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Commercial peppermint (Mentha×piperita L.) teas: Antichlamydial effect and

polyphenolic composition

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DOI: 10.1055/s-0033-1351813

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 Food Research International 53 (2013) 758–766

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Food Research International

journal homepage: www.elsevier.com/locate/foodres

Commercial peppermint (Mentha × piperita L.) teas: Antichlamydial effect and

polyphenolic composition
Karmen Kapp a, Elina Hakala a, Anne Orav b, Leena Pohjala c, Pia Vuorela c, Tõnu Püssa d,
Heikki Vuorela a, Ain Raal e,⁎
a
Division of Pharmaceutical Biology, Faculty of Pharmacy, University of Helsinki, P.O. Box 56 (Viikinkaari 5 E), FI-00014 Helsinki, Finland
b
Institute of Chemistry of Tallinn University of Technology, Akadeemia tee 15, 12628 Tallinn, Estonia
c
Pharmaceutical Sciences Laboratory, Department of Biosciences, Åbo Akademi University, BioCity, Artillerigatan 6 A, FI-20520 Turku, Finland
d
Department of Food Hygiene, Estonian University of Life Sciences, Kreutzwaldi 58A, 51014 Tartu, Estonia
e
Department of Pharmacy, University of Tartu, Nooruse 1, 50411 Tartu, Estonia

a r t i c l e i n f o a b s t r a c t

Article history: The qualitative and quantitative polyphenolic contents in the infusions of the commercial peppermint tea
Received 16 November 2012 (Mentha × piperita L.) samples (n = 27) from different countries were studied using HPLC–UV-MS/MS anal-
Received in revised form 5 February 2013 ysis. The most abundant polyphenolics in the peppermint infusion were eriocitrin, 12-hydroxyjasmonate sul-
Accepted 9 February 2013
fate, luteolin-O-rutinoside and rosmarinic acid. In order to evaluate the content of samples by finding
chemosystematic markers, the essential oil composition was studied by GC. The analyses showed that the
Keywords:
Mentha × piperita
24 (89%) peppermint tea samples contained peppermint, whereas three samples may contain Mentha spicata,
Gram-negative different from that claimed on the package.
Intracellular bacteria The effects of seven peppermint tea extracts against respiratory tract pathogen Chlamydia pneumoniae were
Antibacterial activity investigated in vitro. All seven selected tea extracts were active against C. pneumoniae, the growth inhibition
Polyphenolic compounds ranging from 20.7% to 69.5% at extract concentration of 250 μg/ml. In most cases, the antichlamydial activity
Essential oil was related to the peppermint teas having also high content of luteolin and apigenin glycosides. This study
supports the consumption of peppermint tea to potentially elicit beneficial health effects on acute respiratory
tract infections.
© 2013 Elsevier Ltd. All rights reserved.

1. Introduction antimicrobial, fungicidal, antiviral, insecticidal, radioprotective, and anti-

Tea is a popular and inexpensive beverage, highly valued all around
the world. It is consumed by a range of age groups at all levels of the so-
ciety and about three billion cups of tea are consumed daily worldwide
(Hicks, 2009). During the last decades, there has been a resurgence of in-
terest in herbal teas both in medicinal and non-medicinal purposes. With
concerns about the possible adverse effects of consuming beverages
containing caffeine, the health-oriented people are turning to herbal
teas as alternatives to coffee, cocoa and common tea (Manteiga, Park,
& Ali, 1997; Perumalla & Hettiarachchy, 2012).
Peppermint (Mentha × piperita L.) tea is a popular single ingredi-
ent herbal tea, known for its refreshing taste and aroma. It is an old
medicinal plant species in the Eastern and Western traditions and
the list of peppermint uses as a folk remedy or an alternative medical
therapy includes irritable bowel syndrome, flatulence, indigestion,
nausea, vomiting (Grigoleit & Grigoleit, 2005), cough and bronchitis
(Shkurupii, Odintsova, & Kazarinova, 2006). Furthermore, it is well
documented that the essential oil or extracts of M. × piperita possess
⁎ Corresponding author. Tel.: +372 7375281; fax: +372 7375289.
E-mail address: ain.raal@ut.ee (A. Raal).
oxidant activities (McKay & Blumberg, 2006; Peixoto, Furlanetti, Anibal,
Duarte, & Höfling, 2009; Rita & Animesh, 2011). Perhaps therefore, pep-
permint has been announced as the “medicinal plant of the year” (Saller,
2004).
It is well established that the quality and flavor of an herbal tea are
principally determined by both volatile compounds, contributing to
the property of aroma, and non-volatile compounds, contributing to
the taste (Scharbert & Hofmann, 2005). However, it has been reported
that peppermint infusion may contain only 21% of the original essential
oil of the starting material, while 75% of the original polyphenolic con-
tent is extracted (Duband et al., 1992). For this reason, attention should
be focused on polar compounds such as polyphenolic compounds that
are more stable during boiling and storage (Mimica-Dukic & Bozin,
2008).
Despite advances in antibiotic therapy, respiratory infections remain
one of the most common diseases and even cause of death worldwide
especially among children and elderly. Chlamydia pneumoniae is a
gram-negative intracellular bacterium with a unique developmental
life-cycle, including an infective metabolically inactive elementary
body (EB) and a metabolically active reticulate body (RB). It is established
as an important respiratory pathogen (Saikku, 1992) that infects nearly

