korir et al. (2014) the fortification of tea with sweeteners and milk and its effect on in vitro.pdf

8/9/2019 Korir et al. (2014) The fortification of tea with sweeteners and milk and its effect on in vitro.pdf

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The fortication of tea with sweeteners and milk and its effect on in vitroantioxidant potential of tea product and glutathione levels in an animalmodel

M.W. Korir a , F.N. Wachira b , , J.K. Wanyoko c, R.M. Ngure a , R. Khalid ca Department of Biochemistry and Molecular Biology, Egerton University, P.O. Box 536, Egerton, Kenyab Association for Strengthening Agricultural Research in East and Central Africa (ASARECA), P.O. Box 765, Entebbe, Ugandac Tea Research Foundation of Kenya, P.O. Box 820-20200, Kericho, Kenya

a r t i c l e i n f o

Article history:Received 9 January 2013Received in revised form 6 July 2013Accepted 2 August 2013Available online 11 August 2013

Keywords:TeaMilkSweetenersAntioxidant activityGlutathione

a b s t r a c t

Several studies have demonstrated that tea avonoids protect cells andtissues against freeradicals whichhave been implicated in the etiology of oxidative stress-related disease disorders. However, black tea iscommonly consumed with additives that could otherwise affect the bioavailability of the active tea mol-ecules. In this study, the biochemical parameters of Kenyan teas were determined and the effect of addedmilk and sweeteners on the antioxidant activity of Kenyan teas was investigated. The effect of tea anti-oxidants on glutathione (GSH) was also evaluated in vivo in a time series study using Swiss mice. Greenteas had the highest levels of total polyphenols, total and individual catechins, while black teas had highlevels of total thearubigins, total theaavins and theaavin fractions. The antioxidant activity was high ingreen teas though some of the black teas were as efcacious as the green teas. The addition of milk, sugarand honey signicantly ( p < 0.05) decreased the antioxidant activity of tea in a concentration-dependentmanner. Addition of the sweetener, stevia ( Stevia rebaudiana Bertoni), showed no signicant ( p > 0.05)inuence on the antioxidant activity of tea and therefore can be recommended as a preferred sweetenerfor tea. Signicantly ( p < 0.001) higher levels of GSH were observed in plasma than in other tissues. GSHlevels were generally highest 2 h after tea consumption, which indicates the need to repeatedly take teaevery 2 h to maximise its potential health benets.

2013 Elsevier Ltd. All rights reserved.

1. Introduction

Black (aerated) tea is the most consumed beverage globally. It islargely produced in Africa, with Kenya being the largest producer(Abeywickrama, Ratnasooriya, & Amarakoon, 2011; Cabrera, Reyes,& Rafael, 2006; Economic Review of Agriculture, 2007; Kerio,Wachira, Wanyoko, & Rotich, 2012; Mwaura & Ogise, 2007 ). Blacktea is processed from the tender leaves of Camellia sinensis. Freshtea leaves are richin catechins which form theprincipal polypheno-liccompoundsin green(non-aerated) tea,whileblackteas arerich inoxidation products of the catechins, called theaavins and thearub-igins ( Frei & Higdon 2003; Karori, Wachira, Wanyoko, & Ngure,2007 ). Tea-derived polyphenols have been extensively studied aspromising substances in disease prevention ( Cabrera et al., 2006; Jianbo et al., 2011 ). For example, the chemo-preventive activity of tea against cancer has been demonstrated in several body organs,such as colon, stomach and lung ( Sharangi, 2009; Wang et al.,2010 ). Regular consumption of tea has also been linked with theprotection against harmful UV radiation, and maintenance of skin

structure andfunctions ( Heinrich, Carolyn, Silke, Hagen,& Wilhelm,2011 ). Tea catechins and theaavins have been implicated in theprotectionagainst cardiovascular andkidney ailments through sev-eral mechanisms ( Shinichi, 2008 ). Tea catechins also decrease glu-cose production and hence regulate glucose and insulinconcentrations in the blood ( Wu, Juan, Hwang, Hsu, & Ho, 2004 ).Tea has been demonstrated to ameliorate inammatory conditionsdue to its anti-parasitic, anti-heamolytic and anti-oxidant proper-ties ( Karori, Wachira, Wanyoko, & Ngure, 2008 ). Regular consump-tion of black tea has been associated with low risks of cognitiveimpairment and has been shown to delay or prevent the onset of dementia ( Chen et al., 2010; Ng, Lei, Mathew, Ee, & Keng, 2008 ).Tea polyphenols with the galloyl moiety have particularly beendemonstrated to inhibit the entry of the HIV virus by binding tothe gp41, which is essential for the HIV-1 attachment to the targetcells ( Shuwenet al., 2005 ). The uoride present in tea prevents den-tal decay by inhibiting the demineralization of protective coat en-amel by both pathogenic microorganisms and commensalorganisms (Hamilton-Miller, 2001 ).

The above health effects of tea are largely ascribed to its antiox-idant properties, which enable it to protect cells against oxidativedamage caused by free radicals ( Gupta, Siddique, Beg, Ara, & Afzal,

0308-8146/$ - see front matter 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.foodchem.2013.08.016

Corresponding author. Tel.: +256 722 644279; fax: +256 414 321126.E-mail address: [email protected] (F.N. Wachira).

Food Chemistry 145 (2014) 145153

Contents lists available at ScienceDirect

Food Chemistry

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2008 ). Free radicals are important in normal physiological pro-cesses but they have also been associated with the etiology of chronic and degenerative diseases, including atherosclerosis, Alz-heimers and Parkinsons disease, diabetes, cardiovascular diseases,cancer, allergies and premature aging ( Svetli, Guy, & Heidar-Ali,2010 ). Endogenous antioxidant defence mechanisms are not al-ways sufcient to fully scavenge free radicals from the body anddietary components that have antioxidant properties, such as teaavonoids, are therefore necessary to supplement the activity of endogenous antioxidants. One of the major endogenous antioxi-dants in the body is glutathione (GSH). GSH has many functions,one of which is the protection against oxidative damage by scav-enging free radicals and peroxides produced during normal cellularrespiration, which would otherwise oxidise proteins, lipids and nu-cleic acids ( Giustarini, Rossi, Milzani, Colombo, & Dalle-Donne,2004; Moskaug, Harald, Myhrstad, & Rune, 2005 ).

