Cent. Eur. J. Biol. • 9(9) • 2014 • 888-900 DOI: 10.2478/s11535-014-0322-1

Central European Journal of Biology

Phenolic production and antioxidant properties of some Macedonian medicinal

Research Article Oliver Tusevski1, Aneta Kostovska2, Ana Iloska2, Ljubica Trajkovska2, Sonja Gadzovska Simic1*

1Department of Physiology, Institute of Biology, Faculty of Natural Sciences and Mathematics, University “Ss. Cyril and Methodius”, P.O. Box 162, 1000 Skopje, Macedonia

2Biology Students Research Society (BSRS), Institute of Biology, Faculty of Natural Sciences and Mathematics, University “Ss. Cyril and Methodius”, P.O. Box 162, 1000 Skopje, Macedonia Received 24 September 2013; Accepted 26 December 2013

Abstract: Investigations have been made to study the production of phenolic compounds (total phenolics, flavonoids and phenylpropanoids) and total antioxidant capacity in 27 Macedonian traditional medicinal plants to improve its potential as a source of natural antioxidants. Antioxidant potential of plant extracts was analyzed by five different assays: cupric reducing antioxidant capacity (CUPRAC), phosphomolybdenum method (PM), reducing power (RP), 2,2-diphenyl-1-picrylhydrazyl (DPPH•) and 2,2’-azinobis(3- ethylbenzothiazoline-6-sulphonic acid (ABTS•+) radical scavenging activity. Origanum vulgare extract consistently exhibited the highest content of phenolic compounds and the strongest antioxidant capacity based on the tests performed, and can be proposed as a promising source of natural antioxidants. Melissa officinalis and ringens were also identified as valuable sources of antioxidant compounds. A positive linear correlation between antioxidant activity and total phenolics, flavonoids and phenylpropanoids indicates that these compounds are likely to be the main antioxidants contributing to the observed activities of evaluated plants. These findings suggest that the medicinal plants studied in this paper are good sources of bioactive compounds for the food and pharmaceutical industries.

Keywords: Phenolics • Flavonoids • Phenylpropanoids • Antioxidant activity • Scavenging capacity • Medicinal plants © Versita Sp. z o.o.

Abbreviations: 1. Introduction

AAE - Ascorbic acid equivalents, Reactive oxygen species (ROS) are an integral part of ABTS - 2,2’-azinobis(3-ethylbenzothiazoline-6- normal physiological processes, continuously formed sulphonic acid, as a consequence of aerobic metabolism in eukaryotic CE - Catechin equivalents, cells. ROS at low-to-moderate concentrations play CUPRAC - Cupric reducing antioxidant capacity, important roles in cell physiology, such as regulation DPPH - 2,2-diphenyl-1-picrylhydrazyl, of cell growth, cellular signal transduction pathways, GAE - Gallic acid equivalents, and defence against pathogens [1,2]. In addition to PE - Pyrocatechol equivalents, their biological importance, overproduction of these PM - Phosphomolybdenum method, extremely reactive and unstable oxygen species RP - Reducing power, is considered to be the main contributor to various TE - Trolox equivalents, metabolic and cellular disturbances. These harmful by- TF - Total flavonoid content, products may also induce some oxidative damage to TP - Total phenolic content, functional biomolecules giving rise to oxidative stress. TPP - Total phenylpropanoid content Oxidative stress has been suggested to play a major

* E-mail: [email protected] 888 O. Tusevski et al.

role in the pathogenesis of many degenerative diseases acid, quercetin, catechin, α-tocopherol, BHT and BHA). in humans [3]. In modern medicine, maintaining the This study will be useful to determine the potential of balance between antioxidant defence system and ROS selected traditional medicinal plants as potential sources formation is believed to be a critical concept for healthy of natural antioxidants. biological systems [4]. Recently published data highlight the health benefits of medicinal herbs, fruit, vegetables, grains 2. Experimental Procedures and beverages as dietary antioxidants [5]. Natural plant products, which contribute health benefits to consumers, 2.1 Chemicals and apparatus had stronger antioxidant activity than that of synthetic All chemicals and reagents were of analytical grade antioxidants [6]. Synthetic antioxidants such as butylated and were purchased from Sigma-Aldrich (Germany). hydroxytoluene (BHT), butylated hydroxyanisole (BHA), All spectrophotometric measurements were performed tert-butylhydroquinone (TBHQ) and propyl gallate (PG) on UV–VIS spectrophotometer SpectraMAX 190 are widely used in the food industry to prevent oxidative (Molecular Devices, California, United States). deterioration [7]. However, it has been established that synthetic antioxidants appear to have carcinogenic and 2.2 Plant material tumour-promoting action [8]. Therefore, it is of great Twenty-seven medicinal plant species from twelve importance to find new sources of safe and inexpensive families (Table 1) were collected from various locations antioxidants of natural origin in order to use them in at Galichitsa Mountain, Republic of Macedonia. foods and pharmaceutical formulations. The plant species used in this study belong to the Medicinal plants synthesize antioxidant compounds family (nine tested species), Asteracae as secondary products. These compounds are mainly (four tested spices), Fabaceae, Plantaginaceae (three phenolics that serve as plant defence mechanisms tested spices), Rosaceae, Malvaceae, Gentianaceae, to counteract ROS and avoid oxidative damage. Scrophulariaceae, Linaceae, Caprifoliaceae, Polyphenols possess ideal structural chemistry for Caryophyllaceae and Verbenaceae (one tested species). free radical scavenging activity, and they have been Voucher specimens of collected plants are deposited in shown to be more effective antioxidants in vitro than the Herbarium Collection of Biology Students Research tocopherols and ascorbate. The antioxidant activities of Society (BSRS), Institute of Biology, Faculty of Natural phenolics are due to a number of different mechanisms, Sciences and Mathematics, University “Ss. Cyril and such as free-radical scavenging, hydrogen or electron Methodius”, Skopje. donation, singlet oxygen quenching, metal ion chelation, and acting as scavengers of superoxide, peroxide and 2.3 Extraction procedure hydroxyl radicals [9]. In addition, it has been reported Plant material was air-dried, lyophilized and then that the antioxidant potential of medicinal plants grounded into a fine powder by laboratory mill. correlates with their phenolic compound content [10]. Antioxidant compounds were extracted from There are approximately 3500 powdered plant material (0.02 g) with 80% methanol species in Macedonia, of which 700 have medicinal in an ultrasonic bath for 30 min. The extracts were properties, however only 120 species are generally centrifuged (15 min at 12000 rpm) and the supernatant utilised in folk and official medicine. To our knowledge, was used for quantification of total phenolics, flavonoids there is no available information or systematic survey of and phenylpropanoids, as well for antioxidant activity antioxidant activity in wild-growing Macedonian plants. using different assays. Ascorbic acid (AA), quercetin The aim of this study is to perform a preliminary screen (Q), catechin (C), α-tocopherol (α-T), butylated of antioxidant activities from 27 traditional medicinal hydroxytoluene (BHT) and butylated hydroxyanisole plant species collected on Galichitsa Mountain, Republic (BHA) were used as reference compounds. of Macedonia. Therefore, the main objectives of this Standard chemicals were dissolved in pure methanol study were: 1) to determine total content of phenolics, at a concentration of 0.5 mg mL-1 and prepared prior to flavonoids and phenylpropanoids in methanolic extracts each assay. from selected medicinal plants; 2) to assess antioxidant activity by using a number of chemical in vitro assays; 2.4 Quantification of phenolic compounds and 3) to explore correlation between phenolic contents 2.4.1 Total phenolic content (TP) and antioxidant activity in tested samples. The results Total phenolic (TP) content in methanolic extracts was for antioxidant activity were compared with those determined according to the Folin-Ciocalteu colorimetric obtained with different reference compounds (ascorbic method [11] with the following modifications. An aliquot

