© Copyright by Institute of Animal Reproduction and Food Research of the Polish Academy of Sciences
© 2015 Author(s). This is an open access article licensed under the Creative Commons Attribution-NonCommercial-NoDerivs License
Tea andherbal infusions, which are popular, socially ac-
cepted, andeconomical, drinks [Trevisanato &Young-In
Kim, 2000], can beprepared from any part ofvarious plants,
i.e. roots, ﬂ owers, seeds, berries, or bark, depending on
thesolubility oftheactive constituents included [Apak etal.,
2006]. Itiswell-documented that these infusions, prepared
from valuable parts ofherbs, are among themajor contribu-
tors ofphenolics inour diet [Shahidi, 2000]. Flavonoids, as
theleading polyphenol group present inherbs, have been in-
dicated to provide protection against several forms ofcancer
andcardiovascular diseases, as well as enhance thefunction
oftheimmune system [Craig, 1999]. Brewing tea leaves inhot
water has been reported to release 69–85% ofthebioactive
ﬂavonoids within 3–5 minutes [Keli etal., 1996] which con-
tributes to theintake of80mg ﬂ avonoids per 100mL oftea
consumption [van Dokkum etal., 2008].
Themajority oftheplant materials, that include phyto-
chemicals possessing health-promoting antioxidant activ-
ity, are also used bythebees to collect honey nectar, lead-
ing to thetransfer ofthese bioactive components into honey
[TheNational Honey Board, 2002]. Honey isanatural sweet-
* Corresponding Author: Tel: +90 212 2857340; Fax: +90 212 2857333
E-mail: firstname.lastname@example.org (E.Capanoglu)
ener produced byhoneybees from thenectar ofblossoms
(ﬂ oral (nectar) honey) andfrom secretions ofliving parts
ofplants or excretions ofplant-sucking insects on theliving
part ofplants (honeydew honey) [Persano Oddo etal., 2004].
Honey isreported to bean important source ofantioxidants,
including ﬂ avonoids, phenolic acids, carotenoid derivatives,
organic acids, Maillard reaction products, etc. [Gheldof
&Engeseth, 2002; Aljadi &Kamaruddin, 2004]. Various lit-
erature studies pointed out theantimicrobial [Alvarez-Suarez
etal., 2013; Al-Waili etal., 2011; Alzahrani etal., 2012; Chang
etal., 2011; Israili, 2014], antioxidant [Alvarez-Suarez etal.,
2012a, b; Alzahrani etal., 2012], antiinﬂ ammatory, andan-
titumoral properties [Alvarez-Suarez etal., 2013] ofhoney,
as well as its potential use incombination with conventional
therapy as anovel antioxidant inthemanagement ofchronic
diseases that are mostly related to theoxidative stress [Ere-
juwa etal., 2012].
Theuse ofhoney can besuggested to sweeten tea as
ahealthier way oftea consumption with thepreferred sweet
taste. However, based on our current literature search, there
isno data on how theantioxidant potential ofherbal infu-
sions isaffected bytheaddition ofhoney. Therefore, theaim
ofthepresent work was to determine andcompare theinﬂ u-
ences ofﬂ ower honey (nectar honey) andpine honey (honey-
dew honey) addition on thetotal phenolic andtotal ﬂ avonoid
contents, as well as total antioxidant capacities ofdifferent
Section: Food Chemistry
Pol. J.Food Nutr. Sci., 2015, Vol. 65, No. 2, pp. 127–135
Effects ofHoney Addition on Antioxidative Properties ofDifferent Herbal Teas
Gamze Toydemir1, Esra Capanoglu2*, Senem Kamiloglu2,
Ebru Firatligil-Durmus2, Asli E.Sunay3, Taylan Samanci3, Dilek Boyacioglu2,3
1Department ofFood Engineering, Faculty ofEngineering &Architecture, Okan University, Akﬁ rat-Tuzla,
34959, Istanbul, Turkey
2Department ofFood Engineering, Faculty ofChemical andMetallurgical Engineering, Istanbul Technical University,
34469 Maslak, Istanbul, Turkey
3Scientiﬁ c Bio Solutions LLC., Maslak, Istanbul, Turkey
Key words: herbal tea, honey, temperature, phenolics, ﬂ avonoids, antioxidant capacity
Tea andherbal infusions are among themajor contributors ofphenolic compounds, speciﬁ cally ﬂ avonoids, inour daily diet. Honey isanother
antioxidant-rich food that iswidely used as anatural sweetener. Inthis work, theeffects ofhoney addition on antioxidant properties ofdifferent herbal
teas were investigated. For this purpose, 2 different types ofhoney (ﬂ ower andpine honey) were added into 9 different herbal teas (melissa, green tea,
rosehip, sage, echinacea, fennel, linden, daisy, andginger) at 4 different temperatures (55˚C, 65˚C, 75˚C, and85˚C), andthechanges inthecon-
tent oftotal pheolics, total ﬂ avonoids, andtotal antioxidant capacity were determined. Thetotal phenolic content andthetotal antioxidant capacity
ofthehoney-added-tea samples were found to beincreased (up to 57% for both), especially with pine honey andat higher temperatures ofhoney
addition. Theﬁ ndings ofthis study supported theuse ofhoney as anatural sweetener intea inorder to beable to beneﬁ t from thehealth-enhancing
antioxidative properties ofthese two promising food products.