0963-9969/$ – see front matter © 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.foodres.2013.02.015
 K. Kapp et al. / Food Research International 53 (2013) 758–766
everyone during lifetime with the adult population having a 60–70%
seroprevalence in the Western countries (Grayston, 1992). Most of
the C. pneumoniae symptoms are mild and can be treated with
azithromycin, doxycycline, or rifampicin. However, infection often
transforms to a persistent form that may be involved in severe chronic
diseases such as asthma and atherosclerosis (Cosentini et al., 2008;
Hahn, Peeling, Dillon, McDonald, & Saikku, 2000; Saikku et al., 1988;
Volanen et al., 2006). The chronic infection is difficult to eradicate for
the human immune system or by administering more antibiotics
(Beagley, Huston, Hansbro, & Timms, 2009; Kutlin, Roblin, &
Hammerschlag, 2002). Since, there are no C. pneumoniae selec-
tive antibiotics available, new antichlamydial compounds would be of
great importance.
It has been previously reported that corn mint (Mentha arvensis) is
an inhibitor of C. pneumoniae infection both in vitro and in vivo (Salin
et al., 2011). Also, it has been shown that luteolin, one of the polyphe-
nolic constituents of peppermint, suppresses inflammation in lung
tissue, the development of C. pneumoniae specific antibodies and the
presence of chlamydia in the lung tissue (Törmäkangas et al., 2005).
Thus, the aim of the present study was to research nature-derived
antichlamydial extracts of M. × piperita. Commercial teas were con-
sidered as the main source of peppermint. The infusion method as
proposed by the producer was utilized. The polyphenolic content
of peppermint infusions was determined and in addition, the con-
tent and composition of essential oils as quality indicators were
analyzed.
2. Materials and methods
2.1. Plant material and sample preparation
Commercially available peppermint teas (n = 27) in the form of
crude herb or tea bags were purchased from food markets (European
Pharmacopoeia, 2010), health shops (Brun, Colson, Perrin, & Voirin,
1991) or retail pharmacies (Carson & Riley, 1995). Sample nos. 1–9
and 12–14 were obtained from Estonia (2009–2010), nos. 10–11 from
Egypt (2011), nos. 16–24 from Germany (2011), nos. 25–27 from
Finland (2012) and no. 15 from USA (2010) (Table 1). The infusion
time was tested with wild-grown M. × piperita (Tartu, Estonia) from
1 min to 12 h; the extractant water was at the beginning of the infusion
at 100 °C. Infusion time already 5 min was found to be exhaustive to
polyphenols, whereas longer infusion time did not increase the content.
Therefore, the infusions were made with distilled water according to
the manufacturer's instructions written on the package. For that, the
maximum recommended quantity of herb and extraction time were
used. If the amount of water or plant material was not specified,
200 ml and two teaspoons (2 × 5 ml, 1.0 g) were taken, respectively.
After extraction, the infusions were filtered (Whatman No 1) and
centrifuged at 4000 rpm for 15 min at 20 °C (Eppendorf™ Model
5804R, Hamburg, Germany).
2.2. Isolation of essential oil
The essential oil was isolated from commercial peppermint teas by
the distillation method described in the Ph. Eur. 7th Ed. (European
Pharmacopoeia, 2010). 20 g of the sample and 0.5 ml of xylene were
used to separate the essential oil. Distillation time was 2 h at a rate of
3–4 ml/min. The oils were stored in sealed vials under refrigeration
(−20 °C) prior to analysis.
2.3. Gas chromatographic analysis
The essential oils were analyzed using a Chrom-5 chromatograph
with a flame ionization detector (FID) (Laboratorni Pristoje Praha,
Czechoslovakia) on fused silica capillary columns with stationary
phases: poly (5%-diphenyl-95%-dimethylsiloxane) (SPB™-5. 30 m ×
759
0.25 mm, Supelco, Switzerland) and polar polyethylene glycol (SW-10,
30 m × 0.25 mm, Supelco, Switzerland). The film thickness of both sta-
tionary phases was 0.25 μm. The carrier gas was helium, with a split
ratio of 1:150 and flow rate of 1.2–1.5 ml/min. The temperature pro-
gram was from 50 to 250 °C, 2 °C/min with the injector temperature
of 250 °C. A Clarity Lite chromatography station (DataApex Ltd., Czech
Republic) was used for data processing. The essential oils were diluted
with 0.2 ml of xylene by Ph. Eur. 7th Ed. method. The identification of
the oil components was accomplished by comparing their retention in-
dices (RIs) on two columns with RI values of reference standards, our
RI data bank and with literature data (Davies, 1990; Zenkevich, 1996,
1997, 1999). The percentage content of the oils was calculated from
peak areas (nonpolar column) using normalization method without
correction factors. The relative standard deviation of percentages of oil
components in three repeated GC analyses of a single oil sample did
not exceed 5%.
2.4. HPLC–UV-MS/MS analyses
2.4.1. Chemicals
Organic solvents and reagents used in this section were of analytical
grade. Methanol (MeOH), dimethyl sulfoxide (DMSO), formic acid,
diosmin, salvianolic acid B, gallic acid and jasmonic acid were from
Sigma-Aldrich (Steinheim, Germany). Acetonitrile (ACN) for liquid
chromatography–mass spectrometry (LC–MS) was of ultragradient
grade obtained from Romil (Cambridge, UK). Water used was prepared
by an EASYpure RF compact system (Barnstead, U.S.A). The reference
standards of luteolin, apigenin, narirutin, eriodictyol-7-O-glucuronide
and rosmarinic acid were supplied from Extrasynthese (Genay, France).
Stock solutions of the standard compounds (1 mg/ml) were pre-
pared by dissolving individual compounds in MeOH, in exception of
diosmin in DMSO. Working solutions of all the standards were obtained
by diluting the stock solutions with MeOH.
2.4.2. Identification and quantitation of individual polyphenols
For the identification and quantitation of polyphenols and for
calculation of the content of total polyphenols as gallic acid equiva-
lent (TPGA), the high-performance liquid chromatography hyphen-
ated with UV–vis diode array and ion trap mass spectrometric
detection (LC–DAD-ESI-MS/MS) was performed. A 1100 Series LC/MSD
Trap-XCT equipped with an electrospray interface (ESI) (Agilent Tech-
nologies, Palo Alto, CA, USA) working in negative ionization was used.
The conditions of the MS2 detection were: m/z interval, 50–1000 amu;
target mass, 400 amu; number of fragmented ions, two; maximal accu-
mulation time, 100 ms; compound stability, 100%; drying gas, nitrogen
from generator; and collision gas, helium. The ion trap was connected
to the HPLC instrument consisting of an autosampler, solvent membrane
degasser, binary pump, column thermostat and UV–vis diode array de-
tector. The HPLC 2D ChemStation software with a ChemStation Spectral
SW module was used for the process guidance as well as processing the
results. The compounds were separated on a reversed-phase column
Zorbax 300SB-C18 (150 × 2.1 mm i.d.; 5 μm particle size; Agilent Tech-
nologies, Palo Alto, CA, USA) in the following gradient of 0.1% formic acid
(solvent A) in water (v/v) and acetonitrile (solvent B): 0–5 min — 1% B,
5–60 min a linear gradient of B to 35%, and 60–70 min — 95% B. The col-
umn temperature was 35 °C, eluent rate 0.3 ml/min, injection volume
5 μl.
Phenolic compounds were identified by comparing the retention
times, UV spectra and MS/MS fragmentation spectra either with re-
spective reference standards or with literature data. Quantification
was done using calibration curves built up on the basis of peak
areas using eight different concentrations of the standard com-
pounds (0.003–0.3 mg/ml). The milligrams per gram of herb were
converted into percentage concentrations by summarizing all the
polyphenols and considering the latter sum as a total.
760 K. Kapp et al. / Food Research International 53 (2013) 758–766

