oim-ro,

Leafy herbal teasAntioxidant activityAntimicrobial activityFunctionality

danea,leaf,cte

sumedad, 2

sion of

their anticarcinogenic, antiatherogenic, chemopreventive, antioxi-dant, and antimicrobial activities (Si et al., 2006). Free radicals arereactive oxygen species (ROS) produced in the body as by-productsof cellular aerobic respiration and lead to oxidative stress (Yanai,Shiotani, Hagiwara, Nabetani, & Nakajima, 2008). Antioxidant

noids that account for their biological properties (Exarchou,Nenadis, & Tsimidou, 2002), and the antioxidant and antimicro-bial abilities of LHT are attributed to phenolic compounds (Uhl,2000). The antioxidant ability of phenolic components occursmainly through a redox mechanism and allows the components toact 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 reactivenitrogen species, which include free radicals such as superoxide

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

Contents lists available at

Food Co

lse

Food Control 31 (2013) 403e409E-mail address: han2009@skku.edu (J. Han).hot water, brewed from the leaves, owers, seeds, fruits, and rootsof plant species (Aoshima, Hirata, & Ayabe, 2007). Leafy herbal teas(LHT) are widely known to contain a variety of active phytochem-icals with biological properties that promote human health andhelp reduce the risk of chronic diseases such as allergies, insomnia,headaches, anxiety, intestinal disorders, depression, and high bloodpressure (Craig, 1999). Various studies have reported that LHTextracts exert benecial effects on lifestyle-related diseases due to

radicals through Fentons reaction. LHT extracts generally exhibitboth primary and secondary antioxidant capacities (Chan, Lim,Chong, Tan, & Wong, 2010). Moreover, various herbs and plantshave been receiving greater attention as alternatives to syntheticadditives or preservatives in the food industry (Madsen & Berteisen,1995).

Natural botanical sources contain a diverse array of compoundssuch as phenolic acids, avonoids, tannins, vitamins, and terpe-1. Introduction

Tea is one of the most widely consecond only to water (Muktar & Ahmconsumed in the form of tea, an infu0956-7135/$ e see front matter 2012 Elsevier Ltd.http://dx.doi.org/10.1016/j.foodcont.2012.10.021highest antioxidant activity in all assays except the ferrous ion-chelating assay. Water extracts of greentea and black tea and ethanol extracts of rosemary, mate, and persimmon leaf teas also exhibitedconsiderable antioxidant potential, followed by the green tea ethanol extract. Minimum inhibitoryconcentrations (MIC) and minimum lethal concentrations (MLC) were determined to verify the anti-microbial activities of the LHT extracts against two oral pathogens (Streptococcus mutans and Strepto-coccus sobrinus) and three food-borne pathogens (Listeria monocytogenes, Shigella exneri, and Salmonellaenterica). Among the tested LHTs, green tea ethanol extract had potent antimicrobial activity against allve 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 proper-ties, suggesting their potential application as a health-promoting functional ingredient or naturalpreservative in foods.

2012 Elsevier Ltd. All rights reserved.

beverages worldwide,000). Herbs are mainlydried herbs in warm or

activity is dened as an inhibition of the oxidation of lipids,proteins, DNA or other molecules that occurs by blocking thepropagation step in oxidative chain reactions (Huang, Ou, & Prior,2005). Primary antioxidants directly scavenge free radicals, whilesecondary antioxidants indirectly prevent the formation of freeKeywords:

azinobis-3 ethyl benxothiazoline-6-sulphonic acid (ABTS) radical cation decolorization activity, ferricreducing power, and ferrous ion chelating effect were measured. Green tea ethanol extract showed theAccepted 13 October 2012total avonoid content (TFC), 2,2-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity, 2,2-Antioxidant and antimicrobial activities

Jungmin Oh, Heonjoo Jo, Ah Reum Cho, Sung-Jin KDepartment of Food Science and Biotechnology, Sungkyunkwan University, 2066 Seobu

a r t i c l e i n f o

Article history:Received 29 June 2012Received in revised form6 October 2012

a b s t r a c t

We evaluated the antioxiincluding rooibos, green tpeppermint, persimmonsamples of each were extra

journal homepage: www.eAll rights reserved.f various leafy herbal teas

, Jaejoon Han*

Jangan-gu, Suwon 440-746, Republic of Korea

t and antimicrobial activities of various leafy herbal tea (LHT) extracts,black tea, rosemary, lemongrass, mulberry leaf, bamboo leaf, lotus leaf,and mate tea. To compare the antioxidant activities of various LHTs,

d with 80 C water or 20 C ethanol, and their total phenolic content (TPC),

SciVerse ScienceDirect

ntrol

vier .com/locate/ foodcont

30 min in the dark at room temperature, after which the absor-bance was measured at 517 nm using a spectrophotometer. Results

trolanion (O2), hydroxyl (OH), and nitric oxide (NO), as well as non-free radical species such as hydrogen peroxide (H2O2) and nitrousacid (HNO2) (Zhu, Hackman, & Ensunsa, 2002).

