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1 International Research Journal of Applied and Basic Sciences 2013 Available online at ISSN X / Vol, 6 (7): Science Explorer Publications Phytochemical composition of the essential oil, total phenolic content and antioxidant activity in Salvia mirzayanii Rech. & Esfand. grown wild and cultivated in Iran Farshad Sadeghi 1* and Ardalan Alizadeh 2 1. Department of Horticulture, Faculty of Agriculture, Shiraz Branch, Islamic Azad University, Shiraz, Iran. 2. Department of Horticulture (Biotechnology and Molecular Genetic of Horticultural Crops and Medicinal and Aromatic Plants), Estahban Branch, Islamic Azad University, Estahban, Iran. The research is financed by: Shiraz Branch, Islamic Azad University, Shiraz, Iran. Corresponding Author FS1351@yahoo.com ABSTRACT: Salvia mirzayanii is member of Lamiaceae family, which distributes only in south of Iran. Phytochemical composition of essential oil isolated by hydro distillation from the aerial parts of Salvia mirzayanii Rech. & Esfand. grown wild and cultivated in Iran were determined using Gas Chromatography (GC) and Gas Chromatography/Mass Spectrometry (GC/MS). Forty nine components were identified in the essential oil of wild S. mirzayanii. The major components were, 5- neo-cedranol (15.48%), 1,8-cineol (11.23%), α-terpinyl acetate (9.64%), γ-cadinene (6.64%), bicyclogermacrene (6.47%), -cadinene (5.46%), Globulol (5.12%), α -cadinol (4.58%), epi-α-cadinol (4.20%), Linalool (3.35%), 7-epi- α-selinene (3.23%), Linalyl acetate (2.55%) and α -guaiene (2.23%). Seventy two components were identified in the essential oil of cultivated S. mirzayanii. The major components in cultivated S. mirzayanii were, 5-neo-cedranol (19.42%), spathulenol (7%), α- terpinyl acetate (6.70%), Linalool (6.64%), 1,8-cineol (6.28%), 7-epi- α-selinene (5.46%), α -cadinol (3.73%), (Z)-Nerolidol (3.34%), epi-α-cadinol (2.59%), -cadinene (2.58%), Linalyl acetate (2.54%), camphor (2.42%), globulol (2.36%), bicyclogermacrene (2.05%) and carvacrol (2.03%). The total phenolic content and the antioxidant activity of plant extract was determined by Folin-Ciocalteau and the 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging assays respectively. The highest total phenolic content and antioxidant activities were observed in cultivated S. mirzayanii compare wild growing plants. Keywords: Salvia mirzayanii, essential oil, propagation, medicinal plants. INTRODUCTION In recent years, the essential oils and various extracts of medicinal and aromatic plants have been of great interest as they have been the sources of natural products (Evans, 1996); In this regard, the antioxidant properties of plant secondary metabolites have formed the basis of many applications, including raw and processed preservation, pharmaceuticals, alternative medicine and natural therapies (Reynolds, 1996 and Lis- Balchin and Deans, 1997). The genus Salvia is the largest one among the most important genera of the lamiaceae family consisting of about 900 species. Fifty eight annual or perennial species of the genus are found in Iran of which 17 are endemic (Rechinger, 1982). Several species of Salvia are used in folk medicine as antiseptics, astringents and spasmolytics (Newall and Anderson, 1996). Many studies have shown the antioxidant, antimicrobial and antiviral activities of some Salvia species (Kelen and Tepe, 2008; Fiore, et al., 2006; Wu, et al., 2007 and Damjanoviae-Vratnica et al., 2008). Salvia mirzayanii Rech. f. & Esfand. ( Moor-e-Talkh in Persian) is an endemic plant which is only distributed in south of Iran. This is a perennial and bushy aromatic herb (20-40 cm high) with multiple stems covered with white trichomes and blue white colored flowers, flowering occurred in Mars to April. This species grows wild on the limestone slopes and mountainous rock in Hormozgan region, Southern Iran. In the folk medicine, the aerial parts of S. mirzayanii has been used for diarrhea, stomachache, headache, wound, spasmolytic, sugar and high blood cholesterol by natives of southern parts of Iran (Rechinger, 1982 and

