Corn Mint (Mentha arvensis) Extract Diminishes ... - ACS Publications

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Corn Mint (Mentha arvensis) Extract Diminishes Acute Chlamydia pneumoniae Infection in Vitro and in Vivo Olli Salin,† Liisa T€orm€akangas,§ Maija Leinonen,§ Elise Saario,§ Marja Hagstr€om,# Raimo A. Ketola,# Pekka Saikku,X^ Heikki Vuorela,^ and Pia M. Vuorela*,† †

Pharmaceutical Sciences, Department of Biosciences, Abo Akademi University, BioCity, Artillerigatan 6 A, FI-20520 Turku, Finland National Institute for Health and Welfare (THL), Aapistie 1, FI-90101 Oulu, Finland # Centre for Drug Research, Faculty of Pharmacy, University of Helsinki, P.O. Box 56 (Viikinkaari 5 E), FI-00014 University of Helsinki, Finland X^ Institute of Diagnostics, Department of Medical Microbiology, University of Oulu, P.O. Box 5000, FI-90014 Oulu, Finland ^ Division of Pharmaceutical Biology, Faculty of Pharmacy, University of Helsinki, P.O. Box 56 (Viikinkaari 5 E), FI-00014 University of Helsinki, Finland §

ABSTRACT: Corn mint (Mentha arvensis) provides a good source of natural phenols such as flavone glycosides and caffeic acid derivatives, which may have prophylactic properties against inflammations. This study investigated whether corn mint extract would be beneficial against a universal respiratory tract pathogen, Chlamydia pneumoniae, infection. The extract inhibited the growth of C. pneumoniae CWL-029 in vitro in a dose-dependent manner. The inhibition was confirmed against a clinical isolate K7. The phenolic composition of the extract was analyzed by UPLC-ESI/Q-TOF/MS, the main components being linarin and rosmarinic acid. These compounds were active in vitro against C. pneumoniae. Linarin completely inhibited the growth at 100 μM. Inbred C57BL/6J mice were inoculated with C. pneumoniae K7. M. arvensis extract was given intraperitoneally once daily for 3 days prior to inoculation and continued for 10 days postinfection. The extract was able to diminish the inflammatory parameters related to C. pneumoniae infection and significantly (p = 0.019) lowered the number of C. pneumoniae genome equivalents detected by PCR at biologically relevant amounts. KEYWORDS: Mentha arvensis, polyphenolic compounds, linarin, rosmarinic acid, Chlamydia pneumoniae, antichlamydial effect

’ INTRODUCTION Most of the papers describing the antimicrobial effects of the genus Mentha (mint plants) focus on the mint oils and the compounds within,1,2 which no doubt are responsible for a part of the activity of the fresh and properly dried plants. Various Mentha species have been traditionally used as a tea, serving as an important part of the cuisine in many cultures around the world. Notably, the preparation of a tea might result in different chemical compositions.3 Most of the volatile substances are lost during the preparation of the herbal tea when, for example, prepared uncovered. In these cases the main effective compounds of teas are of a phenolic nature rather than the essential oils. The phenolic content of Mentha species has been shown to comprise multiple compounds, for example, up to 7% of caffeic acid derivatives and multiple flavones and flavone glycosides.4,5 Several phenolic compounds have been recorded from water extracts of Mentha species with eriocitrin, luteolin, and also rosmarinic acid being present in most species in relatively high amounts.5 The phenolic compounds present in Mentha species and in plants in general have been acknowledged as compounds with pharmacological effects against various diseases.6
8 The phenolic compounds are secondary metabolites of plants and have various functions. They have been mostly associated with protection of UV-induced cellular stress, antimicrobial activity, protection against herbivores, and also acting as attractants. This group of compounds is of utmost interest for its antimicrobial properties and capabilities to regulate cellular processes in plants r 2011 American Chemical Society

