Food Research International 53 (2013) 758–766
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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 Institute of Chemistry of Tallinn University of Technology, Akadeemia tee 15, 12628 Tallinn, Estonia 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 b c
a r t i c l e
i n f o
Article history: Received 16 November 2012 Received in revised form 5 February 2013 Accepted 9 February 2013 Keywords: Mentha × piperita Gram-negative Intracellular bacteria Antibacterial activity Polyphenolic compounds Essential oil
a b s t r a c t The qualitative and quantitative polyphenolic contents in the infusions of the commercial peppermint tea (Mentha × piperita L.) samples (n = 27) from different countries were studied using HPLC–UV-MS/MS analysis. The most abundant polyphenolics in the peppermint infusion were eriocitrin, 12-hydroxyjasmonate sulfate, 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 24 (89%) peppermint tea samples contained peppermint, whereas three samples may contain Mentha spicata, different from that claimed on the package. The effects of seven peppermint tea extracts against respiratory tract pathogen Chlamydia pneumoniae were investigated in vitro. All seven selected tea extracts were active against C. pneumoniae, the growth inhibition ranging from 20.7% to 69.5% at extract concentration of 250 μg/ml. In most cases, the antichlamydial activity 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 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 society and about three billion cups of tea are consumed daily worldwide (Hicks, 2009). During the last decades, there has been a resurgence of interest 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 ingredient 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:
[email protected] (A. Raal). 0963-9969/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodres.2013.02.015
antimicrobial, fungicidal, antiviral, insecticidal, radioprotective, and antioxidant activities (McKay & Blumberg, 2006; Peixoto, Furlanetti, Anibal, Duarte, & Höfling, 2009; Rita & Animesh, 2011). Perhaps therefore, peppermint 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 content 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
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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 selective 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 polyphenolic 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 considered 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 content 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 ×
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0.25 mm, Supelco, Switzerland) and polar polyethylene glycol (SW-10, 30 m × 0.25 mm, Supelco, Switzerland). The film thickness of both stationary 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 program 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 indices (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 prepared 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 equivalent (TPGA), the high-performance liquid chromatography hyphenated 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 Technologies, 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 accumulation 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 detector. 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 Technologies, 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 column 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 respective 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 compounds (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.
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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, representative 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 lyophilized 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 Institute 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 Microbiology, Institute of Diagnostics, University of Oulu, Finland and propagated as described by Alvesalo et al. (2006). All infections were preceded by seeding the HL-cells into 24-wellplates (Corning, Inc., USA) with coverslips (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, package
Obtained from
Making of tea
Amount (g) used for 1 cup of tea
Best before, month/year
1 2
EU Germany
Food market Food market
Boiling water (200 ml), 2–3 min Boiling water, 5–8 min
1.0 2.25
05/2011 01/2012
3 4 5 6 7 8 9 10 11 12
Germany Latvia Poland Poland The Netherlands Latvia Poland Egypt Egypt Estonia
1.0 × 20 teabags with strand 2.25 × 20 teabags with strand in paper packages 1.5 × 20 teabags 1.5 × 20 teabags with strand 1.5 × 20 teabags 1.5 × 30 teabags 1.5 × 20 teabags 1.0 × 20 teabags 1.5 × 20 teabags 1.0 × 20 teabags 1.5 × 12 teabags Crude drug, 15.0
Food market Food market Food market Food market Food market Food market Food market Food market Food market Pharmacy
1.5 1.5 1.5 1.5 1.5 1.0 1.5 1.0 1.5 3.0
07/2011 08/2011 09/2011 08/2010 07/2011 06/2011 03/2011 09/2011 10/2009 05/2011
13
Estonia
Crude drug, 15.0
Pharmacy
1.0
10/2011
14 15 16
Estonia USA Germany
Pharmacy Pharmacy Pharmacy
1.0 1.25 1.5
06/2011 12/2012 03/2014
17
Germany
Pharmacy
Boiling water (150 ml), 10–15 min
1.5
02/2014
18
Germany
Crude drug, 30.0 1.25 × 20 teabags 1.5 × 20 teabags with strand in paper packages 1.5 × 20 teabags with strand in paper packages Crude drug, 50.0
