Assessment of phytochemical and agro-morphological variability among different wild accessions of Mentha longifolia L. cultivated in field condition

Industrial Crops & Products 140 (2019) 111698

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Assessment of phytochemical and agro-morphological variability among different wild accessions of Mentha longifolia L. cultivated in field condition

T

Alireza Moshrefi Araghia, Hossein Nematia, , Majid Azizia, , Nasrin Moshtaghib, Mahmood Shoora, Javad Hadianc ⁎

⁎

a

Department of Horticultural Sciences, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran. P.C: 9177948978 Department of Biotechnology and Plant Breeding, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran. P.C: 9177948978 c Department of Agriculture, Medicinal Plants and Drug Research Institute, Shahid Beheshti University, G.C., Evin, 1483963113, Tehran, Iran b

ARTICLE INFO

ABSTRACT

Keywords: Breeding Chemotype Mentha longifolia Piperitenone oxide Pulegone Rosmarinic acid

Mentha longifolia L. is one of the most important aromatic and perennial herbs of the Lamiaceae family, having potential sources of essential oils and compounds with interesting pharmacological and therapeutic properties. In this study, the variability of morphological parameters, essential oil composition, and phenolic compounds such as rosmarinic acid and caffeic acid content were assessed among twenty different accessions of Iranian horsemint at full flowering stage. The average of essential oil content illustrated a high variability, ranging from 0.38% to 4.33% (w/w), and the phytomass amounts varied from 471.5 g/m2 to 860.66 g/m2. Also, the clustering of morphological traits of the studied accessions generated two main groups. The characterization of the essential oils by GC-MS/FID analyses revealed a total of forty-two constituents in the horsemint accessions. Although all the accessions were classified as oxygenated monoterpenes-rich volatile oils (42.38–94.7%), they were divided into seven main chemotypes by cluster and principal component analysis (PCA). The major constituents in the essential oil of the twenty accessions were piperitenone oxide (0.27–75.39%) > pulegone (0.17–69.49%) > trans-piperitone epoxide (0.3–54.39%) > menthone (0.5–48.12%) > 1,8-cineole (1.27–44.7%) > piperitenone (0.35–36.71%) > (Z)-β-ocimene (14.11–21.27%) > menthol (0.13–20.98%), respectively. The rosmarinic acid content of the methanolic extracts using HPLC analysis showed significant quantitative variability, ranging from 36.95 mg/100 g to 302.97 mg/100 g (based on the dry matter). Since all the accessions were subjected to similar soil and climate conditions, it was evident the priority of genetic effects on ecological factors. The high intraspecific variability in Iranian horsemint directs a holistic approach to gain novel and homogeneous genetic materials, required for the breeding programs and pharmaceutical industries.

1. Introduction Since ancient times, various spice plants have been widely used throughout the world as flavoring herbs and preserving agents to improve the nutritional and organoleptic properties (Azizi et al., 2017; Tepe et al., 2007). In recent years, the natural volatile oils and herbal products from different species of medicinal plants have received scientific interests to fulfill the demands of health and safety in the modern food industry (Bozin et al., 2006; Chizzola et al., 2014; Dimitrios, 2006; Geetha and Chakravarthula, 2018; Oroojalian et al., 2010; WHO, 2002). Therefore, the main prerequisite is the use of homogeneous cultivars with prominent and attractive properties for the industry and agriculture (Franz, 2000; WHO, 2002). The Lamiaceae (syn. Labiatae) family contains two main subfamilies

⁎

Lamioideae and Nepetoideae, and Mentha is a very priceless genus in the subfamily Nepetoideae (Beremer et al., 1998). Worldwide the genus Mentha comprises 25 to 30 species, distributed across Eurasia, North America, Australia, and South Africa (Dorman et al., 2003). Mentha longifolia L., commonly known as horsemint or wild mint, is a perennial, rhizomatous, fast-growing and peppermint-scented herb belonging to the Lamiaceae family (Ghahreman, 1994). Its growing habitus is from standing erect to a sprawling status that reaches up to 40–120 cm tall (Brickell and Cathey, 2004). The oil-rich leaves are simple and opposite with 9 cm length and 2.2 cm width. The white or pale purple flowers are small (corolla 0.3-0.5 cm length) which appear during the summer (Shinwari et al., 2011). The species Mentha longifolia L. encompasses a wide range of active components including phenolic acids, flavonoids, ceramides,

Corresponding authors. E-mail addresses: [email protected] (A. Moshrefi Araghi), [email protected] (H. Nemati), [email protected] (M. Azizi), [email protected] (J. Hadian).

https://doi.org/10.1016/j.indcrop.2019.111698 Received 7 May 2019; Received in revised form 14 August 2019; Accepted 17 August 2019 0926-6690/ © 2019 Elsevier B.V. All rights reserved.

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cinnamates, terpenes, and terpenoids (Farzaei et al., 2017; Mikaili et al., 2013). The essential oil of this plant is responsible for carminative, antimicrobial, decongestant, stimulant, antiemetic, and antispasmodic effects in the human, attracted extensive studies due to its economic and therapeutic advantages (Al-Bayati, 2009; Andro et al., 2011; Gulluce et al., 2007; Hajlaoui et al., 2009; Nazem et al., 2019; Okut et al., 2017; Soilhi et al., 2019; Verma et al., 2015). Horsemint has been traditionally consumed for centuries as a beneficial plant for the human immune system and prevention or remedy of secondary infections in folk medicines (Okut et al., 2017; Van Wyk et al., 1997). It is widely used for the treatment of various ailments including convulsive coughs, colds, influenza, headaches, and inflammatory diseases, as well as menstrual problems, respiratory disorders, and digestive disorders (Farzaei et al., 2017; Gulluce et al., 2007; Mikaili et al., 2013; Naghibi et al., 2010; Verma et al., 2015). Moreover, it has externally positive effects on swollen glands and wounds (Asekun et al., 2007; Van Wyk et al., 1997). Although in a variety of literature the essential oil of the genus Mentha composed of 20–60 components, its systematic chemical complexity is still unclear (de Sousa Barros et al., 2015; Jedrzejczyk and Rewers, 2018; Zhao et al., 2013). Horsemint is exclusively enriched by flavonoids and caffeic acid-based compounds such as rosmarinic acid, nepetoidin A and B, and salvianolic acid (Dudai et al., 2005; Padmini et al., 2010; Pereira and Cardoso, 2013; Petersen and Simmonds, 2003). Rosmarinic acid (R-O-caffeoyl-3-4-dihydroxyphenyllacetic acid) includes two aromatic rings with two ortho-position hydroxyl groups that may be used frequently in several activities as anticarcinogenic, antioxidant, antibacterial, anti-HIV, antiallergic and antithrombic agents among other medical applications (Lee et al., 2007; Swarup et al., 2007). Consistent with sustainable agriculture, wild plants (endemic to Iran), which have been adapted to their local environments for many years would be valuable and promising germplasm opposing the genetic erosion (Bahreininejad et al., 2013). In plant domestication processes, the variation could be linked to the several intrinsic or extrinsic factors that affect the volatile constituents and secondary metabolites (Morshedloo et al., 2015; Rodrigues et al., 2013; Shayganfar et al., 2018). Hence, the interaction of different accessions with the agro-climatic condition is of great interest in many aspects for plant breeders to resolve the drawbacks (Patel et al., 2015). Since most of the wild mints are gathered from different natural sources, the introduction of M. longifolia into cultivation should be taking into account. Although there are some studies on the essential oil of Iranian wild-growing accessions of M. longifolia (Saeidi et al., 2016; Zeinali et al., 2005), to the best of our knowledge there have been no comprehensive and systematic evaluations. So that the paucity of information, prompted us to conduct a study concerning the multidirectional insight of chemical and morphological differentiation in M. longifolia accessions from Iran. So the presented study dealt with not only revealing but also characterizing the wealth of diversity among Iranian wild mint accessions. The goal of this research was to detect the interesting chemotypes and analysis of variability from the phytochemical and morphological perspectives, which will exceedingly assist mint breeders to select the superior cultivars according to the future breeding purposes.

analysis indicated the following specifications: pH 7.83; organic matter 1.27%; electric conductivity 485 ms.cm−1; potassium 225 ppm; phosphorus 12.3 ppm; nitrogen 0.07%. Before the onset of cultivation, the soil was supplemented with 25 t/ha of manure to warrant a suitable edaphic condition for mint growth. The seeds of Mentha longifolia L. accessions were provided from the gene bank of the Research Institute of Forest and Rangelands of Iran. The twenty accessions in this study have been coded from G1 to G20, and the geographical attributes of their locations, recorded by GPS device are compiled in Table 1. Also, the distribution of the accessions was depicted on the map along with their different bioclimate and geographical zones, presented in Fig. 1. As can be observed, the studied M. longifolia accessions have occupied very different habitats within latitude 29°23ʹ–38°39ʹ N and longitude 44°34ʹ–58°47ʹ E at an altitude of 61–3415 a.s.l.. The seeds of the twenty accessions were sown in a greenhouse (average indoor temperature: 25 ± 5 °C), and germinated in a bed of coco peat: perlite mixture (75: 25, W: W), in seedling trays. Then, all the uniformly sized seedlings were transplanted into the research field sixty days after seed sowing. Every replication of the three plots had 1.5 × 3 m spacing for each accession, and the distance between the rows was 50 cm. A total of fifteen bushes were established on each furrow with 20 cm distance between two bushes. The accessions were grown under the similar agro-climatic condition from 5 June until the flowering stage in order to diminish the environmental effects on the secondary metabolites and vegetative properties. During the experiment, weeds were constantly removed, and irrigation was performed regularly. There was also no disease, pests, and casualties throughout the experiment. The plants were harvested on 7 Oct after the elimination of 0.5 m from every side of the rows as margin plants, then the aerial parts were put inside the separate envelopes and transferred to the lab for further analysis. 2.2. Phenotypic analysis The voucher specimens were dried using a flat press at the flowering stage and deposited for all the accessions in the Herbarium of Ferdowsi University of Mashhad (H-FUM), Iran. At the end of the second year of experiment 10 main branches from every replication were cut alike at 5 cm above the soil surface to warrant a good morphological comparison. Totally, twenty-seven agro-morphological traits were assessed in the plant materials. To measure dry weight, the samples were ovendried at 70 °C for four days. The number of days, required to reach the full flowering and flower degradation was recorded from the beginning of the cultivation. 2.3. Extraction of essential oil The aerial parts were air-dried for one month in the shade at room temperature to obtain a well-grinded powder. Clevenger-type apparatus was employed to isolate the essential oil from 30 g of the ground leaves and delicate branches in three times for each accession by a hydrodistillation method for 3 h based on the procedure as recommended in British Pharmacopoeia (1993). The essential oil content was calculated as relative percentage units based on the dry weight (W/W). The isolated essential oils were dehydrated over anhydrous sodium sulfate (Na2SO4), and the separated oils were kept in sealed brown vials at 4 °C until further analysis. The essential oil replications of each accession were mixed together and a single sample for each accession was submitted to identify chemical compounds. For gas chromatography-flame ionization detector (GC-FID) and gas chromatography-mass spectrometry (GC–MS), the essential oils were transferred to Medicinal Plant and Drug Research Institution, Shahid Beheshti University, Tehran, Iran.