and also in mammals. These compounds are likely to yield some of the medicinal properties that the herbal drugs have been used for. Chlamydia pneumoniae is a Gram-negative human pathogen that causes respiratory infections. It has been estimated that C. pneumoniae is the causative agent in 5
10% of communityacquired pneumonia, bronchitis, and sinusitis cases.9 This intracellular bacterium also causes chronic infections and prolonged inflammation that have been associated with atherosclerosis,10 asthma,11 lung cancer,12 and Alzheimer’s disease.13 Serological reports indicate that this bacterium is endemic worldwide and infects practically everyone at least once in their lifetime.14 Multiple natural phenols have been shown to possess antichlamydial properties in vitro.15,16 C. pneumoniae seems to be most vulnerable at initial contact with the host cell and thus natural phenols, for example, polyphenols consumed on a daily basis from food, may have prophylactic properties to some extent if consumed in high enough quantities. A tea polyphenol product consisting of (
)-epigallocatechin (18.3%), (
)-epicatechin (8.6%), (
)-epigallocatechin gallate (35.9%), (
)-epicatechin gallate (11.2%), and (
)-gallocatechin gallate (3.5%) has been Received: August 12, 2011 Revised: November 11, 2011 Accepted: November 11, 2011 Published: November 11, 2011 12836

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Journal of Agricultural and Food Chemistry evaluated in vitro against two C. pneumoniae strains with inhibiting concentrations of 0.8 and 1.6 mg/mL.17 A culinary herb, corn mint (Mentha arvensis L.), was selected for this follow-up study on acute C. pneumoniae infection on the basis of our earlier results of screening 93 plant extracts.15 Corn mint extract (40 μg/mL) exerted an inhibition of 100% in the primary in vitro screening. Also, the ethnopharmacological use of corn mint among Native American tribes and in Chinese medicine for fever, inflammation, and chills supported the antiinflammatorial property of this plant. The content of the extract was analyzed with up to date HPLC/DAD and UPLC-ESI/QTOF/MS equipment. The effect of the extract was determined in vitro using both reference and clinical C. pneumoniae strains and compared to the identified pure compounds of the extract. The possible positive health effects of the extract against C. pneumoniae infection were evaluated in an in vivo mouse model.

’ MATERIALS AND METHODS Preparation of Extract. Mentha arvensis (L.), family Lamiaceae (corn mint), was cultivated in Mikkeli, Finland (Karila, Mikkeli, Finland, 61 400 2800 N, 27 130 400 E) and harvested in the flowering stage after a growth period of 3 years. The aerial plant material was obtained from senior scientist Bertalan Galambosi at the MTT Agrifood Research Finland, Mikkeli, Finland. The material was kept in a dark and cool place until used. The extract was prepared by weighing 3  5 g of dry plant parts, ground to a fine texture and homogenized, into a glass vessel. A 100 mL volume of 80% aqueous methanol (MeOH, technical quality, Algol, Finland) was added, and the mixture was sonicated for 10 min and filtered with Whatman 4 filter paper. The extract was rotavaporized to dryness under vacuum, and the residue was lyophilized for 3 days, giving a dry yield of 9% (n = 3). For the in vitro experiments in cell cultures lyophilized extract was first dissolved in DMSO (Merck, Darmstadt, Germany) to a concentration of 128 mg/mL. For the in vivo experiments, the sample was dissolved in DMSO to a concentration of 0.4 g/mL and diluted in water to yield a concentration of 4 mg/mL (1% DMSO). Compounds. The pure compounds used in in vitro experiments or HPLC experiments, acacetin, apigenin, apigenin-7-O-glucoside, linarin, and rosmarinic acid, were purchased from Extrasynthese (Genay, France), whereas caffeic acid and ferulic acid were purchased from Sigma-Aldrich (St. Louis, MO). For cell culture studies, the pure compounds were dissolved in DMSO to a concentration of 100 mM and for analytical purposes in MeOH (Mallinckrodt Baker, Philipsburg, NJ) as described under Animal Experiments. Control antibiotic rifampicin was purchased from Sigma-Aldrich and dissolved in DMSO to a concentration of 12 μM. In Vitro Antichlamydial Assays. HL cells, as described in Salin et al.,18 were grown in RN medium consisting of RPMI 1640 (BioWhittaker, Lonza, Basel, Switzerland) supplemented with 7% fetal bovine serum (FBS) (BioWhittaker, Lonza), 2 mM L-glutamine (BioWhittaker, Lonza), and 20 μg/mL gentamicin (Fluka, Buchs, Switzerland). The C. pneumoniae strain CWL-029, a reference strain from the American Type Culture Collection (ATCC), and the clinical strain K7 were grown and purified as described in Alvesalo et al.16 The in vitro determination of the effect of M. arvensis and of the pure compounds against C. pneumoniae strains CWL-029 and K7 was done as described in Salin et al.18 with two exceptions: the amount of cycloheximide (Sigma-Aldrich) was increased to 1%, and the labeled coverslips were dipped once in 6.7 mM PBS (pH 7.4) solution (BioWhittaker, Lonza) and twice in deionized water.

