F. Ferreres†
*a,
J. Bernardo†b,
P. B. Andradeb,
C. Sousab,
A. Gil-Izquierdoa and
P. Valentão*b
aResearch Group on Quality, Safety and Bioactivity of Plant Foods, Department of Food Science and Technology, CEBAS (CSIC), P.O. Box 164, 30100 Campus University Espinardo, Murcia, Spain. E-mail: federico@cebas.csic.es
bREQUIMTE/LAQV, Laboratório de Farmacognosia, Departamento de Química, Faculdade de Farmácia, Universidade do Porto, Rua de Jorge Viterbo Ferreira, no. 228, 4050-313 Porto, Portugal. E-mail: valentao@ff.up.pt; Fax: +351 226093390; Tel: +351 220428653
First published on 29th April 2015
Herbal teas are consumed for their valuable content of bioactive phytochemicals. This work represents the first attempt to establish a linkage between the chemical composition of pennyroyal (Mentha pulegium L.) infusion and its potential to prevent cellular oxidative stress. The phenolic profile was established by HPLC-DAD-ESI/MSn, twelve out of fifteen compounds being identified for the first time in this species. The infusion presented a phenolic content of 122.92 mg g−1 (lyophilized extract) and a remarkable antiradical activity. A reduced glutathione detoxification mechanism was demonstrated to be involved in human epithelial colorectal adenocarcinoma (Caco-2) cell resistance against tert-butyl hydroperoxide-induced toxicity, while the same was not observed for human epithelial gastric adenocarcinoma (AGS) cells. For the first time, the presence of these phenolic compounds inside the cells was confirmed by 2-aminoethyl diphenylborinate (DPBA) phenol fluorescence dye staining, suggesting a direct antioxidant effect. M. pulegium infusion seems to provide bioactive compounds able to maintain a proper antioxidant balance in gastrointestinal cells.
Mentha pulegium L., commonly known as pennyroyal, is an herbaceous species within the Lamiaceae family and, similarly to the other mints, is strongly aromatic with a characteristic menthol flavor, being used in various culinary preparations.3 In phytotherapy, the infusion of the flowered aerial parts and the essential oil are used to treat and prevent diverse gastrointestinal disorders like lack of appetite, intestinal cramps, low intestinal motility, flatulence and dyspepsia, and to alleviate cold and flu related symptoms.4 However, the possible mechanisms underlying M. pulegium therapeutic applications continue to be unknown.
Different activities, such as insecticidal,5 neuroprotective,6 anti-acetylcholinesterase,7 antioxidant and anti-genotoxic,8,9 have already been reported for this species. Most of the existing works focused on M. pulegium essential oil or alcoholic extracts. In fact, the chemical profile of the essential oil is well established.10
As for the phenolic profile, data is scarce and lacks consistency, as most of the studies available are directed to the quantification of total phenolics and not to their structural characterization, leaves being the most studied material. Phenolic acids, such as caffeic, vanillic, ferulic, rosmarinic and lithospermic, and the flavonoids luteolin, apigenin, naringenin, (+)-catechin, diosmetin 7-O-rutinoside, jaceosidin, pectolinarigenin and pedalitin are some examples of the phenolic compounds already identified in M. pulegium alcoholic extracts.11–16
Dietary intake of polyphenols has revealed to provide innumerous benefits to the pathophysiological development of neurodegenerative and cardiovascular diseases, diabetes, osteoporosis and cancer, probably due to their antioxidant action.17 The combination of these exogenous antioxidants with the endogenous ones, like reduced glutathione (GSH) and GSH-related enzymes, provides a cellular environment able to oppose against adverse oxidative stress, caused either by the normal cellular activities or xenobiotics.18
Our work aimed at the first phenolics characterization of M. pulegium aqueous extract and to establish a correlation between the chemical composition and the physiologic and toxicological responses of two cellular lines, human colon carcinoma cells (Caco-2) and human gastric carcinoma cells (AGS), representative of the human gastrointestinal tract, under quiescent conditions and subjected to oxidative stress induced by tert-butyl hydroperoxide (t-BHP).
