Biochemical parameters and antioxidant property of three Salvia L. taxa endemic in Turkey

Parâmetros bioquímicos e propriedade antioxidante de três espécies de Salvia L. endêmicos da Turquia

Abstract

The aim of the present study was to determine and compare the fatty acids, lipid soluble vitamins, sterols, phenolics, and antioxidant capacities of three endemic Salvia L. taxa (S. euphratica var. Montbret & Aucher ex Bentham leiocalycina (Rech. Fil.) Hedge, S. euphratica var. Montbret & Aucher ex Bentham euphratica (Rech. Fil.) Hedge and S. pseudoeuphratica Rech.Fil.) and to evaluate these results systematically. The fatty acid compositions were determined by using gas chromatography, while the lipid soluble vitamins, sterols, and phenolics were determined by using HPLC. Also, the antioxidant capacities of three Salvia taxa were measured in vitro. Palmitic acid was found as major saturated fatty acid while oleic acid, linoleic acid, α-linolenic acid, and erucic acid were found as major unsaturated fatty acids in the present study. It was found that S. euphratica var. euphratica had lower palmitic acid (8.94 ± 0.71%), total saturated fatty acid (19.16 ± 0.15%), and higher unsaturated fatty acid content (82.08 ± 0.52%) than other studied taxa. Furthermore, it was shown that S. euphratica var. euphratica had different 18.3/18:2 (0.36) unsaturated/saturated fatty acid (4.28) ratios. However, this study demonstrated that Salvia taxa had low lipid soluble vitamins, sterol contents. On the other hand, it was shown that Salvia taxa had similar catechin (509.2 ± 4.21 µg/g and 552.2 ± 9.21 µg/g) and vanillic acid amounts (351.2 ± 2.17 µg/g and 396.8 ± 4.1 µg/g) in this study. And also, it was found that Salvia taxa had high rosmarinic acid content while S. euphratica var. leiocalycina had the highest rosmarinic acid content (1480 ± 7.57 µg/g). On the other hand, it was shown that the two ferulic acid contents of S. euphratica varieties were higher (1175 ±/5.21 µgmg-1740.2 ± 4.82 µg/mg) than the ferulic acid content of S. pseudoeuphratica of which was the lowest (19.2 ± 0.97 µg/mg). The present results suggested that the biochemical results guided the morphological studies, and Salvia taxa have a potent antioxidant capacity.

Keywords:
antioxidant property; biochemical parameters; GC-MS; HPLC; Lamiaceae

Resumo

O objetivo do presente estudo foi determinar e comparar os ácidos graxos, vitaminas lipossolúveis, esteróis, fenóis e capacidades antioxidantes de três espécies endêmicas de Salvia L. (S. euphratica var. Montbret & Aucher ex Bentham leiocalycina (Rech. Fil.) Hedge, S. euphratica var. Montbret & Aucher ex Bentham euphratica (Rech. Fil.) Hedge e S. pseudoeuphratica Rech.Fil.) e avaliar esses resultados sistematicamente. As composições de ácidos graxos foram determinadas por cromatografia gasosa, enquanto as vitaminas lipossolúveis, esteróis e fenóis foram determinadas por HPLC. Além disso, as capacidades antioxidantes das três espécies de Salvia foram medidas in vitro. O ácido palmítico foi encontrado como ácido graxo saturado principal, enquanto o ácido oleico, ácido linoleico, ácido α-linolênico e ácido erúcico foram encontrados como principais ácidos graxos insaturados no presente estudo. Verificou-se que S. euphratica var. euphratica tem menor teor de ácido palmítico (8.94 ± 0.71%) e ácido graxo saturado total (19.16 ± 0.15%) e maior teor de ácidos graxos insaturados (82.08 ± 0.52%) do que as outras espécies estudadas. Além disso, foi demonstrado que a S. euphratica var. euphratica apresentou diferentes proporções 18:3/18:2 (0.36) de ácidos graxos insaturados/saturados (4.28). No entanto, este estudo demonstrou que o gênero Salvia tinha baixo teor de vitaminas lipossolúveis e baixo conteúdo de esteróis. Por outro lado, foi demonstrado que as espécies do gênero Salvia contém quantidades de catequinas (509.2 ± 4,21 µg/mg-552.2 ± 9.21 µg/mg) e ácido vanílico semelhantes (351.2 ± 2.17 µg/mg 396,8 ± 4,1 µg/mg). Descobriu-se também que o gênero Salvia tinha alto conteúdo de ácido rosmarínico enquanto a espécie S. euphratica var. leiocalycina apresentou o maior teor desse ácido (1.480 ± 7.57 µg/g). Por outro lado, foi demonstrado que os teores de ácido ferúlico da espécie S. eupratica foram maiores (1.175 ± 5.21 µg/mg-1740.2 ± 4.82 µg/mg) do que o conteúdo de ácido ferúlico da espécie S. pseudoeuphratica dos quais foi o mais baixo (19.2 ± 0.97 µg/mg). Os resultados atuais sugerem que os resultados bioquímicos orientaram os estudos morfológicos e as espécies de Salvia têm uma potente capacidade antioxidante.

