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Journal of Food Science and Technology logoLink to Journal of Food Science and Technology
. 2016 Dec 26;54(1):219–227. doi: 10.1007/s13197-016-2453-z

Phenolic profile, antioxidant activity and enzyme inhibitory activities of extracts from aromatic plants used in Mediterranean diet

Sandra Gonçalves 1, Elsa Moreira 1, Clara Grosso 2, Paula B Andrade 2, Patrícia Valentão 2, Anabela Romano 1,✉
PMCID: PMC5305718  PMID: 28242919

Abstract

The antioxidant and enzyme inhibitory properties of methanolic extracts from four aromatic plants used in traditional medicine and food [Calamintha nepeta (L.) Savi subsp. nepeta, Helichrysum italicum subsp. picardii Franco, Mentha spicata L. and Origanum vulgare subsp. virens (Hoffmanns. & Link) Bonnier & Layens] were evaluated. The extract from O. vulgare exhibited the strongest DPPH (IC50 of 4.65 ± 0.12 µg/ml) and ABTS (1479.56 ± 12.29 µmolTE/gextract) scavenging capacities, as well as the largest ferric reducing ability (1746.76 ± 45.11 µmolAAE/gextract). This extract also showed the highest total phenolic content (1597.20 ± 24.10 µmolGAE/gextract) and although HPLC–DAD analysis revealed rosmarinic acid as the main compound of the extract, other compounds seem to be involved in the antioxidant activity. Furthermore, the extract from H. italicum, which was found to be rich in caffeoylquinic and dicaffeoylquinic acids and in pinocembrin, showed the highest inhibitory potential against acetylcholinesterase, tyrosinase and α-glucosidase. Overall, the results obtained validate the usefulness of the studied plants as valuable sources of natural agents beneficial for human health.

Keywords: Antioxidants, Curry plant, Lesser calamint, Oregano, Phenolic compounds, Spearmint

Introduction

The demand for aromatic plants as sources of natural antioxidants has been increasing in the last years. Indeed, the use of aromatic plants is highly encouraged by health professionals nowadays in order to reduce the abusive addition of salt in the diet, or by presenting health benefits other than their antioxidant properties (Costa et al. 2015a). Furthermore, many aromatic plants are recognized as safe for human consumption (FDA 2014). Among bioactive compounds in aromatic plants, phenolics are one of the most important and probably the main candidates contributing to the claimed beneficial effects associated with these plants. They have been linked to a number of health benefits, including protection against cardiovascular diseases, cancer, diabetes mellitus (DM) and neurological diseases (Del Rio et al. 2013; Nikousaleh and Prakash 2016). With such a rich array of potential benefits, it is not surprising that phenolic compounds are being sought to replace synthetic antioxidants in the food supply (Fernandes et al. 2016). Natural antioxidants originating from plant sources became a wise option for the management of oxidative stress-related disorders as neurodegenerative diseases and DM (Maritim et al. 2003; Lee et al. 2010). Nowadays, there has been also much interest in the use of phytochemicals with enzyme inhibitory properties for the management of Alzheimer’s disease (AD), Parkinson’s disease (PD) and DM, because some drugs in use are reported to have side effects or low efficacy (Orhan et al. 2015; Zengin et al. 2015; Shanmugam et al. 2016).

Mediterranean diet includes the use of wild plants, such as herbs and aromatic species, harvested and eaten seasonally. The systematic investigation of such plants will help defining their precise pharmacological properties and to determine their value as functional foods or as a source of nutraceutical and pharmaceutical compounds. The knowledge about the health properties of those plants will also contribute to increase their importance as a part of the Mediterranean diet. The aim of this study was to obtain homogeneous and quantitative data on the antioxidant activity and inhibitory capacity against enzymes linked to AD, PD and DM of four wild aromatic plants widely used in traditional medicine and Mediterranean diet (Table 1), namely Calamintha nepeta (L.) Savi subsp. nepeta, Helichrysum italicum subsp. picardii Franco, Mentha spicata L. and Origanum vulgare subsp. virens (Hoffmanns. & Link) Bonnier & Layens. Additionally, the phenolic compounds present in the extracts were identified and quantified by high-performance liquid chromatography with diode-array detection (HPLC–DAD), and their relationships coupled to the biological properties discussed.

Table 1.

Local name, English common name, family, edible tissues and traditional medicinal uses of the four aromatic species studied

Plant species Local name English common name Family Edible tissues Traditional medicinal uses
Calamintha nepeta (L.) Savi subsp. nepeta Erva-das-azeitonas Lesser calamint Lamiaceae Leaves As expectorant and tonic, and to alleviate digestive disorders
Helichrysum italicum subsp. picardii Franco Perpétua-das-areias Curry plant Asteraceae Leaves To relieve inflammation and several skin diseases, including for wound healing
Mentha spicata L. Hortelã-comum Spearmint Lamiaceae Leaves To treat numerous digestive and respiratory problems
Origanum vulgare subsp. virens (Hoffmanns. & Link) Bonnier & Layens Oregão Oregano Lamiaceae Aerial parts As antitussive, expectorant, antiseptic and to alleviate digestive disorders
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Materials and methods