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All of the plates in this study were inspected under a microscope for abnormalities. The effects on host cell viability of the M. arvensis extract and of the pure compounds were determined by a commercial “CellTiter-Glo Luminescent Cell Viability Assay” (Promega, Madison, WI) in 96-well plates (Wallac Isoplate 1450-516, PerkinElmer, Waltham, MA) as described in Salin et al.18 GC/MS Analysis of Essential Oils. The essential oil content of the M. arvensis extract was evaluated by preparing a fresh solution of the extract in MeOH and fractioning the solution with the addition of hexane (Rathburn, Walkerburn, Scotland) (3:1 v/v). This mixture was vortexed for 2 min, and a sample taken from the hexane layer was manually injected to the Hewlett-Packard (HP) gas chromatograph 5890A with a Restek RTX-1 Crossbond 100% dimethyl polysiloxane column (30 m, 0.25 i.d., 0.25 μm df) (Restek Corp., Bellefonte, PA) coupled with HP quadrupole mass selective detector 5970A operated with ionization voltage of 70 eV (EI mode). The oven temperature was programmed to increase from 50 to 250 C at 6 C/min, and the injector and detector temperatures were 250 C. HPLC/DAD Analysis. Extract from M. arvensis was dissolved in MeOH (Mallinckrodt Baker, Philipsburg, NJ) to a concentration of 10 mg/mL and filtered with a Whatman FP 30/0.2 CA-S cellulose acetate filter disk (Schleicher & Schuell, Dassel, Germany). The pure compounds (reference compounds) acacetin, linarin, apigenin-7-O-glucoside, caffeic acid, ferulic acid, and rosmarinic acid were dissolved in MeOH and apigenin in ACN (BDH Prolabo VWR International LLC, West Chester, PA) to a concentration of 0.2 mg/mL. All samples were sonicated for 20 min, and apigenin was heated at 45 C until completely dissolved. Qualitative HPLC analyses were performed using Waters HPLC equipment (autosampler Waters 717, pump Waters 600, controller Waters 600, degasser Waters In-Line Degasser AF; Waters Corp., Milford, MA) coupled with a Waters 2996 photodiode array detector (DAD). Separation was performed on a reverse-phase Hypersil BDSC18 analytical column (250  4.6 mm i.d.), 5 μm (Agilent Technologies, Palo Alto, CA), used without a guardian column. Two different gradient methods were applied to identify the compounds in the extract. For the first method (method 1) the flow rate was 0.8 mL/min and the eluents were (A1) MeOH/H2O/CH3COOH, 10:88:2 v/v/v, and (B1) MeOH/H2O/CH3COOH, 90:8:2 v/v/v. The nonlinear gradient elution program was the following: 0
5 min, 5% B1; 5
10 min, 5
10% B1; 10
15 min, 10
25% B1; 15
18 min, 25% B1; 18
25 min, 25
35% B1; 25
28 min, 35% B1; 28
35 min, 35
100% B1; 35
40 min, 100% B1, returned to initial conditions in 15 min. For the second method (method 2) the flow rate was 0.4 mL/min and the eluents were (A2) 0.2% formic acid (Mallinckrodt Baker) in water and (B2) ACN. The nonlinear gradient elution program was the following: 0
15 min, 15
25% B2; 15
25 min, 25
45% B2; 25
35 min, 45
50% B2; 35
40 min, 50
100% B2; 40
50 min, 100% B2, returned to initial conditions in 15 min. Phenolic compounds were identified and quantified at 280 and 340 nm, and the analyses were performed at room temperature. Detection was carried out with a sensitivity of 0.1 aufs between the wavelengths of 200 and 550 nm for UV spectra. Injection volumes used were 30 and 40 μL for methods 1 and 2, respectively. The amount of phenolic compounds in the M. arvensis extract was determined using calibration curves obtained from six concentrations in the range of 0.5
6 μg/injection (n = 3) of two external standards, rosmarinic acid (0.27x + 0.21, R2 = 0.997) and linarin (0.37x+ 0.04, R2 = 1.000). HPLC method 1 was used for quantification. To examine the presence of glycosidic flavonoids in the extract, the extract and the glucosidic compounds linarin and apigenin-7-O-glucoside were acid hydrolyzed following a method modified from that of Hertog et al.19 Briefly, M. arvensis extract or the pure compound was dissolved in 70% MeOH/1.2 M hydrochloric acid (HCl) (Riedel-deHa€en, Seelze, Germany) (1:1 v/v) to yield a final concentrations of 10 and 0.2 mg/mL, respectively. The extract and the pure compounds were 12837