128 Antioxidative Properties ofHoney-Added Herbal Teas
herbal tea samples. Inaddition, theeffect oftheinfusion tem-
perature, at which honey was added, was also investigated.
Honey andherbal tea samples
Flower honey (from Marmaris, Mugla region ofTurkey)
andpine honey (from Eastern Anatolia region ofTurkey)
samples were collected, induplicates in2013, directly from
beekeepers inTurkey, andtested for their total phenolic con-
tents andtotal antioxidant activities before use. Inorder to
establish thebotanical origins ofhoney (ﬂ oral, pine) samples,
microscopic analysis, pollen andspore determination, con-
ductivity, acidity, humidity, diastase andsugar proﬁ le analy-
sis were performed along with sensory testing. Nectar honey
samples were multiﬂ oral. Herbal tea samples, including me-
lissa, green tea, rosehip, sage, echinacea, fennel, linden, dai-
sy, andginger teas, were supplied from atea manufacturer
inTurkey intheform oftea bags.
Herbal tea infusions were prepared byadding 200mL
offreshly boiled deionised water on atea bag (2 g), andbrew-
ing for 3 min (taking theinstructions on thepackage into
consideration) without additional heating. Tea bags were re-
moved andsubsequently, ﬂ ower honey or pine honey samples
were added to these herbal tea infusions at 85˚C, 75˚C, 65˚C,
and55˚C ofinfusion temperatures, measured using ather-
mometer (ISOLAB Laborgerate GmbH, Germany), andat
aconcentration of7.5 g honey/100mL tea. Infusions without
any honey addition were used as controls. For both thehoney-
-added extracts andthecontrols, theanalyses oftotal pheno-
lics, ﬂ avonoids andantioxidant capacity were conducted after
cooling thesamples to room temperature. All honey-added
andcontrol infusions were prepared intriplicates.
Hydroxymethylfurfural (HMF) content ofhoney sam-
ples was determined using thespectrophotometric method
described inTurkish Honey Standard [TS 3036, 2002].
Themethod was based on thecolorimetric reaction among
p-toluidine, barbituric acid andHMF forming ared col-
ored complex. Theabsorbance was measured at 550nm
andtheHMF was quantiﬁ ed using thefollowing formula:
HMF(mg/kg) = A550 × 192
where A550 istheabsorbance measured at 550nm and192
isatheoretical value linked to themolar extinction coefﬁ cient
Total phenolic (TP) content was determined according to
theFolin-Ciocalteau method described previously byVelioglu
etal. . Inbrief, 0.1mL ofsample was added to 0.75mL
ofFolin-Ciocalteau reagent. Themixture was allowed to
stand for 5 min andthen 0.75mL of6% sodium carbonate
solution was added to themixture. After 2 h ofincubation at
room temperature, absorbance was read at 725 nm. There-
sults were expressed as mg gallic acid equivalent (GAE)/L tea.
Total ﬂ avonoid (TF) content was measured using thecol-
orimetric assay developed byZhishen etal. . At time
zero, 1mL ofsample was mixed with 0.3mL of5% NaNO2
solution. After 5 min, 0.3mL of10% AlCl3 was added. At
the6th min, 2mL of1 mol/L NaOH was added to themixture.
Immediately, 2.4mL ofdistilled water was added andtheab-
sorbance was read at 510 nm. Theresults were given as mg
catechin equivalent (CE)/ L tea.
Total antioxidant capacity (TAC) was estimated using two
invitro tests inparallel. TheDPPH (1,1-diphenyl-2-picryl-
hydrazyl) method was performed as described byKumaran
&Karunakaran . 0.1mL ofeach sample extract was
mixed with 2mL of0.1 mmol/L DPPH inmethanol. After
30 min ofincubation at room temperature, theabsorbance
ofthemixture was measured at 517 nm. TheCUPRAC (Cu-
pric Reducing Antioxidant Capacity) method was applied us-
ing theprotocol reported byApak etal. . 0.1mL ofex-
tract was mixed with 1mL of10 mmol/L CuCl2, 7.5 mmol/L
neocuproine and1 mol/L NH4Ac (pH:7). Immediately, 1mL
ofdistilled water was added to themixture to make theﬁ nal
volume of4.1 mL.After 60 min ofincubation at room tem-
perature, absorbance was read at 450 nm. Theresults were
given as Trolox (6-hydroxy- 2,5,7,8-tetramethylchroman-
2-carboxylic acid) equivalent (TE)/ L tea.
Statistical analysis was applied to thedata obtained from
thesamples that were subjected to each assay intriplicates.
Minitab software (version 16.1.0) was used for theone-way
ANOVA andpairwise comparisons between thetreatments
(honey varieties andtemperatures) were done using Tukey
test with a95% conﬁ dence level. Thecorrelation coefﬁ cients
(R2) for results ofthetwo spectrophotometric assays were
calculated using Microsoft Ofﬁ ce Excel 2011 software (Mi-
crosoft Corporation, Redmond, WA, US).
