Comparison of extraction methods for the assessment of total polyphenol content and in vitro antioxidant capacity of horsemint (Mentha longifolia (L.) L.)

Journal of Applied Research on Medicinal and Aromatic Plants 15 (2019) 100220

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Comparison of extraction methods for the assessment of total polyphenol content and in vitro antioxidant capacity of horsemint (Mentha longifolia (L.) L.)

T

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Katalin Patonaya, , Helga Szalontaia, Julianna Csugányb, Orsolya Szabó-Hudáka, Erika Pénzesné Kónyac, Éva Zámboriné Némethd a

Food and Wine Research Institute, Eszterházy Károly University, 8 Leányka st., H-3300, Eger, Hungary Department of Economics, Eszterházy Károly University, 4 Egészségház st., H-3300, Eger, Hungary Department of Botany and Plant Physiology, Eszterházy Károly University, 8 Leányka st., H-3300, Eger, Hungary d Department of Medicinal and Aromatic Plants, Szent István University, 29-43 Villányi st., H-1118, Budapest, Hungary b c

A R T I C LE I N FO

A B S T R A C T

Keywords: Mentha longifolia Antioxidant Polyphenol Extraction Ultrasound

Horsemint (Mentha longifolia (L.) L.) is a less studied, wild-growing species. Aim of the experiments was to define effective extraction method providing highest antioxidant capacity and polyphenol content from the plant, for long-term goal of food preservation uses. We applied Soxhlet and ultrasonic (US) extraction, both with methanol (MeOH) and ethanol: water in 7:3 (WA) as solvents. Thirty-six accessions were analysed for total phenolic content (TPC) by Folin-Ciocalteu method and antioxidant capacity (AOC) by DPPH and FRAP assays. The TPC of samples showed that the result of extraction methods is dependent on the solvent: MeOH extracts produced higher values by the US method while WA extracts gave better contents by Soxhlet. Concerning radical scavenging activity (DPPH) and ferrous reducing activity (FRAP), both values were higher in samples produced by US extraction method using WA as solvent. Large variability of TPC and AOC of the 36 plant samples indicates high diversity in polyphenol composition.

1. Introduction Horsemint (ML) (Mentha longifolia (L.) L., Lamiaceae) is a perennial wild-growing plant being indigenous in temperate and Mediterranean parts of Eurasia and Africa ML is a spice plant, besides, in ethnomedicine is used for cough, cold and gastrointestinal problems e.g. in Turkey and Arabic countries (Fatiha et al., 2015). Besides collection, ML is cultivated in Tunisia (Hajlaoui et al., 2009) and the taxon M. longifolia var. schimperii syn. ssp. schimperii in Sudan and Saudi Arabia (Younis and Beshir, 2004). Contrary to its abundance, ML is less known and rarely used in Europe and about industrial uses no data have been found. Beside the volatile terpenoids which were described in screenings e.g. of a greater number of Turkish ML accessions (Başer et al., 1999, 2012) ML is rich in phenolic compounds which may enable the use of this species as a source of preservative preparations. Rosmarinic acid has been found as the dominant caffeic acid derivative in this

species. According to the only survey of greater number of ML samples to antiradical activity and major antioxidants (on Israeli accessions) its concentration can reach 20000–80000 mg/kg DW (Dudai et al., 2006). Caffeic acid derivatives are accompanied by various flavonoids. Flavones, mostly apigenin and luteolin derivatives have been detected (Ghoulami et al., 2001; Krzyzanowska et al., 2011; Stanislavljević et al., 2012). Glycosides of flavonols quercetin (Benedec et al., 2013) and kaempferol (Stanislavljević et al., 2012), and flavanones are also present (Shaiq et al., 2002; Hawrył et al., 2016). Quercetin derivatives can also enhance antioxidant activity of a properly processed ML extract. (Patonay et al., 2017) In order to assure a standard quality for applications in industry, evaluation of intraspecific chemical diversity and determining the potential value of the taxa seems to be necessary. For an accurate comparison of samples and development of practical applications, optimization of the extraction procedure cannot be avoided. Thus, also to the

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Corresponding author. E-mail addresses: [email protected] (K. Patonay), [email protected] (H. Szalontai), [email protected] (J. Csugány), [email protected] (O. Szabó-Hudák), [email protected] (E.P. Kónya), [email protected] (É.Z. Németh). https://doi.org/10.1016/j.jarmap.2019.100220 Received 7 February 2019; Received in revised form 9 August 2019; Accepted 28 August 2019 Available online 09 September 2019 2214-7861/ © 2019 Elsevier GmbH. All rights reserved.

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(Eszterházy Károly University, Department of Botany and Plant Physiology), samples JOF, HV1, HV2, HV3, HOV1, HOV2, HOV3, KÜH, JÁV EGR4 by the collector Helga Szalontai, other samples: by Erika Pénzesné Kónya. Identification was based on the manual of the Hungarian vascular flora (Simon, 1994). Deposition: in the herbarium of Esterházy Károly University (EGR) in the chemotaxonomical collection (EGR-CH). In each population, the flowering shoots were cut at approx. 10 cm height from the ground. The plants were harvested in 2016 in full flowering phase, except populations KBÁ and MÁH1 (beginning flowering) as well as MAC and NÁD (end of flowering). 5–20 shoots were harvested depending on the size of the population in order to assure sustainability of the stands. Samples were dried hanged in shady, dry place at room temperature for 21 days. From the dried shoots, leaves and flowers were stripped and stored in plastic bags in a deep freezer (−18 °C) until further processing. For extraction ca. 2 g was taken from frozen dry herb and tempered in desiccator to room temperature.

correct screening of the in vitro antioxidant properties and total polyphenol content of ML samples, optimization of the extraction is a criterion. These assays are important parts of the evaluation of a plant species as a potential source of preservatives, e.g when optimal harvesting time is a question (Varga et al., 2016) or genetically different samples in a species are compared (Dudai et al., 2006). Unfortunately, the available references on this species do not represent a reliable background to establish a standardized procedure. Therefore, the goal of the present work was to define an effective extraction method of horsemint resulting in the highest antioxidant capacity and polyphenol content of the samples. This study also means of the first comprehensive evaluation of greater number of samples of this species with Central European origin. For this purpose, the wellknown Soxhlet method is compared to a three stage extraction performed in a cooled ultrasonic bath. There is a range of studies which conclude that sonication in many cases reduces temperature of extraction and enhances its yield and efficiency, depending on the type of the constituents to extract and on the plant matrix (Bahmania et al., 2018). No reference has been found dealing with extractability of polyphenols of horsemint. Besides, no similar work has been known to deal with extraction methods using a greater number of samples as reliable reference material for comparison.