2.4.3. Determination of total polyphenols
Peppermint infusions were analyzed by LC–UV-DAD-MS/MS and
the net areas under UV–vis chromatograms at 280 nm (AUC280) were
calculated between 1–58 min using HPLC 2D ChemStation software.
In order to convert the areas into milligrams per 1 g of herb, 1 mg/ml
stock solution of gallic acid in water was prepared and AUC280 values
for gallic acid at different concentrations were measured for calibration.
The resulting content of polyphenols (TPGA) is expressed as gallic acid
equivalent (mg GA/g of herb).
2.5. In vitro antichlamydial assays
2.5.1. Preparation of peppermint herbal tea samples
The product nos. 18, 19, 20, 22, 23, 25, 26 were assayed against
C. pneumoniae. For the experiments, the peppermint teas were selected
according to the relatively high and low total polyphenol contents, rep-
resentative of crude herb and tea bags, as well as teas with a different
origin.
Water infusions were made according to the descriptions in
Table 1 and Section 2.1. The infusions were concentrated and the
5 ml residues from a rotary evaporator (Heidolph Instruments Model
VV2000, Schwabach, Germany) (120 rpm; 40 °C) were frozen at
− 20 °C and thereafter lyophilized (Heto LyoPro 3000, Alleroed,
Denmark) for 7 days. For the antichlamydial experiments lyophi-
lized the samples were dissolved in DMSO to a concentration of
100 mg/ml.
2.5.2. Cell culture and C. pneumoniae stock
Human epithelial HL-cells of respiratory tract origin (Kuo &
Grayston, 1990) were grown at 37 °C, 5% CO2 and 95% humidity
to confluence in cell culture flasks (Greiner, Bio-One, Frickenhausen,
Germany) using a medium consisting of Roswell Park Memorial Insti-
tute medium (RPMI) 1640, 2 mM L-glutamine and 7.5% fetal bovine
serum (FBS), all purchased from BioWhittaker, Lonza (Basel, Switzerland)
and with 20 μg gentamicin (Fluka, Buchs, Switzerland) per ml.
C. pneumoniae clinical isolate K7 (Ekman et al., 1993) was obtained
from professor Pekka Saikku, from the Department of Medical Microbi-
ology, Institute of Diagnostics, University of Oulu, Finland and propagat-
ed as described by Alvesalo et al. (2006). All infections were preceded
by seeding the HL-cells into 24-wellplates (Corning, Inc., USA) with cov-
erslips (Menzel-Gläser, Braunschweig, Germany) at density 4 × 105
cells per well and incubated overnight (37 °C).
2.5.3. Infections
The bacteria were diluted in the cell growth medium followed by
inoculation of HL-cell monolayers with the multiplicity of infection

Table 1
Mentha × piperita commercial tea samples.

No. Country of origin Amount (g) and number of teabags, Obtained from Making of tea Amount (g) used for Best before,
package 1 cup of tea month/year