Palavras-chave:
propriedade antioxidante; parâmetros bioquímicos; GC-MS; HPLC; Lamiaceae

1. Introduction

The Lamiaceae, is one of the largest families in the plant kingdom, and includes more than 240 genera that are aromatic (Sharafzadeh and Zare, 2011). Sage, Thymus, Rosemary, Marjoram and Basil are some of the popular aromatic plants belonging to Lamiaceae growing in both the Mediterranean and Asia (Naghibi et al., 2005; Hossain et al., 2010; Khled Khoudja et al., 2014). Sage (Salvia L.), from the subfamily Nepetoideae of the Mentha tribe in the Lamiaceae, is spread throughout different regions of the world and has more than 1000 taxa (Kalaycioğlu et al., 2018). Some species of Salvia L. have the significant pharmaceutical properties and have been used in traditional medicine since ancient times (Asadi et al., 2010; Loizzo et al., 2010). Also, the members of Salvia L. are consumed as herbal tea and also used in cosmetics, flavouring agents, and perfumery industries (Loizzo et al., 2010; Akbulut and Bayramoglu, 2013). The genus is represented by 100 taxa of which 57 are endemic, with Anatolia being a remarkable center for the genus (Celep et al., 2015; Bardakci et al., 2019).

The studies demonstrated that Salvia L. species have antidiabetic, antiviral, antioxidant, anticancer, and anti-inflammatory effects due to possessing the most important biological active compounds including sterols, terpenoids, essential oils, and plyphenolics (Asadi et al., 2010; Ben Farhat et al., 2015a; El Euch et al., 2019). There are several studies related to determining the biochemical characteristics and antioxidant capacity of different Salvia species (Tepe et al., 2006; Kelen and Tepe, 2008; Tosun et al., 2009; Gezek et al., 2019). However, to the best of our knowledge, there is no biochemical and antioxidant report concerning three endemic taxa (S. euphratica var. leiocalycina, S. euphratica var. euphratica and S. pseudoeuphratica). Therefore, the aim of this study is to determine and compare the fatty acid compositions, lipid-soluble vitamins, sterols, flavonoids, phenolic acids, radical scavenging activities, and total phenolics of S. euphratica var. Montbret & Aucher ex Bentham leiocalycina (Rech. Fil.) Hedge, S. euphratica var. Montbret & Aucher ex Bentham euphratica (Rech. Fil.) Hedge and S. pseudoeuphratica Rech.Fil. Another purpose is to compare biochemical results with morphological results. Systematically, Hedge (1982a, b) changed the taxonomic position of S. euphratica after it had been identified by Bentham (1836) from Turkey (Dizkirici et al., 2015). However, S. leiocalycina has been evaluated under a variety of the S. euphratica based on the results of morphological and ecological studies, and in the most recent studies, S. pseudoeuphratica is accepted as being a different species by Kahraman et al. (2010).

2. Material and Methods

2.1. Chemical agents

All the chemicals were provided by Sigma-Aldrich.

2.2. Plant materials

S. euphratica var. Montbret & Aucher ex Bentham leiocalycina (Rech. Fil.) Hedge (B7, Baskil district, marble factory around, railway near, 1330 m, 38° 34’56 °N, 38° 50’23 °E, M. KURŞAT 1610), S. euphratica var. Montbret & Aucher ex Bentham euphratica (Rech. Fil.) Hedge (B7, Elazig-Malatya road, Komurhan district,755 m, 38° 27’10 °N, 38 48’28 °E, M. KURŞAT 1611) and S. pseudoeuphratica Rech. Fil. (B7, Elazığ, Keban-Elazığ road, 3^rd-4^th km, 900 m, 38° 44’46 °N, 38° 47’59 °E, M. KURŞAT 1622) were collected that grow as native plants in Turkey in 2011, which were deposited in FUH (Firat University Herbarium). The identification of collected materials were done by Murat KURSAT. After the collection and identification, the plant materials were immediately extracted, and HPLC and GC-MS analyses were completed.

2.3. Extraction of plant materials

2.3.1. The analysis of fatty acid, lipid soluble, vitamins and sterol

After collection, each plant material was dried at room temperature, grounded and then, extracted with isopropanol/hexane (2:3) as suggested by Hara and Radin, (1978). The lipid samples were centrifuged at 10.000 g, for 5 min. The solvent was evaporated by using a rotary evaporator at 40 °C, and the samples were kept at -25 °C.