2.2. Extraction 2.2.1. Extraction solvents Methanol (MeOH) was taken as a reference, because it is widely used for plant polyphenol extraction, and, for this species, most of investigators use it as solvent, e.g.: Hajlaoui et al. (2009), Gulluce et al. (2007), Iqbal et al. (2013) and Murad et al. (2016). The other solvent was a water-ethyl alcohol mixture in a ratio of 3:7 (WA). This is also applied as a medium of polyphenol rich tinctures (e.g. Tinctura salviae). Although it is in the literature of this species is less mentioned (Stanislavljević et al., 2012; Benedec et al., 2013) in our previous investigation it has shown high efficiency in extraction of polyphenols of

2. Materials and methods 2.1. Plant material and sampling Thirty-six spontaneous populations of ML from Northern Hungary have been sampled. The original locations are shown in Table 1 and Fig. 1. Identification: sample NOSZ, by the collector Jana Táborska Table 1 Origin of the investigated ML samples. Identifier

Location

EGR1 KBÁ EGR2 JOF MBA1 MBA2 DEK SZU MDE1 MDE2 HOV1 HOV2 HOV3 HV1 HV2 HV3 MÁH1 MÁH2 MAC HAS EGR3 EGR4 FET BÜK MDE3 NÁD JÁV KÜH SZD TIB TLE PÉV1 PÉV2 HEA VÁR NOSZ

Eger/Leányka st. Bátonyterenye-Kisbátony, Szarisznyó stream Eger/Zúgó st., isle in Eger stream Jósvafő, Dózsa st. Mátraballa/between a dirt road & railway Mátraballa/Rákóczi st. Railway tracks at ‘Dekics-juss’ railway/highway crossing Near ‘Szurdok’ Hill between Mátraderecske &Mátraballa Mátraderecske, meadows near Balla stream Mátraderecske/Nagyrét Meadow Hosszúvölgy-1 sample Hosszúvölgy-2 sample Hosszúvölgy-3 sample Hór-valley./Tebepuszta Meadow Hór-valley./Rapids on Hór stream Hór-valley./Oszlarét Meadow Mátraháza-1 sample Mátraháza-2 sample Aquifer of Maconka Hasznos, Kövicses stream Eger/At bridge on Eger stream at Zúgó st. Eger/Near bridge on Eger stream at Zalár st. Felsőtárkány/Barát-rét Meadows Bükkszentkereszt/Kaán Károly Springs Mátraderecske/Near bridge o Baláta & Kovácsói streams Nádújfalu Jávorkút ‘Kühne Andor’ tourist pathway, roadside Near Szentdomonkos Tibolddaróc Tarnalelesz Pétervására outskirts-1 sample Pétervására outskirts-2 sample Hevesaranyos Váraszó fish pond Noszvaj Víz-völgy fields

2

N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N

47.903308; 47.952612; 47.890970; 48.483269; 47.986900; 47.989079; 47.974139; 47.964397; 47.954067; 47.941857; 48.013367; 48.016984; 48.012667; 48.029817; 47.996033; 47.979290; 47.865483; 47.864483; 47.993504; 47.929983; 47.890970; 47.903198; 47.992750; 48.083383; 47.949622; 48.010023; 48.097635; 48.098210; 48.088253; 47.931934; 48.048387; 48.003243; 48.003243; 48.019062; 48.085338; 47.926044;

E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E

20.382339 19.815339 20.390122 20.549968 20.021050 20.018066 20.037302 20.051301 20.072450 20.075533 20.493202 20.501000 20.505166 20.552683 20.513726 20.520453 19.980816 19.984433 19.858783 19.736200 20.390122 20.375530 20.460452 20.638888 20.072554 19.969621 20.528104 20.554323 20.178323 20.634509 20.177987 20.099971 20.099971 20.215930 20.094653 20.469540

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Fig. 1. Locations of the investigated ML samples in Northern Hungary.

were determined as mg gallic acid equivalents (GAE) per 1 kg d. h. Calibration was made between 0 and 250 mg/l GAE. All measurements were applied in duplicate.

ML (Patonay et al., 2017). 2.2.2. Extraction methods Soxhlet extraction was performed as follows: 1.5 g dry plant material, herb (further: d. h.) was ground in a coffee mill, scaled, put to the thimble of Soxhlet (Lenz, Germany) apparatus with 250 ml solvent. Extraction was performed in three turns (stages). In case of the MeOH, the water bath was heated to 80 °C. For WA in the first stage the temperature was 85 °C, and 95 °C on the second and third stages, respectively. Extracts were stored in a deep freezer (−18 °C) until analyzed. Ultrasound (US) extraction with homogenation was performed as follows: 0.5 g d. h. was scaled to a 50-ml centrifuge tube, then 25 ml of solvent was added to it. This was treated with IKA Turrax Basic T25 homogenizer (IKA-Werke, Germany) on 6500 rpm for 120 s to provide vigorous mixing and chopping with the exclusion of air. Homogenates were placed to a VWR ultrasonic bath tempered with cooler blocks to 15–17 °C (to avoid overheating of the bath over 30 °C) and treated with 45 kHz for 30 min. During this, bath temperature usually reached 27–30 °C. Then the bath was re-cooled and another 15 min of US treatment was performed. The extract was centrifuged, transferred into a cylinder by pipette, and thereafter the volume measurement was transferred to a capped plastic dish. After adding 25 ml of fresh solvent to the drug, the whole treatment was repeated twice, then the extracts of three stages were combined and stored in a deep freezer (−18 °C) until analysed. During each stage, extracts from previous stages were stored at 4 °C.