Boiling water, 5–8 min Boiling water (150 ml), 3–4 min Boiling water, 5–8 min Boiling water, 5–8 min Boiling water (150 ml), 3–4 min Boiling water (200 ml), 3–5 min Boiling water, 5–7 min Boiling water, (1 cup), 5 min Boiling water (1 cup), 3 min 2–3 g of herb and 1 cup of boiling water, 5–10 min 1–2 teaspoons of herb and boiling water, 5–7 min Not described Boiling water, 3–5 min Boiling water, 10–15 min
Pharmacy
1.5
10/2012
19
Germany
Crude drug, 50.0
Health shop
1.0
12/2013
20
Germany
Crude drug, 50.0
Health shop
0.8
11/2013
21
Germany
Health shop
1.5
07/2013
22
Germany
Food market
Boiling water, 5–6 min
2.25
10/2013
23
Germany
Food market
09/2013
Germany
Food market
1 l of boiling water and 4 teabags, 5–6 min Boiling water, 6 min
2.25
24
1.75
09/2013
25
UK
Health shop
Boiling water, 2–5 min
1.6
06/2013
26
Finland
1.5 × 12 teabags with strand in paper packages 2.25 × 25 teabags with strand in paper packages 2.25 × 25 teabags with strand in paper packages 1.75 × 20 teabags with strand in paper packages 1.6 × 20 teabags with strand in packages Crude drug, 40.0
Boiling water (150 ml) and 1.5 g of herb, 10–15 min 1–2 teaspoons of drug and 1 cup of boiling water, 5–10 min 1 teaspoon of drug and 1 cup (150 ml) of boiling water, 5–10 min Boiling water (150 ml), 10–15 min
Health shop
1.2
02/2013
27
Finland
Crude drug, 30.0
1–2 teaspoons of drug and 1 cup of boiling water, 5–15 min 1 teaspoon of drug is infused 10–20 min in hot water
0.7
08/2012
Health shop
Sample no. 27 contains Mentha × piperita, Mentha spicata and Mentha × dalmatica.
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(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 rifampicin (BioChemika > 97.0% HPLC; Fluka, Buchs, Switzerland) in 1 ml of ethanol (Etax A, Altia Oyj, Finland). In each well the concentration 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 drying them at the room temperature. The staining of host cells and chlamydia inclusions was carried out using Pathfinder Chlamydia culture confirmation system reagent (Bio-Rad Laboratories, France) and the inclusion counts were determined under a fluorescent microscope (Nikon Eclipse TE300, Tokyo, Japan) with a 20× magnification. Inhibition percentage was calculated on the basis of the average number of inclusions per coverslip by comparing the number of inclusions in a treated sample 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 parallel 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 suspension was mixed in the presence of glass beads to release the chlamydia 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 incubated overnight (37 °C) before adding of the seven M. × piperita samples at a final concentration of 250 μg/ml. Then the plate was incubated 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.
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2.6. Statistics Basic statistics and for comparison of groups t-test with independent 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 content 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, respectively 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 presence 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 extraction 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 predominant. Overall, 22 polyphenolic compounds were identified (Table 2, Figure 1 — Supplementary material), of which 12 were determined quantitatively (Table 3). In addition, malic and citric acids were detected. The content of polyphenols varied in a wide scale, but compositional 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 compound 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 concentration 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
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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 second 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.
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. Furthermore, 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%).
3.3.4. 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 significantly 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.
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.
4. Discussion Generally, the essential oils of peppermint teas were rich in menthol and menthone. However, sample no. 10 and two crude drug sample 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 specific 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 percentage concentration of menthol and methyl acetate. This refers to a circumstance 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).
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 concentration 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 concentration 7.5 μg/ml showed inhibition. With sample no. 19 it was 16.8%, no. 26 6.8% and no. 25 1.3%.
Table 2 The LC–DAD-MS/MS characteristics of polyphenols and plant acids identified in the peppermint tea samples. Peak no.
Retention time (tR, min)
m/z characteristic of molecular and fragment ions
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
1.2 1.7 5.0 6.0 16.3 19.4 19.6 20.3 22.8 24.0 26.9 29.4 30.1 31.3 31.7 33.3 34.8 36.0 36.1
133 191 197 315 179 353 305 467 387 357 637 537 595 461 593 579 577 359 717
[M−H]−; MS2: 115; [M−H]−; MS2: 111; [M−H]−; MS2: 179; [M−H]−; MS2: 153; [M−H]−; MS2: 135 [M−H]−; MS2: 173; [M−H]−; MS2: 225; [M−H]−; MS2: 387; [M−H]−; MS2: 207; [M−H]−; MS2: 313; [M−H]−; MS2: 351; [M−H]−; MS2: 339; [M−H]−; MS2: 287 [M−H]−; MS2: 285 [M−H]−; MS2: 285 [M−H]−; MS2: 271 [M−H]−; MS2: 269 [M−H]−; MS2: 223; [M−H]−; MS2: 519;
20 21 22 23 24
36.7 37.2 37.3 40.5 44.5
607 461 493 717 479
[M−H]−; MS2: 299; 284 [M−H]−; MS2: 285 [M−H]−; MS2: 295; 313; 159; 183 [M−H]−; MS2: 519; 321; 339; 393 [M−H]−; MS2: 317
a
Identification based on the literature data.
