Essential Oil Compositions, Antioxidant and Antibacterial Activities of Two Salvia Species ( S. grossheimii Bioss. and S. syriaca L.) Growing in Iran

This article was downloaded by: [Linköping University Library] On: 24 August 2014, At: 16:03 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK Journal of Essential Oil Bearing Plants Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/teop20 Essential Oil Compositions, Antioxidant and Antibacterial Activities of Two Salvia Species (S. grossheimii Bioss. and S. syriaca L.) Growing in Iran Roya Karamiana, Mostafa Asadbegya & Ramtin Pakazada a Department of Biology, Faculty of Science, Bu-Ali Sina University, P. O. Box 65175/4161, Hamedan, Iran Published online: 09 Jun 2014. To cite this article: Roya Karamian, Mostafa Asadbegy & Ramtin Pakazad (2014) Essential Oil Compositions, Antioxidant and Antibacterial Activities of Two Salvia Species (S. grossheimii Bioss. and S. syriaca L.) Growing in Iran, Journal of Essential Oil Bearing Plants, 17:2, 331-345, DOI: 10.1080/0972060X.2014.895156 To link to this article: http://dx.doi.org/10.1080/0972060X.2014.895156 PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. 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Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions http://www.tandfonline.com/loi/teop20 http://www.tandfonline.com/action/showCitFormats?doi=10.1080/0972060X.2014.895156 http://dx.doi.org/10.1080/0972060X.2014.895156 http://www.tandfonline.com/page/terms-and-conditions http://www.tandfonline.com/page/terms-and-conditions Essential Oil Compositions, Antioxidant and Antibacterial Activities of Two Salvia Species (S. grossheimii Bioss. and S. syriaca L.) Growing in Iran Roya Karamian*, Mostafa Asadbegy and Ramtin Pakazad Department of Biology, Faculty of Science, Bu-Ali Sina University, P. O. Box 65175/4161, Hamedan, Iran Abstract: Free radicals and microorganisms are reason of a number of diseases. Plant secondary metabolites may have important role in the prevention of these diseases. Lamiaceae family is potential sources of secondary metabolites that may function as natural antioxidants and antibacterial. The aim of this study is in vitro assessment of total phenol and flavonoid contents and antioxidant activities of the methanolic extracts of two Salvia species, namely S. syriaca and S. grossheimii. In addition, aerial parts of the species were hydrodistilled in a Clevenger-type apparatus and analyzed by GC-MS. Finally, methanolic extracts and essential oils of both species were evaluated for their antibacterial activities. Total phenol and flavonoid contents were measured by Folin-Ciocalteu and AlCl3 assays. Antioxidant activity of the extracts was screened by four complementary test systems, namely DPPH free radical-scavenging, metal chelating activity, anion superoxide radicals scavenging and β-carotene/linoleic acid model system. The antibacterial activity was studied by agar disc diffusion method. Both Salvia species studied were not significantly different in total phenol content. Our results indicated that the Salvia metanolic extracts have effective antioxidant functions. Totally, 40 compounds were characterized in the essential oil of S. grossheimii with germacrene D (12.4 %), α-pinene (11.6 %) and β- pinene (9.5 %) as dominant components and 22 compounds were identified in the essential oil of S. syriaca, with (+) spathulenol (20.5 %), borneol (17.9 %), bicyclogermacrene (11.1 %) and germacrene D (10.7 %) as the major constituents. Both species showed antibacterial activity against the most of bacterial strains tested. Both of Salvia species showed strong antioxidant and antibacterial activities. Then, they may be new health- care foods and drug supplements for special use in the future. Key words: Salvia, phenol, flavonoid, GC-MS, antioxidant activity, antibacterial activity. Introduction The genus Salvia L. with about 900 species belongs to Lamiaceae family. It is wildly distributed throughout the world; however the main centers of its diversity are Mediterranean regions, Central Asia, America and South Africa 1. The genus has 58 annual and perennial species in Iran, 17 of which are endemic 2. Salvia species contain various secondary metabolites such as essential oils 4-6, sterols, flavonoids, sesqui- terpenoids, sesterpenoids 7, diterpenoids 4,7,8, triterpenoids 4,5,7, and flavonoids 8. Free radicals play a crucial role in the develop- ment of tissue damage in various human diseases