1 EU 1.0 × 20 teabags with strand Food market Boiling water (200 ml), 2–3 min 1.0 05/2011
2 Germany 2.25 × 20 teabags with Food market Boiling water, 5–8 min 2.25 01/2012
strand in paper packages
3 Germany 1.5 × 20 teabags Food market Boiling water, 5–8 min 1.5 07/2011
4 Latvia 1.5 × 20 teabags with strand Food market Boiling water (150 ml), 3–4 min 1.5 08/2011
5 Poland 1.5 × 20 teabags Food market Boiling water, 5–8 min 1.5 09/2011
6 Poland 1.5 × 30 teabags Food market Boiling water, 5–8 min 1.5 08/2010
7 The Netherlands 1.5 × 20 teabags Food market Boiling water (150 ml), 3–4 min 1.5 07/2011
8 Latvia 1.0 × 20 teabags Food market Boiling water (200 ml), 3–5 min 1.0 06/2011
9 Poland 1.5 × 20 teabags Food market Boiling water, 5–7 min 1.5 03/2011
10 Egypt 1.0 × 20 teabags Food market Boiling water, (1 cup), 5 min 1.0 09/2011
11 Egypt 1.5 × 12 teabags Food market Boiling water (1 cup), 3 min 1.5 10/2009
12 Estonia Crude drug, 15.0 Pharmacy 2–3 g of herb and 1 cup of boiling 3.0 05/2011
water, 5–10 min
13 Estonia Crude drug, 15.0 Pharmacy 1–2 teaspoons of herb and boiling 1.0 10/2011
water, 5–7 min
14 Estonia Crude drug, 30.0 Pharmacy Not described 1.0 06/2011
15 USA 1.25 × 20 teabags Pharmacy Boiling water, 3–5 min 1.25 12/2012
16 Germany 1.5 × 20 teabags with strand Pharmacy Boiling water, 10–15 min 1.5 03/2014
in paper packages
17 Germany 1.5 × 20 teabags with strand Pharmacy Boiling water (150 ml), 10–15 min 1.5 02/2014
in paper packages
18 Germany Crude drug, 50.0 Pharmacy Boiling water (150 ml) 1.5 10/2012
and 1.5 g of herb, 10–15 min
19 Germany Crude drug, 50.0 Health shop 1–2 teaspoons of drug and 1 cup of 1.0 12/2013
boiling water, 5–10 min
20 Germany Crude drug, 50.0 Health shop 1 teaspoon of drug and 1 cup (150 ml) 0.8 11/2013
of boiling water, 5–10 min
21 Germany 1.5 × 12 teabags with strand Health shop Boiling water (150 ml), 10–15 min 1.5 07/2013
in paper packages
22 Germany 2.25 × 25 teabags with strand Food market Boiling water, 5–6 min 2.25 10/2013
in paper packages
23 Germany 2.25 × 25 teabags with strand Food market 1 l of boiling water and 4 teabags, 2.25 09/2013
in paper packages 5–6 min
24 Germany 1.75 × 20 teabags with strand Food market Boiling water, 6 min 1.75 09/2013
in paper packages
25 UK 1.6 × 20 teabags with strand Health shop Boiling water, 2–5 min 1.6 06/2013
in packages
26 Finland Crude drug, 40.0 Health shop 1–2 teaspoons of drug and 1 cup of 1.2 02/2013
boiling water, 5–15 min
27 Finland Crude drug, 30.0 Health shop 1 teaspoon of drug is infused 0.7 08/2012
10–20 min in hot water