2. Experimental status 2.1. Plant material and growing conditions In order to analysis the accessions independent from their original conditions this study was conducted in the research field with a coordinate of latitude 36°15′N, longitude 59°38′E, and altitude 985 m above the sea level in Iran during 2016 to 2017. Climate data were obtained from the Meteorological Organization (Table 1). The soil samples were taken from the surface layer of 0–30 cm before starting the experiment. The soil texture was silty-loam, and the chemical soil 2

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Table 1 Geographic location and summer climatic conditions of the Iranian M. longifolia accessions. No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 – a

Code G1 G2 G3 G4 G5 G6 G7 G8 G9 G10 G11 G12 G13 G14 G15 G16 G17 G18 G19 G20 ESa

Province Ardabil Kerman Lorestan Chaharmahal Yazd Kerman Alborz W.Azarbaijan Ghazvin N. Khurasan Yazd N. Khurasan Hormuzgan Guilan Kerman Khuzestan S. Khurasan N. Khurasan Chaharmahal Kerman R. Khurasan

Direct. North South West West South South North North North East South East South North South West East East West South East

Longitude ’

’’

48°07 05 E 56°42’44’’E 47°59’00’’E 50°28’14’’E 54°13’00’’E 57°13’07’’E 51°21’15’’E 44°34’40’’E 50°24’29’’E 57°40’38’’E 54°07’35’’E 56°53’58’’E 56°01’28’’E 49°24’58’’E 56°36’30’’E 48°50’33’’E 58°47’07’’E 58°10’26’’E 50°57’57’’E 57°14’03’’E 59°38’00’’E

Latitude ’

’’

38°39 21 N 29°28’06’’N 33°31’00’’N 32°21’16’’N 31°35’00’’N 29°23’39’’N 35°58’17’’N 37°07’08’’N 36°28’49’’N 37°06’52’’N 31°35’32’’N 37°28’01’’N 28°03’05’’N 36°55’15’’N 30°29’30’’N 32°08’38’’N 35°89’31’’N 37°17’03’’N 31°52’37’’N 29°30’06’’N 36°15’00’’N

Altitude (m)

Summer Max Temp (°C)

Summer Min Temp (°C)

Summer Precipitation (mm)

1472 2972 1043 2533 1914 3240 2170 1824 1650 1689 2538 1653 1270 1348 1900 61 1959 1153 2447 3415 985

31.77 38.47 41.43 35.46 39.51 37.40 37.41 34.38 38.15 37.25 39.11 38.47 42.08 37.54 39.71 48.45 38.40 37.25 33.09 37.40 38.30

11.80 11.81 17.10 7.07 18.32 8.88 12.91 10.65 11.37 12.34 16.66 12.48 26.70 17.81 15.43 25.56 10.43 12.34 6.76 8.88 13.47

24.51 10.04 3.43 4.55 0.18 13.25 11.63 32.39 11.76 4.18 0.18 18.44 1.36 8.76 1.96 0 1.13 4.18 2.62 2.65 5.27

ES: Experimental site of the studied accessions under similar soil and climate conditions.

2.4. GC–MS/FID analysis of the volatile constituents

2.6. Preparation of methanolic extract

The GC-FID analysis of the essential oils was performed using a Thermoquest/Finnigan instrument, equipped with a flame ionization detector and a DB-5 fused-silica gel capillary column (30 m (length) × 0.25 mm (internal diameter) × 0.25 μm (film thickness)). The oven temperature program was initiated at 60 °C, then gradually raised to 250 °C with a ramp rate of 4 °C/min, and finally, held isothermally at 250 °C for 10 min. The detector (FID) and injector temperatures were maintained constant at nearly 280 °C, and 250 °C, respectively. Nitrogen (N2) was applied as carrier gas at a linear and constant flow rate of 1.1 ml/min. The samples were injected using a split ratio of 1:50 and a neat injection volume of 1 μl (diluted in hexane). The GC/MS analysis was carried out by a Thermoquest-Finnigan Trace gas chromatography system at the same column characterizations and temperature program as described above. The carrier gas was helium (He) at a linear fixed flow rate of 1.1 mL/min. Furthermore, the mass spectrum was recorded over the scanned mass range of 35–465 atomic mass units in the electron impact ionization (EI) mode with an ionization voltage of 70 eV. Ionization was realized by ion source temperature 200 °C, and interface temperature 250 °C.

The methanolic extraction was followed according to Başkan et al. (2007) method. The dried leaves of 10 main branches were used in three replications to grind into powders for each accession. The ratio of 100 mg of the ground leaves was vigorously mixed with 10 ml MeOH and extracted by sonication (Power-Sonic 450, Hwashin, Korea) in an ultrasonic bath for 30 min. The crude extracts were obtained by filtration and centrifugation and then stored at 4 °C in a refrigerator. 2.7. High-performance liquid chromatography The employed HPLC device was Waters Alliance 2695 Separations Module, USA, equipped with the UV 2487 Dual lambda Absorbance Detector and Sunfire C18 column with the dimension of 4.6 mm × 150 mm and 3.5 μm. The combinations of methanol (HPLC grade, Biochem Chemopharma Co.) + trifluoroacetic acid (TFA) 0.02% (as solvent A) and deionized H2O + TFA 0.02% (as solvent B) was subjected to HPLC analysis as optimal mobile phase. The rosmarinic acid and caffeic acid (Sigma-Aldrich Chemie GmbH, Germany) peaks were monitored at a wavelength of 327 nm as lambda max (Hadian et al., 2010). The injection volume was 20 μl for 45 min with a flow rate of 0.5 μl/min at 25 °C. The linear gradient was initiated with 80% A, and reduced to 20% in 35 min, retained constantly for 7 min, and finished with 80% A in 3 min. Acetonitrile solvent was used for final elution of the column after each injection. Each accession was analyzed in triplicate. The phenolic compounds were identified by comparison of UV spectra and peak retention times with those of analytical standards.

2.5. Identification and quantification of the compounds The retention indices (RIs) was determined by co-injection of homologous series of C6-C24 n-alkanes (Sigma-Aldrich, USA). Identification of components was performed by computer matching of recorded mass spectra with those found in the internal reference massspectral library (Adams and Wiley 7.0). The identification was confirmed by comparison the recorded retention data with available authentic compounds (Merck, Darmstadt, Germany), offered in the literature or database (Adams, 2007; NIST, 2008). The quantification analysis of the composition percentage was calculated by the normalization procedure from the GC-FID peak areas, without using correction factors (Venditti et al., 2015). Most of the non-identified volatile constituents were reported as traces with relative amounts of less than 0.09%. The identified constituents were listed in the order of their elution.

2.8. Statistical analysis The experiment was arranged in a randomized complete block design (RCBD) with three replications (n=3). Data were subjected to analysis of variance (ANOVA) followed by the LSD test at p < 0.05 using SAS v. 9.4 (SAS Institute Inc., Cary, NC, USA). The morphological and phytochemical similarity coefficients were determined based on the SIMINT program of the numerical taxonomy multivariate analysis system NTsys-pc v. 2.02 (Rohlf, 2000). The dendrograms were constructed using SAHN clustering algorithm through the unweighted pair group method with arithmetic averages (UPGMA). As well as, the principal component analysis (PCA) and Pearson’s correlation coefficients were calculated by PAST3 and IBM SPSS® Statistics v. 22.0 (SPSS 3

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Fig. 1. A view of sampling sites of the original plant materials of the twenty M. longifolia L. accessions on different provinces of Iran. Climatic conditions of each geographical directions are available on the separated map (Climatology characterization was based on De Martonne Method and I = aridity index de martonne). Decoding presented in Table 2.