Though numerous studies have been carried out to determinethe health benets associated with antioxidant properties of tea,these efforts have largely been directed towards the biologicalactivity of green tea with very little effort on black tea, which isthe principle product of Africa and which is also widely consumedin the world. Black tea is largely consumed with substantialamounts of milk and added sweeteners, such as sugar, stevia andhoney. The effects of milk and added sweeteners on the antioxi-dant activity of tea have hardly been investigated. This studyaimed to establish whether addition of these secondary additivesaffects the potential health benets that can be derived from theantioxidative potency of tea. It is envisaged that evidence of the ef-fects of these ingredients on the antioxidant activity of tea will im-prove preparation and consumption habits, and promote tea as afunctional beverage and contribute to its increased consumption.

2. Materials and methods

2.1. Tea samples and reagents

Leaf samples from ve tea cultivars (AHP S15/10, TRFK 303/577,TRFK 6/8, BBK35 and TRFK 306/1) were sourced from the Tea Re-search Foundation of Kenya (TRFK) 0 26 S, 37 15 E). Rened whitecane sugar, produced and packaged by Mumias Sugar Company,fresh cows milk, processed and packaged by the Kenya Co-opera-tive Creameries Ltd (KCC) and honey, processed and packaged byBaraka Agricultural Institute, were used in this study. The milkwas ultra-heated with no additives or preservatives. Stevia powderwas bought from a local chemist shop.

2.2. Sample preparation

2.2.1. Processing of tea samplesTea was manufactured in a miniature tea factory at the TRFK,

Kericho, Kenya. Black teas were manufactured using physical with-er for 18 h to attain a moisture content of 5065%. Aeration wascarried out for 2 h at 24 C and the leaf red in a uid bed drierat 120 C for 2025 min. Green teas were manufactured by steam-ing the leaf for 1 min, crushing, tearing and curling and nally r-ing ina uid bed drier at 120 C. The processed teas were packagedinto polythene bags and stored at room temperature prior to fur-ther analysis.

2.2.2. Determination of dry matter content Five grammes (5 g) each of the teaproducts were weighed to the

nearest 0.001 g, placed in pre-weighed aluminiumdishes and dried

in an oven (Oven Memmert, UND300, Germany) at 103 2 C for16 h to constant weight. Percentage dry matter (DM) content for

eachsamplewas calculated fromthe weightdifferencesof thedriedand pre-dried tea samples.

2.3. Preparation of extracts

2.3.1. Extraction of polyphenols and catechinsTea samples of coarse granular structure were ground to ne

powder. A sample of 0.2 0.001 g of tea was weighed into a grad-uated extraction tube (10 ml) and 5 ml of 70% hot water/methanolextraction mixture added using a dispenser (Dispensette BrandGermany), stoppered and mixed on a vortex mixer (Rotamixer,Huck andTucker, England). The mixture was immediately placedin a water bathat 70 C and incubated for 10 min. After incubation,the mixture was removed from the water bath, cooled and centri-fuged at 3500 rpm for 10 min (Heraeus Sepatech, Germany). Thesupernatant was decanted in a graduated tube. The extraction stepwas repeated and both extracts were pooled and the volume ad- justed to 10 ml with cold 70% methanol.

2.3.2. Extraction of theaavins and thearubiginsNine grammes (9 g) of coarse tea sample were weighed, and

placed in a 500 ml thermos ask and 375 ml of hot distiled wateradded .The mixture was then agitated, using a mechanical shakerfor 15 min. The tea infusion was then ltered through cotton woolinto a at-bottomed beaker and allowed to cool to roomtemperature.

2.4. Analysis of total polyphenols

TheFolinCiocalteau reagent methodwas used to determine to-tal polyphenols as per the International Organisation for Standard-ization (ISO) 14502-1 ( ISO 14502-1, 2005 ). From the sampleextract, 1 ml was pipetted into a 100 ml volumetric ask and madeup to the mark with distiled water. One millilitre of the dilutedsample was complexed with 5 ml of 10% FolinCiocalteaus phenolreagent and 4 ml of 7.5% sodium carbonate solution. The mixturewas stoppered, vortexed and the optical densities (OD) measuredafter 1 h in 10 mm length cells against distiled water, using a dig-ital grating spectrophotometer at 765 nm (model Cecil CE 393).The total polyphenol content was expressed as gallic acid equiva-lents (GAE), in g/100 g.

2.5. Analysis of catechins

Catechin analysis was done (HPLC) according to ISO 14502-2,2005 . Reverse phase HPLC was employed. A Shimadzu LC AT HPLCmachine with SIL 20A auto sampler, SPD-20 UVvisible detectorwith a class LC 10 chromatography work station and A Luna TM5 l M C6, 250 mm 4.6 i.d (Phenomenex, Tolerance, CA, USA) col-umn with a Reodyne pre-column lter 7335 model was used. One

millilitre (1 ml) of the extract (2.3.1 above) was diluted to 5 mlwith stabilizing solution (10% v/v acetonitrile with 500 l g/mlEDTA and ascorbic acid), ltered through a 0.45 l m membrane l-ter and 20 l l were injected into the machine. A gradient elutionwas carried out using mobile phases A and B: mobile phase A (ace-tonitrile/acetic acid/double distiled water-9/2/89 v/v/v), and mo-bile phase B (acetonitrile/acetic acid/double distiled water-80/2/18 v/v/v). The mobile phase composition for a binary gradient con-dition started at 100% solvent A for 10 min, then over 15 min a lin-ear gradient to 60% mobile phase A, 32% mobile phase B and held atthis position for 10 min. The condition was reset to 100% mobilephase A and allowed to equilibrate for 10 min before the next sam-pling. The ow rate of the mobile phase was 1 ml per minute andthe temperature of the column was set at 35 0.5 C. The identi-

cation of individual catechins was carried out by comparing theretention times and UV-absorbance of sample peaks with peaks

