Food Control 31 (2013) 403e409

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Food Control
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Antioxidant and antimicrobial activities of various leafy herbal teas

Jungmin Oh, Heonjoo Jo, Ah Reum Cho, Sung-Jin Kim, Jaejoon Han*
Department of Food Science and Biotechnology, Sungkyunkwan University, 2066 Seobu-ro, Jangan-gu, Suwon 440-746, Republic of Korea

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 29 June 2012
Received in revised form
6 October 2012
Accepted 13 October 2012

We evaluated the antioxidant and antimicrobial activities of various leafy herbal tea (LHT) extracts,
including rooibos, green tea, black tea, rosemary, lemongrass, mulberry leaf, bamboo leaf, lotus leaf,
peppermint, persimmon leaf, and mate tea. To compare the antioxidant activities of various LHTs,
samples of each were extracted with 80
C water or 20
C ethanol, and their total phenolic content (TPC),
total avonoid content (TFC), 2,2-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity, 2,2azinobis-3 ethyl benxothiazoline-6-sulphonic acid (ABTS) radical cation decolorization activity, ferric
reducing power, and ferrous ion chelating effect were measured. Green tea ethanol extract showed the
highest antioxidant activity in all assays except the ferrous ion-chelating assay. Water extracts of green
tea and black tea and ethanol extracts of rosemary, mate, and persimmon leaf teas also exhibited
considerable antioxidant potential, followed by the green tea ethanol extract. Minimum inhibitory
concentrations (MIC) and minimum lethal concentrations (MLC) were determined to verify the antimicrobial activities of the LHT extracts against two oral pathogens (Streptococcus mutans and Streptococcus sobrinus) and three food-borne pathogens (Listeria monocytogenes, Shigella exneri, and Salmonella
enterica). Among the tested LHTs, green tea ethanol extract had potent antimicrobial activity against all
ve pathogens, and the mate tea water extract was the most effective against Gram-positive bacteria.
Consequently, green tea ethanol extracts had the most powerful antioxidant and antimicrobial properties, suggesting their potential application as a health-promoting functional ingredient or natural
preservative in foods.
2012 Elsevier Ltd. All rights reserved.

Keywords:
Leafy herbal teas
Antioxidant activity
Antimicrobial activity
Functionality

1. Introduction
Tea is one of the most widely consumed beverages worldwide,
second only to water (Muktar & Ahmad, 2000). Herbs are mainly
consumed in the form of tea, an infusion of dried herbs in warm or
hot water, brewed from the leaves, owers, seeds, fruits, and roots
of plant species (Aoshima, Hirata, & Ayabe, 2007). Leafy herbal teas
(LHT) are widely known to contain a variety of active phytochemicals with biological properties that promote human health and
help reduce the risk of chronic diseases such as allergies, insomnia,
headaches, anxiety, intestinal disorders, depression, and high blood
pressure (Craig, 1999). Various studies have reported that LHT
extracts exert benecial effects on lifestyle-related diseases due to
their anticarcinogenic, antiatherogenic, chemopreventive, antioxidant, and antimicrobial activities (Si et al., 2006). Free radicals are
reactive oxygen species (ROS) produced in the body as by-products
of cellular aerobic respiration and lead to oxidative stress (Yanai,
Shiotani, Hagiwara, Nabetani, & Nakajima, 2008). Antioxidant

* Corresponding author. Tel.: 82 31 290 7803; fax: 82 31 290 7882.

E-mail address: han2009@skku.edu (J. Han).
0956-7135/$ e see front matter 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.foodcont.2012.10.021

activity is dened as an inhibition of the oxidation of lipids,

proteins, DNA or other molecules that occurs by blocking the
propagation step in oxidative chain reactions (Huang, Ou, & Prior,
2005). Primary antioxidants directly scavenge free radicals, while
secondary antioxidants indirectly prevent the formation of free
radicals through Fentons reaction. LHT extracts generally exhibit
both primary and secondary antioxidant capacities (Chan, Lim,
Chong, Tan, & Wong, 2010). Moreover, various herbs and plants
have been receiving greater attention as alternatives to synthetic
additives or preservatives in the food industry (Madsen & Berteisen,
1995).
Natural botanical sources contain a diverse array of compounds
such as phenolic acids, avonoids, tannins, vitamins, and terpenoids that account for their biological properties (Exarchou,
Nenadis, & Tsimidou, 2002), and the antioxidant and antimicrobial abilities of LHT are attributed to phenolic compounds (Uhl,
2000). The antioxidant ability of phenolic components occurs
mainly through a redox mechanism and allows the components to
act as reducing agents, hydrogen donors, singlet oxygen quenchers,
and metal chelators (Rice Evans, Miler, & Paganga, 1997). Therefore,
phenolic compounds can prevent the formation of ROS and reactive
nitrogen species, which include free radicals such as superoxide

