Chemical characterization and bioactive properties of two aromatic plants : Calendula o ffi cinalis L . ( fl owers ) and Mentha cervina L . ( leaves )

The chemical composition and bioactive properties of two plants (Calendula officinalis L. and Mentha cervina L.) were studied. Their nutritional value revealed a high content of carbohydrates and low fat levels, and very similar energy values. However, they presented different profiles in phenolic compounds and fatty acids; C. officinalis presented mainly glycosylated flavonols and saturated fatty acids, while M. cervina presented mainly caffeoyl derivatives and polyunsaturated fatty acids. M. cervina showed the highest concentration of phenolic compounds while C. officinalis presented higher amounts of sugars, organic acids and tocopherols. The highest antioxidant and cytotoxic activities were obtained for the hydromethanolic extract of M. cervina, which presented the lowest values of EC50 and exhibited cytotoxicity against the four tumor cell lines tested. Infusions showed no cytotoxicity for the tumor cell lines, and none of the extracts showed toxicity against non-tumor cells. This study contributes to expand the knowledge on both natural sources and therefore their use.


Introduction
Since ancient times, plants have been used as food and as medicine.Traditional medicine systems have used them for different therapies, some of them are still in use today, and have led to some important drugs.At present, natural products and their derivatives represent more than 50% of all drugs in clinical use.Nowadays, the use of traditional medicine is increasing, as more and more consumers believe that the use of medicinal plants could contribute to their health and wellness.These circumstances increase the interest in searching natural products which could lead to new drugs, nutraceuticals and functional foods.All in all, it is very important to ensure the quality, effectiveness and safety of these products, by toxic and antigenotoxic dose-dependent, 17 antioxidant 4,[18][19][20] and antitumoral effects. 7,21,22entha cervina L. (Lamiaceae) also known as Hart's pennyroyal, is an aromatic herb, found mainly in Eurasia and Africa.This species of mint grows on edges of flooded areas, sometimes temporarily and has been cultivated in Central Europe since the sixteenth century, used as a medicinal herb in part because of its fine flavor. 23It is used traditionally as a food seasoning, mainly in fish recipes, fish soup, together with M. pulegium L. or as a substitute.It has also been used for its medicinal properties in the prevention of various gastric disorders and inflammation of the respiratory tract, and its essential oil has industrial applications in food conservation. 24,25he chemical composition of M. cervina essential oil has been reported to be constituted mainly by monoterpenoids ( pulegone, isomenthone, menthone and limonene) with antibacterial and antifungal activities, resulting in an alternative of other mints for therapeutic purposes because of its lower level of pulegone, a terpenoid ketone which is toxic to the liver. 25The total phenolic content and total antioxidant capacity of M. cervina aqueous extract have been reported, and seven phenolic compounds have been identified ( protocatechuic acid, p-coumaric acid, caffeic acid, chlorogenic acid, epicatechin, orientin and rutin). 26n the present work, C. officinalis and M. cervina were chemically characterized regarding their nutritional/energy values, free sugars, organic acids, fatty acids and tocopherols of the dry plants and their infusions, as commonly consumed preparations.Furthermore, the phenolic compounds and the bioactive properties (antioxidant and cytotoxic properties for tumor and non-tumor cells) of the hydromethanolic extracts and infusions of these plants were studied and compared.To the best of author's knowledge, the available data about the phytochemical characterization and bioactivity of these plants are limited, especially for M. cervina.

Plant material and preparation of the extracts
C. officinalis (air-dried flowers) and M. cervina (air-dried leaves) samples were purchased from two companies, Soria Natural® from Soria, Spain, and Cantinho das Aromáticas® from Vila Nova de Gaia, Portugal, respectively.Both companies have their own organically grown crops.Each sample was reduced to a fine dried powder (20 mesh) and stored in a desiccator, protected from light, until further analysis.
To prepare the infusions, each sample (1 g) was added to 200 mL of boiled distilled water and kept for resting at room temperature for 5 min followed by subsequent filtration through a Whatman No. 4 paper.
For hydromethanolic extract preparation, each sample (1 g) was extracted by stirring in 30 mL of methanol/water (80 : 20 v/v, at 25 °C at 150 rpm) for 1 h and subsequently filtered through a Whatman paper No. 4. The residue was then extracted with an additional portion of 30 mL of the hydro-methanolic mixture.The combined extracts were evaporated under reduced pressure (rotary evaporator Büchi R-210, Flawil, Switzerland) until the complete removal of methanol, and afterwards the aqueous phase was frozen and lyophilized (FeeeZone 4.5, Labconco, Kansas City, MO, USA).

