Investigation of phenolic profiles and antioxidant activities of some Salvia species commonly grown in Southwest Anatolia using UPLC-ESI-MS / MS

The genus Salvia is the largest member of the Lamiaceae family with nearly 1000 species spread throughout the various regions of the World mainly central and south America, western Asia (especially Turkey, Oran, Russia) and eastern Asia. Recently, 99 species of the genus Salvia have been identified in Turkey and 52 (52%) of them are endemic to Turkey (Alziar, 1988; Celep et al., 2014). Some members of the Salvia genus are commercially important and used for flavouring agents in foods as well as cosmetics, perfumery and the pharmaceutical industries with its biological activities (Chalchat et al., 1998; Villa et al., 2009).


Introduction
The genus Salvia is the largest member of the Lamiaceae family with nearly 1000 species spread throughout the various regions of the World mainly central and south America, western Asia (especially Turkey, Oran, Russia) and eastern Asia.Recently, 99 species of the genus Salvia have been identified in Turkey and 52 (52%) of them are endemic to Turkey (Alziar, 1988;Celep et al., 2014).Some members of the Salvia genus are commercially important and used for flavouring agents in foods as well as cosmetics, perfumery and the pharmaceutical industries with its biological activities (Chalchat et al., 1998;Villa et al., 2009).
The traditional medical practices of the Salvia species have been studied all over the world (Martínez-Francés et al., 2017;Li et al., 2013).Ot is known that Salvia species have been used as infusions against simple diseases in Anatolian traditional medicine applications (Baytop, 1999).Many Salvia species have been reported for use in the treatment of diseases such as epilepsy, colds, bronchitis and tuberculosis (Dweck, 2000) as well as biological activities such as antioxidant, antimicrobial (Kelen & Tepe, 2008), anti-inflammatory (González-Chávez et al., 2017), antidiabetic (Eidi & Eidi, 2009), antitumor (Fiore et al., 2012), anti cancer (Jiang et al., 2017) ve antiviral activities (Šmidling et al., 2008).Additionally, Scholey et al. (2008) examined the administration of a standardised Salvia extract to improve cognitive function in healthy older individuals and reported enhancement in cognative performance.Recently, Lopresti (2017) published a useful review about potential cognitive-enhancing and protective effects of Salvia and Miroddi et al. (2014) has reviewed clinical trials assessing pharmacological properties of Salvia species on memory, cognitive impairment and alzheimer's disease.
Today, it is well known that free radicals cause many diseases.Antioxidants have great importance in the fight against free radicals, which can damage biological molecules with different mechanisms of action (Young & Woodside, 2001) and the interest in the usage of antioxidants in the food, pharmaceutical and cosmetic industries is constantly increasing.Nowadays, synthetic antioxidants such as butylated hydroxyanisole (BHA), Butylated hydroxytoluene (BHT) and natural antioxidants are used as preservatives in many industries, especially in food industry.However, the concerns about the safety and toxicity of synthetic antioxidants have not been overcome yet (Kahl, 1984;Pokorný, 2007;Oto et al., 1986).Therefore, the need for new and safer antioxidant sources is still maintained.
As one of the plants used as natural antioxidant source is the genus Salvia and the antioxidant activities of Salvia extracts have been associated mainly with their total phenolic contents (Farhat et al., 2013;Dudonné et al., 2009).Plants with phenolic content are used especially in oily food because of their significant functions such as dealing with undesirable fragrances, prolonging thier shelf life, delaying the formation of toxic oxidation products, increasing nutritional value and preventing microbial growth.(Tepe et al., 2006;Karpinska et al., 2001;Rota et al., 2004).Phenolic compounds are known to be extremely beneficial in terms of human nutrition (Du & Kwok, 2004), cosmetic (Magnani et al., 2014) and pharmacological (Galati & D'Brien, 2004).
A wide variety of Salvia species have been studied as novel phenolic compound sources and qualitative and quantitative analyzes of phenolic compounds have been carried out using various techniques.(Drhan et al., 2012;Kamatou et al., 2010;Lu & Foo, 2002).Ot is known that the phenolic compositions and antioxidant activities of Salvia species vary depend on species.For example, Şenol et al. (2010) have examined the radical-scavenging activities of 55 Salvia species include S. nydeggeri using DPPH • and FRAP assays and found S. fruticosa and S. cilicica as most active species while Asadi et al. (2010) reported S. hydrange had higher FRAP (ferric reducing antioxidant power) activity than S. lachnocalyx.A number of studies have been done to determine antioxidant activities and phenolic contents of Salvia species (Erdemoglu et al., 2006;Tosun et al., 2009;Akkol et al., 2008;Alimpić et al., 2017).To the best of our knowledge, there is no any study on determination of phenolic profile of S. nydeggeri, S. albimaculata and S. potentilifolia using UPLC-MS/MS.On this study, phenolic profiles of three edible and commercially valuable Salvia species were analyzed using UPLC-ESO-MS/MS with new extractions techniques.Quantitative determinations were made using calibration curves.Total phenolic and flavonoid concentrations were determined for each plant.The antioxidant activity of plant extracts was determined using three complimentary methods (β-carotene-linoleic acid bleaching, DPPH • free radical scavenging and ABTS •+ cation radical scavenging).Correlations between phenolic and flavonoid content and antioxidant activity results were determined.