such as cancer, aging, neurodegenerative disease, malaria and arteriosclerosis, and pathological events in living organisms 9. Antioxidants may have an important role in the prevention of these diseases 10-13. Infections due to bacterial species also remain a serious therapeutic problem. Emerging resistance of these species is seriously decreasing the number of effective antibiotics. Plant extracts and essential oils can be used as potentially useful sources of antimicrobial and ISSN Print: 0972-060X ISSN Online: 0976-5026 *Corresponding author (Roya karamian) E-mail: < [email protected] > © 2014, Har Krishan Bhalla & Sons TEOP 17 (2) 2014 pp 331 - 345 331 Received 23 May 2013; accepted in revised form 17 September 2013 D ow nl oa de d by [ L in kö pi ng U ni ve rs ity L ib ra ry ] at 1 6: 03 2 4 A ug us t 2 01 4 antioxidant compounds. Literature search did not reveal any references to previous spacious work on the antioxidant and antibacterial activities of these two species. However, results from a previous study on the essential oils of S. grossheimii and S. syriaca collected from different localities in Iran showed that germacrene D (45.4 %), β-caryophyllene (22.4 %) and bicyclogermacrene (7.1 %) in the former and germacrene B (34.8 %) and germacrene D (29.2 %) in the later were the major constituents 14,15. The aim of this study is in vitro assessment of total phenol and flavonoid contents and their antioxidant activities of two Salvia species, namely S. syriaca and S. grossheimii. Essential oil compositions of the two species were also determined by GC-MS method. In addition, methanolic extracts and essential oils of both species were evaluated for their antibacterial activities. Materials and methods Chemicals reagents All of the chemicals used in this work were purchased from Merck (Germany), with exception of DPPH (2, 2'-diphenyl-1- picrylhydrazyl), which was purchased from Sigma (USA). Plant material Aerial parts of S. syriaca were collected randomly from Ardabil Province (Susahab, 1400 m a.s.l., 4 March 2010, Ranjbar 19447) and those of S. grossheimii from Qazvin Province (Ziyaran, 1200 m a.s.l., 25 Feb 2010, Ranjbar 9452), NW and W Iran, respectively. The taxonomic identification of plant species were confirmed by Dr. Ranjbar and voucher specimens were kept in the Bu-Ali Sina University herbarium, Hamedan, Iran. The dried aerial parts were powdered and then used for extraction. Preparation of the extracts Briefly, 25 g of powdered aerial parts were continuously extracted in absolute methanol for 12 h using a Soxhlet apparatus. The extracts were concentrated to dryness using a rotary evaporator (Lab Tech, Ev 311, Italy). Isolation of the essential oil A portion (100 g) of dried and ground aerial parts of S. grossheimii and S. syriaca were submit- ted to water-distillation for 3 h using a Clevenger- type apparatus (British pharmacopoeia type). Determination of total phenol content Total phenol content was determined by Folin- Ciocalteu reagent 16. A diluted extract of each species (0.5 mL of 1:10 g/mL) or gallic acid (standard phenolic compound) was mixed with Folin Ciocalteu reagent (5 mL, 1:10 diluted with distilled water) and aqueous Na2CO3 (4 mL, 1 M). The mixtures were allowed to stand for 15 min and the total phenols were determined by spectrophotometrically at 765 nm. The standard curve (y = 0.002 x + 0.0177, R2 = 0.978) was prepared using 0, 50, 100, 150, 200 and 250 mg/ L solutions of gallic acid in methanol: water (50:50, v/v). Total phenol values are expressed in terms of gallic acid equivalent (mg/g of DW), which is a common reference compound. Determination of total flavonoids Aluminum chloride colorimetric method was used for flavonoid determination 17. Each plant extract (0.5 mL of 1:10 g/mL) in methanol were separately mixed with 1.5 mL of methanol, 0.1 mL of 10 % aluminum chloride, 0.1 mL of 1 M potassium acetate and 2.8 mL of distilled water. It remained at room temperature for 30 min; the absorbance of the reaction mixture was measured at 415 nm by a double beam Perkin Elmer UV/ visible spectrophotometer (USA). The calibration curve (y = 0.0091 x + 0.0206, R2 = 0.995) was prepared by quercetin solution at different concentrations from 12.5 to 100 g/mL in methanol. Antioxidant activity DPPH radical scavenging assay In order to determine radical scavenging ability of the extracts, the method reported by Mensor et al., 18 was used. Briefly, 0.3 mM alcohol solution of DPPH (1 mL) was