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Journal of Agricultural and Food Chemistry heated for 2 h at 80 C in sealed 10 mL test tubes. The analysis was performed with HPLC method 2. UPLC-ESI/Q-TOF/MS or MS/MS Analysis. UPLC-ESI/Q-TOF MS and MS/MS analyses were performed to confirm the compounds detected in HPLC-DAD analyses. LC was performed with a Waters Acquity UPLC (Waters Corp.) and a Waters Acquity UPLC BEH Shield RP18 (2.1  100 mm, 1.7 μm) analytical column (Waters Corp.). A gradient elution was utilized with eluent A of water with 0.1% of formic acid and eluent B of 100% ACN. The gradient elution program was the following: 0
1 min, 10
60% B; 1
2 min, 60% B; 2
4 min, 60
95% B; 4
5 min, 95% B; 5
5.1 min, 95
10% B; 5.1
6 min, 10% B. The flow rate of eluent was 0.5 mL/min. The injection volume was 5 μL with partial loop with needle overfill. The mass spectrometer used was a Waters Xevo quadrupole-time-of-flight (Q-TOF) mass spectrometer equipped with an electrospray ionization (ESI) source (Waters Corp.). ESI capillary voltage was 3 kV (in positive ion mode), sampling cone was 35 V, extraction cone was 2.9 V, source temperature was 120 C, and desolvation temperature was 300 C. Nitrogen was used as a desolvation gas with a flow rate of 850 L/h. TOF mass spectra were collected in the mass range of m/z 100
1000 with a 0.1 s scan time. The mass axis was calibrated using sodium fluoride, the mass resolution obtained was over 10000, and the mass accuracy was below 5 ppm when using leucine enkephalin as a lock reference compound. In MS/MS measurements argon was used as a collision gas with the following collision energies: rosmarinic acid, 5 eV; acacetin, 15 eV; and linarin, 30 eV. Animal Experiments. Mice were inoculated with C. pneumoniae isolate Kajaani 7 (K7) free of mycoplasma and diluted in sucrose phosphate glutamic acid (SPG) buffer. The inoculum dose was estimated by culturing serial dilutions of the stock in duplicate in HL cells. The cell cultures were done as described earlier.20 Inbred C57BL/6J female mice were purchased from Harlan Netherlands at the age of 6 weeks. Treatments with either M. arvensis extract (20 mg/kg) or placebo (1% DMSO) were started at the age of 8 weeks. The treatments were given intraperitoneally (ip) once daily for 3 days prior to inoculation and continued for 10 days postinfection (pi). On the fourth day after starting the treatments, the mice were inoculated intranasally with C. pneumoniae isolate K7 (7  105 IFU/mouse) under inhaled methoxyflurane (Medical Developments, Springvale, Australia) anesthesia. Samples were taken on days 3, 6, 10, 13, and 20 pi. A third group was inoculated with SPG instead of chlamydia (uninfected, untreated controls), and samples from these mice were taken on days 3 and 10 pi. The Animal Care and Use Committee of National Public Health Institute, Helsinki, Finland, approved all procedures involving animals. C. pneumoniae antibody detection, culture of lung tissue, and detection of chlamydial DNA were done as described earlier,21 with the exception that no analysis of the RNA content was done. Histopathology of lungs was done as described earlier.20 Statistical Analyses. In the in vivo studies, the differences between the groups after C. pneumoniae antibody, culture, and DNA analyses were tested with nonparametric Mann
Whitney U-tests. The lung histopathology scores between the groups were compared with the chisquare test for trend. The statistical analyses were performed using SPSS version 11.5.1.