Chemicals and reagents

2,2′-Azinobis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) tablets, 2,2-diphenyl-1-picrylhydrazyl (DPPH), methanol, formic acid, trichloroacetic acid (TCA), K2S2O8, acetylthiocholine iodide (ATCI), 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB), acetylcholinesterase (AChE) (Electric-eel, EC 3.1.1.7, Type VIS), mushroom tyrosinase (EC 1.14.18.1), 3,4-dihydroxy-L-phenylalanin (L-DOPA), porcine pancreatic α-amylase (EC 3.2.1.1), yeast α-glucosidase (EC 3.2.1.20), p-nitrophenyl-α-D-glucopyranoside, starch, galanthamine hydrobromide, kojic acid, acarbose, quercetin, pinocembrin, caffeic acid and 3,4-dihydroxybenzoic acid were purchased from Sigma-Aldrich (St. Louis, MO, USA). Rosmarinic acid, quercetin-3-O-rutinoside, 5-O-caffeoylquinic acid, gallic acid, catechin, epicatechin, hesperetin-7-O-rutinoside and p-hydroxybenzoic acid were from Extrasynthèse (Genay, France). 3-O-Caffeoylquinic, 4-O-caffeoylquinic, 3,4-di-O-caffeoylquinic, 3,5-di-O-caffeoylquinic, 1,5-di-O-caffeoylquinic and 4,5-di-O-caffeoylquinic were purchased from Chengdu Biopurity Phytochemicals Ltd. (Schuan, China). Folin–Ciocalteu’s phenol reagent (F–C reagent), gallic acid, Na2CO3, NaCl and FeCl3 were acquired from VWR (Leuven, Belgium). Potassium ferricyanide, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) and butylated hydroxytoluene (BHT) were purchased from Acros Organics (Geel, Germany). Methanol Lichrosolv was from Merck (Darmstadt, Germany).

Plant material and extraction procedure

Randomly selected leaves of C. nepeta subsp. nepeta, H. italicum subsp. picardii, M. spicata and O. vulgare subsp. virens were collected from plants growing wild in the Algarve region (South Portugal). A representative sample of each plant was authenticated by JM Rosa Pinto from the herbarium of the University of Algarve (Faro, Portugal). The plant material was dried at 40 °C until constant weight (24–48 h) and powdered in a blender to achieve a mean particle size lower than 2 mm. Dried plant material was extracted twice by maceration with 80% methanol (1:20, w/v) during 24 h at room temperature and the resulting extracts were filtered through a 5–13 µm membrane. Finally, the extracts were concentrated to dryness in a rotary evaporator at 40 °C and under reduced pressure, and stored at −20 °C. The dried extract was later re-suspended in the appropriate solvent for each biological activity test, methanol in the case of DPPH assay and HPLC–DAD analyses, and the specific buffer (indicated below) used in each of the other assays.

HPLC–DAD analysis

The extracts (50 mg/ml) were analyzed on an analytical HPLC unit (Gilson), using a Spherisorb ODS2 column (4.6 × 250 mm, 5 µm particle size). The solvent system used was a gradient of water-formic acid (19:1) (A) and methanol (B), starting with 5% methanol and using a gradient of 15% B at 3 min, 25% B at 13 min, 30% B at 25 min, 35% B at 35 min, 45% B at 39 min, 45% B at 42 min, 55% B at 47 min, 75% B at 56 min, 100% B at 60 min, at a solvent flow rate of 0.9 ml/min. Detection was achieved with a Gilson DAD. Spectral data from all peaks were accumulated in the range 200–400 nm. Chromatograms were recorded at 280 nm for hydroxybenzoic acids, flavan-3-ols and flavanones, at 320 nm for hydroxycinnamic acids and at 350 nm for the other flavonoids. The data were processed on an Unipoint® System software (Gilson Medical Electronics, Villiers le Bel, France). The compounds in each extract were identified by comparing their retention times and UV–Vis spectra in the 200–400 nm range with authentic standards injected in the same conditions. Extracts were also spiked with the standards of the identified compounds in order to verify the identification. Phenolic compounds quantification was achieved by the absorbance recorded in the chromatograms relative to external standards, by using the following equation:

Cc=AcAst×Cst

where, C(c) and A(c) are the concentration and the area of the compound in the sample, respectively, and C(st) and A(st) are the concentration and the area of the standard, respectively.

Determination of the total phenolic contents

The total phenolic content of each extract was measured using a modified F–C colorimetric method (Ainsworth and Gillespie 2007). Briefly, 200 µl of 10% (v/v) F–C reagent was mixed with 100 µl of each extract in phosphate buffer (75 mM, pH 7.0). Then 800 µl of 700 mM Na2CO3 were added and the reaction was incubated at room temperature for 2 h. Instead of the plant extracts, gallic acid was used as a positive control or phosphate buffer as a negative control. Two hundred microliters were transferred from each reaction to a clear 96-well microplate (NUNC, Rochester, New York, USA) and the absorbance was measured at 765 nm. A standard curve was calculated using several gallic acid concentrations and the results were expressed as gallic acid equivalents per gram of extract (µmolGAE/gextract).

Antioxidant activity

DPPH free radical scavenging assay

The ability of the extracts to scavenge DPPH radicals was determined according to the procedure described by Soler-Rivas et al. (2000) with slight modifications. One hundred microliters of 90 µM DPPH methanolic solution was added to 10 µl of sample solution at different concentrations, and the mixture was diluted with 190 µl of methanol in a clear 96-well microplate. The extract was replaced with solvent for the control. After 30 min, the reduction of DPPH radicals was measured at an absorbance of 515 nm. BHT was used as the reference antioxidant in this assay. The radical scavenging activity was expressed in terms of the amount of sample necessary to decrease the initial absorbance by 50% (IC50).

ABTS•+ radical cation decoloration assay

The ABTS free radical-scavenging activity of each sample was determined as described by Re et al. (1999) and the results were expressed in terms of Trolox equivalent antioxidant capacity (TEAC). The 7 mM ABTS•+ stock solution was prepared using potassium persulfate as the oxidizing agent. The absorbance was determined at 734 nm, 1 min after mixing. The sample dilution diluted in phosphate buffer (75 mM, pH 7) that achieved 20–80% inhibition of the blank absorbance was used to calculate the TEAC values and the results were expressed as trolox equivalents per gram of extract (µmolTE/gextract).