Hydroxymethylfurfural (HMF) content ofhoney samples
TheHMF contents oftheﬂ ower andpine honey sam-
ples were determined to check for an acceptable quality
ofthehoney samples that were subjected to high tempera-
tures. Themaximum value for HMF content ofhoney after
processing and/or blending, isﬁ xed as 40 mg/kg bytheCodex
standard [Codex Stan 12–1981, Rev 2 2001]. Theconcentra-
tions found inthecurrent study were very low (below theCo-
dex limit) both inﬂ ower andpine honey samples, ranging
between 4.6–8.1 mg/kg.
Changes intotal phenolic andtotal ﬂ avonoid contents
oftea samples added-with-honey
Theresults obtained for TP andTF contents were repre-
sented inTable 1. Melissa, green tea, androsehip were theﬁ rst
three teas determined to have thehighest TP contents (549,
465, and397mg GAE/L tea, respectively) thantheir controls,
followed bysage, echinacea, fennel, linden, daisy, andginger,
respectively (71–268mg GAE/L tea). Theﬂ ower honey led to
signiﬁ cant increases (p<0.05) inTP content ofsage tea at all
G.Toydemir etal. 129
infusion temperatures ofhoney addition. On theother hand,
ﬂ ower honey-added green tea andlinden tea gave signiﬁ -
cantly higher (p<0.05) values at 85˚C, rosehip tea at 65˚C
and75˚C, echinacea tea at 55˚C, 65˚C, and85˚C, andfen-
nel tea at 75˚C ofhoney addition temperatures. Flower honey
did not result inany signiﬁ cant changes (p>0.05) inTP con-
tent ofmelissa, daisy, andginger tea samples.
Thepine honey addition into sage, linden, daisy, andgin-
ger tea resulted insigniﬁ cantly higher (p<0.05) TP contents,
incomparison to their controls, at all temperatures. While sig-
niﬁ cant increases were obtained inTP contents ofgreen tea at
75˚C and85˚C androsehip tea at 55˚C, pine honey addition
did not make asigniﬁ cant change inTP contents ofmelissa,
echinacea, andfennel tea samples, at any temperature.
TheTF contents ofmelissa, sage, androsehip tea were
found to bethehighest among theanalysed tea samples
(3705, 1421, and962mg CE/L tea, respectively), followed
byechinacea, green tea, ginger, fennel, linden, anddaisy
teas, respectively (145–799mg CE/L tea). TheTF content
results obtained for theﬂ ower honey addition indicated sig-
niﬁ cant increases (p<0.05) inTF content ofmelissa tea at
55˚C andsage tea at 75˚C, only. On theother hand, ﬂ ower
honey-added green tea, rosehip, echinacea, fennel, andgin-
ger tea samples were determined to have signiﬁ cantly reduced
(p<0.05) TF contents at any temperature ofhoney addition.
TF contents ofdaisy andlinden tea did not change signiﬁ -
cantly with ﬂ ower honey.
TheTF content measurements for pine honey-added tea
samples revealed that pine honey addition resulted insig-
niﬁ cant increases (p<0.05) inTF contents ofmelissa tea at
55˚C, 65˚C, and75˚C, sage tea at 55˚C, 65˚C, and85˚C,
fennel tea at 65˚C, linden tea at 65˚C, 75˚C, and85˚C,
anddaisy tea at all 4 infusion temperatures ofhoney addi-
tion. On theother hand, signiﬁ cant reductions were obtained,
with pine honey addition, ingreen tea at 65˚C andinrosehip
tea at 85˚C.TF contents ofechinacea andginger tea were not
affected signiﬁ cantly with theaddition ofpine honey.