889 Antioxidant properties of Macedonian medicinal plants

TP TF TPP Plant species (Familly) Plant part Common name (mg GAE/g DW)b (mg CE/g DW)c (mg PE/g DW)d

Achillea holosericea Sibth. & Sm. (Asteraceae) Aerial parts / 24.52 ± 1.50 11.11 ± 0.96 9.84 ± 0.31 Agrimonia eupatoria L. Aerial parts Common agrimony 59.57 ± 0.08 20.14 ± 0.20 18.34 ± 1.05 (Rosaceae) Alcea pallida Waldst. et Kit. Flowers Hollyhock 10.90 ± 0.36 1.42 ± 0.19 1.13 ± 0.12 (Malvaceae) Anthyllis vulneraria L. Aerial parts Woundwort 12.02 ± 0.42 2.22 ± 0.15 1.81 ± 0.19 (Fabaceae) Astragalus glycyphyllos L. Aerial parts Wild liquorice 15.93 ± 0.47 1.62 ± 0.40 0.94 ± 0.08 (Fabaceae) Centaurium erythraea Rafn. Aerial parts Common centaury 22.28 ± 1.07 4.48 ± 0.13 3.56 ± 0.38 (Gentianaceae) Cichorium intybus L. Aerial parts Chicory 33.36 ± 0.14 8.06 ± 0.24 10.25 ± 0.79 (Asteraceae) Clinopodium vulgare L. Aerial parts Wild basil 57.07 ± 4.15 46.33 ± 2.22 35.13 ± 2.75 (Lamiaceae) Digitalis ferruginea L. Aerial parts Rusty foxglove 13.24 ± 1.28 2.86 ± 0.54 1.81 ± 0.18 (Plantaginaceae) Digitalis lanata Ehrh. Aerial parts Woolly Foxglove 22.16 ± 1.02 11.19 ± 1.38 6.66 ± 0.75 (Plantaginaceae) Galega officinalis L. Aerial parts Goat’s rue 32.53 ± 2.80 8.95 ± 0.13 7.69 ± 1.06 (Fabaceae) Gratiola officinalisL. Leaves Hedge hyssop 26.92 ± 0.95 13.57 ± 0.61 9.88 ± 0.62 (Plantaginaceae) Helichrysum zivojinii Cernj. & Soska Aerial parts Strawflower 29.57 ± 0.34 6.24 ± 0.61 5.06 ± 0.61 (Asteraceae) Inula britannica L. Flowers British yellowhead 37.26 ± 5.78 20.33 ± 1.95 16.91 ± 1.90 (Asteraceae) Linaria angustissima (Loisel.) Borbás Aerial parts Italian toadflax 21.44 ± 1.36 3.52 ± 0.13 2.47 ± 0.04 (Scrophulariaceae) Linum hirsutum L. Aerial parts Hairy flax 18.99 ± 0.75 6.54 ± 1.32 6.72 ± 0.04 (Linaceae) Melissa officinalisL. Leaves Lemon balm 70.86 ± 1.01 45.71 ± 0.40 40.21 ± 1.87 (Lamiaceae) Origanum vulgare L. Aerial parts Wild majoram 123.41 ± 8.77 72.60 ± 5.66 58.97 ± 2.06 (Lamiaceae) Salvia nemorosa L. Aerial parts Woodland sage 47.98 ± 1.63 26.95 ± 1.34 21.47 ± 0.13 (Lamiaceae) Salvia ringens Sibth. & Sm. Aerial parts Rumanian sage 69.42 ± 1.36 49.43 ± 1.35 33.19 ± 1.15 (Lamiaceae) Salvia sclarea L. Aerial parts Clary sage 48.75 ± 4.38 25.78 ± 1.55 26.75 ± 2.28 (Lamiaceae) Sambucus ebulus L. Flowers Danewort 44.09 ± 0.75 14.86 ± 0.13 12.85 ± 1.76 (Caprifoliaceae) Saponaria officinalis L. Aerial parts Common soapwort 12.72 ± 0.89 1.24 ± 0.01 1.42 ± 0.13 (Caryophyllaceae) Sideritis raeseri Boiss et Heldr. Aerial parts Mountain tea 52.60 ± 8.75 23.52 ± 2.42 16.79 ± 2.68 (Lamiaceae) Stachys thymphaea Hausskn. Aerial parts / 21.11 ± 1.84 8.10 ± 0.13 6.72 ± 0.93 (Lamiaceae) Thymus tosevii Vel. Aerial parts Wild thyme 42.05 ± 1.94 21.33 ± 0.67 18.67 ± 0.42 (Lamiaceae) Verbena officinalisL. Aerial parts Common vervain 31.01 ± 3.74 9.78 ± 2.76 4.72 ± 0.04 (Verbenaceae)