Ferric reducing antioxidant power (FRAP)

The reducing properties of the extracts were determined using FeCl3 as described by Pulido et al. (2000) with some modifications. Briefly, 100 µl of plant extract was mixed with 250 µl sodium phosphate buffer (200 mM, pH 6.6) and 250 µl 1% K3[Fe(CN)6] and was incubated at 50 °C for 20 min. After the addition of 250 µl 10% TCA the mixture was centrifuged at 650 rpm for 10 min. Then 100 µl of the supernatant were mixed with 100 µl of water and 20 µl 0.1% FeCl3 in a 96-well microplate. Instead of the plant extracts, ascorbic acid was used as a positive control and phosphate buffer as a negative control. Reducing activity was measured by determining the absorbance at 700 nm and the results were expressed as ascorbic acid equivalents (µmolAAE/gextract).

Enzyme inhibitory activities

Acetylcholinesterase inhibition

The evaluation of AChE inhibitory activity was based on Ellman’s method (Ellman et al. 1961), using a 96-well microplate reader. Firstly, 3 mM DTNB, 15 mM substrate (ATCI), 100 mM phosphate buffer (pH 8.0) and extract at 10 mg/ml, buffer or galanthamine at 5 µg/ml (standard inhibitor) were mixed. Finally, AChE (0.28 U/ml) was added and the absorbance was read at 405 nm for 5 min. The reaction enzyme activity was calculated as a percentage of the velocities compared to that of the assay using buffer without any inhibitor. Inhibitory activity was calculated from 100 subtracted by the percentage of enzyme activity.

Tyrosinase inhibition

Inhibition of tyrosinase was determined using the modified dopachrome method as described by Masuda et al. (2005) Assays were conducted in 96-well microplates, an aliquot of the extracts at 10 mg/ml with 80 µl of phosphate buffer (pH 6.8), 40 µl of tyrosinase, and 40 µl of L-DOPA were mixed. The absorbance was measured at 475 nm and the tyrosinase inhibitory activity was expressed as percentage inhibition. Kojic acid (200 µg/ml) was used as the reference.

α-Amylase inhibition

α-Amylase inhibition assay was performed according to the procedure described by Kwon et al. (2008) with slight modifications. A total of 500 µl of plant extract at 10 mg/ml and 500 µl of 0.02 M sodium phosphate buffer (pH 6.9 with 0.006 M sodium chloride) containing porcine pancreatic α-amylase (0.5 mg/ml) were incubated at 25 ºC for 10 min. After pre-incubation, 500 µl of a 1% starch solution in phosphate buffer was added to each tube. After incubation at 25 °C for 10 min the reaction was stopped with 1.0 ml of dinitrosalicylic acid color reagent. The test tubes were then incubated in a boiling water bath for 5 min and cooled to room temperature. The reaction mixture was diluted with distilled water and absorbance was measured at 540 nm. A control with buffer instead of plant extract was performed and the α-amylase inhibitory activity was expressed as percentage inhibition. Acarbose at 300 µg/ml was used as reference.

α-Glucosidase inhibition

α-Glucosidase inhibition assay was performed according to the procedure described by Kwon et al. (2008) with slight modifications. A volume of 50 µl of plant extract at 10 mg/ml or acarbose (1000 µg/ml) were mixed with 100 µl of 0.1 M phosphate buffer (pH 6.9) containing yeast α-glucosidase solution (1.0 U/ml) in 96-well plates and were incubated at 25 °C for 10 min. After pre-incubation, 50 µl of 5 mM p-nitrophenyl-α-D-glucopyranoside solution in phosphate buffer was added to each well. The reaction mixtures were incubated at 25 °C for 5 min and then absorbance readings were recorded at 405 nm and compared to a control which had 50 µl of buffer solution in place of the extract. The α-glucosidase inhibitory activity was expressed as percentage inhibition.

Statistical analysis

All the experiments were carried out three times using triplicate samples. The data were presented as the mean ± standard error and were processed by one-way analysis of variance (ANOVA). Significant differences between means were identified using Duncan’s New Multiple Range Test (p < 0.05), and correlations were calculated using Pearson’s test. All statistical analysis was carried out using the SPSS statistical package for Windows (release 18.0; SPSS Inc., Chicago, IL, USA).

Results and discussion

Phenolic composition

The values of extraction yield obtained in this study, expressed as weight of extract relative to the weight of the initial plant material, were in the same range for the four species, 23.89, 24.86, 26.24 and 27.95% for C. nepeta, H. italicum, M. spicata and O. vulgare, respectively.

The phenolic composition of the extracts was analysed by HPLC–DAD and several phenolic acids and flavonoids were identified (Table 2; Fig. 1). H. italicum and M. spicata extracts contained the highest amount of the identified phenolic compounds (51.65 and 47.93 mg/g of dry extract, respectively). Rosmarinic acid was the major compound identified in M. spicata and O. vulgare extracts (35.63 and 23.53 mg/g of dry extract, respectively) similarly to the observed in other Lamiaceae species (Costa et al. 2013), which is not surprising as this is a taxonomic marker of this botanical family (Cantino and Sanders 1986; Janicsák et al. 1999). Substantial amounts of the flavanone hesperetin-7-O-rutinoside and the flavan-3-ol (−)-epicatechin were also found respectively in M. spicata and O. vulgare extracts (Table 2). The extract from C. nepeta (Lamiaceae) also contained rosmarinic acid but in much lower amount (1.44 mg/g of dry extract). This extract contained 4-O-caffeoylquinic acid (14.69 mg/g of dry extract) as the major compound as well as considerable amount of quercetin-3-O-rutinoside (Table 2). Pacifico et al. (2015) recently reported qualitative and quantitative variations in the phytochemical constitution of C. nepeta (L.) Savi during the life cycle. For rosmarinic acid the authors observed a strong variation in its content throughout the year. 3,5-di-O-Caffeoylquinic acid (14.48 mg/g of dry extract) was the predominant phenolic compound in H. italicum extract followed by pinocembrin (13.98 mg/g of dry extract). In addition, another caffeoylquinic acid (5-O-caffeoylquinic acid) and three dicaffeoylquinic acids (3,4-, 1,5- and 4,5-di-O-caffeoylquinic acids) were identified in this extract (Table 2). Some caffeoylquinic and dicaffeoylquinic acids were also identified in other species of the same genus (Barroso et al. 2014). Furthermore, Scognamiglio et al. (2015) also identified 5-O-caffeoylquinic acid (chlorogenic acid) and dicaffeoylquinic acids in Helichrysum italicum G. Don. Studies reporting the phenolic composition of extracts from the subspecies investigated in this study, C. nepeta subsp. nepeta, H. italicum subsp. picardii and O. vulgare subsp. virens, are scarce although there are some studies describing the chemical composition of the essential oils or volatile extracts (Vale-Silva et al. 2012; Costa et al. 2015b). Conversely, there are reports describing the phenolic compounds of M. spicata extracts from different origins (Dorman et al. 2003; Fatiha et al. 2015). Similarly to what was observed in this study, rosmarinic acid was one of the main compounds identified in the extracts analyzed by most authors but in variable amounts, possibly reflecting the effect of edafoclimatic factors, and the application of distinct extraction and analytical techniques. Indeed, Mentha species have been considered promising sources of rosmarinic acid, which has attracted a great deal of attention due to its reported health benefits (Fatiha et al. 2015).