Compoundsa | Regression equation | R2 | Range of concentrations (mg mL−1) | LODb (mg mL−1) | LOQc (mg mL−1) |
---|---|---|---|---|---|
a Caffquin: caffeoylquinic; Ac: acid; Lut: luteolin; Apig: apigenin; Gluc: glucoside.b Limit of detection.c Limit of quantification. | |||||
5-Caffquin Ac | 7.2 × 108x − 3.1 × 106 | 0.994 | 1.2 × 10−1 − 1.5 × 10−2 | 4.3 × 10−4 | 1.3 × 10−3 |
Caffeic Ac | 1.5 × 109x + 7.5 × 106 | 0.992 | 1.6 × 10−1 − 8.0 × 10−3 | 6.4 × 10−4 | 1.9 × 10−3 |
Ferulic Ac | 1.4 × 109x + 6.2 × 106 | 0.994 | 1.3 × 10−1 − 6.0 × 10−3 | 4.7 × 10−4 | 1.4 × 10−3 |
Rosmarinic Ac | 8.2 × 108x + 6.1 × 106 | 0.994 | 2.1 × 10−1 − 1.1 × 10−2 | 1.3 × 10−3 | 1.4 × 10−3 |
Lut-7-Gluc | 4.6 × 108x + 2.0 × 106 | 0.999 | 1.20 × 10−1 − 6.0 × 10−3 | 1.1 × 10−4 | 3.4 × 10−4 |
Apig-7-Gluc | 8.5 × 108x − 1.5 × 106 | 0.997 | 1.4 × 10−1 − 1.7 × 10−2 | 4.3 × 10−4 | 1.3 × 10−3 |
The HPLC-DAD-ESI/MSn analysis of the aqueous extract of M. pulegium at 330 nm (Fig. 1) showed the presence of diverse compounds, the majority of them with an ultra violet (UV) spectrum characteristic of cinnamoyl derivatives (1–3, 7, 11–13 and 15) (Table 2). Compounds 1 and 3 exhibited the deprotonated molecular ion at m/z 353 (caffeoylquinic acid) but different MS fragmentations, corresponding to 3-caffeoylquinic acid and 4-caffeoylquinic acid, respectively (Table 2), according to that reported before.23 Compound 2, with [M − H]− ion at m/z 311 and MS fragmentation showing the loss of 132 amu (pentosyl moiety) originating the ion at m/z 179 ([caffeic acid − H]−), can possibly be labelled as caffeoylpentoside. The MS2 of compound 7 presented losses of −162 (hexosyl moiety) and −(162 + 18), corresponding to an interglycosidic linkage, and of −(162 + 132) (hexopentosyl moiety) over a caffeic acid (179, [caffeic acid − H]−) (Table 2). Moreover, its MS3[(M − H) → (M − H − 162)]− was similar to the MS2 of compound 2; as so, compound 7 should be a derivative of compound 2 with an additional hexose over the pentose, being here characterized as a caffeoyl hexosylpentoside. Compound 12 exhibited an UV spectrum similar to that of 7. Both the deprotonated molecular ion and the fragmentation pattern of compound 12 differed from those of compound 7 in 14 amu: since [feruloyl acid − H]− (193 amu) was observed instead of the ion at m/z 179 [caffeic acid − H]−, compound 12 was identified as feruloyl hexosylpentoside.