Table 1. Total phenolic (TP), flavonoid (TF) and phenylpropanoid (TPP) content of 27 plant extracts.a

a Data are expressed as the mean of triplicate ± SD. bmg GAE/g DW: data expressed as grams of gallic acid equivalents (GAE) per gram dry weight (DW). cmg CE/g DW: data expressed as grams of catechin equivalents (CE) per gram dry weight (DW). dmg PE/g DW: data expressed as grams of pyrocatechol equivalents (PE) per gram dry weight (DW).

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of the diluted extract (100 μL) was mixed with 500 μL FeCl3. The intensity of blue-green colour was measured Folin–Ciocalteu reagent (previously diluted with water spectrophotometrically at 700 nm after incubation at 1:9 (v/v)) and 400 μL 0.7 M L-1 sodium carbonate. room temperature for 10 min. A standard curve was Samples were incubated for 5 min at 50ºC and then prepared using various concentrations of ascorbic acid cooled for 5 min at room temperature. Absorbance (1–1000 μM L-1). The RP of samples was expressed was measured spectrophotometrically at 765 nm. as μmol of ascorbic acid equivalents (AAE) per g dry The concentration of TP was calculated using gallic weight (μmol AAE/g DW). acid (0-20 mg mL-1) as a standard. The results were expressed as mg gallic acid equivalents (GAE) per g dry 2.5.2 Phosphomolybdenum method (PM) weight (mg GAE/g DW). The total antioxidant activity of plant extracts was evaluated by phosphomolybdenum (PM) method 2.4.2 Total flavonoid content (TF) according to the procedure of Prieto et al. [15]. Total flavonoid (TF) content was determined by using An aliquot of 100 μL of plant extract diluted in methanol a method described by Makris et al. [12]. A 100 μL was combined with 900 μL of reagent solution (0.6 M L-1 -1 -1 aliquot of appropriately diluted extract was mixed H2SO4, 28 mM L Na3PO4 and 4 mM L (NH4)2MoO4) and with 400 μL distilled water, then 30 μL 5% NaNO2 was assayed against a blank containing 100 μL methanol added, and allowed to react for 5 min. Following this, mixed with 900 μL of reagent solution. The samples

30 μL 10% AlCl3 was added and the mixture stood for were incubated at 95°C for 90 min and then cooled to a further 5 min. Finally, to the reaction mixture 200 μL room temperature. The absorbance of the green PM of 1 M Na2CO3 and 240 μL distilled water were added, complex was measured at 695 nm against the blank. and the absorbance at 510 nm was obtained against Ascorbic acid (0–50 mg×mL-1) was used as a standard. a blank prepared similarly, but by replacing the extract The total antioxidant capacity of extracts was expressed with distilled water. TF content was calculated from as mg ascorbic acid equivalent (AAE) per gram dry a calibration curve using catechin (0-10 mg mL-1) weight (mg AAE/g DW). as a standard. The results were expressed as mg catechin equivalents (CE) per gram dry weight (mg CE/g DW). 2.5.3 Cupric reducing antioxidant capacity assay (CUPRAC) 2.4.3 Total phenylpropanoid content (TPP) The cupric reducing antioxidant capacity (CUPRAC) of Total phenylpropanoid (TPP) content was determined plant extracts was determined according to the method using a 96-well microtiter spectrophotometric method at of Apak et al. [16]. In a test tube the following were added -1 -1 525 nm, modified from Fraisse et al. [13]. An aliquot of and mixed; 1 mL 10 mM L CuCl2, 1 mL 7.5 mM L -1 150 μL of diluted plant extract was mixed with 50 μL neocuproine and 1 mL 1 M L CH3COONH4 buffer (pH

0.5 M HCl, 50 μL of Arnow reagent (5% NaNO2 and 5% 7.0). (x) mL plant extract followed by (1.1-x) mL water -1 Na2MoO4) and 50 μL 2 M L NaOH. After incubation at were then added (total volume, 4.1 mL) and mixed well. room temperature for 10 min, absorbance was read Absorbance of the mixture was recorded against a blank at 525 nm against a blank. The concentration of TPP at 450 nm after 30 min incubation at room temperature. in the samples was calculated using pyrocatechol Total antioxidant capacity of plant samples was (0-4 mg mL-1) as a standard. The results were expressed calculated using the molar extinction coefficient of trolox as mg pyrocatechol equivalents (PE) per g dry weight (ε=1.67x104 L×mol-1×cm-1). CUPRAC values for total (mg PE/g DW). antioxidant capacity of plant extracts were expressed as μmol trolox equivalents (TE) per g dry weight 2.5 Quantification of total antioxidant and (μmol TE/g DW). scavenging capacity 2.5.1 Reducing power assay (RP) 2.5.4 ABTS radical scavenging activity Reducing power (RP) of the plant extracts was The ABTS radical scavenging activity of plant extracts determined according to the method of Oyaizu [14] was determined using the method proposed by Re with the following modifications. The reaction mixture et al. [17]. ABTS radical cation (ABTS•+) was generated contained 50 μL of diluted sample, 100 μL phosphate by reacting 7 mM L-1 aqueous ABTS [2,2’-azinobis(3- buffer (0.2 M, pH 6.6) and 100 μL of 1% K3[Fe(CN)6]. ethylbenzothiazoline-6-sulphonic acid) diammonium

After incubation at 50ºC for 20 min, 100 μL of 10% TCA salt] and 2.45 mM K2S2O8 in a volume ratio of 1:1 was added to the mixture followed by centrifugation (v/v). The mixture was incubated overnight in the dark at 1000 rpm for 10 min. Finally, 100 μL of supernatant (12–16 h) at room temperature. On the day of analysis, was combined with 100 μL methanol and 20 μL 0.1% the generated blue-green ABTS•+ solution was diluted