’ RESULTS Inhibition of C. pneumoniae Growth by M. arvensis Extract. The antichlamydial dose
response relationship of M.

arvensis extract was first assayed against C. pneumoniae reference strain CWL-029 (Figure 1). The extract inhibited the growth of C. pneumoniae strain CWL-029 in a dose-dependent manner, and the highest concentration used (256 μg/mL) inhibited the formation of chlamydial inclusions by 73% (SEM = 3.5%, n = 12). The antichlamydial effect of M. arvensis extract in vitro was

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Figure 1. Inhibition of the growth of C. pneumoniae strains CWL-029 and K7 by M. arvensis (lines). Data represent the mean ( SEM of 12 observations. The effect of the M. arvensis extract on host cell viability (bars) represents the mean ( SD of three replicates.

confirmed against the clinical isolate K7, which was to be used in the mouse model. The extract showed a dose-dependent activity against strain K7 similar to, but slightly higher than, that shown against strain CWL-029 (Figure 1). The maximum inhibition of formation of chlamydial inclusions was 90% (SEM = 3.5%, n = 12) at the concentration of 256 mg/mL. Identification of the Most Prevalent Compounds in M. arvensis Extract. No essential oil components were found in the GC/MS analysis of M. arvensis. Three major peaks with retention times of 38.52, 41.27, and 41.52 min were detected by HPLC analysis (method 1). The retention times and UV spectra acquired with PDA detection of compounds in the extract were compared to an in-house library and to retention times and UV spectra of freshly prepared control compounds. The first major peak was identified to be rosmarinic acid. The UV spectra of the second and third major peaks resembled the pure compound linarin (acacetin-7-O-rutinoside). The latter had exactly the same retention time as the reference compound linarin. On the basis of the literature, the second major peak of the extract eluting before the linarin peak was determined to be acacetin-acetylglucosiderhamnoglycoside.22,23 These three peaks covered 85% of the phenolic compounds (calculated from the total peak areas) in the M. arvensis extract according to the performed HPLC-analysis. HPLC analysis with method 2 also showed three major peaks in the M. arvensis extract. To verify the results and to compare the retention times of pure compounds to the retention times of the compounds in the extract, the extract was spiked with pure compounds. The concentrations of spiked samples were 5 mg/mL of extract and 0.1 mg/mL of pure compound. The spiked samples supported the presence of rosmarinic acid and linarin in the extract. HPLC analysis of the acid-hydrolyzed samples supported the existence of acacetin glycosides in the M. arvensis extract. Mass spectrometric analysis using UPLC-ESI/Q-TOF was performed to confirm the structural identification of compounds detected by HPLC from the extract. Table 1 shows the compounds identified from the sample using ultrahigh separation and accurate mass measurements. The quantitative HPLC analysis (n = 3) indicated that the extract contained 5.2% rosmarinic acid (dry weight) and 6.0% linarin. The extract also contained 2.5% acacetin-acetylglucosiderhamnoglycoside and minor amounts of acacetin. The 128 μg/mL amount of the extract used in the C. pneumoniae inhibition assay contained 18 μM (7.2 μg/mL) rosmarinic acid and 13 μM (7.7 μg/mL) linarin. The corresponding values for 256 μg/mL extract were 36 and 26 μM, respectively. 12838

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Journal of Agricultural and Food Chemistry

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Table 1. Compounds Identified from the Mentha arvensis Extract by Accurate Measurements with UPLC-ESI/Q-TOF [M + H]+

error

retention compound

a

time (min)

exact mass

measured mass

mDa

ppm

molecular formula

rosmarinic acid

1.66

361.0923

361.0929


0.6


1.7

C18H16O8

acacetin

3.61

285.0763

285.0775


1.2


4.2

C16H12O5

linarin

1.61

593.1870

593.1866

0.4

0.7

C28H32O14

acacetin-acetylglucoside-rhamnoglycoside

naa

797.2504

797.2512


0.8


1.0

C36H44O20

na, not available.

Table 2. Antichlamydial Effect of Pure Compounds on Strain CWL-029 (n = 12) and Effect on Host Cell Viability at Concentrations of 100 and 10 μM (n = 3) host cell viability ( SD (%)

compound linarin

inhibition ( SD (%, 100 μM)

100 μM

10 μM 97 (1.3)

100 (1.7)

99 (2.0)

acacetin

97 (2.6)

97 (4.2)

94 (2.3)

rosmarinic acid

73 (14.6)

100 (1.8)

101 (2.8)

Figure 3. Geometric means and SD of the C. pneumoniae-specific IgG antibody responses in the study groups measured by the microimmunofluorescence method. Values on the x-axis indicate the time postinfection.