In the present study, we measured the antimicrobial activity ofLHTs against two common oral pathogens and three food-bornemicroorganisms. There are numerous studies showing that thepolyphenols and tannins extracted from teas inhibit a broad spec-trum of bacteria (Sreeramulu, Zhu, & Knol, 2000). It has beendemonstrated that the antimicrobial effects of the plant-derivedpolyphenols cause structural or functional damage to the bacte-rial cell membrane (Yoo, Murata, & Duarte, 2011). Several studieshave concluded that the functional hydroxyl groups and conjugateddouble bonds in LHTextractsmay be involved in binding to cell wallcomponents. Microbial cells are negatively affected by plant-derived substances via various mechanisms of actions that attackthe phospholipid bilayer of the cell membrane and disrupt enzymesystems (Proestos, Boziaris, Kapsokefalou, & Komaitis, 2008).Furthermore, a common type of dental disease and caries areassociated with microorganisms present on the surface of teeth.Mutans streptococci play a signicant role in the formation ofdental biolm and the initiation of dental caries and includebacteria such as Streptococcus mutans, Streptococcus sobrinus,Streptococcus cricetus, Streptococcus rattuce, and Streptococcus ferus.Accordingly, various LHT extracts are expected to be effective notonly in maintaining food safety, but also in preventing the growthof oral microorganisms. The current research aims to monitor theantioxidant and antimicrobial properties of various LHT extracts.Few studies have investigated and compared the potential prop-erties of various LHTs, although the biological effects of green andblack tea have been well documented.

The objectives of this study were (i) to examine the antioxidantcapacities of aqueous and ethanol extracts of 11 LHTs, (ii) todemonstrate a correlation between TPC and the antioxidant activ-ities of LHT, and (iii) to characterize the antimicrobial activity of LHTagainst 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-(4-phenylsulphonic acid)-1,2,4-triazine], and potassium persulfatewere 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 DaejungChemical & Metals Co. (Shiheung, Korea). Ferrous chloride waspurchased from Kanto Chemicals Co. (Tokyo, Japan). All othersolvents and reagents used in the analysis were of analytical grade.

2.2. Herbal teas and preparation of extracts

Eleven LHTs, including rooibos tea (Aspalathus linearis), greentea (Camellia sinensis), black tea (C. sinensis), rosemary tea (Ros-marinus ofcinalis), lemongrass tea (Cymbopogon citrates), mulberryleaf tea (Morus alba. Linne), bamboo leaf tea (Sasa borealis), lotusleaf tea (Nelumbo nucifera), peppermint tea (Mentha piperita),persimmon leaf tea (Diospyros kaki), and mate tea (Ilex para-guariensis) were obtained from Gaialand Corp. (Seoul, Korea), Tea-fresh Corp. (Busan, Korea) or Fusae Corp., Ltd. (Seoul, Korea).

Individual extracts of the 11 LHTs were prepared using water orethanol (90%, v/v) as solvents. We used water as a solvent tosimulate the general condition of preparation for consumption.Ethanol was used to improve the solubility efciency of both polar

J. Oh et al. / Food Con404and non-polar functional compounds in the herbal teas. Water-are expressed as mg ascorbic acid equivalents (AAE) per g dry-matter of herbal tea.

