Chemical compositions, radical scavenging capacities and antimicrobial activities in seeds of Satureja hortensis L. and Mentha spicata L. subsp. spicata from Turkey

Distributed: February 28, 2021 Abstract The present study determined some biological compounds, radical scavenging activity and antimicrobial capacity in seeds of Satureja hortensis L. and Mentha spicata L. subsp. s picata . Alpha-linolenic acid (C18:3 n3) has been found to be the major polyunsaturated fatty acid of Satureja hortensis L. (66.24 ± 1.24%) and Mentha spicata L. subsp. s picata (48.17 ± 1.01%). Linoleic acid (C18:2 n6) is identified as the second major polyunsaturated fatty acid in the present study and oleic acid (C18:1 n9) is determined as the major monounsaturated fatty acid. Current study showed that Satureja hortensis L. and Mentha spicata L. subsp. s picata have low levels of saturated fatty acids. It has been demonstrated that ergosterol (263.1 ± 2.14 µg/g), stigmasterol (39.07 ± 0.91 µg/g) and beta-sitosterol (14.64 ± 0.49 µg/g) have been found in Mentha spicata L. subsp. s picata , while ergosterol (69.41 ± 1.75 µg/g) and beta-sitosterol (19.81 ± 1.14 µg/g) have been determined in Satureja hortensis L. Also, this study determined that Satureja hortensis L. and Mentha spicata L. subsp. s picata have low lipide-soluble vitamin content. Furthermore, it has been found that Satureja hortensis L. contains naringenin (612.57 ± 2.57 µg/g), morin (86.97 ± 1.12 µg/g), quercetin (22.87 ± 0.75 µg/g), and kaempferol (20.11 ± 0.94 µg/g) while naringenin (135.91 ± 1.91 µg/g), naringin (61.23 ± 2.15 µg/g) and quercetin (47.51 ± 1.17 µg/g) have been detected as major flavonoids in the seeds of Mentha spicata L. subsp. s picata. The results of the present study suggest that methanol extracts of Satureja hortensis L. and Mentha spicata L. subsp. s picata have significant free radical scavenging activity. The present results revealed that Satureja hortensis L. and Mentha spicata L. subsp. s picata showed major activity against gram-positive and gram-negative


Introduction
Medicinal plants include a large variety of substances named phytochemicals that posses antioxidant activity (Giao et al., 2007;Tepe, 2008;Yesiloglu et al., 2013). Typical compounds that exhibit antioxidant activity comprise vitamins, carotenoids and phenolic compounds (Chanwitheesuk et al., 2005). Since synthetic antioxidants may lead to toxicity and carcinogenity interest in natural antioxidants has been rised last years (Pandini et al., 2018). Many herbs, particularly members of Lamiaceae family show strong antioxidant activity (Javanmardi et al., 2003).
Satureja, which is from Lamiaceae, is represented by 15 species of which the endemism ratio is 33% in Flora of Turkey (Davis, 1982;Gören et al., 2003;Satil and Kaya, 2007). Many members of Satureja have aromatic and medicinal characteristics (Eminagaoglu et al., 2007;Abd El Tawab et al., 2014). The leaves, flowers and stems of Satureja are used for herbal tea and it has been reported that Satureja species posses antimicrobial, antifungal, antiinflammatory (Güllüce et al., 2003;Gören et al., 2003;Boroja et al., 2018). Mentha, the other genus studied, distributed thoroughout temperate regions of Eurasia, Australia and South Africa (Gulluce et al., 2007). The genus includes fifteen taxa belonging to eight species in the flora of Turkey (Aksit et al., 2013). Leaves, flowers and the stem of Mentha species are frequently used in herbal teas or as additives in commercial spice mixtures for many foods to offer aroma and flavour (Moreno et al., 2002;Gulluce et al., 2007). It has been reported that the aeiral parts of Mentha have tonics, antispasmodic, stomachic and anti-inflammatory effects in the traditional medicine (Benabdallah et al., 2016(Benabdallah et al., , 2018. This is the report about fatty acid, vitamin, sterol, radical scavenging and antimicrobial activity in seeds of Satureja hortensis and Mentha spicata L. subsp. spicata. The goals of present study are i) to detect fatty acid compositions, vitamin and sterol contents; ii) to evaluate flavonoid contents and radical scavening properties in the seeds of Satureja hortensis and Mentha spicata L. subsp. spcicata; iii) in addition, the aim of this research is to investigate the antimicrobial activities of fatty acids, vitamins and flavonoid contents in the seeds which such a study has not been found in the literature.