2.3.1.1. Preparation of fatty acid methyl esters

In order to determine fatty acids analyses in lipids using gas chromatography, they must be converted into methyl esters derivatives. The FAME analysis was conducted based on the method by Christie (1990). Fatty Acid Methyl Esters (FAME) are produced from vegetable oils by transesterification. In this process, a glyceride reacts with an alcohol in the presence of a catalyst, forming a mixture of fatty acids esters and an alcohol (Schuchardta et al., 1998). Hexane was used to extract FAME. To prepare methyl ester, the lipid extract in the hexane/isopropanol phase (3:2, v/v) was taken into 30 mL non-leakage test tubes. 5 mL of 2% methanolic sulfuric acid was added to the extraction and mixed using a vortex. This mixture was left to be methylated in a 50 °C oven for 15 hours, and then the tubes were cooled to room temperature and mixed well by adding 5 mL of 5% NaCl. The fatty acid methyl esters formed were extracted with 5 mL hexane, and the hexane phase was taken, and treated with 5 mL of 2% KHCO₃. The extracts were left for four hours to separate phases and the solvent in the mixture including the methyl esters was evaporated at 45ºC and under nitrogen flow. Then, the samples were dissolved with 1 mL of hexane and taken into 2 mL otosampler vials to analyze with gas chromatography (Bahsi, 2008).

2.3.1.2. Fatty acid methyl esters in the gas chromatography

After obtaining the methyl esters from fatty acids, they were analyzed by SHIMADZU (Kyoto, Japan) GC 17 Ver. 3 Gas Chromatography. GC Capillary Column Permabond CW 20 M-DEG ID: 0.25 mm, film thickness: 0.25 µm length: 25 (Macherey-Nagel® Germany) was used for the analysis. The column temperature was adjusted between 120-220 °C, the injection temperature was kept at 240 °C, and the detector temperature was at 280 °C during analysis. Prior to the analysis of the fatty acid methyl esters, the retention times of each fatty acid were determined by injecting mixtures of standard fatty acid methyl esters, and an analysis of fatty acid methyl esters mixtures was performed. Each of the total fatty acids were calculated as a percentage amount. Results were calculated by using the GC Solution 2.3 program.

2.3.1.3. Chromatographic analysis and quantification of lipid soluble vitamins and sterols

The Shimadzu SPD UV detector VP series HPLC apparatus equipped with a DAD detector was used to determine the sterols (ergosterol, sitosterol and beta-sitosterol) and to perform a lipid-soluble vitamins analysis according to the Sánchez-Machado et al. (2002)’ method. The samples were homogenized in a 2 mL acetonitrile/methanol (3/1, v/v) mixture for one minute. The extracts were centrifuged at 6000 g for 10 min. at 4 °C, and 1 mL supernatant was taken into vials and analyzed on HPLC. The extracts were treated with acetonitrile/methanol (75/25 v/v). The flow rate of the mobile phase was determined to be 1 mL/min., and the temperature of the analytical column was kept at 40 °C. Supelcosil LC 18 DB (250 × 4.6 mm, 5 µm; Sigma, USA) colon was used as a reverse phase column. Class Vp 6 1 Software supplied by the Shimadzu Corporation was used to determine the amount of each compound in the samples, and results were given as µg/mL. The detection of retinol and retinol acetate were done at 320 nm, while α-tocopherol, vitamin D, a-tocopherol, α-tocopherol acetate was detected at 215 nm, and phytosterols were detected at 202 nm, and vitamin K1 and K2 were detected at 265 nm (López-Cervantes et al., 2006). Class Vp 6.1 software was used to calculate the amounts in the study.

2.4. The extraction procedure

2 g seed was ground and treated with 5 mL 80% methanol to homogenization for flavonoids and phenolic acids analyses. The homogenization was performed at 5000 rpm at +4º C. And rotary evaporation was used to obtain a supernatant. Lastly, extracts were suspended by 2 mL dimethyl sulphoxide (DMSO) (Kursat et al., 2011).

2.4.1. Chromatographic conditions for phenolics

The flavonoids and phenolic acids were determined based on the method by Zu et al. (2006). Two g of plant materials were homogenized with a 5 mL 80% methanol solution, and extracts were centrifuged at 5000 rpm at +4 °C, and dimethyl sulphoxide (DMSO) was used to supply a stock solution. The column was a PREVAIL C18 reversed-phase column (15 × 4.6 mm, 5 µm, USA), and the mobile phase was methanol/water/acetonitrile (46/46/8, v/v/v) comprised of 1.0% acetic acid in the chromatographic analysis (Zu et al., 2006). The flow ratio was 1.05 mL/min., and the injection volume was 10 μL. The chromatographic peaks were confirmed by determining the retention times with those of the standards. Rutin, myricetin, morin, quercetin, and vanillic acid at 254 nm; kaempferol at 264 nm; catechin, naringin and cinnamic acid at 280 nm; naringenin at 285 nm; resveratrol at 306 nm; and caffeic acid, ferulic acid and rosmarinic acid at 330 nm were determined by DAD following the RP-HPLC (Shimadzu SPD UV detector VP series HPLC). The chromatographic studies were done at 25 °C.