2.3.4. ABTS radical cation decolorization activityABTS radical cation decolorization activity was assayed by the

method of Re et al. (1999). ABTS radical cations were generated byreacting 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 roomtemperature. The ABTS radical cation solution was then dilutedwith ethanol to give an absorbance of 1.0e1.2 at 734 nm. LHTextract or ascorbic acid as a standard solution (200 ml) was mixedwith diluted ABTS radical cation solution (1 ml). The mixture wasvortexed and left to stand at room temperature for 1 h. Theabsorbance of the resulting solutionwas measured at 734 nm usinga spectrophotometer. Results are shown as mg AAE per g dry-soluble extracts were prepared under conditions similar to thosegenerally used for tea drinking. Finely ground samples (50 g) wereextracted in 80 C hot water (1 L) and stirred gently on a magneticstirrer for 10 min. For preparation of ethanol extracts, each LHTpowder (50 g) was soaked in ethanol (1 L) and mixed with a labstirrer (MS280, Misung Scientic Co., Korea) for 12 h at roomtemperature (20 C). Each extract was then ltered through no. 2Whatman lter paper (Whatman Inc., Clifton, NJ, USA). Thecombined ltrate was then evaporated using a rotary evaporator(Rotavapor RE121, Bchi, Switzerland), and the extra solvent wasremoved with a freeze dryer (Heto FD 3, Heto Lab Equipment,Holten, Denmark). The dried extract sample was weighed tocalculate the soluble constituent yield. Samples were kept in an air-tight container at 20 C until further analysis.

2.3. Determination of antioxidant activities of LHT

2.3.1. TPCTPC 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 standardsolution was mixed with 2 ml of 2% (w/v) sodium carbonate solu-tion. The mixture was then incubated for 3 min, after which 100 mlof FolineCiocalteu reagent was added. After standing for 30 min atroom temperature for color development, absorbance wasmeasured at 750 nm using a spectrophotometer (UV mini-1240,Shimadzu, Kyoto, Japan). Results are expressed as mg gallic acidequivalents (GAE) per g dry-matter of herbal tea.

2.3.2. TFCTFC 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 wasdiluted with 1.25 ml of distilled water. Exactly 75 ml of NaNO2solution (5%) was added to the mixture, followed by a 6-minincubation at room temperature. Then, 150 ml of AlCl3$6H2O (10%)was added to the mixture, followed by a 5-min incubation and theaddition of 500 ml of NaOH (1 M). Absorbance was measured at510 nm using a spectrophotometer. TFC is shown as ()-catechinequivalents (CTE) per g dry-matter of herbal tea.

2.3.3. DPPH radical scavenging activityDPPH radical scavenging activity was performed by the method

of Cheung, Cheung, and Ooi (2003). An aliquot (1 ml) of 0.2 mMDPPH radical in methanol was mixed with 200 ml of LHT extract orascorbic acid standard solution. The mixture was incubated for

31 (2013) 403e409matter of herbal tea.

2.3.5. Ferric reducing powerThe reducing power of the LHT extracts was determined by the

method of Oyaizu (1986). An aliquot (250 ml) of sample or distilledwater (negative control), 250 ml of sodium phosphate buffer(200 mM, pH 6.6), and 250 ml of potassium ferricyanide (1%) weremixed and incubated in a water bath at 50 C for 20 min. Thereaction was terminated by adding 250 ml of trichloroethanoic acidsolution (10%, w/v). Then, the mixtures were centrifuged at5000 rpm for 5 min. The supernatant (500 ml) was mixed with anequal volume of distilled water and 100 ml of ferric chloride solution(0.1%, w/v). The intensity of the Prussian blue color was measuredat 700 nm using a spectrophotometer. Results are expressed as themean absorbance value.

Chelating effect%1

AbssampleAbscontrol

100

2.4. Determination of antimicrobial activities of LHT

The microorganisms and complex culture media used in thisstudy are shown in Table 1. Microorganisms were kept frozen at80 C in broth containing glycerol (15%, v/v). Before the test, stockcultures were inoculated into the broth and incubated at 37 C for24 h. Cultures were transferred to the broth three times at 24 hintervals. All mediawere obtained fromDifco Co. (Sparks, MD, USA).

The broth micro-dilution test was performed according to themodied method of Hufford and Clark (1988) to determine the MICof each extract. Briey, 100 ml of LHT extract was diluted with brothin a sterile 96-well microtiter plate. The same volume (100 ml) ofovernight bacterial culture, at a density of 106 CFU/ml, was added tothe wells, and the culture plates were placed in an incubator at37 C for 24 h. Two-fold serial dilutions were produced in broth togive nal concentrations of 2.44 mg/mle10 mg/ml. The wells werevisually examined for the lowest concentration of extract thatinhibited microbial growth (indicated by clear wells) after 24 h. Thelowest concentration of extract at which growth did not occur wasnoted as the MIC.

The MLC was determined after reading the results of the MIC bystreaking 10 ml of suspension in the well at concentrations above

Analysis of variance and Duncans multiple-range tests were

Table 1List of microorganisms and culture media.