Sample no. 27 contains Mentha × piperita, Mentha spicata and Mentha × dalmatica.
 K. Kapp et al. / Food Research International 53 (2013) 758–766
(MOI) 0.2. The infections were all done in the presence of 1 μg/ml of
cycloheximide (Sigma-Aldrich, St. Louis, MO, USA). After inoculating
the cells, the plates were centrifuged (Eppendorf centrifuge 5810 R,
Hamburg, Germany) at 550 ×g for 1 h at 4 °C and then incubated at
37 °C for 1 h. Thereafter the infection medium was removed from
the wells and fresh medium supplemented with 1 μg/ml cycloheximide
containing the extracts or controls was added as three replicates.
Nontreated (infected or non-infected) samples were used as controls,
in addition to an infected control treated with 0.009 μg of antibiotic ri-
fampicin (BioChemika > 97.0% HPLC; Fluka, Buchs, Switzerland) in
1 ml of ethanol (Etax A, Altia Oyj, Finland). In each well the concen-
tration of DMSO was adjusted to 0.25%. Plates were incubated in the
cell culture conditions described above. After 72 h post infection, the
wells were washed with phosphate buffered saline (PBS) (BioWhittaker,
Lonza, Basel, Switzerland) and fixed with methanol (Sigma-Aldrich,
St. Louis, MO, USA). The coverslips were removed and stained after dry-
ing them at the room temperature. The staining of host cells and chla-
mydia inclusions was carried out using Pathfinder Chlamydia culture
confirmation system reagent (Bio-Rad Laboratories, France) and the in-
clusion counts were determined under a fluorescent microscope (Nikon
Eclipse TE300, Tokyo, Japan) with a 20× magnification. Inhibition per-
centage was calculated on the basis of the average number of inclusions
per coverslip by comparing the number of inclusions in a treated sam-
ple to the number of inclusions in infected control samples. The sample
concentration was 250 μg/ml unless otherwise stated. The extract nos.
19, 25, and 26 that showed the highest activity (≥50 ± 0.5% inhibition)
at 250 μg/ml, were chosen for the dose–response experiments, with a
dilution series consisting of the following concentrations: 250, 125,
62.5, 30.0, 15.0 and 7.5 μg/ml.
2.5.4. Infectious progeny assay
In order to show that the extracts are able to diminish the amount
of infectious progeny at the second round of infection more than after
one 72 h infection period as described by Hammerschlag (1994), the
method was carried out as described by Kuo and Grayston (1988).
Assaying the effect of the samples on the production of infectious
progeny, the infection protocol was conducted as follows: two paral-
lel replicates were used and after the 72 h infection period two of the
coverslips were fixed and stained to count the inclusions and thus
confirm the inhibitory effect of the sample. From the other two wells
the medium was removed, 200 μl of fresh medium supplemented with
1 μg/ml cycloheximide was added, and cells were scraped off. This sus-
pension was mixed in the presence of glass beads to release the chla-
mydia by breaking of the host cells. Then the suspension was used to
infect fresh HL-cell monolayers according to the infection protocol.
After a second round of 72 h infection, i.e., at 144 h, two wells
corresponding to each sample were fixed and stained to determine
the amount of infectious progeny in the second passage of infection.
2.5.5. Host cell viability assay
The host cell viability was determined by a resazurin assay in which
the signals reflect the amount of viable cells that are able to reduce
resazurin to a highly fluorescent derivate, resorufin. The assay was
performed as described by Karlsson et al. (2012). Briefly, HL-cells were
seeded into a 96-wellplate at density of 6 × 105 cells per well and incu-
bated overnight (37 °C) before adding of the seven M. × piperita
samples at a final concentration of 250 μg/ml. Then the plate was in-
cubated at 37 °C for 72 h. After the exposure period the medium was
removed and resazurin (Sigma-Aldrich, St. Louis, MO, USA), diluted
in PBS to 20 μM, was added into the wells. The plate was incubated
at 37 °C for 2 h and the fluorescence was measured with VarioSkan™
Flash plate reader (Thermo Fischer Scientific, Vantaa, Finland) at
570/590 nm (excitation/emission) and 22 °C. Wells containing no cells
filled with medium were used as a blank. Concentration of DMSO in
the sample wells was 0.25%. Usnic acid (Aldrich, Switzerland) was used
as a positive control at a concentration 50 μM.
761
2.6. Statistics
Basic statistics and for comparison of groups t-test with indepen-
dent samples were carried out by using IBM® SPSS® Statistics version
21.0. The figures were produced in MS Excel 2010.
3. Results
3.1. Content and composition of essential oils
Among all the 27 analyzed peppermint samples, less than a half
(n = 12) exceeded the Ph. Eur. 7th Ed. limit of 0.9% for the total oil
content (Table 2 — Supplementary material). From peppermint teas
packed in teabags (n = 19), 8 fulfilled the requirement. From herbs
available as a crude drug (n = 8), 4 samples met the standard. The
yield of essential oil ranged from 0.4 to 2.2%. The minimum oil con-
tent of 0.4% was found in tea no. 20 and no. 27, while the maximum
2.2% in sample no. 15.
For the identification purpose, 66 compounds were identified in the
peppermint oils, representing 96.5–99.7% of the total oil. The main
components of all tea samples are presented in Table 2- Supplementary
material. Ph. Eur. 7th Ed. gives limits for menthol percentage content as
30.0–55.0% and for menthone 14.0–32.0%.
Most of the teas fulfilled the criteria for menthol and menthone.
The highest percentage content of menthol (57.6%) was observed in
sample no. 20, remarkably lower in sample nos. 10 and 27, respec-
tively 11.0% and 15.0%. In peppermint tea no. 14, menthol was not
present. In product no. 12, menthol was detected only as traces. In
these latter four teas the same trend was also observed for the pres-
ence of menthone.
The percentage content of carvone was very high in tea nos. 10, 12,
14 and 27. In the sample no. 14, the percentage content of carvone was
up to 71-fold higher than the Ph. Eur. 7th Ed. limit (max. 1%). The two
teas, nos. 12 and 14 differed from others by the absence or low yield
of menthofuran (limits 1.0–9.0%) and menthyl acetate (limits 2.8–
10.0%). The tea no. 12 did not contain isomenthone (limits 1.5–10.0%).
3.2. Polyphenolic constituents in peppermint tea
Preliminary studies on infusions with hot water (data not shown)
showed that 5–15 min extraction time is sufficient for exhaustive ex-
traction of polyphenolic compounds. Prolongation of the extraction
time does not increase the content of total polyphenols.
The infusions of commercial peppermint contained many secondary
metabolites, having glycosides of flavanones and flavones as predomi-
nant. Overall, 22 polyphenolic compounds were identified (Table 2,
Figure 1 — Supplementary material), of which 12 were determined quan-
titatively (Table 3). In addition, malic and citric acids were detected.
The content of polyphenols varied in a wide scale, but composi-
tional profile of all the infusions was similar. The main phenolic
compound in all teas, except no. 12 and no. 14, was eriocitrin, which
was found to be the highest in tea no. 17 (61.4%) and the lowest in
the sample no. 12 (8.8%). The second most abundant phenolic com-
pound was luteolin-O-rutinoside, found in a range of 3.2–28.9%.
Luteolin was also represented by two other glycosides: O-glucuronide
and di-O-glucuronide, whereas the latter glycoside was absent in tea
nos. 11, 12 and 14. Rosmarinic acid was also found in a large concentra-
tion range (2.1–54.2%). The content of salvianolic acid B, quantified for
the first time in peppermint, was in the range of 1.0–9.7%. Narirutin,
diosmin, eriodictyol and apigenin-O-rutinoside were detected in
minor amounts.
Eight compounds were identified for the first time in the genus
Mentha, whereby 12-hydroxyjasmonate sulfate was identified on the
basis of the results of Gidda et al. (2003)(see Table 2). The lowest
amount (3.2%) of 12-hydroxyjasmonate sulfate was recorded in the
tea no. 17 and the highest (39.3%) in the tea no. 11. Based on literature
762 K. Kapp et al. / Food Research International 53 (2013) 758–766