Figure 2. Inhibitory effect of pure compounds on the growth of C. pneumoniae strain K7 (n = 3, error bars ( SD).

Inhibition of C. pneumoniae Growth by Pure Compounds. The antichlamydial effect of rosmarinic acid and linarin, as well as its aglycone acacetin, was determined against the reference strain CWL-029 (Table 2). Acacetin-acetylglucoside-rhamnoglycoside was not available as a pure compound. Linarin and acacetin completely inhibited the growth of strain CWL-029 at 100 μM. The activity of pure compounds against strain K7 was determined in a dose
response manner (Figure 2). Both of the main components of the extract, rosmarinic acid and linarin, inhibited the growth of strain K7 by >60% at a concentration of 100 μM. Acacetin inhibited the growth of C. pneumoniae by 80% at 100 μM. During the visual inspection of the plates under a microscope, no abnormalities were found in the controls or the treated wells. The effect of the M. arvensis extract and the pure compounds on host cell viability was measured as the change in the intracellular ATP concentration. The effect of the extract was dose-dependent (Figure 1). The pure compounds did not affect the host cells at concentrations of 10 and 100 μM (Table 2). In Vivo Experiments. The IgG antibody levels against C. pneumoniae in the mice were significantly higher (p = 0.002)

in the untreated control group than in the group treated with M. arvensis extract at 20 days pi (Figure 3). At the same time, the lung histopathology findings and/or the presence of C. pneumoniae in the lung tissue indicated successful inoculation of the mice treated with the M. arvensis extract, despite the low IgG levels. The presence of C. pneumoniae in the lung tissue of the mice was detected by chlamydial culture and by the analysis of chlamydial DNA. The overall C. pneumoniae positivity was determined as a percentage of the mice in a study group positive for C. pneumoniae by culture and/or PCR detection. On days 6, 10, and 13 pi, the C. pneumoniae positivity was lower in the group treated with M. arvensis extract than in the untreated control group. Especially on day 10 pi, only 38% (3/8) of the mice in the treated group were positive, whereas in the untreated control group 88% (7/8) of the mice were found positive (Figure 4). At this time point (day 10 pi), one mouse in both of these study groups showed no increase in C. pneumoniae IgG antibody levels or changes in lung histopathology. Because culture and PCR findings were negative as well, it is possible that inoculation had been unsuccessful in these two mice. The number of viable C. pneumoniae particles was analyzed by lung tissue culture, and the number of C. pneumoniae genome equivalents in 50 mg of lung tissue was analyzed by PCR. Statistically significant differences were not found in the lung tissue culture (Figure 5A). However, on day 13 pi significantly (p = 0.019) lower numbers of C. pneumoniae genome equivalents were detected by PCR in the group treated with M. arvensis extract (Figure 5B). Viable C. pneumoniae or chlamydial DNA was not found in the lung tissue of mice in the uninfected, untreated control group. The lung histopathology changes were evaluated on a scale from 0 to 4. As shown in Figure 6, mean inflammation grades in 12839

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Journal of Agricultural and Food Chemistry the lung tissue of the mice treated with M. arvensis extract were lower on days 6, 10, and 13 pi, yet no statistical significances were found.

’ DISCUSSION Polyphenols and phenolic acids have been strongly associated with various health effects including the prevention of cancers and heart diseases by multiple mechanisms (see, e.g., refs 6, 24, and 25) and via anti-inflammatory actions.26 In addition to the straight physiological mechanisms the polyphenols have in mammals, they are likely to serve as antimicrobial compounds as well (see, e.g., refs 8, 15, and 24) and thus prevent cellular stress and inflammation caused by infections. This type of protection is especially important in chronic infections that cause persistent inflammation of tissues, typical of Chlamydia.9,10 The M. arvensis extract showed >60% inhibition in vitro of both C. pneumoniae strains used in this study at a concentration of 128 μg/mL. The extract inhibited the growth of strain K7 at a concentration of 256 μg/mL by 90% without affecting the level of intracellular ATP in host cells, a delicate indicator of cell viability. The low toxicity is in agreement with the general opinion that species of the genus Mentha as a traditional herbal drug are almost completely nontoxic, unlike the mint oils that may contain pulegone, a natural hepatotoxin.27 The presence and amount of compounds in an extract depend highly on several environmental factors such as the time of harvest,2 and thus the pharmacological effects of the plant

Figure 4. Overall C. pneumoniae positivity indicating the number of mice found to be positive for C. pneumoniae either by lung tissue culture or by DNA analysis or both. Values on the x-axis indicate the time postinfection.