Compoundsb | Rt (min) | UV (nm) | [M − H]−, m/z | MS2[M − H]−, m/z (%) | MS3, m/z (%) | |
---|---|---|---|---|---|---|
a Main observed fragments. Other ions were found but they have not been included.b Ac: acid; Caff: caffeoyl; Fer: feruloyl; Quin: quinic; Pent: pentosyl; Hex: hexosyl; Gluc: glucuronoyl; Lut: luteolin; Apig: apigenin; Chrys: chrysoeriol; Diosmt: diosmetin; Salv: salvianolic; Rosm: rosmarinic; Lith: lithospermic; Rut: rutinoside.c MS3(537 → 359): 197(40), 179(40), 161(100).d MS3(535 → 359): 197(20), 179(35), 161(100). | ||||||
1 | 3-CaffQuin Ac | 3.1 | 300sh, 328 | 353 | 191(100), 179(45) | |
2 | CaffPent | 4.0 | 300sh, 328 | 311 | 179(65), 149(100) | |
3 | 4-CaffQuin Ac | 4.9 | 300sh, 328 | 353 | 173(100) | |
4 | Salv Ac H | 10.3 | 252, 286, 314sh, 344 | 537 | 493(15), 339(100) | 295(80), 229(100) |
5 | Lut-7-Rut | 11.1 | 256, 266sh, 348 | 593 | 285(100) | |
6 | Lut-7-Gluc | 12.0 | 256, 266sh, 348 | 461 | 285(100) | |
7 | CaffHexPent | 12.3 | 300sh, 328 | 473 | 311(100), 293(60), 179(10) | 179(50), 149(100) |
8 | Salv Ac E isomer | 13.3 | 256, 284, 316, 348 | 717 | 519(100), 475(35) | 475(100) |
9 | Chrys/Diosmt-7-Rut | 14.4 | 256, 268sh, 348 | 607 | 299(100), 284(30) | |
10 | Apig-7-Gluc | 14.8 | 267, 337 | 445 | 400(10), 269(100) | |
11 | Rosm Ac | 15.0 | 300sh, 328 | 359 | 197(30), 179(40), 161(100) | |
12 | FerHexPent | 16.1 | 300sh, 328 | 487 | 325(100), 307(57), 293(85), 193(30) | 193(100), 149(4) |
13 | Lith Ac | 17.1 | 298sh, 325 | 537 | 493(100), 359(35)c | 359(100) |
14 | Salv Ac C isomer | 19.2 | 262, 320 | 491 | 311(100) | |
15 | Fer Rosm Ac | 20.0 | 300sh, 328 | 535 | 359(90), 177(100)d |
With a similar UV spectrum (Table 2) and a deprotonated molecular ion at m/z 359, compound 11 presented in its MS2 fragmentation losses of 3,4-dihydroxyphenyllactic acid (danshensu (DSS), −198 amu), caffeic acid (−180 amu) and caffeic acid − 18 (−162 amu), with formation of ions at m/z 161, 179 and 197, respectively. These are characteristic of dimers, trimers and tetramers of caffeic acid.24 This fragmentation pattern is in agreement with the one reported for rosmarinic acid,25 and the co-elution of the extract with a standard allowed the confirmation of compound 11 as rosmarinic acid.
Compound 15 also displayed a similar UV spectrum (Table 2) and its MS fragmentation showed a loss of 176 amu (feruloyl moiety), with formation of the ion at m/z 359. As the fragmentation (MS3[535 → 359]−) observed matched with the one of rosmarinic acid, compound 15 was tentatively identified as a feruloyl rosmarinic acid derivative.
Compounds 4, 13 and 14 are trimers. Compound 4 presented [M − H]− ion at m/z 537, losses of DSS (−198, m/z 339 base peak) and of a carbonyl group (−44) coincident with the fragmentation observed for salvianolic acid H (SAH).23 In addition, its UV spectrum, different from those of the other caffeic acid derivatives above described, has already been reported.26
As for compound 13, an isomer of compound 4, it was observed a characteristic UV spectrum of caffeic acid derivatives (Table 2). Both the similar MS fragmentation, showing a base peak corresponding to the loss of a carbonyl group (−44, m/z 493), and chromatographic mobility compared to rosmarinic acid were reported for lithospermic acid;24 therefore, it was tentatively labelled as lithospermic acid. Compound 14, exhibited the same deprotonated molecular ion of salvianolic acid C (m/z 491), also with the same chromatographic behavior, eluting at the end of the chromatogram. However, the loss of the DSS (−198) fragment with formation of the base peak reported before was not observed;24 instead, the fragmentation originated the base peak at m/z 311 by loss of a caffeic acid moiety (−180), as reported previously,26 either caused by different analytical conditions or by the presence of an isomer. Thus, it was tentatively labelled as salvianolic acid C isomer.
Compound 8 is a tetramer ([M − H]− at m/z 717). Its MS fragmentation showed the loss of DSS, giving rise to the base peak (m/z 519), as reported for the tetramers salvianolic acids E, B and L.27 Nevertheless, taking into account its lower chromatographic mobility, compared with that of rosmarinic acid, and strong resemblance with the fragmentation pattern and UV spectrum of a salvianolic acid E isomer described before,26 compound 8 may correspond to this molecule.
Other compounds with UV spectra of cinnamoyl derivatives were noticed at Rt 6.0 and 15.3 min (Ac, Fig. 1), though their structures could not be determined.