Extraction of seed oils
Seed materials have been finely ground in a mill and then extracted with hexane/isopropanol (3:2 v/v) (Hara and Radin, 1978). The lipid extracts have been centrifuged at 10.000 g for 5 minutes and filtered, and the solvent has been then removed on a rotary evaporator at 40 °C. The extracted lipids have been stored under -25 °C until further analysis.

Fatty acids analyses
2% sulphuric acid (v/v) in methanol has been used to obtain the fatty acid methyle esters in the lipid extracts based on Christie' (1990) method. The methyl esters have been separated and quantified by gas chromatography and flame-ionization detection (Shimadzu GC 17 Ver.3) coupled to a Glass GC 10 after the fatty acid methyl esters have been treated with n-hexane and. Chromatographical conditions have been done with a capillary column (25 m in length and 0.25 mm in diameter, Permabound 25, Macherey-Nagel, Germany) using nitrogen as a carrier gas (flow rate 0.8 mL/min.). The temperatures of the column, detector and injection valve are adjusted to 130-220, 240, and 280 °C, respectively. It has been used authentic standart mixtures to obtain the methyle esters.

Chromatographic analysis and quantification of lipid soluble vitamins and sterols
Lipide-soluble vitamins and phytosterols have been extracted from the lipid fraction by the method of Sánchez-Machado et al. (2002) with minor modifications. The extracted lipids of seed material have been dissolved in acetonitrile/methanol (75/25 v/v) and have been injected 50 μL to HPLC (Shimadzu, Kyota Japan). The used column is a Supelcosil TM LC18 (250 × 4.6 mm, 5 μm, Sigma, USA) and the mobile phase is acetonitrile/methanol (75/25, v/v). The elution has been performed at a flow-rate of 1 mL/min and the temperature of analytical column is kept constant at 40 °C. the detection has been performed at 320 nm for retinol (vitamin A) and retinol acetate, and 215 nm for δ-tocopherol, vitamin D, α-tocopherol, α-tocopherol acetate, 265 nm for vitamin K1 and 202 nm for phytosterols (López-Cervantes et al., 2006). Class Vp 6.1 software assisted at workup of the data. The results of analysis have been expressed as μg/g for samples. (1,1-diphenyl-2picrylhydrazyl) radical scavenging capacity 2.4.1. Preparation of the extracts Homogenization of two g seed materials is done in 5 mL 80% methanol. Homogenates have been centrifuged at 5000 rpm at +4 °C. The supernatant is concentrated by reduced-pressure rotary evaporation after centrifugation. Each extract is re-suspended in dimethyl sulphoxide (DMSO) to produce a reserve solution (Kursat et al., 2011).

Chromatographic conditions for flavonoids
A PREVAIL C18 reversed-phase column (15 × 4.6 mm, 5 µm, USA) is used to do chromatographic analysis and methanol/water/acetonitrile (46/46/8, v/v/v) containing 1.0% acetic acid has been used as the mobile phase (Zu et al., 2006). The mobile phase has been filtered through a 0.45 µm membrane filter (Millipore), then de-aerated ultrasonically prior to use. Catechin (CA), naringin (NA), kaempferol (KA), naringenin (NAR), resveratrol (RES), myricetin (MYR), morin (MOR), quercetin (QU) and rutin (RU) have been measured by DAD separation at 280 nm for CA and NA, 254 nm for RU, MYR, MOR and QU, 306 nm for RES, and 265 nm for KA. Flow rate and injection volume have been adjusted to 1.0 mL/min and l0 µL, respectively. The chromatographic peaks of the extracts have been evaluated by comparing their retention time with that of the reference standards. All chromatographic operations have been done at a temperature of 25 °C.

Antioxidant assay by DPPH radical scavenging activity
The free radical scavenging effects of extracts have been measured by the decoloration of a methanolic solution of DPPH • based on the Liyana-Pathirana and Shahidi' (2005) method. A solution of 25 mg/L DPPH in methanol has been solved and 4.0 mL solution is mixed with 50, 100 and 250 µL of extract in DMSO. Then, mixture has been stored in darkness at room temperature for 30 minutes. The absorbance of the mixture has been evaluated spectrophotometrically at 517 nm. 1 µM quercetin is used as a reference (Kursat et al., 2011).
The scavenging capacity of DPPH radicals have been determined by the following Equation 1: Abs control Abs sample DPPH radical scavenging activity Abs control x100 where: Abs control is the absorbance of DPPH radical + methanol; Abs sample is the absorbance of DPPH radical + sample extract /standard (Kursat et al., 2011 Antimicrobial tests have been done by using the well agar method (100 µL of suspension containing 10 6 cells/mL of bacteria, 10 4 cells/mL yeast and cells/mL dermatophyta fungi as per McFarland standard, inoculated into Mueller Hinton Agar (Difco), Malt Extract Agar (Difco), and Sabouroud Dextrose Agar (Oxoid), respectively). Wells have been prepared in the plates with the help cork-borer (0.85 cm). 10 µL of the flavonoids, vitamins and fatty acids in the seeds have been added in to the well. Steril petri dishes (9 cm diameter) have been placed at 4 °C for 2h. Then, the inoculated plates have been incubated at 37 ± 0.1 °C at 24 h for bacterial strains and also at 25 ± 0.1 °C at 72 h for yeast and dermatophyta fungi. Antimicrobial activity has been observed by measuring the zone of inhibition against the test organisms (Collins and Lyne, 1987). Wells injected with methanol and hexane served as negative controls.