4

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Table 2 Mean comparison of some different morphological and essential oil traits in studied accessions of M. longifolia. Code

Leaf lengtha (cm)

G1 G2 G3 G4 G5 G6 G7 G8 G9 G10 G11 G12 G13 G14 G15 G16 G17 G18 G19 G20 CV (%)

3.83 5.63 5.55 3.64 3.24 6.08 4.57 4.26 3.93 5.53 4.15 5.81 7.13 4.30 3.87 5.83 5.41 2.70 2.63 4.44 5.24

Code

Inflorescence length (cm) 8.54 ± 0.36d 9.75 ± 0.26b 5.67 ± 0.28jk 4.77 ± 0.24l 6.04 ± 0.02ij 8.28 ± 0.30de 7.32 ± 0.21gh 7.18 ± 0.17h 7.06 ± 0.05h 10.31 ± 0.21a 5.63 ± 0.12k 6.41 ± 0.37i 7.69 ± 0.18fg 5.04 ± 0.04l 3.83 ± 0.18m 8.19 ± 0.28de 9.12 ± 0.11c 7.99 ± 0.02ef 8.44 ± 0.24d 8.45 ± 0.39d 3.12

G1 G2 G3 G4 G5 G6 G7 G8 G9 G10 G11 G12 G13 G14 G15 G16 G17 G18 G19 G20 CV (%) a

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.07fgb 0.01c 0.43c 0.19g 0.43h 0.03b 0.14d 0.10def 0.03efg 0.33c 0.13def 0.36bc 0.23a 0.29de 0.09efg 0.09bc 0.28c 0.27i 0.27i 0.29d

Leaf width (cm) 2.03 1.43 2.01 1.39 1.07 1.40 1.33 3.12 2.15 2.75 1.46 2.49 2.83 1.98 1.28 2.49 2.55 1.15 1.55 0.92 8.68

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.02d 0.02ef 0.11d 0.14ef 0.20gh 0.02ef 0.13efg 0.12a 0.02d 0.22bc 0.05e 0.22c 0.24b 0.14d 0.20efg 0.20c 0.03bc 0.09fgh 0.26e 0.24h

Inflorescence diameter (cm) 0.68 0.89 0.83 0.51 0.67 0.67 0.81 0.78 0.85 0.70 0.66 0.64 0.74 0.55 0.79 0.87 0.78 0.81 0.69 0.82 3.17

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.01fgh 0.02a 0.02bcd 0.02i 0.02gh 0.02gh 0.01cd 0.01d 0.10abc 0.02fg 0.02gh 0.01h 0.02ef 0.02i 0.01d 0.02ab 0.02de 0.02cd 0.02fgh 0.00bcd

Plant height (cm) 72.17 65.83 75.50 72.83 71.50 77.83 70.33 72.83 70.50 70.50 66.00 75.83 85.33 64.17 79.33 84.17 74.17 77.83 77.83 75.83 4.32

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

3.33de 4.75fg 4.77cde 2.36de 0.87ef 2.84cd 2.52ef 2.36de 4.77ef 0.50ef 5.00fg 0.29ce 4.04a 3.55g 1.53bc 3.55ab 3.55cde 2.84cd 2.84cd 2.75cde

Full Flowering (day) 52.67 70.00 97.67 74.00 62.33 66.67 62.33 63.00 94.67 97.33 65.67 69.00 103.0 52.33 96.33 93.00 94.33 64.67 51.67 72.67 3.97

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

2.08f 5.00cd 3.79b 2.65c 3.21e 3.79de 2.52e 2.65e 0.58b 2.52b 1.15de 3.61cd 2.00a 2.52f 2.52b 3.00b 1.15b 4.73de 1.53f 3.06c

Shoot diameter (mm) 1.94 1.84 2.24 1.91 1.71 2.15 2.14 1.78 2.48 2.12 1.39 2.60 2.84 2.07 2.22 2.64 2.33 2.65 1.36 1.48 6.97

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.19efg 0.01fg 0.01cd 0.27efg 0.08gh 0.10de 0.30de 0.07g 0.13bc 0.28de 0.01i 0.12ab 0.04a 0.07def 0.01d 0.22ab 0.02cd 0.04ab 0.13i 0.04hi

Flower degradation (day) 107.67 132.67 138.67 117.67 116.33 131.33 132.67 132.33 137.33 116.00 117.00 119.00 148.67 112.00 137.33 140.00 132.00 114.67 112.67 132.33 2.35

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

2.52g 4.04cd 4.73bc 3.06ef 2.31ef 3.21d 3.06cd 2.52cd 2.52bcd 5.29ef 2.00ef 2.65e 2.31a 2.65fg 2.52bcd 5.00b 2.65d 3.51ef 5.51efg 3.21cd

Internode length (cm) 7.66 8.66 6.37 7.29 8.78 5.60 6.46 5.42 6.87 7.48 6.52 6.61 6.59 6.44 8.69 6.89 6.79 5.60 7.84 6.59 3.35

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.20bc 0.20a 0.30f 0.15c 0.29a 0.16g 0.30df 0.10 0.12de 0.28bc 0.23df 0.26df 0.39df 0.32ef 0.05 0.13d 0.13def 0.16g 0.15b 0.31def

Essential oil content (% w/w)

Dry yield (gr/m2)

0.29def 0.23b 0.69cde 0.10f 0.17f 0.18def 0.23f 0.55c 0.29ab 0.01cd 0.17g 0.06cde 0.46a 0.29def 0.23ef 0.87ab 0.17c 0.12def 0.23g 0.12g

647.16 ± 27.79cd 846.83 ± 38.78a 760.16 ± 31.37b 548.66 ± 25.69e 535.5 ± 24.63e 604.83 ± 24.11d 471.5 ± 30.78f 859.83 ± 28.53a 845.5 ± 23.06a 732.33 ± 26.27b 549.93 ± 23.79e 646.86 ± 24.09cd 860.66 ± 31.14a 668.5 ± 26.45c 653.33 ± 24.58cd 855.33 ± 20.00a 768.66 ± 24.11b 659.83 ± 37.07c 472.66 ± 23.58f 636.5 ± 25.11cd 4.02

1.92 ± 3.55 ± 2.24 ± 1.45 ± 1.55 ± 1.79 ± 1.48 ± 2.61 ± 3.75 ± 2.36 ± 0.47 ± 2.21 ± 4.33 ± 1.78 ± 1.65 ± 4.10 ± 2.71 ± 1.82 ± 0.38 ± 0.56 ± 16.07

Flower color

Leaf shape

Hairy leaf

Pink Purple Pink Purple Purple Pink Violet Violet Violet Purple Pink Pink Pink Violet white Pink Pink Purple Purple Pink

Ovate Long Ovate Ovate Ovate Long Long Ovate Twisted Ovate Long Ovate jagged Ovate Ovate Long Long Ovate Spear Long

Medium Medium Low Medium Low Medium Low Medium High Low Low Low Low Medium Low Low Medium Low Medium Medium

leaf length and width data were measured by the average of middle leaves from ten branches in triplicate. In each column, the means which are common at least in one alphabet, based on the LSD test, is not significant at the 5% probability level.

b

Inc., Chicago, IL, USA). The climatology map of the original sites of the accessions was drawn by using ArcGIS 10.4.

0.38% (w/w, based on the dry weight) in G19 to 4.33% in G13. The high essential oil content for the other accessions was seen in the order in accessions G16 (4.10%), G9 (3.75%), and G2 (3.55%), which revealed propitious efficiency. Previous reports demonstrated a significant oil content variability, ranging from 0.05 to 2.28% (Nazem et al., 2019; Zeinali et al., 2005), occurring in different accessions of M. longifolia. Zhao et al. (2013) demonstrated that the essential oil content of forty-five populations of M. haplocalyx and M. spicata was in the range of 0.5–3.0% (v/w). The present results showed that the studied accessions of M. longifolia had higher essential oil content than the other species reported. The essential oil production tends to increase in mint as a mechanism of adaptation to the growing condition through the higher density of oil glandular trichomes (Azizi et al., 2009; Figueiredo et al., 2008; Moshrefi Araghi et al., 2018; Vokou et al., 1993). Our results indicated that the high accumulation of essential oil for some accessions was observed in the lower altitudes and warm habitats. Accordingly, the high content of essential oil was observed in the accessions G16, G13, and G17, originating from the lower altitudes (61, 1270, and 1959 m a.s.l. resp.) and the arid regions with lower seasonal rainfall of 0–1.36 mm per summer (Table 1, and 2). In this study, due to

3. Results and discussion 3.1. Morphological and essential oil content variability Regarding the importance of aerial parts of medicinal plants on volatile oil properties, researchers attempt to bring these factors into focus. The accessions in the present study represented a significant difference in the phytomass and morphological descriptors (Table 2). Among the accessions, the essential oil content and the leaf width had the most portion of variation (CVs = 16.07% and 8.68%, respectively), and the full flowering degradation and the inflorescence length showed the minimum variability (CVs = 2.35% and 3.12% resp.). Also, the accessions G13 (860.66 ± 28.53 gr/m2), G8 (859.83 ± 28.53 gr/m2), G16 (855.33 ± 20.00 gr/m2) and G2 (846.83 ± 38.78 gr/m2) were ones which exhibited significantly the maximum amount of dry matter, respectively. In the studied accessions, the essential oil content varied from 5

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Fig. 2. The GC/MS (A) and three samples of GC/FID chromatograms in three accessions of G2 (B), G11 (C), and G13 (D) from M. longifolia L. essential oil. The peak numbers correspond to: (1) α-pinene, (2) β-pinene, (3) 1,8-cineole, (4) cis-sabinene hydrate (5) Menthone, (6) Isopulegone, (7) citronellol, (8) pulegone, (9) transepiperitone epoxide, (10) neoiso-isopulegyle acetate, (11) piperitenone, (12) piperitenone oxide, (13) (E)-β -caryophyllene, (14) caryophyllene oxide.