2.4. Measurement of antiradical activity by DPPH assay EC50 of extracts against 0.1 mM ethanolic DPPH solution were determined at room temperature with normal lighting, in a 30 min reaction time. Dilutions from samples were made with ethanol to 1.0 ml as the final volume. To this, 5 ml of DPPH solution was added. After 30 min absorption spectra were collected between 200–800 nm against ethanol; maximum of 517 ± 1 nm were evaluated. A solution made up with 1 ml ethanol, with absorbance of A(517) = 1,0 ± 0025 was used as a blank. Carvacrol, 3,5-di-tert-butyl-4-hydroxytoluene (BHT) and 6hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (trolox) were measured as positive controls. All measurements were applied in duplicate. 2.5. Measurement of ferrous reducing activity In case of all samples we carried out also the modified method of Benzie and Strain (Benzie and Strain, 1996) for determination of ferrous reducing activity of plasma (FRAP). To avoid contamination of the FRAP reagent all solutions used here were prepared with ultrapure water. 50 ml buffer (46.3 ml 0.2 M acetic acid and 3.7 ml of 0.2 M Naacetate filled to 100 ml) was measured in a screw-capped plastic dish and 5 ml 10 mM 2,4,6-s-tripyridyl-triazine (TPTZ) acidified with 84 μl cc HCl/25 ml solution. Then 5 ml 20 mM FeCl3 was added to the mixture and the dish was closed. After the reaction of 50 μl of sample with 1450 μl FRAP reagent for 5 min, spectra were collected and maximum of absorption at 596 ± 1 nm evaluated. Results were determined as mg ascorbic acid equivalents (AAE) per kg d. h. For calibration, 0, 4, 8, 12, 16, 20 ppm aqueous ascorbic acid solutions were used. Measurements were carried out in triplicate.

2.3. Measurement of total polyphenol content (TPC) Folin and Ciocalteu’s assay was used. The method is based on the microscale protocol of the assay given by Waterhouse (Waterhouse, 2002) but it is modified due to adaptation to a larger series of samples with shortened incubation time (1 h instead of the original 2). Shortly thereafter, 1 ml of 2 N Folin-Ciocalteu reagent was added to 5 ml deionized water, followed by 200 μl of sample and immediately 1 ml saturated Na2CO3 solution (30.72 g anhydrous salt/100 ml water). After 60 min of reaction, absorption spectra were collected in a 1 cm quartz cuvette between 200 and 800 nm against water as a reference. Absorption maxima at 765 ± 1 nm were used for calculations. Results

2.6. Statistical evaluation SPSS 16.0 and MS Excel 2013 were used for calculations. For each data series of assays mean, extrema, quartiles (first, Q1; median; third, 3

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Q3), and coefficient of variation (CV) were calculated. The Kolmogorov-Smirnov test was performed to test the normality of distribution and the Levene’s test was used to check the homogeneity of the variance. For determining the significant differences between extract types in cases of homogeneous variance independent samples t-test were chosen to perform, and in cases of inhomogeneous variance Welch test was performed, according to the result of the corresponding Levene’s test. Thus for each investigation of significant differences, cases of both homogeneous and inhomogeneous variances were marked. Wilcoxon’s signed rank test was used to compare matched samples of the observed TPC and the antioxidant values. Correlations between the TPC and antioxidant properties were examined with Pearson’s correlation coefficient (two-tailed). Evaluation of the strength of correlations: R ≤ 0.4 weak, 0.4 < R ≤ 0.6 medium, R > 0.6 strong. Homogeneity of antioxidant parameters and TPC of samples were characterized with the coefficient of variation (CV), as: CV ≤ 10%: extremely homogeneous, 10% < CV ≤ 20% homogeneous, 20% < CV ≤ 45%: heterogeneous, CV > 45% extremely heterogeneous.

Table 2 Total polyphenol content (TPC) of ML samples calculated from the different extracts. Measurements were performed in duplicate, s.d. was calculated from the 2 repetitions. TPC mg GAE/kg d. h. Identifier

EGR1 KBÁ EGR2 JOF MBA1 MBA2 DEK SZU MDE1 MDE2 HOV1 HOV2 HOV3 HV1 HV2 HV3 MÁH1 MÁH2 MAC HAS EGR3 EGR4 FET BÜK MDE3 NÁD JÁV KÜH SZD TIB TLE PÉV1 PÉV2 HEA VÁR NOSZ