data, six more new polyphenols were discovered and identified for the
mint family. Compounds detected were: prolithospermic acid, salvianolic
acid H/I, salvianolic acid E, isosalvianolic acid A and medioresinol. Fur-
thermore, a compound characterized by m/z 467 and a constant neutral
loss of 80 Da indicating to sulfate moiety was found to be medioresinol
sulfate. Danshensu, previously detected in Mentha haplocalyx (She, Xu,
Liu, & Shi, 2010), was recorded for the first time in the peppermint.
The total polyphenolic content (TPGA) varied largely among the 27
peppermint tea infusions (Table 3). High yields of polyphenols were
found in tea no. 17 (7.7%), no. 19 (8.2%) and no. 27 (7.6%). However,
the extraordinarily high score was found in no. 21 (21.8%) whereas,
the smallest score was present in the sample no. 5 (1.0%).
3.3. In vitro antichlamydial activity
3.3.1. Inhibition of C. pneumoniae by M. × piperita herbal tea extracts
The ability of the seven M. × piperita extracts to inhibit the growth
of C. pneumoniae was determined at 250 μl/mg (Fig. 1). These tea
samples diminished the chlamydial growth in the range of 20.7–
69.5%. Tea no. 23 had the lowest inhibition percentage and no. 19
had the highest. The inhibition proved to be statistically significant
at p ≤ 0.01 and p ≤ 0.001 (t-test) for the extracts of sample nos. 19,
20, 25 and 26. The three extracts with the inhibition percentage of
≥ 50 ± 0.5% were considered as active and were taken for further
dose–response experiments.
3.3.2. Dose–response experiments
The measured half maximal inhibitory concentration (IC50, mean ±
standard error of the mean, SEM) for the tea extract nos. 19, 25 and
26 are 224 ± 26.9 μg/ml, 168 ± 14.5 μg/ml and 98 ± 5.5 μg/ml
(see Fig. 1), respectively. Extract nos. 19, 25 and 26 inhibited growth
of C. pneumoniae in a dose-dependent manner. The highest concentra-
tion of no. 26 (250 μg/ml) had an inhibition of 62.9%, the corresponding
percentages for no. 25 and no. 19 were 55.1% and 52.6%, respectively. In
all of these three dose–response experiments even the lowest concen-
tration 7.5 μg/ml showed inhibition. With sample no. 19 it was 16.8%,
no. 26 6.8% and no. 25 1.3%.
3.3.3. The effect of M. × piperita extracts on the amount of infectious
progeny
Seven peppermint tea extracts were assayed for their effect on the
inclusion counts at the second passage of infection as described in
Materials and methods (Fig. 2). After one 72 h acute infection period
the chlamydial inhibition was from 22.0% to 68.0%. Sample no. 18 had
the lowest inhibition, and sample no. 19 had the highest. After the sec-
ond round of infection, the inhibition percentage was in the range of
7.8–78.1%. The tea no. 20 and no. 22 had the lowest values, whereas,
the highest values were found in tea nos. 19, 25 and 26.
3.3.4. Host cell viability assay
The viability of host cells was determined upon exposure to each
of the seven extracts at the concentration of 250 μg/ml. Fig. 3 shows
that the host cell viability after the 72 h exposure to the peppermint
herbal teas ranged from 82.4% to 99.4%. Most of the peppermint tea
extracts had no effect on host cell viability, except sample no. 26
that statistically significantly decreased the viability by 17.6%, which
can however be regarded as a mild effect. Usnic acid was used as a
positive control and it decreased the cell viability statistically signifi-
cantly by 60.6%, the viability being 30.4%. The control 0.25% DMSO
had a viability of 100.3%, which shows that this DMSO concentration
does not have a significant effect on the viable cells.
4. Discussion
Generally, the essential oils of peppermint teas were rich in men-
thol and menthone. However, sample no. 10 and two crude drug sam-
ple nos. 12 and 14 had a very high percentage content of carvone. This
is atypical oil composition for M. × piperita. It indicates that 3 teas out
of 27 consisted of Mentha spicata as high content of carvone is a spe-
cific marker for this given mint species (Hussain, Anwar, Nigam,
Ashraf, & Gilani, 2010; Lawrence, 1978). In addition, the crude herb
sample no. 20 showed low amount of essential oil and high percent-
age concentration of menthol and methyl acetate. This refers to a cir-
cumstance that the herb might have been harvested after flowering
from older plants (Aflatuni, Uusitalo, & Hohtala, 2006; Brun et al.,
1991; Rohloff, 1999; Voirin & Bayet, 1996).

Table 2
The LC–DAD-MS/MS characteristics of polyphenols and plant acids identified in the peppermint tea samples.

Peak no. Retention time m/z characteristic of molecular and fragment ions Identification
(tR, min)

1 1.2 133 [M−H]−; MS2: 115; 73; 87; 89 Malic acid

2 1.7 191 [M−H]−; MS2: 111; 173 Citric acid
3 5.0 197 [M−H]−; MS2: 179; 73 Danshensua (Sheet al., 2010)
4 6.0 315 [M−H]−; MS2: 153; 109 Protocatechuic acid glucoside
5 16.3 179 [M−H]−; MS2: 135 Caffeic acid
6 19.4 353 [M−H]−; MS2: 173; 179; 135 Chlorogenic acid
7 19.6 305 [M−H]−; MS2: 225; 97 12-Hydroxyjasmonate sulfatea (Gidda et al., 2003)
8 20.3 467 [M−H]−; MS2: 387; 241; 207 Medioresinol sulfatea
9 22.8 387 [M−H]−; MS2: 207; 163; 369 Medioresinola (Vaquero et al., 2012)
10 24.0 357 [M−H]−; MS2: 313; 269; 159 Prolithospermic acida (Xu et al., 2007)
11 26.9 637 [M−H]−; MS2: 351; 285 Luteolin-di-O-glucuronide
12 29.4 537 [M−H]−; MS2: 339; 229; 493; 295 Salvianolic acid H/Ia (Liu et al., 2007; Xu et al., 2007)
13 30.1 595 [M−H]−; MS2: 287 Eriocitrin
14 31.3 461 [M−H]−; MS2: 285 Luteolin-O-glucuronide
15 31.7 593 [M−H]−; MS2: 285 Luteolin-O-rutinoside
16 33.3 579 [M−H]−; MS2: 271 Narirutin
17 34.8 577 [M−H]−; MS2: 269 Apigenin-O-rutinoside
18 36.0 359 [M−H]−; MS2: 223; 197; 161 Rosmarinic acid
19 36.1 717 [M−H]−; MS2: 519; 537; 321; 339 Salvianolic acid Ea (Liu et al., 2007; Ruan, Li, Li, Luo,
& Kong, 2012)
20 36.7 607 [M−H]−; MS2: 299; 284 Diosmin
21 37.2 461 [M−H]−; MS2: 285 Luteolin-O-glucuronide
22 37.3 493 [M−H]−; MS2: 295; 313; 159; 183 Isosalvianolic acid Aa (Ruan et al., 2012)
23 40.5 717 [M−H]−; MS2: 519; 321; 339; 393 Salvianolic acid B
24 44.5 479 [M−H]−; MS2: 317 Myricetin-O-glucoside
a
Identification based on the literature data.
 K. Kapp et al. / Food Research International 53 (2013) 758–766 763

Table 3
Concentration of individual polyphenolic compounds in Mentha × piperita herbal tea samples (%).