borneol (Hussain, 2009); cis-carveol (Zeinali et al., 2005). Although all the studied accessions were classified as oxygenated monoterpenes-rich volatile oils (42.4–94.7%), they have displayed a wide variation in their chemical profiles. So, the accessions were separated by numerical cluster and principal component analysis into the chemical batches in order to investigate the similarities and differences. As shown in Fig. 3, results of cluster analysis agglomerated the accessions into seven groups, representing seven chemotypes in agreement with the major components, whereas two chemotypes have been previously reported in the species M. longifolia from Israel, Tunisia, and Southern Africa (Segev et al., 2012; Soilhi et al., 2019; Viljoen et al., 2006). The chemotypes of menthone (Oyedeji and Afolayan, 2006), piperitenone oxide/piperitone oxide (Ghoulami et al., 2001), menthofuran, and piperitone oxide/piperitenone oxide (Viljoen et al., 2006) for Mentha longifolia L. have been already asserted. In the current study, the principal constituent of chemotypes I and II in the essential oil of seven accessions (G1, G19, G4, G11, G3, G15, and G17), was pulegone. These seven accessions according to the other major oil compounds were separated into two various chemotypes that out of them, four accessions (G1, G19, G4, and G11) belonged to the chemotype I. The essential oils of chemotype I (piperitenone oxide/ pulegone) were characterized by high amounts of piperitenone oxide (24.79–50.8%) and pulegone (3.31–35.03%), while piperitenone oxide as an oxygenated monoterpene was absent in the chemotype II. The highest amount of pulegone (35.03%), in the essential oil of accession G11, differentiated it within the chemotype I. The cis-piperitone epoxide (1.63–10.25%) and 1,8-cineole (1.67–7.29%) were also the other main oil components of this chemotype. Previous studies have indicated that piperitenone oxide and pulegone along with the other active ingredients could constitute the antioxidant and multifunctional characteristics of the essential oil (Abootalebian et al., 2016; de Sousa

the similarity of soil and climate condition for all the cultivated accession, it could be an inference that the diversity of essential oil content was attributed to interior genetic properties of the plants (Heydari et al., 2019). 3.2. Phytochemical variation among the essential oils The GC–MS chromatogram and three samples of GC-FID chromatogram of the isolated essential oils from the accessions (G2, G11, and G13) were presented in Fig. 2. A total of forty-two various compounds were identified in the studied accessions, accounting for 82.8–99.6% of the total essential oil compositions (Table 3). The number of identified compounds for each essential oil ranged from 14 to 26. The major components in the essential oil of the studied accessions were rich in piperitenone oxide (0.27–75.39%) > pulegone (0.17–69.49%) > transpiperitone epoxide (0.3–54.39%) > menthone (0.5–48.12%) > 1,8-cineole (1.27–44.7%) > piperitenone (0.35–36.71%) > (Z)-β-ocimene (14.11–21.27%) > menthol (0.13–20.98%), respectively (Table 4). The major constituents of the essential oil of the species M. longifolia, originating from different regions, confirmed in recent reports are pulegone (Abedi et al., 2015; Asekun et al., 2007; Golparvar et al., 2017; Hajlaoui et al., 2009; Nazem et al., 2019; Okut et al., 2017; Saeidi et al., 2016; Soilhi et al., 2019); menthone (Asekun et al., 2007; Hajlaoui et al., 2009; Okut et al., 2017; Saeidi et al., 2016); 1,8-cineole (Abedi et al., 2015; Asekun et al., 2007; Golparvar et al., 2017; Hajlaoui et al., 2009); piperitone oxide (Andro et al., 2011; Golparvar et al., 2017; Hussain, 2009; Verma et al., 2015); piperitenone oxide (Golparvar et al., 2017; Hussain, 2009; Saeidi et al., 2016; Verma et al., 2015); piperitone (Džamić et al., 2010; Okut et al., 2017); dihydrocarvone (Džamić et al., 2010; Okut et al., 2017); menthol (Al-Bayati, 2009); piperitenone (Nazem et al., 2019); (Z)-β-ocimene (Andro et al., 2011); 6

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Table 3 The composition of the isolated essential oils by hydrodistillation from the twenty M. longifolia accessions of Iran. No.

Formula

Compound name and class

RIEXPa

RILITb

IDc

Content [%] Chemotype Ιd

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36

C10H16 C10H16 C10H16 C10H16 C10H16 C8H18 C10H16 C10H18O C10H18O C10H16 C10H18O C10H18O C10H20O2 C10H18O C10H18O C10H20O C10H16O C10H20O2 C10H16O C10H20O C10H16O C10H16O C10H16O2 C10H16O2 C10H14O2 C12H20O2 C12H20O2 C12H20O2 C10H14O C12H16O2 C10H14O2 C15H24 C15H24 C15H25O C15H24O

Pinene < α- > camphene Sabinene Pinene < β- > Myrcene Octanol < 2- > Limonene Cineole < 1,8- > Sabinene hydrate < cis- > Terpinolene Linalool Menth-2-en-1-ol < cis-ρ- > Octanol acetate < 3- > Menthone Borneol Menthol Isopulegone Octanol acetate Carveol < trans- > Citronellol Pulegone Piperitone Piperitone epoxide < cis- > Piperitone epoxide < trans > Carvone oxide < trans- > Bornyl acetate Isopulegyl acetate < neoiso > Dihydrocarveol acetate < iso > Piperitenone Thymol acetate Piperitenone oxide Caryophyllene < (E)-β- > Germacrene D Caryophyllene oxide Humulene epoxide II Monoterpene hydrocarbons Oxygenated monoterpenes Sesquiterpene hydrocarbons Oxygenated Sesquiterpenes Other Total identified

939 948 972 977 989 995 1029 1031 1068 1086 1101 1121 1127 1156 1166 1172 1179 1197 1211 1221 1244 1254 1256 1263 1277 1286 1313 1328 1345 1354 1380 1421 1482 1586 1612

932 946 969 974 988 994 1024 1026 1065 1086 1095 1118 1120 1148 1223 1172 1185 1195 1211 1223 1233 1249 1250 1252 1273 1284 1312 1326 1340 1349 1366 1417 1484 1582 1608

No.

Formula

Compound name and class

RIEXP

RILIT

Chemotype ΙI

G1

G19

G4

G11

G3

G15

G17

3.36 2.47 3.37 4.12 3.21 – 3.76 9.73 – – – – – – 6.04 3.65 3.05 3.06 – – 3.31 – 6.41 10.01 – – – – – – 28 3.7 – – – 20.29 73.26 3.7 0 0 97.25

0.89 0.16 0.85 1.62 0.69 – 1.26 9.39 – – – 8.67 – 0.89 8.83 3.68 0.95 0.83 – – 8.15 4.53 1.63 11.23 – 0.24 – 0.17 3.6 – 24.79 1.14 – 1.35 – 5.47 87.58 1.14 1.35 0 95.54

1.19 – 1.62 2.25 1.58 – 2.56 17.29 – 0.51 – – 0.28 0.72 – – – 0.6 – – 8.2 – 6.38 – – – – – 0.63 – 50.8 1.05 0.44 0.93 – 9.71 84.9 1.49 0.93 0 97.03

0.46 – 0.3 0.8 0.29 0.16 0.96 1.67 0.91 – – – – 4.31 0.3 0.13 1.83 – – 0.41 35.03 2.27 10.25 – 0.17 – 0.54 0.12 0.74 0.33 31.5 0.83 – 3.46 0.14 2.81 90.51 0.83 3.6 0.16 97.91

2.89 2.36 3.09 3.78 2.8 2.41 3.8 7.17 – 2.33 – – – 9.38 – 2.44 2.59 3.07 – – 27.63 – – – – – – – – – – 2.37 – 4.17 2.36 21.05 52.28 2.37 6.53 2.41 84.64

2.16 – 2.05 2.81 1.95 – 2.62 8.23 – – 1.44 – – 8.23 – – 2.28 1.9 1.84 1.57 9.48 3.57 3.04 21.24 – 1.5 – 1.46 5.21 – – 2.56 1.84 2.72 – 11.59 70.99 4.4 2.72 – 89.7

0.98 0.13 1.04 1.85 0.72 – 1.07 11.59 – – 8.67 – – 29.14 1.54 0.63 0.97 – – 14.4 9.06 0.13 8.85 – 0.36 – – 2.83 – 4.2 0.66 – 0.82 – 5.79 92.37 0.66 0.82 0 99.64

Content [%]

RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS Std Std Std Std RI, MS RI, MS RI, MS RI, MS Std Std RI, MS RI, MS RI, MS Std RI, MS RI, MS Std RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS Std RI, MS RI, MS

ID

ChemotypeΙΙΙ

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

C10H16 C10H16 C10H16 C10H16 C8H18 C10H16 C10H18O C10H18O C10H16O C10H20O2 C10H18O C10H20O C10H16O C10H20O2 C10H16O C10H16O C10H10O C10H16O2 C10H16O2 C12H20O2 C10H14O C12H20O2

Pinene < α- > Sabinene Pinene < β- > Myrcene Octanol < 2- > Limonene Cineole < 1,8- > linalool Thujone < cis- > Octanol acetate < 3- > Menthone Menthol Isopulegone Octanol acetate Carveol < trans- > Pulegone Piperitone Piperitone epoxide < cis- > Piperitone epoxide < trans > Bornyl acetate Thymol Dihydro carveol acetate < iso >

939 972 977 989 995 1029 1031 1101 1103 1127 1156 1172 1179 1197 1211 1244 1254 1256 1263 1286 1293 1328

932 969 974 988 994 1024 1026 1095 1101 1120 1148 1172 1185 1195 1211 1233 1249 1250 1252 1284 1289 1326

G5

G7

G14

G9

G18

G12

G10

1 1.11 1.34 1.19 – 1.15 2.26 – – 0.81 – – – 1.01 – 1.48

0.4 0.53 0.65 0.66 0.32 1.28 2.33 – 0.42 0.16 – – – 0.98 – 0.91 – 0.45 54.39 – – –

1.07 0.82 1.67 0.93 0.39 5.47 4.52 – – 0.19 0.94 0.19 – – 0.16 0.63 13.63 0.69 32.52 – 0.13 –

– 0.29 1.02 0.38 – 0.4 2.68 – – – 7.24 1.68 0.63 0.39 – 4.11 0.15 – 24.65 – – –

0.29 0.24 0.55 0.19 – 1.26 1.27 – – – 0.5 – – 0.29 – 0.81 – 2.15 29.12 – – 0.19

0.95 0.71 1.54 0.44 – 1.4 3.11 0.11 – – 0.75 – 0.14 0.25 – 2.22 – 0.34 34.44 0.3 – 0.2

0.62 0.44 0.97 0.52 – 0.69 1.91 – – – – – – 0.23 – 0.17 – 0.16 15.9 – – 0

2.17 38.95 – – –

RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS Std Std Std RI, MS RI, MS RI, MS Std Std RI, MS RI, MS Std RI, MS Std Std RI, MS

(continued on next page) 7

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Table 3 (continued) No.