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J. Oh et al. / Food Control 31 (2013) 403e409

anion (O2), hydroxyl (OH), and nitric oxide (NO), as well as nonfree radical species such as hydrogen peroxide (H2O2) and nitrous
acid (HNO2) (Zhu, Hackman, & Ensunsa, 2002).
In the present study, we measured the antimicrobial activity of
LHTs against two common oral pathogens and three food-borne
microorganisms. There are numerous studies showing that the
polyphenols and tannins extracted from teas inhibit a broad spectrum of bacteria (Sreeramulu, Zhu, & Knol, 2000). It has been
demonstrated that the antimicrobial effects of the plant-derived
polyphenols cause structural or functional damage to the bacterial cell membrane (Yoo, Murata, & Duarte, 2011). Several studies
have concluded that the functional hydroxyl groups and conjugated
double bonds in LHT extracts may be involved in binding to cell wall
components. Microbial cells are negatively affected by plantderived substances via various mechanisms of actions that attack
the phospholipid bilayer of the cell membrane and disrupt enzyme
systems (Proestos, Boziaris, Kapsokefalou, & Komaitis, 2008).
Furthermore, a common type of dental disease and caries are
associated with microorganisms present on the surface of teeth.
Mutans streptococci play a signicant role in the formation of
dental biolm and the initiation of dental caries and include
bacteria such as Streptococcus mutans, Streptococcus sobrinus,
Streptococcus cricetus, Streptococcus rattuce, and Streptococcus ferus.
Accordingly, various LHT extracts are expected to be effective not
only in maintaining food safety, but also in preventing the growth
of oral microorganisms. The current research aims to monitor the
antioxidant and antimicrobial properties of various LHT extracts.
Few studies have investigated and compared the potential properties of various LHTs, although the biological effects of green and
black tea have been well documented.
The objectives of this study were (i) to examine the antioxidant
capacities of aqueous and ethanol extracts of 11 LHTs, (ii) to
demonstrate a correlation between TPC and the antioxidant activities of LHT, and (iii) to characterize the antimicrobial activity of LHT
against oral and food-borne pathogens.
2. Materials and methods
2.1. Chemicals
DPPH, potassium ferricyanide, gallic acid, ()-catechin, ABTS,
FolineCiocalteus reagent, ferrozine [3-(2-pyridyl)-5,6-bis-(4phenylsulphonic acid)-1,2,4-triazine], and potassium persulfate
were purchased from SigmaeAldrich Chemical Co. (St. Louis, MO,
USA). Ascorbic acid was obtained from Duksan Pure Chemical Co.,
Ltd. (Ansan, Korea). Ferric chloride was provided from Daejung
Chemical & Metals Co. (Shiheung, Korea). Ferrous chloride was
purchased from Kanto Chemicals Co. (Tokyo, Japan). All other
solvents and reagents used in the analysis were of analytical grade.

soluble extracts were prepared under conditions similar to those

generally used for tea drinking. Finely ground samples (50 g) were
extracted in 80
C hot water (1 L) and stirred gently on a magnetic
stirrer for 10 min. For preparation of ethanol extracts, each LHT
powder (50 g) was soaked in ethanol (1 L) and mixed with a lab
stirrer (MS280, Misung Scientic Co., Korea) for 12 h at room
temperature (20
C). Each extract was then ltered through no. 2
Whatman lter paper (Whatman Inc., Clifton, NJ, USA). The
combined ltrate was then evaporated using a rotary evaporator
(Rotavapor RE121, Bchi, Switzerland), and the extra solvent was
removed with a freeze dryer (Heto FD 3, Heto Lab Equipment,
Holten, Denmark). The dried extract sample was weighed to
calculate the soluble constituent yield. Samples were kept in an airtight container at 20
C until further analysis.

2.3. Determination of antioxidant activities of LHT

2.3.1. TPC
TPC of the LHT extracts was analyzed spectrometrically
according to the FolineCiocalteu method (Dewanto, Wu, Adom, &
Liu, 2002). Briey, 100 ml of LHT extract or gallic acid standard
solution was mixed with 2 ml of 2% (w/v) sodium carbonate solution. The mixture was then incubated for 3 min, after which 100 ml
of FolineCiocalteu reagent was added. After standing for 30 min at
room temperature for color development, absorbance was
measured at 750 nm using a spectrophotometer (UV mini-1240,
Shimadzu, Kyoto, Japan). Results are expressed as mg gallic acid
equivalents (GAE) per g dry-matter of herbal tea.
2.3.2. TFC
TFC of the LHT extracts was determined according to the
procedure described by Zhishen, Mengcheng, and Jianming (1999).
Briey, 250 ml of LHT extract or ()-catechin standard solution was
diluted with 1.25 ml of distilled water. Exactly 75 ml of NaNO2
solution (5%) was added to the mixture, followed by a 6-min
incubation at room temperature. Then, 150 ml of AlCl3$6H2O (10%)
was added to the mixture, followed by a 5-min incubation and the
addition of 500 ml of NaOH (1 M). Absorbance was measured at
510 nm using a spectrophotometer. TFC is shown as ()-catechin
equivalents (CTE) per g dry-matter of herbal tea.