added to samples (2.5 mL) containing different extracts. The samples were first kept in a dark place at room temperature and their absorbance was read at 517 nm after 30 min. The percentage of DPPH radical Roya Karamian et al., / TEOP 17 (2) 2014 331 - 345 332 D ow nl oa de d by [ L in kö pi ng U ni ve rs ity L ib ra ry ] at 1 6: 03 2 4 A ug us t 2 01 4 scavenging ability was measured with the help of the following formula, % DPPH radical scavenging =1 – (As – Ab)/ Ac×100 Blank samples contained 1 mL methanol and 2.5 mL from various concentrations of extract; control sample containing 1 mL of 0.3 mM DPPH and 2.5 mL methanol. The optic density of the samples, the control and the empty samples were measured in comparison with methanol. The discoloration was plotted against the sample concentration in order to calculate the IC50 value, which is the amount of sample necessary to decrease the absorbance of DPPH by 50 %. Fe2+-Ferrozine test system for iron chelating The chelating activity for ferrous ions was measured according to the method of Dinis et al., 19. The reaction mixture contained 0.5 mL of various concentrations of test compounds, 1.6 mL of deionized water and 0.05 mL of 2 mM of FeCl2+ solution. After 30 s, 0.1 mL of 5 mM ferrozine solution was added. Fe2+-Ferrozine magenta complex was very soluble and stable in water. After 10 min at room temperature, the absorbance at 562 nm was measured. The percentage ferrozine-Fe2+ complex formation ability was measured with the help of the following formula, % Metal chelating activity = (A control – A sample)/ A control × 100. Ascorbic acid was used as positive control. βββββ-Carotene/linoleic acid assay The antioxidant activity of the extracts was evaluated using β-carotene/linoleic acid system according to the modified literature procedure 20,21. In short, 1 mL of β-carotene solution in chloroform (0.2 mg/mL) was pipetted into a round-bottom flask. To the solution, 20 mg of linoleic acid and 200 mg of Tween 40 were added. After removing chloroform in a rotary evaporator at room temperature, 50 mL of aerated distilled water was added to the oily residue with vigorous stirring. Aliquots (5 mL) of thus obtained emulsion were transferred to a series of tubes containing 2 mg of extract or 0.5 mg of BHT (positive control) dissolved in 1 mL of 0.4 % (w/ v) Tween 40 solution. A tube with 1 mL of 0.4 % Tween 40 solution to which no antioxidant was added, served as water control (negative control). Solution of 2 mg of an extract in 6 mL of Tween 40 solution served as blank for the corresponding extract. After addition of the emulsion to the tubes, they were placed in a water bath at 50°C for 2 h. During that period, the absorbance of each sample was measured at 470 nm at 15 min intervals, starting immediately after sample preparation (t = 0 min) until the end of the experiment (t = 120 min). The antioxidant activity was calculated from the absolute changes in absorbance at t = 60 min and t = 120 min (AA-60 and AA-120, respectively) 21. The results were normalized using both controls: the water control and the positive control. The first should offer no protection against oxidation of β-carotene in emulsion, while the other should offer maximum protection over the time course of the assay. The percentage of inhibition β-carotene bleaching activity was measured with the help of the following formula, AA%= 1– [(A E t-=-0 – AE t-=-t)/ (Aw t-=-0 – Aw t-=-t) + (ABHT t-=-0 – ABHT t-=-t)] × 100. Where AE t=0 is the absorbance of the extract at t = 0 min, AE t=t is the absorbance of the extract at t = 60 or t = 120 min, AW t=0 is the absorbance of the water control at t = 0 min, AW t=t is the absorbance of the water control at t = 60 min or t = 120 min, ABHT t=0 is the absorbance of BHT at t = 0 min and ABHT t=t is the absorbance of BHT sample at t = 60 or t = 120 min. Anion superoxide scavenging activity Measurement of superoxide anion scavenging activity of the extracts was performed with NBT/ NADH/PMS system based on the Nishimiki et al., 22, method. About 1 mL of NBT (Nitroblue tetrazolium) solution (156 μM in 100 mM phosphate buffer, pH 7.4), 1 mL NADH solution (468 ìM in 100 mM phosphate buffer, pH 7.4) and 0.1 mL of sample solution (0.4, 0.6, 0.8 and Roya Karamian et al., / TEOP 17 (2) 2014 331 - 345 333 D ow nl oa de d by [ L in kö pi ng U ni ve rs ity L ib ra ry ] at 1 6: 03 2 4 A ug us t 2 01 4 1 mg/mL) in methanol were mixed. The reaction started by adding 100 μL of