No LDG LR LG1 LG2 N ER E D SAB AR RA 12-HJS TPGA

1 4.8 24.6 4.5 1.1 1.7 33.2 3.2 2.2 2.4 2.9 7.7 11.8 3.0
2 5.9 15.0 0.3 0.7 0.8 48.9 2.3 0.9 1.2 1.4 3.7 18.9 1.5
3 6.2 19.1 3.5 0.9 1.4 44.3 5.8 1.4 nd 2.0 4.5 10.9 1.3
4 4.8 15.1 3.5 1.0 1.4 38.1 6.6 1.5 2.1 1.7 9.7 14.7 1.2
5 3.0 10.1 2.6 1.0 2.2 44.2 13.2 2.1 3.4 2.4 5.2 10.7 1.0
6 5.6 15.9 3.1 1.2 1.6 42.3 7.8 1.8 2.5 2.1 6.4 9.7 1.5
7 5.7 17.0 3.2 0.9 1.6 40.6 4.7 1.7 2.7 2.4 8.7 10.8 2.3
8 4.1 11.2 4.2 1.6 4.3 44.9 2.1 3.9 nd 4.1 4.5 15.1 1.8
9 2.4 9.2 2.5 0.9 2.0 47.0 13.8 1.9 3.3 1.9 5.4 9.8 1.2
10 4.1 11.4 5.1 3.2 1.7 24.2 nd 11.7 7.4 1.9 19.1 10.2 4.6
11 nd 7.8 3.5 2.0 1.3 25.1 nd 8.3 2.3 1.4 8.9 39.3 1.3
12 nd 3.2 1.3 1.3 nd 8.8 nd 6.8 9.7 nd 54.2 14.6 1.5
13 2.5 23.0 3.1 0.6 2.2 30.0 1.0 2.3 8.4 5.8 2.7 18.4 2.8
14 nd 20.1 1.5 nd 3.4 20.1 9.9 3.5 9.5 3.0 7.0 21.9 1.2
15 9.9 20.3 3.1 1.2 1.8 42.1 3.4 1.4 2.8 2.0 3.1 9.0 7.3
16 5.9 17.6 2.7 0.8 0.8 53.8 3.5 0.7 2.1 0.9 5.4 5.9 4.0
17 3.1 13.4 1.5 0.4 0.5 68.1 3.0 0.4 1.1 0.8 4.4 3.2 7.7
18 0.9 17.7 0.7 0.3 3.6 48.0 3.0 1.0 2.6 1.2 6.7 14.1 4.3
19 4.7 25.4 4.0 0.7 1.8 38.2 2.3 2.0 1.5 5.1 2.1 12.1 8.2
20 13.0 15.3 4.7 1.3 1.3 19.9 2.1 1.4 3.0 1.7 20.9 15.4 7.1
21 4.7 12.8 1.6 0.4 0.4 61.4 1.7 0.5 1.0 0.9 5.1 9.4 21.8
22 2.3 12.6 2.0 0.5 0.8 56.1 9.6 0.7 1.4 0.9 6.1 6.5 1.0
23 3.1 14.7 2.4 0.5 0.8 48.8 8.0 0.8 1.7 1.0 8.0 9.9 1.1
24 4.1 15.4 1.9 0.6 0.7 59.5 3.7 0.7 1.2 1.0 3.2 8.1 3.7
25 6.5 11.4 3.7 1.0 1.4 36.6 6.7 1.4 6.4 1.8 8.4 14.8 2.6
26 1.4 28.9 1.8 0.4 4.5 30.0 3.9 1.7 2.7 4.8 5.9 14.1 3.0
27 4.8 17.6 4.4 2.2 2.4 27.3 1.9 3.0 5.2 2.3 16.3 12.5 7.6

Key: LDG, luteolin-di-O-glucuronide; LR, luteolin-O-rutinoside; LG1, luteolin-O-glucuronide; LG2, luteolin-O-glucuronide; N, narirutin; ER, eriocitrin; E, eriodictyol; D, diosmin; SAB,
salvianolic acid B; AR, apigenin-O-rutinoside; RA, rosmarinic acid; 12-HJS, 12-hydroxyjasmonate sulfate; TPGA, content of total polyphenols (mg GA); nd, not detected.