Formula

Compound name and class

RIEXP

RILIT

Content [%]

ID

ChemotypeΙΙΙ

23 24 25 26 27 28 29

No.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38

C10H14O C10H14O2 C15H24 C15H24 C15H24 C15H24 C15H24O

Formula

C10H16 C10H16 C10H16 C10H16 C10H16 C8H18 C10H16 C10H18O C10H16 C10H18O C10H16 C10H18O C10H18O C10H20O2 C10H18O C10H18O C10H18O C10H20O C10H16O C10H14O C10H20O2 C10H16O C10H20O C10H16O C10H16O C10H16O2 C10H16O2 C10H14O2 C12H20O2 C10H14O C12H20O2 C10H14O C12H16O2 C10H14O2 C15H24 C15H24 C15H24O C15H24O

Piperitenone Piperitenone oxide Caryophyllene < (E)-β- > Hummulene < α- > Germacrene D Bicyclogermacrene Caryophyllene oxide Monoterpene hydrocarbons Oxygenated monoterpenes Sesquiterpene hydrocarbons Oxygenated Sesquiterpenes Other Total identified Compound name and class

Pinene < α- > camphene Sabinene Pinene < β- > Myrcene Octanol < 2- > Limonene Cineole < 1,8- > Ocimene < (Z)-β- > Sabinene hydrate < cis- > Terpinolene Linalool Menth-2-en-1-ol < cis-ρ- > Octanol acetate < 3- > Menth-3-en-8-ol < ρ- > Menthone Borneol Menthol Isopulegone Myrtenal Octanol acetate Carveol < trans- > Citronellol Pulegone Piperitone Piperitone epoxide < cis- > Piperitone epoxide < trans > Carvone oxide < trans- > Bornyl acetate Thymol Isopulegyl acetate < neoiso > piperitenone Thymol acetate Piperitenone oxide Caryophyllene < (E)-β- > Germacrene D Caryophyllene oxide Humulene epoxide II Monoterpene hydrocarbons Oxygenated monoterpenes Sesquiterpene hydrocarbons Oxygenated Sesquiterpenes Other Total identified

1345 1380 1421 1455 1482 1502 1586

RIEXP

939 948 972 977 989 995 1029 1031 1035 1068 1086 1029 1121 1127 1152 1156 1166 1172 1179 1197 1211 1214 1221 1244 1254 1256 1263 1277 1286 1293 1313 1345 1354 1380 1421 1482 1586 1612

1340 1366 1417 1452 1484 1500 1582

RILIT

932 946 969 974 988 994 1024 1026 1032 1065 1086 1024 1118 1120 1145 1148 1165 1172 1185 1195 1211 1215 1223 1233 1249 1250 1252 1273 1284 1289 1312 1340 1349 1366 1417 1484 1582 1608

G5

G7

G14

G9

G18

G12

G10

1.11 24.54 2.07 – 1.03 – 1.62 5.79 72.33 3.1 1.62 0 82.84

0.35 25.86 4.85 0.18 1.98 0.3 1.97 3.52 85.85 7.31 1.98 0.32 98.98

1.61 30.19 1.76 – – – 0.67 9.96 85.4 1.76 0.67 0.39 98.18

0.58 50.82 – – – – 1.36 2.09 92.93 0 1.36 0 96.38

0.39 59.98 0.81 – – – 0.86 2.53 94.7 0.81 0.86 0 98.9

2.86 45.04 1.03 – 0.12 – 2.12 5.04 89.76 1.15 2.12 0 98.07

0.66 75.39 0.49 – – – 1.04 3.24 94.42 0.49 1.04 0 99.19

Content [%]

ID

ChemotypeIV

ChemotypeV

ChemotypeVI

G13

G16

G8

2.42 0.31 2.78 4.42 2.11 0.61 1.74 31.34 – – 0.18 – – 0.16 – – – – 0.19 0.2 1.69 – – 0.63 – – – 0.55 – 0.2 – 36.71 – 10.56 0.52 – 0.32 – 5.79 92.37 0.66 0.82 0 99.64

a

RI, MS RI, MS RI, MS Std Std Std RI, MS

1.85 0.28 2.68 4.01 1.68 – 0.52 44.7 – – 0.21 – – – – 28.66 – 0.95 – – 3.72 – – 8.84 – – – – – – – – – – 0.53 – – – 2.22 94.49 0.18 0 0.4 97.29

0.34 – 0.41 0.67 0.37 0.4 0.43 4.76 – – – – – – – 48.12 – 20.98 0.47 – – 0.47 – 18.86 – 0.18 – 0.22 – – – – 0.16 0.27 – 0.18 – – 7.38 89.73 0.18 0 0.4 97.29

ChemotypeVII G2

G6

G20

0.99 – 0.64 1.97 0.31 0.24 0.49 – 21.27 0.1 0.21 0.14 – – 1.43 – 0.25 – 0.85 – – 1.15 – 59.59 – – – – – – – 0.51 0.33 – – – 1.02 – 5.47 87.58 1.14 1.35 0 95.54

0.75 0.1 0.42 1.33 0.19 0.17 0.45 – 15.31 – 0.12 0.17 – – 2.9 – 0.26 – 2.85 – 0.47 – – 69.3 – – – – 0.1 – 0.23 0.84 1.02 – – – – 0.82 9.71 84.9 1.49 0.93 0 97.03

0.85 0.12 0.12 1.53 0.13 – 0.47 – 14.11 0.28 – – – – 2.05 – 0.2 – 1.48 – 0.29 0.13 69.49 – – 0.3 – – – 0.12 0.82 0.92 1.06 0.19 0.14 – 1.73 2.81 90.51 0.83 3.6 0.16 97.91

RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS RI, MS Std RI, MS Std Std Std Std Std RI, MS RI, MS RI, MS RI, MS Std Std Std RI, MS RI, MS RI, MS Std RI, MS RI, MS Std Std RI, MS RI, MS RI, MS RI, MS RI, MS Std RI, MS RI, MS

RIEXP: Retention indices determined relative to n-alkanes (C6–C24) on a DB-5 column. RILIT: (Adams, 2007) alphabetical listing of compounds with their retention time and arithmetic retention index on DB-5. c Identification methods: MS, comparing of the mass spectrum with those found in the computer mass libraries Adams, Wiley and NIST 08; RI, comparing of retention indices with those declared in literature; Std, comparing of mass spectrum and retention time of available authentic standard. d Chemotype I piperitenone oxide/pulegone; II, cineole < 1,8- > /pulegone; III, piperitenone oxide/piperitone epoxide < trans > ; IV, piperitenone/ Cineole < 1,8- > ; V, menthone/Cineole < 1,8- > ; VI, Menthone/Menthol; VII, pulegone/Ocimene < (Z)-β- > for a detailed description of the accessions, cf. Table 1. b

8

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Table 4 Major compounds of the EOs profile among the twenty studied accessions of M. longifolia L. from Iran. Provinces

Code

Major compounds content [%]a

Ardabil Kerman Lorestan Chaharmahal Yazd Kerman Alborz W.Azarbaijan Ghazvin N. Khurasan Yazd N. Khurasan Hormuzgan Guilan Kerman Khuzestan S. Khurasan

G1 G2 G3 G4 G5 G6 G7 G8 G9 G10 G11 G12 G13 G14 G15 G16 G17

N. Khurasan Chaharmahal

G18 G19

Kerman

G20

piperitenone oxide (28) > trans piperitone epoxide (10.01) > 1,8-cineole (9.73) > cis piperitone epoxide (6.41) > borneole (6.04) pulegone (59.59) > (Z)-β-ocimene (21.27) pulegone (27.63) > menthone (9.38) > 1,8-cineole (7.17) piperitenone oxide (50.8) > 1,8-cineole (17.29) > pulegone (8.2) > cis piperitone epoxide (6.38) trans piperitone epoxide (38.95) > piperitenone oxide (24.54) pulegone (69.3) > (Z)-β-ocimene (15.31) trans piperitone epoxide (54.39) > piperitenone oxide (25.86) menthone (48.12) > menthol (20.98) > pulegone (18.86) piperitenone oxide (50.82) > trans piperitone epoxide (24.65) > menthone (7.24) piperitenone oxide (75.39) > trans piperitone epoxide (15.9) pulegone (35.03) > piperitenone oxide (31.5) > cis piperitone epoxide (10.25) piperitenone oxide (45.04) > trans piperitone epoxide (34.44) piperitenone (36.71) > piperitenone oxide (10.56) > 1,8-ineole (31.34) trans piperitone epoxide (32.52) > piperitenone oxide (30.19) > piperitone (13.63) > limonene (5.47) trans-Piperitone epoxide (21.24) > Pulegone (9.48) > 1,8-cineole (8.23) = menthone (8.23) 1,8-cineole (44.7) > menthone (28.66) > pulegone (8.84) borneole (29.14) > pulegone (14.4) > 1,8-cineole (11.59) > piperitone (9.06) > menth-2-en-1-ol < cis-ρ- > (8.67) > trans piperitone epoxide (8.85) piperitenone oxide (59.98) > trans piperitone epoxide (29.12) piperitenone oxide (24.79) > trans piperitone epoxide (11.23) > 1,8-cineole (9.39) >) > borneole (8.83) > cis-ρ-menth-2-en-1-ol (8.67) > pulegone (8.15) pulegone (69.49) > (Z)- β-ocimene (14.11)

a Within the total oil composition of each accession compounds with a content more than 5% introduced as a major compound; contents are presented in their front parentheses by percent.