Plant materials
Salvia albimaculata Hedge & Hub.-Mor.samples were collected in the region between Burdur, 15. km from Tefenni to Söğüt, Turkey.Salvia potentillifolia Boiss.& Heldr.ex Bentham.samples were collected in the region between Burdur, 15. km from Gölhisar to Altınyayla, Turkey and Salvia nydeggeri Hub.-Mor.samples were collected in the region between Muğla-Fethiye, 8. and 9. km from Fethiye to Antalya, Turkey.All Salvia species were endemic to their region and collected in August, 2015.Samples were dried under shadow in ambient temperature (25 °C) for the extraction procedures.Authentication of the plant materials were performed by Dr. Ergun Kaya from Department of Molecular Biology and Genetics, Faculty of Science, Muğla Sıtkı Koçman University, Muğla (Turkey).

Extraction of Salvia species
On the determination of total phenolic content, total flavonoid content and antioxidant activities of Salvia species, methanol, hexane, ethyl acetate and water extracts were used.The Salvia samples were extracted five times for 24 h at room temperature with methanol, filtered through Whatman no 4 and solvents were evaporated (Heidolph, Hei-VAP Precision).Then dry plant extracts were dissolved in distilled water and subjected to liquid-liquid extraction with hexane and ethly acetate, respectively.For each plant, hexane and ethyl acetate extracts were evaporated to dryness under vacuum.The plants remaining after extraction with methanol were used for the water extraction.For this purpose, the plants remaining were extracted with water at 80 °C, filtered through Whatman no 4 and the water extracts were lyophilized (Christ Freeze Dryer, Alpha 1-4 LD plus, Germany).All extracts were stored in deepfreeze (-18 °C) for further analysis.

Determination of total phenolic and flavonoid concentration
The total concentrations of phenolic content of extracts were determined using the Folin-Ciocalteu Reagent (FCR) according to the method described by (Slinkard & Singleton, 1977;Singleton et al., 1999) and results expressed as microgrammes of pyrocatechol equivalents (PEs).Briefly, the sample solution (1 mL) dissolved in methanol was added to distilled water (46 mL) and mixed with FCR (1 mL).After 3 min, 3 ml of Na 2 CD 3 (2%) was added to the mixture and this mixture was kept in room temperature for 2 h by shaken intermittently.The absorbance was read at 760 nm.The concentration of total phenolic compounds was calculated according to the following equation (Equation 1) that was obtained from the standard pyrocatechol graph: (1) Quantification of total flavonoid concentrations of the extracts were determined as the aluminum nitrate method described by Moreno et al. (2000) as quercetin equivalents (QEs). 1 mL of solution containin 1 mg of sample in ethanol was mixed with 10% aluminum nitrate (100 µL) followed by 1 M potassium acetate (100µL), and 80% ethanol (3.8 mL) in test tubes.Mixtures were kept in room temperature for 40 min and then absorbance was measured at 415 nm.The total concentrations of flavonoid contents were calculated according to the following equation (Equation 2) that was obtained from the standard quercetin graph:

Antioxidant activity of the extracts β-Carotene-Linoleic Acid Bleaching Assay
The total antioxidant activity was determined using β-carotene-linoleic acid assay based on the detection of inhibition of conjugated dien hydroperoxides due to oxidation of linoleic acid (Miller, 1971).This method uses the bleaching of β-carotene.Briefly, β-carotene (0.5 mg) was dissolved in 1 mL of chloroform.Tween 40 (200 mg) and linoleic acid (20 µL) were added to this mixure.Chloroform was completely evaporated using a vacuum evaporator.Then, 100 mL of distilled water, saturated with oxygen, was added by vigorous shaking.4 mL of this emulsion was mixed with 1 mL extract solutions at different concentrations ranging 500 µg to 4000 µg.Zero time absorbances were measured at 470 nm just after emulsions were transfered to each tube. 1 mL of methanol was used as control.The emulsion systems were incubated at 50 °C untill the color of β-carotene was disappeared after 120 min.The results were given as 50% inhibition concentration (OC 50 ).