Standards and reagents
Acetonitrile (99.9%), n-hexane (95%) and ethyl acetate (99.8%) were HPLC grade and obtained from Fisher Scientific (Lisbon, Portugal) and the other solvents used were of analytical grade and purchased from common sources.Water was obtained from a Millipore Direct-Q purification system (TGI Pure Water Systems, Greenville, SC, USA).

Chemical characterization of the plant dry material and infusions
2.3.1.Nutritional and energy values.Ash, proteins, fat and carbohydrate contents ( proximate composition) were analysed in the samples (dry plant and infusions), through standard procedures. 27To estimate the crude protein content (N × 6.25) a macro-Kjeldahl method was applied; crude fat was determined by using a Soxhlet apparatus with petroleum ether; ash content was determined by incineration at 600 ± 15 °C and total carbohydrates were calculated by difference.The energy value was calculated according to the following equation: Energy (kcal) = 4 × (g protein + g carbohydrate) + 9 × (g fat).For infusion, total carbohydrates were calculated on the basis of total free sugars (section 2.3.2) and the energy value was calculated taking into account those results.
2.3.2.Free sugars.Free sugars were determined by high performance liquid chromatography coupled to a refraction index detector (HPLC-RI; Knauer, Smartline system 1000, Berlin, Germany), as previously described by the authors. 28dentification of sugar was made by comparing the relative retention times of sample peaks with standards (D(-)-fructose, quantification was based on the RI signal response of each standard, using the internal standard (IS, melezitose) method or the external standard method for infusions, and by using calibration curves obtained from the commercial standards of each compound.Results were expressed in g per 100 g of dry weight or in g per 100 mL of infusion.
2.3.3.Organic acids.Organic acids namely oxalic, quinic, malic, ascorbic and citric acids were determined following a procedure previously described by Barros et al. 28 and the analysis was performed by ultra-fast liquid chromatography coupled to photodiode array detection (UFLC-PDA; Shimadzu Corporation, Kyoto, Japan), using 215 nm and 245 nm (for ascorbic acid) as the preferred wavelengths; the quantification was performed by comparison of the area of the peaks recorded at the corresponding wavelength with calibration curves obtained from the commercial standards of each compound.The organic acids found were quantified by comparison of the area of their peaks with the calibration curves obtained from the commercial standards of each compound: oxalic acid (y = 9 × 10 6 x + 377 946; R 2 = 0.994); quinic acid (y = 612327 x + 16 563; R 2 = 1); malic acid (y = 863548 x + 55 591; R 2 = 0.999); ascorbic acid (y = 1 × 10 8 x + 751 815; R 2 = 0.999) and citric acid (y = 1 × 10 6 x + 16 276; R 2 = 1).The results were expressed in mg per 100 g of dry weight or in mg per 100 mL of infusion.
2.3.4.Tocopherols.Tocopherols were determined following a procedure previously described by Barros et al., 28 using a HPLC system (Knauer, Smartline system 1000, Berlin, Germany) coupled to a fluorescence detector (FP-2020; Jasco, Easton, USA) programmed for excitation at 290 nm and emission at 330 nm; the identification was performed by chromatographic comparisons with authentic standards (α-, β-, γ-, and δ-isoforms), while the quantification was based on the fluorescence signal response of each standard, using the IS (tocol) method and by using calibration curves obtained from the commercial standards of each compound.The results were expressed in μg per 100 g of dry weight or μg per 100 mL of infusion.
2.3.5.Fatty acids.Fatty acids were determined in the crude lipid fraction, after a trans-esterification process, by gas-liquid chromatography with flame ionization detection (GC-FID; DANI model GC 1000 instrument, Contone, Switzerland) as previously described by Barros et al. 28 Fatty acid identification was made by comparing the relative retention times of FAME peaks from samples with standards.The results were recorded and processed using Clarity Software (DataApex, Prague, The Czech Republic) and expressed as the relative percentage of each fatty acid.