2 Zargari, 1990). The information on the essential oil of S. mirzayanii is very scarce. Javidnia et al., (2002). reported that, spathulenol, δ-cadinene, linalool, α-terpinyl acetate, α-cadinol, β-eudesmol, cubenol and linalyl acetate were the main components of S. mirzayanii essential oil. Mirza et al., (2003) reported that, linalool, linalyl acetate, 1,8-cineol, terpinenyl acetate were the main constituents of the S. mirzayanii essential oil. In another study by Yamini et al., (2008) linalyl acetate, 1,8-cineole, linalool and 8-acetoxy linalool were reported to be the major components in S. mirzayanii essential oil. Endemically and limitation of geographical habitat in Iran, excessive medicinal properties, increscent of harvest in natural habitat and reduction of rain fall in previous years, cause reduction of S. mirzayanii population in natural habitat in Iran. This study focuses on feasibility of S. mirzayanii propagation and essential oil composition, phenolic content and antioxidant activity of this plant under different habitats. Knowledge of the factors that influence on yield and essential oil composition in medicinal plants is insufficient, and these factors seems to play an important role in drug yield and phytochemical in medicinal and aromatic plants. This is the first report of propagation in S. mirzayanii. Up to our knowledge, the phytochemical compositions and biological activities of cultivated S. mirzayanii has not been investigated yet. The result of this study can provide useful information in medicinal and aromatic plant propagation and determine the effects of different environmental conditions on secondary metabolites and active substances in medicinal and aromatic plants. Experimental Chemical reagents Chemical reagents DPPH (2,2-diphenyl-1-picrylhydrazyl), gallic acid monohydrate (3,4,5- trihydroxybenzoic acid) and sodium carbonate were purchased from Sigma-Aldrich chemicals (St. Louis, USA). Folin Ciocalteu reagent and methanol were purchased from Merck (Darmstadt, Germany). Quercetin hydrate was purchased from Across organics (New Jersey, USA). Plant Materials Seeds of Iranian S. mirzayanii were obtained from natural habitat in Hormozgan province, in South of Iran. The seeds were grown in medicinal and aromatic plants experimental garden in Estahban branch, Islamic Azad University in soil containing clay, sand and peat moss (1:1:1, v:v:v) under standard conditions. Plants were harvested in flowering stages. Also the aerial parts of S. mirzayanii were obtained from natural habitat in the same location in Hormozgan province at flowering stage on mother plants. Every plant in each habitat was weighed, bulked and placed in paper bag and dried at room temperature for 15 days. Dry plants were stored in a dark and dry place until analysis. Essential oil extraction The dried aerial parts of S. mirzayanii in each habitat were subjected to hydro-distillation for 3 hours using a Clevenger-type apparatus. The essential oil obtained was separated from water and dried over anhydrous sodium sulfate and stored in sealed amber flasks at 4 C till analysis. Gas Chromatography (GC) Gas Chromatography analysis was performed on an Agilent technologist model (7890A) equipped with flame ionization detector and capillary column DB-5 (30 m 0.32 mm, 0.25 μm film thicknesses). The chromatographic conditions were as follows: The oven temperature increased from 60 to 210ºC at a rate of 3ºC/min then 210 to 240 ºC at a rate of 20ºC/min. The injector and detector temperatures were 280 and 290ºC, respectively. N2 used as the carrier gas (1 ml/min). Gas Chromatography-Mass spectrometry (GC-MS) Essential oil was also analyzed by Hewlett- Packard GC-MS (model 6890 series II) operating at 70e V ionization energy. Equipped with a DB-5 capillary column (phenyl methyl siloxane (30 m, 0.25 mm, 0.25 μm film thickness) with He as the carrier gas and a split ratio of 1:50. The retention indices for all the components were determined according to the Van Den Doll method using n-alkanes as standard (Van Den Dool and Kratz, 1963). The compounds were identified by comparison of retention indices (RRI- AP-5) with those reported in the literature and by comparison of their mass spectra with the Wiley and mass finder 3 libraries or with the published mass spectra (Adams, 2001). Extract preparation Grounded air dried plant material (7.5 g) was weighed in a glass and then defatted with light petroleum for 3 h. Each Sample was extracted with 200 ml of 90% aqueous methanol, for 2 days with one change of solvent after 1 day. Supernatants were combined and evaporated to dryness using a rotary evaporator to a volume of about 1 ml. These concentrated extracts were freeze-dried and weighed to determine the yield, and 978