Table 2.

Phenolic compounds identified in the extracts (mg/g of dry extract)

Peak Compound RT (min) λ (max) C. nepeta H. italicum M. spicata O. vulgare
1 Gallic acid 6.48 270.7 – – – 1.19 ± 0.43
2 3,4-Dihydroxybenzoic acid 9.47 259.3, 294.1 – – – 1.97 ± 0.33
3 3-O-Caffeoylquinic acid 9.95 249.1, 296.4, 324.2 3.80 ± 0.13 – – –
4 (+)-Catechin 12.35 279.3 – – – 1.03 ± 0.10
5 p-Hydroxybenzoic acid 13.43 254.7 – 1.88 ± 0.09 – –
6 4-O-Caffeoylquinic acid 15.43 249.7, 296.9, 325.9 14.69 ± 0.28 – 1.52 ± 0.10 –
7 5-O-Caffeoylquinic acid 17.38 248.7, 297.8, 325.7 2.76 ± 0.22 6.89 ± 0.86 – –
8 Caffeic acid 17.84 249.1, 295.9, 323.3 – – 1.00 ± 0.03 0.06 ± 0.002
9 (−)-Epicatechin 18.98 278.8 – – – 4.65 ± 2.20
10 3,4-di-O-Caffeoylquinic acid 36.00 251.0, 296.9, 324.2 – 2.03 ± 0.44 – –
11 3,5-di-O-Caffeoylquinic acid 38.58 251.9, 298.3, 327.0 – 14.48 ± 2.84 – –
12 Hesperetin-7-O-rutinoside 40.00 287.0, 337.7 – – 9.78 ± 0.45 –
13 1,5-di-O-Caffeoylquinic acid 40.78 250.5, 299.2, 328.4 – 3.53 ± 0.92 – –
14 Rosmarinic acid 40.99 254.2, 289.0, 329.5 1.44 ± 0.08 – 35.63 ± 2.09 23.53 ± 0.47
15 Quercetin-3-O-rutinoside 42.85 257.4, 265.2, 299.1, 355.6 5.20 ± 0.60 – – –
16 4,5-di-O-Caffeoylquinic acid 43.51 251.0, 299.7, 327.9 – 8.86 ± 1.60 – –
17 Pinocembrin 55.08 290.4, 334.4 – 13.98 ± 0.88 – –
Σ 27.89 51.65 47.93 32.43
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Values are expressed as mean ± SD (n = 3); –: not detected

Fig. 1.

Fig. 1

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HPLC–DAD chromatograms of C. nepeta (a), H. italicum (b), M. spicata (c) and O. vulgare (d). Detection at 320 nm except in D (280 nm). Identity of compounds as in Table 2

Total phenolic contents and antioxidant activity

Plant-based products capable of protecting humans against oxidative damage have recently attracted interest worldwide. Phenolic compounds, the main class of plant antioxidants, can reduce the oxidative stress in the body maintaining a balance between oxidants and antioxidants due to their reducing, free radical scavenging or metal chelating properties (Aguilera et al. 2016). The total phenolic contents of the extracts from the four aromatic species were determined by F–C assay and the results ranged from 549.27 ± 23.86 to 1597.20 ± 24.10 µmolGAE/gextract. The highest phenolic level was observed in the extract of O. vulgare, whereas C. nepeta extract contained relatively low levels of phenolic compounds.

The free radical-scavenging capacity of the four extracts was investigated by two methods, DPPH and ABTS (Table 3). Furthermore, the reducing capacity of the extracts was evaluated by determining their ability to reduce ferricyanide/Fe3+ complex to the corresponding Fe2+ type (Table 3). The results of DPPH assay were expressed as IC50 values and ranged from 4.65 ± 0.12 to 13.27 ± 0.46 µg/ml. The results of ABTS assay, expressed as trolox equivalents, varied between 877.93 ± 22.38 and 1479.56 ± 12.29 µmolTE/gextract. Among the four extracts O. vulgare was the most efficient at neutralizing both ABTS•+ and DPPH•+ radicals. This extract also had the highest ferric reducing ability expressed as ascorbic acid equivalents, 1746.76 ± 45.11 µmolAAE/gextract. There are some reports describing the antioxidant activity of phenolic extracts from M. spicata and O. vulgare subsp. virens but the comparison between results from different investigations is not always possible because of the different assay formats, experimental conditions and data presentation strategies. The DPPH assay is recurrently used and overall the IC50 values obtained in this work were lower for both species (Barros et al. 2010; Fatiha et al. 2015). Studies describing the antioxidant activity from the subspecies of H. italicum and C. nepeta studied in this work are limited and mainly focused on essential oils and volatile extracts (Conforti et al. 2012; Costa et al. 2015b).

Table 3.