Finally, four other peaks (5, 6, 9 and 10) found in trace amounts and with UV and MS spectra of flavonoids were observed. Compounds 5, 6 and 9 presented an UV spectrum characteristic of flavones with di-substitution at the B ring (Table 2). The MS fragmentation of compounds 5 and 6 showed the ion at m/z 285 ([luteolin − H]−) as base peak, thus being identified as luteolin derivatives. Compound 5 fragmentation, with loss of 308 amu (rhamnohexosyl moiety) and without ions characteristic of interglycosyl linkage cleavage, suggested the presence of a rhamnosyl(1→6)hexoside linkage. As so, it was tentatively labelled as luteolin 7-O-rhamnosyl(1→6)glucoside (luteolin 7-O-rutinoside). Moreover, the loss of 176 amu (glucuronoyl moiety) by fragmentation of compound 6 led to its identification as luteolin 7-O-glucuronide. As it happened for compound 5, in the MS fragmentation of compound 9 it was observed the loss of 308 amu, resulting in the formation of the deprotonated aglycone ion (m/z 299), pointing to a methyl-luteolin, probably chrysoeriol (5,7,4′-OH-3′-OMe-flavone, 3′-Me-luteolin) or diosmetin (5,7,3′-OH-4′-OMe-flavone, 4′-Me-luteolin). Thus, compound 9 was tentatively labelled as chrysoeriol/diosmetin 7-O-rutinoside. Compound 10 was identified as apigenin 7-O-glucuronide due to the UV spectrum characteristic of an apigenin derivative (Table 2) and because the fragmentation of its deprotonated molecular ion ([M − H]−, 445) resulted in the deprotonated molecular ion of apigenin ([apigenin − H]−, 269), the base peak, by loss of a 176 amu fragment (glucuronoyl moiety).
With the exceptions of rosmarinic and lithospermic acids and of diosmetin 7-O-rutinoside, all the other twelve compounds are identified for the first time in M. pulegium.
Compoundsa | mg g−1 Lyophilized infusionb | |
---|---|---|
a Ac: acid; Caff: caffeoyl; Fer: feruloyl; Quin: quinic; Pent: pentosyl; Hex: hexosyl; Gluc: glucuronoyl; Lut: luteolin; Apig: apigenin; Chrys: chrysoeriol; Diosmt: diosmetin; Salv: salvianolic; Rosm: rosmarinic; Lith: lithospermic; Rut: rutinoside.b Results are expressed as means ± standard deviations of three independent determinations.c Sum of the quantified compounds. | ||
1 | 3-CaffQuin Ac | 4.16 ± 0.05 |
2 | CaffPent | 2.44 ± 0.02 |
3 | 4-CaffQuin AC | 2.64 ± 0.04 |
4 | Salv Ac H | 7.60 ± 0.76 |
5 | Lut-7-Rut | 0.64 ± 0.02 |
6 | Lut-7-Gluc | 1.40 ± 0.06 |
7 | CaffHexPent | 16.20 ± 1.36 |
8 | Salv Ac E isomer | 1.88 ± 0.12 |
9 | Chrys/Diosmt-7-Rut | 3.40 ± 0.13 |
10 | Apig-7-Gluc | 1.32 ± 0.09 |
11 | Rosm Ac | 66.08 ± 5.19 |
12 | FerHexPent | 3.48 ± 0.10 |
13 | Lith Ac | 7.52 ± 0.69 |
14 | Salv Ac C isomer | 1.84 ± 0.03 |
15 | Fer Rosm Ac | 2.24 ± 0.07 |
Σc | 122.92 |
As for the identified flavonoids, chrysoeriol/diosmetin 7-O-rutinoside (compound 9) was the most abundant one (3%).
Concentration-dependent responses were observed, although total scavenging activity was not reached for ˙NO radical, due to insolubility interference of high infusion's concentrations. The EC50 values obtained were 23, 39 and 226 μg mL−1 against DPPH˙, O2˙− and ˙NO radicals, respectively (data not shown). A similar result was obtained in a previous work, in which the hot water extract revealed to be very effective against DPPH˙.10 As for the other radicals, no previous results were found. As referred above, phenolic compounds are extremely effective in neutralizing radical species. Therefore, the results here obtained can be, at least partially, explained by the phenolic content of the tested infusion (Table 3).
