The Antioxidant and Antimicrobial Capacities of Phenolic Profiles of Some Salvia L. Seeds Grown in Turkey

The aim of current study is to show phenolics, antioxidant capacities and antimicrobial activities of seeds of five Salvia L. ( S. frigida Boiss., S. candidissima subsp. candidissima Vahl., S. virgata Jacq., S. verticillata L. var. verticillata and S. russellii Benth.) taxa grown in Turkey. The flavonoid and phenolic acid contents were measured by using HPLC whilst the antioxidant capacities were determined by using different methods. In addition, agar well diffusion method was used to determine the antimicrobial activities of Salvia species in this study. It was found that S. frigida , S. verticillata var. verticillata and S. russellii have the highest catechin contents and S. frigida and S. verticillata var. verticillata have high rosmarinic acid while S. frigida , S. candidissima subsp. candidissima and S. verticillata var. verticillata have high vanilic acid. Also, it was determined that S. frigida and S. verticillata var. verticillata have high DPPH radical scavenging activities in 150 and 250 µL while S. frigida and S. verticillata var. verticillata have highest ABTS radical scavenging activity in all concentrations apart from 25 µL for S. frigida . Furthermore, S. frigida and S. verticillata var. verticillat a have high total phenolic contents. On the other hand, Salvia species have similar lipid peroxidation inhibitions. However, the metal chelating activities of Salvia species are different. And also, it was demonstrated that Salvia taxa have antimicrobial activity.


INTRODUCTION
Herbs from the Lamiaceae have been used in traditional medicine for more than 2000 years to treat different diseases such as cancer, diabetes, depression, memory enhancement and infection throughout the world (Shekarchi et al., 2012;Lopresti, 2017). Lamiaceae, contains most popular aromatic plants including marjoram, sage, basil and thyme, have strong antioxidant and antimicrobial activity due to rich in biologically effective components as caffeic

MATERIAL and METHODS
The plants were collected from natural habitats. The plant samples and seeds were deposited in Firat University Herbarium (FUH). The localities of studied Salvia L. taxa were given in Table 1.

DPPH Radical Scavenging Activity
25, 50, 100, 150 and 250 µL of extracts were treated with 25 mg/L DPPH solved in methanol (4.0 mL). The DPPH radical protocol was performed based on Liyana-Pathiranan and Shahidi (2005)'s method in the current study. The absorbances were measured at 517 nm after the samples were stored in the dark for 30 minutes. 1 µM quercetin was used as reference. The formula (1) was used for the DPPH radical scavenging potential is following: (1) The absorbance of control was represented as Ab(control) and the absorbance of sample was represented as Ab(sample).

ABTS Radical Scavenging Activity
ABTS [2,2-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt] assay was determined according to the Ree et al. (1999) methods. 7 mM ABTS and 2.45 mM potassium persulphate were mixed to form ABTS •+ solution. The solution was stored at room temperature approximately 12-16 h. And ABTS was dissolved with water to provide an absorbance of 0.700 ± 0.020. Lastly, three mL of diluted ABTS were mixed with 25, 50, 100, 150 and 250 µL of extract and absorption was determined in the 6 min at 734 nm (Skotti et al., 2014). The formula was used for the DPPH radical scavenging potential is following (2): (2) The absorbance of control was represented as Ab(control) and the absorbance of sample was represented as Ab(sample).

Determination of Total Phenolics
Folin-Ciocalteu method was used to evaluate total phenolics (Singleton et al., 1999). 100 µL extracts were mixed with 3.16 mL of H2O and 200 µL of Folin-Ciocalteu solution. The samples were stored at room temperature about 3 min. Later, the extracts were treated with anhydrous sodium carbonate (20% w/v) and total phenolic content was observed at 765 nm after two hours in room temperature (Robya et al., 2013). The total phenolic amount was evaluated by using gallic acid equivalents (µgGAE/mg).

Chelating Effects of Ferrous Ions
The chelating activities of samples were evaluated method by Dinis et al. (1994). 50 µL of 2 mM FeCl2 was injected to extracts (50, 100, 250 and 500 µg/mL). 5 mM ferrozine (0.2 mL) mixed with extracts to start the reaction. The extracts were shaken vigorously and stored at room temperature approximately 10 min. The absorbances of samples were measured at 562 nm. The inhibition (%) of ferrozine-Fe 2+ complex was evaluated based on following formula (3) The absorbance of sample was represented as Abs and the absorbance of control was represented as Abc where 100 where Na2EDTA was used as positive control.