Total phenolic contents determined by Folin–Ciocalteu assay and antioxidant activity evaluated by 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azinobis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) and ferric reducing antioxidant power (FRAP) assays, of extracts from the four aromatic species studied

Plant species Total phenolics Antioxidant activity
F–C
(µmolGAE/gextract)		 DPPH
(IC50, µg/ml)		 ABTS
(µmolTE/gextract)		 FRAP
(µmolAAE/gextract)
C. nepeta 549.27 ± 23.86 c 13.27 ± 0.46 c 1050.31 ± 81.45 b 582.83 ± 6.59 c
H. italicum 814.23 ± 35.14 b 13.27 ± 0.89 c 947.52 ± 8.68 bc 638.28 ± 22.04 c
M. spicata 870.62 ± 45.69 b 8.93 ± 0.27 b 877.93 ± 22.38 c 996.88 ± 13.08 b
O. vulgare 1597.20 ± 24.10 a 4.65 ± 0.12 a 1479.56 ± 12.29 a 1746.76 ± 45.11 a
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Values are expressed as mean ± SE (n = 3). In each column values followed by different letters are significantly different at p < 0.05. Reference in DPPH assay, BHT: IC50 = 462.24 ± 0.03 μg/ml

The correlation between antioxidant activity and the total phenolic content was evaluated by calculating Pearson’s correlation coefficients and, in accordance with a wide range of reports, high correlation coefficients were found for all the used assays (FC-DPPH: 0.909; FC-ABTS: 0.809; FC-FRAP: 0.955). The outstanding antioxidant activity of O. vulgare extract could be partially attributed to the higher content in rosmarinic acid, the main phenolic compound found in this extract, which has been described as a potent antioxidant compound (Kim et al. 2015). However, other active compounds, as (+)-catechin and (−)-epicatechin, which are only present in this extract, certainly play an important role for the antioxidant activity of the extract. This is supported by the fact that M. spicata extract, while containing the highest amount of rosmarinic acid, did not show the greatest antioxidant activity.

Although phenolic compounds are the most abundant and potent antioxidants in plants, there is also a pool of non-phenolic compounds that contribute to the overall antioxidant activity of the extracts. Different phenolic compounds also vary in their antioxidant activities, and may work cooperatively, antagonistically or synergistically with other compounds present in crude extracts. Additionally, it is important to note that results of F–C assay should be interpreted carefully as it is nonspecific to phenolic compounds and other substances, including nonphenolic organic substances and some inorganic substances, could be oxidised by the F–C reagent (Huang et al. 2005).

Enzyme inhibitory activities

The cholinergic neurotransmitter acetylcholine is reduced in AD patients and cholinesterase inhibitors have been shown to augment the activity of surviving cholinergic neurons in patients with the disease, resulting in improvement of memory and cognition (Massoud and Gauthier 2010). Phytochemical substances have demonstrated AChE inhibitory activity and thus could be beneficial in the treatment of neurodegenerative disorders, such as AD. Evaluation of the effects of the extracts (at 10 mg/ml) studied in this work on AChE enzyme are listed in Table 4. H. italicum and M. spicata extracts showed the highest inhibitory percentages (78.29 and 72.33%, respectively). As far as our literature survey could ascertain the AChE inhibitory activity of H. italicum is reported for the first time. The HPLC–DAD analysis showed that H. italicum extract is rich in pinocembrin and results from Liu et al. (2014) indicated that this compound is inactive against AChE although it improves cognition and protects the neurovascular unit in Alzheimer related deficits (Liu et al. 2014). Thus, H. italicum extract can be effective for the prevention and/or treatment of AD through different mechanisms.

Table 4.

Inhibitory activity (%) of methanol extracts (at 10 mg/ml) from the four aromatic species studied against acetylcholinesterase (AChE), tyrosinase, α-amylase and α-glucosidase

AChE Tyrosinase α-Amylase α-Glucosidase
C. nepeta 48.78 ± 5.21 b 38.45 ± 4.06 c 16.45 ± 0.94 a 66.62 ± 1.61 c
H. italicum 78.29 ± 2.44 a 74.13 ± 3.79 a 10.03 ± 1.87 b 96.65 ± 1.34 a
M. spicata 72.33 ± 2.65 a 57.90 ± 1.44 b 6.81 ± 0.65 b 79.64 ± 0.69 b
O. vulgare 20.11 ± 3.56 c 69.65 ± 1.45 ab 6.79 ± 0.57 b 58.41 ± 1.97 d
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Values are expressed as mean ± SE (n = 3). In each column values followed by different letters are significantly different at p < 0.05. References: α-amylase, acarbose (300 µg/ml) = 72.47%; α-glucosidase, acarbose (1000 µg/ml) = 80.71%; AChE, galanthamine (5 µg/ml) = 67.78%; tyrosinase, kojic acid (200 µg/ml) = 94.46%

Tyrosinase is a copper-containing polyphenol oxidase that converts l-tyrosine to L-DOPA and oxidizes L-DOPA to form dopachrome, which induces the production of melanin pigments (Seo et al. 2003). Melanin production is critical in preventing UV damage to skin, hair and eyes, however its overproduction is associated with pigment variations and also with neurodegenerative disorders namely PD (Khan 2012). Tyrosinase is also responsible for browning in some fruits and vegetables thus it is frequent the treatment of plant-based foods with tyrosinase inhibitors (Loizzo et al. 2012). Concerns over the toxicity and side effects of synthetic inhibitors have led to a search for new safe and effective tyrosinase inhibitors particularly from natural sources (Zengin et al. 2015). The extracts (at 10 mg/ml) of the four species studied in this work were tested against tyrosinase activity and, although all the extracts inhibit the enzyme, the highest inhibitory activity was again obtained with H. italicum extract (74.13%). According to our literature survey, tyrosinase inhibitory activity of this species has never been studied up to now. This activity might be associated to the high amount of hydroxycinnamic acids, particularly dicaffeoylquinic acids in this extract (Iwai et al. 2004) (Table 2). Additionally, the tyrosinase inhibitory capacity of plant extracts is also frequently associated to the presence of flavonoids (Fatiha et al. 2015; Orhan et al. 2015) that can act as cofactor and/or substrate of tyrosinase or by active site chelation. This may lead to the assumption that the tyrosinase inhibitory action of H. italicum extract might also be related to the presence of pinocembrin, the only flavonoid identified in the extract. O. vulgare and M. spicata extracts also considerably inhibit this enzyme and rosmarinic acid is probably involved in that effect (Tundis et al. 2015).

α-Amylase and α-glucosidase enzymes are responsible for carbohydrate digestion and have been identified to be the therapeutic targets for the management of postprandial hyperglycemia (Shobana et al. 2009). The inherently associated adverse side effects of the synthetic inhibitors necessitate further efforts to find natural and safer alternatives. In this study the methanolic extracts (at 10 mg/ml) of the four aromatic plants were evaluated for their α-amylase and α-glucosidase inhibitory capacity. All the extracts showed a weak activity against α-amylase (Table 4). On the other hand, all the extracts were considerably more effective against α-glucosidase, with inhibition percentages in the range of 58.41 to 96.65%. The selective inhibition of α-glucosidase is the preferred effect for plant extracts to control glucose uptake because simultaneous inhibition of both enzymes would result in abnormal bacterial fermentation in the colon due to the presence of undigested carbohydrates (Costamagna et al. 2016). Indeed, plant extracts from many plants, including plants traditionally used to control diabetes or hyperglycemia, were reported to exert strong inhibition of α-glucosidase and moderate or negligible effect on α-amylase activity (Pradeep and Sreerama 2015). The best α-glucosidase inhibition result was once more obtained with H. italicum extract (Table 4). Since caffeoylquinic acids possess strong inhibition potential against α-glucosidase (Xiao et al. 2013) these compounds are almost certainly implicated in the α-glucosidase inhibitory properties of H. italicum extract.

Conclusion

The present study evaluated the phenolic profile, and the antioxidant and enzyme inhibitory properties of extracts from four aromatic species used in traditional medicine and Mediterranean diet. Phenolic acids and flavonoids are the main identified phenolic compounds in the extracts. The O. vulgare extract showed the highest antioxidant activity and the H. italicum extract exhibited the highest enzyme inhibitory potential. Overall, the results obtained demonstrate that the studied plants represent promising sources of food rich in natural antioxidants and of active ingredient agents for the pharmaceutic and cosmetic industries.

Acknowledgements

This work received financial support from the European Union (FEDER funds through COMPETE) and National Funds (FCT/MEC, Fundação para a Ciência e Tecnologia/Ministério da Educação e Ciência) through project UID/QUI/50006/2013, co-financed by European Union (FEDER under the Partnership Agreement PT2020), and from Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF) (project NORTE-01-0145-FEDER-000024). S. Gonçalves acknowledges a grant from FCT (SFRH/BPD/84112/2012) and C. Grosso thanks FCT for the FCT Investigator (IF/01332/2014).

References

  1. Aguilera Y, Martin-Cabrejas MA, de Mejia EG. Phenolic compounds in fruits and beverages consumed as part of the mediterranean diet: their role in prevention of chronic diseases. Phytochem Rev. 2016;15:405–423. doi: 10.1007/s11101-015-9443-z. [DOI] [Google Scholar]
  2. Ainsworth EA, Gillespie KM. Estimation of total phenolic content and other oxidation substrates in plant tissues using Folin–Ciocalteu reagent. Nat Protoc. 2007;2:875–877. doi: 10.1038/nprot.2007.102. [DOI] [PubMed] [Google Scholar]
  3. Barros L, Heleno SA, Carvalho AM, Ferreira ICFR. Lamiaceae often used in Portuguese folk medicine as a source of powerful antioxidants: vitamins and phenolics. LWT-Food Sci Technol. 2010;43:544–550. doi: 10.1016/j.lwt.2009.09.024. [DOI] [Google Scholar]
  4. Barroso MR, Barros L, Dueñas M, Carvalho AM, Santos-Buelga C, Fernandes IP, Barreiro MF, Ferreira ICFR. Exploring the antioxidant potential of Helichrysum stoechas (L.) Moench phenolic compounds for cosmetic applications: chemical characterization, microencapsulation and incorporation into a moisturizer. Ind Crops Prod. 2014;53:330–336. doi: 10.1016/j.indcrop.2014.01.004. [DOI] [Google Scholar]
  5. Cantino PD, Sanders RW. Subfamilial classification of Labiatae. System Bot. 1986;11:163–185. doi: 10.2307/2418955. [DOI] [Google Scholar]
  6. Conforti F, Marrelli M, Statti G, Menichini F, Uzunov D, Solimene U, Menichini F. Comparative chemical composition and antioxidant activity of Calamintha nepeta (L.) Savi subsp. glandulosa (Req.) Nyman and Calamintha grandiflora (L.) Moench (Labiatae) Nat Prod Res. 2012;26:91–97. doi: 10.1080/14786419.2010.545356. [DOI] [PubMed] [Google Scholar]
  7. Costa P, Gonçalves S, Valentão P, Andrade PB, Romano A. Accumulation of phenolic compounds in in vitro cultures and wild plants of Lavandula viridis L’Hér and their antioxidant and anticholinesterase potential. Food Chem Toxicol. 2013;57:69–74. doi: 10.1016/j.fct.2013.03.006. [DOI] [PubMed] [Google Scholar]
  8. Costa DC, Costa HS, Albuquerque TG, Ramos F, Castilho MC, Sanches-Silva A. Advances in phenolic compounds analysis of aromatic plants and their potential applications. Trends Food Sci Technol. 2015;45:336–354. doi: 10.1016/j.tifs.2015.06.009. [DOI] [Google Scholar]
  9. Costa P, Loureiro JM, Teixeira MA, Rodrigues AE. Extraction of aromatic volatiles by hydrodistillation and supercritical fluid extraction with CO2 from Helichrysum italicum subsp. picardii growing in Portugal. Ind Crops Prod. 2015;77:680–683. doi: 10.1016/j.indcrop.2015.09.042. [DOI] [Google Scholar]
  10. Costamagna MS, Zampini IC, Alberto MR, Cuello S, Torres S, Pérez J, Quispe C, Schmeda-Hirschmann G, Isla MI. Polyphenols rich fraction from Geoffroea decorticans fruits flour affects key enzymes involved in metabolic syndrome, oxidative stress and inflammatory process. Food Chem. 2016;190:392–402. doi: 10.1016/j.foodchem.2015.05.068. [DOI] [PubMed] [Google Scholar]
  11. Del Rio D, Rodriguez-Mateos A, Spencer JPE, Tognolini M, Borges G, Crozier A. Dietary (poly) phenolics in human health: structures, bioavailability, and evidence of protective effects against chronic diseases. Antioxid Redox Signal. 2013;18:1818–1892. doi: 10.1089/ars.2012.4581. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Dorman HJD, Koşar M, Kahlos K, Holm Y, Hiltunen R. Antioxidant properties and composition of aqueous extracts from Mentha species, hybrids, varieties, and vultivars. J Agric Food Chem. 2003;51:4563–4569. doi: 10.1021/jf034108k. [DOI] [PubMed] [Google Scholar]
  13. Ellman GL, Courtney KD, Andres V, Feather-Stone RM. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem Pharmacol. 1961;7:88–95. doi: 10.1016/0006-2952(61)90145-9. [DOI] [PubMed] [Google Scholar]
  14. Fatiha B, Didier H, Naima G, Khodir M, Martin K, Léocadie K, Caroline S, Mohamed C, Pierre D. Phenolic composition, in vitro antioxidant effects and tyrosinase inhibitory activity of three Algerian Mentha species: M. spicata (L.), M. pulegium (L.) and M. rotundifolia (L.) Huds (Lamiaceae) Ind Crops Prod. 2015;74:722–730. doi: 10.1016/j.indcrop.2015.04.038. [DOI] [Google Scholar]
  15. Fernandes RPP, Trindade MA, Tonin FG, Lima CG, Pugine SMP, Munekata PES, Lorenzo JM, de Melo MP. Evaluation of antioxidant capacity of 13 plant extracts by three different methods: cluster analyses applied for selection of the natural extracts with higher antioxidant capacity to replace synthetic antioxidant in lamb burgers. J Food Sci Technol. 2016;53:451–460. doi: 10.1007/s13197-015-1994-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Food and Drug Administration (FDA) (2014) Code of Federal Regulations (CFR). Title 21: Food and Drugs. Chapter I e Food and Drug Administration, Department of Health and Human Services (Continued), Subchapter B e Food for Human Consumption, Part 182 e Substances Generally Recognized as Safe (GRAS), Subpart A—General Provisions, Subpart 182.10—Spices and other natural seasonings and flavourings and Subpart A—General Provisions, Subpart 182.20—Essential oils, oleoresins, and natural extractives. Office of the Federal Register, Washington, DC (Revised April, 2014a)
  17. Huang D, Ou B, Prior RL. The chemistry behind antioxidant capacity assays. J Agric Food Chem. 2005;53:1841–1856. doi: 10.1021/jf030723c. [DOI] [PubMed] [Google Scholar]
  18. Iwai K, Kishimoto N, Kakino Y, Mochida K, Jujita T. In vitro antioxidative effects and tyrosinase inhibitory activities of seven hydroxycinnamoyl derivatives in green coffee beans. J Agric Food Chem. 2004;52:4893–4898. doi: 10.1021/jf040048m. [DOI] [PubMed] [Google Scholar]
  19. Janicsák G, Máthé I, Miklóssy-Vári V, Blunden G. Comparative studies of the rosmarinic and caffeic acid contents of Lamiaceae species. Biochem Syst Ecol. 1999;27:733–738. doi: 10.1016/S0305-1978(99)00007-1. [DOI] [Google Scholar]
  20. Khan MT. Novel tyrosinase inhibitors from natural resources—their computational studies. Curr Med Chem. 2012;19:2262–2272. doi: 10.2174/092986712800229041. [DOI] [PubMed] [Google Scholar]
