Journal of Applied Botany and Food Quality 88, 115 - 119 (2015), DOI:10.5073/JABFQ.2015.088.016 1 Institute of Botany and Botanical Garden “Jevremovac”, Faculty of Biology, University of Belgrade, Belgrade, Serbia 2 Laboratory for Molecular Biomedicine, Institute of Molecular Genetics and Genetic Engineering, University of Belgrade, Belgrade, Serbia 3 Institute of Biology, Faculty of Natural sciences and mathematics, University “Ss. Cyril and Methodius”; Macedonian Academy of Sciences and Arts, Skopje, Macedonia 4 Department of Biology, Faculty of Arts and Science, Al-Mergeb University, Al-Khoms, Libya The in vitro antioxidative and cytotoxic effects of selected Salvia species water extracts Ana Alimpić 1*, Nikola Kotur 2, Biljana Stanković 2, Petar D. Marin 1, Vlado Matevski 3, Najat Beleed Al Sheef 4, Sonja Duletić-Laušević 1 (Received October 10, 2014) * Corresponding author Summary The current paper presents antioxidant and cytotoxic activities and total phenolic and flavonoid content of the selected species of ge- nus Salvia (Lamiaceae) growing wild in Macedonia (S. jurisicii Košanin, S. amplexicaulis Lam., S. ringens Sibth. & Sm.) and Libya (S. fruticosa Mill. and S. lanigera Poir.). Crude water extracts, ob- tained from aerial parts, were yielded from 6.50 to 14.32 %. Total phenolic content was the highest in water extracts of S. amplexi- caulis and S. ringens (226.30 and 189.01 mg GAE/g, respectively), while the flavonoids were the most abundant in S. jurisicii extract (32.36 mg QE/g). Antioxidant activities of extracts were measured using DPPH, ABTS and FRAP assays. S. amplexicaulis and S. rin- gens extracts showed the strongest antioxidant activity, measured using DPPH (14.21 and 23.44 μg/mL, respectively) and ABTS assays (2.91 and 2.42 mg AAE/g, respectively). In FRAP assay, S. amplexi- caulis and S. fruticosa extracts exhibited strongest activity (1406.73 and 1191.51 μmol Fe(II)/g). Water extract of S. amplexicaulis and S. ringens performed the strongest cytotoxic activity against K562 cells (151.07 and 173.06 μg/mL, respectively). Based on these fin- dings, it can be concluded that S. amplexicaulis and S. ringens water extracts could be considered as possible source of antioxidant and cytotoxic agents. Introduction The genus Salvia (Lamiaceae) represents an enormous and cosmo- politan assemblage of nearly 1000 species worldwide of which 36 were found in Europe (HEDGE, 1972) and 10 in Libya (JAFRI and EL-GADI, 1985). The representatives of Salvia genus are widely cul- tivated and used in flavoring and folk medicines. Sage and rosemary from the Lamiaceae family were shown to have similar patterns of phenolic compounds and the antioxidant activi- ty had been attributed mainly to carnosic, rosmarinic acid and their isomers. Additional classes of active compounds include terpenoids, flavonoids and other phenolic acids (LU and FOO, 2001). Majority of these compounds, excluding terpenoids, are water-soluble and pre- sent in aqueous extract obtained using common techniques of extrac- tion (TIWARI et al., 2011). The genus Salvia was the research topic of numerous chemical, medicinal and pharmacological studies. Species of Salvia showed diverse biological activities of plant material and/or isolated essen- tial oil/extracts due to the presence of large number of different com- pounds. The antioxidant (COULADIS et al., 2003; JANICSÁK et al., 2010; ORHAN et al., 2013; ALIMPIĆ et al., 2014), cytotoxic (FIORE et al., 2006; KAMATOU et al., 2005; JANICSÁK et al., 2007; ABU- DAHAB et al., 2012), antimicrobial (KAMATOU et al., 2005; HAWAS and EL-ASNARI, 2006), antiinflammatory (KAMATOU et al., 2005; 2010), antimalarial (KAMATOU et al., 2005), anticholinesterase (ŞENOL et al., 2010, ORHAN et al., 2012; 2013), etc. effects were reported. S. jurisicii Košanin, perennial herb inhabiting arid habitats, is an endemic species in the central part of Republic of Macedonia (HEDGE, 1972). It was previously investigated for antioxidant activi- ty (JANICSÁK et al., 2010). S. amplexicaulis Lam. is a perennial plant, distributed on Balkan Peninsula (HEDGE, 1972). It was investigated for vasodepressor (ULUBELEN, 2003), antioxidant, and neurobiological activity (OR- HAN et al., 2012) of extracts. S. ringens Sibth. & Sm. is a hardy herbaceous perennial plant, dis- tributed in South and Eastern parts of Balkan peninsula (HEDGE, 1972). Total acetone extract and some isolated compounds showed cytotoxic activity against HeLa cells (JANICSÁK et al., 2007), while ethanol extracts performed strong antioxidant activity (COULADIS et al., 2003). S. lanigera Poir. is perennial herb with thick woody rootstock dis- tributed in North Africa, from northern Egypt and Arabia, to the south of Turkey and Iran (JAFRI and EL-GADI, 1985). There are a few reports on phenolic composition, antimicrobial and cytotoxic activi- ty of S. lanigera extracts (HAWAS and EL-ASNARI, 2006; SHAHEEN et al., 2011). S. fruticosa Mill. (S. triloba L.) is subshrub distributed in Canary Islands and North Africa. It is sometimes used for flavouring tea and cultivated as an ornamental plant (JAFRI and EL-GADI, 1985). Jor- danian S. fruticosa ethanol extract performed significant cytotoxic activity (ABU-DAHAB et al., 2012). The objective of this study was to investigate antioxidative and cyto- toxic activity as well as phenolic/flavonoid contents in water extracts of selected Macedonian and Libyan Salvia species. Material and methods Plant material Aerial parts of the Salvia species were collected in flowering pe- riod from their natural populations at localities in Macedonia and Libya and were identified by Prof. P.D. Marin and Prof. V. Matevski. Plant material was dried and kept in shadow at room temperature for further processing. Total sample for each species consisted of at least 20-50 individuals (about 500 g of dry material, depending of species). Voucher specimens were deposited in the Herbarium of the Institute of Botany and Botanical Garden “Jevremovac”, Facul- ty of Biology, University of Belgrade. Collection data and voucher numbers of investigated Salvia species are given in Tab. 1. Preparation of water extracts Extracts were prepared of whole aerial plant parts using classic maceration procedure. Dry plant material (5 g), randomly taken 116 A. Alimpić, N. Kotur, B. Stanković, P.D. Marin, V. Matevski, N.B. Al Sheef, S. Duletić-Laušević from whole collected sample of each species, was grounded in small pieces (2-6 mm) and extracted by 50 ml of boiling distilled water (10 % w/v). Extraction was performed during 24 h at room tempe- rature. The mixture was exposed to ultrasound 1 h before and after 24 h-maceration. Subsequently, extracts were filtered through a pa- per filter (Whatman No.1) and evaporated under reduced pressure by the rotary evaporator (Buchi rotavapor R-114). The obtained crude extracts (Tab. 2) were stored in the fridge at +4 °C for further ex- periments. as a control. BHA, BHT and ascorbic acid were used as positive con- trols (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 equa- tion: Inhibition of DPPH radical (%) = [(AC-AS)/AC] * 100% where Ac is the absorbance of control (without test sample) and As is the absorbance of the test samples at different concentrations. IC50 values (µg/mL) (concentrations of the test samples and standard an- tioxidants providing 50% inhibition of DPPH radicals) were calcula- ted from DPPH absorption curve at 517 nm. ABTS assay ABTS assay is performed according to procedure of MILLER et al. (1993) with some modifications. Fresh ABTS+ solution was prepa- red 12-16 hours before use by dissolving ABTS in 5 ml of 2.46 mmol potassium-persulfate to obtain concentration of 7 mmol/L and stored in the dark at room temperature. The ABTS+ solution was dissol- ved by distilled water to achieve an absorbance of working solution 0.700 ± 0.020 at 734 nm. 50 μL of test samples (1 mg/mL) and/or standards (0.1 mg/mL) were mixed with 2 mL of diluted ABTS+ so- lution and incubated for 30 min at 30 ºC. Absorbance was recorded at 734 nm using JENWAY 6305UV/Vis spectrophotometer. Distilled water was used as blank. BHA and BHT dissolved in methanol were used as standards. ABTS activity was calculated from ascorbic acid calibration curve (0-2 mg/L) and expressed as ascorbic acid equi- valents per gram of dry extract (mg AAE/g). Ferric-reducing ability of plasma (FRAP) assay The FRAP assay was performed according to BENZIE and STRAIN (1996) procedure with slight modifications. FRAP reagent was pre- pared freshly to contain sodium acetate buffer (300 mmol/L, pH 3.6), 10 mmol/L TPTZ in 40 mmol/L HCl and FeCl3*6H2O solution (20 mmol/L), i.e. in proportion 10:1:1(v/v/v), respectively. Working FRAP solution was warmed to 37 °C prior to use. 100 μL of test sample (500 μg/mL) were added to 3 mL of working FRAP rea- gent and absorbance was recorded at 593 nm after 4 min using the Tab. 1: Collection data and voucher numbers of examined Salvia species Salvia species Collection data Voucher No. Locality Date Geographical data S. jurisicii Štip N 41°50.324' Košanin (Macedonia) 11.7.2011 E 22°08.289' BEOU; 16674 Alt. 420 m S. amplexicaulis Pletvar N 41°22.169' Lam. (Macedonia) 12.7.2011 E 21°39.180' BEOU; 16673 Alt. 1010 m S. ringens Krivolak N 41°53.33' Sibth. & Sm. (Macedonia) 2.7.2012 E 21°41.08' BEOU; 16675 Alt. 229 m S. lanigera Zintan N 31°55.50' Poir. (Libya) 22.3.2010 E 12°14.54' BEOU; 16880 Alt. 694 m S. fruticosa Biadda N 32°45.59' Mill. (Libya) 10.3.2010 E 21°44.30' BEOU; 16702 Alt. 624 m Tab. 2: The yield, total phenolic and flavonoid content of Salvia water ex- tracts. Values are presented as mean ± standard deviation. Water extracts Extract yielda Total phenolic Total flavonoid contentb contentc S. jurisicii 9.50 81.03 ± 0.216 32.36 ± 0.731 S. amplexicaulis 14.32 226.30 ± 1.179 17.87 ± 0.089 S. ringens 6.50 189.01 ± 1.699 22.64 ± 0.898 S. lanigera 7.32 58.47 ± 0.200 17.18 ± 0.544 S. fruticosa 7.62 67.68 ± 0.001 21.73 ± 0.163 a % of weight of dry plant material b mg GAE/g dry extract c mg QE/g dry extract Evaluation of antioxidant activity Antioxidant activity was evaluated using three spectrophotometric assays: DPPH, ABTS, and FRAP. Stock solutions of dry extracts were prepared in the distilled water in concentration of 1000 μg/ mL (w/v). DPPH assay For evaluation of antioxidant activity of extracts, 2,2-dyphenyl-1- picrylhydrazyl (DPPH) free radical scavenging method (BLOIS, 1958) with slight modifications was used. Stock extract solution was diluted with methanolic solution of DPPH (40 μg/mL) to adjust the final volume of reaction mixture (2000 μL) of the test tube. Methanol was used as a blank, while methanol with DPPH solution was used Antioxidative and cytotoxic effects Salvia water extracts 117 JENWAY 6305UV/Vis spectrophotometer. Blank was prepared to contain distilled water instead of extract. Ascorbic acid, BHA and BHT dissolved in methanol in concentration 0.1 mg/mL were used as standards. The same procedure was repeated for standard solution of FeSO4 * 7H2O (200-1600 μmol/L) in order to construct calibration curve. FRAP values of sample was calculated from standard curve equation and expressed as μmol Fe (II)/g dry extract). Determination of total phenolic content The total phenolic content was measured using spectrophotometric method (SINGLETON and ROSSI, 1965). The reaction mixture was prepared by mixing 0.2 mL of extract solution in concentration of 1 mg/mL, 1 mL of 10 % Folin-Ciocalteu reagent and 0.8 mL of 7.5 % Na2CO3. 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 spectrophotome- ter. Phenolic content in samples was calculated from standard curve equation and expressed as gallic acid equivalents (mg GAE/g dry extract). Determination of flavonoid concentration Flavonoid concentrations of samples were measured spectrophoto- metrically according to procedure of PARK et al. (1997). The reaction mixture was prepared by mixing 1 mL of extract solution in concen- tration 1 mg/mL, 4.1 mL of 80 % ethanol, 0.1 mL of 10 % Al(NO3)3 x 9 H2O and 0.1 mL 1M CH3COOK. 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. Concentration of flavonoids in samples (mg/ml) was calculated from standard curve equation and expressed as quercetin equivalents (mg QE/g dry extract). Cytotoxic assay − MTT assay To assess cytotoxic effect of sage water solutions, 3-(4,5-dime- thylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed (DRAKULIĆ et al., 2012). It is a colorimetric assay which detects conversion of yellow tetrazolium salt into purple formazon. The conversion is catalyzed by cellular enzymes and its rate repre- sents measure of cells viability. K562 cells are human immortalised myelogenous leukemia line. K562 cells are of the erythroleukemia type. The line is derived from a CML patient in blast crisis. K562 cells were maintained in essential minimal medium (MEM) sup- plemented with 10 % FCS. Cells were treated in 96 well plates for 48 h with 50, 100, 150, 200, 300 and 400 μg/mL sage water extracts in MEM. After addition of MTT solution (0.5 mg/mL), plate was incubated for additional 3 h. Acidified isopropanol was added to dis- solve tetrazolium salts. Absorbance was measured at 620 nm. 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 cytotoxic and antioxidant assays and interpreted according to TAYLOR (1990). Calculations and constructing of the charts were performed using the MS Office Excel, 2007. Results and discussion The yield of extract, total phenolic and flavonoid content The yields were expressed as percent of dry extract of dry plant material. The yields of obtained water extract were ranged from 14.32 % for S. amplexicaulis to 6.50 % for S. ringens extract (Tab. 2). Considering the uniform extraction procedure applied in this study, variations in extract’s yields can be attributed to diffe- rences of plant material (species). In some previous studies resear- chers have observed that type of plant material, choice of extrac- tion solvent and extraction procedure affects composition, activity and possible future use of the obtained extract (TIWARI et al., 2011). ŞENOL et al. (2010) obtained that yield of methanolic extract of 55 Salvia taxa varied between 2.88 and 13.41 % and our results are in agreement with these findings. Total phenolic and flavonoid contents of water extracts were eva- luated spectrophotometrically and expressed as gallic acid equi- valents/g dry extract and flavonoids concentrations as quercetin equivalents/g dry extract. As can be seen in Tab. 2, the highest amount of phenolics was measured in water extracts of S. amplexi- caulis and S. ringens (226.30 and 189.01 mg GAE/g, respectively), while the content of phenolics in the other extracts was lower than 100 mg GAE/g. On the contrary to phenolics, flavonoids were the most abundant in S. jurisicii extract (32.36 mg QE/g). When the ob- tained results are compared to the values for total phenolic content of Camellia sinensis green tea (140.11 mg GAE/g) and Ginkgo biloba (140.18 mg GAE/g) standardized extract (STANKOVIĆ et al., 2010), it can be seen that S. amplexicaulis and S. ringens have higher values. Our results are in agreement with the literature data for ethanol ex- tracts of fourteen Turkish Salvia species (57.10-218.09 mg GAE/g for total phenols and 8.29-108.78 mg QE/g for flavonoids) obtained by ORHAN et al. (2013). STAGOS et al. (2012) found total phenolic content of 267 and 190 mg GAE/g for methanolic and water ex- tract of S. fruticosa from Greece, respectively. As many researchers reported, amount of extracted phenolics and flavonoids depends on selection of extraction solvent. Evaluation of antioxidant activity Oxygen sometimes can be fatal for the organisms although euka- ryotic organisms cannot exist without it, which is known as oxygen paradox. A free radical is any species capable of independent existence that contains one or more unpaired electrons. Reactive oxygen species (ROS) may damage important cellular molecules such as DNA, proteins and lipids, causing cancer, cardiovascular, neurodegenerative and other diseases (HALLIWELL, 2006). Antioxidant activities of Salvia species water extracts measured using DPPH, ABTS and FRAP assays are presented in Tab. 3. DPPH and ABTS assays were applied to examine scavenging activity of extracts, while FRAP assay measured ability of extracts to reduce Fe (III) to Fe (II) ion. Water extract of S. amplexicaulis showed marked- ly the strongest antioxidant activity in all