3. Results and discussion 3.1. TPC of plant samples calculated from the different extracts TPCs of the plant samples calculated from the different types of extracts are shown in Table 2 and its basic descriptive statistics in Table 3. Based on the Kolmogorov-Smirnov test the normality of TPC data is assumed. Significant differences were observed between the results from different extracts as follows: MeOH Soxhlet vs. MeOH US (p < 0.001, variance: homogeneous), MeOH Soxhlet vs. WA Soxhlet (p < 0.001, variance: inhomogeneous) and MeOH US vs WA US (p < 0.025, variance: inhomogeneous). No significant difference was found however, between TPC measured from the WA Soxhlet and WA US extracts (p = 0.662, variance: inhomogeneous). The difference concerning TPC between the extraction methods is dependent on the solvent: in MeOH extracts higher values were reached by the US method in 27 cases out of 36 while in the WA extracts the US method showed better results only in 16 cases out of the 36. Concerning the solvents however, in both methods WA exctracts assured higher TPC: by the UH method it was registered in 22 samples out of the 36 and by the Soxhlet method in 30 cases out of 36. The highest TPC value of the total series was reached by the WA Soxhlet method (in sample TLE: 87 024 mg GAE/ kg d. h.) while the lowest one by the MeOH Soxhlet method (in accession EGR3: 6295 mg GAE/ kg d. h.). It was demonstrated that in some cases the results of the different extracts of the same sample may produce large differences like e.g. in accessions HAS (3.69 fold) or PEV1 (3.19 fold), (Table 2). All TPC data series are heterogeneous based on their CVs. CV (MeOH Soxhlet) = 31.4%, CV (MeOH US) = 28.8% CV (WA Soxhlet) = 30.1%, CV (WA US) = 19.4%. It anticipates a highly variable polyphenol composition of the plant samples both in qualitative and quantitative aspects. Results of the TPC match the range reported in literature data about polyphenol content of ML. Former investigations report a wide interval between approx. 10 000 and 90 000 mg GAE/kg drug. Extraction of wild collected ML was performed using an unspecified three-stage method with a 27% yield and resulted in 10 222 mg GAE/kg drug TPC (Motamed and Naghibi, 2010). Iqbal et al. (2013) examined Soxhlet extracts prepared in sequence with three different solvents of increasing polarity: n-hexane, dichloromethane and MeOH (4 h/solvent). They produced a high TPC from the MeOH extract: 71.43 mg GAE calculated in g drug. None of our TPC results from MeOH extracts exceeded this value and only two Soxhlet samples from our WA extracts showed higher values than these (TLE and PÉV1, 87024 and 83407 mg GAE/kg d. h. respectively). These latter results are also close to the highest TPC

MeOH Soxhlet

MeOH US

WA Soxhlet

WA US

Mean

s. d.

Mean

s. d.

Mean

s. d.

Mean

s. d.

21162 34430 17274 24855 39771 49033 26318 35268 43907 42887 23336 31718 42459 42229 41966 35478 29425 37791 23272 17772 6295 44170 12087 37946 36277 39040 45849 40208 47798 23565 32957 26163 34197 41072 28209 40964

263 1267 207 324 161 312 1385 107 2675 2143 976 495 3640 641 530 1825 1818 1212 1743 995 120 662 148 262 422 118 292 786 1300 1093 1943 2143 1355 3532 716 1572

45335 31872 30425 25113 41595 49705 24863 55330 50477 31809 36695 42405 42848 39729 63623 59009 44063 44666 45064 59107 7105 52720 30997 54622 57615 66923 57690 55202 67952 37733 60325 53770 58047 51458 39614 53433

646 550 2097 142 1402 2115 339 2207 1684 1279 151 529 2657 35 1982 5577 445 3024 1745 2088 398 3006 433 929 2955 3023 185 3440 3720 552 669 883 2166 2549 780 2686

47297 27634 32527 57052 61280 65650 35587 69452 42580 58663 38271 48575 43884 61566 62750 55289 61254 58412 37666 56118 18381 39762 28118 52153 49929 69255 68380 51808 58337 38334 87024 83407 57371 46723 65165 47206

2830 1366 1601 1922 3172 2100 540 468 1708 2581 2461 2994 299 783 875 2809 2166 2129 101 673 611 2528 223 4037 2608 1250 3194 1457 752 1051 2039 185 746 2072 2263 3659

54919 42506 50651 56545 56108 67640 60039 63190 55485 58499 50754 55231 47011 50052 58278 43375 37191 47738 47893 65504 23390 46238 36490 45878 40464 60820 56758 47522 59711 34526 67910 63175 55650 52614 41006 48206

2096 260 298 4809 123 267 1896 1069 2058 1155 316 272 1273 3180 940 2268 3846 2516 2952 1769 605 724 196 993 1483 1626 1169 1589 1726 1282 2570 2439 531 3267 1352 532

Table 3 Basic descriptive statistics of TPC of ML samples calculated from the different extracts. All data are given in mg GAE/kg d. h. except CV. CV was calculated from n = 36.

Q1 Median Q3 Mean Min Max CV, %

MeOH Soxhlet

MeOH US

WA Soxhlet

WA US

25836 35373 41295 33254 6295 49033 31.4

39143 47520 55901 46359 7105 67952 28.8

41875 53721 61351 52301 18381 87024 30.1

46148 51684 58333 51360 23390 67910 19.4

which was found in literature of this species: 89.1 mg GAE/g drug (89 100 mg GAE/kg) detected by Hajlaoui and co-workers (Hajlaoui et al., 2009). It is noteworthy that almost all of the available literature data refer to a single charge of ML thus, the variability of TPC in individual ML samples cannot be evaluated from any of these studies. 3.2. Radical scavenging activity of ML extracts EC50 values are shown in Table 4, basic statistics in Table 5. Compared to carvacrol (EC50 = 1776 mg/l) all extracts were found to be stronger, except MeOH Soxhlet extract of EGR3 (EC50 = 3186 mg/ l). However, none of the extracts are stronger than BHT 4

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Based on the Kolmogorov-Smirnov test the normality of EC50 data is assumed except MeOH Soxhlet data. This last one approximates it because EGR3, EGR2 and FET cause right-skewed distribution. The radical scavenging activity of all types of experimental ML extracts is in strong and significant (to all of them p < 0.001) correlation with the TPC values. The following Pearson correlation coefficients were determined: MeOH Soxhlet R (EC50/TPC) = -0.7631, MeOH US R (EC50/TPC) = -0.8143, WA Soxhlet R (EC50/TPC) = -0.8727 and WA US R (EC50/TPC) = -0.8585. These tests showed significant differences between each of the experimental treatments as follows: MeOH US vs WA US (p < 0.001 variance: inhomogeneous), MeOH Soxhlet vs MeOH US (p < 0.025 variance: inhomogeneous), WA Soxhlet vs WA US (p < 0.01 variance: homogeneous) and MeOH Soxhlet vs WA Soxhlet (p < 0.001 variance: inhomogeneous). In the case of both of the solvents, US extraction assured higher scavenging activity: with MeOH in 23 cases out of the 36 ones and in WA solvent in 24 cases out of the 36 ones. Concerning the solvents, WA assured better antioxidant activity by both methods: By the Soxhlet method it was experienced in 28 cases out of the 36 and by US method it was demonstrated in 31 cases out of the 36 cases. Highest antiradical activity was observed in WA Soxhlet extract of sample SZD: EC50 = 168.5 mg/l). On the other hand, a MeOH Soxhlet extract was the weakest: sample EGR3 which showing also the minimum of TPC in the total series (see Sect 3.1). In some samples, large differences among the EC50 values of the treatments were observed, i.e. EGR2 (4.88 fold) EGR3 (3.94 fold), FET (3.083 fold). All EC50 data series could be evaluated as heterogeneous or extremely heterogeneous: CV (MeOH Soxhlet) = 70.8%, CV (MeOH US) = 48.2%, CV (WA Soxhlet) = 44.4% CV (WA US) = 31.6%. Surveying the publications studying ML by similar reaction conditions of DPPH assay, we find our results are in accordance with most literature data. Mint extracts prepared with polar solvents have high antiradical activity but they are usually weaker than BHT (Hajlaoui et al., 2009; Gulluce et al., 2007). Nevertheless other results were also published: Iqbal et al. (2013) described EC50 = 6.7 mg/l in a ML MeOH Soxhlet extract while the corresponding value of BHT was EC50 = 9.9 mg/l. Dichloromethane and n-hexane extracts tested in the same study showed weaker activities (EC50 are 21.2 and 33.3 mg/l, respectively) than MeOH ones which findings also show the importance of the proper choice of the solvent for extraction of the antioxidants of ML.

Table 4 EC50 values of the different ML extracts against DPPH. Measurements were performed in duplicate, s.d. was calculated from the 2 repetitions. DPPH EC50 mg/l extract Identifier

EGR1 KBÁ EGR2 JOF MBA1 MBA2 DEK SZU MDE1 MDE2 HOV1 HOV2 HOV3 HV1 HV2 HV3 MÁH1 MÁH2 MAC HAS EGR3 EGR4 FET BÜK MDE3 NÁD JÁV KÜH SZD TIB TLE PÉV1 PÉV2 HEA VÁR NOSZ Carvacrol BHT Trolox

MeOH Soxhlet

MeOH US

Meang

s.d.

Mean

962.8 685.7 1613.0 862.0 583.1 417.5 853.2 465.0 353.3 374.2 636.0 767.4 503.9 479.4 555.5 471.3 655.5 441.3 788.7 797.5 3186.0 460.3 1513.7 610.6 688.3 468.5 425.7 573.4 385.6 669.6 520.0 623.4 504.8 449.3 641.9 607.0 1776.0 87.4 493.9

2.3 12.2 35.8 39.9 3.4 19.4 27.0 8.0 12.4 1.6 9.6 3.5 22.7 23.7 32.1 9.0 11.8 6.7 4.1 22.3 47.6 2.7 30.0 60.0 3.4 1.5 1.7 11.7 4.5 4.4 4.7 6.1 1.4 2.2 41.7 10.6 29.8 4.5 4.5

611.6 748.0 609.7 908.5 526.2 501.6 1144.0 474.8 486.4 647.8 695.5 568.6 705.2 520.2 291.7 388.6 400.6 407.2 606.9 385.6 1736.0 442.0 538.9 520.1 517.3 397.8 456.9 443.4 312.7 459.6 342.1 507.7 376.7 356.7 427.1 350.6 1776.0 87.4 493.9

WA Soxhlet

WA US

s.d.

Mean

s.d.

Mean

s.d.

48.6 32.1 9.6 0.2 37.9 17.5 30.7 27.3 18.2 29.7 57.5 11.5 48.5 6.5 5.2 1.9 5.5 17.6 24.2 0.3 129.7 6.6 3.6 1.6 35.1 8.5 2.2 1.5 7.4 8.2 6.3 6.7 7.0 19.2 3.7 0.5 29.8 4.5 4.5

500.9 871.7 541.2 338.1 317.0 302.3 682.8 267.9 396.7 380.0 589.1 437.1 424.8 304.1 345.5 322.6 288.4 356.2 476.5 298.5 983.4 448.8 934.9 424.6 434.5 299.3 305.8 362.1 168.5 524.8 210.7 249.5 392.1 334.8 272.2 464.4 1776.0 87.4 493.9

12.7 35.2 29.6 13.1 4.4 0.1 5.6 6.1 2.5 24.2 43.5 0.2 1.9 8.4 4.7 10.9 5.2 7.3 1.6 31.6 31.0 15.0 89.3 6.7 15.0 2.9 1.7 2.2 1.8 5.1 2.4 4.6 21.7 14.3 1.4 17.4 29.8 4.5 4.5

367.5 438.1 330.3 338.4 374.7 299.8 393.9 289.9 355.8 316.0 343.0 368.3 396.9 358.9 307.7 406.9 431.3 354.7 343.2 252.3 806.4 369.0 490.6 391.9 417.4 226.9 272.7 372.9 262.9 363.7 336.4 341.2 339.3 262.9 363.7 336.4 1776.0 87.4 493.9

1.1 0.6 4.1 13.2 3.8 11.5 16.7 2.7 0.8 1.8 7.2 3.1 0.9 3.4 17.5 2.1 4.5 4.0 1.9 9.2 8.9 9.0 8.6 5.6 4.9 4.8 5.3 0.8 3.5 4.6 2.0 12.8 18.0 3.5 4.6 2.0 29.8 4.5 4.5

Table 5 Basic descriptive statistics EC50 values of the different ML extracts. All data are given in EC50 mg/l with exception of CV. CV was calculated from n = 36.

Q1 Q3 Median Mean Min Max CV, %

MeOH Soxhlet

MeOH US

WA Soxhlet

WA US

466.7 727.8 583.1 713.8 353.3 3183.0 70.81

403.9 608.3 501.6 556.1 291.7 1736.0 48.16

303.2 462.6 362.1 422.5 168.5 983.4 44.41

323.1 383.3 355.8 362.4 226.9 806.4 31.63

3.3. Ferrous reducing activity of ML samples Results of FRAP assay are shown in Tables 6 and 7 with basic statistics. All FRAP data series passed the Kolmogorov-Smirnov normality test. Compared to the AO capacities of the controls, ML samples are intermediate in reducing ability and are weaker than expected based on their strong antiradical activity and relatively high TPCs. We found that all of the FRAP values close to that of BHT (> 11,000 mg AAE/kg) were obtained from WA extracts, predominantly from WA US. None of the MeOH extracts reach or approximate the activity of BHT. If the extracts are compared to carvacrol (9662 mg AAE/kg), the overall view is similar: none of the MeOH extracts reach the activity of this control. According to t or Welch tests, significant differences were observable between the results of the following extracts: MeOH Soxhlet vs. WA Soxhlet (p < 0.001 variance: inhomogeneous); MeOH US vs WA US (p < 0.001 variance: inhomogeneous) and WA Soxhlet vs WA US (p < 0.001 variance: inhomogeneous). No significant difference was found between MeOH US vs. MeOH Soxhlet extracts (p = 0.278 variance: homogeneous). In case of both solvents, the US extraction method showed higher reducing activity than Soxhlet: with MeOH, it was observable in 21 samples and WA extracts in 22 samples out of the 36. For the solvents,

(EC50 = 87.4 mg/l). Comparing the results with the value of trolox (EC50 = 493.9 mg/l) a more detailed overall view may be available. In case of MeOH Soxhlets, 14 of 36 extracts and among MeOH US extracts, 16 samples out of the 36 are stronger than this control. Contrary to MeOH extracts, a majority of the WA Soxhlets, namely 28 samples are stronger than trolox while in the case of the WA US extracts, 34 of 36 samples demonstrated stronger activity than of trolox. This data refers to the fact that if antiradical activity of extracts are in focus, WA solvent and US extraction method could be more efficient than other treatments. Interestingly, the TPCs calculated from the two types of WA extracts do not differ significantly from each other (see Sect. 3.1.). 5

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extracts of the same sample may produce large differences, i.e. in accession HAS (3.080fold), DEK (3.048 fold) and JOF (2.991fold). However, for the majority of the samples, the differences are lower than those observed in the data series of antiradical activity. Similarly to the TPC and DPPH data, the FRAP data series was also heterogeneous. CV (MeOH Soxhlet) = 19.11%, CV (MeOH US) = 24.10%, CV (WA Soxhlet) = 27.38% CV (WA US) = 22.92%. Reducing activity shows a strong, significant (to all extract types, p < 0.001) correlation with TPC. The following correlation coefficients were calculated: MeOH Soxhlet R (FRAP/TPC) = 0.7232, MeOH US R (FRAP/TPC) = 0.7174, WA Soxhlet R (FRAP/TPC) = 0.8169, WA US R (FRAP/TPC) = 0.7389. According to our knowledge, FRAP assay on a larger number of samples, has not been published before neither for ML nor for other Mentha taxa although the method is widely used in characterization of medicinal and spice plants or their preparations. Thus, FRAP results given here may represent the first screening of activity of polyphenolics in ferric/ferrous system from simple solvent extracts of horsemint.

Table 6 FRAP values of ML samples calculated from the different extracts. Measurements were performed in triplicate, s.d. was calculated from the 3 repetitions. FRAP mg AAE/kg d. h. Identifier

EGR1 KBÁ EGR2 JOF MBA1 MBA2 DEK SZU MDE1 MDE2 HOV1 HOV2 HOV3 HV1 HV2 HV3 MÁH1 MÁH2 MAC HAS EGR3 EGR4 FET BÜK MDE3 NÁD JÁV KÜH SZD TIB TLE PÉV1 PÉV2 HEA VÁR NOSZ Carvacrol BHT Trolox

MeOH Soxhlet

MeOH US

WA Soxhlet

WA US

Mean

s. d.

Mean

s. d.

Mean

s. d.

Mean

s. d.

5213 4841 4149 4725 5422 6761 5555 5826 6743 7052 6264 5125 6666 7463 6167 6911 5879 6371 4690 4072 3264 6794 5518 6373 5259 5708 6394 6805 7216 5213 6565 5430 6563 7266 6118 8386 9662 12169 20367

83 251 131 185 226 124 43 36 340 383 190 424 187 271 346 113 207 142 165 264 74 261 601 302 65 473 182 193 235 208 319 353 636 179 235 98 297 342 1004

6502 3603 4540 3565 4599 5455 3097 7398 6188 4588 6214 5746 6356 6275 7557 7743 6542 6487 5345 6445 2875 5541 5917 6099 5804 5921 7748 7573 7913 7135 8149 7204 7862 6436 9101 7959 9662 12169 20367

72 62 111 95 89 27 83 67 64 43 122 173 101 73 70 210 85 129 115 275 30 20 231 148 357 68 57 402 39 170 121 147 226 466 128 101 297 342 1004

6675 4538 5489 10662 9247 10322 5582 9318 6386 7961 5567 7127 7129 9939 8017 8548 11610 7635 5421 8441 3441 6499 4007 8207 7394 10087 10241 9189 10011 8652 11969 10482 7235 7542 10183 7385 9662 12169 20367

123 161 47 135 144 53 48 41 138 136 390 96 105 401 153 310 168 312 466 203 150 125 96 77 269 234 228 258 129 439 459 266 231 310 187 101 297 342 1004

9359 7604 8807 10022 9079 11419 9441 11509 10186 10967 11774 11371 10237 11155 12200 9701 5671 6111 7372 12543 6903 7715 6454 6398 5837 8477 9102 6974 9550 5883 10657 10076 8325 7833 6512 8085 9662 12169 20367

65 448 387 568 107 39 222 306 238 250 273 222 202 207 201 76 65 52 328 118 90 65 555 146 157 291 651 380 146 84 193 154 43 140 89 199 297 342 1004

4. Conclusions Based on our experimental data it was demonstrated that TPC and AO capacity of ML samples strongly depends both on solvent type and extraction method. It was established that WA is a better extraction medium for horsemint to obtain extracts of high content of potent antioxidant polyphenols than MeOH, although – based on references – the latter is used much more frequently. The difference between MeOH and WA extracts was manifested in TPC and also in the results of antioxidant assays. As for the two different WA extraction methods in our study, when focusing on better antioxidant capacity of a product of ML, WA US extraction is more efficient than WA Soxhlet. At the same time the two WA extract types do not differ in their TPC. Based on these findings can be presumed that the polyphenol composition of the WA Soxhlet and WA US experimental extracts differ from each other. From the technological side, it was demonstrated that an extraction method operating with no circulation of boiling solvent can be as efficient or even better for processing ML, as the widely referred Soxhlet method. On the other hand, the WA as an extraction solvent is observed to be more efficient than MeOH. It can be an advantage from the viewpoint of processing because MeOH is frequently mentioned in scientific papers, its use is highly restricted in food or preservative industry. The maximum levels set by the European Commission are 10 mg/kg in general (32/2009/EC, 2009The European Parliament and Council, 200932/2009/EC, 2009) and 1.5 mg to flavorings from natural flavoring materials (59/2010/EC, 2010The European Parliament and Council, 201059/2010/EC, 2010). Thus, it may not represent an optimal solution for product development. In contrary, both of components of WA are applicable in the food industry of the EU with no upper limits of residues if the good manufacturing practices are followed (32/ 2009/EC, 2009The European Parliament and Council, 200932/2009/ EC, 2009). As for the investigated plant materials, the data of accessions showing best antioxidant activities (MBA2, SZU, HV1, HV2, SZD, TLE) are summarized in Table 8. However, the establishment of their industrial uses obviously require further investigations on their polyphenol composition, terpenoid spectrum and the environmental effects.

Table 7 Basic descriptive statistics of FRAP values. All data are given in mg AAE/kg d. h. with exception of CV. CV calculated from n = 36.

Q1 Q3 Median Mean Min Max CV, %

MeOH Soxhlet

MeOH US

WA Soxhlet

WA US

5236 6704 6118 5896 3264 7463 19.11

5498 7301 6275 6157 2875 9101 24.10

6587 9975 8017 8021 3441 11969 27.38

7173 10447 9102 8949 5671 12543 22.92

in case of both methods WA assured extracts of higher antioxidant capacity: this was the result of 30 out of the 36 of the cases treated by the US method and in 29 cases out of the 36 cases treated by the Soxhlet method. The highest ferrous reducing activity in the total series (Table 4) was observed in a WA US extract (accession HAS: 12,453 mg AAE/kg d. h.) and the lowest activity is shown by the MeOH US extract of EGR3 (2875 mg AAE/kg d. h.). Similarly to the TPC and antiradical activity values, none of the accessions could be chosen as the best one concerning FRAP values. In some cases reducing activity of the four

Funding The research was supported by the project EFOP-3.6.1-16-201600001‚ Complex improvement of research capacities and services at Eszterhazy Karoly University’, provided by the European Social Fund (ESF) of the European Union. 6

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Table 8 Data of ML samples of strongest antioxidant activities (mean values), given from WA extracts. Identifier