Pathogens Microorganisms Culture Media

Oral pathogens Streptococcus sobrinusKCTC 3308

Brain heart infusion or agar

Streptococcus mutansKCTC 3065

Brain heart infusion or agar

Food-borne pathogens Listeria monocytogenesKCTC 3710

Brain heart infusion or agar

Shigella exneriKCTC 22192

Nutrient broth or agar

Salmonella entericaKCTC 2514

Nutrient broth or agar

radicgingAE/g

0 0 1 0 0 0 0 0 0

J. Oh et al. / Food Control 31 (2013) 403e409 4052.3.6. Ferrous ion chelating effectThe ferrous ion chelating effect of the extracts was investigated

with a method slightly modied from that of Dinis, Madeira, andAlmeida (1994). LHT extract or control (1 ml of distilled deionizedwater) was reacted with 2 mM of ferrous chloride (100 ml) for10 min, and 5 mM ferrozine (100 ml) was added. After 10 min, thesolvent of the sample (3 ml) was mixed with the mixture andreacted for another 10 min. The absorbance of the mixture wasmeasured at 562 nm using a spectrophotometer. Ferrous ionchelating capacity was calculated by the follow equation.

Table 2Antioxidant contents and activities of various leafy herbal teas.

Total phenoliccontent(mg GAE/g tea)

Total avonoidcontent (mgCTE/g tea)

DPPHscaven(mg A

Water extractsRooibos tea 38.66 0.11c 11.14 0.23e 9.06Green tea 82.21 1.76a 16.42 0.17c 82.54Black tea 82.86 3.18a 14.89 0.59d 66.65Rosemary tea 30.84 0.93d 14.94 0.21d 15.06Lemongrass tea 13.67 1.01f 4.22 0.20g 5.84Mulberry leaf tea 11.64 0.99f 3.62 0.21g 5.35Bamboo leaf tea 11.50 0.82f 1.83 0.09i 2.63Lotus leaf tea 20.17 0.37e 6.76 0.27f 11.12Peppermint tea 75.31 3.58b 19.75 0.64a 29.73

Persimmon leaf tea 14.72 0.26f 2.51 0.11h 12.35 0Mate tea 27.93 0.84d 17.34 0.32b 17.90 0Ethanol extractsRooibos tea 16.72 0.48g 6.49 0.17g 14.42 1Green tea 144.52 5.36a 29.27 1.35b 290.60 1Black tea 29.32 0.62f 5.30 0.03g 28.91 2Rosemary tea 39.44 0.92d 20.83 0.09d 37.02 3Lemongrass tea 17.32 0.32g 5.79 0.50g 8.32 0Mulberry leaf tea 10.98 0.38h 4.85 0.11g 5.56 0Bamboo leaf tea 14.93 0.82g 6.05 0.03g 6.54 0Lotus leaf tea 32.28 0.39ef 12.39 0.35e 21.13 1.39de 41.41 0.53e 0.67 0.03e 27.34 2.45cdPeppermint tea 33.68 0.44e 24.69 0.50c 30.56 1 cd e d cdePersimmon leaf tea 46.42 0.95c 9.89 0.36f 30.67 1Mate tea 66.86 0.66b 48.33 2.16a 58.24 0

All values are mean standard deviation of triplicates.Values in columns with same extraction method that are not followed by the same lette.67 40.70 0.42 0.73 0.01 26.72 3.26

.72cd 70.30 0.88c 0.77 0.01c 25.61 0.49cdef

.58b 80.51 0.17b 0.95 0.01b 33.50 1.05aemployed to statistically analyze all results. Differences betweenmeans were considered signicant when p 0.05. SAS Analytics(SAS Institute, Cary, NC, USA) was used for the analysis.

al

tea)

ABTS radicalcation scavenging(mg AAE/g tea)

Reducing power(200 mg/ml)(Abs700)

Chelating effect(1 mg/ml) (%)