891 Antioxidant properties of Macedonian medicinal plants

with methanol to an absorbance of 0.70 (±0.05) at from 10.90 to 123.41 mg GAE/g DW, which represents 734 nm. All extracts were diluted with methanol to give a variation of approximately 11-fold. Our results on TP 20–80% inhibition of the blank absorbance. The reaction content were within the range of values reported by was initiated by the addition of 1990 μL of ABTS•+ solution Shan et al. [19] for 26 spices originating from Asian and to 10 μL of each plant extract and mixed thoroughly. Western countries. Interestingly, Surveswaran et al. Control samples contained methanol instead of plant [20] reported very high variation (695-fold) in TP for 133 extract. The mixture was allowed to stand for 6 min at Indian medicinal plants, however, it is not appropriate room temperature and the absorbance was immediately to directly compare these data with the results from our recorded at 734 nm. Trolox solution (1–1000 μM L-1) study due to differences in the families of plant species, was used as a reference standard. The absorbance of and heterogeneity of the plant parts tested. Results samples was compared to that of trolox standards and showed that Origanum vulgare possessed the highest the results were expressed as μmol trolox equivalents TP (>100 mg GAE/g DW), which is in accordance with (TE) per g dry weight (μmol TE/g DW). previously reported data for plants collected in Turkey [21]. Melissa officinalis and Salvia ringens extracts 2.5.5. DPPH radical scavenging activity also showed high TP (70.86 and 69.42 mg GAE/g DW, Ability of the extracts to scavenge the stable free respectively) in comparison with other evaluated plant radical 2,2-diphenyl-1-picrylhydrazyl (DPPH•) was species. On the other hand, Saponaria officinalis, Anthyllis determined following the procedure described by Brand- vulneraria and Alcea pallida extracts showed very low TP Williams et al. [18] with the following modifications. (12.72; 12.02 and 10.90 mg GAE/g DW, respectively), In the presence of an antioxidant, the purple colour of which is in agreements with previous reports [21,22]. DPPH• decays, and the change in absorbance can be In contrast, Godevac et al. [23] noticed relatively high followed spectrophotometrically at 517 nm. The reaction TP content in the methanolic extracts of A. vulneraria. mixture consisted of 10 μL extract and 290 μL Significant differences in TP contents could be due to 0.25 mM L-1 DPPH methanolic solution. A control sample genotypic and environmental variations (climate, location, was included, in which extract was replaced by methanol. temperature, fertility and diseases) within species, plant The reaction for scavenging DPPH• was carried out at part tested, harvesting time and extraction procedure room temperature in the dark for 10 min, and then the [19,24]. Moreover, it is known that the Folin-Ciocalteu reduction in absorbance was recorded at 517 nm against method gives variable results depending on the chemical the methanol blank. A calibrated trolox standard curve structures of phenolic compounds [25]. (1–1000 μM L-1) was also made. The scavenging capacity Results for TF content in plant extracts showed of plant extracts was expressed as μmol trolox equivalents variation of about 58-fold, ranging from 1.24 to (TE) per g dry weight (μmol TE/g DW). 72.60 mg CE/g DW (Table 1). Patel et al. [26] reported a similar variation (65-fold) in TF of selected medicinal 2.6 Statistical analysis plants from the western region of India. In contrast, Results were expressed as means ± standard deviation Bouayed et al. [27] reported lower amounts of TF for (SD) of three measurements. Correlations among data plants collected from Iran, while Zhang et al. [28] obtained were calculated using MS Excel software noticed higher TF for Chinese traditional plants. It is not correlation coefficient statistical option. All statistical appropriate to directly compare these data with our tests were considered significant at p < 0.05. results owing to the differences in the procedure for quantification of TF and reference compounds used (e.g. quercetin and rutin). From the plant extracts 3. Results and Discussion tested, the highest TF were found in O. vulgare (72.60 mg CE/g DW). Relatively high TF levels 3.1 Phenolic compounds content (40-50 mg CE/g DW) were observed in S. ringens, In this study, we measured the total phenolic compound Clinopodium vulgare and M. officinalis extracts. content (phenolics, flavonoids and phenylpropanoids) In addition to these results, moderate levels of TF and we tested the complex matrices of these compounds (20-30 mg CE/g DW) were found in Salvia nemorosa, that could contribute to the total antioxidant status of Salvia sclarea, Sideritis raeseri, Thymus tosevii, plant species. Among the plant species used in this Inula britannica and Agrimonia eupatoria extracts. study, we observed large variations in the content of It is noteworthy that while relatively high TF was antioxidant compounds (Table 1). noticed for S. ringens (49.43 mg CE/g DW), other As shown in Table 1, there is a large variation in TP Salvia species, e.g. S. nemorosa and S. sclarea content of the plant species examined. The values varied extracts showed moderate levels of TF content (26.95

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and 25.78 mg CE/g DW, respectively). This finding is conditions; consequently, in different methods, supported by Miliauskas et al. [29] who found variable particular antioxidants have varying contributions to total amounts of flavonoids in Salvia pratensis, Salvia antioxidant potential [34]. Therefore, no single method is officinalis, S. sclarea and Salvia glutinosa. Because of sufficient and more than one type of antioxidant capacity the high amounts of TF in the extracts, S. ringens may measurement needs to be performed to take into become a promising source of flavonoid production. account the various modes of action of antioxidants [35]. Similarly, our results showed inter-species variation Furthermore, the chemical complexity of extracts, often in TF for two examined Digitalis species, i.e. Digitalis a mixture of various compounds with different functional ferruginea and Digitalis lanata (2.86 and 11.19 mg groups, polarity and chemical behaviour, could lead to CE/g DW, respectively). According to Jiang et al. scattered results, depending on the antioxidant assay [30], this heterogeneity in the amount of TF may be employed. Another important aspect is the selection attributed to inter-species or inter-varietal variation, as of appropriate reference compounds to compare the well as environmental conditions such as location and antioxidant activity of plant samples. Consequently, harvesting season. It is known that TF content can vary extracts showing poor antioxidant properties with one in different plant parts of the same plant species [31]. concrete method should not be discarded as poor On the other hand, Astragalus glycyphyllos, A. pallida sources of antioxidants without having been tested with and Saponaria officinalis extracts showed very low TF other methods and compared with different reference levels (1.62; 1.42 and 1.24 mg CE/g DW, respectively), standards. An approach that utilizes multiple assays for (Table 1). Our results reveal that TP and TF show evaluating the antioxidant potential of extracts is more similar ranking tendency in the plant species evaluated. informative and arguably necessary. Therefore, we Specifically, O. vulgare is a potentially rich source used five different methods for in vitro evaluation of the of phenolics and flavonoids, while A. pallida extract total antioxidant properties of 27 Macedonian traditional is a poor source of these metabolites. These data medicinal plants. Methods used in this study were suggest that different plants contain similar levels of TF selected as the most appropriate for rapid screening and as a portion of TP compounds. estimation of total antioxidant potential of plant extracts. Results from our study showed a wide variation The reducing power (RP) of selected plant species in TPP content of plant species tested (0.94 was determined by the potassium ferricyanide method to 58.97 mg PE/g DW), which represents a variation and this assay represents a very important parameter of approximately 63-fold (Table 1). These data are for estimation of total antioxidant activity. The plant similar to those reported by Fraisse et al. [13] for certain extracts examined in this study demonstrated good mountain grassland plants. The highest amounts of TPP RP, thereby acting as efficient reductones. Reductones (>50 mg PE/g DW) were found in extracts of O. vulgare are reported to be terminators of free radical chain (58.97 mg PE/g DW). As shown in Table 1, the extracts reactions [36]; thus, the antioxidant activity of plant of M. officinalis, C. vulgare, S. ringens, S. sclarea and extracts may be related to their RP. Results from S. nemorosa also showed remarkably high amounts of our study showed that different plant extracts gave TPP (20-40 mg PE/g DW). The lowest concentration RP values ranging from 26.72 to 1108.82 μmol of TPP (<1 mg PE/g DW) was found in extracts of AAE/g DW, representing variation of approximately A. glycyphyllos (Table 1). Of the 12 families tested in 42-fold (Table 2). As shown in Table 2, O. vulgare this study, only Lamiaceae species exhibited high levels possessed the strongest RP (1108.82 μmol AAE/g of total phenolic compounds. The Lamiaceae family DW), followed by S. ringens (805.88 μmol AAE/g DW) includes many common traditional medicinal plants, and M. officinalis extracts (677.21 μmol AAE/g DW). e.g. peppermint, sage, thyme, oregano, lemon balm and Our results indicate that these plant species may well basil. Recent studies showed that these plant species act as electron donors and they can react with free contain high TP content and exhibit strong antioxidant radicals to convert them into more stable products. effects [32,33]. From the results summarised in Table 1, In the RP assay, electron-donation capacity of samples we conclude that O. vulgare possess the highest was compared to that of tested reference compounds. amounts of phenolic compounds of the species tested, Results showed that extracts possessed some degree including phenolics, flavonoids and phenylpropanoids. of electron donation capacity, but the capacities were, as expected, inferior to positive controls. Namely, 3.2 Antioxidant activity and scavenging ascorbic acid, as a strong reducing agent, showed more capacity pronounced RP (>10 000 μmol AAE/g DW) than that of Methods used for antioxidant activity determination the plant extracts examined. Ascorbic acid is a strong differ in terms of their assay principles and experimental reductone that could readily donate a hydrogen atom

893 Antioxidant properties of Macedonian medicinal plants

RP PM CUPRAC ABTS DPPH Plant species (μmol AAE/g DW)b (mg AAE/g DW)c (μmol TE/g DW)d (μmol TE/g DW)d (μmol TE/g DW)d

Achillea holosericea 156.99 ± 30.68 20.36 ± 0.40 174.88 ± 14.37 204.04 ± 3.64 129.84 ± 6.11

Agrimonia eupatoria 456.25 ± 45.23 33.27 ± 0.95 419.26 ± 1.63 504.71 ± 39.90 357.07 ± 30.54

Alcea pallida 26.72 ± 5.01 11.91 ± 0.79 52.89 ± 1.08 51.52 ± 6.99 56.73 ± 0.68

Anthyllis vulneraria 31.62 ± 6.24 17.36 ± 0.91 88.91 ± 9.21 113.64 ± 13.57 68.02 ± 0.83

Astragalus glycyphyllos 73.77 ± 5.22 22.32 ± 0.11 63.23 ± 3.52 134.34 ± 11.43 49.27 ± 7.57

Centaurium erythraea 110.29 ± 3.12 50.09 ± 0.98 105.64 ± 4.61 152.53 ± 9.64 79.29 ± 1.22

Cichorium intybus 271.32 ± 8.32 18.75 ± 1.87 211.16 ± 5.70 364.31 ± 3.82 154.40 ± 0.20

Clinopodium vulgare 562.87 ± 1.56 47.79 ± 5.03 525.99 ± 15.99 489.90 ± 39.57 213.57 ± 17.65

Digitalis ferruginea 97.79 ± 11.84 21.66 ± 0.40 62.98 ± 1.17 83.84 ± 1.43 64.02 ± 0.95

Digitalis lanata 135.29 ± 10.40 22.72 ± 0.88 152.78 ± 1.97 111.11 ± 4.63 113.31 ± 3.05

Galega officinalis 220.59 ± 25.99 26.04 ± 0.41 191.23 ± 4.61 326.77 ± 22.14 118.64 ± 3.94

Gratiola officinalis 209.19 ± 20.28 20.55 ± 0.69 169.26 ± 1.35 189.39 ± 2.14 121.47 ± 4.00

Helichrysum zivojinii 155.15 ± 8.32 15.49 ± 2.05 139.69 ± 10.57 221.55 ± 12.18 106.74 ± 1.76

Inula britannica 250.74 ± 5.20 19.55 ± 1.48 252.55 ± 37.67 232.83 ± 3.57 140.62 ± 10.86