Changes intotal antioxidant capacity oftea samples
Thechanges intotal antioxidant capacity (TAC) values
ofdifferent tea samples, with ﬂ ower honey andpine honey ad-
TABLE 1. Thechanges intotal phenolic andtotal ﬂ avonoid contents of9 different herbal teas with ﬂ ower honey andpine honey addition at 4 different
Total phenolic content (mg GAE/L)
Melissa Green tea Rosehip Sage Echinacea Fennel Linden Daisy Ginger
Flower-55˚C 596±41a533±78ab 493±43ab 343±26a389±85ab 163±27ab 134±29ab 55±13b58±2d
Flower-65˚C 584±96a543±36ab 521±54a327±23a449±101a152±38ab 116±10ab 58±17b60±2d
Flower-75˚C 601±53a547±68ab 525±61a343±19a361±64abc 174±28a107±27ab 60±25b57±4d
Flower-85˚C 600±65a563±63a 436±77ab 333±23a378±63ab 166±33ab 144±16a73±25b93±10bc
Pine-55˚C 659±101a544±61ab 530±59a336±8a291±61bc 155±18ab 135±15a134±33a127±25a
Pine-65˚C 680±111a544±37ab 476±108ab 336±17a279±51bc 145±38ab 140±16a132±28a128±11a
Pine-75˚C 640±57a552±21a460±56ab 313±23a293±65bc 134±22ab 136±32a141±28a 111±16ab
Pine-85˚C 584±72a553±32a417±68ab 340±12a308±53bc 160±34ab 152±33a138±22a132±11a
Total ﬂ avonoid content (mg CE/L)
Melissa Green tea Rosehip Sage Echinacea Fennel Linden Daisy Ginger
Control 3705±170c726±40a962±88a1421±121b799±87a219±35b179±29c145±7de 323±43a
Flower-55˚C 4104±135ab 550±40de 743±74cd 1675±122ab 627±75bcd 157±9c181±23bc 131±11e249±10bcd
Flower-65˚C 4018±72abc 541±63e702±51d1583±74ab 644±66bcd 162±9c200±31abc 145±7cde 258±16bcd
Flower-75˚C 3876±156bc 571±51cde 803±97bcd 1703±53a575±103d166±17c216±34abc 146±8cde 244±7cd
Flower-85˚C 3858±447bc 563±64cde 686±76d1650±279ab 623±99cd 164±6c181±20bc 154±11bcd 223±17d
Pine-55˚C 4128±107ab 629±28bcd 846±48abc 1768±161a780±79ab 240±25ab 220±32abc 175±12a293±23abc
Pine-65˚C 4271±262a637±22bc 900±58ab 1851±203a762±84abc 262±12a262±49a170±13ab 290±46abc
Pine-75˚C 4171±129ab 682±29ab 880±87abc 1564±197ab 759±81abc 245±22ab 262±44a162±9abc 308±34ab
Pine-85˚C 3977±211abc 658±22ab 786±36bcd 1756±141a777±46abc 235±22ab 243±44ab 162±8abc 304±48abc
* Data represent average values ± standard deviation ofthree independent samples. Different letters inthecolumns represent statistically signiﬁ cant
differences (p<0.05). Control samples were tea samples with no added-honey. GAE: gallic acid equivalent; CE: catechin equivalent.
130 Antioxidative Properties ofHoney-Added Herbal Teas
dition at 4 different infusion temperatures (55˚C, 65˚C, 75˚C,
and85˚C), were determined using 2 invitro tests inparallel, which
were DPPH andCUPRAC methods (Table 2). Thehighest TAC
values, determined with DPPH method, were measured for me-
lissa, green tea, androsehip tea samples (1111, 962, and684mg
TE/L, respectively), followed bysage, echinacea, ginger, fennel,
linden, anddaisy tea samples, respectively (43–441mg TE/L)
(Table 2). Theresults ofDPPH method indicated that ﬂ ower
honey led to asigniﬁ cant increase (p<0.05) inTAC ofgreen tea
andsage tea at 85˚C, linden tea at 55˚C and65˚C, andginger
tea at 75˚C.There was no change observed inTAC ofﬂ ower
honey-added melissa, rosehip, echinacea, fennel, anddaisy tea
samples, incomparison to their control infusions.
Thedifferences inTAC ofpine-honey added tea infusions,
measured using DPPH method, indicated signiﬁ cantly higher
values (p<0.05) inpine honey-added sage tea at 65˚C, fennel
tea at 55˚C, 75˚C, and85˚C, linden tea at 65˚C and75˚C,
anddaisy tea at all four temperatures ofhoney addition,
incomparison to their control samples. Whereas, theTAC
ofmelissa, green tea, rosehip, echinacea, andginger tea did not
show any signiﬁ cant change with theinclusion ofpine honey.
Thehighest TAC values, determined using CUPRAC
method, were again inmelissa, green tea, androsehip tea
(2212, 1813, and1424mg TE/L), followed bysage, echi-
nacea, ginger, fennel, daisy, andlinden, respectively (215–
911mg TE/L). Flower honey increased theTAC ofsage tea
signiﬁ cantly (p<0.05) at all infusion temperatures. Inaddi-
tion, substantial increases were also obtained for ﬂ ower hon-
ey-added fennel tea at 55˚C, 75˚C, and85˚C, andlinden tea
at 55˚C and65˚C.TheTAC values oftheremaining 6 tea
samples, analysed with CUPRAC method, were not found to
beaffected signiﬁ cantly with ﬂ ower honey addition at differ-
Pine honey-added sage, echinacea, fennel, andlinden
tea samples, analysed using CUPRAC method, were found
to have signiﬁ cantly higher (p<0.05) TAC values at all
temperatures ofhoney addition, compared to their control
samples. Moreover, pine honey also provided signiﬁ cant in-
creases inTAC ofgreen tea at 85˚C, daisy tea at 55˚C, 65˚C,
and75˚C, andginger tea at 75˚C.TheTAC ofrosehip tea
did not change signiﬁ cantly with theinclusion ofpine honey
at 4 different infusion temperatures, whereas melissa tea was
TABLE 2. Thechanges intotal antioxidant capacity of9 different herbal teas with ﬂ ower honey andpine honey addition at 4 different tea temperatures.