.35g 39.31 0.64cde 0.78 0.03c 66.54 0.51d

.46a 187.36 9.62a 1.01 0.02a 47.88 0.82f

.55b 118.53 4.06b 0.90 0.03b 59.78 1.03e

.57e 35.25 3.65de 0.78 0.02c 61.20 0.85e

.36h 25.36 4.59ef 0.29 0.01f 82.44 0.98a

.90h 25.53 5.38ef 0.27 0.01f 82.69 0.72a

.15i 15.75 1.78f 0.22 0.01g 79.43 2.25b

.43f 30.82 5.05ef 0.65 0.03d 61.34 0.86e

.20c 50.08 1.70c 0.54 0.02e 71.42 0.59c

.22f 36.16 4.93de 0.56 0.01e 25.10 0.10h

.61d 44.46 7.21cd 0.79 0.03c 41.36 0.45g

.83ef 19.42 0.17f 0.76 0.00c 22.45 1.26ef3.84a 400.12 5.07a 2.22 0.03a 23.21 0.25def.15cd 45.38 0.96d 0.64 0.00f 31.89 0.56ab.53c 50.48 0.59f 0.69 0.01e 22.15 1.85f.55f 20.24 0.30f 0.32 0.00g 29.26 0.67bc.05f 16.41 0.30f 0.24 0.01h 22.74 1.09ef.19f 18.53 0.22f 0.21 0.01i 24.13 0.82defthe MIC. Then, the subcultured agar plates were incubated over-night at 37 C. The MLC was dened as the lowest concentration ofextract that resulted in no bacterial growth on agar.

2.5. Statistical analysisr are signicantly different (p 0.05).

trol3. Results and discussion

3.1. Determination of antioxidant content

3.1.1. TPCPhenolic compounds are a class of chemical constituents con-

taining one or more acidic hydroxyl residues attached to anaromatic arene (phenyl) ring. They are one of the most effectiveantioxidative constituents that contribute to the antioxidantactivity of plant food (Velioglu, Mazza, Gao, & Oomah,1998). Hence,it is important to quantify phenolic content and to assess itscontribution to antioxidant activity. The TPC results are shown inTable 2 (2nd column). TPC varied widely in the LHT extracts,ranging from 10.98 to 144.52 mg GAE/g herb tea. Among the waterextracts, 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/gherb tea) extracts had signicantly (p 0.05) higher concentrationsof phenolic compounds than the other tea extracts. In the ethanolextracts, green tea displayed the highest phenolic content(144.52 mg GAE/g herb tea), followed by mate tea (66.86 mg GAE/gherb tea) and persimmon leaf tea (46.42 mg GAE/g herb tea).Overall, the results indicate that ethanol extracts contain morephenolic 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 andethanol extracts. This result may be attributed to the high antiox-idant activity of tea catechins that are abundant in green teaextracts. Catechins belong to the avonoid family and are alsoreferred to as avan-3-ols. According to a study by Stewart, Mullen,and Crozler (2005), the greatest antioxidant contribution to greentea comes from avan-3-ols, which account for approximately 68%of its total antioxidant potential. The four major catechins presentin green tea are epigallocatechin gallate (EGCG), epigallocatechin,epicatechin gallate, and epicatechin (Mckay & Blumberg, 2002),which have relatively high antioxidant capacity among variouspolyphenolic compounds (Apak et al., 2007). While green tea isproduced from fresh leaves of C. sinensis, black tea can be obtainedthrough enzymatic aerobic oxidation of C. sinensis leaves (Harvowy& Banlentine, 1997). During this process, avan-3-ols in the tealeaves are converted to complex condensation products, theaavinsand thearubigins. Del Rio et al. (2004) reported that the proportionof avan-3-ols in green tea phenolics was 77.1%, whichwas reducedto 3.3% in black tea phenolics. Instead, the amount of thearubiginsincreased by 54.8% in black tea, which was not detected beforefermentation of fresh leaves of C. sinensis. A study conducted byStewart et al. (2005) indicates that the trolox equivalent antioxi-dant capacity values of theaavins are much lower than those ofavan-3-ols. This may be a possible explanation for the differencein phenolic content between green and black tea ethanol extracts.

3.1.2. TFCFlavonoids constitute a special class of phenolic compounds

with a structure based on the diphenylpropane (C6eC3eC6) carbonskeleton. In general, avonoids contain multiple hydroxyl groupsand exhibit higher antioxidant activities than phenolic acids(Robards, Prenzler, Tucker, Swatsitang, & Glover, 1999). Many well-known antioxidant compounds, such as catechin, epicatechin, andquercetin, 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 teacontained signicantly (p 0.05) high concentrations of avonoidsin both water and ethanol extracts as compared to the other herbalteas. As shown, the ethanol extract of mate tea showed the highestavonoid content according to this assay (48.33mg CTE/g herb tea).

J. Oh et al. / Food Con406The lowest avonoid value occurred in bamboo leaf water extract(1.83 mg CTE/g herb tea). The high level of avonoid content inmate tea ethanol extract might be due to its richness in caffeoylderivatives. The caffeoyl derivatives found in mate tea includecaffeic acid, chlorogenic acid, and dicaffeoylquinic acid. Thesecompounds are the primary constituents that account for theantioxidant capacity of mate tea (Filip, Lotito, Ferraro, & Fraga,2000). Though the TFC results were not entirely consonant withthose of TPC, it was demonstrated that LHT samples showing highlevels of phenolic content also contained large amounts ofavonoids.