Phenolic compound characterization in the hydromethanolic extracts and infusions
Chromatographic analyses were carried out on a Spherisorb S3 ODS-2 C 18 column (3 μm, 4.6 × 150 mm, Waters, Milford, MA, EUA), thermostatted at 35 °C.The mobile phase consisted of two solvents: (A) 0.1% formic acid in water and (B) acetonitrile, using a gradient as follows: 15% B for 5 min, 15% B to 20% B over 5 min, 20-25% B over 10 min, 25-35% B over 10 min, 35-50% B for 10 min, and re-equilibration of the column, with a flow rate of 0.5 mL min −1 and the injection volume 100 µL.The spectral data for all peaks were recorded at 280 and 370 nm as preferred wavelengths.The HPLC-DAD-MS/ESI analyses were carried out using a Hewlett-Packard 1100 series chromatograph (Hewlett-Packard 1100, Agilent Technologies, Santa Clara, CA, USA) equipped with a diode-array detector (DAD) and a mass detector (API 3200 Qtrap, Applied Biosystems, Darmstadt, Germany) connected to the HPLC system via the PDA cell outlet. 29The identification of the different phenolic compounds was performed by comparison with available commercial standard compounds, or were tentatively identified using reported data from the literature.For quantitative analysis, a calibration curve for each available phenolic standard (caffeic acid, 5-O-caffeoylquinic acid, quercetin-3-Orutinoside, isorhamnetin-3-O-rutinoside, kaempferol-3-O-rutinoside, quercetin-3-O-glucoside, isorhamnetin-3-O-glucoside, rosmarinic acid) was constructed based on the UV signal or when no commercial standard was available, a similar compound from the same phenolic group was used as a standard.The results were expressed in mg per g of extract or mg per mL of infusion.

Evaluation of bioactive properties of hydromethanolic extracts and infusions
2.5.1.In vitro antioxidant activity assays.Hydromethanolic extracts were redissolved in methanol/water (80 : 20 v/v) to the final concentration of 20 mg mL −1 , and infusions (5 mg mL −1 ) were further diluted to different concentrations to be subjected to the following assays.DPPH radical-scavenging activity (RSA) was evaluated by using an ELX800 microplate reader (Bio-Tek Instruments, Inc.; Winooski, VT, USA), and calculated as a percentage of DPPH discolouration using the formula: % RSA = [(A DPPH − A S )/A DPPH ] × 100, where A S is the absorbance of the solution containing the sample at 515 nm, and A DPPH is the absorbance of the DPPH solution.Reducing power was evaluated by the capacity to convert Fe 3+ into Fe 2+ , measuring the absorbance at 690 nm in the microplate reader mentioned above.Inhibition of β-carotene bleaching was evaluated though the β-carotene/linoleate assay; the neutralization of linoleate free radicals avoids β-carotene bleaching, which is measured by the formula: (β-carotene absorbance after 2 h of assay/initial absorbance) × 100.Lipid peroxidation inhibition in porcine brain homogenates was evaluated by the decrease in thiobarbituric acid reactive substances (TBARS); the color intensity of the malondialdehyde-thiobarbituric acid (MDA-TBA) was measured by its absorbance at 532 nm; the inhibition was calculated using the following formula: Inhibition ratio (%) = [(A − B)/A] × 100, where A and B were the absorbance of the control and the sample solution, respectively. 28The final results were expressed in EC 50 values (mg mL −1 ); sample concentration providing 50% of antioxidant activity or 0.5 of absorbance in the reducing power assay.Trolox was used as the positive control.
2.5.2.Cytotoxicity in tumor cell lines and in non-tumor primary cells.Hydromethanolic extracts (final concentration 8 mg mL −1 , redissolved in water) and infusions (5 mg mL −1 ) were further diluted to different concentrations to be subjected to in vitro antitumor activity and hepatotoxicity evaluation.
The human tumor cell lines used were: HeLa (cervical carcinoma), HepG2 (hepatocellular carcinoma), MCF-7 (breast adenocarcinoma) and NCI-H460 (non-small cell lung cancer).Each of the cell lines were plated in a 96-well plate, at an appropriate density (7.5 × 10 3 cells per well for MCF-7 and NCI-H460 and 1.0 × 10 4 cells per well for HeLa and HepG2) and were allowed to attach for 24 h.Afterwards, various extract concentrations were added to the cells and incubated for 48 h.Afterwards, cold trichloroacetic acid (TCA 10%, 100 μL) was used in order to bind the adherent cells and further incubated for 60 min at 4 °C.After the incubation period, the plates were washed with deionised water and dried and sulforhodamine B solution (SRB 0.1% in 1% acetic acid, 100 μL) was then added to each plate well and incubated for 30 min at room temperature.The plates were washed with acetic acid (1%) in order to remove the unbound SRB and then air dried; the bound SRB was solubilised with Tris (10 mM, 200 μL) and the absorbance was measured at 540 nm using an ELX800 microplate reader (Bio-Tek Instruments, Inc.; Winooski, VT, USA). 28The results were expressed in GI 50 values; the sample concentration that inhibited 50% of the net cell growth.Ellipticine was used as the positive control.
For hepatotoxicity evaluation, a freshly harvested porcine liver, obtained from a local slaughter house, was used in order to obtain the cell culture, designated as PLP2.The liver tissues were rinsed in Hank's balanced salt solution containing penicillin (100 U mL −1 ), streptomycin (100 µg mL −1 ) and divided into 1 × 1 mm 3 explants.A few of these explants were transferred to tissue flasks (25 cm 2 ) containing DMEM supplemented with fetal bovine serum (FBS, 10%), nonessential amino acids (2 mM), penicillin (100 U mL −1 ) and streptomycin (100 mg mL −1 ) and incubated at 37 °C under a humidified atmosphere (5% CO 2 ).The medium was changed every two days and the cell cultivation was continuously monitored using a phase contrast microscope.When confluence was reached, the cells were sub-cultured and plated in a 96-well plate (density of 1.0 × 10 4 cells per well) containing DMEM supplemented with FBS (10%), penicillin (100 U mL −1 ) and streptomycin (100 µg mL −1 ).The growth inhibition was evaluated using the SRB assay, previously described. 28The results were expressed in GI 50 values; the sample concentration that inhibited 50% of the net cell growth.Ellipticine was used as the positive control.