Antioxidant Activity against TBARS
The antioxidant activity of samples was measured according to Shimoi et al. (1994)' method. The samples were prepared by using DMSO (dimethyl sulfoxide). The Fe 2+ (FeCl2.2H2O) and hydrogen peroxide were used in the experiments. Also, oleic acid (3.35 mM), linoleic acid (9.01 mM) and linolenic acid (2.30 mM) were used dissolved in DMSO. Sage extracts, control and Fenton reagent groups were formed. The control group contained 0.5 mL of fatty acid and a buffer (pH=7.4; 0.05 M Tris HCl; 0.2% Tween, 20; 0.15 M KCl) whilst the fenton group contained buffer; hydrogen peroxide (0.01 mM); 0.5 mL of fatty acid and FeCl2.2H2O (50 μM) and the extracts comprised FeCl2 (50 μM), 0.25 mL sage extract, 0.5 mL of fatty acid and hydrogen peroxide (0.01 mM). 0.1 mL of a 4% (w/v) BHT was added to all groups to protect the more oxidation and the examples were stored at the 37 °C approximately 24 h. After that, 1 mL of samples from three groups was taken and treated with 1 mL of 0.6% TBA and the samples were stored at 90 °C for 30 min. Finally, 4 mL butan-1-ol was injected to tubes, blended and centrifuged at 4250 rpm for 10 min. The absorbance of the supernatant was measured at 532 nm. MDA standard curves were formed by 1,1,3,3-tetramethoxypropane, and TBARS were written as mg MDA/kg dry matter (Keser et al., 2014).

Antimicrobial Activity
Antimicrobial activities were evaluated agar well diffusion method according to Collins and Lyne (1987)' method. Agar contained Sabouroud Dextrose Agar (Oxoid), Mueller Hinton Agar (Difco) and Malt Extract Agar (Difco) and McFarland standard. And also, bacteria (10 6 cells/mL), dermatopyhte and yeast (10 4 cells/mL), were found in 100 µL suspension. Phenolics (10 µL) were added to the well after the wells were filled with cork-borer (0.85 cm) and plates. After that, incubation for bacteria was conducted at 37±0.1°C for 24 h and for yeast and dermatophyta fungi were conducted at 25±0.1°C for 72 h. The inhibiton zone was referenced to decide the antimicrobial activity.

Statistical Analysis
All analysis were performed by using SPSS 21.0 packet program. The simple lineer regression model was used to found the correlation between antioxidant capacity (ABTS, DPPH and metal chelating) and total phenolic contents. Data obtained from present study represented as mean values ± standard deviation. Also, to evaluate the significance of the observed differences, the least significant difference (LSD) test was used in the antimicrobial activity. The conclusions were expressed as mean ± S.D. p<0.0001, p<0.001 and p>0.05 have been conceived significant when compared to the control group (ampicillin sulbactam, mycostatin). All samples were analysed in triplicate.
Moreover, the present study demonstrated that phenolic contents of Salvia L. taxa represented different antimicrobial activities (Table 6). It was showed that S. verticillata subsp. verticillata represented higher antimicrobial activity against B. megaterium, C. albicans and S. aureus than other studied Salvia taxa. And also, it was found that only S. frigida exhibited antimicrobial activity against E. coli while only S. candidissima subsp. candidissima exhibited antimicrobial activity against C. glabrata. On the other hand, it was determined that studied Salvia taxa don't show antimicrobial activity against Epidermophyton sp. and Trichopyton sp. (Table 6). It was reported that Salvia taxa have potent antimicrobial activity study by done Bayar and Genc (2016). They showed that the methanolic extracts of S. candidisssima have significant antifungal capacity (Bayar & Genc, 2018). In another study by done Akin et al. (2010). S. russellii is effective against micororganisms. And also, Kunduhoglu et al. (2011) suggested that S. verticillata exhibited antimicrobial activity.

CONCLUSION
The present study demonstrated that the catechin amounts of S. frigida, S. verticillata subsp. verticillata and S. russellii are high whilst the the rutin and naringin content of S. verticillata subsp. verticillata are high. Also, the current study showed that S. frigida and S. verticillata subsp. verticillata have high rosmarinic acid and S. frigida (64.74±1.21 µg/mg), S. candidissima (65.4±1.34 µg/mg) and S. verticillata subsp. verticillata (84.12±0.97 µg/mg) have high vanilic acid content. On the other hand, it was found that Salvia taxa have high ABTS (in 100, 150 and 250 µL) and DPPH (in 250 µL) except for S. candidissima subsp. candidissima) radical scavenging activities. Moreover, it was demostrated that S. frigida and Salvia verticillata subsp. verticillata have high total phenolic content. And also, Salvia taxa represented antimicrobial activity.