DPPH • Free Radical Scavenging Assay
The free radical scavenging activities of plant extracts were determined using DPPH (2,2-Diphenyl-1-picrylhydrazyl) free radical assay (Blois, 1958) with slight modifications.Briefly, 4 mL of 0.004% DPPH • solution in ethanol was added to the extract solutions (1 mL) at concentrations ranging from 500 μg to 4000 μg. 1 mL of ethanol was used as a control.After 30 min of incubation at room temperature, the absorbance was measured at 517 nm.Absorbance values of the samples were evaluated against the control.The free radical scavenging activity (RSA) was calculated using the following equation (Equation 3): Where A Sample is the absorbance of the solution containing the sample and A Control is the absorbance of the DPPH • solution.

ABTS •+ Cation Radical Decolorization Assay
The cation radical scavenging activities of the extracts were determined using ABTS (2,2-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt) (Re et al., 1999).Briefly, 7 mM ABTS in water and 2.45 mM K 2 SD 8 reacted to form 5 mL of ABTS •+ radical.The mixture was stored in the dark at room temperature for 16 h to allow cation radical formation.1 mL of this radical solution was adjusted by diluting with ethanol to give an absorbance about 0.700.Then 4 mL of the ethanol-prepared ABTS •+ solution was added onto 1 mL of the sample at concentrations ranging from 500 μg to 4000 μg.Ethanol (1 mL) was used as control.After incubation at room temperature for 10 min, the absorbance was measured at 734 nm.Absorbance values of the samples were evaluated against the control.ABTS •+ cation radical removal activity was calculated using the following equation (Equation 4): ( ) Where A Sample is the absorbance of remaining concentration of ABTS •+ in the presence of sample and A Control is the initial concentration of the ABTS •+ .

Determination of phenolic compounds using UPLC-ESI-MS/MS
Phenolic compounds of plant samples were analyzed using high-throughput instrument, a Waters UPLC-ESO-MS/MS and C18 column (Acquity UPLC BEH C18 100 mm × 2.1 mm, 1.7-μm particle size) and the separation of phenolic compounds was performed by gradient elution at 40 °C.The mobile phases were composed of solvent A (0.5% acetic acid in water) and solvent B (0.5% acetic acid in methanol), and the flow rate was 0.650 mL/min.
Approximately 20 g of each plant sample was frozen with 200 mL of liquid nitrogen and then lyophlized.A mixture of 30 mL of acetone:water (80:20) was added to lyophlized powder and the mixture was allowed to extraction for 6 h at -86 °C.Then, ultrasonic extraction was applied for 15 min., the extract was centrifuged at 4000 rpm for 10 min at 20 °C and filtered using Whatman No 4. The residue was extracted twice more with 30 mL of acetone:water mixtures (Kıvrak et al., 2013(Kıvrak et al., , 2017;;Kıvrak & Kıvrak, 2016;Kıvrak, 2015), extracts were combined, the solvents in the combined extracts was evaporated at 40 °C (Rotary Evaporator Heidolph Basis Hei-VAP ML).The aqueous phase was washed 3 times with 30 mL of n-hexane and 3 times with 30 mL of ethyl acetate for liquid-liquid extraction.The organic phases were combined and evaporated to dryness at 40 °C.The residue was redissolved in a mixture of water:methanol (80:20).The solution (2 µL) was passed through Macherey-Nagel Chromafil Xtra PTFE-20/25 0.20 μm filters and injected to UPLC-ESO-MS/MS (Waters Acquity Ultra Performance LC, Xevo TQ-S MS-MS).On this present study, method parameters of UPLC-ESO-MS/MS for the phenolic compounds analysis were applied according to our previous literatures (Kıvrak et al., 2013(Kıvrak et al., , 2017;;Kıvrak & Kıvrak, 2016;Kıvrak, 2015) (Table 1).All extractions techniques and UPLC-ESO-MS/MS analysis methods used in the analysis of phenolic compounds are original.