It is well known that a homeostatic cell environment is necessary to maintain proper physiological functions. Dietary polyphenols intake may display an important role on balancing potential toxic environments, caused by a vast number of free radicals: reactive oxygen species (ROS), including either oxygen radicals like superoxide (O2˙−), hydroxyl (˙OH), and peroxyl (RO˙2) radicals, or non-radical oxidizing agents, such as hydrogen peroxide (H2O2) and hypochlorous acid (HOCl), easily converted into radicals; reactive nitrogen species (RNS) and reactive sulphur species (RSS), which are formed by thiols' reaction with ROS. Besides the antioxidant activity, it is accepted that these compounds can display other combined complex bioactivities. A deeper understanding over the ability to activate phase II enzymes, to act as anti-inflammatory agents, and to interact with the intestinal flora will certainly explain and sustain the importance of a phenolic-rich diet to prevent diverse human pathologies.29,30
A preliminary experiment was conducted in order to assess the range of concentrations for which the exposure to the infusion was not able to affect cellular viability (Fig. 2), since phenolic compounds can exert dose-dependent cytotoxicity.31
When comparing the two cell lines, AGS cells demonstrated to be more sensitive, since viability started to significantly decrease when exposed to 0.31 mg mL−1 of infusion, while for Caco-2 this happened only at 1.25 mg mL−1 (Fig. 2). Moreover, the results obtained by the MTT reduction assay were more expressive than the ones by LDH leakage for both cell lines, thus suggesting that mitochondrial damage happens prior to membrane's damage.
The following step was to evaluate the potential of M. pulegium infusion to protect the cells against the toxicity caused by t-BHP. This hydroperoxide is frequently applied to induce oxidative stress, the toxicity to hepatocytes being a result from either the metabolism through cytochrome P450 or reduction of GSH.32 Thus, several biomarkers can be monitored, like the increased levels of alanine transaminase, aspartate aminotransferase, LDH and malondialdehyde, and also GSH depletion.32
In this work, cells were treated with M. pulegium infusion for 24 h prior to t-BHP exposure (0.5 mM, 6 h). Cellular viability was again determined by MTT reduction and LDH leakage assays (Fig. 3). The infusion clearly exerted a dose-dependent protective effect in the MTT reduction assay. In fact, total protection, when compared to controls, was almost achieved with the infusion at the concentrations of 1.25 and 0.63 mg mL−1 for Caco-2 and AGS cells, respectively (Fig. 3), despite that at this concentrations the infusion induced cytotoxicity by itself (Fig. 2).
Hence, it can be assumed that from the pre-treatment with the infusion and post exposure to the toxicant did not result a synergic toxic effect.
Contrarily to the expected, no increase of LDH leakage was observed in either cell lines (Fig. 3). In a previous work, using LDH leakage as a biomarker of necrosis and DNA fragmentation as a biomarker of apoptosis, it was reported that a concentration of t-BHP of 0.4–0.5 mM provides a transition point, below which apoptosis is favored and beyond which necrosis is favored.33 Taking this into consideration, it can be inferred that for the experimented t-BHP exposure conditions (0.5 mM, 6 h) cell death was triggered by apoptosis, thus not being observed the increase of extracellular LDH, which is related with cellular membrane damage.
The phenolic composition of M. pulegium infusion here reported (Table 2) can be implicated in the antioxidant protective effect observed. Since apoptosis can be induced by a ROS-dependent mitochondrial pathway, phenolic compounds may also display a protective role by reducing toxic levels of reactive species.34 Other flavonoids, like rutin and quercetin, also tested in a similar pre-treatment experimental model, were very effective at preventing Caco-2 DNA damage induced by t-BHP.35
The authors attributed such results to the metal iron chelating and free radical scavenging properties of those compounds. With HepG2 cells, but using a co-exposition model, it was demonstrated that other phenolics like luteolin, quercetin, luteolin 7-O-glucoside, caffeic and rosmarinic acids also protected the cells against t-BHP toxicity, by preventing lipid peroxidation and GSH depletion.36
This known regulator mechanism of cellular resistance to oxidants may ultimately lead to the enhancement of the γ-glutamylcysteine synthetase enzyme transcription, the rate limiting enzyme responsible for glutathione synthesis, which is known to be more active in response to cellular treatments with phenolic compounds, leading to de novo synthesis.18
As expected, after t-BHP exposure, higher GSSG levels were observed when compared to the respective control, and it was demonstrated that the infusion was able to prevent GSH oxidation (Fig. 4).