Fatty acids, vitamins and sterol contents in the seeds of S. hortensis and M. spicata subsp. spicata
Essential fatty acids, are called polyunsaturated fatty acids, can not be produced by body and they must be taken from dietary sources (Singh, 2005). The intake of polyunsaturated fatty acids have been shown to reduce the risk of coronary artery, other cardiovascular and some chronic diseases (Campos et al., 2008;Rajaram, 2014). The Lamiaceae is characterized by high percentage of unsaturated fatty acids (Azcan et al., 2004). Similarly, present study demonstrated that S. hortensis and M. spicata subsp. spicata from Lamiaceae have high polyunsaturated fatty acids.

Flavonoid contents and radical scavenging capacities in the seeds of S. hortensis and M. spicata subsp. spicata
Phenolics, are one of main group herbal compounds, have potent to high antioxidant capacity against free radical damage (Benabdallah et al., 2016). It has been indicated that species from Lamiaceae have strong antioxidant capacity mostly due to phenolic compounds (Hossain et al., 2010). The variety of phenolics reduce cancer growth by capturing cancer cells in the certain phases of the cell cycle, heart disease and diabetes (Berdowska et al., 2013;  Shahidi and Ambigaipalan, 2015). Flavonoids are most abundant compounds of phenolics in the plants and contained 6000 chemicals (Gomaa et al., 2015). Total nine flavonoids (rutin, myricetin, morin, quercetin, kaempferol, catechin, naringin, naringenin, resveratrol) are studied in this study (Table 3). It has been found that S. hortensis contained naringenin (612.57 ± 2.57 µg/g), morin (86.97 ± 1.12 µg/g), quercetin (22.87 ± 0.75 µg/g), and kaempferol (20.11 ± 0.94 µg/g). However, myricetin, catechin and naringin aren't identified in the seeds of S. hortensis. Literatures showed that Satureja has natural phenolic compounds (Zheng and Wang, 2001;Zeljkovic et al., 2015). It has been determined that Satureja has luteolin and naringenin contents studies done by different researchers (Skoula et al., 2005;Kosar et al., 2005). Oke et al. (2009) suggested that the amounts of total phenols found in the Satureja methanolic extract are very high. Also, Tepe and Cilkiz (2016)  The results related to the radical-scavenging potential of S. hortensis and M. spicata subsp. spicata are summarized in Table 3. It has been found that 25 and 50 µL methanolic extracts of S. hortensis (89.62 ± 1.17%, 85.24 ± 1.24%, respectively) exhibited higher radical scavenging activity than those of M. spicata subsp. spicata (66.85 ± 1.01%, 89.91 ± 2.12%, respectively); this might be due to the high flavonoid concentration of S. hortensis. Several studies indicated that methanol extracts of Satureja species exhibited high antioxidant activity (Eminagaoglu et al., 2007;Oke et al., 2009;Alonso-Carrillo et al., 2017). Dorman and Hiltunen (2004) suggested that the crude and ethyle acetate extracts of Satureja are capable of scavenging reactive free radical species. It has been suggested that the extracts of S. hortensis may be able to defend sensitive constituents such as amino acids, DNA, lipoproteins, polyunsaturated fatty acids, sugars and proteins from oxidative stress (Dorman and Hiltunen, 2004;Zahedifar and Najafian, 2015). Besides, different studies demonstrated that Mentha species represent strong antioxidant activity and high phenolic constituent (Tawaha et al., 2007;Benabdallah et al., 2016;Tang et al., 2016). Unver et al. (2009) andConforti et al. (2008) indicated that Mentha has high free radical scavenging capacity. Also, Sytar et al. (2018) found that Mentha spicata subsp. spicata has phenolic content and high antioxidant capacity. Furthermore, Motamed and Naghibi (2010) indicated that Mentha (93.68%) and Satureja species (93.39%) have the highest DPPH radical scavenging activity. These results suggested that methanol extracts of S. hortensis and M. spicata subsp. spicata have significant free radical scavenging activity.