Total phenolic and flavonoid concentrations
Methanol, hexane, ethyl acetate and water extracts of Salvia species were examined for the determination of total phenolic and flavonoid contents.The results expressed as pyrocatechol and quercetin equivalents, respectively (Table 2).According to the study, ethyl acetate extract of S. albimaculata showed the highest amount of total flavonoid content (296.8 ± 1.4 µg QEs/mg) and the lowest content (29.1 ± 0.7 µg QEs/mg) of total flavonoid were found belonging to hexane extract of S. nydeggeri.
Dn the other hand, total phenolic content analysis of Salvia species revealed that ethyl acetate extract of S. potentillifolia had the highest concentration (62.4 ± 0.1 µg PEs/mg) of total phenolics among all extracts and species while the water extract of S. nydeggeri had the lowest amount (36.3 ± 0.8 µg PEs/mg) of total phenolic content.For all species, the results indicated that ethyl acetate extracts had the highest total phenolic and The values were given as averages of 3 parallel measurements p<0.05.flavonoid content while the lowest amounts of total phenolics and flavonoids determined in water extracts and hexane extracts, respectively.

Individual phenolic compounds
On this study, individual phenolic compounds of three Salvia species were determined using UPLC-ESO-MS/MS.Thirty two of phenolic compounds were identified and 21 of them were detected and quantitated in all Salvia samples.Pyrogallol, galantamin, catechin hydrate, epicatechin, catechin gallate, trans-2-hydroxycinnamic acid, resveratrol, quercetin, chlorogenic acid and gallic acid were not detected.All results were summarized in Table 3 and total ion chromatograms (TOC) of most abundant phenolic compunds of Salvia species were given in Figure 1.

Antioxidant activity
The antioxidant activity of plant extracts was determined three complimentary methods (β-carotene-linoleic acid bleaching, DPPH • free radical scavenging and ABTS •+ cation radical scavenging).According to the total phenolic and flavonoid determination results, the ethyl acetate extracts were presented higher total phenolic and flavonoid content in all species.With this reason, ethyl acetate fractions of Salvia extracts were choosen for comparision of antioxidant activities instead of methanol, water and hexane extracts.Antioxidant activity were examined and the 50% inhibition concentration (OC 50 ) of ethyl acetate extract was found to be highest as 26.1 ± 0.6 μg/mL for S. potentillifolia in the β-carotene-linoleic acid method.The antioxidant activities of the Salvia species and standartds were decreased in the following order: S. potentillifolia > S. albimaculata > BHA > S. nydeggeri > BHT > α-Tocopherol.The total antioxidant activity results consistent with total phenolic content results (S. potentillifolia > S. albimaculata > S. nydeggeri).
Oncreased concentrations in all samples also indicate increased inhibition.
According to the β-Carotene-linoleic acid and ABTS •+ assays, increasing in total phenolic content and antioxidant activity was in positive correlation.These results suggest that the major part of the antioxidant activities in Salvia species selected in this study is a result of the phenolic compounds.This observation has been in accordance with previous literatures, exhibited similar correlations between total phenolic content and antioxidant activity of various plants (Kıvrak et al., 2017;Kumar et al., 2014;Mammadov et al., 2012;Piluzza & Bullitta, 2011).On general, the differences between the results obtained in this study and in previous reports is considered to be related by the conditions of experiments, the instrument used and the growing areas and collection times.

Conclusion
Onvestigation of phenolic compositions and antioxidant properties of plants or their different extracts revealed remarkable data since the interest on the use of natural sources for food, pharmaceutical and cosmetic industries increased tremendouly in the last decade.On this study, individual phenolic compounds of S. albimaculata, S. nydeggeri and S. potentillifolia were analyzed and quantitated using UPLC for the first time.The studied three Salvia species presented rich phenolic content.On addition, up to now the lack of information about antioxidant activity studies using three complemantry method on S. albimaculata, S. nydeggeri and S. potentillifolia makes this present study unique and important.Considering the results obtained in this study and the large number of researches on Salvia species previously reported, all selected plants have great potential for use in pharmaceutical, cosmetics and many other industrial fields, especially in the food industry.On the light of findings of the present study, particularly remarkable antioxidant activities and rich phenolic contents of plants could trigger the scientists.We also think that the results obtained in this study provide useful of information for researchers who want to study various biological activities of these three Salvia species and encourage entrepreneurs to use them for commercial purposes with the respect of biodiversity conversation.

Figure 1 .
Figure 1.Total ion chromatograms of major phenolic compounds determined in Salvia species.

Table 2 .
Total phenolic and flavonoid concentrations of Salvia samples.

Table 1 .
Method parameters for the phenolic compounds analysis using UPLC-ESO-MS/MS.
nd: not detected.

Table 4 .
Antioxidant activities of ethyl acetate extracts of Salvia samples.OC 50 and SC 50 values represent the means ± SD of three parallel measurements; BHA; Butylated hydroxyanisole, BHT; Butylated hydroxytoluene.