2.5. Antioxidant activity

2.5.1. DPPH Radical scavenging capacity

The DPPH radical scavenging capacity was measured based on the method by Liyana-Pathirana and Shahidi (2005). 4.0 mL DPPH solution was mixed with 25, 50, 100, 150, and 250 µL of extract. The complex was kept in darkness for 30 minutes at room temperature (Liyana-Pathirana and Shahidi, 2005). The absorbances were measured at 517 nm, using Shimadzu UVmini-1240 (UV-VIS Spectrophometer). Quercetin (1 µM) was used as reference standard. The results were determined by using Formula 1:

$$\begin{array}{l}DPPHradicalscavengingcapacity\left(\%\right)=\\ \left[\left(Abs\_control–Abs\_sample\right)\right]/\left(Abscontrol\right)]x100\end{array}$$ (1)

Abs_control is the absorbance of the DPPH radical + methanol; Abs_sample is the absorbance of DPPH radical + sample extract/standard.

2.5.2. ABTS assay

The 2,2-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) method was used in accordance to Re et al. (1999). The ABTS (7 mM) was mixed with 2.45 mM potassium persulphate and used to obtain ABTS radical cation (ABTS•⁺). The solution was stored for 12-16 h at room temperature. The (ABTS•⁺) solution was dissolved with water to measure an absorbance of 0.700 ± 0.020 at 734 nm. The 3 mL ABTS solution was treated with 25, 50, 100, 150 and 250 µL extracts, and the absorption was detected at 6 min. The absorbance of the control (3.0 mL (ABTS^•+) solution with 30 L water) was written as Acontrol (Skotti et al., 2014) (Formula 2).

$$\begin{array}{l}ABTSradicalcationscavengingcapacity\left(\%\right)=\\ \left[\left(Abs\_control–Abs\_sample\right)\right]/\left(Abs\_control\right)]x100\end{array}$$ (2)

2.5.3. Metal chelating activity

The chelating capacity of the plant materials were assessed based on the Dinis et al.’s (1994) method. According to this method, 50 µL of 2 mM FeCl₂ was added to several concentrations including 50, 100, 250, and 500 µg/mL of extracts. 0,2 mL ferrozine was added to the mixture to start the reaction. The solution was kept at room temperature for 10 minutes after the solution was firmLy mixed. 562 nm was used as absorbance (Dinis et al., 1994). The percentage inhibiting of the ferrozine–Fe²⁺ complex was found as the following Formula 3:

$$\begin{array}{l}TheFerrousionchelatingactivity\left(\%\right)=\\ \left[1-\left(As/Ac\right)\right]\times 100\end{array}$$ (3)

where A_c is the absorbance of the control, and As is the absorbance of the extract/ standard (Kizilpinar-Temizer et al., 2017). Positive control is Na₂EDTA.

2.5.4. Determination of antioxidant activity by MDA/TBARS formation

The antioxidant activity of materials was done using the method proposed by Shimoi et al. (1994). The extracts were prepared by using DMSO (dimethyl sulfoxide). Oleic acid (3.35 mM), linoleic acid (9.01 mM), linolenic acid (2.30 mM) dissolving in the DMSO, Fe²⁺ (FeCl₂.2H₂O) and hydrogen peroxide are used in the experiment. The Control, Fenton reagent group, and the group of sage extracts were formed. The control group contained 0.5 mL of fatty acid and a buffer solution (0.2% Tween 20/0.05 M Tris HCl/0.15 M KCl, pH=7.4), whilst the fenton group contained 0.5 mL of fatty acid, a buffer solution, FeCl₂.2H₂O (50 μM), and hydrogen peroxide (0.01 mM). Also, the sample extracts contained 0.5 mL of fatty acid, a buffer, FeCl₂ (50 μM), hydrogen peroxide (0.01 mM), and 0.25 mL sage extract. All the groups were stored at 37 °C for 24 h for incubation by adding 0.1 mL of a 4% (w/v) BHT to protect the greater oxidation. After that, 1 mL was taken from the samples in all the three groups and 1 mL of 0.6% TBA was added to the reaction mixture and samples incubated at 90 °C for 30 min. Finally, 4 mL butan-1-ol was added to the tubes, mixed, and centrifuged at 4250 rpm for 10 min. The absorbance of the supernatant was determined at 532 nm using a spectrophotometer Shimadzu UV mini–1240. MDA standard curves were formed by 1,1,3,3-tetramethoxypropane, and thiobarbituric acid-reactive substances (TBARS) were expressed as mg MDA/kg dry matter (Keser et al., 2014).