three assays. Tab. 3: DPPH, ABTS and FRAP activity of Salvia water extracts. Water extracts DPPH assay ABTS assay FRAP assay (IC50, μg/ml) (mg AAE/g) (μmol Fe(II)/g) S. jurisicii 212.24 1.15 ± 0.062 290.14 ± 6.880 S. amplexicaulis 14.21 2.91 ± 0.019 1406.73 ± 8.055 S. ringens 23.44 2.42 ± 0,019 615.44 ± 6.720 S. lanigera 230.87 1.77 ± 0.085 79.13 ± 5.255 S. fruticosa 48.11 1.98 ± 0.005 1191.51 ± 8.109 Positive controls BHT 17.94 2.75 ± 0.021 445.34 ± 5.772 BHA 13.37 2.82 ± 0.011 583.72 ±5.255 Ascorbic acid 5.11 / 180.81 ± 8.607 118 A. Alimpić, N. Kotur, B. Stanković, P.D. Marin, V. Matevski, N.B. Al Sheef, S. Duletić-Laušević 200 μg/ml), followed by S. fruticosa and S. lanigera (IC50 200- 400 μg/ml) and S. jurisicii (> 400 μg/ml). Some of the species examined in this study were partially investi- gated before for their cytotoxic activity. Ethanol extract of S. fruti- cosa collected in Jordan showed lower IC50 values (17.43-38.91 μg/ ml) against breast cancer cell lines measured by Sulphorhodamine B assay (ABU-DAHAB et al., 2012) than those in our study. Water extract of Libyan S. lanigera in this study was less active than se- veral extracts of Egyptian S. lanigera (IC50 values from 9.83 to over 100 μg/ml), especially obtained by acetone (SHAHEEN et al., 2011). JANICSÁK et al. (2007) reported on cytotoxic activity of Bulgarian S. ringens extract and its isolated components. The literature data on cytotoxic activity of S. ringens, S. jurisicii and S. amplexicaulis were not available. Previous studies have demonstrated that South African and Jordanian Salvia species showed IC50 values from ap- proximately 20 to above 100 μg/ml (KAMATOU et al., 2005) and from 90 to 400 μg/ml (FIORE et al., 2006), respectively. Our findings are in accordance with previous reports. Correlation between cytotoxic and antioxidant activities and to- tal phenolic and flavonoid content Pearson’s correlation coefficients were calculated between total phe- nolics and flavonoid content of the Salvia water extracts and their antioxidant and cytotoxic activities (Tab. 4). In this study, interpre- tation of correlation coefficients according to TAYLOR (1990) was chosen. On the contrary to ABTS and FRAP activity, cytotoxic and DPPH activity of extracts were negatively correlated to phenolic and positively to flavonoid content because of presenting of results as IC50 values (Tab. 4). Correlation between total phenolic and flavo- noid contents was negative (data not presented). It was previously reported that water soluble flavonoids (mostly anthocyanins) have no antimicrobial significance and water soluble phenolics were only important as antioxidant compounds (TIWARI et al., 2010). However, obtained results indicated that biological activities of extracts were negatively correlated to flavonoid content in extracts. Previously, BEN FARHAT et al. (2014) reported on negative correlation of some flavonoids from Salvia officinalis extracts and antioxidant activity evaluated by DPPH, ABTS and FRAP assays. Our findings are in agreement with various studies which showed that antioxidant acti- vity of rosemary and sage, both belonging to the family Lamiaceae, is mostly manifested by presence of the phenolic acids (rosmarinic and carnosic acids and their derivates) and then terpenoids, flavo- noids and other phenolic acids (LU and FOO, 2001; KAMATOU et al., 2010; ORHAN et al., 2012). Tab. 4: Linear correlation coefficients (r) of cytotoxic and antioxidant ac- tivities versus total phenolic and flavonoid content of Salvia water extracts Total phenolic content Flavonoid content Cytotoxic activity -0,9514c 0,4886b DPPH activity -0,7338c 0,3248a ABTS activity 0,8394c -0,6972c FRAP activity 0,5512b -0,3169a 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 Fig. 1: Cytotoxic activity of water extracts of tested Salvia species tested against K562 cell line Conclusions Based on these findings, it can be concluded that some of the exa- mined extracts could be taken into consideration as possible anti- DPPH activity of extracts was assessed as good (IC50<30 μg/ml) for S. amplexicaulis and S. ringens, as moderate 3080 μg/ml) for S. jurisicii and S. lanigera according to KAMATOU et al. (2010). S. amplexicaulis water extract showed the best DPPH radical scavenging activity (14.21 μg/ml), higher than activity of the synthetic antioxidant BHT (17.94 μg/ml) and commercially used green tea (20.62 μg/ml) (STANKOVIĆ et al., 2010) and very close to BHA (13.37 μg/ml). Be- sides, water extract of S. amplexicaulis performed stronger activity than ethanol and methanol extracts (28.74 and 21.28 μg/ml, respec- tively) as previously reported by ALIMPIĆ et al. (2014). S. fruticosa water extract showed lower DPPH activity than those measured by STAGOS et al. (2012) for methanolic and water S. fruticosa extracts, 22 and 16 μg/ml, respectively. JANICSÁK et al. (2010) previously found low antioxidant activity of S. jurisicii aqueous-methanol ex- tract (191.2 μg/ml), as confirmed in this study. In ABTS assay, S. amplexicaulis and S. ringens showed the stron- gest activity (> 2 mg AAE/g), while other extracts were quite weaker than aforementioned (Tab. 3). Some researchers preferred to express ABTS activity by IC50 value (KAMATOU et al., 2010; STAGOS et al., 2012), and more frequently using standard equivalents (Trolox, as- corbic acid, etc.). In this study, the ascorbic acid calibration curve was chosen for presentation of obtained results. LI et al. (2008) mea- sured wide range of ABTS activity of selected Chinese medicinal plants (0.97-265.43 μmol Trolox/g dry plant material). S. fruticosa showed activity of 13 μg/ml for methanolic and 29 μg/ml for water extract (STAGOS et al., 2012). S. amplexicaulis followed by S. fruticosa water extract showed FRAP activity over 1000 μmol Fe(II)/g. In contrast, S. lanigera wa- ter extract performed FRAP activity lower than 100 μmol Fe(II)/g (Tab. 3). BENZIE and STRAIN (1996) suggested expressing of re- sults using Fe(II) calibration curve. Some researches preferred to present results by IC50 values or as Trolox equivalents. The lacking of a unique form of presenting results makes comparison of results obtained in various studies rather complicated. LI et al. (2008) mea- sured FRAP ability of methanol extracts of 45 Chinese medicinal plants, and results ranged as 1.23-453 μmol Fe(II)/g. Ethyl-acetate extracts of selected Turkish Salvia species exhibited quite low FRAP activity, whereas methanol extracts were highly active (ORHAN et al., 2012). Cytotoxic activity of extracts Cytotoxic activity of extracts was tested using MTT assay against K562 cell line over 48 h. Obtained results, expressed as IC50, are presented in Fig. 1. All tested water extracts exhibited cytotoxic ef- fect on K562 cells. Among examined Salvia species, S. amplexicau- lis and S. ringens showed the strongest antioxidant activity (IC50 < Antioxidative and cytotoxic effects Salvia water extracts 119 oxidant and cytotoxic agents. The results showed that certain species have optimal ratio of the yield, total phenolic and flavonoid content and performed antioxidant and cytotoxic activities, such as Mace- donian S. amplexicaulis, S. ringens and Libyan S. fruticosa. Taking into account non-toxicity of the water as extraction solvent, their application in prevention and treatment of some free radical caused disorders such as cancer, cardiovascular and neurodegenerative di- seases could be proposed. Future studies will provide data on quali- tative composition of phenolics and other possible biologically ac- tive components of the water extracts (research in progress). Acknowledgments Authors are grateful to the Ministry of Education, Science and Tech- nological Development of Serbia for financial support (Project No. 173029 and 41004). 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Address of corresponding author: University of Belgrade, Faculty of Biology, Institute of Botany and Botanical Garden “Jevremovac”, Takovska 43, 11000 Belgrade, Serbia E-mail: alimpic.ana@bio.bg.ac.rs © The Author(s) 2015. This is an Open Access article distributed under the terms of the Creative Commons Attribution Share-Alike License (http://creative- commons.org/licenses/by-sa/4.0/).