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obtained from the mixed known catechin standards under thesame conditions. The standards used were catechin(C), epicatechin(EC), epigallocatechin (EGC), epicatechin gallate (ECG) and epigal-locatechin gallate (EGCG) purchased from Sigma Aldrich, UK. Thequantication of catechins was performed at 278 nm and wasachieved using a caffeine standard with a calibration curver 2 = 0.9992 in conjunction with the consensus individual catechinrelative response factor (RRF) values with respect to caffeine calcu-lated on a dry matter basis. All determinations were done in trip-licate. Total catechin, as percentage by mass, on a sample drymatter was given on the summation of individual catechins:

% Total catechin % ECG % C % EC % EGCG % EGC

Caffeine content was quantied as follows:

% Caffeine Asample Aintercept RRFstd V d 100Slope caffeine m 1000 DM

where;Asample Peak area of the individual component in the testsample

Aintercept Peak area at the point of interception on Y -axisSlope caffeine Caffeine calibration line slopeV Sample extraction volumed Dilution factorm Mass in grammes of test sampleDM Dry matter content of test sample

2.6. Analysis of theaavin fractions

One millilitre (1 ml) of the infusion ((2.3.2. above) was dilutedwith distiled water (1:1) and 20 l l of the diluted sample were injected into the HPLC C18 ODS column. A gradient elution was car-ried out, using mobile phases A (acetic acid/double distiled water1/99 v/v) and B (acetonitrile/double distiled water 80/20 v/v).

The mobile phase composition for a binary gradient conditionstarted at 92% solvent A and 8% mobile phase B for 49 min, thenover a linear gradient to 69% mobile phase A and 31% mobile phaseB and held at this for 7 min. The ow rate of the mobile phase was1.5 ml per minute; the temperatures were maintained at35 0.5 C. The identication and quantication of the theaavinfractions were performed at 378 nm, by comparing the retentiontimes and UV-absorbance of the sample peaks with peaks obtainedfrom a mixture of known standards of simple theaavin (TF), thea-avin 3 monogallate (TFMG), theaavin 3monogallate (TF3MG)and theaavin 33 digallate (TFDG), (Sigma Aldrich,UK) under thesame conditions.

2.7. Analysis of total theaavin (TFs) and thearubigins (TRs)

Total theaavins (TFs) and total thearubins (TRs) contents werequantied using a digital grating spectrophotometer (Model CecilCE 393). Total theaavin content was determined at 625 nm, usingthe Flavagnost method of Hilton (1973) . Ten millilitres of the infu-sion were mixed with 10 ml of isobutylmethylketone-IBMK (SigmaAldrich, USA) and allowed to stand until the layers separated. Twomillilitres of the upper layer were transferred into labelled testtubes in triplicate. Four millilitres of ethanol, followed by 2 ml of Flavognost reagent were added and the absorbance read againstan IBMK/ethanol blank (1:1 v/v). The Flavagnost reagent formeda green complexwith the cis-h,i-dihydroxybenzene ring associatedwith TFs. Total theaavin was calculated using the formula;

TFs (l mol/g) = A 625 479 100/DM, where A 625 is the

absorbance at 625 nm and DM is the dry matter of the sample(Hilton, 1973 ).

Total thearubigins content was determined using the method of Roberts and Smith (1961) . Six millilitres of 1% aqueous solution of anhydrous disodium hydrogen orthophosphate were mixed with6 ml of the cooled tea infusion prepared as described above. Themixture was extracted with 10 ml of ethyl acetate and the aqueouslayer was drained off. Ten millilitres of the ethyl acetate extractwere diluted with 25 ml of methanol (solution A). One millilitreof infusion was mixed with 9 ml of distiled water and the volumewas made up to 25 ml with methanol (solution B). One millilitre of the infusion was mixed with 1 ml of aqueous 10% oxalic acid, fol-lowed by 8 ml of distiled water and the volume was made to25 ml with methanol (solution C). Each sample was extracted intriplicate. The absorbancies of solutions A, B and C were read at380 nm, 460nm and 380nm, respectively. TFS and TRS valueswere calculated using the formula;

TFs% = 2.25 A DM%; TRs% = 7.06 (4C-B) DM% whereDM is the dry matter of the sample ( Robert & Smith, 1961 ).

2.8. Determination of antioxidant activity of tea, with and without secondary additives, using DPPH radical-scavenging activity

Different tea infusions were prepared without and with differ-ent levels of secondary additives as follows: (a) green and blacktea with no additives, (b) green and black tea with milk and nosweetener, (c)black tea with milk and sugar (d) black teawith milkand stevia and (e) black tea with milk and honey. Each infusionwas prepared by infusing ve grammes of coarse tea samples in100 ml of boiling distiled water in a 100 ml volumetric ask andstirring with a magnetic stirrer on a hot plate for 10 min. Tea infu-sions with milk were made using 5 g of tea and different concen-trations of milk (2%, 4%, 6%, 8%, 10%, 20% and 40% (v/v milk/water). Infusions with milk and sugar were brewed with 3 g and10 g of sugar plus the several concentrations of milk, and thosewith milk and stevia (0.1 g and 0.3 g) were prepared. Similarly, teaswith several concentrations of milk plus 3 g and 10 g honey wereprepared. All the above tea infusions were strained through cottonwool to remove the tea particles and left to cool to room temper-ature. The antioxidant activity of the prepared tea extracts wasdetermined using the 2, 2 0-diphenyl-1-picryhydrazyl radical(DPPH) method, as described by Karori et al. (2007) . The antioxi-dant activities of sugar, stevia and honey were also assayed usingthe DPPH method.

2.9. Animal s tudies

2.9.1. AnimalsSwiss albino mice (female, 68 weeksoldand weighing 2135 g

were used in this study. The animals were housed in standard mice

cages in a controlled environment and provided ad libitum withfood and water. Animal care protocols and procedures were re-viewed and approved by the Institutional Animal Care and UseCommittee. The animals were treated once using 1% ivermectinat a dose of 0.01 ml per kg/body weight duringthe rst week to ex-clude any endoparasites and ectoparasites. Twenty grammes (20 g)of the processed tea fromselectedcultivars TRFK303/577 (for Blackand Green tea) and TRFK 306/1 (for Purple tea) were separatelyweighed and 1 L of hot distiled water added. Infusions with milk(2% v/v) were also prepared, using black and purple teas. The ani-mals were randomly allocated into 5 groups, each with three miceper group, and each group of mice being housed separately. The 5groups represented sampling intervals (0 h; 30 min; 2 h; 4 h and8 h) while the three mice were replications of the same treatments.