2.2. Herbal teas and preparation of extracts

2.3.3. DPPH radical scavenging activity

DPPH radical scavenging activity was performed by the method
of Cheung, Cheung, and Ooi (2003). An aliquot (1 ml) of 0.2 mM
DPPH radical in methanol was mixed with 200 ml of LHT extract or
ascorbic acid standard solution. The mixture was incubated for
30 min in the dark at room temperature, after which the absorbance was measured at 517 nm using a spectrophotometer. Results
are expressed as mg ascorbic acid equivalents (AAE) per g drymatter of herbal tea.

Eleven LHTs, including rooibos tea (Aspalathus linearis), green

tea (Camellia sinensis), black tea (C. sinensis), rosemary tea (Rosmarinus ofcinalis), lemongrass tea (Cymbopogon citrates), mulberry
leaf tea (Morus alba. Linne), bamboo leaf tea (Sasa borealis), lotus
leaf tea (Nelumbo nucifera), peppermint tea (Mentha piperita),
persimmon leaf tea (Diospyros kaki), and mate tea (Ilex paraguariensis) were obtained from Gaialand Corp. (Seoul, Korea), Teafresh Corp. (Busan, Korea) or Fusae Corp., Ltd. (Seoul, Korea).
Individual extracts of the 11 LHTs were prepared using water or
ethanol (90%, v/v) as solvents. We used water as a solvent to
simulate the general condition of preparation for consumption.
Ethanol was used to improve the solubility efciency of both polar
and non-polar functional compounds in the herbal teas. Water-

2.3.4. ABTS radical cation decolorization activity

ABTS radical cation decolorization activity was assayed by the
method of Re et al. (1999). ABTS radical cations were generated by
reacting 7.4 mM ABTS with 2.45 mM potassium persulfate (1:1, v/
v). The mixture was left to stand for 12e16 h in the dark at room
temperature. The ABTS radical cation solution was then diluted
with ethanol to give an absorbance of 1.0e1.2 at 734 nm. LHT
extract or ascorbic acid as a standard solution (200 ml) was mixed
with diluted ABTS radical cation solution (1 ml). The mixture was
vortexed and left to stand at room temperature for 1 h. The
absorbance of the resulting solution was measured at 734 nm using
a spectrophotometer. Results are shown as mg AAE per g drymatter of herbal tea.

J. Oh et al. / Food Control 31 (2013) 403e409

Table 1
List of microorganisms and culture media.

Chelating effect

Pathogens

Microorganisms

Culture Media

Oral pathogens

Streptococcus sobrinus
KCTC 3308
Streptococcus mutans
KCTC 3065
Listeria monocytogenes
KCTC 3710
Shigella exneri
KCTC 22192
Salmonella enterica
KCTC 2514

Brain heart infusion or agar

Food-borne pathogens

405


 


Abssample
 100
% 1
Abscontrol

2.4. Determination of antimicrobial activities of LHT

Brain heart infusion or agar

The microorganisms and complex culture media used in this

study are shown in Table 1. Microorganisms were kept frozen at
80
C in broth containing glycerol (15%, v/v). Before the test, stock
cultures were inoculated into the broth and incubated at 37
C for
24 h. Cultures were transferred to the broth three times at 24 h
intervals. All media were obtained from Difco Co. (Sparks, MD, USA).
The broth micro-dilution test was performed according to the
modied method of Hufford and Clark (1988) to determine the MIC
of each extract. Briey, 100 ml of LHT extract was diluted with broth
in a sterile 96-well microtiter plate. The same volume (100 ml) of
overnight bacterial culture, at a density of 106 CFU/ml, was added to
the wells, and the culture plates were placed in an incubator at
37
C for 24 h. Two-fold serial dilutions were produced in broth to
give nal concentrations of 2.44 mg/mle10 mg/ml. The wells were
visually examined for the lowest concentration of extract that
inhibited microbial growth (indicated by clear wells) after 24 h. The
lowest concentration of extract at which growth did not occur was
noted as the MIC.
The MLC was determined after reading the results of the MIC by
streaking 10 ml of suspension in the well at concentrations above
the MIC. Then, the subcultured agar plates were incubated overnight at 37
C. The MLC was dened as the lowest concentration of
extract that resulted in no bacterial growth on agar.