phenazine metho- sulphate solution (60 μM in 100 mM phosphate buffer, pH 7.4) to the mixture and BHT was used as a standard. The reaction mixture was incubated at 25°C for 5 min, and the absorbance at 560 nm was measured against blank samples. Decreased absorbance of the reaction mixture indicated increased superoxide anion scavenging activity. The percentage of superoxide anion scavenging activity was measured with the help of the following formula, % Anion superoxide scavenging activity = [(Ablank – Asample) /Ablank] × 100. Where Ablank is the absorbance of the blank in absence of sample, and Asample is the absorbance in the presence of the sample. The IC50 value for each sample defined as the concentration of the test sample leading to 50 % reduction of the initial anion superoxide radical concentration. Quantitative and qualitative analyses of essential oils The composition of the volatile constituents was established by GC-MS analysis, using a Agilent 6890N Network GC System, and Mass Selective Detector S973 Network fitted with a capillary column (30 m× 250 μm (i.d.), film thick- ness: 0.25 μm). Detector and injector tempera- tures were set at 275°C. The temperature program for the column was 50°C (1 min) to 100°C at a rate of 5°C/min and then holding at 100°C for 3 min. Then the temperature was going to 240°C at a rate of 10°C/min and then holding at 240°C for 10 min. Then the temperature was going to 280°C at a rate of 30°C/min and then holding at 280°C for 10 min. Helium was used as a carrier gas at a flow rate of 1 mL/min (split 1:20 mL/ min) and the injection volume of each sample was 1 μL. The ionization energy was set at 70 ev. Qualitative analysis was based on the comparison of retention times and retention indices and the computer mass spectra library using Wiley GC-MS Library. The percentages of compounds were computed from the GC peak areas. Determination of antibacterial activity Bacterial strains Methanolic extracts and essential oils of the studied Salvia species were tested against a panel of microorganisms including Escherichia coli (Wild), Bacillus cereus (PTCC 1247), Proteus vulgaris (PTCC 1079), Staphylococcus aureus (Wild), Bacillus megaterium (PTCC 1017) and Serratia marcescens (PTCC 1111). Stock cultures were maintained on nutrient agar media at 40°C, then subcultured in nutrient Broth media at 37°C, prior to each antibacterial test. Disc diffusion assay Methanolic extracts were dissolved in DMSO to a final concentration of 100 mg/mL and sterilized by filtration by 0.45 μm millipore filter. Antibacterial activities of the extracts and essential oils were examined by disc diffusion method 23, using 10 mL of suspension containing 1.5×108 of bacteria in mL and spread on nutrient agar media. Negative control was prepared by using DMSO. Gentamicin, penicillin, neomycin and nitrofurantion were used as positive reference standards. Statistical analysis Analysis of variance was performed by Excel and SPSS procedures. Statistical analysis was performed using Student’s t-test, and p value < 0.05 was regarded as significant. Data obtained from antibacterial and antioxidant assays are the average of triplicate analyses and recorded as means ± standard deviation. Results and discussion Total phenol and flavonoid contents The extract yields (w/w) were 21.38 % for S. grossheimii and 22.5 % for S. syriaca. Results showed that the Salvia species are not significantly different in total phenol content. However, S. grossheimii possessed a higher content of total flavonoids (4.62 ± 0.35 mg QES/ g dw) than that of S. syriaca (Table 1). This acti- vity is believed to be mainly due to its redox pro- perty, which plays an important role in adsorbing and neutralizing free radicals, quenching singlet and triplet oxygens, or decomposing peroxides Roya Karamian et al., / TEOP 17 (2) 2014 331 - 345 334 D ow nl oa de d by [ L in kö pi ng U ni ve rs ity L ib ra ry ] at 1 6: 03 2 4 A ug us t 2 01 4 24,25. Salvia species have been investigated parti- cularly as a source of natural antioxidants 26-30. Results from previous studies have shown that some flavonoid components such as quercetin have anticancer activities and are able to inhibit cancer cell growth 31,32. Gallic acid was reported as a free radical scavenger and as an inducer of differentiation and apoptosis in leukemia, lung cancer 33. It seems that flavonoids are one of important constituents in the extracts of Salvia species show antioxidant activity and a part of pharmacological effects can be attributed to the presence of these compounds. However, further works are required to establish if quercetin or other flavonoids have any roles in prevention of the cancerous growth and development phenolics have attracted increasing attention for their antioxidant behavior and beneficial health- promoting effects. It is assumed that many antioxidative phenolic compounds in plants are usually presented in a covalently-bound form 24. DPPH free radical scavenging Antioxidant properties, especially radical scavenging activities, are very important due to the deleterious role of free radicals in foods and biological systems 34. Literature search did not reveal any references to previous works on the DPPH radical scavenging activity of the studied species. Both species exhibit very good DPPH radical scavenging activity. Assessment of antioxidant activities of the extracts of both species showed the excellent DPPH radical scavenging activity in comparison with ascorbic acid (82.61 %), (Fig 1). The higher antioxidant activity is reflected in a lower IC50. The values of IC50 were in the order: Salvia species studied < ascorbic acid as synthetic antioxidant. However, there were no significant differences between IC50 of the methanolic extracts of two species (Table 1). The effectiveness of antioxidants as DPPH radical scavengers ranged in the following descending order: S. syriaca (IC50 = 0.11 mg/mL) = S. grossheimii (IC50 = 0.11 mg/mL) > ascorbic acid (IC50 = 0.13 mg/mL). Fe2+-Ferrozine test system for iron chelating There are no previous reports on metal Ta bl e 1. T ot al p he no l a nd fl av on oi d co nt en ts a nd a nt io xi da nt a ct iv ity o f t he s tu di ed S al vi a ex tr ac ts a nd p os iti ve c on tr ol s IC 50 v al ue (m g/ m l) To ta l p he no ls To ta l f la vo no id s ββββ β- C ar ot en e/ lin ol ei c D PP H a ss ay A ni on s up er ox id e M et al (m g/ g D w ) (m g/ g D w ) a ci d as sa y (% ) a ss ay ch el at in g (% ) S. s yr ia ca 5. 89 ±0 .2 6a 2. 15 ±0 .2 8a 60 .0 0± 2. 6a 0. 11 ±0 .0 0a 0. 39 ±0 .0 0a 26 .0 5± 2. 2a S. g ro ss he im ii 5. 29 ±0 .2 1a 4. 62 ±0 .3 5b 55 .1 8± 1. 8b 0. 11 ±0 .0 0a 0. 24 ±0 .0 1b 36 .1 9± 2. 1b A sc or bi c ac id - - - 0. 13 ±0 .0 1b - 21 .8 0± 3. 3a B H T - - - - 0. 43 ±0 .0 1a - Ex pe rim en t w as p er fo rm ed in tr ip lic at e an d ex pr es se d as m ea n ± SD . Va lu es in e ac h co lu m n w ith d iff er en t s up er sc rip ts a re si gn ifi ca nt ly d iff er en t ( P < 0. 05 ) Roya Karamian et al., / TEOP 17 (2) 2014 331 - 345 335 D ow nl oa de d by [ L in kö pi ng U ni ve rs ity L ib ra ry ] at 1 6: 03 2 4 A ug us t 2 01 4 chelating activity of the studied Salvia species. Analysis of metal-chelating properties showed that all the extracts studied were capable of chelating iron and did so in a concentration- dependent manner and also strong than ascorbic acid as synthetic antioxidant (Fig 2). Chelating agent can interfere with the formation of ferrous and ferrozine complexes and thus decrease the red color. There was no significant difference between metal chelating activities of S. syriaca (13.26 %) and ascorbic acid (12.28 %), however S. grossheimii showed more metal activity (22.55 %) than S. syriaca (Table 1, Fig 2). Chelating agents may inhibit radical generations by stabili- zing transition metals consequently reducing free radical damage. In addition, some phenolic compounds exhibit antioxidant activity through the chelating of metal ions 35. In the metal chelating assay, ferrozine can quantitatively form complexes with Fe2+. In the presence of other chelating agents, the complex formation is disrupted with consequent decrease in the intensity of the red color of the complex 24. Fig 1. DPPH radical scavenging abilities of the studied Salvia extracts in different concentrations Fig 2. Metal chelating effects on ferrous ions of the studied Salvia extracts and ascorbic acid in different concentrations Roya Karamian et al., / TEOP 17 (2) 2014 331 - 345 336 D ow nl oa de d by [ L in kö pi ng U ni ve rs ity L ib ra ry ] at 1 6: 03 2 4 A ug us t 2 01 4 βββββ-Carotene/linoleic acid assay Antioxidant activity of Salvia species using β- carotene/linoleic acid assay has been previously studied and reported between 29.0 and 75.8 % 36- 38. Literature search did not reveal any references to previous works on the β-carotene/linoleic acid assay on the studied species. Both extracts studied significantly inhibited bleaching of β-carotene in comparison with control. Our results showed that S. grossheimii (60.0 ± 2.6 %) was more active than another species (55.18 ± 1.8 %) (Table1). According to Amarowicz et al. 21, the normalized antioxidant activities of plant extracts at t = 60 min and t = 120 min of incubation (AA-60 and AA-120) are very good. The change in absorb- ance (In t = 60 and t = 120) of β-carotene/linoleic emulsion in the presence and absence of extracts and BHT are shown in Figure 3. β-carotene undergoes rapid discoloration in the absence of an antioxidant. This is because of the coupled oxidation of β-carotene and linoleic acid, which generates free radicals. The linoleic acid free radical formed upon the abstraction of a hydrogen atom from one of its diallylic methylene groups attacks the highly unsaturated β-carotene molecules. As a result, β-carotene will be oxidized and broken down in part. Subsequently, the system looses its chromophore and character- istic orange color, which can be monitored spectrophometrically. Anion superoxide scavenging activity Formation and accumulation of ROS is believed to be one of the mechanisms of myocardial damage by ischemia/reperfusion. During reperfusion, xanthine oxidase converts oxygen into superoxide anion, which in turns dismutase into H2O2, and generates hydroxyl radicals through the Fenton reaction, resulting in cell damages. Electron spin resonance (ESR) study showed that ROS, mainly the superoxide anion and semiquinone radicals, and partly H2O2, were involved in myocardial reperfusion. When treated with SM, ESR signal of the superoxide anion was disappeared 39. A literature search did not reveal any reference to previous work on the superoxide scavenging activity of S. grossheimii and S. syriaca. In the present study, we investi- gated the scavenging or preventive capacities of two Salvia species against superoxide anion free radicals for the first time. The scavenging effects of the crude methanolic extracts increased with increasing concentration (Fig 4). In addition, superoxide anion scavenging abilities of both species were more than that of BHT (55.19 %), and S. grossheimii (84.95 %) was found to be more active than S. syriaca (64.86 %) (Table 1, Fig 4). The higher antioxidant activity is reflected in a lower IC50. The values of IC50 were in the order: Both Salvia species studied < BHT as synthetic antioxidant (Table 1). The effectiveness Fig 3. Inhibition of bleaching of β-carotene/linoleic acid emulsion by the studied Salvia extracts and BHT over 120 min. Roya Karamian et al., / TEOP 17 (2) 2014 331 - 345 337 D ow nl oa de d by [ L in kö pi ng U ni ve rs ity L ib ra ry ] at 1 6: 03 2 4 A ug us t 2 01 4 Fig 4. Superoxide anion radical-scavenging activities of the studied Salvia extracts and BHT in different concentrations of antioxidants as anion superoxide free radical scavenging ranged in the following descending order: S. grossheimii (IC50 = 0.24 mg/mL) > S. syriaca (IC50 = 0.39 mg/mL) > BHT (IC50 = 0.43 mg/mL). In total according to Kikuzaki and Nakatani 40, methanolic extracts may include phenolic and hydrox-phenolic compounds with acid, alcohol, sugar or glycoside and part of the antioxidative activity may be due to these com- ponents or flavonoids. GC-MS analysis of essential oils The components identified are given in Table 2 with retention times and indices and also their relative percentages. The essential oil (v/w) yields were 1.0 % for S. grossheimii and 0.7 % for S. syriaca. In total, 40 compounds were charact- erized in the oil of S. grossheimii, making up 92.4 % of the oil and 22 compounds identified in the oil of S. syriaca that representing 100 % of the oil. Sesquiterpenes were the main class of essential oil of both species studied in this research. The oil of S. grossheimii collected from Ardebil Province, NW Iran was characterized by germacrene D (12.4 %) α-pinene (11.6 %) and β-pinene (9.5 %) as major constituents. Our results are in agreement with Mirza and Baher Nik 14, work that reported a high content of sesquiterpenes such as germacrene D (45.4 %), β-caryophyllene (22.4 %) and bicyclogermacrene (7.1 %), which composed 88.8 % of the oil of S. grossheimii collected from Azerbaijan Province, W Iran. In addition, the main components des- cribed in the oil of S. syriaca were (+) spathulenol (20.5 %), borneol (17.9 %), bicyclogermacrene (11.1 %) and germacrene D (10.7 %). In another study on S. syriaca, germacrene B (34.8 %) and germacrene D (29.2 %) were