(Z)-β-ocimene) was found to be pulegone (59.59–69.49%, the highest among all the studied accessions), followed by (Z)-β-ocimene (14.11–21.27%), separating the accessions G6, G20, and G2 from the others. In contrast to the chemotype III, the amounts of trans-piperitone epoxide and piperitenone oxide did not exceed 0.3% and 1.06% of the total volatile oil composition of the chemotype VII. A literature survey showed that the antifungal effects of herbal oils are directly attributed to the presence of pulegone (the precursor of menthol, menthone, and menthofuran) in their active ingredients (de Sousa Barros et al., 2015). Menthofuran, a hepatotoxic compound with fatal complications on the liver and cytochrome p450, is also metabolized from pulegone (Amini et al., 2014; Asekun et al., 2007; Shahverdi et al., 2007). In the present study, the principal component analysis was implemented in order to identify the chemotypes by determining the relationships among the data set (20 accessions × 42 components) (Fig. 3B). The biplot depicted by graphical PCA expounded the first two principal components (PCs) with an explication of 72.8% of the total variance. The components affording the highest loading factors in the data variation were piperitenone oxide (values of eigenvectors: 31.22; 22.52) and pulegone (values of eigenvectors: -32.21; 27.53) in the first component (PC1) and 1,8-cineole (values of eigenvectors: -1.72; 22.14) in the second component (PC2), mainly determined the phytochemical separation. The first component (PC1) accounted for 52.54% of the total variance and possessed positive correlation with piperitenone oxide and trans-piperitone epoxide, and negative correlation with (Z)-βocimene, ρ-menth-3-en-8-ol, pulegone, and thymol acetate. The second component (PC2) with a justification of 20.26% of the total variance, consisted of sabinene, β-pinene, 1,8-cineole, and octanol acetate as negative components. The third component (PC3) included negative correlation with cis-thujene, (E)-β-caryophyllene, α-humulene, germacrene D, and bicyclogermacrene, contributed to 11.52% of the total variance. Each chemotype formed a united group of accessions, characterized by higher quantities of specific components. Therefore, The PCA allowed classifying the studied M. longifolia accessions into different chemotypes. As reported by previous studies, the considerable variability of the chemical compounds might be attributed to the plant section (Uyanik and Gurbuz, 2014), ontogeny and plant growth phase (Saeb and Gholamrezaee, 2012), genotype and gene flow (Sari and Ceylan, 2002), harvesting location and time (Morshedloo et al., 2018), geographical

Barros et al., 2015; Gulluce et al., 2007). It is worth to note that piperitenone oxide is also the main integral component that is associated with insecticidal activity (Cordero et al., 2012; Mikaili et al., 2013). Besides, chemotype II (pulegone/1,8-cineole) was composed of three accessions (G3, G15, and G17), having essential oils with pulegone (9.48–27.63%) and 1,8-cineole (7.17–11.59%) as major compounds. With respect to the compounds of chemotype II, anti-inflammatory and antinociceptive properties of 1,8-cineole have been previously reported (Santos and Rao, 2000). Another separation within chemotype II out of the three accessions was related to G17, having an essential oil with borneol (29.14%) as the most abundant compound. The existence of such new oil composition (borneol) of the species M. longifolia was not described before from this region. The seven accessions of chemotype III (piperitenone oxide/transpiperitone epoxide), i.e., G5, G7, G14, G9, G18, G12, and G10, had Piperitenone oxide and trans-piperitone epoxide content of (24.54–75.39%) and (15.9–54.39%), respectively, for which 1,8-cineole (1.27–4.52) was the other main essential oil component of this chemotype. In this respect, Gulluce et al. (2007) provided evidence that M. longifolia possessed broad-spectrum antimicrobial activities among microorganisms due to the existence of the high amounts of piperitone epoxide (18.4%), pulegone (15.5%), and piperitenone oxide (14.7%). Moreover, piperitone isolated from M. longifolia var. chorodictya enhanced the antimicrobial activity of nitrofuran drugs, used for the remedy of urinary tract infections (Shahverdi et al., 2004). Therefore, with respect to the side effects of chemical drugs, these chemotypes are believed to be extremely useful for the above-mentioned issues. By comparison of the principal essential oil components of chemotypes (IV, V, and VI), the high content of piperitenone (36.71%) for G13, menthone (28.66%) for G16, and menthone (48.12%) accompanied by menthol (20.98%) for G8, separated the essential oil of these accessions into three distinct chemotypes. A previous study revealed that menthol might be used as an antimicrobial, antifungal, and anticandidal agent (Mikaili et al., 2013; Şahin et al., 2003). Nowadays, strong repellency of menthol against dental plaque has also been documented (Al-Bayati, 2009). Furthermore, the volatile oil of M. longifolia may act expressively as a scavenger of free radicals due to the existence of the monoterpene ketone (menthone) (Mimica-Dukić et al., 2003). Finally, the first main oil component of chemotype VII (pulegone/ 9

Industrial Crops & Products 140 (2019) 111698

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Fig. 3. Classification of the twenty accessions of M. longifolia L. based on phytochemical properties (A), Biplot of the first two principal components (PCs) for the studied accessions of M. longifolia L. based on the phytochemical compounds (B).

distribution (Aghaei et al., 2013), analytical method, and environmental factors (Bistgani et al., 2019; Hadian et al., 2011). Therefore, it seems that adaptation to the environmental condition due to obtaining special characters has been found to strongly influence the separation in the cluster analysis. Since the accessions G6, G20 and G2 (chemotype VII; Table 3) have originated from the sites with short geographical distances and the high altitudes (3240, 3415 and 2972 m a.s.l. respectively, Table 1), thus they could be categorized in the same cluster. The accessions G16 and G13 have inhabited in the lowest altitudes (61 and 1270 m a.s.l.) with the warm and dry climate (48.45 and 42.08 °C maximum temperature in the summer, Fig. 1, Table 2) that these conditions may bring them closer together in the chemotypes IV and V. Similarly, not only the accessions (G10, G12, and G18) or (G7, G9, and G14) geographically close to each other but also they have the same climate, forming a homogenous cluster in the chemotype III. In

addition, since the accessions G13 and G8 grow in distinct geographical zones, located on the Northwest and South Iran and possessing a unique climate with 1.36 to 32.39 mm rainfall per summer (Table 1), it might be that these fluctuations have impressive roles on their chemical differentiation in the chemotypes IV and VI. Indeed, the accessions G5 and G11 were geographically close together with similar arid climate but partly at different altitudes (1914 and 2538 m a.s.l. resp.), clustered distinctly into separate chemotypes I and III (Fig. 1). Nevertheless, the western accessions including G4 and G19, geographically farther away than G11 and G1, but having a similar climatic condition, classified a unit cluster (Chemotypes I; Fig. 3). However, it has been already reported that the variability of phytochemical characteristics was not always in agreement with the climatological properties, and in some cases seemed to be affiliated with the genetic factors, acting on the chemical variation (Croteau and Gershenzon, 1994; Moghaddam and 10

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Fig. 4. Classification of the twenty accessions of M. longifolia L. based on morphological properties.

Fig. 5. Leaf dimension (cm) variability of some studied accessions arrived from the cluster group A (A), and group B (B), the introduction of Mentha longifolia L. into cultivation in the field condition (C), a cultivated sample of Mentha longifolia L. (D).

11

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Table 5 Principal component analysis (PCA) of studied morphological traits in twenty M. longifolia accessions. Traits

Component

Leaf fresh weight Leaf dry weight Leaf surface Leaf length Leaf width Leaf width /Leaf length Plant height Node number Internode length Leaf number Stem diameter Leaves Dry Weight Inflorescence fresh weight Inflorescence dry weight Inflorescence diameter Inflorescence length Stem dry weight Dry yield Half Flowering Full Flowering Half Flower degradation Full Flower degradation Lateral branch number Lateral branch length Essential oil content Essential oil yield Flower Color Leaf Shape Hairy Leaf Eigenvalue % of variance Cumulative %

1

2

3

4

5

6

7

0.53** 0.66** 0.38 0.47 0.71** −0.41 0.58** 0.44 −0.18 0.19 0.78** 0.67** 0.09 0.19 0.30 0.30 −0.21 0.87** 0.44 0.62** 0.30 0.32 −0.50 −0.13 0.89** 0.88** 0.01 0.03 −0.04 7.03 24.27 24.27

0.31 0.45 0.18 0.29 −0.11 0.43 0.38 0.29 −0.05 0.12 0.08 0.44 0.60** −0.21 0.76** 0.27 −0.28 0.23 0.66** 0.44 0.89** 0.87** −0.05 0.02 0.31 0.33 −0.15 0.17 0.09 4.68 16.14 40.41

0.32 0.12 0.69** 0.40 0.43 −0.02 0.10 0.72** −0.89** 0.86** 0.25 0.23 0.09 0.10 −0.09 0.06 0.12 0.21 0.37 0.32 0.16 0.21 −0.17 −0.15 0.10 0.10 −0.13 −0.33 −0.12 3.71 12.81 53.22