Brain heart infusion or agar

Nutrient broth or agar
Nutrient broth or agar

2.3.5. Ferric reducing power

The reducing power of the LHT extracts was determined by the
method of Oyaizu (1986). An aliquot (250 ml) of sample or distilled
water (negative control), 250 ml of sodium phosphate buffer
(200 mM, pH 6.6), and 250 ml of potassium ferricyanide (1%) were
mixed and incubated in a water bath at 50
C for 20 min. The
reaction was terminated by adding 250 ml of trichloroethanoic acid
solution (10%, w/v). Then, the mixtures were centrifuged at
5000 rpm for 5 min. The supernatant (500 ml) was mixed with an
equal volume of distilled water and 100 ml of ferric chloride solution
(0.1%, w/v). The intensity of the Prussian blue color was measured
at 700 nm using a spectrophotometer. Results are expressed as the
mean absorbance value.
2.3.6. Ferrous ion chelating effect
The ferrous ion chelating effect of the extracts was investigated
with a method slightly modied from that of Dinis, Madeira, and
Almeida (1994). LHT extract or control (1 ml of distilled deionized
water) was reacted with 2 mM of ferrous chloride (100 ml) for
10 min, and 5 mM ferrozine (100 ml) was added. After 10 min, the
solvent of the sample (3 ml) was mixed with the mixture and
reacted for another 10 min. The absorbance of the mixture was
measured at 562 nm using a spectrophotometer. Ferrous ion
chelating capacity was calculated by the follow equation.

2.5. Statistical analysis

Analysis of variance and Duncans multiple-range tests were
employed to statistically analyze all results. Differences between
means were considered signicant when p  0.05. SAS Analytics
(SAS Institute, Cary, NC, USA) was used for the analysis.

Table 2
Antioxidant contents and activities of various leafy herbal teas.
Total phenolic
content
(mg GAE/g tea)

Total avonoid
content (mg
CTE/g tea)

DPPH radical
scavenging
(mg AAE/g tea)

ABTS radical
cation scavenging
(mg AAE/g tea)

Reducing power
(200 mg/ml)
(Abs700)

Chelating effect
(1 mg/ml) (%)

Water extracts
Rooibos tea
Green tea
Black tea
Rosemary tea
Lemongrass tea
Mulberry leaf tea
Bamboo leaf tea
Lotus leaf tea
Peppermint tea
Persimmon leaf tea
Mate tea

38.66
82.21
82.86
30.84
13.67
11.64
11.50
20.17
75.31
14.72
27.93













0.11c
1.76a
3.18a
0.93d
1.01f
0.99f
0.82f
0.37e
3.58b
0.26f
0.84d

11.14
16.42
14.89
14.94
4.22
3.62
1.83
6.76
19.75
2.51
17.34













0.23e
0.17c
0.59d
0.21d
0.20g
0.21g
0.09i
0.27f
0.64a
0.11h
0.32b

9.06
82.54
66.65
15.06
5.84
5.35
2.63
11.12
29.73
12.35
17.90













0.35g
0.46a
1.55b
0.57e
0.36h
0.90h
0.15i
0.43f
0.20c
0.22f
0.61d

39.31
187.36
118.53
35.25
25.36
25.53
15.75
30.82
50.08
36.16
44.46













0.64cde
9.62a
4.06b
3.65de
4.59ef
5.38ef
1.78f
5.05ef
1.70c
4.93de
7.21cd

0.78
1.01
0.90
0.78
0.29
0.27
0.22
0.65
0.54
0.56
0.79













0.03c
0.02a
0.03b
0.02c
0.01f
0.01f
0.01g
0.03d
0.02e
0.01e
0.03c

66.54
47.88
59.78
61.20
82.44
82.69
79.43
61.34
71.42
25.10
41.36













0.51d
0.82f
1.03e
0.85e
0.98a
0.72a
2.25b
0.86e
0.59c
0.10h
0.45g

Ethanol extracts
Rooibos tea
Green tea
Black tea
Rosemary tea
Lemongrass tea
Mulberry leaf tea
Bamboo leaf tea
Lotus leaf tea
Peppermint tea
Persimmon leaf tea
Mate tea