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extracts should be compared to the effects of the main components of the extract. The essential oil composition of the M. arvensis extract was analyzed by GC/MS, but no essential oils were found, as expected on the basis of the method used in the preparation of the extract. This indicates that the biological effects derive from the phenolic content of the extract. The phenolic composition of the M. arvensis extract was analyzed by HPLC/DAD, UPLC-ESI/Q-TOF MS, and MS/MS, and the main components present were identified as rosmarinic acid and linarin, in accordance with previous studies.7,22,23 Two different gradient HPLC methods were used to detect possible smaller peaks eluting at the same retentions times as the main peaks. The hydrolyzation of the extract indicated the presence of two glycosylated flavonols that yielded acacetin when hydrolyzed. Acacetin was also detected from the extract in a small amount. This was in accordance with the literature and supported that one was linarin and that the other flavonol eluting before linarin was acacetin-acetylglucoside-rhamnoglycoside.22,23 In this study, it was shown that rosmarinic acid, linarin, and acacetin, the aglycone of linarin, were efficient growth inhibitors of both C. pneumoniae strains investigated. The bioavailability of polyphenols needs to be elaborated when the medical potential of plant products such as M. arvensis and other mint species is evaluated.25 It has been generally estimated that polyphenolic concentrations of 5 μM in plasma could be achieved from nutrition.28 Tea polyphenols, when consumed in high quantities, have been shown to be present in human prostate tissue even when not being detectable from the plasma of the same

Figure 6. Averages of inflammatory grades per study group at different times. Grades: 0, no inflammation; 1, mild changes with lymphocyte and plasma cell infiltration; 2, moderate; 3, marked; 4, severe changes. Values on the x-axis indicate the time postinfection.

Figure 5. Quantitative detection of (A) viable chlamydia in lung tissue by culture and (B) the number of chlamydial genomes detected per 50 mg of lung tissue of the mice still infected with C. pneumoniae. Dots at lines represent geometric means per study groups. On day 13 there was a significant difference (p = 0.019) of the M. arvensis treated group compared to the infected and untreated control groups measured by PCR (asterisk). Values on the x-axis indicate the time postinfection. 12840

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Journal of Agricultural and Food Chemistry individuals.29 The bioavailability of the flavone luteolin indicates that it has biological effects also in vivo, as reviewed by LopezLazaro.30 Luteolin has also showed an antichlamydial effect in our earlier in vitro and in vivo studies.16,21 Several epidemiological studies support that bioavailability profiles similar to that of luteolin can be expected for other flavonoid aglycones as well,31 although there are still multiple aspects that are unclear about the bioavailability of polyphenolic compounds from nutrition.32 In the antichlamydial assays in our study, concentrations higher than 100 μM were not used for the pure compounds as such high concentrations would have been of little biological relevance from the nutritional point of view. The slight differences in the responses of the two C. pneumoniae strains assayed with the phenolic compounds at 100 μM indicate small differences in the sensitivities of the strains to phenolic compounds, as also shown for, for example, betulin derivatives.18 The effect of the pure compounds rosmarinic acid, linarin, and acacetin against the clinical strain K7 in vitro suggests that these compounds alone could inhibit C. pneumoniae infection in humans if present in sufficient amounts at the site(s) of the infection. The amount of the main components and their derivatives present in the extract could explain the antichlamydial effect of the M. arvensis. Thus far, no antibacterial activity has been reported for linarin, whereas rosmarinic acid has been reported as a moderately active antibacterial agent, but with little effect on Gram-negative microorganisms.33 The antibacterial properties of acacetin have been reported as weak, but it has shown moderate effects on viruses.8 The role of animal models in C. pneumoniae research is especially important, because most genetic tools used in bacterial studies generally are not available for chlamydia. Well-established animal models for C. pneumoniae infection are available20,34 and even models for connection to, for example, atherosclerosis.35 Thus, animal models are crucial in showing the effect of any antichlamydial extract or compound on tissue and organ level consequences of C. pneumoniae, such as inflammatory damage. The in vivo results were gained with a concentration that was estimated to be biologically valid as the 20 mg/kg of extract given to mice ip would result in 1.4 g for a 70 kg human (i.e., if no metabolism or degradation). A higher amount would also not have been relevant in the evaluation of the usage of M. arvensis as part of the human diet or as a nutrition supplement. Although on the basis of the in vitro results, the amount of mint extract required to achieve rapid inhibition of C. pneumoniae in vivo should have been roughly 6
12 times higher than the used concentration, statistically significant differences were already achieved with the lower concentration used. In the mouse study, M. arvensis extract administered ip was able to decrease C. pneumoniae-induced inflammatory responses. As shown previously, mice are able to clear primary C. pneumoniae infection in 3
5 weeks16 and, therefore, the differences between the treatment groups at the earlier time points are more representative. The C. pneumoniae-specific IgG antibody response was significantly diminished at 3 weeks pi. The inflammation detected in the lung tissue histopathology was milder, especially on day 10 pi, although this difference was not statistically significant (p = 0.08). These findings are similar to our earlier report of a polyphenolic compound, luteolin, being anti-inflammatory.21 The effects of the M. arvensis extract, or luteolin as shown previously,21 on the presence and growth of viable C. pneumoniae in mice are not comparable to the effects induced by antibiotic treatments.20 However, the number of mice found positive for