Linaria angustissima 43.75 ± 8.36 43.92 ± 1.39 80.61 ± 5.70 137.37 ± 4.63 62.34 ± 2.24

Linum hirsutum 99.26 ± 16.64 16.16 ± 1.17 120.34 ± 12.19 104.04 ± 22.86 101.36 ± 0.68

Melissa officinalis 677.21 ± 12.48 54.00 ± 1.14 542.28 ± 0.54 775.25 ± 3.57 406.03 ± 13.57

Origanum vulgare 1108.82 ± 18.72 71.22 ± 1.37 1068.58 ± 57.99 1240.74 ± 3.82 714.15 ± 12.22

Salvia nemorosa 403.31 ± 46.27 38.33 ± 0.91 378.25 ± 7.05 283.33 ± 17.86 213.09 ± 14.25

Salvia ringens 805.88 ± 6.24 57.56 ± 0.09 630.42 ± 20.05 809.60 ± 43.57 329.72 ± 29.86

Salvia sclarea 455.15 ± 12.15 40.41 ± 3.27 381.06 ± 34.14 309.09 ± 43.57 223.41 ± 19.34

Sambucus ebulus 247.30 ± 35.30 31.26 ± 1.50 220.55 ± 10.03 283.33 ± 0.71 140.38 ± 13.91

Saponaria officinalis 37.87 ± 6.40 16.56 ± 0.22 60.04 ± 0.27 64.65 ± 7.14 56.87 ± 1.83

Sideritis raeseri 484.19 ± 11.96 41.88 ± 4.44 304.67 ± 48.78 377.27 ± 35.00 160.54 ± 4.41

Stachys thymphaea 152.57 ± 3.64 15.59 ± 0.71 160 ± 4.61 130.30 ± 1.43 102.42 ± 14.11

Thymus tosevii 314.34 ± 8.84 30.95 ± 1.04 414.79 ± 0.27 287.88 ± 15.71 223.25 ± 6.79

Verbena officinalis 197.79 ± 20.18 40.59 ± 0.18 120.72 ± 7.59 178.45 ± 7.45 88.64 ± 0.07

AAe 12552.42 ± 773.22 817.22 ± 14.65 3009.68 ± 281.06 8138.86 ± 219.15 5418,92 ± 54,48

Qe 8032.67 ± 647.89 360.45 ± 13.96 9336.63 ± 142.40 5959.22 ± 72.71 8026.54 ± 183.58

Ce 8218.70 ± 896.80 645.63 ± 7.38 9164.84 ± 97.55 7842.46 ± 635.68 4938.14 ± 140.51

α-Te 2687.33 ± 157.85 477.22 ± 15.53 2488.81 ± 156.93 2685.04 ± 117.64 2221.83 ± 30.39

BHTe 6493.24 ± 206.25 519.81 ± 22.58 4599.41 ± 134.16 1356.02 ± 43.05 559.17 ± 28.75

BHAe 9319.34 ± 947.05 606.62 ± 19.86 6422.31 ± 142.80 2476.47 ± 154.10 4317.85 ± 179.08

Table 2. Reducing power (RP), phosphomolybdenum method (PM), cupric reducing antioxidant capacity (CUPRAC), 2,2’-azinobis(3- ethylbenzothiazoline-6-sulphonic acid (ABTS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity.a

aData are expressed as the mean of triplicate ± SD. bµmol AAE/g DW: data expressed as micromoles of ascorbic acid equivalents (AAE) per gram dry weight (DW). cmg AAE/g DW: data expressed as milligrams of ascorbic acid equivalents (AAE) per gram dry weight (DW). dµmol TE/g DW: data expressed as micromoles of trolox equivalents (TE) per gram dry weight (DW). eAscorbic acid (AA), quercetin (Q), catechin (C), α-tocopherol (α-T), butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA) were used as reference compounds.

894 O. Tusevski et al.

to a free radical, thus terminating free radical reactions demonstrated similar CUPRAC value ranges as those [37]. However, the RP of selected plant species was previously reported by Apak et al. [44], for aqueous herbal lower than that of the positive controls most likely due to extracts (50-1639 μmol TR/g DW). Results showed that the fact that the extracts contain antioxidant compounds only O. vulgare extracts had very strong CUPRAC activity at much lower quantities. (>1000 μmol TR/g DW). Moderate CUPRAC activity Phosphomolybdenum (PM) assay has been (>500 μmol TR/g DW) was noticed for S. ringens and routinely used to evaluate total antioxidant capacity C. vulgare (630.42 and 525.99 μmol TR/g DW, of plant extracts. In the presence of extracts, Mo (VI) respectively), while A. pallida extracts possessed is reduced to Mo (V) and forms a green-coloured the lowest CUPRAC values (52.89 μmol TR/g DW). phosphomolybdenum V complex at acidic pH, which CUPRAC values of the plant samples examined were shows maximum absorbance at 695 nm [15]. Because not comparable to those of reference compounds. This is of its simplicity and the low cost of the reagents used, understandable, since these compounds are already in the PM assay is a good alternative to the methods a pure form, while the crude plant extracts still need to be already available for evaluation of total antioxidant processed in order to isolate the compounds responsible capacity [38]. Results from our study show that values for their antioxidant activity. It is worth mentioning that for the PM assay vary from 11.91 to 71.22 mg AAE/g the CUPRAC assay is useful for determining antioxidant DW (Table 2). The variation observed between different capacity in a wide variety of polyphenols, including plant extracts could be explained by the fact that the PM phenolic acids, flavonoids, carotenoids, anthocyanins, assay evaluates the antioxidant activity of polyphenols as well as for thiols (glutathione), synthetic antioxidants, and other non-phenolic hydrophilic and lipophylic vitamins C and E. Furthermore, the results obtained compounds [15]. Among the selected plant species from these in vitro measurements may be more relevant tested in this study, O. vulgare showed the highest to the possible in vivo reactions of antioxidants because value in the PM assay (71.22 mg AAE/g DW). Different the CUPRAC reaction is carried out at pH 7.0, close to groups studying the antioxidant activity of oregano physiological pH [16]. alcohol extracts revealed that strong antioxidant activity A combination of rapid, sensitive, and reproducible of O. vulgare might be attributed to the presence of methods, preferably requiring small sample amounts, phytochemicals such as rosmarinic acid, caffeic acid, should be used whenever antioxidant activity screening tocopherols, carvacrol and thymol [39-41]. In this study, is designed. A rapid estimation of radical scavenging relative high PM values (50-60 mg AAE/g DW) were activity, by using the nitrogen-centered free radicals also noticed in S. ringens, M. officinalis and Centaurium DPPH and ABTS, could save much laboratory work and erythraea extracts. In contrast, the lowest PM value be very helpful in lead-finding of novel antioxidants in was found in A. pallida extracts (11.91 mg AAE/g DW). phytochemical screening procedures. None of the plant extracts used in this assay were as In this study, we selected the DPPH method as effective as the positive controls. Several studies have one of the most effective assays for evaluating radical- been carried out assessing the antioxidant potential of scavenging capacity of plant extracts by the chain- medicinal plants using the PM method [33,42]. Marwah breaking mechanism. A freshly prepared DPPH● et al. [43] studied the antioxidant capacity of 19 edible solution exhibits a deep purple colour with an absorption and wound-healing herbs from Oman using the PM maximum at 517 nm [45]. This purple colour generally assay and revealed that traditional uses of these plants disappears when an antioxidant is present in the are rationalised based on their antioxidant capacity. medium. Thus, antioxidant molecules can quench The CUPRAC method for total antioxidant DPPH● free radicals (2,2-diphenyl-1-picrylhydrazyl) and capacity is based on the absorbance measurement of convert them to a colourless product (2,2-diphenyl-1- chromophore, Cu(I)-neocuproine (Nc) chelate, formed picrylhydrazine), resulting in a decrease in absorbance. as a result of a redox reaction of antioxidants with Cu(II)- Plant extracts used in this study showed varying levels neocuproine reagent. The CUPRAC assay has been of DPPH scavenging activity ranging from 49.27 to widely used to determine antioxidant capacities of plant 714.15 μmol TE/g DW (Table 2). Specifically, O. vulgare extracts as it requires relatively standard equipment extracts exhibited the highest DPPH radical scavenging and delivers fast and reproducible results [16]. A wide activity, with a value of 714.15 μmol TE/g DW. Studies range of CUPRAC antioxidant capacity was observed in of O. vulgare DPPH scavenging activity showed that the plant extracts analysed, as shown in Table 2. Plant protocatechinic acid, caffeic acid and rosmarinic acid are extracts gave CUPRAC values ranging between 52.89 the major phenolic compounds with antioxidant properties and 1068.58 μmol TR/g DW, representing variation in oregano extracts [41,46,47]. In our study, extracts from of approximately 20-fold. Results from our study M. officinalis, A. eupatoria and S. ringens also showed

895 Antioxidant properties of Macedonian medicinal plants

high DPPH values (> 300 μmol TE/g DW). Previously hydrogen-donating groups (-OH, -NH, -SH) in the reports suggest that the antioxidant property of methanolic aromatic ring and glycosylation [9], it is possible that this extracts of lemon balm (M. officinalis) is due to the high could also be responsible for the degree of variation in portion of phenolic acids, such as rosmarinic acid, the scavenging activity of extracts. Results show that protocatechuic acid, caffeic acid and their methyl esters 9 of the 27 plant species tested contained more than [48,49]. In our study, alcoholic extracts of A. eupatoria also 300 μmol TE/g DW, and only 3 species were below exhibited high DPPH values, which might be due to the 100 μmol TE/g DW. The majority of tested plants showed presence of flavonoids, including flavan-3-ols, flavonols, mid-level ABTS capacity, i.e. 15 species exhibited flavones and phenolic acids [50,51]. The results for values from 100 to 300 μmol TE/g DW. In this study, DPPH scavenging activity of Salvia species examined in O. vulgare extracts showed the highest antioxidant capacity this study were inconsistent with that previously reported (1240.74 μmol TE/g DW). Our results are similar to by Miliauskas et al. [29]. Various Salvia species were ABTS values previously reported for oregano extracts shown to demonstrate remarkable scavenging activity [19,21,41]. In addition, Wojdylo et al. [55] pointed out that against DPPH radicals [52-54]. In contrast, results from O. vulgare antioxidant activity is a result of the presence our study showed that Saponaria officinalis, A. pallida of caffeic acid and p-coumaric acid. Our comparative and A. glycyphyllos had the lowest DPPH scavenging study showed that for the 3 Salvia species tested, activity (< 70 μmol TE/g DW). It is worth noting that strong S. ringens had the strongest ABTS scavenging DPPH scavenging activity was found in plant extracts properties (809.60 μmol TR/g DW), followed by moderate rich in phenolic contents. Namely, O. vulgare proved to activity of S. sclarea and S. nemorosa (309.09 and have the highest DPPH scavenging activity and also 283.33 μmol TR/g DW, respectively). Similarly, possess the highest phenolic (123.41 mg GAE/g DW), Miliauskas et al. [29] reported strong ABTS scavenging flavonoid (72.60 mg CE/g DW) and phenylpropanoid capacity by S. officinalis, compared to moderate activity (58.97 mg PE/g DW) contents. The DPPH values of of S. pratensis, S. glutinosa and S. sclarea extracts. O. vulgare extracts were significantly lower than those Results from this study revealed that D. ferruginea, of reference compounds, but higher than that of BHT Saponaria officinalis and A. pallida possess the lowest (Table 2). These results suggest that O. vulgare extracts ABTS values compared to other species tested. contain different classes of antioxidant phenolics with As shown in Table 2, plant extracts demonstrate high hydrogen-donating capacity to scavenge DPPH remarkably lower ABTS scavenging activity than the radicals. In contrast, A. glycyphyllos had the lowest reference compounds. DPPH scavenging activity, which correlates with its lower In this study, the results for ABTS and DPPH phenolic (15.93 mg GAE/g DW), flavonoid (1.62 mg CE/g scavenging activity were expressed as the same unit DW) and phenylpropanoid (0.94 mg PE/g DW) contents. (μmol TE/g DW), in order to directly compare the values It is noteworthy that the DPPH radical scavenging from these two methods. Antioxidant activity measured assay measures only the activity of the water-soluble by DPPH● showed similar trends to those observed fraction of plant extracts. As such, the ABTS●+ assay is using the ABTS●+ method, however the values were more versatile as both the polar and non-polar samples lower, suggesting that the kinetics of radical scavenging (lipophilic and hydrophilic antioxidants) can be assessed reactions in these two systems differ. The results from for their scavenging activity. both radical-scavenging assays show that extracts The ABTS free-radical scavenging assay overcomes from medicinal plants tested in this study might be limitations of the DPPH method such as solubility used in prevention of biomolecules (lipoproteins, fatty and problems of spectral interference. In particular, acids, DNA, amino acids, proteins and sugars) from the spectral interference is minimised as the long reactive radical attack in susceptible biological systems. wavelength absorption maximum at 734 nm eliminates It is worth noting that traditional medicinal plants from colour interference in plant extracts [17]. In this study, our study could be used as a promising source of natural all plant extracts tested showed capacity to scavenge antioxidants. the ABTS●+ radical (Table 2). ABTS values ranged from 51.52 to 1241.92 μmol TE/g DW and differences 3.3 Correlation between the contents of phenolic in scavenging activity were quite large, up to 24-fold. compounds and antioxidant capacity The scavenging effects may result from the action of The relationship between antioxidant activity of plant antioxidant metabolites present in the corresponding extracts and their phenolic composition is very difficult medicinal plants. However, as the radical scavenging to describe due to the fact that antioxidant properties of activity of phenolics (e.g. flavonoids, phenylpropanoids) single compounds within the group can vary remarkably, mainly depends on the number and position of and therefore equal levels of phenolics do not necessarily