16.72
144.52
29.32
39.44
17.32
10.98
14.93
32.28
33.68
46.42
66.86













0.48g
5.36a
0.62f
0.92d
0.32g
0.38h
0.82g
0.39ef
0.44e
0.95c
0.66b

6.49
29.27
5.30
20.83
5.79
4.85
6.05
12.39
24.69
9.89
48.33













0.17g
1.35b
0.03g
0.09d
0.50g
0.11g
0.03g
0.35e
0.50c
0.36f
2.16a

14.42
290.60
28.91
37.02
8.32
5.56
6.54
21.13
30.56
30.67
58.24













1.83ef
13.84a
2.15cd
3.53c
0.55f
0.05f
0.19f
1.39de
1.67cd
1.72cd
0.58b

19.42
400.12
45.38
50.48
20.24
16.41
18.53
41.41
40.70
70.30
80.51













0.17f
5.07a
0.96d
0.59f
0.30f
0.30f
0.22f
0.53e
0.42e
0.88c
0.17b

0.76
2.22
0.64
0.69
0.32
0.24
0.21
0.67
0.73
0.77
0.95













0.00c
0.03a
0.00f
0.01e
0.00g
0.01h
0.01i
0.03e
0.01d
0.01c
0.01b

22.45
23.21
31.89
22.15
29.26
22.74
24.13
27.34
26.72
25.61
33.50













1.26ef
0.25def
0.56ab
1.85f
0.67bc
1.09ef
0.82def
2.45cd
3.26cde
0.49cdef
1.05a

All values are mean  standard deviation of triplicates.

Values in columns with same extraction method that are not followed by the same letter are signicantly different (p  0.05).

406

J. Oh et al. / Food Control 31 (2013) 403e409

3. Results and discussion

3.1. Determination of antioxidant content
3.1.1. TPC
Phenolic compounds are a class of chemical constituents containing one or more acidic hydroxyl residues attached to an
aromatic arene (phenyl) ring. They are one of the most effective
antioxidative constituents that contribute to the antioxidant
activity of plant food (Velioglu, Mazza, Gao, & Oomah, 1998). Hence,
it is important to quantify phenolic content and to assess its
contribution to antioxidant activity. The TPC results are shown in
Table 2 (2nd column). TPC varied widely in the LHT extracts,
ranging from 10.98 to 144.52 mg GAE/g herb tea. Among the water
extracts, the green tea (82.21 mg GAE/g herb tea), black tea
(82.86 mg GAE/g herb tea), and peppermint tea (75.31 mg GAE/g
herb tea) extracts had signicantly (p  0.05) higher concentrations
of phenolic compounds than the other tea extracts. In the ethanol
extracts, green tea displayed the highest phenolic content
(144.52 mg GAE/g herb tea), followed by mate tea (66.86 mg GAE/g
herb tea) and persimmon leaf tea (46.42 mg GAE/g herb tea).
Overall, the results indicate that ethanol extracts contain more
phenolic compounds than water extracts, except for rooibos tea,
black tea, mulberry leaf tea, and peppermint tea.
Green tea showed the highest value of TPC in both water and
ethanol extracts. This result may be attributed to the high antioxidant activity of tea catechins that are abundant in green tea
extracts. Catechins belong to the avonoid family and are also
referred to as avan-3-ols. According to a study by Stewart, Mullen,
and Crozler (2005), the greatest antioxidant contribution to green
tea comes from avan-3-ols, which account for approximately 68%
of its total antioxidant potential. The four major catechins present
in green tea are epigallocatechin gallate (EGCG), epigallocatechin,
epicatechin gallate, and epicatechin (Mckay & Blumberg, 2002),
which have relatively high antioxidant capacity among various
polyphenolic compounds (Apak et al., 2007). While green tea is
produced from fresh leaves of C. sinensis, black tea can be obtained
through enzymatic aerobic oxidation of C. sinensis leaves (Harvowy
& Banlentine, 1997). During this process, avan-3-ols in the tea
leaves are converted to complex condensation products, theaavins
and thearubigins. Del Rio et al. (2004) reported that the proportion
of avan-3-ols in green tea phenolics was 77.1%, which was reduced
to 3.3% in black tea phenolics. Instead, the amount of thearubigins
increased by 54.8% in black tea, which was not detected before
fermentation of fresh leaves of C. sinensis. A study conducted by
Stewart et al. (2005) indicates that the trolox equivalent antioxidant capacity values of theaavins are much lower than those of
avan-3-ols. This may be a possible explanation for the difference
in phenolic content between green and black tea ethanol extracts.
3.1.2. TFC
Flavonoids constitute a special class of phenolic compounds
with a structure based on the diphenylpropane (C6eC3eC6) carbon
skeleton. In general, avonoids contain multiple hydroxyl groups
and exhibit higher antioxidant activities than phenolic acids
(Robards, Prenzler, Tucker, Swatsitang, & Glover, 1999). Many wellknown antioxidant compounds, such as catechin, epicatechin, and
quercetin, are members of the avonoid family (Iwashina, 2000).
Total avonoid levels ranged from 2.51 to 48.33 mg CTE/g herb tea
(3rd column of Table 2). Green tea, peppermint tea, and mate tea
contained signicantly (p  0.05) high concentrations of avonoids
in both water and ethanol extracts as compared to the other herbal
teas. As shown, the ethanol extract of mate tea showed the highest
avonoid content according to this assay (48.33 mg CTE/g herb tea).
The lowest avonoid value occurred in bamboo leaf water extract