3 kept at -20 C until used. Shortly before each experiment, the lyophilized powder was dissolved in methanol at the desired concentration, and tested for antioxidant activity and total phenolic content. Total phenolic content Total phenolic content in S. mirzayanii extracts was determined by the Folin-Ciocalteau colorimetric method, as described previously (Singleton and Rossi, 1965). Briefly, 200 μl of plant extract dissolved in methanol (1 mg/ml) were mixed with 2.5 ml of Folin-Ciocalteu reagent (diluted 10 times in distilled water) in glass tubes in triplicate. The samples were incubated at room temperature for 5 min and vortex mixed at least 2 times. Then, 2 ml of Na 2 CO 3 (7.5%) was added and the glass tubes were incubated in the dark for 90 min with continuous shaking. The absorbances of samples were measured at 765 nm using a spectrophotometer (LABOMED. INC. UV/VIS double beam PC. UVD. 2980). A methanolic solution of Gallic acid was tested in parallel to obtain a standard curve. The amount of the total phenolic contents were determined as milligrams of Gallic acid equivalent per gram of dry weight (mg GAE/g DW) using the standard curve. Free radical scavenging capacity The 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging effects were evaluated according to the method employed by Brand-Williams et al., Briefly, 4 different concentrations of the plant extract dissolved in methanol were incubated with a methanolic solution of DPPH 100 M in a total volume of 4 ml. After 30 min of incubation at room temperature in the dark, the absorbance at 517 nm was measured using a spectrophotometer (LABOMED. INC. UV/VIS double beam PC.UVD.2980). After 30 min of incubation at room temperature, the absorbance was recorded at 517 nm. Methanol was used as blank and all measurements were carried out in triplicate. Quercetin was used as reference compounds. All solutions were made daily. The percent inhibition of DPPH free radical was calculated by the formula: Percentage inhibition (%I) = [(A blank -A sample ) / A blank] 100 Where, A blank is the absorbance of the control reaction (DPPH alone), and A sample is the absorbance of DPPH solution in the presence of the plant extract. IC 50 values denote the concentration of the sample, required to scavenge 50% of DPPH free radicals. Statistical analysis All experiments were carried out with three replicate. All data were expressed as mean ± standard deviation. Analysis of variance was performed by ANOVA by the software SAS (version 9.2 for windows). Significant differences between means were determined by Duncan's new multiple-range test. A significant difference was considered at the level of P < Correlation analyses of antioxidant activity versus the total phenolic content were carried out using the Microsoft Office Excel program. IC 50 values of the antioxidant activity were calculated by the software Sigma Plot (version 8.0 for Windows). RESULT AND DISCUSSION Essential oil composition The chemical composition of the essential oil of S. mirzayanii harvested in each habitat and the retention indices are presented in Table 1. The essential oil isolated by hydro distillation of the aerial parts of S. mirzayanii harvested in natural habitat was a purple yellow oil, with a yield of 1.75 % (w/w), based on dry weight. In total, forty nine components were identified in the essential oil of wild S. mirzayanii that represented 99.09% of the oils. The major components were, 5-neo-cedranol (15.48%), 1,8-cineol (11.23%), α-terpinyl acetate (9.64%), γ-cadinene (6.64%), bicyclogermacrene (6.47%), -cadinene (5.46%), Globulol (5.12%), α - cadinol (4.58%), epi-α-cadinol (4.20%), Linalool (3.35%), 7-epi- α-selinene (3.23%), Linalyl acetate (2.55%) and α -guaiene (2.23%). Other components were present in amounts less than 2% (Table 1). Seventy two components were identified in the essential oil of cultivated S. mirzayanii that represented 98.17% of the oils. The major components in cultivated S. mirzayanii were, 5-neo-cedranol (19.42%), spathulenol (7%), α-terpinyl acetate (6.70%), Linalool (6.64%), 1,8-cineol (6.28%), 7-epi- α-selinene (5.46%), α -cadinol (3.73%), (Z)- Nerolidol (3.34%), epi-α-cadinol (2.59%), -cadinene (2.58%), Linalyl acetate (2.54%), camphor (2.42%), globulol (2.36%), bicyclogermacrene (2.05%) and carvacrol (2.03%). According to the Table 1, the number of identified components in cultivated S. mirzayanii was higher than wild S. mirzayanii essential oil. The major components in essential oil in wild and cultivated S. mirzayanii were nearly