The animals were then orally fed with 0.5 ml of the prepared andcooled tea infusions.

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p < 0.05) and + catechin ( r = 0.30, p < 0.05), showed a negativecorrelation with the antioxidant activity.

3.3. Effects of added milk and sweeteners on antioxidant activity of green and black teas

The antioxidant activity signicantly ( p < 0.05) decreased withincrease in milk concentration in both green and black teas, thoughthe activity was generally higher in green teas than in black teas. Asignicant decrease in antioxidant activity ( p < 0.05) was notedafter addition of 10% milk in both green and black teas ( Table 3 ).

Theantioxidantactivitiesof sugar,steviaandhoneywerealsoas-sayed in this study. Honey hadsignicantly( p < 0.05) higherantiox-idant activity (10.6%) than had sugar (6.77%) or stevia (4.33%)though these were comparatively lower than those of the tea. Anti-oxidant activity determinedon plain black tealiquorsandthose for-tied with milk and sweeteners showed signicant variations as

shown in Table 4 . However, the addition of 0.1 g and 0.3 g of steviahad no signicant inuence on the antioxidant activity of the plainteas or those fortied with milk. Antioxidant activity of tea liquorswith honey and sugar varied signicantly ( p < 0.05) in a concentra-tion-dependentmanner. Teasfortiedwith honeyshowedless anti-oxidant activity than did other sweeteners.

3.4. Glutathione levels

Glutathione (GSH) levels in different tissues were measured toinvestigate the effects of the tea bioactive molecules on GSH afterfeeding the mice with green, black and purple teas. GSH concentra-

tions varied signicantly ( p < 0.001) in different tissues, as shownin Fig. 1 (a). The highest levels of GSH were recorded in plasma after2 h of tea consumption. The GSH levels due to various types of teadiffered signicantly ( p < 0.001) in the same tissue at different timeintervals, as shown in Fig. 1 (bd). Highest GSH levels were re-corded for black tea fortied with 2% milk in plasma, as comparedwith the other teas. However, in kidney and brain tissues, higherGSH levels were recorded for plain black teas after 2 h and 4 h of tea consumption, respectively. The GSH levels in the liver weresimilar to those in the brain but the peak in the liver was after30 min.

4. Discussion

The analysed tea samples showed signicant variations of teachemical parameters. The variations in the biochemical parametersassayed in the teaproducts canbe ascribed to the wide genetic var-iationin the tea cultivars used in the study. These variations enablethe selection of tea cultivars with specic biochemical proles. Thephenolic composition of test tea products varied greatly after aer-ation, demonstrating that the oxidation process during black teaprocessing resulted in distinctly different polyphenol proles. Inac-tivation of phenol oxidases by steaming during green tea manufac-ture prevented oxidation of the catechins and therefore the higherlevels of total and individual catechins in green than in black teas.Individual catechins varied signicantly, with EGCG being the mostpredominant followed by EGC, ECG and nally EC in the green teas.The results obtained in this study are similar to those reported byothers ( Cabrera et al., 2006; Ferruzzi, 2010; Kerio et al., 2012 ). Inblack tea processing, the leaf is macerated to initiate oxidationby the enzyme polyphenol oxidase (PPO) in the presence of molec-ular oxygen. Quinones from the oxidation of B-ring dihydroxlatedcatechins (non-gallated) condense with quinones from the trihydr-

oxylated catechins (gallated) to form the dimeric theaavins andpolymeric thearubigins ( Owuor & Obanda, 2007 ). Consequently,the levels of total and individual catechins were signicantly lowerin black teas than in green teas, whichconrmed that the oxidationprocess during black tea manufacture determines the chemicalcomposition of the end-product. The gallated catechins are de-pleted faster than are the non-gallated ones during the aerationprocess and this probably explains why the levels of EGCG andECG were lower in the black tea samples than in green tea whencompared to EGC, EC and (+C). These results are comparable withthose of Mendel et al. (2005) and Ferruzzi (2010) . Total theaavinlevels were also signicantly higher in black teas than in green teasconrming the oxidation process of catechins. Variations in theaf-lavins fractions were signicant, with theaavin-3, 3 0-digallate

(TFDG) being the most predominant, followed by theaavin-3-monogallate, theaavin-3 0-monogallate and nally the simple

Table 2

Mean values of antioxidant activity of green and black teas with no additives.

Tea samples Antioxidant activity (%)

1 92.4 abc

2 92.9 a

3 92.6 ab

4 91.8 cd

5 91.8 cd

6 91.8 cd7 92.1 bc

8 91.3 d

9 90.2 e

10 91.2 d

C.V (%) 0.44LSD( p = 0.05) 0.69

Different superscripts in the samecolumn indicate that activities differ signicantlyat p = 0.05 by the LSD test. Tea samples Nos. 15 (Green teas), 610 (Black teas) asdescribed in the footnote for Table 1.

Table 3

Mean values of antioxidant activity of green and black with different milkconcentrations. No sweetener added.

Milk concentrations (%) Antioxidant activity

Green Black

0 92.3 a 91.3 a

2 91.7 ab 90.6 a

4 91.2 abc 90.1 ab

6 90.5 bc 88.9 abc

8 90.0 bc 88.0 bc

10 89.5 c 86.9 c

20 87.1 d 81.4 d

40 79.6 e 56.1 e

Mean 88.9 84.2C.V. (%) 1.21 1.78LSD( p = 0.05) 1.76 2.44

Means with different letters within each column are signicantly ( p < 0.05) differ-ent by LSD test.

Table 4

Mean values of antioxidant activity of sweetened plain and milk-fortied black teas.

Tea treatment Antioxidant activity

Plain tea Milk fortied tea

No sweetener 91.6 a 84.2 a

0.1 g stevia/100ml 91.6 a 85.0 a

0.3 g stevia/100ml 90.5 a 84.6 a

3 g sugar/100 ml 84.1 b 75.2 b

10 g sugar/100 ml 46.5 d 44.7 d

3 g honey/100 ml 54.2 c 46.5 c

10 g honey/100 ml 37.1 e 33.1 d

C.V (%) 2.8 3.75LSD 3.2 4.07

Means within a column followed by the same letters are not signicantly differentat p < 0.05 by LSD test.

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theaavin fraction. These observations indicate that, indeed, the oxi-dation of the individual catechin precursors, which happens at dif-ferent rates, determines the end-product of the oxidation process(Obanda, Owuor, & Mangoka, 2001 ). EGCG and ECG, the precursorsof TFDG are signicantly higher in fresh tea leaves which results inhigh levels of TFDG. Similar results were obtained by Owuor et al.(2006) in a study to investigate the relationship between chemicalparameters and sensory evaluations for plain black tea.

Though black tea is consumed without secondary ingredients insome countries, in the majority of cases, it is consumed with a sub-stantial amount of milk and sweeteners, to reduce its astringency.Studies have demonstrated that tea exerts numerous health effectswhich are ascribed to its antioxidant properties ( Cabrera et al.,2006; Svetli et al., 2010 ). Theaavins and catechins in black andgreen tea, respectively, contribute to the antioxidant characteris-tics of tea, which is key to the health benets associated with thisbeverage. In this study, free radical-scavenging potential of black

and green tea extracts, with and without milk, sugar, honey andstevia, was evaluated. The antioxidant activity in green teas wassignicantly higher than that in black teas. Nonetheless, some of the black teas, such as those processed from cultivars TRFK 303/577, and TFRK 6/8, had antioxidant activities that were comparableto some of the green teas. This could be explained by the initialhigh levels of total catechins, EGCG and ECG in the leaves of thethese cultivars which positively correlated with levels of totaltheaavin, theaavin monogallates, theaavin digallate and gallicacid, all of which, in turn, correlated positively with antioxidantactivity in the black tea products. These results were consistentwith those of Cabrera et al. (2006), Henning et al. (2006) , andKarori et al. (2007) . The high antioxidant activity noted in blackteas in this study implies that black tea from appropriate tea culti-

vars canpotentially be as efcacious in its antioxidative capacity asgreen tea.

Although the antioxidant capacity was not statistically differentbetween teas fortied withmilk at 0%(v/v) and 2% (v/v), the antiox-idant activity generally decreased signicantly ( p < 0.05) with in-creased concentrations of milk above the 2% threshold and it wassignicantly lower in black teas than in green tea. The decrease inantioxidant activitywith addition of milk reveals an interaction be-tweenteapolyphenols andmilk proteins. These ndings arecompa-rable to those obtained by Lorenz et al. (2007) who showed that theadditionof milk toblack teainhibited the biologicalactivity of tea incardiovascularendothelial functions while teawithout milk hadpo-sitive effects on the same. The major proteins in milk are casein ( a -casein, b-casein, k-casein), a -lactalbumin, b-lactoglobulin, immu-noglobulin and serum albumin ( Etzel, 2004; William, 2004 ). In anexperimentto investigate theinteraction of individualmilk proteinswith catechins, thecaseinproteins were foundto lower thecatechinlevels though no effect was reported with the other milk proteins(Lorenz et al., 2007 ). The lowering of tea antioxidant potency by

addedmilk maythereforebe ascribedto theinteraction andbindingof tea catechins by casein proteins. Catechins correlated positivelywith antioxidant activity in this study and, therefore, it can be ex-pected that the addition of casein-rich milk to tea would decreaseits antioxidant activity. The interaction and binding of polyphenolsto milk proteins depends both on the types of both polyphenol andprotein. Polyphenols with a galloylmoiety, high numberofhydroxylgroups and large molecular size have strong binding afnity to themilk proteins ( Imed et al., 2011; Jianbo et al., 2011; Svetli et al.,2010 ). Binding of the tea molecules by the milk proteins reducestheir ability to donatehydrogenatoms ( Svetli et al., 2010 ), that bindand stabilize the free radicals and hence the decline in antioxidantpotency. Theantioxidant activitycorrelated highlywith thegallatedcatechins ingreenteas, andTF-3-MG, TFDG,totalTFand TRsin black

tea. Black teas possess high levels of TFs and TRs that have largermolecular size, more galloyl groups andhydroxyl groupsandhence

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GSH levels in various tissues at different time intervals

c

GSH levels due to different teas in the kidney

b d

GSH levels due to different teas in plasma GSH levels due to different teas in mice brain

Kidney Brain Plasma Liver 0

3

6

9

12


G S H ( m

/ l )

Fig. 1. (ad) GSHlevels in various mice tissues after consumption of various teas at different time intervals. Untreated-Mice on water only;Purple-black teaprocessed from apurple leaf coloured tea cultivar rich in anthocyanins; Purple m-black tea from a purple leaf tea cultivar fortied with 2% milk; Black m-milk (2%) fortied black tea.



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high binding afnity to the milk proteins as compared to the cate-chingreenteasandthis could explainthe lower antioxidant activityinmilk-fortiedblack teas than ingreenteas with milk. Proteins richin proline have particularly been shown to have a strong afnity forthe hydroxyl groups of avonoids and therefore casein proteins canbe expected to bind the tea polyphenols, unlike the other milk pro-teins that lack or have very low contents of proline residues ( Imedetal., 2011 ). Proline-richproteins haveexiblesecondary structuresand tend to readily form hydrogen bonds due to increased accessi-bilityof the peptidebond and the carbonyl group of tertiary amides(Poncet-Legrand, Gatier,Cheynier, & Imberty, 2007 ). These proteinshave particularly high binding afnity towards galloylated mole-cules, including EGCG and TFDG that signicantly contribute totheantioxidant activityof green andblack tea,respectively. Bindingof the polyphenols by milk proteins masks the active sites such thatthey do not attain their optimum radical-scavenging capacity ( Artset al., 2002; Svetli et al., 2010 ).

Besides milk, black tea consumers prefer to sweeten their tea.The concentrations of the sweeteners used in this study were setto be similar to those that are normally added to tea during con-sumption. Stevia, which is 300 times sweeter than sugar andknown to exhibit some antioxidant activity ( Abou-Arab, Azza, &Ferial, 2010 ), had no signicant inuence on the antioxidant activ-ityof plain and milk black teas. This implies that there areeither nosignicant interactions between the stevia glycosides and tea mol-ecules in the plain teas or any interaction with the milk teas andtherefore the plain and milk black teas with different stevia con-centrations were not statistically different. However, addition of table sugar and honey to plain black teas signicantly decreasedthe antioxidant activity of the latter in a concentration-dependentmanner and the decrease was more in teas with milk. These obser-vations reveal that the antioxidant capacity of tea is not only af-fected by the milk proteins but also by other additives, such assucrose, which is a major constituent of both honey and table su-gar. Complex compounds, such as pentagalloylglucose (PGG),tetragalloylglucose (4GG) and trigalloylglucose (3GG), are likelyto be formed as glucose interacts with the gallic acid in tea whereby the glucose hydroxyl groups are serially substituted by gallicacid ( Zhang, Li, Sung-Hoon, Ann, & Junxuan, 2009 ). Bovine serumalbumin and other proline-rich proteins present in milk, throughhydrophobic, hydrogen and covalent bonding, ultimately interactwith these glucose complexes altering the biological activity of tea molecules ( Monthana, Ho, Wang, & Huang, 2007 ). This couldprobably explain the decrease in antioxidant activity of black teasafter addition of table sugar and honey and also of teas with milk.On its own, honey, though chemically similar to sugar was estab-lished to signicantly have a higher antioxidant activity than theother sweeteners. This could be due to the processing methodsfor table sugar that strip off all the vitamins, proteins and other ac-tive biomolecules that might otherwise contribute to the antioxi-

dant activity in sugar. Though rich in sugars, honey also containscompounds such as chrysin, pinobanksin, vitamin C, catalase, andpinocembrin that are thought to function as antioxidants ( Valen-tini, Miquel, & Pierre, 2010 ), and which could be linked to itsslightly higher antioxidant activity. Though the antioxidant activ-ity of honey was, in this study, expected to positively affect theantioxidant activity of tea, it was found to be the most inhibitingsweetener in both plain and black teas with milk. The mechanismby which sucrose inhibits the antioxidant activity in tea is still notfully understood though, as postulated, this may involve the for-mation of glucosegallic complexes. There is a need for more re-search on this so as to elucidate the interactions betweensucrose, proteins and the bioactive molecules in tea.

The potential health effects of tea depend, not only on the

amount consumed, but on bioavailability ( Joshua & Chung, 2003 ).Increase in the antioxidant capacity in the plasma is indirect

evidence of absorption through the gut barrier after consumptionof polyphenol-rich foods ( Augustin & Gary, 2000 ). Besides othermechanisms, tea polyphenols are known to scavenge free radicalsdirectly since they are capable of donating hydrogen atoms fromthe hydroxyl groups ( Frei & Higdon, 2003 ). The ultimate antioxi-dant potential in vivo is dependent on the absorption, metabolism,distribution and excretion of the bioactive compounds within thebody after ingestion ( Spencer, 2003 ). In this study, the effect of tea antioxidants in vivo was investigated by measuring the gluta-thione (GSH) levels. GSH is the most abundant intracellular thiol-based antioxidant, present in all living aerobic cells, and providespowerful antioxidant protection to body systems heavily exposedto reactive oxygen species ( Turgut, Ya sar, Bunyamin, Sebahat, &Osman, 2006 ). Tea is one of the well-known antioxidant-rich andwidely-consumed beverages and its antioxidant activity after con-sumption is therefore expected to aid the antioxidant capacity inthe body system. The results obtained from this study clearly dem-onstrate the enhancing effect of tea antioxidants on GSH. Thoughthe specic bioactive tea molecules responsible for these effectswere not evaluated, different teas, namely, green, black and purpleteas, showed increased and signicant variations of GSH levels indifferent tissues. Overall, higher levels were found in plasma whilelower levels were seen in the liver, explaining the fact that theabsorption and distribution of the tea molecules varied in the dif-ferent tissues. According to Henning, Jung, and Heber (2008) , gal-lated tea molecules, such as EGCG and ECG, mainly occur inplasma and urine in the free form, whereas the majority of nongal-lated catechins, including EC, EGC, occur in the conjugated form.Gallated tea molecules correlated signicantly with the antioxi-dant activity and this could have led to the higher levels of GSHin plasma than in other tissues. Besides variation between tissues,GSH levels also varied in the same tissues due to various types of teas and also at different time intervals after tea consumption.The inuence of tea consumption on GSH in the brain indicatesthat indeed the tea molecules are able to cross the brain blood bar-rier where they can scavenge free radicals that are continuouslyproduced under normal physiological and pathological conditions.

Catechins, which are largely found in green tea, are rapidlymetabolised and eliminatedand this could explain the lower levelsof GSH due to green tea consumption when compared to the othertypes of tea (black and purple). It has been demonstrated that thelevels of EGCG, which is the most physiologically active and abun-dant catechin in green tea, decrease in the intestine due to thealkaline pH that favours its oxidation, thereby reducing the antiox-idant activity of green tea ( Spencer, 2003 ). Absorption and metab-olism of biomolecules of black and purple teas, that are rich intheaavins, thearubigins and anthocyanins, respectively, are, how-ever, not well researched. The metabolism and excretion of theselatter molecules, through the kidneys, seem to be lower than thoseof the green teas, probably due to their high molecular sizes, which

could explain the high levels of GSH due to black and purple teaconsumption noted in this study. In a study to investigate the bio-availability of tea polyphenols, higher levels of theaavins thanEGCG in prostrate tissue were reported and this could also explainthe higher levels of GSH due to consumption of black teas ratherthan green teas noted in this study ( Henning et al., 2006 ). The highlevels of GSH due to black tea when compared to green tea con-sumption could also be due to the rapid elimination of gallated cat-echins via the bile to the colon, as documented by Manach, Gary,Christine, Augustin, and Christian (2005) , and McKay and Jeffrey(2007) . Results on antioxidant activity have demonstrated thatthe antioxidant activity of black teas in vitro with 2% milk was sim-ilar to that of plain black tea. Consumption of black teas with milkresulted in higher GSH levels in plasma than did consumption of

plain black teas. However, in liver, kidney and brain tissues, theGSH levels due to consumption of plain black teas were higher



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than that due to black teas with milk. The binding of tea polyphe-nols by the milk proteins in the milk-fortied black teas could havehindered the bioavailability of these tea molecules in these lattertissues and therefore the lowGSH levels due to black tea with milk.The GSH levels were signicantly different at different time inter-vals. Although the variations were noticeable in different tissues,overall, the GSH levels peaked at 2 h after tea consumption. Thisimplies that consumption of tea every 2 h would be necessary tomaximise the bioavailability of the tea molecules in the body sys-tem and hence their health benets. At 8 h after consumption of tea, the GSH levels were low in all the tissues, meaning that almostall the tea molecules had been metabolised and excreted. These re-sults are consistent with those obtained by Henning et al. (2006) .

5. Conclusion

The bioactive molecules assayed in black teas in this studyclearly established that Kenyan black teas possess antioxidantproperties comparable with those of green teas. Addition of sec-ondary ingredients, especially milk and sucrose, lowers the antiox-idative capacity of tea, which is associated with the positive healtheffects of the beverage and therefore tea preparation and con-sumptionhabits should be rationalised so as to maximise potentialhealth benets. Stevia, a non-caloric sweetener, is a better alter-native to sugar according to the ndings in this study. Regular con-sumption of tea, probably every 2 h, will ensure the bioavailabilityof the bioactive tea molecules in order to maximise health effectsin the body system. Further studies are, however, required toinvestigate the specic tea molecules and their optimum intakelevels, since metabolism of these compounds differs in differentbody tissues. This will enable the processing of teas with specicactive biomolecules that can impact specic biological activity inspecic body organs and at the same time contribute to increasedconsumption for better health.

Acknowledgement

We wish to acknowledge the Tea Research Foundation of Kenya(TRFK) for funding this work.

References

Abeywickrama, K., Ratnasooriya, W., & Amarakoon, A. (2011). Oral hypoglycaemic,antihyperglycaemic and antidiabetic activities of Sri Lankan Broken OrangePekoe Fannings (BOPF) grade black tea ( Camellia sinensis L .) in rats. Journal of Ethnopharmacology, 135 , 278286 .

Abou-Arab, A., Azza, A., & Ferial, A. (2010). Physico-chemical assessment of naturalsweeteners steviosides produced from Stevia rebaudiana bertoni plant. African Journal of Food Science, 4 , 269281 .

Arts, M., Haenen, G., Wilms, C., Beetstra, S., Heijnen, C., & Voss, H. (2002).

Interactions between avonoids and proteins: Effect on the total antioxidantcapacity. Journal of Agricultural and Food Chemistry, 50 , 11841187 .Augustin, S., & Gary, W. (2000). Dietary intake and bioavailability of polyphenols.

Journal of Nutrition, 130 , 20732085 .Cabrera, C.,Reyes, A.,& Rafael, G. (2006). Benecial effectsof green tea. Journal of the

American College of Nutrition, 25 , 7999 .Chen, W., Xiao-Ling, Z., Dong-Lan, W., Shu-Tian, L., Yue, H., Yan, H., et al. (2010).

Effects of epigallocatechin-3-gallate on behavioral impairments induced bypsychological stress in rats. Experimental Biology and Medicine, 235 , 577583 .

Economic Review of Agriculture, Central planning and project monitoring unit,(2007). Ministry of Agriculture, Kenya, 5 , 3436 .

Etzel, M. (2004). The emerging role of dairy proteins and bioactive peptides innutrition and health manufacture and use of dairy protein fractions. The Journalof Nutrition, 134 , 9961002 .

Ferruzzi, M. (2010). The inuence of beverage composition on delivery of phenoliccompounds from coffee and tea. Physiology and Behavior, 100 , 3341 .

Frei, B., & Higdon, J. (2003). Antioxidantactivityof teapolyphenols in vivo : Evidencefrom animal studies. Journal of Nutrition, 133 , 32753284 .

Giustarini, D., Rossi, R., Milzani, A., Colombo, R., & Dalle-Donne, I. (2004). S-

glutathionylationfrom redox regulation of protein functions to human diseases. Journal of Cell Molecular and Medicine, 8 , 201212 .

Gupta, J., Siddique, Y., Beg, T., Ara, G., & Afzal, M. (2008). A review on benecialeffects of tea polyphenols on human health. International Journal of Pharmacology, 5 , 314338 .

Hamilton-Miller, J. (2001). Anticariogenic properties of tea ( Camellia sinesis ). Journalof Medical Microbiology, 50 , 299302 .

Heinrich, U., Carolyn, E., Silke, H., Hagen, T., & Wilhelm, S. (2011). Green teapolyphenols provide photoprotection, increase microcirculation and modulateskin properties of women. The Journal of Nutrition, 141 , 12021208 .

Henning, S., Aronson, W., Yantao, N., Conde, F., Nicolas, H., Navindra, P.,et al.(2006).Tea polyphenols and theaavins are present in prostate tissue of humans andmice after green and black tea consumption. The Journal of Nutrition, 136 ,18391843 .

Henning, S., Jung, J., & Heber, D. (2008). Nongallated compared withgallated avan-3-ols in green and black tea are more bioavailable. The Journal of Nutrition, 138 ,15291534 .

Hilton, P. (1973). Tea In: Snell F. and Ettre L. Encyclopedia of Industrial Chemical Analysis, 8 , 416455 .

Imed, H., Philippe, B., Saber, H., Guy, S., Robert, C., & Heidar-Ali, T. (2011).Interaction of milk a- and b-caseins with tea polyphenols. Food Chemistry, 126 ,630639 .

ISO 14502-1, (2005). Determination of substances characteristic of green and blacktea. Part 1: Content of total polyphenols in tea. Colorimetric method usingFolin-Ciocalteu reagent.

ISO 14502-2, (2005). Determination of substances characteristic of green and blacktea. Part 2:Contents of catechins in green tea Method using High PerformanceLiquid Chromatography.

Jianbo, X., Fangfang, M., Fan, Y., Yalu, Z., Chao, Z., & Koichiro, Y. (2011). Structureafnity relationship and inuencing bioactivity aspects. Molecular Nutrition andFood Research, 55 , 16371645 .

Joshua, D., & Chung, S. (2003). Cancer chemo preventive activity and bioavailabilityof tea and tea polyphenols. Mutation Research, 523 , 201208 .