Total antioxidant capacity, DPPH Method (mg TE/L)
Melissa Green tea Rosehip Sage Echinacea Fennel Linden Daisy Ginger
Control 1111±179ab 962±162b 684±113a441±29b273±51abc 78±22b64±13c43±12c100±15b
Flower-55˚C 989±114ab 1069±207ab 613±137a522±67ab 261±28abc 82±8ab 105±14ab 23±9c133±32ab
Flower-65˚C 975±123ab 1106±121ab 629±135a541±21ab 220±13c87±14ab 97±12ab 33±9c125±19ab
Flower-75˚C 940±126b1100±68ab 637±140a545±59ab 220±13bc 97±13ab 89±14abc 44±10bc 157±17a
Flower-85˚C 937±94b1181±181a586±119a642±72a231±24bc 77±8b76±9bc 37±9c124±16ab
Pine-55˚C 1058±157ab 1131±43ab 594±32a544±51ab 316±28ab 107±9a85±20abc 60±5ab 98±15b
Pine-65˚C 1102±132ab 1079±30ab 584±65a575±46a348±80a98±13ab 102±11ab 61±4ab 100±17b
Pine-75˚C 1179±130ab 1114±81ab 604±47a527±48ab 313±54ab 107±11a110±34a70±6a106±19b
Pine-85˚C 1221±124a1055±76ab 519±56a544±58ab 287±73abc 104±16a89±21abc 63±7ab 102±17b
Total antioxidant capacity, CUPRAC Method (mg TE/L)
Melissa Green tea Rosehip Sage Echinacea Fennel Linden Daisy Ginger
Control 2212±84a1813±277b1424±182ab 911±126b610±48b238±34b215±21c219±40c256±47b
Flower-55˚C 2300±105a1859±176ab 1484±82ab 1202±84a660±35ab 299±24a283±41ab 265±57abc 276±26ab
Flower-65˚C 2390±78a1895±281ab 1428±96ab 1119±100a667±57ab 294±41ab 281±27ab 267±49abc 280±29ab
Flower-75˚C 2262±106a1852±104ab 1611±107a1217±67a661±67ab 358±23a252±24bc 266±80abc 276±49ab
Flower-85˚C 2329±28a1810±314ab 1326±100ab 1155±112a674±39ab 308±34a272±26abc 251±53bc 271±27ab
Pine-55˚C 2161±285ab 1929±188ab 1382±124ab 1167±78a730±29a339±39a322±51a339±18a314±45ab
Pine-65˚C 2145±171ab 1892±166ab 1428±260ab 1142±111a706±67a345±41a339±54a309±25ab 297±42ab
Pine-75˚C 2155±202ab 2067±70ab 1389±176ab 1103±123a702±38a333±49a337±49a301±26ab 339±44a
Pine-85˚C 1956±224b2199±103a1214±242b1128±85a729±29a303±30a302±34ab 278±21abc 296±62ab
* Data represent average values ± standard deviation ofthree independent samples. Different letters inthecolumns represent statistically signiﬁ cant
differences (p<0.05). Control samples were tea samples with no added-honey. DPPH: 1,1-diphenyl-2-picrylhydrazyl; Cupric Reducing Antioxidant
Capacity; TE: Trolox (6-hydroxy- 2,5,7,8-tetramethylchroman-2-carboxylic acid) equivalent.
G.Toydemir etal. 131
measured to have reduced TAC values when pine honey was
added at 85˚C.
Thecorrelations between spectrophotometric assay results
Thelinear correlation coefﬁ cients (R2) were calculated
for plots ofTP versus TF, TP versus DPPH, TP versus CU-
PRAC, TF versus DPPH, TF versus CUPRAC, andDPPH
versus CUPRAC assay results. Thelowest correlation was
observed between TP andTF content values (R2=0.30118),
while ahighly linear relationship was determined between
theresults ofDPPH andCUPRAC methods (R2=0.90073)
(Figure 2A). Additionally, thelinear curves obtained for CU-
PRAC versus TP (R2=0.73632) (Figure 2B) andCUPRAC
versus TF (R2=0.54313) (Figure 2C) results had higher cor-
relation coefﬁ cients than those observed for DPPH versus TP
(R2=0.70238) andDPPH versus TF (R2=0.44324) results.
Theeffect ofdifferent types ofhoney
Flower honey andpine honey addition either led to signiﬁ -
cant increases (p<0.05) or did not signiﬁ cantly change theTP
contents ofthehoney-added tea samples compared to their
controls. Flower honey provided asigniﬁ cantly higher (p<0.05)
TP content value inechinacea tea at 65˚C incomparison to
thevalue obtained with pine honey addition at thesame temper-
ature. On theother hand, pine honey-added daisy andginger
teas were measured to besigniﬁ cantly higher intheir TP con-
tents, at all temperatures ofhoney addition, compared to their
ﬂ ower-honey added counterparts (Table 1). Itcould belinked
to thefact that honeys with darker color, as inthecase ofpine
honey, have been reported to possess higher amounts oftotal
phenolic compounds inrecent research studies [Alvarez-Suarez
etal., 2010; Escuredo etal., 2013; Kus etal., 2014 Wilczynska,
2010]. Kus etal.  determined theTP contents oflighter
honeys investigated intheir study (black locust, goldenrod,
rapeseed, andlime) to range inbetween 142.8–192.5mg GAE/
kg; while this range for darker honeys (heather andbuckwheat)
was found to befrom 306.2 to 1113.0mg GAE/kg. Inanother
study, thehighest TP contents were again measured for darker
colored honeys, including chestnut honey (1313mg GAE/kg)
andheather honey (1789mg GAE/kg) [Escuredo etal., 2013].
TheTP contents oftheﬂ ower andpine honeys that we used
inour work were 510 and680mg GAE/kg, respectively.
Thereason for not obtaining thesame effect ofpine honey
for all theanalysed tea samples could berelated with thedif-
ferent phenolic proﬁ les oftheherbal tea samples or thelack
ofthespeciﬁ city oftheFolin-Ciocalteau method for phenolic
compounds [Capanoglu etal., 2008]. TheFolin-Ciocalteau
method was reported to besuffering from anumber ofin-
terfering substances, including speciﬁ cally sugars, aromatic
amines, ascorbic acid [Box, 1983], andamino acids andpro-
teins [Meda etal., 2005] that can also react with Folin-Ciocal-
teau reagent. Thus, itwas strongly suggested that corrections
for those interfering substances should bemade inorder to
establish auniformly acceptable method ofTP to compare
theobtained results rationally [Prior etal., 2005].
Theaddition ofpine andﬂ ower honey, at 4 infusion tem-
peratures, was determined to affect theTF contents ofdiffer-
ent tea samples indifferent ways, including theeffects ofall
signiﬁ cant increases/decreases or not any signiﬁ cant changes
(p<0.05). Itwas remarkable that ﬂ ower honey addition led
to signiﬁ cant decreases (p<0.05) inTF contents ofﬁ ve (out
ofnine) tea samples, including green tea, rosehip, echinacea,
fennel, andginger, at all temperatures ofhoney addition.
On theother hand, pine honey addition did not signiﬁ cantly
change or even increased theTF contents oftea samples (ex-
cept for green tea at 55˚C and65˚C, androsehip tea at 85˚C)
(Table 1). Silici etal.  reported catechin andepicate-
chin as theonly compounds that were determined as thekind
ofﬂ avonoids inhoneydew honey samples, which were also
determined to constitute thelargest content (53% ofdetected
total phenolics) oftotal phenolics intheanalysed honeydew
honey samples. On theother hand, thecontribution ofcate-
chin andepicatechin components to theTP content ofnectar
honeys was found to be33% ofthedetected phenolics [Silici
etal., 2013]. This could have an inﬂ uence on these higher TF
contents ofpine honey-added-tea samples inour study, since
theresults for TF content analysis have been expressed as cat-
echin equivalents (Table 1).
Theresults obtained byDPPH method, for thechanges
inTAC ofdifferent tea samples added-with-ﬂ ower honey re-
vealed signiﬁ cant increases (p<0.05) inTAC ofgreen tea (at
85˚C), sage tea (at 85˚C), linden tea (at 55˚C and65˚C),
andginger tea (at 75˚C). On theother hand, again with
thesame method pine honey was observed to lead to signiﬁ -
cant increases (p<0.05) inTAC ofsage tea (at 65˚C), fennel
tea (at 55˚C, 75˚C, and85˚C), linden tea (at 65˚C and75˚C)
anddaisy tea (at all temperatures). When theﬂ ower honey
andpine honey were compared for their inﬂ uences on TAC,
at thesame temperature ofhoney addition, pine honey was
found to differ from ﬂ ower honey with its signiﬁ cantly higher
(p<0.05) contribution to theTAC ofechinacea tea (at 65˚C),
fennel tea (at 85˚C), anddaisy tea (at all 4 temperatures). For
theother tea samples, pine honey andﬂ ower honey did not
signiﬁ cantly differ (p>0.05) (Table 2).
TheTAC values measured with CUPRAC method in-
dicated signiﬁ cantly increased (p<0.05) TAC bytheeffect
ofpine honey addition inﬁ ve (out ofnine) tea samples,
including sage, echinacea, fennel, linden, anddaisy, inde-
pendent from theinfusion temperatures tested (except for
thedaisy tea at 85˚C). Flower honey was found to contribute
signiﬁ cantly to theTAC ofsage tea (at all temperatures), fen-
nel tea (at 55˚C, 75˚C, and85˚C), andlinden tea (at 55˚C
and65˚C). Inaddition, thecomparison oftheﬂ ower honey-
-added andpine honey-added tea samples, at thesame tem-
perature ofhoney addition, revealed no signiﬁ cant differences
regarding their TAC measured byCUPRAC method (except
for melissa tea at 85˚C andlinden tea at 75˚C). On theother
hand, pine honey had agreater contribution to theTAC val-
ues oftea samples incomparison to their respective control
tea samples (Table 2). These relatively higher TAC values pro-
vided bypine honey could beexplained based on theﬁ ndings
ofother studies, which have pointed out that honey samples
that are darker intheir color have higher antioxidant capaci-
ties ingeneral [Alvarez-Suarez etal., 2010; Kus etal., 2014;
Wilczynska, 2010] since honey color depends on thepotential
alkalinity andash content, as well as on theantioxidatively
132 Antioxidative Properties ofHoney-Added Herbal Teas
active pigments, such as carotenoids andﬂ avonoids [Frankel
etal., 1998]. Alvarez-Suarez etal.  reported theTAC
values ofhoneys tested intheir study to range inbetween
1035 and2945 μmol TE/kg which was linearly correlated
with thecolor range ofthehoneys changing from light to am-
ber. Accordingly, theTAC ofthedarker-colored pine honey
(4075mg TE/kg), used inthis work, was higher incompari-
son to theTAC ofthelighter ﬂ ower honey (3545mg TE/kg).
Theeffect ofdifferent infusion temperatures ofhoney
Theresults obtained for TP contents ofhoney-added-tea
samples pointed out that thehighest values, although not all
were statistically different from control samples, were gener-
ally obtained with theaddition ofﬂ ower/pine honey at infu-
sion temperatures of75˚C and85˚C (except for echinacea tea
for ﬂ ower honey addition, andmelissa androsehip teas for
pine honey addition). TheTF contents ofﬂ
ed-tea samples were again mainly higher at 75˚C and85˚C
ofhoney addition temperatures compared to theother in-
fusion temperatures ofhoney addition. However, itshould
beemphasized that these relatively higher values obtained
at 75˚C/85˚C, incomparison to theother infusion tem-
peratures, ofﬂ ower honey addition were mostly signiﬁ cantly
lower or were not signiﬁ cantly different from therespective
control tea samples (Table 1). On theother hand, when pine
honey was added into tea samples at 65˚C and75˚C ofinfu-
sion temperatures, itprovided relatively higher TF contents
incomparison to theother infusion temperatures ofhoney
addition. Whereas some ofthese TF content values, obtained
for 65˚C/75˚C ofpine honey addition temperatures, were
still lower than thevalues obtained for respective control tea
samples (including green tea, rosehip, andginger tea sam-
ples) (Table 1).
TheTAC ofﬂ ower/pine honey added-tea samples, deter-
mined using DPPH method, were again found to behigher
at 75˚C and85˚C ofhoney addition temperatures com-
pared to theother infusion temperatures ofhoney addition.
On theother hand, themeasurement ofTAC values with
CUPRAC method gave higher values at 65˚C and75˚C
ofinfusion temperatures for ﬂ ower honey addition, andat
55˚C and65˚C ofinfusion temperatures for pine honey ad-
dition (Table 2).
When all theresults were evaluated ingeneral, itcould
beconcluded that theaddition ofﬂ ower/pine honey into dif-
ferent tea samples at 75˚C and(to alesser extent) at 85˚C
gave relatively high values ofTP andTF contents, as well
as TAC, incomparison to theother tested temperatures.
Thepercent changes inTP content (Figure 1A) andTAC
values, determined using CUPRAC method (Figure 1B),
obtained with ﬂ ower/pine honey addition at 75˚C are given
inFigure 1 as therepresentative graphs. Theﬂ ower andpine
honey additions into tea samples at 75˚C were determined
to lead up to 41% and57% increases inTP contents (Figure
1A), andup to 50% and57% increases inTAC values, de-
termined using CUPRAC method (Figure 1B), respectively.
These higher values at higher temperatures may depend on
theformation ofMaillard reaction products, melanoidins,
which have been reported to act as antioxidants [Brudzynski
&Miotto, 2011a,b,c; Turkmen etal., 2006]. Turkmen etal.
, who studied theeffect ofheating honey to 50˚C,
60˚C, and70˚C on theantioxidant activity andbrown pig-
ment formation due to Maillard reaction, determined that
both ofthemeasured values increased with theincreased
temperature. Theauthors evaluated that theincrease
inbrown pigment formation, due to theformation ofMail-
lard reaction products, was accompanied with theincrease
inantioxidant activity, which was more remarkable inheat-
ed honey samples at 70˚C than those at 50˚C and60˚C
[Turkmen etal., 2006]. Inaddition, these Maillard reaction
products were also reported to react with Folin-Ciocal-
teau reagent [Verzelloni etal., 2007] which could explain
thehigher TP content values inhoney added tea samples,
speciﬁ cally at higher temperatures ofhoney addition. Inan-
other study, Brudzynski &Miotto [2011a] hypothesized
that phenolics inhoney may becomponents ofmelanoidin
structure, andthey tested themelanoidin fractions ofun-
heated andheat-treated honey samples for their total phe-
% change, TP
bb b bb b
greentea roschip sage echinasea fennel linden daisy ginger
% change, CUPRAC
control pine honey-addedflower honey-added
bab b b b c
greentea roschip sage echinasea fennel linden daisy ginger
FIGURE 1. Thepercent changes in(A) Total phenolic (TP) contents
and(B) Total antioxidant capacity (TAC) values, determined using Cu-
pric Reducing Antioxidant Capacity (CUPRAC) assay, oftheanalysed
tea samples with ﬂ ower andpine honey addition at 75˚C.Different let-
ters on thecolumns represent thestatistically signiﬁ cant (p<0.05) differ-
ences observed with ﬂ ower or pine honey addition into aherbal infusion
at 75˚C. (See Table 1 (for TP content data) andTable 2 (for TAC data
obtained via CUPRAC method) for thecomplete statistical data).
G.Toydemir etal. 133
nolic contents. Their results indicated asigniﬁ cant increase
intheTP content inmelanoidin fractions oftheheat-treated
honeys as compared to theTP content inmelanoidin frac-
tions oftheir unheated counterparts. This could also explain
thereaction between Maillard reaction products andtheFo-
Because ofthefact that heating ofhoney leads to
theformation ofHMF (5-hydroxymethylfurfural), as are-
sult ofthehexose dehydration inacid media [Belitz &Gro-
sch, 1999], we also checked theHMF contents ofthehoney
samples that were subjected to high temperatures inour
study, andconﬁ rmed that theHMF contents were all below
thelimit value (40 mg/kg). HMF isconsidered as an im-
portant quality parameter for honey bymeans ofevaluating
thefreshness andtheheating andstorage history [Karabour-
nioti &Zervalaki, 2001; Fallico etal., 2004]. HMF forma-
tion inhoney could beinﬂ uenced bythechemical proper-
ties ofhoney, including pH, total acidity, mineral content,
etc., which are dependent on theﬂ oral source from which
thehoney sample has been extracted [Anam &Dart, 1995;
Bath &Singh, 1999]. So, theinclusion ofhoney samples
obtained from different ﬂ oral sources can provide different
levels ofHMF contents.
Therelationships between theresults oftheapplied
Thecorrelation coefﬁ cients (R2) calculated between
theapplied spectrophotometric methods showed that there-
sults oftheCUPRAC assay correlated better with theTP
andTF contents ofdifferent herbal tea samples, compared
to theDPPH assay results. Besides, good correlations were
also observed between theresults ofDPPH andCUPRAC
assays (Figure 2). Inaccordance with our results, CUPRAC
method was proved to correlate well with ABTS andFolin-Ci-
ocalteau assays inherbal plant infusions [Apak etal., 2006],
apricot [Guclu etal., 2006], andkiwifruit [Park etal., 2006]
extracts. Apak etal.  reported theCUPRAC assay as
themost consistent method oftotal antioxidant measurement
inrelation to Folin reagent-responsive TP content, since this
method issuitable for andreacts with avariety ofantioxidant
compounds regardless ofchemical type or hydrophilicity.
Additionally, thelinear correlation determined between CU-
PRAC andABTS assays (R2=0.8) has been linked to thefact
that these methods are similar electron transfer-based antiox-
idant assays [Apak etal., 2007], which can also beevaluated
for thehigh correlation found out between DPPH andCU-
PRAC assays (R2=0.90073) inthis present work. How-
ever, itisworth to remark that although there are anumber
ofmethods that have been developed to assess theantioxi-
dant capacity ofeither pure antioxidant compounds or prod-
ucts containing complex mixture ofantioxidants, there isstill
lack ofcorrelation between theresults obtained for thesame
compound/product bydifferent assays, as well as bythesame
assay indifferent laboratories [Niki, 2011].
On theother hand, lower correlation coefﬁ cients were ob-
tained between TF assay results andtheresults oftheother
three assays. Similarly, Park etal.  andMeda etal.
 reported low correlations between ABTS, CUPRAC
or TP content results andTF contents, which was linked to
thenature ofthemeasurement technique used for total ﬂ avo-
noids. Thealuminum chloride (AlCl3) colorimetric test used
for ﬂ avonoid analysis has been pointed out to besensitive
only for ﬂ avonoid groups that possess thecharacteristic che-
lating functional groups for Al binding (i.e. ﬂ avones andﬂ a-
vonols), while this method does not measure theﬂ avonoids
that do not include these functional groups (i.e. ﬂ avanones).
This leads to theunderestimation oftheTF content byusing
this aluminum chloride method [Chang etal., 2002].
Thecomparison on theeffect ofﬂ ower andpine honey
addition into 9 different herbal tea samples at 4 different
temperatures revealed that theTP content andTAC values
CUPRAC (mg TE/L)
DPPH (mg TE/L)
TP (mg GAE/L)
TF (mg CE/L)
0 200 400 800600 1000
0 1000 2000 40003000 5000
CUPRAC (mg TE/L)
CUPRAC (mg TE/L)
FIGURE 2. Thelinear correlation coefﬁ cients (R2) calculated for plots
of(A) 1,1-diphenyl-2-picrylhydrazyl (DPPH) versus Cupric Reduc-
ing Antioxidant Capacity (CUPRAC), (B) Total phenolics (TP) versus
CUPRAC, and(C) Total ﬂ avonoids (TF) versus CUPRAC assay results.
134 Antioxidative Properties ofHoney-Added Herbal Teas
ofthehoney-added-tea samples were generally higher than
those ofthecontrol tea samples, speciﬁ cally with pine honey
addition andat higher temperatures. These ﬁ ndings support
theuse ofhoney as anatural sweetener intea drink inorder
to beable to beneﬁ t from thehealth-enhancing antioxidative
properties ofthese two promising food products.
This project was ﬁ nancially supported byIstanbul Devel-
opment Agency “Center ofExcellence inBee Products” proj-
ect (Project Number: TR10/14/YEN/0028). Fatma Damla
Bilen, Diyar Selimoglu, Gulbeyaz Cevik, andOznur Aydın
are acknowledged for analytical support.
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Submitted: 22 July 2014. Revised: 31 October and28 November
2014. Accepted: 26 January 2015. Published on-line: 15 April 2015.