896 O. Tusevski et al.

(R)a TPb TFb TPPb RPc PMc CUPRACc DPPHc

TFb 0.949*** TPPb 0.955*** 0.986***

RPc 0.976*** 0.974*** 0.967***

PMc 0.806*** 0.800*** 0.773*** 0.806***

CUPRACc 0.974*** 0.974*** 0.976*** 0.975*** 0.775***

DPPHc 0.960*** 0.908*** 0.930*** 0.933*** 0.725*** 0.963***

ABTSc 0.964*** 0.931*** 0.929*** 0.965*** 0.785*** 0.954*** 0.953***

Table 3. Correlation analysis between antioxidant contents (TP, TF, TPP), antioxidant capacity (RP, PM, CUPRAC) and radical scavenging assays (DPPH, ABTS) of 27 plant extracts (n=27 samples).

aR, Correlation coefficient. ***Significance level at p < 0.001. bAbbreviations correspond to those in Table 1. cAbbreviations correspond to those in Table 2. correspond to the antioxidant response. In addition, phenolic constituents with antioxidant properties. different methods used to determine the antioxidant In accordance with previous reports [42,44,57-59], these activity are based on different mechanisms of reaction, findings suggest that phenolic compounds significantly so that they often give different results. Furthermore, contribute to the antioxidant activity in medicinal plants plant extracts are complex mixtures of many different used in this study. Moreover, antioxidant activity of compounds with antioxidant and prooxidant properties, phenolic compounds is mainly due to their redox showing synergic actions in comparison to individual properties, which allow them to act as reducing agents, compounds [56]. hydrogen donors, heavy metal chelators and radical In this study, correlation analyses of values scavengers [9,60,61]. for total antioxidant capacity (CUPRAC, PM, RP), radical scavenging activity (ABTS, DPPH) and phenolic compounds (TP, TF, TPP) were performed. 4. Conclusions The correlation coefficients (R) are listed in Table 3. Significantly positive linear correlations (Table 3) In conclusion, antioxidant potential and phenolic were established between total phenolics and RP contents of 27 Macedonian traditional medicinal plants (R=0.976; ***p<0.001), PM (R=0.806; ***p<0.001), were evaluated for the first time. Antioxidant properties CUPRAC (R=0.974; ***p<0.001), DPPH and ABTS and total phenolic amounts differed significantly (R=0.960 and R=0.964; ***p<0.001, respectively). among the selected plant species. Among these plant Strong positive correlations (Table 3) were also found extracts, O. vulgare showed the strongest antioxidant between total flavonoids and RP (R=0.974; ***p<0.001), activity and the highest total phenolic content. From PM (R=0.800; ***p<0.001), CUPRAC (R=0.974; the results presented for S. ringens, it can be proposed ***p<0.001), DPPH and ABTS scavenging activity that this poorly studied plant is a promising source of (R=0.908 and R=0.931; ***p<0.001, respectively). The natural antioxidants. A significant correlation between contents of total phenylpropanoids showed remarkably antioxidant properties and phenolic contents was found positive correlations with RP (R=0.967; ***p<0.001), PM indicating that flavonoids and phenylpropanoids are the (R=0.773; ***p<0.001), CUPRAC (R=0.976; ***p<0.001), major contributor to the antioxidant properties of selected DPPH and ABTS assays (R=0.930 and R=0.929; plants. The investigation of inter-relationship between ***p<0.001, respectively). Results from this study also phenolics and antioxidant activity will be a promising revealed significantly positive correlations (Table 3) field to understand and elucidate possible mechanisms of phenolics with flavonoids (R=0.949; ***P<0.001) and for utilization of selected medicinal plants as sources of phenylpropanoids (R=0.955; ***P<0.001), as well as bioactive compounds in the food and pharmaceutical between flavonoids and phenylpropanoids (R=0.986; industries. Through our systematic comparative study, ***P<0.001). Significant positive correlations between certain traditional Macedonian plants, especially those phenolic contents (TP, TF and TPP) and antioxidant belonging to the Lamiaceae family, can be considered assays (CUPRAC, RP, PM, ABTS, DPPH) indicated as excellent free-radical scavengers and potent natural that flavonoids and phenylpropanoids are the main antioxidants for commercial exploration.

897 Antioxidant properties of Macedonian medicinal plants

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