same, although some components as spathulenol, (Z)-Nerolidol, camphor and carvacrol in cultivated S. mirzayanii essential oil and γ-cadinene and α guaiene in wild S. mirzayanii essential oil were not common in both essential oils as major components. In our study the number of major components in S. mirzayanii essential oil was higher than previous reports in this plant. Also the main components observed in wild and cultivated S. mirzayanii essential oils were different to those obtained by Javidnia et al., (2002), Mirza et al., (2003) and Yamini et al., (2008). However, some major components as, linalool, linalyl acetate, α-terpinyl acetate, 1,8-cineole, α-cadinol and δ-cadinene, were 979

4 common between all studies. These differences in the essential oil compositions can be attributed to several environmental factors such as climatic, seasonal and geographical or ontogenesis variations (Alizadeh et al., 2011, 2013b and Sadeghi et al., 2012). Total Phenolic Content and Antioxidant Activity Free radicals may cause many disease conditions such as cancer and coronary heart disease in human (Javanmardi et al., 2003; Löliger, 1991). Phenolic compounds are a class of antioxidant agents which act as free radical scavengers and are responsible for antioxidant activity in medicinal plants (Shahidi and Wanasundara, 1992). The total phenolic content in wild and cultivated S. mirzayanii were measured by Folin Ciocalteau reagent and expressed as gallic acid equivalent (standard curve equation: y = x , R 2 = data not shown). According to the findings presented in the Table 2, the highest total phenolic content was observed in cultivated plants (42.33 mg GAE/g DW) contrasted with wild plants (35.44 mg GAE/g DW) and this difference was significant. These findings indicate that the different growth conditions are significant effect on S. mirzayanii phenolic content. The antioxidant activity of the methanolic extracts of the aerial parts of S. mirzayanii in wild and cultivated conditions were assessed by the DPPH (2, 2-diphenyl-1-picryl hydrazyl) free radical scavenging method. The effect of different growth conditions on radical scavenging capacity of S. mirzayanii is shown in Table 2. The highest antioxidant activity was observed in cultivated plants (34.64 μg/ml), while the lowest value was detected in wild condition (38.44 μg/ml). Also our results show that, S. mirzayanii extract has high antioxidant activity compare quercetin as artificial antioxidant (Table 2). The total phenolic contents of S. mirzayanii showed good correlations with the results of DPPH (Y= x , R 2 = , data not shown). According to the Table 2, S. mirzayanii has the highest phenolic content in cultivated condition and highest antioxidant activity in this time. In fact, with increasing in total phenolic content, IC 50 value in DPPH radical scavenging decreased and antioxidant activity, was increased. These results suggest that the major part of the antioxidant activity in S. mirzayanii results from the phenolic compounds. This is in line with the observation of other authors who found similar correlations between total phenolic content and antioxidant activity of various plants (Alizadeh et al., 2010; Alizadeh, 2013a; Javanmardi et al., 2003 and Nencini, et al., 2007). CONCLUSION Salvia mirzayanii Rech. f. & Esfand. is an endemic medicinal plant which is only distributed in south of Iran and used in the Iranian folk medicine for various purposes. Whereas these plants are extinction, propagation and conservation of this plant is so important. The results of this study confirmed the possibility of propagation of this plant. Also our research show that, essential oil yield, chemical compositions, total phenolic content and antioxidant activities in S. mirzayanii, changed importantly depending on growth medium conditions. The highest essential oil yield, total phenolic content and antioxidant activities in S. mirzayanii were observed in cultivated plants compare wild grown plants. ACKNOWLEDGEMENTS This study was supported financially by the Scientific Research Unit (Project No: ) of Shiraz Branch, Islamic Azad University. Shiraz, Iran. REFERENCES Adams RP/2001. Identification of essential oils components by gas chromatography/ quadrupole mass spectroscopy. Allured Publishing Corporation, Carol. Stream, IL, USA, 456. Alizadeh A, Alizadeh O, Amari G, Zare M b. 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5 Fiore G, Nencini C, Cavallo F, Capasso A, