Industrial Crops and Products 76 (2015) 702–709 Contents lists available at ScienceDirect Industrial Crops and Products journal homepage: www.elsevier.com/locate/indcrop Composition and biological effects of Salvia ringens (Lamiaceae) essential oil and extracts Ana Alimpić a,∗ , Dejan Pljevljakušić b , Katarina Šavikin b , Aleksandar Knežević a , Milena Ćurčić c , Dragan Veličković d , Tatjana Stević b , Goran Petrović e , Vlado Matevski f , Jelena Vukojević a , Snežana Marković c , Petar D. Marin a , Sonja Duletić-Laušević a a Institute of Botany and Botanical Garden “Jevremovac”, Faculty of Biology, University of Belgrade, Takovska 43, 11000 Belgrade, Serbia Institute for Medicinal Plant Research “Dr. Josif Pančić”, Tadeuša Košćuška 1, 11000 Belgrade, Serbia Department of Biology and Ecology, Faculty of Science, University of Kragujevac, Radoja Domanovića 12, 34 000 Kragujevac, Serbia d College of Agriculture and Food Technology, Ćirila and Metodija 1, 18400 Prokuplje, Serbia e Department of Chemistry, Faculty of Natural Sciences and Mathematics, University of Niš, Višegradska 33, 18000 Niš, Serbia f Institute of Biology, Faculty of Natural Sciences and Mathematics, University “Ss. Cyril and Methodius” and Macedonian Academy of Sciences and Arts, Blvd. Goce Delcev 9, 1000 Skopje, Macedonia b c a r t i c l e i n f o Article history: Received 6 March 2015 Received in revised form 25 June 2015 Accepted 26 July 2015 Keywords: Salvia ringens Essential oil Extracts Phenolics Flavonoids Biological activities a b s t r a c t This comprehensive study was carried out in order to investigate composition and biological activities of essential oil and extracts of Salvia ringens Sibth. & Sm. (Lamiaceae) originating from Macedonia. Major components of the oil, analyzed using GC-FID and GC–MS, were monoterpenes 1.8-cineole (31.99%), camphene (17.06%), borneol (11.94%) and ␣-pinene (11.52%). HPLC analysis showed presence of 17 phenolic components, mainly in methanol and ethyl acetate, followed by ethanol, water and dichloromethane extracts. Total phenolics and ﬂavonoids as well as DPPH, ABTS, and FRAP activities were measured spectrophotometrically. Essential oil, ethanol, and water extracts showed antimicrobial activity using microdilution method. Ethanol and water extracts performed cytotoxic activity against colon carcinoma HCT-116 cell line using MTT assay. According to the obtained results, S. ringens herb can be considered as the potential source of the essential oil and/or raw material for the extraction and isolation of natural compounds with a range of biological activities. © 2015 Elsevier B.V. All rights reserved. 1. Introduction The Lamiaceae family comprises aromatic plants widely used as spices and medicinal plants, such as rosemary, basil, sage, lavender, thyme, mint, and oregano. The ﬂavor of herbs and spices derives from essential oil components which make food more pleasant and, at the same time, show a wide spectrum of biological activities (Miguel, 2010). Some of the Lamiaceae species were reported as a rich source of phenolic compounds possessing strong antioxidant activity, and therefore, can be applied in prevention and therapy of free-radical associated diseases such as atherosclerosis, cancer, cardio-vascular disease, immune-system decline, brain dysfunction, cataracts, skin diseases (Asadi et al., 2010; Kamatou et al., 2010; Li et al., 2008) and may also serve as natural food preservatives (Miguel, 2010). ∗ Corresponding author. Fax: +381 113246655. E-mail address: [email protected] (A. Alimpić). http://dx.doi.org/10.1016/j.indcrop.2015.07.053 0926-6690/© 2015 Elsevier B.V. All rights reserved. The genus Salvia is the largest member of the family Lamiaceae which comprises about 1000 worldwide distributed species. In Flora of Europe, the genus is represented by 36 species grouped into 7 sections (Hedge, 1972). In vitro pharmacological investigations showed its antioxidant, antibacterial, antifungal, antiviral, cytotoxic, neuroprotective, antiinﬂammatory, and tumorigenesispreventing as well as ecological signiﬁcance such as pest-toxic and repellent and other activities (Asadi et al., 2010; Baričević and Bartol, 2000; Ben Farhat et al., 2009; Kamatou et al., 2010; Orhan et al., 2012; Veličković et al., 2002). Aerial parts of these plants usually contain ﬂavonoids and triterpenoids as well as essential oils with volatile compounds such as monoterpenoids, while diterpenoids are the main compounds in roots (Baričević and Bartol, 2000). It is a rich source of polyphenols, with an excess of 160 polyphenols having been identiﬁed, some of which are unique to the genus (Lu and Foo, 2002). Salvia ringens Sibth. & Sm. is a hardy herbaceous perennial herb, heights of up to 60 cm. Speciﬁc epithet, ringens, refers to the wide open two-lipped ﬂowers. It inhabits dry stony and grass-covered A. Alimpić et al. / Industrial Crops and Products 76 (2015) 702–709 places of South and Eastern parts of Balkan Peninsula, just extending to southeast Romania (Hedge, 1972). This is drought tolerant and long lived and highly valued as ornamental and melliferous plant species due to ponderous leaf rosette, attractive purple ﬂowers, and pleasant intense fragrance. Previous researchers have partially investigated composition and biological activities of S. ringens essential oil and/or extracts. Monoterpenes 1.8-cineole and ␣-pinene have been recognized as the major constituents of S. ringens essential oil from Greece and Macedonia (Šavikin et al., 2008; Tzakou et al., 2001) and camphor and borneol in Bulgarian S. ringens (Georgiev et al., 2013). The oil and isolated main compounds showed signiﬁcant antimicrobial activity (Šavikin et al., 2008; Tzakou et al., 2001). Among 27 Macedonian medicinal plants chosen from different plant families, Origanum vulgare, Melissa ofﬁcinalis, and Salvia ringens showed the strongest antioxidant activity and highest amount of total phenolics, ﬂavonoids, and phenylpropanoids (Tusevski et al., 2014). Many researchers pointed out that strong antioxidant activity of S. ringens extracts probably was correlated to high amount of polyphenols (Coisin et al., 2012; Nikolova, 2011; Tusevski et al., 2014). Extracts and some isolated compounds from S. ringens root performed signiﬁcant cytotoxic activity against several human carcinoma cell lines (Janicsák et al., 2007, 2011), while literature data on antimicrobial activity of extracts were not available till now. Taking into account the lack of comprehensive research data on of S. ringens herb, especially those growing wild in Macedonia, the aim of the present study was to investigate chemical composition and biological activities of its essential oil and extracts. 2. Material and methods 2.1. Standards and reagents Methanol, ethanol, distilled water, glacial acetic acid, hydrochloric acid, hexane, dichlormethane, and ethyl acetate were purchased from Zorka Pharma, Šabac (Serbia). Gallic acid, quercetin, ascorbic acid, 2(3)-t-butyl-4-hydroxyanisole (BHA), 3,5-di-tert-butyl4-hydroxytoluene (BHT) 2,2-dyphenyl-1-picrylhydrazyl (DPPH), 2,2 -azino-bis (3-ethylbenzothiazoline-6-sulfonic acid diammonium salt) (ABTS), 2,4,6-tripyridyl-s-triazine (TPTZ), potassium acetate (C2 H3 KO2 ), potassium-persulfate (K2 S2 O8 ), sodium carbonate anhydrous (Na2 CO3 ), alluminium nitrate nonahydrate (Al(NO3 )3 × 9H2 O), sodium acetate (C2 H3 NaO2 ), iron(III) chloride (FeCl3 ), iron(II)-sulfate heptahydrate (FeSO4 × 7H2 O) and Folin–Ciocalteu phenol reagent were purchased from Sigma Chemicals Co. (USA). The phenolic compounds standards were from Merck (Germany). All chemicals used in experimental procedure were of analytical grade purity. 2.2. Plant material Aerial parts of the Salvia ringens Sibth. & Sm. are collected during the ﬂowering period in July of 2012 at Krivolak locality (Macedonia). Voucher samples are stored in the Herbarium of the Institute of Botany and Botanical Garden “Jevremovac”, Faculty of Biology, University of Belgrade BEOU; voucher No. (16671). 2.3. Essential oil isolation Air-dried aerial parts of S. ringens were grounded. Essential oil was isolated by hydrodistillation using a Clevenger type apparatus, according to the procedure I of the Yugoslavian Pharmacopoeia (1984). 703 2.4. Preparation of the extracts Extracts were prepared from whole aerial plant parts using two parallel extraction procedures. Dry plant material was grounded into small pieces (2–6 mm) in the cylindrical crusher. First, portion of 10 g of plant material was successively extracted by 100 mL of dichloromethane, ethyl acetate, and methanol, according to procedure of Şenol et al. (2010) and Orhan et al. (2013). Second, portion of 10 g of plant material was individually extracted by 100 mL of solvent (ethanol and hot distilled water). In both cases, extraction was performed by classic maceration during 24 h at room temperature (10% w/v). The mixture was exposed to ultrasound 1 h before and after 24 h of maceration to improve extraction process (Veličković et al., 2007; Glišić et al., 2011). Subsequently, extracts were ﬁltered through a ﬁlter paper (Whatman No. 1) and evaporated under reduced pressure by the rotary evaporator (Buchi rotavapor R-114). After evaporation of the solvent, the obtained crude extracts were stored in the fridge at +4 ◦ C for further experiments. 2.5. Essential oil analysis Qualitative and quantitative analysis was carried out using GCFID and GC–MS. In the ﬁrst instance model HP-5890 Series II gas chromatograph equipped with a split-splitless injector, HP-5 capillary column (25 m × 0.32 mm, ﬁlm thickness 0.52 m) and a ﬂame ionization detector (FID), was employed. Hydrogen was used as carrier gas (1 mL min−1 ). The injector was heated at 250 ◦ C, the detector at 300 ◦ C, while the column temperature was linearly programmed from 40 to 260 ◦ C (4 ◦ C/min). GC–MS analyses were carried out under almost the same analytical conditions, using HP G 1800C Series II GCD analytical system, equipped with HP-5MS column (30 m × 0.25 mm × 0.25 m). Helium was used as carrier gas. The transfer line (MSD) was heated at 260 ◦ C. The EI mass spectra (70 eV) were acquired in the scan mode in the m/z range 40–400. In each case, 1 L of sample solution in ethanol (10 L/mL) was injected in split mode (1:30). The identiﬁcation of constituents was performed by matching their mass spectra and retention indices with those obtained from authentic samples and/or NIST/Wiley spectra libraries, using different types of search (PBM/NIST/AMDIS) and available literature data (Adams, 2001; Hochmuth, 2006). The percentage compositions were obtained from electronic integration measurements using ﬂame ionization detection (FID; 250 ◦ C). 2.6. HPLC analysis of extracts The HPLC analyses of phenolic components were performed using the Agilent 1100 Series and UV-DAD (UV-diode array detector) according to procedure Veit et al. (1995). The column was an Agilent Eclipse XDB-C18, 5 m, 150 × 4.6 mm, 80 Å. Injection volume was 15 L of extracts in concentration of 10 mg/mL. Peak detection in UV region at 350 nm was used. The mobile phase was composed of solvent (A) 0.15% (w/v) phosphoric acid in water: methanol mixture (77:23, v/v, pH 2) and solvent (B) methanol as follows: isocratic 0–3.6 min 100% A; 3.6–24 min 80.5% A; 24–30 min isocratic; linear 30–60 min 51.8% A; 60–67.2 min 100% B. The ﬂow rate of mobile phase was set to the 1 cm3 /min and temperature to 15 ◦ C. Phenolic compounds in the samples were identiﬁed by comparing their retention times and spectra with retention time and spectrum of standards for each component. Identiﬁcation of the glycoside components was based on Rf values in the HPLC chromatogram. 2.7. Determination of total phenolic content The total phenolic content of was measured using spectrophotometric method (Singleton and Rossi, 1965). The reaction mixture 704 A. Alimpić et al. / Industrial Crops and Products 76 (2015) 702–709 was prepared by mixing 0.2 mL of extract solution in concentration of 1 mg/mL and 1 mL of 10% Folin–Ciocalteu reagent and after 6 min was added 0.8 mL of 7.5% Na2 CO3 . Blank was prepared to contain distillated water instead of extract. Absorbance was recorded at 740 nm after 2 h incubation at room temperature using JENWAY 6305UV–vis spectrophotometer. The same procedure was repeated for standard solution of water solution of gallic acid in order to construct calibration curve. Phenolic content in samples was calculated from standard curve equation and expressed as gallic acid equivalents (mg GAE/g dry extract). 2.8. Determination of ﬂavonoid content in 5 mL of 2.46 mM potassium-persulfate and stored in the dark at room temperature. The ABTS+ solution was dissolved by distilled water to obtain an absorbance of working solution 0.700 ± 0.020 at 734 nm. 50 L of test samples (1 mg/mL) were mixed with 2 mL of diluted ABTS+ solution and incubated for 30 min at 30 ◦ C. Absorbance was recorded at 734 nm using JENWAY 6305UV–vis spectrophotometer. Distilled water was used as blank. BHA and BHT dissolved in methanol in concentration 0.1 mg/mL were used as standards. ABTS activity was calculated from ascorbic acid calibration curve (0–2 mg/L) and expressed as ascorbic acid equivalents per gram of dry extract (mg AAE/g). • FRAP assay Flavonoid concentrations of samples were measured spectrophotometrically according to procedure of Park et al. (1997). The reaction mixture was prepared by mixing 1 mL of extract solution in concentration 1 mg/mL, 4.1 mL of 80% ethanol, 0.1 mL of 10% Al(NO3 )3 × 9H2 O and 0.1 mL 1 M dilution CH3 COOK. Blank was prepared to contain 96% ethanol instead of extract. After 40 min of incubation at room temperature, absorbance was measured at 415 nm using JENWAY 6305UV–vis spectrophotometer. The same procedure was repeated for 96% ethanol solution of standard antioxidant quercetin in order to construct calibration curve. Concentration of ﬂavonoids in samples was calculated from standard curve equation and expressed as quercetin equivalents (mg QE/g dry extract). 2.9. Evaluation of antioxidant activity For testing of antioxidant activity, crude extracts were dissolved in methanol. FRAP assay evaluates total antioxidant power of the sample using reduction of ferric tripyridyltriazine (Fe(III)-TPTZ) complex to the ferrous tripyridyltriazine (Fe(II)-TPTZ) by a test sample at low pH. The FRAP assay was performed according to Benzie and Strain (1996) procedure with slight modiﬁcations. FRAP reagent was prepared freshly to contain sodium acetate buffer (300 mmol/L, pH 3.6), 10 mmol/L TPTZ in 40 mmol/L HCl and FeCl3 × 6H2 O solution (20 mmol/L) in proportion 10:1:1 (v/v/v), respectively. Working FRAP solution was warmed to 37 ◦ C prior to use. 100 L of test sample (500 g/mL) were added to 3 mL of working FRAP reagent and absorbance was recorded at 593 nm after 4 min using the JENWAY 6305UV–vis spectrophotometer. Blank was prepared to contain methanol instead of extract. BHA, BHT, and ascorbic acid dissolved at concentration of 0.1 mg/mL were used as standards. The same procedure was repeated for standard solution of FeSO4 × 7H2 O (0.2–1.6 mmol/L) in order to construct calibration curve. FRAP values of sample was calculated from standard curve equation and expressed as mol (FeSO4 × 7H2 O/g dry extract). (a) DPPH assay 2.10. Antimicrobial assays For evaluation of antioxidant activity of extracts, 2,2-dyphenyl1-picrylhydrazyl (DPPH) free radical scavenging method (Blois, 1958) with slight modiﬁcations was used. This assay is spectrophotometric and uses stable DPPH radical as reagent. Stock solutions of dry extracts were prepared in concentration of 1000 g/mL (w/v) and then were diluted with methanolic solution of DPPH (40 g/mL) to adjust the ﬁnal volume of reaction mixture (2000 L) of the test tube (extract concentrations 10–300 g/mL (v/v)). Methanol was used as a blank, while methanol with DPPH solution was used as a control. BHA, BHT, and ascorbic acid were used as positive controls (standards). Each blank, samples and standards’ absorbances were measured in triplicate. Absorbance of the reaction mixture was measured after 30 min in the dark at room temperature at 517 nm using the JENWAY 6305UV–vis spectrophotometer. The decrease of absorption of DPPH radical at 517 nm was calculated using equation: Inhibition of DPPH radical (%) = (Ac − As ) × 100% Ac where Ac is the absorbance of control (without test sample), and As is the absorbance of the test samples at different concentrations. IC50 values (g/mL) (concentrations of the test samples and standard antioxidants providing 50% inhibition of DPPH radicals) were calculated from DPPH absorption curve at 517 nm. • ABTS assay (a) Antibacterial assay The antibacterial activity of essential oil and ethanol/water extracts was tested against six Gram-negative: Esherichia coli (ATCC 25922), Salmonella typhimurium (ATCC 14028), Salmonella enteritidis (ATCC 13076), Pseudomonas tolaasii (NCTC 387), Pseudomonas aeruginosa (ATCC 27853), Proteus mirabilis (ATCC 14273) and ﬁve Gram-positive bacteria: Staphylococcus aureus (ATCC 25923), Bacillus cereus (ATCC 10876), Micrococcus ﬂavus (ATCC 14452), Sarcina lutea (ATCC 10054) and Listeria monocytogenes (ATCC 15313). In order to investigate the antimicrobial activity of extracts, a modiﬁed version of the microdilution technique was used (Daouk et al., 1995; Hanel and Raether, 1988). Determination of MIC (minimum inhibitory concentrations) was performed by a microdilution technique using 96-well microtiter plates. Serial dilutions of stock solutions of extracts in broth medium (Muller–Hinton broth for bacteria) were prepared in a 96-wells microtiter plate. The microbial suspensions were adjusted with sterile saline to a concentration of 1 × 105 CFU/mL. The microplates plates were incubated at 37 ◦ C during 48 h. The lowest concentrations without visible growth were deﬁned as concentrations that completely inhibited bacterial growth (MICs). The standard antibiotic streptomycin (1 mg/mL DMSO) was used to control the sensitivity of the tested bacteria. • Antifungal assay In this test, antioxidant activity of samples was tracked spectrophotometrically, using change of ABTS solution colour in presence of antioxidants. ABTS assay is performed according to procedure Miller et al. (1993) with some modiﬁcations. Fresh ABTS+ solution was prepared 12–16 h before use by dissolving of ABTS Antifungal activity of extracts was tested against pathogenic micromycetes (human isolates): Candida krusei (Castell.) Berkhout, Candida albicans (C.P. Robin) Berkhout, Candida parapsilosis (Ashford) Langeron & Talice, Aspergillus glaucus (L.) Link, Aspergillus A. Alimpić et al. / Industrial Crops and Products 76 (2015) 702–709 fumigatus Fresen., Aspergillus ﬂavus Link and Trichophyton mentagrophytes (C.P. Robin) Sabour. Cultures were maintained on Sabouraud Dextrose Agar (SDA) at 4 ◦ C in the culture collection of the Institute of Botany, Faculty of Biology, University of Belgrade (BEOFB). Antifungal activity of extracts was studied by microdilution method using 96-well plates (Sarker et al., 2007). Spore suspensions were prepared by washing of SDA surface using sterile 0.9% saline containing 0.1% Tween 20 (v/v). Turbidity was determined spectrophotometrically at 530 nm and spore number was adjusted to 106 CFU/mL (NCCLS, 1998). Ethanol and water extracts were dissolved in 5% DMSO in stock concentration. Series of double dilutions of extract and essential oil (64–0.25 mg/mL and 4–0.125 mg/mL, respectively) in Sabouraud liquid medium were analyzed. Each well contained Sabouraud liquid medium, spore suspension, resazurine, and extract or essential oil of deﬁned concentration. The mixture without extract was used as the negative control, while the positive control contained commercial antimycotic, ketoconazole, instead of extract. Incubation was continued for another 48 h, and results were recorded using binocular microscope. The lowest concentration of extract or essential oil without visible fungal growth was deﬁned as minimal inhibitory concentration (MIC). The lowest concentration of extract or essential oil which inhibited fungal growth after re-inoculation on SDA was deﬁned as minimal fungicidal concentration (MFC). 2.11. Cytotoxic activity HCT-116 cells were seeded in a 96-well plate (104 cells per well). After 24 h of cells incubation, the medium was replaced with 100 L medium containing various doses of ethanol and water extracts of S. ringens at different concentrations (1, 10, 50, 100, 250, and 500 g/mL). Untreated cells were used as the control. After 24 and 72 h of treatment the cell viability was determined by MTT assay (Mosmann, 1985). Solution of MTT (ﬁnal concentration 5 mg/mL in PBS) was added to each well and incubated at 37 ◦ C in 5% CO2 for 2–4 h. The colored crystals of produced formazan were dissolved in 150 L of DMSO. The absorbance was measured at 570 nm on Microplate Reader (ELISA 2100C). Cell proliferation was calculated as the ratio of absorbance of treated group divided by the absorbance of control group, multiplied by 100 to give a percentage proliferation. 2.12. Statistical analysis All experimental measurements were carried out in triplicate and are expressed as average of three measurements ± standard deviation. Pearson’s correlation coefﬁcients were calculated between on one hand total phenolics and ﬂavonoids and on the other hand antioxidant assays and interpreted according to Taylor (1990). Calculations and constructing of the charts were performed using the MS Ofﬁce Excel, 2007. 3. Results and discussion 3.1. Essential oil analysis The aerial parts of S. ringens yielded 0.19% of the yellowish essential oil. Chemical composition of the essential oil is presented in Table 1. Of 39 detected compounds, representing 99.62% of the total oil, 36 were identiﬁed. The most abundant classes of terpenes were monoterpenes (93.60%) including oxygenated monoterpenes and monoterpene hydrocarbons represented with 56.89% and 36.74%, respectively. The main components of the oil were 1.8-cineole (31.99%), camphene (17.06%), borneol (11.94%), ␣-pinene (11.52%), camphor (5.16%) and bornyl acetate (4.52%). Monoterpenes were 705 Table 1 Chemical constituents in the Salvia ringens essential oil. Peak Compound RIa m/m (%) 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. 39. Tricyclene ␣-Thujene ␣-Tinene Camphene Thuja-2,4(10)-diene ß-Pinene 1-Octen-3-ol Myrcene ␣-Phellandrene ␣-Terpinene p-Cymene 1,8-Cineole cis-Thujone 1-Octen-3-yl acetate endo-Fenchol ß-Phellandrene ␣-Campholenal trans-Pinocarveol Camphor Camphene hydrate Borneol Terpinen-4-ol ␣-Terpineol trans-Piperitol Bornyl acetate ␣-Cubebene ␣-Copaene ß-Bourbonene cis-Caryophyllene 2-epi-Beta-funebrene ␣-Humulene 9-epi-trans-Caryophyllene ␣-Muurolene n.i. n.i. ␥-Cadinene n.i. trans-Calamenene Humulene epoxide II 918.8 925.3 931.0 945.3 951.1 973.2 986.0 991.6 1003.7 1015.8 1024.5 1030.4 1105.4 1110.4 1114.5 1122.9 1126.6 1138.9 1142.3 1145.4 1167.8 1177.7 1194.0 1208.6 1285.3 1354.0 1367.0 1375.2 1410.0 1415.5 1444.8 1450.2 1491.2 1496.9 1500.1 1512.8 1509.1 1523.2 1608.5 0.82 0.03 11.52 17.06 0.11 3.69 0.73 3.16 0.11 0.05 2.96 31.99 0.35 0.20 0.12 0.19 0.32 0.39 5.16 0.16 11.94 1.15 0.39 0.20 4.52 0.17 0.31 0.16 0.35 0.21 0.10 0.13 0.14 0.11 0.16 0.26 0.11 0.32 0.16 Aliphatic hydrocarbons Aromatic hydrocarbons Total hydrocarbons Monoterpene hydrocarbons Oxygenated monoterpens Total monoterpenes Sesquiterpene hydrocarbons Oxygenated sesquiterpenes Total sesquiterpenes Total identiﬁed 0.73 2.96 3.69 36.74 56.89 93.60 2.16 0.16 2.32 99.62 a Retention index relative to n-alkanes on HP-5 capillary column; n.i., not identiﬁed. previously recognized as dominant class of S. ringens oil from different localities. Dominant components in the oil of S. ringens from Greece were 1.8-cineol, ␣-pinene, bornyl acetate, and ␤-pinene (Tzakou et al., 2001), 1.8-cineole, ␣-pinene and myrcene in S. ringens var. baldacciana from Dautica Mt. (Macedonia) (Šavikin et al., 2008) and camphor and borneol in leaves and ﬂowers of S. ringens from Bulgaria (Georgiev et al., 2013). Our ﬁndings are in agreement with these studies with exception of particularly high percent of camphene (17.06%). Differences in the chemical composition could be derived from several factors such as plant age, plant part, development phase, growing place, harvesting period, chemotype (Ben Farhat et al., 2009; Miguel, 2010). 3.2. The yield of extracts, total phenolic and ﬂavonoid content S. ringens extracts were obtained using individual and successive extraction procedure and yields of extracts are presented in Table 2. The yields of ethanol and methanol extracts were the high- 706 A. Alimpić et al. / Industrial Crops and Products 76 (2015) 702–709 Table 2 The yield, total phenolic content (TPC), ﬂavonoid content (FC) and antioxidant activities evaluated by DPPH, ABTS, and FRAP assays of S. ringens. Sample Yielda TPCb FCc DPPHd ABTSe FRAPf Methanol Dichloromethane Ethyl acetate Ethanol Essential oil BHA BHT Ascorbic acid 6.94 2.39 1.37 7.20 0.19 – – – 185.05 ± 1.471 58.10 ± 0.510 248.38 ± 0.455 208.27 ± 1.113 – – – – 27.31 ± 0.588 32.31 ± 0.428 66.67 ± 1.464 30.41 ± 0.640 – – – – 20.29 ± 0.263 266.22 ± 4.208 22.25 ± 0.571 17.26 ± 0.412 654.33 ± 6.522 17.94 ± 0.168 13.37 ± 0.430 5.11 ± 0.143 1.19 ± 0.026 0.58 ± 0.021 2.36 ± 0.030 2.44 ± 0,028 nt 2.75 ± 0.021 2.82 ± 0.011 – 274.85 ± 13.192 191.13 ± 11.020 969.80 ± 25.238 1088.30 ± 17.655 ± 17.655 nt 445.34 ± 5.772 583.72 ± 5.255 180.81 ± 8.607 nt-not tested. a Percentage of yield (%). b mg GAE/g dry extract. c mg QE/g dry extract. d IC50 , g/ml. e mg AAE/g. f mol Fe(II)/g. est (7.70 and 6.94%, respectively), while dichloromethane and ethyl acetate extracts showed lower yield. Previous researchers pointed out that polar alcoholic extracts, such as ethanol and methanol, showed higher yield than less and/or non-polar solvents extracts (Akkol et al., 2008; Orhan et al., 2012). Total phenolic content (TPC) and ﬂavonoid content (FC) were measured using spectrophotometric assays and results are presented in Table 2. Ethyl acetate extract showed the largest total phenolic content (248.38 mg GAE/g), whereas dichloromethane extract was the poorest in total phenolics (58.10 mg GAE/g). Flavonoid contents of extracts ranged from 27.31 mg QE/g for methanol to 66.67 mg QE/g for ethyl acetate extract. Total phenolic and ﬂavonoid content were previously reported for many Salvia species from Turkey (Akkol et al., 2008; Orhan et al., 2012), South Africa (Kamatou et al., 2010), Iran (Asadi et al., 2010) and Greece (Stagos et al., 2012). Our ﬁndings were congruent with above mentioned studies, as well as with polyphenolic content of S. ringens herb collected in Bulgaria (Nikolova, 2011) and Romania (Coisin et al., 2012). 3.3. Phenolic composition of the extracts Phenolic composition of S. ringens extracts was determined using HPLC and components were classiﬁed according to Neveu et al. (2010) (Table 3). Methanol and ethyl acetate extracted most of the components, followed by ethanol, water and dichloromethane. As previously reported, the efﬁciency of extraction of phenolic components was rising with increasing polarity of the extraction solvent (Akkol et al., 2008; Orhan et al., 2012). Among phenolic acids, gallic, caffeic and rosmarinic acids were present. Caffeic acid was present in all extracts (0.18–8.27 %), excluding dichloromethane. Rosmarinic acid was present only in methanol extract (3.59%), although it was reported as the most common derivate of caffeic acid in Lamiaceae family (Lu and Foo, 2002) and the most abundant phenolic acid in Salvia genus with strong antioxidant activity (Akkol et al., 2008; Ben Farhat et al., 2009; Coisin et al., 2012; Kamatou et al., 2010; Orhan et al., 2012). The absence of rosmarinic acid in the ethanol extract could be attributed to the extraction procedure applied. Flavonoids, including ﬂavones and ﬂavonols, were identiﬁed in examined extracts whereby the ﬂavonols were present in a higher percentage. In Table 3 Phenolic constituents of S. ringens extracts (%). Extractsa Constituents Phenolic acids Gallic acid Caffeic acid Caffeic acid methyl ether Rosmarinic acid Flavonoids Flavones Apigenin Apigenin 5-O-glucoside Apigenin 4 -O-glucoside Genkwanin 5-O-glucoside Luteolin Flavonols Kaempferol 3-O-7-O-diglucoside Kaempferol 3-O-glucoside-7-O-rhamnoside Kaempferol 3-O-(6 -O-acetilglucoside)-7-O-rhamnoside Kaempferol 3-O-rhamnoside Rutin Quercetin Hyperoside Other polyphenols