73; 87; 89 173 73 109 179; 97 241; 163; 269; 285 229;
135 207 369 159 493; 295
197; 161 537; 321; 339
Identification Malic acid Citric acid Danshensua (Sheet al., 2010) Protocatechuic acid glucoside Caffeic acid Chlorogenic acid 12-Hydroxyjasmonate sulfatea (Gidda et al., 2003) Medioresinol sulfatea Medioresinola (Vaquero et al., 2012) Prolithospermic acida (Xu et al., 2007) Luteolin-di-O-glucuronide Salvianolic acid H/Ia (Liu et al., 2007; Xu et al., 2007) Eriocitrin Luteolin-O-glucuronide Luteolin-O-rutinoside Narirutin Apigenin-O-rutinoside Rosmarinic acid Salvianolic acid Ea (Liu et al., 2007; Ruan, Li, Li, Luo, & Kong, 2012) Diosmin Luteolin-O-glucuronide Isosalvianolic acid Aa (Ruan et al., 2012) Salvianolic acid B Myricetin-O-glucoside
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 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27
4.8 5.9 6.2 4.8 3.0 5.6 5.7 4.1 2.4 4.1 nd nd 2.5 nd 9.9 5.9 3.1 0.9 4.7 13.0 4.7 2.3 3.1 4.1 6.5 1.4 4.8
24.6 15.0 19.1 15.1 10.1 15.9 17.0 11.2 9.2 11.4 7.8 3.2 23.0 20.1 20.3 17.6 13.4 17.7 25.4 15.3 12.8 12.6 14.7 15.4 11.4 28.9 17.6
4.5 0.3 3.5 3.5 2.6 3.1 3.2 4.2 2.5 5.1 3.5 1.3 3.1 1.5 3.1 2.7 1.5 0.7 4.0 4.7 1.6 2.0 2.4 1.9 3.7 1.8 4.4
1.1 0.7 0.9 1.0 1.0 1.2 0.9 1.6 0.9 3.2 2.0 1.3 0.6 nd 1.2 0.8 0.4 0.3 0.7 1.3 0.4 0.5 0.5 0.6 1.0 0.4 2.2
1.7 0.8 1.4 1.4 2.2 1.6 1.6 4.3 2.0 1.7 1.3 nd 2.2 3.4 1.8 0.8 0.5 3.6 1.8 1.3 0.4 0.8 0.8 0.7 1.4 4.5 2.4
33.2 48.9 44.3 38.1 44.2 42.3 40.6 44.9 47.0 24.2 25.1 8.8 30.0 20.1 42.1 53.8 68.1 48.0 38.2 19.9 61.4 56.1 48.8 59.5 36.6 30.0 27.3
3.2 2.3 5.8 6.6 13.2 7.8 4.7 2.1 13.8 nd nd nd 1.0 9.9 3.4 3.5 3.0 3.0 2.3 2.1 1.7 9.6 8.0 3.7 6.7 3.9 1.9
2.2 0.9 1.4 1.5 2.1 1.8 1.7 3.9 1.9 11.7 8.3 6.8 2.3 3.5 1.4 0.7 0.4 1.0 2.0 1.4 0.5 0.7 0.8 0.7 1.4 1.7 3.0
2.4 1.2 nd 2.1 3.4 2.5 2.7 nd 3.3 7.4 2.3 9.7 8.4 9.5 2.8 2.1 1.1 2.6 1.5 3.0 1.0 1.4 1.7 1.2 6.4 2.7 5.2
2.9 1.4 2.0 1.7 2.4 2.1 2.4 4.1 1.9 1.9 1.4 nd 5.8 3.0 2.0 0.9 0.8 1.2 5.1 1.7 0.9 0.9 1.0 1.0 1.8 4.8 2.3
7.7 3.7 4.5 9.7 5.2 6.4 8.7 4.5 5.4 19.1 8.9 54.2 2.7 7.0 3.1 5.4 4.4 6.7 2.1 20.9 5.1 6.1 8.0 3.2 8.4 5.9 16.3
11.8 18.9 10.9 14.7 10.7 9.7 10.8 15.1 9.8 10.2 39.3 14.6 18.4 21.9 9.0 5.9 3.2 14.1 12.1 15.4 9.4 6.5 9.9 8.1 14.8 14.1 12.5
3.0 1.5 1.3 1.2 1.0 1.5 2.3 1.8 1.2 4.6 1.3 1.5 2.8 1.2 7.3 4.0 7.7 4.3 8.2 7.1 21.8 1.0 1.1 3.7 2.6 3.0 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 peppermint herbal teas was 12-hydroxyjasmonate sulfate. According to Miersch and co-authors, 12-hydroxyjasmonic acid and its derivatives 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 polyphenol 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 remarkably 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 glycosides 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 hydrocarbons 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
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