3. Results and Discussion

This study showed that the main saturated fatty acids in Salvia taxa were palmitic acid (16:0) and stearic acid (18:0) (Table1). The palmitic acid contents of the studied Salvia taxa were between 8.94 ± 0.71% (S. euphratica var. euphratica) and 32.57 ± 1.29% (S. euphratica var. leiocalycina). The stearic acid was the second major saturated fatty acid in the studied Salvia taxa (3.04 ± 0.22%-7.89 ± 0.86%). The other saturated fatty acids were myristic acid (14:0) and arachidic acid (20:0). Caprylic acid (8:0), capric acid (10:0), undecylic acid (11:0), behenic acid (22:0) and lignoceric acid (24:0) were either absent or in low amounts (Table 1). Azcan et al. (2004) showed that palmitic acid and stearic acid were major saturated fatty acids in Salvia. Also, different studies demonstrated that Salvia species have palmitic acid and stearic acid as saturated fatty acids (Bagci et al., 2004; Kilic et al., 2005; Alipour-Gough and Asgarpanah, 2015; Moazzami Farida et al., 2016). However, the results of Habibvash et al. (2007) conflict with the present study because they found that palmitic acid and stearic acid content of Salvia taxa were lower than in this study (Habibvash et al., 2007). Moreover, they determined that some Salvia taxa had a high arachidic acid content (14.82-26.91%) (Habibvash et al., 2007). Also, Delange et al. (2012) determined that Salvia had a higher stearic acid and arachidic acid content than palmitic acid content. Furthermore, Bakoglu et al. (2016) determined that Salvia had higher stearic acid (2.88-22.54%) and arachidic acid (4.32-13.9%) content than the present study. Furthermore, Kilic (2018) found that the stearic acid (36.33%) content of S. euphratica var. leiocalycina were higher than in the present study while he found that it has a similar palmitic acid content with current study (Kilic, 2018). On the other hand, α-linolenic acid (18:3 n3), linoleic acid (18:2 n6), oleic acid (18:1 n9) were determined as a major unsaturated fatty acids in this study (Table 1). S. pseudoeuphratica had the highest α-linolenic acid (29.45 ± 1.32%), is ω-3 fatty acid, while S. euphratica var. euphratica had the lowest α-linolenic acid (11.53 ± 1.19%) in this study. The current study also showed that the α-linolenic acid content of S. euphratica var. leiocalycina was 16.23 ± 1.01%. Also, it was found that S. euphratica var. euphratica had the highest linoleic acid content (31.58 ± 1.16%), ω-6 fatty acids, and its ω-6 fatty acid content was different from S. euphratica var. leiocalycina (12.06 ± 0.98%). Also, it was found that the linoleic acid content of S. pseudoeuphratica was 7.48 ± 0.63%. Essential fatty acids (EFAs) are polyunsaturated fatty acids and cannot be synthesized by humans but must be taken from food, and they are divided into two groups, omega-6 and omega-3 (Turan et al., 2013). Similarly, previous studies showed that linoleic acid, oleic acid and linolenic acid were primary unsaturated fatty acids in Salvia (Azcan et al., 2004; Kilic et al., 2005; Alipour-Gough and Asgarpanah, 2015; Ben Farhat et al., 2015b). Furthermore, it was indicated that an 18:3/18:2 ratio should be used as a taxonomic tool in Lamiaceae (Azcan et al., 2004; Goren et al., 2006) and it was found that the 18:3/18:2 ratio of S. euphratica var. euphratica (0.36) was different from S. euphratica var. leiocalycina (1.34) and S. pseudoeuphratica (3.93) in the present study. Besides, S. euphratica var. leiocalycina has higher oleic acid content (11.82 ± 1.24%) than S. euphratica var. euphratica (9.07 ± 1.13%) and S. pseudoeuphratica (4.71 ± 0.49%). Palmitoleic acid (16:1 n7) and erucic acid (22:1) were the other dominant unsaturated fatty acids in the three Salvia taxa. S. euphratica var. euphratica had higher erucic acid (22:1; 20.13 ± 1.47%) than S. euphratica var. leioclaycina (2.42 ± 0.3%) and S. pseudoeuphratica (5.87 ± 0.59%), but it had the lowest palmitoleic acid (16:1 n7) content in the study. Moreover, myristoleic acid (14:1), pentadecanoic acid (15:1), heptadecanoic acid (17:1), eicosadienoic acid (20:2 n6) and eicosapentaenoic (20:5 n3) contents were absent or low in the present study. Also, this study showed that the ratio of unsaturated fatty acids (61.38 ± 0.67%-82.08 ± 0.52%) were more than the ratio of saturated fatty acids and it was determined that the proportion of unsaturated fatty acids were larger than the saturated fatty acids in Lamiaceae (Azcan et al., 2004). And it was found that the ratio of unsaturated fatty acid to saturated fatty acid (U/S) of S. euphratica var. euphratica (4.28) was different from S. euphratica var. leiocalycina (1.32) and S. pseudoeuphratica (1.67) (Table 1).

Table 1
Fatty acid, lipid soluble vitamins, sterols content (µg/mg) in polar extracts from three Turkish Salvia taxa