Statistical analysis
Three samples were used for each species and all the assays were carried out in triplicate.Results were expressed as mean values and standard deviation (SD) and analysis was performed through a Student's t-test with α = 0.05, using the SPSS v. 22.0 program.

Nutritional value and chemical characterization of C. officinalis and M. cervina dry material and infusions
The results of the nutritional and estimated energy values obtained in the dry plants and infusions of C. officinalis and M. cervina are shown in Table 1.Carbohydrates, calculated by difference for the dry plant, were the most abundant macronutrients and M. cervina showed the highest values, both in the infusions (0.05 g per 100 mL) and in dry material (86 g per 100 g dw).Mentha cervina also revealed a higher protein content (6 g per 100 g dw), while C. officinalis showed higher ash and fat levels (14 g per 100 g dw and 6 g per 100 g dw, respectively).The infusion prepared using both plants did not reveal the presence of fat, ash and proteins, therefore the energy value was calculated taking into account total carbohydrates, calculated by the total free sugar content.The energy value calculated for the dry plants (376 kcal per 100 g dw, on average) did not show significant differences between C. officinalis and M. cervina ( p > 0.05).
The composition of free sugars, organic acids and tocopherols of the dry plants and infusions is presented in Table 2.There is scarce information about C. officinalis, except regarding its use as a cosmetic ingredient 6 and its composition in water-soluble polysaccharides, 30 while no information about M. cervina was found.Five free sugars were identified in C. officinalis (xylose, fructose, glucose, sucrose and trehalose), while xylose could not be found in M. cervina.Calendula officinalis showed higher levels of fructose (5 g per 100 g plant dw, 19 mg per 100 mL infusion), sucrose (4 g per 100 g plant dw, 14 mg per 100 mL infusion), and total free sugars

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(12 g per 100 g plant dw, 49 mg per 100 mL infusion), while M. cervina gave higher levels of glucose (4 g per 100 g plant dw, 16 mg per 100 mL infusion).Regarding dry plants, no significant differences ( p > 0.05) were found in the content of trehalose (0.6 g per 100 g plant dw on average), although this sugar was not detected in the infusion of M. cervina.Oxalic, quinic, malic, citric and fumaric acids were identified in C. officinalis and M. cervina.The highest total content was found in C. officinalis (2830 mg per 100 g plant dw, 3.0 mg per 100 mL infusion), and the most abundant one was citric acid (963 mg per 100 g plant dw, 2.6 mg per 100 mL infusion).In the C. officinalis infusion, only oxalic, citric and fumaric acids were detected, while citric acid was the only organic acid identified in M. cervina, probably due to degradation of some of these compounds by heat during the preparation procedure.
Calendula officinalis presented three tocopherol isoforms (α, β and γ-tocopherols), while M. cervina presented only two (α and δ-tocopherols).In infusions, only α-tocopherol was detected, the low tocopherol concentration in infusion samples could also be due to the extraction procedure (water extraction), and not only due to thermal treatment during the extraction process.Calendula officinalis showed the highest concentration of tocopherols (23 mg per 100 g plant dw, 0.9 mg per 100 mL infusion) and α-tocopherol was the major isoform (19 mg per 100 g marigold plant dw; 2 mg per 100 mL Hart's pennyroyal plant).

