods from 10 minutes up to 24 hours are of interest to the producer. AP values inclusive their reaction time of green tea beverages brewed at a temperature of 50 C and a dwell time of 10 minutes and stored for different times are listed in Table 5. The tea beverages were stored at temperatures between 4-8 C (refrigerator temperature). As a result we see only a small decrease of the AP over time. After 24 hours the antioxidant capacity and reactivity is nearly unchanged. This insight is relevant for products as food and dietary supplements as well as cosmetics containing green tea as free 82

14 radical scavenger. The high antioxidant power together with its high stability qualifies green tea for a broad application as powerful antioxidant. References [1] Henry, J.P., Stephens-Larson,P., Reduction of chronic psychosocial hypertension in mice by decaffeinated tea, Hypertension, 6 (1984) [2] Ding, Z.Y., Chen, Y., Zhou, M., & Fang, Y.Z., Inhibitory effect of green tea polyphenol and on the oxidative modification of low-density lipoprotein, Chin. J. Pharmacology Toxicology, 6 (1992) [3] Leung, L.K., Su, Y., Chen, R., Zhang, Z., Huang, Y., & Chen, Z.Y., Theaflavins in black tea and catechins in green tea are equally effective antioxidants, J. Nutrition, 131 (2001) [4] Hertog, M.G., Feskens, E.J., Hollmann, P.C., Katan, M.B., & Kromhout, D., Dietary antioxidant flavonoids and risk of coronary heart disease: the Zutphen Elderly Study, Lancet, 342 (1993) [5] Li, H.L., Huang, Y., Zhang, C.N., Liu, G., Wei, Y.S., Wang, A.B., Liu, Y.Q., Hui, R.T., Wei, C., Williams, G.M., Liu, D.P., & Liang, C.C., Epigallocathechin-3 gallate inhibits cardiac hypertrophy through blocking reactive oxidative species-dependent and -independent signal pathways, Free Radical Biology & Medicine, 40 (2006) [6] Shi, S.T., Wang,Z.Y., Smith, T.J., Hong, J.Y., Chen, W.F., Ho, C.T., & Yang, C.S., Effects of green tea and black tea on 4-(methylnitrosamino)-1-(3- pyridyl)-1-butanone bioactivation, DNA methylation, and lung tumorigenesis in A/J mice, Cancer Research, 54 (1994) [7] Liu, Q., Wang, Y., Christ, K.A., Wang, Z.Y., Lou, Y.R., Huang, M.T., Conney, A.H., & You, M., Effect of green tea on p53 mutation distribution in ultraviolet B radiation-induced mouse skin tumors, Carcinogenesis, 19 (1998) [8] Imai, K., & Nakachi, K., Cross sectional study of effects of drinking green tea on cardiovascular and liver disease, Biochem. Med. J., 310 (1985) [9] Yang, M., Wang, C., & Chen, H., Green, oolong and black tea extracts modulate lipid metabolism in hyperlipidemia rats fed high-sucrose diet, J. Nutr. Biochem., 12 (2001) [10] Pellegrini, Re.R., Protteggente, A., Pannala, A., Yang, M., & Rice-Evans, C., Antioxidant activity applying an improved ABTS radical cation decolorization assay, Free Radic. Biol. Med., 26 (1999) [11] Wang, L.F. Kim., D.M., & Lee, C.Y., Effects of heat processing and storage on flavanols 83

15 and sensory qualities of green tea beverage, J. Agric. Food Chem., 48 (2001) [12] Jung, K., Richter, J., Kabrodt, K., Lucke, I.M., Schellenberg, I., & Herrling, T., The antioxidative power AP A new quantitative time dependent (2D) parameter for the determination of the antioxidant capacity and reactivity of different plants, Spectrochim. Acta A. Mol. Biomol. Spectrosc., 63, (2006) [13] Manfredini, S., Vertuani, S., Manfredi, B., Rossoni, G., Calviello, G., & Palozza, P., Novel antioxidant agents deriving from molecular combinations of vitamins C and E analogues: 3,4-dihydroxy- 5(R), Bioorg. Med. Chem., 8 (2000) [14] Palozza, P., Piccioni, E., Avanzi, L., Vertuani, S., Calviello, G., & Manfredini, S., Design, synthesis, and antioxidant activity of FeAOX-6, a novel agent deriving.g from a molecular combination of the chromanyl and polyisoprenyl moieties, Free Radic. Biol. Med., 33 (2002) [15] Blois, M.S., Antioxidant determinations by the use of a stable free radical, Nature, 181 (1958) [16] Brand-Williams, W., Cuvelier, M.E., & Berset, C., Use of free radical method to evaluate antioxidant activity, Lebensmittel-Wiss. u. -Technol., 28 (1995) [17] Calliste, C.A., Trouillas, P., Allais, D.P., Simon, A. & Duroux, J.L., Free radical scavenging activities measured by electron spin resonance spectroscopy and B16 cell antiproliferative behaviors of seven plants, J. Agric. Food Chem., 49 (2001) [18] Calliste, C.A., Trouillas, P., Allais, D.P. & Duroux, J.L., Castanea sativa Mill. leaves as new sources of natural antioxidant: an electronic spin resonance study, J. Agric. Food Chem., 26 (2005) [19] Buettner, G., The pecking order of free radicals and antioxidants: lipid peroxidation, alpha-tocopherol, and ascorbate, Arch. Biochem. Biophys., 300 (1993) , Review [20] Schlesier, K., Harwat, M., Böhm,V., & Bitsch, R., Assessment of antioxidant activity by using different in vitro methods, Free Radic. Res., 36 (2000) Abbreviations used AP Antioxidative Power ARP Antiradical Power AU Antioxidative Unit DPPH 2,2-Diphenyl-1-picrylhydrazil ESR Electron Spin Resonance RA Reduction Amplitude ROS Reactive Oxygen Species 84

16 Dr. Katinka Jung Marietta Seifert GEMATRIA Test Lab Berlin Pestalozzistrasse Berlin, Germany Dr. Thomas Herrling Department of Medical Physics University of Applied Sciences TFH Berlin Luxemburger Str Berlin, Germany 85