Coumarin Total of identiﬁed constituents Number of identiﬁed constituents Number of non-identiﬁed constituents a DCM ETAC – – – – – ETOH W 0.05 0.51 0.30 3.59 – 1.28 – – – 8.27 – – – – – – – 1.64 – 0.03 1.39 2.68 0.13 3.31 1.71 – 0.60 1.32 – – – 2.21 – – 1.40 – – – – – – – – 12.81 1.18 – 12.00 0.71 0.25 1.44 2.64 – 1.67 28.71 0.20 17.31 – 5.99 – – 46.46 – 7.74 – 2.83 – – 48.19 – 8.54 – – – 12.81 1 8 1.35 25.49 12 25 2.90 66.98 14 22 1.72 63.56 7 8 – 66.39 4 11 0.18 – – DCM (dichloromethane), ETAC (ethyl acetate), MEOH (methanol), ETOH (ethanol), W (water); – not identiﬁed. MEOH A. Alimpić et al. / Industrial Crops and Products 76 (2015) 702–709 Table 4 Pearson’s correlation coefﬁcients (r) of antioxidant activities versus total phenolic content (TPC) and ﬂavonoid content (FC) of S. ringens extracts. TPC FC DPPH vs. ABTS DPPH vs. FRAP ABTS vs. FRAP TPC vs. FC DPPH ABTS FRAP −0.945c −0.236a 0.886c 0.504b −0.780c −0.636b 0.977c 0.530b 0.770c 0.490b 707 pound is extracted in the greatest extent by the non-polar solvent (dichloromethane), which could be explained by applying of successive extraction, also reported by Askun et al. (2012) and Orhan et al. (2013). 3.4. Evaluation of antioxidant activity According to Taylor (1990): a r ≤ 0.35 weak correlation. b 0.36 < r < 0.67 moderate correlation. c 0.68 < r < 1 strong correlation. our study, ﬂavonol kaempferol 3-O-(6”-O-acetilglucoside)-7-Orhamnoside was present in the highest amounts (12.00–48.19 %), especially in water and ethanol extracts. Recent data reported on powerful antioxidant activity of some ﬂavonols such as kaempferol rhamnoside derivatives (Tatsimo et al., 2012). The majority of ﬂavonoids identiﬁed in the present study, such as luteolin, apigenin, kaempferol, rutin, quercetin and its glycosides, were recognized previously in methanol extract of S. ringens from Romania (Coisin et al., 2012) as well as in the extracts of other Salvia species (Akkol et al., 2008; Ben Farhat et al., 2009; Kamatou et al., 2010; Lu and Foo, 2002; Orhan et al., 2012). Hyperoside–glycoside, polar com- Antioxidant activity of S. ringens extracts was measured using three parallel test assays, i.e., DPPH, ABTS for the evaluation of free-radical scavenging activity of extracts, and FRAP assay for measuring the total ferric-reducing power of extracts (Table 2). DPPH scavenging activity of extracts, presented by IC50 value, ranged from 17.26 g/mL for ethanol to 266.22. g/mL for dichloromethane extract. Due to the very small amount of the essential oil obtained, it was tested only by DPPH assay, and performed extremely weaker activity than extracts (IC50 value of 654.33 g/mL). Against ABTS radicals the most powerful was ethanol extract (2.44 mg AAE/g, respectively) on the contrary to the dichloromethane extract (0.58 mg AAE/g). Similarly, ethanol extract showed the most expressive ability to reduce Fe(III) to Fe(II) ion (1088.30 mol Fe(II)/g), unlike dichloromethane with 191.13 mol Fe(II)/g. In all three assays, ethanol extract exhibited activity close to references antioxidants BHA and BHT. Our ﬁndings are congruent with previous studies on antioxidant activity of Table 5 Antimicrobial activity of the S. ringens ethanol (ETOH), water (W), essential oil (EO) and reference substances. Extracts Bacteria Gram-negative bacteria Esherichia coli ATCC 25922 Salmonella typhimurium ATCC 14028 Salmonella enteritidis ATCC 13076 Pseudomonas tolasii NCTC 387 Pseudomonas aeruginosa ATCC 27853 Proteus mirabilis ATCC 14273 Gram-positive bacteria Staphylococcus aureus ATCC 25932 Bacillus cereus ATCC 10876 Micrococcus ﬂavus ATCC 14452 Sarcina lutea ATCC 10054 Listeria monocytogenes ATCC 15313 Micromycetes Candida krusei Candida albicans Candida parapsilosis Aspergillus glaucus Aspergillus fumigatus Aspergillus ﬂavus Trichophyton mentagrophytes EO Streptomycin Ketoconazole 25 – 20 – 20 – 30 – 30 – 25 – 14.25 – 14.25 – 11.40 0.012 – 0.010 – 0.010 14.25 0.016 17.10 0.016 17.10 0.005 – – – – – – – – – – – 5 – 10 – 10 – 15 – 5 – 15 – 20 – 20 – 25 – 15 – 9.50 – 9.50 – 9.50 – 11.40 – 9.50 – 0.016 – 0.005 – 0.010 64 NA 64 NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA 16 NA NA NA NA NA NA NA 0.125 3.000 0.125 3.000 0.125 3.000 0.125 3.000 3.000 NA 0.25 NA 0.75 1.50 – – – – – – – – – – – – – – ETOH W MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC 15 – 10 – 15 – 20 – 20 – 15 – MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC MIC MFC MIC MFC MIC MFC MIC MFC MIC MFC MIC MFC MIC MFC 0.012 – 0.010 – – – – – – – – – 0.0078 0.0156 0.0078 0.0156 0.0078 0.0156 0.0078 0.0078 0.0078 0.0156 0.0078 0.0078 0.0019 0.0039 708 A. Alimpić et al. / Industrial Crops and Products 76 (2015) 702–709 methanol extracts of S. ringens herb from Bulgaria (Nikolova, 2011) and Galičica Mt., Macedonia (Tusevski et al., 2014). Table 6 Cytotoxic activity presented as IC50 values (g/mL) of S. ringens extracts against HCT-116 and SW480 cell lines. 