SZU HV1 HV2 HAS SZD TLE

WA Soxhlet

ingredients. Official Journal of the European Union L 225, 10–12. Fatiha, B., Didier, H., Naima, G., Khodir, M., Martin, K., Kamagaju, L., Stévigny, C., Mohamed, C., Pierre, D., 2015. 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). Industrial Crops and Products 74, 722–730. https://doi.org/10.1016/j.indcrop.2015.04.038. Ghoulami, S., Il-Idrissi, A., Fkih-Tetouani, S., 2001. Phytochemical study of Mentha longifolia of Morocco. Fitoterapia 72, 596–598. https://doi.org/10.1016/S0367-326X (01)00279-9. Gulluce, M., Sahin, F., Sokmen, M., Ozer, H., Daferera, D., Sokmen, A., Polissiou, M., Adiguzel, A., Ozkan, H., 2007. Antimicrobial and antioxidant properties of essential oils and methanolic extract from Mentha longifolia ssp. longifolia. Food Chemistry 103, 1449–1456. https://doi.org/10.1016/j.foodchem.2006.10.061. Hajlaoui, H., Trabelsi, N., Noumi, E., Snoussi, M., Fallah, H., Ksouri, R., Bakhrouf, A., 2009. Biological activities of the essential oils and methanol extract of two cultivated mint species (Mentha longifolia and Mentha pulegium) used in the Tunisian folkloric medicine. World Journal of Microbiology and Biotechnology 25, 2227–2238. https:// doi.org/10.1007/s11274-009-0130-3. Hawrył, M.A., Niemiec, M., Słomka, K., Waksmundzka-Hajnos, M., Szimczak, G., 2016. Micro-2D-TLC Separation of phenolics in some species of mint and their fingerprints on diol bonded polar stationary phase. Acta Chromatographica 28, 119–127. https:// doi.org/10.1556/AChrom.28.2016.1.9. Iqbal, T., Hussain, A.I., Shahid Chatha, S.A., Naqvi, S.A.R., Hussain Bokhari, T., 2013. Antioxidant activity and volatile and phenolic profiles of essential oil and different extracts of wild mint (Mentha longifolia) from the Pakistani flora. Journal of Analytical Methods in Chemistry 2013, 536490. https://doi.org/10.1155/2013/ 536490. Krzyzanowska, J., Janda, B., Pecio, L., Stochmal, A., Oleszek, W., 2011. Determination of polyphenols in Mentha longifolia and M. piperita field-grown and in vitro plant samples using UPLC-TQ-MS. Journal of the AOAC International 94, 43–50 DOI: N/A, PMID: 21391480. Motamed, S.M., Naghibi, F., 2010. Antioxidant activity of some edible plants of the Turkmen Sahra region in northern Iran. Food Chemistry 119, 1637–1642. https:// doi.org/10.1016/j.foodchem.2009.09.057. Murad, H.A.S., Abdallah, H.M., Ali, S.S., 2016. Mentha longifolia protects against aceticacid induced colitis in rats. Journal of Ethnopharmacology 190, 354–361. Patonay, K., Korózs, M., Murányi, Z., Pénzesné Kónya, E., 2017. Polyphenols in northern Hungarian Mentha longifolia (L.) L. treated with ultrasonic extraction for potential oenological uses. Turkish Journal of Agriculture and Forestry 41, 208–217. https:// doi.org/10.3906/tar-1701-61. Shaiq, A.M., Saleem, M., Ahmad, W., Parvez, M., Yamdagni, R., 2002. A chlorinated monoterpene ketone, acylated glycosides of beta-sitosterol and a flavonoid glycoside of Mentha longifolia. Phytochemistry 59, 889–895 DOI: N/A. PII: S0031-9422(01) 00490-3. Simon, T., 1994. A magyarországi edényes flóra határozója: Harasztok - virágos növények. Nemzeti Tankönyvkiadó Rt, Budapest. (Handbook of the Hungarian vascular flora. Ferns and seed plants. Hungarian. ISBN: 963-186-093-0.). Stanislavljević, D.M., Stojičević, S.S., Đorđević, S.M., Zlatković, B.P., Veličković, D.T., Karabegović, I.T., 2012. Antioxidant activity, the content of total phenols and flavonoids in the ethanol extracts of Mentha longifolia L. Hudson dried by the use of different techniques. Chemical Industry & Chemical Engineering Quarterly 18, 411–420. https://doi.org/10.2298/CICEQ110919017S. Younis, J.M.H., Beshir, S.M., 2004. Carvone-rich essential oils from Mentha longifolia(L.) Huds. ssp.schimperii Briq. and Mentha spicata L. grown in Sudan. Journal of Essential Oil Research 16, 539–541. https://doi.org/10.1080/10412905.2004.9698792. Varga, L., Engel, R., Szabó, K., Abrankó, L., Gosztola, B., Zámboriné Németh, É, Sárosi, S., 2016. Seasonal variation in phenolic content and antioxidant activity of Glechoma hederacea L. harvested from six Hungarian populations. Acta Alimentaria Hungarica 45, 268–276. https://doi.org/10.1556/066.2016.45.2.14. Waterhouse, A.L., 2002. Determination of total phenolics. Current Protocols in Food Analytical Chemistry I1.1.1–I1.1.8.

WA US

EC50 mg/l

FRAP mg AAE/kg

TPC mg GAE/kg

EC50 mg/l

FRAP mg AAE/kg

TPC mg GAE/kg

267.9 345.5 304.1 298.5 168.5 210.7

9319 9939 8017 8441 10012 11969

69452 50052 62750 56118 58337 87024

289.9 358.9 307.7 252.3 245.1 219.1

11509 11155 12200 12543 9549 10657

63190 61566 58278 65403 59711 67910

Declaration of Competing Interest None. Acknowledgement The authors gratefully acknowledge the valuable support of the Directorate of Bükk National Park in this project. References Bahmania, L., Aboonajmia, M., Arabhosseini, A., Mirsaeedghazi, H., 2018. Effects of ultrasound pre-treatment on quantity and quality of essential oil of tarragon (Artemisia dracunculus L.) leaves. Journal of Applied Research on Medicinal and Aromatic Plants 8, 47–52. https://doi.org/10.1016/j.jarmap.2017.10.002. Başer, K.H.C., Kürkçüoğlu, M., Tarɩmcɩlar, G., Kaynak, G., 1999. Essential oils of Mentha species from Northern Turkey. Journal of Essential Oil Research 11, 579–588. https://doi.org/10.1080/10412905.1999.9701218. Başer, K.H.C., Kürkçüoğlu, M., Demirci, B., Özek, T., Tarɩmcɩlar, G., 2012. Essential oils of Mentha species from Marmara region of Turkey. Journal of Essential Oil Research 24, 265–272. https://doi.org/10.1080/10412905.2012.676775. Benedec, D., Vlase, L., Oniga, I., Mot, A.C., Silaghi-Dumitrescu, R., Hanganu, D., Tiperciuc, B.T., Crisan, G., 2013. LC-MS analysis and antioxidant activity of phenolic compounds from two indigenous species of Mentha. Note I Farmacia 61, 262–267 DOI: N/A. Benzie, I.F.F., Strain, J.J., 1996. The ferric reduction ability of plasma (FRAP) as a measure of “Antioxidant Power”: the FRAP assay. Analytical Biochemistry 239, 70–76. https://doi.org/10.1006/abio.1996.0292. Dudai, N., Segev, D., Havkin-Frenkel, D., Eshel, A., 2006. Genetic variation of phenolic compounds content, essential oil composition and anti-oxidative activity in Israelgrown Mentha longifolia L. Proc 1st IS on Natural Preserv. in Food Systems. Acta Horticulturae 709, 69–78. https://doi.org/10.17660/ActaHortic.2006.709.8. The European Parliament and Council, 2009. Directive 2009/32/EC of the European Parliament and Council of 23 April 2009 on the approximation of the laws of the Member States on extraction solvents used in the production of foodstuffs and food ingredients. Official Journal of the European Union L 141, 3–7. The European Parliament and Council, 2010. Directive 2010/59/EC of the European Parliament and Council of 26 August 2010 amending Directive 2009/32/EC of the European Parliament and of the Council on the approximation of the laws of the Member States on extraction solvents used in the production of foodstuffs and food

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