The comparison between total essential oil concentration and
total content of polyphenols indicated that high amount of oil does
not necessarily point to high amounts of polyphenols. In the analyses
of polyphenols, similarly to the previous studies, eriocitrin was found
to be the dominating compound (Areias, Valentão, Andrade, Ferreres,
& Seabra, 2001; Atoui, Mansouri, Boskou, & Kefalas, 2005; Dorman,
Kosar, Kahlos, Holm, & Hiltunen, 2003; Duband et al., 1992; Fecka &
Turek, 2007; Guédon & Pasquier, 1994; Sroka, Fecka, & Cisowski,
2005). However, the second most abundant compound in pepper-
mint herbal teas was 12-hydroxyjasmonate sulfate. According to
Miersch and co-authors, 12-hydroxyjasmonic acid and its deriva-
tives are constituents of various organs of many plant species (Miersch,
Neumerkel, Dippe, Stenzel, & Wasternack, 2008). Thus, it is surprising
that 12-hydroxyjasmonate sulfate has earlier not been reported in
Mentha spp.
The preliminary study on the infusion time showed that when the
herb and water were in contact for a very short period, the total poly-
phenol content was low. The infusion time 5–15 min, recommended
on most of the herbal tea packages, was found to be optimal. Similar
findings are reported for chamomile (Raal et al., 2012), lemon balm
(Katalinic, Milos, Kulisic, & Jukic, 2006) and black tea infusions
(Hertog, Hollman, & Putte van de, 1993).
The herbal tea products of peppermint were assayed for the first
time against C. pneumoniae. Herbal teas were found to have inhibiting
effect on the chlamydial growth, whereas a half of them were re-
markably active. The extract no. 19, that had the highest inhibition,
was also the one having the highest content of luteolin and apigenin
glycosides. Interestingly, apigenin and luteolin have been shown to
be very active against C. pneumoniae, both with a 100% inhibition at
50 μM (13.5 and 14.3 mg/l resp.) concentration. In addition, it has
been found that among flavones and flavonols, structure is related
to activity. Compounds with 50% or less inhibition contain a glycoside
moiety or moieties as substituents, whereas none of the more active
compounds (over 70% inhibition) contain glycoside (Alvesalo et al.,
2006). In peppermint tea infusions, luteolin and apigenin were found as
glycosides. Therefore, the presence of these phenolic compounds as gly-
cosides may contribute to the weaker activity of the tested tea extracts
compared to the previous reports on M. arvensis or the pure aglycones
(Alvesalo et al., 2006; Salin et al., 2011). This phenomenon can be
explained, at least partly, by easier penetration of more hydrophobic
polyphenols aglycones into biomembranes and crossing the membranes
(D'Archivio, Filesi, Vari, Scazzocchio, & Masella, 2010).
Among peppermint teas tested against chlamydia, sample no. 19
had also the highest content of total essential oil. When peppermint is
used as an infusion, up to 21% of the original essential oil is found in
the infusion, whereas during the extraction, there is loss of hydrocar-
bons and increase of oxygen containing compounds (Duband et al.,
1992). In general, oxygenated monoterpenes have been found to be
more antimicrobial than hydrocarbon monoterpenes (Carson & Riley,
1995). Thus, the antichlamydial effect of peppermint tea extracts may
be influenced by the presence of essential oil.
Consumption of polyphenol-rich fruits, vegetables, and beverages
derived from plants has been shown to be beneficial for human health.
IC50 values ***
No 19. 224 ± 26.9 µg/ml
No 25. 168 ± 14.5 µg/ml
No 26. 98 ± 5.5 µg/ml
***
** **
**
*** p<0.001; ** p<0.01;* p<0.05; t-test, compared to non-treated control
Fig. 1. Inhibition of C. pneumoniae growth by the peppermint tea extracts. Extracts at
250 μg/ml and rifampicin (0.009 μg; positive control) [mean ± standard error of the
mean (SEM); n = 3]. The measured half maximal inhibitory concentration (IC50)
(mean ± SEM, n = 3) values of peppermint tea extract no. 19, no. 25 and no. 26.
764 K. Kapp et al. / Food Research International 53 (2013) 758–766

** * *
*

* *

***
**
*
**

*** p<0.001; ** p<0.01; * p<0.05; t-test

Fig. 2. The effect of seven peppermint teas at the concentration of 250 μg/ml on the chlamydial growth after 72 h and 144 h compared to the infected control HL-cells [mean ± (SEM);
n = 2].

There are many possibilities to acquire effective preparations from 5. Conclusions

plants, but the most common is the preparation of water extracts in
the form of herbal tea infusions. Peppermint has been found to be a po-
tential source for bioactive compounds having notable antimicrobial ac-
tivities (McKay & Blumberg, 2006; Peixoto et al., 2009; Rita & Animesh,
2011). In this study, it was shown for the first time that a common bev-
erage like peppermint tea possesses antichlamydial properties and even
in low concentrations. Although the activity was tested in vitro experi-
ments, previous study on mice with M. arvensis extract (Salin et al.,
2011) indicates that peppermint is likely to yield an inhibiting effect
on C. pneumoniae also in vivo.
The qualitative and quantitative polyphenolic contents of com-
mercial herbal tea (M. × piperita L.) samples were studied using
HPLC–UV-MS/MS analysis. Also, for the determination of the content
of the samples, the essential oil analyses were needed. The analyses
showed that the 24 peppermint tea samples contained peppermint,
whereas three samples may contain M. spicata, different from that
claimed on the package.
The results indicate that the content of peppermint tea samples
varies and the high essential oil content may accompany with the

***

***

*** p<0.001; t-test, compared to control (Ctrl)

Fig. 3. The effect of seven peppermint tea extracts on host cell viability. HL cells were exposed to the extracts at 250 μg/ml or usnic acid (50 μM; positive control) for 72 h. The data
represents [mean ± (SEM) results, n = 8].
 K. Kapp et al. / Food Research International 53 (2013) 758–766
high polyphenolic content. The seven tested peppermint herbal teas
showed activity against the respiratory tract pathogen C. pneumonia,
whereby the teas characterized by high content of luteolin and apigenin
glycosides showed high activity. Hence, M. × piperita could serve as a po-
tential source of health promoting agent against C. pneumoniae infection
when consumed in sufficient amounts. The results of the present study
may help in specification of the everyday diet and the give further rea-
sons to continue the investigation of peppermint as an antichlamydial
agent.
Supplementary data to this article can be found online at http://
dx.doi.org/10.1016/j.foodres.2013.02.015.
Acknowledgments
Karmen Kapp acknowledges the FinPharma Doctoral Programme
(FPDP) for financial support. This work has been funded by the Acad-
emy of Finland grant to Leena Pohjala (project no. 252216), as well as
by support from the Drug Discovery and Chemical Biology (DDCB)
network of Biocenter Finland to Pia Vuorela. The authors wish to
thank Elmar Arak, Aile-Ly Mardim and Kairi Aia for their kind help
at the laboratory.
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