Antimicrobial activities of lipid soluble vitamins, flavonoids and fatty acids in the seeds of S. hortensis and M. spicata subsp. spicata
The antimicrobial capacities of seed extracts have many practices comprising pharmaceuticals, food protection, natural therapies and alternative medicine (Reynolds, 1996;Lis-Balchin and Deans, 1997;Kelen and Tepe, 2008). The antimicrobial activities of vitamins, flavonoids and fatty acids of the studied species, negative control group and standart antibiotics have been showed in Table 4. It has been found that the extracts of vitamins and flavonoids in seeds have antibacterial and antifungal activity against the microorganisms tested but it seems that the antimicrobial activities of fatty acids extracts of seeds are lower than flavonoids and vitamins extracts ( Table 4). Table 4 shows that the vitamin extracts of S. hortensis have the maximum antimicrobial activity against all of the tested microorganisms: E. coli (12.1 ± 0.1 mm), K. pneumoniae  of Satureja hortensis showed antibacterial activity against E. coli (11.2 ± 0.1 mm), K. pneumoniae (8.2 ± 0.4mm), S. aureus (13.1 ± 0.3 mm), and B. megaterium (8.3 ± 0.2 mm) ( Table 4). Several studies revealed that Satureja species showed major activity against the gram positive and gram microorganisms, fungi and yeast (Dikbas et al., 2009;Choulitoudi et al., 2016;Tepe and Cilkiz, 2016;Valdivieso-Ugarte et al., 2019;Vitanza et al., 2019). On the other hand, the results obtained from study of Sahin et al. (2003) showed that hexane extract of S. hortensis don't have antifungal, but they observed antibacterial activity against four strains of three Bacillus species. Furthermore, the vitamin extracts in the seeds of M. spicata subsp. spicata have strongly antimicrobial effect over some tested microrganisms; K. pneumoniae (25 mm), B. megaterium (25 mm), C. albicans (24 mm), Epidermophyton sp. (24 mm), S. aureus (21 mm), C. glabrata (18 mm), Trichophyton sp. (18 mm) and E. coli (15 mm) (Table 4). Also, the extracts of flavonoids in the seeds of M. spicata have antimicrobial activity on E. coli, K. pneumoniae, S. aureus, B. megaterium, C. albicans, C. glabrata and Trichophyton sp. (13 mm, 23 mm,13 mm, 35 mm, 17 mm, 11 mm and 19 mm zone of inhibition respectivelly) while it has not antimicrobial activity on Epidermophyton sp. However; the fatty acids in the seeds of M. spicata subsp. spicata exhibited antimicrobial effect over K. pneumoniae (13 mm) and B. megaterium (11 mm), where as those have not antimicrobial effect on the other tested microrganisms: E. coli, S. aureus, C. albicans, C. glabrata, Epidermophyton sp. and Trichophyton sp. It has been reported that chemical compositions of Mentha species have antifungal properties against human pathogens (Malassezia furfur, Trichophyton rubrum, and Trichosporon beigelii) and inhibited efficiently antimicrobials (Yadegarinia et al., 2006;Mahboubi and Haghi, 2008;Scherer et al., 2013;Biswas et al., 2014;Singh et al., 2015;Alexa et al., 2018). On the contrary, Gulluce et al. (2007) reported the methanol extract from aerial parts of Mentha showed no antimicrobial activities.

Conclusions
The present study found that α-linolenic acid (C18:3 n3) is the major polyunsaturated fatty acid of S. hortensis (87.1 ± 0.61%) and M. spicata subsp. spicata (84.4 ± 0.59%). It has been showed that the saturated fatty acids of S. hortensis and M. spicata subsp. spicata low. Also, ergosterol has been found to be a major sterol in the studied taxa. However, it has been found that S. hortensis and M. spicata subsp. spicata have low lipide-soluble vitamin content. On the other hand, naringenin and quercetin have been identified as the predominant flavonoids in S. hortensis and M. spicata subsp. spicata. Furthermore, present results suggested that methanol extracts of S. hortensis and M. spicata subsp. spicata display significant free radical scavenging activity. In addition, the present results indicated that S. hortensis and M. spicata subsp. spicata showed major activity against gram-positive and gram-negative microorganisms, fungi and yeast. ABD EL TAWAB, A.M., SHAHIN, N.N. and ABDELMOHSEN, M.M., 2014. Protective effect of Satureja montana extract on cyclophosphamide-induced testicular injury in rats. vol. 224,