  21. Kim G-D, Park YS, Jin Y-H, Park C-S. Production and applications of rosmarinic acid and structurally related compounds. Appl Microbiol Biotechnol. 2015;99:2083–2092. doi: 10.1007/s00253-015-6395-6. [DOI] [PubMed] [Google Scholar]
  22. Kwon Y-I, Apostolidis E, Shetty K. In vitro studies of eggplant (Solanum melongena) phenolics as inhibitors of key enzymes relevant for type 2 Diabetes and hypertension. Bioresour Technol. 2008;99:2981–2988. doi: 10.1016/j.biortech.2007.06.035. [DOI] [PubMed] [Google Scholar]
  23. Lee HP, Zhu X, Casadesus G, Castellani RJ, Nunomura A, Smith MA, Lee H, Perry G. Antioxidant approaches for the treatment of Alzheimer’s disease. Expert Rev Neurother. 2010;10:1201–1208. doi: 10.1586/ern.10.74. [DOI] [PubMed] [Google Scholar]
  24. Liu R, Li JZ, Song JK, Zhou D, Huang C, Bai XY, Xie T, Zhang X, Li YJ, Wu CX, Zhang L, Li L, Zhang TT, Du GH. Pinocembrin improves cognition and protects the neurovascular unit in Alzheimer related deficits. Neurobiol Aging. 2014;35:1275–1285. doi: 10.1016/j.neurobiolaging.2013.12.031. [DOI] [PubMed] [Google Scholar]
  25. Loizzo MR, Tundis R, Menichini F. Natural and synthetic tyrosinase inhibitors as antibrowning agents: an update. Comp Rev Food Sci Food Saf. 2012;11:378–398. doi: 10.1111/j.1541-4337.2012.00191.x. [DOI] [Google Scholar]
  26. Maritim AC, Sanders RA, Watkins JB. Diabetes, oxidative stress and antioxidants: a review. J Biochem Mol Toxicol. 2003;17:24–38. doi: 10.1002/jbt.10058. [DOI] [PubMed] [Google Scholar]
  27. Massoud F, Gauthier S. Update on the pharmacological treatment of Alzheimer’s Disease. Curr Neuropharmacol. 2010;8:69–80. doi: 10.2174/157015910790909520. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Masuda T, Yamashita D, Takeda Y, Yonemori S. Screening for tyrosinase inhibitors among extracts of seashore plants and identification of potent inhibitors from Garcinia subelliptica. Biosci Biotechnol Biochem. 2005;69:197–201. doi: 10.1271/bbb.69.197. [DOI] [PubMed] [Google Scholar]
  29. Nikousaleh A, Prakash J. Antioxidant components and properties of dry heat treated clove in different extraction solvents. J Food Sci Technol. 2016;53:1993–2000. doi: 10.1007/s13197-015-2113-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Orhan IE, Senol FS, Erdem SA, Tatli II, Kartal M, Alp S. Tyrosinase and cholinesterase inhibitory potential and flavonoid characterization of Viola odorata L. (sweet violet) Phytother Res. 2015;29:1304–1310. doi: 10.1002/ptr.5378. [DOI] [PubMed] [Google Scholar]
  31. Pacifico P, Galasso S, Piccolella S, Kretschmer N, Pan S-P, Marciano S, Bauer R, Monaco P. Seasonal variation in phenolic composition and antioxidant and anti-inflammatory activities of Calamintha nepeta (L.) Savi. Food Res Int. 2015;69:121–132. doi: 10.1016/j.foodres.2014.12.019. [DOI] [Google Scholar]
  32. Pradeep PM, Sreerama YN. Impact of processing on the phenolic profiles of small millets: evaluation of their antioxidant and enzyme inhibitory properties associated with hyperglycemia. Food Chem. 2015;169:455–463. doi: 10.1016/j.foodchem.2014.08.010. [DOI] [PubMed] [Google Scholar]
  33. Pulido R, Bravo L, Saura-Calixto F. Antioxidant activity of dietary polyphenols as determined by a modified ferric reducing/antioxidant power assay. J Agric Food Chem. 2000;48:3396–3402. doi: 10.1021/jf9913458. [DOI] [PubMed] [Google Scholar]
  34. Re R, Pellegrini N, Proteggente A, Pannala A, Yang M, Rice-Evans C. Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radical Bio Med. 1999;26:1231–1237. doi: 10.1016/S0891-5849(98)00315-3. [DOI] [PubMed] [Google Scholar]
  35. Scognamiglio M, D’Abrosca B, Esposito A, Fiorentino A (2015) Chemical composition and seasonality of aromatic Mediterranean plant species by NMR-based metabolomics. J Anal Methods Chem:Article ID 258570 [DOI] [PMC free article] [PubMed]
  36. Seo SY, Sharma VK, Sharma N. Mushroom tyrosinase: recent propects. J Agric Food Chem. 2003;51:2837–2853. doi: 10.1021/jf020826f. [DOI] [PubMed] [Google Scholar]
  37. Shanmugam S, Baby JP, Chandran R, Thankarajan S, Thangaraj P. Maesa indica: a nutritional wild berry rich in polyphenols with special attention to radical scavenging and inhibition of key enzymes, α-amylase and α-glucosidase. J Food Sci Technol. 2016;53:2957–2965. doi: 10.1007/s13197-016-2263-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Shobana S, Sreerama YN, Malleshi NG. Composition and enzyme inhibitory properties of finger millet (Eleusine coracana L.) seed coat phenolics: mode of inhibition of α-glucosidase and pancreatic α -amylase. Food Chem. 2009;115:1268–1273. doi: 10.1016/j.foodchem.2009.01.042. [DOI] [Google Scholar]
  39. Soler-Rivas C, Espín JC, Wichers HJ. An easy and fast test to compare total free radical scavenger capacity of food stuffs. Phytochem Anal. 2000;11:330–338. doi: 10.1002/1099-1565(200009/10)11:5&#x0003c;330::AID-PCA534&#x0003e;3.0.CO;2-G. [DOI] [PubMed] [Google Scholar]
  40. Tundis R, Loizzo MR, Bonesi M, Menichini F. Potential role of natural compounds against skin aging. Curr Med Chem. 2015;22:1515–1538. doi: 10.2174/0929867322666150227151809. [DOI] [PubMed] [Google Scholar]
  41. Vale-Silva L, Silva M-J, Oliveira D, Gonçalves M-J, Cavaleiro C, Salgueiro L, Pinto E. Correlation of the chemical composition of essential oils from Origanum vulgare subsp. virens with their in vitro activity against pathogenic yeasts and filamentous fungi. J Med Microbiol. 2012;61:252–260. doi: 10.1099/jmm.0.036988-0. [DOI] [PubMed] [Google Scholar]
  42. Xiao J, Kai G, Yamamoto K, Chen X. Advance in dietary polyphenols as α-glucosidases inhibitors: a review on structure-activity relationship aspect. Crit Rev Food Sci Nutr. 2013;53:818–836. doi: 10.1080/10408398.2011.561379. [DOI] [PubMed] [Google Scholar]
  43. Zengin G, Uysal S, Ceylan R, Aktumsek A. Phenolic constituent, antioxidative and tyrosinase inhibitory activity of Ornithogalum narbonense L. from Turkey: a phytochemical study. Ind Crops Prod. 2015;70:1–6. doi: 10.1016/j.indcrop.2015.03.012. [DOI] [Google Scholar]

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