reported as the major constituents of the oil 15. These variations in the essential oil composition might have arisen from climatic, seasonal, geographical or geological factors. In addition, studies on other Salvia species in Iran reported that caryophyllene was the major component in the oils of S. nemorosa, S. virgata, S. atropatana, and S. multicaulis 15,41-43, while (E)-ocimene, gurjunene and germacrene D were the major constituents in S. reuterana 42,44, (E)-ocimene, caryophyllene and isopentyl isovalerate in S. spinosa 45, and germacrene D, linalyl acetate and linalool in S. limbata 46. It is possible that the chemical differentiation of essential oils for Salvia species is correlated to the existence of many chemotypes, provoked either by different climatic factors, or as a result of pollination caused by genetic differences (intraspecific or intrapopulation crosspollination). The considerable differences among Salvia species may depend on the extrac- tion procedure, the season, the stage of development and the distinct habitat in which the Roya Karamian et al., / TEOP 17 (2) 2014 331 - 345 338 D ow nl oa de d by [ L in kö pi ng U ni ve rs ity L ib ra ry ] at 1 6: 03 2 4 A ug us t 2 01 4 Table 2. Chemical composition of S. grossheimii and S. syriaca essential oils (%) No Compound S. grossheimii S. syriaca RT* RI* Content (%)* Content (%)* 1 α-Thujene 6.60 927 0.4 - 2 α-Pinene 6.82 935 11.6 1.6 3 Camphene 7.15 947 1.3 0.4 4 Sabinene 7.85 963 5.2 0.3 5 β-Pinene 7.94 973 9.5 - 6 α-Terpinene 8.99 1015 0.5 - 7 p-Cimene 9.23 1025 1.4 - 8 1,8-Cineole 9.42 1035 5.0 - 9 γ-Terpinene 10.19 1068 1.9 - 10 α-Terpinolene 11.04 1078 1.7 - 11 trans-Pinocarveol 12.70 1137 0.5 - 12 trans-(+)-Carveol 12.91 1151 1.5 - 13 Borneoll 13.77 1164 2.2 17.9 14 α-Terpinol 14.57 1188 0.6 - 15 Hexyl isovalerate 16.33 1236 - 1.3 16 Bicycloelemene 18.57 1327 0.5 - 17 ND 18.79 1336 0.6 - 18 α-Cubebene 18.81 1348 - 1.5 19 α-Copaene 19.32 1375 1.5 5.6 20 β-Bourbonene 19.50 1386 0.5 - 21 β-Gurjurene 19.59 1395 0.9 - 22 β-Elemene 19.61 1405 - 3.6 23 α-Gurjunene 19.94 1407 0.5 - 24 trans-Caryophyllene 20.12 1410 - 2.5 25 (+)-Epi-bicyclosesquiphellandrene 20.27 1434 1.5 0.5 26 Aromadendrene 20.43 1448 0.7 - 27 β-Cedrene 20.66 1452 1.7 - 28 α-Humulene 20.67 1470 - 1.6 29 γ-Cadinene 21.05 1475 0.7 0.6 30 Germacrene D 21.14 1489 12.4 10.7 31 Bicyclogermacrene 21.35 1518 5.0 11.1 32 Torreyol 21.67 1521 - 4.8 33 δ-Cadinene 21.69 1523 1.7 - 34 Exo-1,5-Epoxysalvial-4(14)-ene 22.35 1548 0.5 - 35 Palustrol 22.36 1557 - 1.6 36 (+) Spathulenol 22.51 1571 5.9 20.5 37 Caryophyllene oxide 22.58 1575 0.9 4.3 38 Ledol 22.83 1581 - 2.1 39 (-) Spathulenol 22.92 1584 - 0.6 40 Isospathulenol 23.26 1636 - 1.8 41 Selina-6-en-4-ol 23.95 1647 - 5.1 42 ND 24.53 1664 5.1 - 43 Fenthion O-analog 26.13 1812 6.0 - Roya Karamian et al., / TEOP 17 (2) 2014 331 - 345 339 D ow nl oa de d by [ L in kö pi ng U ni ve rs ity L ib ra ry ] at 1 6: 03 2 4 A ug us t 2 01 4 table 2.(continued). No Compound S. grossheimii S. syriaca RT* RI* Content (%)* Content (%)* 44 (12S,13R)-8,13-epoxy-14-labden-12-ol 28.08 2050 0.4 - 45 n-Tetracosane 31.58 2400 1.5 - 46 n-Pentacosane (CAS) 33.44 2500 1.3 - 47 n-Hexacosane 35.81 2600 1.7 - 48 n-Heptacosane 38.62 2700 1.5 - 49 n-Octacosane 39.99 2800 0.7 - 50 n-Nonacosane (CAS) 41.33 2900 1.6 - 51 n-Triacontane 42.83 3000 1.4 - RI is the retention index relative to C8-C32 n-alkanes on the HP-5ms column RT: retention time ND: not determined Content: Values are the percent of constituents in total oils plant has been collected. It can be concluded that the composition of oils varies greatly with respect to the geographical proximity (different species collected in the same region have similar compo- sitions), mainly for the proportion of aliphatic compounds and terpenoids. All of these differences suggest further investigations on other Salvia species may further confirm their biodiversity. Antibacterial activity The potential antibacterial effects of the methanolic extracts were investigated against 3 Gram-positive bacteria, namely Bacillus cereus, Staphylococcus aureus and Bacillus megaterium and 3 Gram negative bacteria, namely Escherichia coli, Proteus vulgaris, and Serratia marcescens and also of the essential oils against 2 Gram-positive and negative bacteria, namely Proteus vulgaris and Bacillus cereus. As it is well known, S. aureus, E. coli and Bacillus species, especially B. cereus, are agents of food poisoning. The most interesting area of