0.34 0.34 0.06 0.58** −0.13 0.59** 0.14 0.12 0.00 0.03 0.05 0.33 0.39 0.81** 0.10 0.74** 0.03 0.04 0.10 0.01 −0.01 0.09 −0.14 0.08 0.12 0.17 −0.13 0.62** −0.03 2.93 10.11 63.33

−0.13 0.26 0.01 0.25 0.03 0.08 0.30 0.16 −0.01 0.30 0.20 0.24 0.50 0.33 −0.14 −0.03 −0.12 −0.16 0.35 0.34 0.01 0.06 −0.37 0.22 −0.03 −0.05 −0.86** −0.17 −0.69** 2.55 8.82 72.15

0.16 0.31 0.46 −0.07 0.32 −0.42 0.25 0.02 0.26 0.01 0.13 0.29 0.28 0.02 0.03 −0.16 −0.81** 0.05 0.10 0.00 0.07 0.13 0.38 −0.04 −0.03 0.01 0.14 0.31 −0.24 1.97 6.79 78.93

−0.01 0.04 0.16 0.07 0.32 −0.13 −0.40 −0.32 0.06 −0.13 −0.18 −0.06 −0.03 −0.01 0.28 0.40 0.07 0.05 −0.11 −0.07 −0.06 −0.15 0.52** 0.82** −0.13 −0.13 0.02 −0.36 −0.24 1.92 6.65 85.58

Eigenvalues ** are significant ≥0.52. Table 6 Rosmarinic acid and caffeic acid content (mg /100 g DW) variability in twenty M. longifolia accessions. No

Code

Min

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

G1 G2 G3 G4 G5 G6 G7 G8 G9 G10 G11 G12 G13 G14 G15 G16 G17 G18 G19 G20

Rosmarinic acid 258.8 68.72 203.23 33.44 58.37 109.46 92 70.63 72.96 37.09 177.81 40.68 52.53 78.48 129.78 72.54 36.95 43.09 150.87 52.06

Max Caffeic acid 5.41 5.82 5.81 8.06 5.57 6.13 7.3 6.26 6.23 6.33 6.84 7.34 6.74 6.83 5.66 5.45 4.67 4.8 5.39 5.01

Rosmarinic acid 264.53 124.53 302.97 44.43 61.78 145.82 148.17 119.13 77.22 101.03 184.14 69.1 79.19 92.32 177.06 127.43 59.9 61.09 156.23 66.27

Mean Caffeic acid 8.16 8.97 8.45 8.09 8.01 7.87 8.65 9.02 8.23 7.99 8.97 9.55 7.98 9.51 7.95 8.06 8.16 7.97 7.95 7.89

Pirbalouti, 2017). Therefore, to prove precisely this claim, it was indispensable to cultivate the various accessions of M. longifolia under a similar environmental condition (Esmaeili et al., 2019).

Rosmarinic acid 261.33 87.29 253.77 39.6 60.44 128.3 110.76 95.54 75.75 69.72 181.64 55.56 66.53 86.07 154.09 100.65 49.09 53.09 153.57 60.17

CV (%) Caffeic acid 6.87 7.43 7.18 8.2 6.87 7.06 8.05 7.73 7.32 7.22 7.98 8.52 7.44 8.23 6.85 6.85 6.5 6.48 6.72 6.48

Rosmarinic acid 1.12 36.95 19.66 14.17 3.01 14.2 29.25 25.41 3.19 45.88 1.86 25.67 20.11 8.16 15.36 27.29 23.49 17.27 1.75 12.17

Caffeic acid 20.09 21.27 18.38 2.68 17.9 12.46 8.57 17.98 13.8 11.63 13.41 13.03 8.6 16.28 16.79 19.27 26.92 24.54 19.05 22.38

accessions into two principal groups A and B (Fig. 4). The first cluster (A) contained eleven accessions (G1, G14, G18, G6, G15, G4, G5, G7, G20, G11, and G19). The second cluster (B) covered nine accessions (G2, G9, G8, G13, G16, G3, G17, G10, and G12). Among the morphological descriptors, the leaf size variability of some cultivated accessions from the two main clusters was compared in Fig. 5, and it showed clearly that in the most cases the leaf dimension of the group A was smaller than the group B. To characterize the degree of agro-

3.3. Morphological and essential oil variability The result of cluster analysis using the UPGMA method based on Euclidean distances of the morphological traits scattered the studied 12

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Fig. 6. HPLC diagram that shows the relative values of caffeic acid (1) and rosmarinic acid (2) in three samples of the studied accessions (A, G2; B, G7; C, G14) of M. longifolia L.

morphological variability, a principal component analysis (PCA) was implemented through a correlation matrix of all the morphological traits (Table 5). The first seven components of the PCA, representing 85.6% of the total variation. The first principal component (PC1) clarifying 24.3% of the total variance, belonged to ten vegetative traits and essential oil factors, including the highest eigenvalues of essential oil content, essential oil yield, and yield traits. The second principal component (PC2) explained 16.14% of the total variance, consisted of five reproductive traits, containing half and full flower degradation, and inflorescence diameter. In the following, the third principal component (PC3) revealed 12.8% of the total variance, comprising internode distance, leaf number, and node number traits. The morphological variability studied in previous reports elucidated that the variation may be linked to genetic factors and inhomogeneity (Pouyanfar et al., 2018; Selseleh et al., 2019) or geographical distribution (Heidari et al., 2016; Mohammadi and Asadi-Gharneh, 2018). The present results showed that the morphological variability acquired from the clustering of the M. longifolia accessions in some cases has no concordant with the geographical distribution. So discrepancies between the morphological cluster and the geographical location of the accessions could be explained by the priority of genetic factors on geographical origins. Although there was a rarely direct relation between clusters based on the phytochemical profile (Fig. 3) and morphological variability (Fig. 4),

there were some similarities between them. The phytochemical clustering of the accessions G13, G16, G8, G1, G4, and G19 were in similar to the morphological clustering results. In our current study, it was well known that phytochemical characteristics had been more influenced by geographical distribution than morphological traits, strengthening the fact that the phytochemical profile might be a more strong representation of the original location (Pirbalouti et al., 2013). 3.4. Rosmarinic acid and caffeic acid content The HPLC analysis of MeOH extracts showed a remarkable variability of rosmarinic acid and caffeic acid content (CV = 17.29% and CV = 16.25%, respectively, Table 6) among the M. longifolia accessions. Interestingly, this study indicated that rosmarinic acid was found to be a predominant phenolic compound in all the extracts, and caffeic acid had low-level content in comparison with rosmarinic acid (Fig. 6). On the contrary, Tahira et al. (2011) investigating the 15 cultivars of Mentha, Melissa, and Nepeta declared that rosmarinic acid had a lower content than caffeic acid in M. longifolia. Among all the accessions, the highest CV for rosmarinic acid was seen in accession G10 (45.88%), and for this value, accession G1 was ranked the lowest (1.12%). The highest CV for caffeic acid was in accession G17 (26.92%), and accession G4 had the lowest (2.68%). The rosmarinic acid content evaluated by HPLC 13

14

1 0.75** 0.47* 0.58** 0.44* 0.52** 0.58** −0.35 0.47* 0.32 0.17 0.40* 0.46* 0.56** 0.53** 0.63** 0.53** −0.04 −0.22

1 0.58** 0.70** 0.54** 0.71** 0.58** −0.19 0.63** 0.68** 0.36 0.53** 0.50* 0.78** 0.69** 0.73** 0.65** −0.11 −0.24

LDW (cm)

1 0.57** 0.76** 0.39* 0.65** −0.54** 0.46* 0.36 0.13 0.17 0.24 0.58** 0.47* 0.44* 0.56** −0.13 0.32

LS (cm2)

1 0.41* 0.49* 0.59** −.042* 0.52** 0.61** 0.56** 0.25 0.72** 0.63** 0.52** 0.64** 0.58** −0.19 −0.01

LL (cm)

1 0.39* 045* −0.34 0.57** 0.12 0.06 0.13 0.21 0.50* 0.19 0.59** 0.73** −0.13 0.12

LW (cm)

1 0.66** −0.08 0.71** 0.54** 0.26 0.3 0.25 0.60** 0.67** 0.65** 0.55** −0.14 −0.28

PH (cm)

1 −0.78** 0.66** 0.38* 0.29 0.26 0.17 0.67** 0.62** 0.60** 0.56** −0.01 −0.01

NN (no.)

1 −0.35 −0.05 −0.15 −0.03 −0.06 −0.44* −0.26 −0.30 −0.32 −0.03 −0.21

INL (cm)

1 0.27 0.39* 0.26 0.19 0.64** 0.46* 0.76** 0.69** −0.28 −0.02

SD (cm)

1 0.37 0.41* 0.39* 0.46* 0.66** 0.32 0.25 −0.09 −0.26

IFW (g)

1 −0.01 0.53** 0.12 0.03 0.21 0.15 −0.12 −0.07

IDW (g)

1 0.46* 0.39* 0.65** 0.44* 0.44* 0.03 −0.46*

ID (cm)

1 0.27 0.29 0.36 0.36 −0.29 −0.26

IL (cm)

1 0.63** 0.68** 0.60** −0.14 −0.15

FF (day)

1 0.61** 0.51* −0.07 −0.08

FFD (day)

1 0.91** −0.17 −0.01

EOC (%[w/ w])

1 −0.17 −0.02

DY (g.m-2)

1 −0.11

RA (%[w/ w])

1

CA (%[w/ w])

Abbreviations; LFW, Leaf fresh weight; LDW, Leaf dry weight; LS, Leaf surface; LL, Leaf length; LW, Leaf width; PH, Plant height; NN, Node number; INL, Internode length; SD, Stem diameter; IFW, Inflorescent fresh weight; IDW, Inflorescence dry weight; ID, Inflorescence length; FF, Full flowering; FFD, Full flower degradation; EOC, Essential oil content; DY, Drug yield; RA, Rosmarinic acid; CA, Caffeic acid. The correlation coefficients have been calculated with 20 pairs of values from the accessions. * and ** indicated significant correlations with p < 0.05 and p < 0.01 respectively.

LFW (cm) LDW (cm) LS (cm2) LL (cm) LW (cm) PH (cm) NN (no.) INL (cm) SD (cm) IFW (g) IDW (g) SD (cm) ID (cm) FF (day) FFD (day) EOC(%[w/w]) DY (g) RA(%[w/w]) CA(%[w/w])

LFW (cm)

Table 7 Pearson’s Correlation Coefficient of important traits studied among different Iranian accessions of M. longifolia L.

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analysis had high variation, ranging from 33.44 mg/100 g dry weight (G4) to 302.97 mg/100 g dry weight (G3), while the mean percentage of rosmarinic acid content varied from 39.6 mg/100 g dry weight (G4) to 261.33 mg/100 g dry weight (G1). The caffeic acid content varied from 4.67 mg/100 g dry weight (G17) to 9.55 mg/100 g dry weight (G12), while the average of caffeic acid content ranged from 6.48 mg/ 100 g dry weight (G18, and G20) to 8.52 mg/100 g dry weight (G12). The rosmarinic acid content in the other accessions also ranged from 36.95 to 264.53 mg/100 g dry weight and caffeic acid from 4.8 to 9.51 mg/100 g dry weight. these variations may be affiliated with the geographical source and different genome of the accessions used in the present study (Hadian et al., 2014; Tahira et al., 2011). However, Fletcher et al. (2010) stated that environmental factors did not affect the rosmarinic acid biosynthesis in mint clones. In a previous study, rosmarinic acid was found in the range of 0.001 to 0.93% in different species of subfamily Nepetoideae (Janicsák et al., 1999). Rosmarinic acid content has been previously reported in Ocimum basilicum (0.306%), Satureja montana (0.260%), Mentha longifolia (0.208%), and Mentha piperita (0.143%) (Adham, 2015; Zgórka and Głowniak, 2001). Different concentrations of rosmarinic acid of the accessions in Iran and other countries can be exploited for mint breeding projects. Also, an investigation on the horsemint indicated that the antioxidant activity of the methanolic extract is much better than the essential oil (Gulluce et al., 2007). Recently, a study revealed that chronic kidney disease (CKD), an important human health problem and concern in underdeveloped countries, is meaningfully subdued by rosmarinic acid, which is effective against cisplatin (CP)-induced nephrotoxicity (Oroojalian et al., 2017). By keeping into view the other effects of rosmarinic acid, its healing potencies in human immunodeficiency virus type 1 (HIV-1) and irritable bowel syndrome (IBS) have been reported to date (Karaman et al., 2003; Mazumder et al., 1997). Therefore, aforesaid natural extracts can be suggested for further pharmaceutical researches.

menthol were the major essential oil compounds. Cluster analysis grouped all the accessions into seven chemotypes, embraced chemotype I (piperitenone oxide/pulegone), chemotype II (1,8-cineole/pulegone), chemotype III (piperitenone oxide/trans-piperitone epoxide), chemotype IV (piperitenone/1,8-cineole), chemotype V (menthone/1,8-cineole), chemotype VI (menthone/menthol), chemotype VII (pulegone/ (Z)-β-ocimene). Moreover, in the analysis of MeOH extracts, rosmarinic acid was the predominant phenolic compound in comparison with caffeic acid. The morphological analysis of the studied accessions also generated two main clusters. With regard to the phytochemical and morphological clustering, in some cases the separation of accessions was not concordant with the geographical distribution, indicating the priority of genetic factors on geographical origin. Outstanding diversity in the studied characteristics among the accessions enabled the introduction of the elite accessions such as G13, G16, and G2 in terms of having the most biomass and essential oil content. These appropriate selections donate to breeder a gene pool for breeding programs, which led to desired and homologous cultivars in accordance with industry needs. Declaration of Competing Interest The authors declare no conflicts of interest. Acknowledgments The investigation was funded by Ferdowsi University of Mashhad code no. 32019 and student affairs organization. References Abedi, R., Golparvar, A.R., Hadipanah, A., 2015. Identification of the essential oils composition from four ecotypes of Mentha longifolia (L.) Huds. Growing wild in Isfahan province, Iran. J. BioSci. Biotech. 4, 117–121. Abootalebian, M., Keramat, J., Kadivar, M., Ahmadi, F., Abdinian, M., 2016. Comparison of total phenolic and antioxidant activity of different Mentha spicata and M. Longifolia accessions. Ann. Agric. Sci. 175–179. Adams, R., 2007. Identification of Essential Oil Components by Gas Chromatography/ Quadrupole Mass Spectroscopy, 4th edn. Allured Publishing Corporation, Carol Stream, IL. Adham, A.N., 2015. Comparative extraction methods, phytochemical constituents, fluorescence analysis and HPLC validation of rosmarinic acid content in Mentha piperita, Mentha longifolia and Ocimum basilicum. J. Pharmacogn. Phytochem. 130–139. Aghaei, Y., Mirjalili, M.H., Nazeri, V., 2013. Chemical diversity among the essential oils of wild populations of Stachys lavandulifolia Vahl (Lamiaceae) from Iran. Chem. Biodivers. 262–273. Al-Bayati, F.A., 2009. Isolation and identification of antimicrobial compound from Mentha longifolia L. leaves grown wild in Iraq. Ann. Clin. Microbiol. Antimicrob. 20. Amini, S., Azizi, M., Joharchi, M.R., Shafei, M.N., Moradinezhad, F., Fujii, Y., 2014. Determination of allelopathic potential in some medicinal and wild plant species of Iran by dish pack method. Theor. Exp. Plant Physiol. 26, 189–199. Andro, A.R., Atofani, D., Boz, I., Zamfirache, M., Burzo, I., Toma, C., 2011. Studies concerning the histo-anatomy and biochemistry of Mentha longifolia (L.) Huds. During vegetative phenophase. Analele Stiintifice ale Universitatii“ Al. I. Cuza” din Iasi 57, 25. Asekun, O., Grierson, D., Afolayan, A., 2007. Effects of drying methods on the quality and quantity of the essential oil of Mentha longifolia L. subsp. Capensis. Food Chem. 101, 995–998. Azizi, A., Yan, F., Honermeier, B., 2009. Herbage yield, essential oil content and composition of three oregano (Origanum vulgare L.) populations as affected by soil moisture regimes and nitrogen supply. Ind. Crops Prod. 29, 554–561. Azizi, M., Kaboli Farshchi, H., Oroojalian, F., Orafaee, H., 2017. Green synthesis of silver nano-particles using kelussia odoratissima mozaff. Extract and evaluation of its antibacterial activity. J. Agric. Sci. Technol. 681–691. Bahreininejad, B., Razmjou, J., Mirza, M., 2013. Influence of water stress on morphophysiological and phytochemical traits in Thymus daenensis. Int. J. Plant Prod. 151–166. Başkan, S., Öztekin, N., Erim, F.B., 2007. Determination of carnosic acid and rosmarinic acid in sage by capillary electrophoresis. Food Chem. 101, 1748–1752. Beremer, K., Chase, M.W., Stevens, P.F., 1998. An ordinal classification for the families of flowering plants. Ann. Mo. Bot. Gard. 531–553. Bistgani, Z.E., Hashemi, M., DaCosta, M., Craker, L., Maggi, F., Morshedloo, M.R., 2019. Effect of salinity stress on the physiological characteristics, phenolic compounds and antioxidant activity of Thymus vulgaris L. and Thymus daenensis Celak. Ind. Crops Prod. 311–320. Bozin, B., Mimica-Dukic, N., Simin, N., Anackov, G., 2006. Characterization of the

3.5. Correlations among traits The trait-to-trait correlation coefficients presented in Table 7, showed that there were some positive and negative relationships between the quantitatively tested traits. The essential oil content had the positive correlation with leaf fresh weight (r = 0.63), leaf dry weight (r = 0.73), leaf length (r = 0.64), leaf width (r = 0.59), leaf surface (r = 0.44), plant height (r = 0.65), yield (r = 0.91), node number (r = 0.60) and stem diameter (r = 0.76). Most of these traits are ascribed to the parts of essential oil storage of M. longifolia. One of the determining factors in the essential oil content is the growth of drug fraction of plants using the nutrition, irrigation, and light (Heidari et al., 2014). On the other hand, it was observed that the coefficient of inflorescent fresh weight (r = 0.32), inflorescent dry weight (r = 0.21), inflorescent length (r = 36) and internode length (r=-0.30) had no significant correlation with the essential oil production, and all related to reproductive characteristics. Therefore, it can be concluded that vegetative organs have much more effect on essential oil content than the reproductive organs in M. longifolia. This matter may be due to the distribution of essential oil storage organs, requiring further study. There was also no significant correlation between rosmarinic acid and studied traits, except a reverse correlation (r=-0.46) between caffeic acid and inflorescence diameter among the accessions. 4. Conclusions The results of GC-MS/FID and HPLC analysis revealed a wide range of variability among the twenty different accessions of M. longifolia. A total of forty-two compounds were characterized. The oxygenated monoterpenes including piperitenone oxide, pulegone, trans-piperitone epoxide, menthone, 1,8-cineole, piperitenone, (Z)-β-ocimene, and 15

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