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C. pneumoniae was decreased in the groups treated with the M. arvensis extract. The numbers of C. pneumoniae genome equivalents or viable chlamydia particles in the mice still positive were rather similar compared to the untreated mice. Further animal studies with larger treatment groups and different doses would be needed to more precisely show the antichlamydial effects of the M. arvensis extract and the natural phenols in vivo. In conclusion, it seems that M. arvensis and its main components alone could serve as a potential source of health-promoting agents against C. pneumoniae infection when consumed in sufficient amounts or as part of a polyphenol-rich diet. More importantly, the extract was able to diminish the inflammatory parameters related to C. pneumoniae infection in vivo at a biologically relevant dose and thus could help to prevent some of the long-term harmful effects of C. pneumoniae infection.

’ AUTHOR INFORMATION Corresponding Author

*Phone: +358 (0)2 215 4267. Fax: +358 (0)2 230 5018. E-mail: pia.vuorela@abo.fi. Funding Sources

This study was part of the ERA-NET PathoGenoMics ECIBUGproject (European Initiative to Fight Chlamydial Infections by Unbiased Genomics) and was funded by the Academy of Finland (119804). P.V. acknowledges the “Tor, Joe och Pentti Borgs minnesfond” foundation for financial support, and O.S. thanks the Finnish Cultural Foundation (Elli Turunen) for funding.

’ ACKNOWLEDGMENT We thank Into Laakso for excellent assistance and guidance with HPLC and GC analyses. ’ ABBREVIATIONS USED ATCC, American Type Culture Collection; ip, intraperitoneally; pi, post infection; SPG, sucrose phosphate glutamic acid; UPLC, ultraperformance liquid chromatography. ’ REFERENCES (1) Iscan, G.; Kirimer, N.; Kr€ukc€uoglu, M.; Baser, K.; Demirci, F. Antimicrobial screening of Mentha piperita essential oils. J. Agric. Food Chem. 2002, 50, 3943–3946. (2) Hussain, A. I.; Anwar, F.; Nigam, P.; Ashraf, M.; Gilani, A. H. Seasonal variation in content, chemical composition and antimicrobial and cytotoxic activities of essential oils from four Mentha species. J. Sci. Food Agric. 2010, 90, 1827–1836. (3) J€ager, S.; Beffert, M.; Hoppe, K.; Nadberezny, D.; Frank, B.; Scheffler, A. Preparation of herbal tea as infusion or by maceration at room temperature using mistletoe tea as an example. Sci. Pharm. 2011, 79, 145–155. (4) Fecka, I.; Turek, S. Determination of water-soluble polyphenolic compounds in commercial herbal teas from Laminaceae: peppermint, melissa, and sage. J. Agric. Food Chem. 2007, 55, 10908–10917. (5) Kosar, M.; Dorman, D.; Baser, H.; Hiltunen, R. Screening of free radical scavenging compounds in water extracts of Mentha samples using a postcolum derivatization method. J. Agric. Food Chem. 2004, 52, 5004–5010. (6) Link, A.; Balaguer, F.; Goel, A. Cancer chemoprevention by dietary polyphenols: promising role for epigenetics. Biochem. Pharmacol. 2010, 80, 1771–1792. 12841

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