Chemical composition	S. euphratica var. leiocalycina	S. euphratica var. euphratica	S. pseudoeuphratica
Saturated fatty acids*
(8:0)	-	0.39 ± 0.01	-
10:0	-	-	1.01 ± 0.45
11:0	-	0.42 ± 0.01	-
14:0	1.37±0.14	0.69 ± 0.01	1.25 ± 0.27
16:0	32.57±1.29	8.94 ± 0.71	24.16 ± 1.42
18:0	7.12±0.57	3.04 ± 0.22	7.89 ± 0.86
20:0	1.7±0.32	1.07±0.1	2.24 ± 0.27
22:0	-	2.00±0.11
24:0	-	2.61±0.1
Total	42.76± 0.58	19,16± 0.15	36.55 ± 0.65
Unsaturated fatty acids*
14:1	1.3±0.1	-	-
15:1	-	1.26±0.1	4.28±0.37
16:1 n7	12.9±1.21	2.9±0.1	9.59±0.65
17:1	-	2.27±0.12	-
18:1 n9	11.82±1.24	9.07±1.13	4.71±0.49
18:2 n6	12.06±0.98	31.58±1.16	7.48±0.63
18:3 n3	16.23 ±1.01	11.53±1.19	29.45±1.32
18:3 n6	-	0.7±0.01	-
20:2 n6	-	0.39±0.1	-
20:5 n3	-	1.67±0.24
22:1	2.42±0.3	20.13±1.47	5.87±0.59
Total	56.73 ± 0.8	82.08 ± 0.52	61.38 ± 0.67
18:3/18:2	1.34	0.36	3.93
Unsaturated/Saturated(U/S)	1.32	4.28	1.67
Lipid soluble vitamin and sterol**
K1	4.05±0.15	7.87 ±0.14	2.7±0.2
K2	0.25±0.01	0.68±0.01	0.65±0.01
R-tocopherol	0.22±0.01	1.04±0.01	0.2±0.01
D2	1.2±0.12	1.34±0.01	1.5±0.35
D3	0.32±0.01	0.35±0.01	1.15±0.32
a-tocopherol	25.05±1.14	6.14±0.37	19.5±0.97
Retinol	-	-	0.25±0.01
Retinol-acetate	1.1±0.1	2.1±0.32	1.35±0.14
Stigmasterol	44.21±1.22	80.14±1.34	66.4±1.27
B-sitosterol	42.45±1.41	102.17±1.24	87.15±1.53

• ^*
  By Gas chromatography. **By High pressure liquid chromatography.

The current study showed the studied three Salvia taxa low lipid soluble vitamins and sterol content (Table 1). It was determined that S. euphratica var. leiocalycina and S. pseudoeuphratica had higher α-tocopherol (25.05 ± 1.14 µg/g, 19.5 ± 0.97 µg/g, respectively) than S. euphratica var. euphratica (6.14 ± 0.37 µg/g). Also, it was found that K1 vitamin contents of three Salvia taxa were determined as 4.05 ± 0.15 µg/g (S. euphratica var. leiocalycina), 7.87 ± 0.14 µg/g (S. euphratica var. euphratica) and 2.7 ± 0.2 µg/g (S. pseudoeuphratica). Similarly, Sari et al. (2009) found that Salvia taxa had a low lipid soluble vitamin content and they determined that D2, D3, α-tocopherol acetate and K1 content of S. euphratica were 15.0 µg/g, 18.0 µg/g, 7.4 µg/g and 7.8 µg/g, respectively. Sterols are structural elements of the cell membrane and play a significant role in the regulation of membrane fluidity and permeability (Trautwein and Demonty, 2007). Also, they are used as therapeutic agents to reduce plasma cholesterol concentration (St-Onge and Foo, 2003). This study determined that the ß-sitosterol content of the studied Salvia taxa were between 42.45 ± 1.41 µg/g (S. euphratica var. leiocalycina) and 102.17 ± 1.24 µg/g (S. euphratica var. euphratica) and their stigmasterol content were between 44.21 ± 1.22 µg/g (S. euphratica var. leiocalycina) and 80.14 ± 1.34 µg/g (S. euphratica var. euphratica; Table 1).

Recent studies have shown that phenolic compounds from medicinal plants are important because of the various benefits, especially their antioxidant effect, and these studies supported significant steps for the discovery of new drugs (Tungmunnithum et al., 2018; Najjaa et al., 2020). It was found that Salvia taxa had high catechin content (509.2 ± 1.21 µg/mg-552.2 ± 9.21 µg/mg; Table 2) in the present study. It was suggested that catechin has free radical scavenging effects, inhibit the extracellular matrix degradation caused by UV, and have anti allergenic and anti-inflammatory effects (Bae et al., 2020). Also, catechin plays a significant role against cancer, diabetes, obesity, cardiovascular diseases, infections, and neurodegenerative diseases (Isemura, 2019).

Table 2
Flavonoids and phenolic acid content (µg/mg) in polar extracts from three Turkish Salvia taxa

Compound	S. euphratica var. leiocalycina	S. euphratica var. euphratica	S. pseudoeuphratica
Flavonoids*
Rutin	32 ± 1.12	328.4 ± 4.27	308.6 ± 5.01
Myricetin	-	8.9±0.47	1.8±0.2
Morin	3.2±0.21	6.2±0.1	14±0.32
Querecetin	0.2±0.01	-	0.6±0.01
Kaempherol	-	-	-
Catechin	552.2±9.21	509.2±4.21	539±7.62
Naringenin	92.8±2.17	-	7.01±0.34
Resveratrol	2.2±0.01	2.5±0.11	10.8±0.37
Phenolic acid*
Vanilic acid	382.1± 3.41	351.2 ± 2.17	396.8 ± 4.1
Cinnamic acid	-	0.6±0.1	1.6±0.15
Caffeic acid	16.8±1.12	10.4±0.17	153.2±2.13
Ferulic acid	1740.2±4.82	1175±5.21	19.2±0.97
Rosmarinic acid	1480±7.57	989±4.92	546.2±7.61

• ^*
  By High pressure liquid chromatography.