(1.83 mg CTE/g herb tea). The high level of avonoid content in

mate tea ethanol extract might be due to its richness in caffeoyl
derivatives. The caffeoyl derivatives found in mate tea include
caffeic acid, chlorogenic acid, and dicaffeoylquinic acid. These
compounds are the primary constituents that account for the
antioxidant capacity of mate tea (Filip, Lotito, Ferraro, & Fraga,
2000). Though the TFC results were not entirely consonant with
those of TPC, it was demonstrated that LHT samples showing high
levels of phenolic content also contained large amounts of
avonoids.
3.2. Determination of antioxidant capacity
3.2.1. DPPH and ABTS radical scavenging activities
Phenolic compounds exhibit their antioxidant activity through
their radical scavenging effects. Radical scavenging activity is very
important owing to the deleterious role of free radicals in biological
systems and generally proceeds via hydrogen atom transfer or
donation of electrons (Niki & Noguchi, 2000). To determine free
radical scavenging activity of LHT extracts, we used two types of
radicals, DPPH and ABTS.
Both DPPH and ABTS radical scavenging assays are performed to
estimate the free radical scavenging activity of a sample and are
based on the reduction of these radicals. However, they have an
important difference in their response to antioxidants. DPPH can
only be solubilized in organic media (especially in alcohol), not in
aqueous media, which is a signicant limitation when interpreting
the role of hydrophilic antioxidants. In contrast, ABTS has a exible
usage in multiple media, allowing its use in the determination of
antioxidant capacity of both hydrophilic and lipophilic compounds
(Awika, Rooney, Wu, Prior, & Cisneros-Zevallos, 2003).
As shown in Table 2 (4th and 5th columns), a similar tendency
was observed in both types of radical scavenging activity assays.
Among the water extracts, green tea, black tea, and peppermint tea
extracts displayed superior scavenging activity, and the ethanol
extracts of green tea, mate tea, rosemary tea, and persimmon leaf
tea showed relatively high radical scavenging activity in both assays
(p  0.05). In particular, the green tea ethanol extract exhibited the
highest radical scavenging activity than the other LHT extracts
regardless of extraction method or radical type. This can be
attributed to the higher TPC of green tea extract. There is a close
correlation between radical scavenging activity and TPC of extracts
obtained from various natural sources (Erkan, Ayranci, & Aryranci,
2008).
3.2.2. Ferric reducing power
The reducing capacity of a sample is regarded as a signicant
indicator of its potential antioxidant activity. The reducing power
values of the LHT extracts (200 mg/ml) are presented in Table 2 (6th
column). The results of the reducing power assay showed a similar
tendency to those of the TPC and radical scavenging assays. Water
and ethanol extracts of green tea had the highest reducing power,
followed by the mate tea ethanol extracts and black tea water
extracts (p  0.05). These results reveal that the extracts of green
tea, mate tea, and black tea could act as electron donors and could
also react with free radicals by converting them to more stable
products and terminating the radical chain reaction (Yen & Chen,
1995). On the other hand, the lowest reducing capacity was
observed in bamboo leaf tea both in water and ethanol extracts.
3.2.3. Chelating effect on ferrous ions
Iron is essential for oxygen transport, respiration, and enzyme
activity and is a reactive metal that catalyzes oxidative damage in
cells. Furthermore, among the transition metals, iron is regarded as
the most important pro-oxidant because of its high reactivity

J. Oh et al. / Food Control 31 (2013) 403e409

b
DPPH RSA ethanol extracts
(mg AAE/g tea)

a
DPPH RSA water extracts
(mg AAE/g tea)

100
R = 0.7911
75
50
25
0
0

50
TPC of water extracts
(mg GAE/g tea)

300
R = 0.9318
200

100

0
0

100

200
R = 0.6672
150
100
50
0
0

50
TPC of water extracts
(mg GAE/g tea)

150

450
R = 0.935
300

150

100

50
100
TPC of ethanol extracts
(mg GAE/g tea)

150

100

Chelating effect of ethanol

extracts(%)

Chelating effect of water

extracts(%)

50
100
TPC of ethanol extracts
(mg GAE/g tea)

d
ABTS RSA ethanol extracts
(mg AAE/g taea)

ABTS RSA water extracts

(mg AAE/g taea)

R = 0.023
75
50
25
0
0

50
TPC of water extracts
(mg GAE/g tea)

20
10
0
50
100
TPC of ethanol extracts
(mg GAE/g tea)

150

h
R = 0.4692

0.8

0.4

0
0

R = 0.0002
30

Reducing power of ehtnaol

extracts (Abs700)

1.2

40

100

g
Reducing power of water
extracts (Abs700)

407

20
40
60
80
TPC of water extracts
(mg GAE/g tea)

100

2.4
R = 0.9273
1.8
1.2
0.6
0
0

50
100
TPC of ethanol extracts
(mg GAE/g tea)

150

Fig. 1. Correlations between total phenolic content (TPC) and other antioxidant capacity assays in herbal teas. RSA: radical scavenging activity.

(Miller, 1996). Therefore, ferrous chelating ability can be an indicator of antioxidant activity of LHT extracts and was monitored by
measuring the formation of the ferrous ioneferrozine complex.
Table 2 (7th column) shows the chelating effect (%) of different LHT
extracts on ferrous ions.

Water extracts of mulberry leaf tea (82.96%), lemongrass tea

(82.44%), and bamboo leaf tea (79.43%) showed signicantly
(p  0.05) higher chelating effects than those of other extracts. This
means that these extracts include chelating compounds that
disrupt the formation of the ferrouseferrozine complex by

408

J. Oh et al. / Food Control 31 (2013) 403e409

capturing ferrous ion precursor. It has been reported that the

chelators that form a s bond with a metal are effective as secondary
antioxidants, since they reduce the redox potential, thereby stabilizing the oxidized form of the metal ion (Gordon, 1990). No
correlation was observed between ferrous ion chelating effect and
any other assay performed in this study. Water extracts of green tea
and peppermint tea, which showed outstanding antioxidant
capacities in the above-mentioned experiments, only exhibited low
chelating effect values of 47.88% and 71.42%, respectively.
3.3. Correlations among measurements
To examine the relationships between TPC and various analytical methods used to determine the antioxidant potential of LHT
extracts, we calculated their correlations, and the results are presented in Fig. 1. In ethanol extracts of LHT, the highest correlation
value was estimated in the ABTS radical scavenging activity assay
(r2 0.9350). The DPPH radical scavenging activity assay and
ferrous reducing power assay were well correlated with TPC, but
their correlation coefcients were slightly lower (r2 0.9318 and
0.9273, respectively) than that of the ABTS assay. In contrast, the
correlation values of the water extracts exhibited a different
tendency from the ethanolic extracts. The DPPH assay had better
correlation with TPC compared with other antioxidant assays
(r2 0.7911). The r2-values in the ABTS and reducing power assays
were 0.6672 and 0.4692, respectively, indicating a relatively weak
correlation. In the case of the chelating effect on ferrous ions assay,
no signicant correlation was found in either the ethanol or water
extracts (r2 0.0002 and 0.023, respectively). High correlation
between these four measurements has also been observed in other
studies. Dudonn, Vitrac, Coutire, Woillez, and Mrillon (2009)
found high correlation among FolineCiocalteu, DPPH, ABTS, and
reducing power assays in 30 aqueous plant extracts. These results
indicate a relationship between phenolic compound concentration
in LHT extracts and their free radical scavenging and ferric reducing
capacities. Therefore, the presence of phenolic compounds in LHT
extracts contributes signicantly to their antioxidant potential. In
addition, when compared to the correlation factors for the water
extracts, those of ethanol extracts showed much higher values.
Judging from this, it can be said that ethanol extracts of LHT contain
higher levels of phenolic compounds, which are mainly both watersoluble and insoluble compounds that are responsible for their
antioxidant capacities, compared to the levels in the water extracts.
As opposed to radical scavenging activities and reducing power,
there was no obvious relationship between TPC and the chelating
effect on ferrous ions. This can be demonstrated by the difference in
mechanisms between the chelating effects and the other analyses
for measuring antioxidant capacity. The FolineCioclateu, DPPH and
ABTS radical scavenging, and reducing power assays are methods
based on transfer of electrons. In these assays, the capacity of an
antioxidant is measured by reduction of an oxidant, which changes
color when reduced (Apak et al., 2007). However, the iron chelating

effects are related to the reactivity of antioxidant compounds with

iron, and this mechanism is basically different from redox reactions.
3.4. Antimicrobial activity
As the MIC is the lowest concentration of an agent that prevents
visible growth of bacteria, there is a limit to turbidity determination
by experimenters. The MIC was determined only to identify the
MLC value (MIC data not shown). The LHT extracts were evaluated
for growth inhibitory activity against oral pathogens (S. mutans and
S. sobrinus) and food-borne pathogens (Listeria monocytogenes,
Shigella exneri, and Salmonella enterica) (Table 3). Among the 11
LHTs, only the green tea ethanol extract inhibited the growth of all
tested pathogens. Green tea and black tea extracts showed a similar
tendency in the antioxidant activity results, but their antimicrobial
activities were different. Black tea had no inhibition against any of
the ve pathogens, and this may result from the changes in
composition due to the manufacturing process. While green tea is
produced by dehydration of plant material, black tea is manufactured using a fermentation process that leads to changes in the
major compounds. It has been reported that EGCG, which is one of
the major compounds of green tea, has antimicrobial activities
against several pathogens, including the ve pathogens tested in
this study (Gordon & Wareham, 2010). After the fermentation
process, theaavins, the major compound of black tea, are formed,
and catechins, including EGCG, disappear (Bancirova, 2010). The
different results between green tea and black tea regarding antimicrobial activities may result from the changing of chemical
compounds. The water extract of mate tea demonstrated antimicrobial activity against S. mutans, S. sobrinus, and L. monocytogenes
at MLCs of 5.83, 5.42, and 6.88 mg/ml, respectively, thus showing
strong bactericidal activity against Gram-positive bacteria. The
major antimicrobial compound of mate tea is nerolidol, which has
shown inhibitory activity against Gram-positive bacteria, including
S. mutans (Kubo, Muroi, & Himejima, 1993). According to our
results, both water and ethanol extracts of mate tea showed antimicrobial activity against Gram-positive bacteria, which might be
caused by nerolidol. Tsai, Tsai, Chien, Lee, and Tsai (2008) tested the
antimicrobial activities of several herbal teas against cariogenic
bacteria, and only rosemary tea extract demonstrated inhibitory
activity against S. mutans and S. sobrinus. Of the major compounds
of rosemary tea extract, myricetin has the highest concentration. In
a study by Cai and Wu (1996), it showed that myricetin has antimicrobial activity against S. mutans. Hence, the data from Table 3
for rosemary tea may result from myricetin. Overall, the ethanol
extracts show higher values of MLC compared to the water extracts,
and this may be because the solubility of antimicrobial compounds
in ethanol is better than that of water. In general, Gram-negative
bacteria are more resistant to the plant-originating antimicrobials
because of their lipopolysaccharide outer membrane, which
restricts diffusion of hydrophobic compounds. However, this does
not mean that Gram-positive bacteria are always more vulnerable

Table 3
Minimum lethal concentration (MLC) of water and ethanol extracts of some herbal teas against both oral pathogens and food-borne pathogens.
MLC (mg/ml)

Water extracts
Green tea
Mate tea
Ethanol extracts
Green tea
Rosemary tea
Mate tea
a

e : Not detectable.

S. mutans

S. sobrinus

L. monocytogenes

S. exneri

S. enterica

10  0.00
5.83  1.95

10  0.00
5.42  2.34

ea
6.88  2.50

e
e

e
e

10  0.00
10  0.00
10  0.00

10  0.00
10  0.00
10  0.00

10  0.00
10  0.00
10  0.00

10  0.00
e
e

10  0.00
e
e

J. Oh et al. / Food Control 31 (2013) 403e409

(Burt et al., 2007). Moreover, there is still an unanswered question

regarding the relationship between antimicrobial activity and plant
polyphenols. In the MLC value of comparing green tea with its TPC,
it seems like there was a correlation between antimicrobial activity
and TPC. In addition, the TPC values of ethanol extracts that
demonstrated antimicrobial activities in this study were relatively
high. However, in the case of black tea and peppermint tea, there
seems to be relatively little relationship between TPC and antimicrobial activity. Hence, more in-depth research about this bioactivity relationship is needed.
4. Conclusions
Commonly consumed teas from 11 leafy herbs were studied for
their antioxidant and antimicrobial properties. The results demonstrate that green tea extracts have signicantly higher TPC and
antioxidant activity compared to extracts of the other herbal teas. In
addition, there were signicant correlations among TPC and DPPH,
ABTS, and reducing power assays. The correlations were higher in
the ethanol extracts than in the water extracts. Ethanol or aqueous
extracts of some LHTs, including green tea, showed efcient antimicrobial activity against the tested oral and food-borne pathogens.
LHT that inhibited oral pathogens via the water extraction method
could improve dental health simply by consumption. Eliminating
cariogenic bacteria from the oral cavity by drinking teas may be an
efcient alternative for preventing dental caries and maintaining
dental health. Moreover, these LHT extracts exhibited antibacterial
activity against food-borne pathogens, implying a possible application in the food industry as a natural antimicrobial preservative.
Acknowledgments
This research was supported by the High Value-added Food Technology Development Program of the Ministry for Food, Agriculture,
Forestry and Fisheries, Republic of Korea (No. 111138-03-1-SB010).
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