Analysis of phenolic compounds in C. officinalis and M. cervina hydromethanolic extracts and infusions
The phenolic compounds found in C. officinalis and M. cervina infusions and hydromethanolic extracts are listed in Tables 4  and 5, and their HPLC profiles can be observed in Fig. 1.The studied samples presented completely different profiles; C. officinalis presented thirteen different compounds, mainly glycosylated flavonols, while M. cervina presented eleven compounds, mainly caffeoyl derivatives (caffeic acid dimers, trimers and tetramers).In the literature there are several studies regarding the phenolic composition of C. officinalis, 22,[31][32][33][34][35][36] while for M. cervina only one study was found. 26Nevertheless, the phenolic profile of the infusions, the most common form of consumption of this plant, are limited.
Isorhamnetin-3-O-rhamnosylrutinoside (typhaneoside; peak 6) and isorhamnetin-3-O-rutinoside (narcissin, peak 11) were the most abundant phenolics in the analyzed extracts and infusions of marigold (Table 5).These compounds were also reported as the main phenolic compounds in C. officinalis flowers. 35s mentioned above, the phenolic composition of M. cervina was characterised by the presence of caffeoyl derivatives, namely caffeic acid dimers, trimers and tetramers, compounds which have not been reported before in this species.To the best of our knowledge, the only study that reports on the phenolic composition of M. cervina was published by Politi et al., 26 who identified four phenolic acids ( protocatechuic acid, p-coumaric acid, caffeic acid and chlorogenic acid) and three flavonoids (epicatechin, orientin and rutin).The only common compound between the ones reported in that study and those detected herein was caffeic acid (compound 2′).This latter and trans-rosmarinic acid (compound 9′) were positively identified according to their retention times, mass and UV-vis characteristics by comparison with commercial standards.Compound 5′ ([M − H] − at m/z 521) yielded a fragment at m/z 359 (rosmarinic acid) from the loss of 162 mu (hexoside moiety), as well as other fragments identical to those observed for compound 9′, which allowed its tentative identification as rosmarinic acid hexoside.Furthermore, compound 8′ with similar characteristics to compound 9′ should correspond to a rosmarinic acid isomer that was tentatively identified as cis-rosmarinic acid.
ions at m/z 179 [caffeic acid-H] − and 135 [caffeic acid-CO 2 -H] − .Nevertheless, no definite structure could be matched to the molecular mass of the compound that remains as an unidentified caffeic acid derivative.Compound 3′ presented a pseudomolecular ion [M − H] − at m/z 537, the UV spectrum and fragmentation pattern being consistent with the caffeic acid trimer lithospermic acid A. This compound can easily lose the 8″-carboxyl group (−44 u) releasing a fragment at m/z 493 that further breaks down to form the fragment ions at m/z 313 and 295.However, peak 3′ showed a different retention time compared to lithospermic acid A, which is expected to elute later than trans-rosmarinic acid, as previously observed in other Lamiaceae analyzed in our laboratory. 29,38Other compounds with the same molecular weight are salvianolic acids H or I, although they showed different fragmentation patterns. 39,40A compound with similar characteristics was found in a sample of Melissa officinalis and identified as a lithospermic acid A isomer, 29 an identity that has been tentatively assumed for peak 3′ detected herein.Compounds 4′ and 6′ ([M − H] − at m/z 539) presented the same pseudomolecular ion and similar fragmentation pattern and UV spectra, coherent with those of yunnaneic acid D as described by Chen et al. 39 in Salvia miltiorrhiza, based on which they were identified as yunnaneic acid D isomers.Compound 7′ showed a pseudomolecular ion [M − H] − at m/z 719 and an MS 2 majority fragment at m/z 359 corresponding to [M − 2H] 2− ; these mass characteristics coincided with those of sagerinic acid, a rosmarinic acid dimer, reported by us in other plant samples. 29,38Finally, compounds 10′ and 11′ also presented the same pseudomolecular ion [M − H] − at m/z 493, which together with the characteristic fragment ions at m/z 313, 295 and 197 and UV spectra allowed assigning them as salvianolic acid A isomers, e.g., isosalvianolic acid A and salvianolic acid A, as previously described by Ruan et al. 40 Rosmarinic acid was the most abundant phenolic compound in M. cervina.This compound had not been identified in the only report previously published on the phenolic composition of this species, 26 which described a completely different phenolic profile.