3.5. Correlation between antioxidant assays, total phenolic and ﬂavonoid contents Cell line Type of extract 24 h 72 h HCT-116 Antioxidant activity measured by DPPH, ABTS, and FRAP assays on the one hand and content of total phenolic (TPC) and ﬂavonoid content (FC) on the other hand, were correlated in different ways (Table 4). Antioxidant assays were moderate to strongly correlate between each other. Antioxidant assays were more strongly correlated to total phenolic than to ﬂavonoid content and these ﬁndings are in agreement with previous studies (Asadi et al., 2010; Ben Farhat et al., 2009; Li et al., 2008; Stagos et al., 2012). SW480 ETOH W ETOH W 31.83 ± 1.89 9.83 ± 0.28 >500 >500 179.30 ± 4.12 406.71 ± 4.61 >500 412.36 ± 2.31 3.6. Antimicrobial activity Antimicrobial (antibacterial and antifungal) activities of S. ringens essential oil and ethanol and water extracts were investigated using microdilution method and data were presented in Table 5. These extracts were selected for testing of the antimicrobial activity and later for cytotoxic activity since the mixtures of ethanol/water and water extracts are mostly used in phytotherapy (Miguel, 2010; Stagos et al., 2012). Antibacterial activity was tested against six Gram-negative and ﬁve Gram-positive bacteria. Results of the present study showed that the essential oil possess the strongest antibacterial activity (MICs 9.50–17.10 mg/mL), followed by ethanol (MICs 5–20 mg/mL) and water extract (MICs from 15 to 30 mg/mL), which is in accordance with ﬁndings previously reported by Tepe et al. (2004). Similar to the previous study published by Šavikin et al. (2008) dealing with S. ringens var. baldachiana from Macedonia, Grampositive strains were more sensitive. Unlike the aforementioned, Tzakou et al. (2001) found that inhibitory effects of S. ringens oil was stronger against Gram-negative bacteria and mainly attributed to presence of 1,8-cineole as dominant component. Comparing to streptomycin (MICs ranged as 0.005–0.016 mg/mL), our samples exhibited weaker activity. The most sensitive bacteria were S. aureus and L. monocytogenes while the most resistant bacteria were P. tolasii and P. aeruginosa, as reported before (Šavikin et al., 2008; Tepe et al., 2004; Veličković et al., 2002). S. ringens essential oil showed stronger antifungal activity against seven tested mycromicetes (MICs 0.125–3 mg/mL) compared to ethanol and water extracts. Fungicidal effects were not observed only for A. fumigatus and A. ﬂavus, which were previously reported as very resistant micromycetes (Veličković et al., 2002). Ethanol extract inhibited the growth of C. krusei and C. albicans at the highest applied concentration of 64 mg/mL. Similar as in previous studies (Šavikin et al., 2008; Tepe et al., 2004; Tzakou et al., 2001; Veličković et al., 2002), Candida species were generally sensitive to Salvia essential oil and extracts. 3.7. Cytotoxic activity The ethanol and water extracts of S. ringens were tested for their cytotoxic activity against human colon carcinoma HCT-116 and SW480 cell lines using the MTT assay. Results were recorded after 24 and 72 h of treatment and presented as IC50 values (g/mL) in Table 6. Our results showed that S. ringens extracts exhibited more signiﬁcant cytotoxic activity after 24 h than after 72 h on HCT-116 cells. The water extract showed IC50 values lower than 30 g/mL, which were considered as very good cytotoxic activity according to National Cancer Institute criteria for cytotoxic activity of crude extracts (Suffness and Pezzuto, 1990). HCT-116 cell line was more sensitive than SW480 cell line (IC50 values above 500 g/mL). Cytotoxic effects of S. ringens methanol extract on skin cancer cell lines (A431) were evaluated as strong, while some isolated abietane diterpenes from S. ringens root displayed marked concentration-dependent effects on human cervix adenocarcinoma (HeLa) cells (Janicsák et al., 2007, 2011). In current study, as the main phenolic constituent was found kaempferol 3-O-(6”-Oacetilglucoside)-7-O-rhamnoside, especially in water and ethanol extracts. Some kaempferol glycosides were previously reported as strong cytotoxic agents against lung and melanoma cancer cell lines (Moon et al., 2010). 4. Conclusions According to results of this study, it can be concluded that essential oil and extracts of Salvia ringens showed strong antioxidant and cytotoxic activity and promising antimicrobial effects. Essential oil was composed mainly from monoterpenes 1.8-cineole, camphene, borneol, and ␣-pinene. 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