Karori, S., Wachira, F., Wanyoko, J., & Ngure, R. (2007). Antioxidant capacity of different types of tea products. African Journal of Biotechnology, 6 , 22872296 .

Karori, S., Wachira, F., Wanyoko, J., & Ngure, R. (2008). Different types of teaproducts attenuate inammation induced in Trypanosome brucei infected mice.Parasitology International, 57 , 325333 .

Kerio, C., Wachira, F., Wanyoko, J., & Rotich, M. (2012). Characterization of anthocyanins in Kenyan teas: Extraction and identication. Food Chemistry,131 , 3138 .

Lorenz, M., Nicoline, J., Amelie-von, K., Peter, M., Gert, B., Karl, R., et al. (2007).Addition of milk prevents vascular protective effects of tea. European Heart Journal, 28 , 219223 .

Manach, C., Gary, W., Christine, M., Augustin, S., & Christian, R. (2005).Bioavailability and bioefcacy of polyphenols in humans. I. Review of 97bioavailability studies. The American Journal Clinical Nutrition, 81 , 230242 .

McKay, D., & Jeffrey, B. (2007). Roles for epigallocatechin gallate in cardiovasculardisease and obesity:An introduction. Journal of the American College of Nutrition,

26 , 362365 .Mendel, F.,Soo-yeun, K.,Sin-jung, L.,Gyeong-phil, H.,Jae-sook, H.,Kap-rang, L., et al.

(2005). Distribution of catechins, theaavins, caffeine and theobromine in 77teas consumed in the United States. Journal of Food Science, 70 , 550559 .

Monthana, C., Ho, C., Wang, X., & Huang, Q. (2007). Monitoring the bindingprocesses of black tea thearubigin to bovine serum albumin surface usingQuartz Crystal Microbalance with dissipation monitoring. Journal of Agricultureand Food Chemistry, 55 , 1011010116 .

Moskaug, J., Harald, C., Myhrstad, W., & Rune, B. (2005). Polyphenols andglutathione synthesis regulation. American Journal of Clinical Nutrition, 81 ,277283 .

Mwaura, F., Ogise, M. (2007). Tea farming enterprise contribution to smallholderswell-being in Kenya. African Association of Agricultural Economists ConferenceProceeding 307313.

Ng, T., Lei, F., Mathew, N., Ee, H., & Keng, B. (2008). Tea consumption and cognitiveimpairment and decline in older Chinese adults. American Journal of ClinicalNutrition, 88 , 224231 .

Obanda, M., Owuor, O., & Mangoka, R. (2001). Changes in the chemical and sensoryquality parameters of black tea due to variations of fermentation time andtemperature. Food Chemistry, 75 , 395404 .

Owuor, P., & Obanda, M. (2007). The useof green tea( Camellia sinensis ) leaf avano-3-ol composition in predicting plain black tea quality potential. Food Chemistry,100 , 874884 .

Owuor, O., Obanda, M., Hastings, E., Nicholas, I., Louwrance, P., & Zeno, A. (2006).The relationship between some chemical parameters and sensory evaluationsfor plain black tea ( Camellia sinensis ) produced in Kenya and comparison withsimilar teas from Malawi and South Africa. Food Chemistry, 97 , 644653 .

Poncet-Legrand, C., Gatier, C., Cheynier, V., & Imberty, A. (2007). Interactionsbetween avan-3-ols and poly ( L -proline) studied by isothermal titrationcalorimetry: Effect of the tannin structure. Journal of Agriculture and FoodChemistry, 55 , 92359240 .

Rahman, I., Arnna, K., & Saibal, K. (2007). Assay for quantitative determination of glutathione and glutathione disulphide levels using enzymatic recyclingmethod. Nature Protocols, 1 , 31593165 .

Roberts, E., & Smith, R. (1961). Spectrophotometric determinationof theaavins andthearubigins in black tealiquors in assessmentsof quality in tea. The Analyst, 86 ,9498 .

Sharangi, B. (2009). Medicinal and therapeutic potentialities of tea ( Camelliasinensis ). Food Research International, 42 , 529535 .

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Shinichi, K. (2008). The relation between green tea consumptionand cardiovasculardisease as evidenced by epidemiological studies. The Journal of Nutrition, 138 ,15481558 .

Shuwen, L., Hong, L., Qian, Z., Yuxian, H., Jinkui, N., Asim, K., et al. (2005). Theaavinderivatives in black teaand catechin derivatives in green teainhibit HIV-1 entryby targeting gp41. Biochimica et Biophysica Acta, 1723 , 270281 .

Spencer, J. (2003). Metabolism of tea avonoids in the gastrointestinal tract. Journalof Nutrition, 133 , 32553261 .

Svetli, D., Guy, S., & Heidar-Ali, T. (2010). Dual effect of milk on the antioxidantcapacity of green, Darjeeling, and English breakfast teas. Food Chemistry, 122 ,539545 .

Turgut, G., Ya sar, E., Bunyamin, K., Sebahat, T., & Osman, G. (2006). Changes in thelevels of MDA and GSH in mice serum, liver and spleen after aluminumadministration. Eastern Journal of Medicine, 11 , 712 .

Valentini, A.,Miquel, C., Pierre, T. (2010) DNA barcoding forhoney biodiversityISSN14242818.

Wang, P., William, J., Min, H., Yanjun, Z., Ru-Po, L., David, H., et al. (2010). Green teapolyphenols and metabolites in prostatectomy tissue: Implications for cancerprevention. Cancer Prevention Research, 3 , 985993 .

William, R. (2004). Bioactive properties of milk proteins with particular focus onanticariogenesis. The Journal of Nutrition, 134 , 989995 .

Wu, L., Juan, C., Hwang, L., Hsu, Y., & Ho, L. (2004). Green tea supplementationameliorates insulin resistance and increases glucose transporter IV content in afructose-fed rat model. European Journal of Nutrition, 43 , 116124 .

Zhang, J., Li, L., Sung-Hoon, K., Ann, H., & Junxuan, L. (2009). Anti-cancer, anti-diabetic and other pharmacologic and biological activities of penta-galloyl-glucose. Pharmaceutical Research, 26 , 127 .

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korir et al. (2014) the fortification of tea with sweeteners and milk and its effect on in vitro.pdf

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