Bioactivity of C. officinalis and M. cervina hydromethanolic extracts and infusions
The in vitro antioxidant and cytotoxic properties of C. officinalis and M. cervina, hydromethanolic extracts and infusions were evaluated, and the results are given in Table 6.The highest antioxidant activity was observed for M. cervina; its hydromethanolic extract showed the lowest EC 50 values in all the assays, except in the β-carotene bleaching inhibition, where C. officinalis infusion gave higher antioxidant activity.These differences could be related with the different phenolic profiles of each plant (Table 5).Whereas M. cervina showed phenolic acid derivatives as the major compounds, C. officinalis proved to have flavonoids as the main phenolic molecules.9][20] The results of the extracts studied revealed a DPPH radical scavenging activity lower than that of aqueous extracts and hydromethanolic extracts obtained by Ćetković et al. 19 (EC 50 : 0.30-0.90mg mL −1 ).All the publications confirmed the antioxidant capacity of C. officinalis, suggesting that many of its therapeutic activities are due to that capacity.
Regarding antitumor potential, the most promising results were obtained for the M. cervina hydromethanolic extract, which exhibited cytotoxicity against the four tested tumor cell lines, being more active against cervical carcinoma (HeLa, GI 50 = 223 µg mL −1 ).The hydromethanolic extract of C. officinalis revealed selectivity against cervical (HeLa, GI 50 = 256 µg mL −1 ) and hepatocellular (HepG2, GI 50 = 330 µg mL −1 ) carcinoma.The infusions of both plants did not show effects on the tumor cell lines, however, and none of the extracts revealed toxicity against non-tumor cells (PLP2).In contrast to the lack of studies with M. cervina in this regard, there are some reports that evaluate the antitumor activity of C. officinalis extracts and isolated compounds by using in vitro and in vivo models. 7,21,22The antitumor activity of triterpene glycosides isolated from marigold was shown by Ukiya et al., 7 and the results obtained by Matić et al. 22 on marigold infusion against HeLa (GI 50 = 750 µg mL −1 ) and other tumor cell lines, are consistent with the present study.
In summary, C. officinalis (marigold flowers) and M. cervina (Hart's pennyroyal leaves) contain phytochemicals that are of great interest due to their potential antioxidant and antitumor activities.Overall, the present study extends the knowledge of C. officinalis and provides innovative results for M. cervina regarding chemical characterization and bioactive properties, contributing to extend their use as functional ingredients, and for medical purposes.The antioxidant activity was expressed as EC 50 values, which means that higher values correspond to lower reducing power or antioxidant potential.EC 50 : extract concentration corresponding to 50% of antioxidant activity or 0.5 of absorbance in reducing power assay.Trolox EC 50 values: 41 µg mL −1 (reducing power), 42 µg mL −1 (DPPH scavenging activity), 18 µg mL −1 (β-carotene bleaching inhibition) and 23 µg mL −1 (TBARS inhibition).GI 50 values correspond to the sample concentration achieving 50% of growth inhibition in human tumor cell lines or in liver primary culture PLP2.Ellipticine GI 50 values: 1.42 µg mL −1 (HCT-15) and 2.06 µg mL −1 (PLP2).In each row, p < 0.05 means significant difference.

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Table 1
Nutritional and energy values of plant dry material and infusions of C. officinalis and M. cervina Published on 12 April 2016.Downloaded by Instituto Politecnico de Braganca on 30/11/2016 15:39:38.

Table 2
Chemical composition in free sugars, organic acids and tocopherols of plant dry material and infusions of C. officinalis and M. cervina Published on 12 April 2016.Downloaded by Instituto Politecnico de Braganca on 30/11/2016 15:39:38.

Table 3
Fatty acid composition in the dry material of C. officinalis and M. cervina

Table 4
Retention time (Rt), wavelengths of maximum absorption in the visible region (λ max ), mass spectral data and identification of phenolic compounds in C. officinalis and M. cervina hydromethanolic extracts and infusions