Taking together these results with the ones obtained for the MTT reduction assay (Fig. 3), it can be noticed that the infusion prevented GSH oxidation even at concentrations at which it was not able to protect against the induced toxicity (0.04 to 0.16 mg mL−1). As so, this mechanism is certainly important, but not the only one behind the protective effect observed. No statistical significance was found for the direct action of the infusion over the GSSG variation levels (Fig. 4).
In contrast, under the same experimental conditions, the infusion was not able to exert the same effect in AGS cells (Fig. 4). GSx levels were not significantly altered by either the infusion or toxicant exposure. However, for the concentration of 0.31 mg mL−1, a significant decrease of the GSSG levels was observed (Fig. 4), in accordance with the verified viability increase (Fig. 3). The results indicate that for AGS cells, t-BHP reduction by GSH was not the most relevant toxicity mechanism. Nevertheless, for both cell lines, taking into account the possible toxicity mechanisms exerted by t-BHP and the antioxidant potential of phenolic compounds, it is possible that the cellular protection was also achieved either by the activity of glutathione peroxidase (GPx), leading to the conversion of the aggressor into a less toxic alcohol,38 by chelation of metallic ions, namely iron that proved to be essential for t-BPH-induced toxicity,39 by down-regulation of the apoptotic ROS-dependent mitochondrial pathway,34 or even by high level of DNA repaired synthesis that demonstrated to be involved in the protection mediated by pretreatment with the flavonoid baicalin.40
To sustain whether the phenolic compounds present in M. pulegium infusion were able to enter into the cells, a DPBA staining technique was applied. Nuclear staining with DAPI was also used to check nuclear morphology. Based on the phenolic profile here reported, phenolic acids were the major represented class, rosmarinic acid being the one at the highest concentration (Table 3). So, in addition to the positive apigenin control (25 μM), as described in a previous work,43 a rosmarinic acid control (25 μM) was also tested. The results showed that after 24 h of exposure to the infusion, the phenolic compounds could yet be targeted inside Caco-2 and AGS cells (Fig. 5).
Based on the positive controls, the detected fluorescence was derived by DPBA linkage to flavonoids and phenolic acids (Fig. 5). As advanced in a previous work with leukemia cells,43 for the assayed conditions, it is possible that when incubated with apigenin, AGS and Caco-2 cells may undergo an apoptotic process in which chromatin condensation and vacuolization are clearly observed (Fig. 5).
As for rosmarinic acid control, in comparison to the respective negative control (cells and DAPI) some slight morphological alterations can be observed, but in a smaller scale when compared to apigenin (Fig. 5). It has been reported that despite its antioxidant properties, rosmarinic acid can also act as antiproliferative and as an apoptotic inducer agent.44–46 The viability assays did not demonstrate cellular toxic effects for the experimented infusion concentrations (Fig. 2). However, nuclear morphology alterations may be earlier visible than other toxicological end-points.47
This work also sustains that the phenolic compounds are able to pass through the cellular membrane, even without prior hydrolyses.48 At the incubation conditions no chemical hydrolyses occurs: the compounds present are O-heterosides, requiring very acid pH conditions and high temperature in order to break the link between the sugar moieties and the aglycones.
Furthermore, no enzymes were present in the culture medium nor added to it. As so, the compounds could only have been taken upon the cell on their natural forms. It is known that when compared with the aglycones, glycosylated compounds are able to enter the cell, even if it occurs in a smaller scale.22
More important, since the protective effect was observed under a pre-treatment model, the phenolic compounds were still inside the cells when exposed to t-BHP and the 24 h incubation period may have enhanced the induction of Nrf2/ARE-dependent transcription. Besides GSH defenses, phenolic compounds might also act directly over the toxicant, and down-regulate the apoptotic ROS-dependent mitochondrial pathway.
Footnote |
† Ferreres and Bernardo are co-first authors on this work. |
This journal is © The Royal Society of Chemistry 2015 |