application for plant extracts and oils is the inhibition of growth and reduction in numbers of the more serious food- borne pathogens 47. Our results showed that antibacterial activities of the essential oil and methanolic extract of S. syriaca were much higher than those of S. grossheimii. S. syriaca extract showed a strong antibacterial activity against Proteus vulgaris at concentration of 100 mg/mL (Table 3). Generally, the methanolic extracts of both species represented more antimicrobial activities than the essential oils against bacteria tested. The essential oils showed more anti- bacterial activities against the Gram-positive bacteria; while the extracts were more active against Gram-negative ones. In general, both extracts and essential oils were more active than those of some reference discs. Although all bacteria strains tested are resistant to penicillin and E. coli to gentamicin (Table 5), but the antibacterial effects of extracts and essential oils were higher than that of penicillin (Tables 3-5). Our results indicated that the studied species may be used in the treatment of diseases caused by tested microorganisms tested. This is due to the fact that medicinal plants are of natural origin, which means more safety for consumers, and they might present low risk of resistance development by pathogenic microorganisms. Although the present work is the first report on antibacterial activities of the extracts and essential oils of S. grossheimii and S. syriaca from Iran, but other members of the genus have been subjected to antimicrobial activity evaluation 44,48-58. Conclusion The results from present study clearly demonstrated that S. syriaca and S. grossheimii Roya Karamian et al., / TEOP 17 (2) 2014 331 - 345 340 D ow nl oa de d by [ L in kö pi ng U ni ve rs ity L ib ra ry ] at 1 6: 03 2 4 A ug us t 2 01 4 Ta bl e 3. A nt ib ac te ri al a ct iv ity o f S . g ro ss he im ii an d S. s yr ia ca e xt ra ct s us in g di sc d iff us io n m et ho d In hi bi tio n zo ne (m m ) M ic ro or ga ni sm S. g ro ss he im ii S. s yr ia ca C on tr ol (- ) 10 0 50 25 12 .5 10 0 50 25 12 .5 D M SO P. v ul ga ri s (- ) 10 ±0 .2 8a 8± 0. 33 b 7± 0. 26 c N A 17 ±1 .2 0a 11 ±0 .6 8b 10 ± 0. 24 c 8 ±0 .2 1d N A E. c ol i ( -) 10 ±0 .4 4a 8 ±0 .5 7b N A N A 12 ±0 .5 7a 10 ± 0. 24 b 7 ±0 .3 3c N A N A B. c er eu s ( +) 11 ±0 .3 3a 9± 0. 51 b 7± 0. 00 c 7 ±0 .1 6c 11 ±0 .2 2a 9 ±0 .4 4b 7 ±0 .1 6c N A N A S. a ur eu s ( +) 7 ±0 .0 7a N A N A N A 9 ±0 .3 3a 7 ±0 .0 0b N A N A N A B. m eg at er iu m (+ ) N A N A N A N A 7 ±0 .0 0a N A N A N A N A S. m ar ce sc en s ( -) N A N A N A N A N A N A N A N A N A Ex pe ri m en t w as p er fo rm ed in tr ip lic at e an d ex pr es se d as m ea n ± SD . V al ue s i n ea ch c ol um n w ith d if fe re nt su pe rs cr ip ts a re si gn if ic an tly d if fe re nt (P < 0 .0 5) ; N A : n o ac tiv e. Roya Karamian et al., / TEOP 17 (2) 2014 331 - 345 341 D ow nl oa de d by [ L in kö pi ng U ni ve rs ity L ib ra ry ] at 1 6: 03 2 4 A ug us t 2 01 4 Table 4. Antibacterial activities of S. grossheimii and S.syriaca essential oils Inhibition zone (mm) Microorganism S. grossheimii S. syriaca Negative control 10 % 30 % 10 % 30 % Hexane Proteus vulgaris (−) NA NA NA NA NA Bacillus cereus (+) NA 8±0.16 7 ±0.00b 10 ±0.33a NA Experiment was performed in triplicate and expressed as mean ± SD Values in each column with different superscripts are significantly different (P < 0.05) Table 5. Antibacterial activities of four antibiotics Microorganism Inhibition zone (mm) Gentamicin Penicillin Nitrofurantion Neomycin Proteus vulgaris (−) 30±0.66 NA 15±0.33 22±0.55 Escherichia coli (−) NA NA 25±0.57 20±0.26 Bacillus cereus (+) 25±0.57 NA 10±0.18 20±0.57 Staphylococcus aureus (+) 35±0.20 NA 30±0.22 25±0.28 Bacillus megaterium (+) 25±0.44 NA 20±0.16 20±0.66 Serratia marcescens (−) 27±0. 26 NA 18±0.44 22±0.33 Experiment was performed in triplicate and expressed as mean ± SD. extracts and oils include important phyto- chemicals that may be potential sources of antibacterial and antioxidant agents and helpful in preventing the progress of various diseases. Also, they can be used as alternative system of medicine. Of course, deleterious effects of the extracts and essential oils must be studied on the human body. Determination of the natural antioxidant and antibacterial compounds of plants will help to develop new drug candidates for antioxidant and antimicrobial therapy. 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