3.2. Determination of antioxidant capacity

3.2.1. DPPH and ABTS radical scavenging activitiesPhenolic compounds exhibit their antioxidant activity through

their radical scavenging effects. Radical scavenging activity is veryimportant owing to the deleterious role of free radicals in biologicalsystems and generally proceeds via hydrogen atom transfer ordonation of electrons (Niki & Noguchi, 2000). To determine freeradical scavenging activity of LHT extracts, we used two types ofradicals, DPPH and ABTS.

Both DPPH and ABTS radical scavenging assays are performed toestimate the free radical scavenging activity of a sample and arebased on the reduction of these radicals. However, they have animportant difference in their response to antioxidants. DPPH canonly be solubilized in organic media (especially in alcohol), not inaqueous media, which is a signicant limitation when interpretingthe role of hydrophilic antioxidants. In contrast, ABTS has a exibleusage in multiple media, allowing its use in the determination ofantioxidant 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 tendencywas observed in both types of radical scavenging activity assays.Among the water extracts, green tea, black tea, and peppermint teaextracts displayed superior scavenging activity, and the ethanolextracts of green tea, mate tea, rosemary tea, and persimmon leaftea showed relatively high radical scavenging activity in both assays(p 0.05). In particular, the green tea ethanol extract exhibited thehighest radical scavenging activity than the other LHT extractsregardless of extraction method or radical type. This can beattributed to the higher TPC of green tea extract. There is a closecorrelation between radical scavenging activity and TPC of extractsobtained from various natural sources (Erkan, Ayranci, & Aryranci,2008).

3.2.2. Ferric reducing powerThe reducing capacity of a sample is regarded as a signicant

indicator of its potential antioxidant activity. The reducing powervalues of the LHT extracts (200 mg/ml) are presented in Table 2 (6thcolumn). The results of the reducing power assay showed a similartendency to those of the TPC and radical scavenging assays. Waterand ethanol extracts of green tea had the highest reducing power,followed by the mate tea ethanol extracts and black tea waterextracts (p 0.05). These results reveal that the extracts of greentea, mate tea, and black tea could act as electron donors and couldalso react with free radicals by converting them to more stableproducts and terminating the radical chain reaction (Yen & Chen,1995). On the other hand, the lowest reducing capacity wasobserved in bamboo leaf tea both in water and ethanol extracts.

3.2.3. Chelating effect on ferrous ionsIron is essential for oxygen transport, respiration, and enzyme

activity and is a reactive metal that catalyzes oxidative damage incells. Furthermore, among the transition metals, iron is regarded as

31 (2013) 403e409the most important pro-oxidant because of its high reactivity

AcerxHighlight

ntrolR = 0.7911100

acts

a

J. Oh et al. / Food Co(Miller, 1996). Therefore, ferrous chelating ability can be an indi-cator of antioxidant activity of LHT extracts and was monitored bymeasuring the formation of the ferrous ioneferrozine complex.Table 2 (7th column) shows the chelating effect (%) of different LHTextracts on ferrous ions.

0

25

50

75

0 50 100

DPP

H R

SA w

ate

r ex

tr(m

g AAE

/g tea

)

TPC of water extracts(mg GAE/g tea)

R = 0.6672

0

50

100

150

200

0 50 100

ABT

S R

SA w

ate

r ex

trac

ts

(mg

AA

E/g

taea

)

TPC of water extracts(mg GAE/g tea)

R = 0.023

0

25

50

75

100

0 50 100

Che

latin

g ef

fect

of w

ate

r ex

trac

ts(%

)

TPC of water extracts(mg GAE/g tea)

R = 0.4692

0

0.4

0.8

1.2

0 20 40 60 80 100

Red

ucin

g po

wer

o

f wa

ter

extr

acts

(Abs

700)

TPC of water extracts(mg GAE/g tea)

Red

ucin

g po

wer

o

f eht

naol

c

e

g

Fig. 1. Correlations between total phenolic content (TPC) and other antioxR = 0.9318300

ract

s

b

31 (2013) 403e409 407Water 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. Thismeans that these extracts include chelating compounds thatdisrupt the formation of the ferrouseferrozine complex by

0

100

200

0 50 100 150

DPP

H R

SA et

han

ol e

xt

(mg A

AE/g

tea)

TPC of ethanol extracts(mg GAE/g tea)

R = 0.935

0

150

300

450

0 50 100 150

ABT

S R

SA et

hano

l ex

trac

ts

(mg A

AE/g

taea

)

TPC of ethanol extracts (mg GAE/g tea)

R = 0.0002

0

10

20

30

40

0 50 100 150

Che

latin

g ef

fect

of e

than

ol

extr

acts

(%)

TPC of ethanol extracts(mg GAE/g tea)

R = 0.9273

0

0.6

1.2

1.8

2.4

0 50 100 150

extr

acts

(Abs

700)

TPC of ethanol extracts(mg GAE/g tea)

h

f

d

idant capacity assays in herbal teas. RSA: radical scavenging activity.

capturing ferrous ion precursor. It has been reported that thechelators that form a s bondwith a metal are effective as secondaryantioxidants, since they reduce the redox potential, thereby stabi-lizing the oxidized form of the metal ion (Gordon, 1990). Nocorrelation was observed between ferrous ion chelating effect andany other assay performed in this study. Water extracts of green teaand peppermint tea, which showed outstanding antioxidantcapacities in the above-mentioned experiments, only exhibited lowchelating effect values of 47.88% and 71.42%, respectively.

3.3. Correlations among measurements

effects are related to the reactivity of antioxidant compounds withiron, and thismechanism is basically different from redox reactions.

3.4. Antimicrobial activity

As the MIC is the lowest concentration of an agent that preventsvisible growth of bacteria, there is a limit to turbidity determinationby experimenters. The MIC was determined only to identify theMLC value (MIC data not shown). The LHT extracts were evaluatedfor growth inhibitory activity against oral pathogens (S. mutans andS. sobrinus) and food-borne pathogens (Listeria monocytogenes,

eas

J. Oh et al. / Food Control 31 (2013) 403e409408To examine the relationships between TPC and various analyt-ical methods used to determine the antioxidant potential of LHTextracts, we calculated their correlations, and the results are pre-sented in Fig. 1. In ethanol extracts of LHT, the highest correlationvalue was estimated in the ABTS radical scavenging activity assay(r2 0.9350). The DPPH radical scavenging activity assay andferrous reducing power assay were well correlated with TPC, buttheir correlation coefcients were slightly lower (r2 0.9318 and0.9273, respectively) than that of the ABTS assay. In contrast, thecorrelation values of the water extracts exhibited a differenttendency from the ethanolic extracts. The DPPH assay had bettercorrelation with TPC compared with other antioxidant assays(r2 0.7911). The r2-values in the ABTS and reducing power assayswere 0.6672 and 0.4692, respectively, indicating a relatively weakcorrelation. In the case of the chelating effect on ferrous ions assay,no signicant correlation was found in either the ethanol or waterextracts (r2 0.0002 and 0.023, respectively). High correlationbetween these four measurements has also been observed in otherstudies. Dudonn, Vitrac, Coutire, Woillez, and Mrillon (2009)found high correlation among FolineCiocalteu, DPPH, ABTS, andreducing power assays in 30 aqueous plant extracts. These resultsindicate a relationship between phenolic compound concentrationin LHT extracts and their free radical scavenging and ferric reducingcapacities. Therefore, the presence of phenolic compounds in LHTextracts contributes signicantly to their antioxidant potential. Inaddition, when compared to the correlation factors for the waterextracts, those of ethanol extracts showed much higher values.Judging from this, it can be said that ethanol extracts of LHT containhigher levels of phenolic compounds, which aremainly bothwater-soluble and insoluble compounds that are responsible for theirantioxidant 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 chelatingeffect on ferrous ions. This can be demonstrated by the difference inmechanisms between the chelating effects and the other analysesfor measuring antioxidant capacity. The FolineCioclateu, DPPH andABTS radical scavenging, and reducing power assays are methodsbased on transfer of electrons. In these assays, the capacity of anantioxidant is measured by reduction of an oxidant, which changescolor when reduced (Apak et al., 2007). However, the iron chelating

Table 3Minimum lethal concentration (MLC) of water and ethanol extracts of some herbal t

MLC (mg/ml)

S. mutans S. sobrinus

Water extractsGreen tea 10 0.00 10 0.00Mate tea 5.83 1.95 5.42 2.34Ethanol extractsGreen tea 10 0.00 10 0.00Rosemary tea 10 0.00 10 0.00Mate tea 10 0.00 10 0.00

a e : Not detectable.Shigella exneri, and Salmonella enterica) (Table 3). Among the 11LHTs, only the green tea ethanol extract inhibited the growth of alltested pathogens. Green tea and black tea extracts showed a similartendency in the antioxidant activity results, but their antimicrobialactivities were different. Black tea had no inhibition against any ofthe ve pathogens, and this may result from the changes incomposition due to the manufacturing process. While green tea isproduced by dehydration of plant material, black tea is manufac-tured using a fermentation process that leads to changes in themajor compounds. It has been reported that EGCG, which is one ofthe major compounds of green tea, has antimicrobial activitiesagainst several pathogens, including the ve pathogens tested inthis study (Gordon & Wareham, 2010). After the fermentationprocess, theaavins, the major compound of black tea, are formed,and catechins, including EGCG, disappear (Bancirova, 2010). Thedifferent results between green tea and black tea regarding anti-microbial activities may result from the changing of chemicalcompounds. The water extract of mate tea demonstrated antimi-crobial activity against S. mutans, S. sobrinus, and L. monocytogenesat MLCs of 5.83, 5.42, and 6.88 mg/ml, respectively, thus showingstrong bactericidal activity against Gram-positive bacteria. Themajor antimicrobial compound of mate tea is nerolidol, which hasshown inhibitory activity against Gram-positive bacteria, includingS. mutans (Kubo, Muroi, & Himejima, 1993). According to ourresults, both water and ethanol extracts of mate tea showed anti-microbial activity against Gram-positive bacteria, which might becaused by nerolidol. Tsai, Tsai, Chien, Lee, and Tsai (2008) tested theantimicrobial activities of several herbal teas against cariogenicbacteria, and only rosemary tea extract demonstrated inhibitoryactivity against S. mutans and S. sobrinus. Of the major compoundsof rosemary tea extract, myricetin has the highest concentration. Ina study by Cai and Wu (1996), it showed that myricetin has anti-microbial activity against S. mutans. Hence, the data from Table 3for rosemary tea may result from myricetin. Overall, the ethanolextracts show higher values of MLC compared to thewater extracts,and this may be because the solubility of antimicrobial compoundsin ethanol is better than that of water. In general, Gram-negativebacteria are more resistant to the plant-originating antimicrobialsbecause of their lipopolysaccharide outer membrane, whichrestricts diffusion of hydrophobic compounds. However, this doesnot mean that Gram-positive bacteria are always more vulnerable

against both oral pathogens and food-borne pathogens.

L. monocytogenes S. exneri S. enterica

ea e e

6.88 2.50 e e

10 0.00 10 0.00 10 0.0010 0.00 e e10 0.00 e e

dental health. Moreover, these LHT extracts exhibited antibacterial

This research was supported by the High Value-added Food Tech-

59(10), 987e990.

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4. Conclusions

Commonly consumed teas from 11 leafy herbs were studied fortheir antioxidant and antimicrobial properties. The results demon-strate that green tea extracts have signicantly higher TPC andantioxidant activity compared to extracts of the other herbal teas. Inaddition, there were signicant correlations among TPC and DPPH,ABTS, and reducing power assays. The correlations were higher inthe ethanol extracts than in the water extracts. Ethanol or aqueousextracts of some LHTs, including green tea, showed efcient anti-microbial activity against the tested oral and food-borne pathogens.LHT that inhibited oral pathogens via the water extraction methodcould improve dental health simply by consumption. Eliminatingcariogenic bacteria from the oral cavity by drinking teas may be anefcient alternative for preventing dental caries and maintaining

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Antioxidant and antimicrobial activities of various leafy herbal teas1. Introduction2. Materials and methods2.1. Chemicals2.2. Herbal teas and preparation of extracts2.3. Determination of antioxidant activities of LHT2.3.1. TPC2.3.2. TFC2.3.3. DPPH radical scavenging activity2.3.4. ABTS radical cation decolorization activity2.3.5. Ferric reducing power2.3.6. Ferrous ion chelating effect

2.4. Determination of antimicrobial activities of LHT2.5. Statistical analysis

3. Results and discussion3.1. Determination of antioxidant content3.1.1. TPC3.1.2. TFC

3.2. Determination of antioxidant capacity3.2.1. DPPH and ABTS radical scavenging activities3.2.2. Ferric reducing power3.2.3. Chelating effect on ferrous ions

3.3. Correlations among measurements3.4. Antimicrobial activity

4. ConclusionsAcknowledgmentsReferences