Also, it was determined that S. euphratica var. euphratica and S. pseudoeuphratica had high rutin content (328.4 ± 4.27 µg/mg and 308.6 ± 5.01 µg/mg, respectively). However, it was seen that the rutin amount of S. euphratica var. leiocalycina was the lowest (32 ± 1.12 µg/mg). On the other hand, it was showed that the naringenin amount of S. euphratica var. leiocalycina was higher (92.8 ± 2.17 µg/mg) than the other studied taxa (Table 2). The myricetin, morin, quercetin, kaempferol, and naringin amounts of the studied taxa were either absent or in low amounts in the present study (Table 2). In a study conducted by Kivrak et al. (2019), it was reported that Salvia had kaempferol, naringenin, rutin, vanillic acid, caffeic acid, and ferulic acid content. However, they found that Salvia did not have myricetin, resveratrol and quercetin (Kivrak et al., 2019). Also, Zengin et al. (2018) reported that in Salvia species including S. euphratica var. leiocalycina possess rutin, apigenin, kaempferol, luteolin, protocatechuic acid, rosmarinic acid, caffeic acid, and 3- O-caffeoylquinic acid. Conversely, the present study showed that the vanillic acid contents of Salvia taxa were between 351.2 ± 2.17 µg/mg and 396.8 ± 4.1 µg/mg. The results obtained from this study found that S. euphratica var. leiocalycina and S. euphratica var. euphratica had a higher rosmarinic acid content (1480 ± 7.57 µg/mg, 989 ± 4.92 µg/mg, respectively) than S. pseudoeuphratica (546.2 ± 7.61 µg/mg; Table 2). It was reported that rosmarinic acid is the most prominent compound in Salvia species, and it is chiefly responsible for the antioxidant activity in Salvia (Lu and Yeap, 2002). Meanwhile, it was reported that vanillic acid has pharmacological effects against cancer, cardiovascular disease, inflammation, oxidative stress, and is used as a food additive (Dandekar and Wasewar, 2020). In addition, two S. euphratica taxa had the highest ferulic acid content (1740.2 ± 4.82 µg/mg and 1175 ± 5.21 µg/mg) whereas it was found that S. pseudoeuphratica had the lowest ferulic acid (19.2 ± 0.97 µg/mg). It was reported that ferulic acid contributed to the reduction in oxidative stress in the ß-cells and stimulated insulin secretion, and it has anticarcinogenic activity by stimulating cytoprotective enzymes against free radical damage (Kumar and Pruthi, 2014). However, S. pseudoeuphratica had higher caffeic acid content (153.2 ± 2.13 µg/mg) than S. euphratica taxa, while the cinnamic acid content of the studied Salvia taxa had the lowest or had trace amounts (Table 2). A study conducted by Yumrutas et al. (2012), found that rosmarinic acid and caffeic acid were dominant phenolics in two varieties of S. euphratica, whereas the present study showed that S. euphratica var. leiocalycina does not have caffeic acid. Adimcilar et al. (2019) determined that the rosmarinic acid content of S. euphratica var. leiocalycina was 9.81 ± 0.23 mg/g. Similarly, some studies showed that Salvia taxa had high rosmarinic acid content (Tepe, 2008; Erdogan-Orhan et al., 2012; Kocak et al., 2016).

Furthermore, this study demonstrated that Salvia taxa had high DPPH and ABTS radical scavenging activities (Figures 1 and 2). Tepe et al. (2006) indicated that S. euphratica var. euphratica had more active plants based on the DPPH radical scavenging activity. Similarly, Senol et al. (2010) found that methanol extracts of Salvia taxa including S. euphratica var. leiocalycina (89.02 ± 0.80%-91.28 ± 0.44%) and S. euphratica var. euphratica (88.85 ± 0.22%-92.39 ± 0.22%) had high DPPH radical scavenging capacity. Also, Senol et al. (2010) indicated that the ferrous ion chelating activity of S. euphratica var. leiocalycina (23.23 ± 0.84%) and S. euphratica var. euphratica (12.9 ± 1.32%) were higher than the present results. Furthermore, Yumrutas et al. (2012) showed that the DPPH radical scavenging capacity of S. euphratica var. leiocalycina was higher than in S. euphratica var. euphratica. The literatures reported that phenolic compounds were responsible for the antioxidant capacities in Lamiaceae including Salvia (Tosun et al., 2009; Roby et al., 2013; Kocak et al., 2016).

Figure 1
DPPH % Radical Scavenging Activities of three Salvia taxa.

Figure 2
ABTS % Radical Scavenging Activities of three Salvia taxa.

This study demonstrated that S. euphratica var. leiocalycina and S. euphratica var. euphratica had high metal chelating activity (82.22 ± 1.29%-84.4 ± 1.1%, respectively) while it was found that S. pseudoeuphratica had low metal chelating activity (30.33 ± 0.39%). Some studies reported that Salvia taxa comprising of S. euphratica var. leiocalycina had strong metal chelating capacity (Topcu et al., 2007; Zengin et al., 2018). In contrast, a study done by Senol et al. (2010) suggested that S. euphratica var. euphratica, and S. euphratica var. leiocalycina had low metal chelating activity. Additionally, it was observed that lipid peroxidation inhibitions of S. euphratica var. leiocalycina (2.1 ± 0.1 mg/kg) and S. euphratica var. euphratica ((3.47 ± 0.2 mg/kg) are quite similar. But the lipid peroxidation inhibition of S. pseudoeuphratica calculated as 15.32 ± 0.41 mg/kg lipid peroxidation. Sari et al. (2012) found that lipid peroxidation inhibition of Salvia taxa was between 0.58mg/l and 0.92 mg/l. Also, Khlifi et al. (2006) indicated that Salvia had significant inhibition of oxygen consumption. Furthermore, Giamperi et al. (2012) suggested that Salvia taxa had significant antioxidant activity against lipid peroxidation.

Furthermore, it was demonstrated that the biochemical results in the current study supported the morphological studies. In the flora of Turkey, S. pseudoeuphratica formerly has taken its place as a synonym of S. euphratica var. euphratica (Hedge, 1982a, b). However, it was shown that S. pseudoeuphratica apparently diverged from S. euphratica according to its many morphological characters, and it was demonstrated that former was clustered differently from latter in the dendrogram (Kahraman et al., 2010). In particular, it was found that some biochemical parameters (such as palmitic acid, linoleic acid, α-linolenic acid, 18:3/18:2, rutin, naringenin, caffeic acid, ferulic acid and metal chelating) of S. pseudoeuphratica were different from the those of S. euphratica var. euphratica in the present study. Moreover, Kahraman et al. (2010) found that the populations of S. euphratica var. euphratica was clustered in a different position than the populations of S. euphratica var. leiocalycina in the dendrogram constructed using morphological characters. Also, Yilmaz et al. (2019) indicated that two varieties of S. euphratica had difference hair cover on the stem, bracts, calyx and pedicel, and Bagherpour (2010) reported that they had differences in glabrous inflorescence, the bracts, and the calyx. Similarly, it was reported that the current biochemical results (palmitic acid, oleic acid, linoleic acid, α-linolenic acid, γ-linolenic acid, 18:3/18:2, U/S, α-tocopherol, rutin, quercetin, naringenin, resveratrol, vanillic acid, caffeic acid, ferulic acid, and rosmarinic acid) of both variety were different from each other in the current study. Although it was stated that the difference in biochemical content is due to environmental conditions, soil structure, variation in sunlight hour, altitude, temperature and exposure to UV-B (Çeti̇nkaya et al., 2017; Çoklar, 2017), the difference in the biochemical content of these two varieties was thought to be caused not only by environmental conditions but also by genetic differences.

4. Conclusion

The current study showed that palmitic acid was found as a major saturated fatty acid, and S. euphratica var. euphratica had lower palmitic acid (8.94 ± 0.71%) and total saturated fatty acid (19.16 ± 0.15%) content than the two studied taxa. In addition, S. euphratica var. euphratica had the highest unsaturated fatty acid content (82.08 ± 0.52%), and oleic acid, linoleic acid, α-linolenic acid, and erucic acid were found as major unsaturated fatty acids. Furthermore, it was found that 18.3/18:2 (0.36) and the unsaturated/saturated fatty acid (4.28) ratios of S. euphratica var. euphratica were different from the two other studied taxa. It can be concluded that the fatty acids could be used as systematical tool, and the fatty acid content of S. euphratica var. euphratica diverged from the other two taxa. However, this study demonstrated that Salvia taxa had low lipid soluble vitamins and sterol contents. On the other hand, the present study demonstrated that the three studied Salvia taxa were found to have similar catechin (509.2 ± 4.21 µg/g and 552.2 ± 9.21 µg/g) and vanillic acid amounts (351.2 ± 2.17 µg/g and 396.8 ± 4.1 µg/g). Also, the studied Salvia taxa had high rosmarinic acid content; in particular, S. euphratica var. leiocalycina had the highest level of rosmarinic acid (1480 ± 7.57 µg/g). On the other hand, two S. euphratica varieties had the highest ferulic acid content (1175 ± 5.21 µg/mg-1740.2 ± 4.82 µg/mg) while it was found the ferulic acid content of S. pseudoeuphratica was the lowest (19.2 ± 0.97 µg/mg). Moreover, this study showed that Salvia taxa have a potent antioxidant capacity, and it was shown that the biochemical results were supported in the morphological studies.

Acknowledgements

Authors are grateful to Prof. Okkes Yilmaz for assistance in the reading on the HPLC and GC-MS devices.

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Publication Dates

• Publication in this collection
  28 June 2021
• Date of issue
  2022

History

• Received
  13 June 2020
• Accepted
  16 Nov 2020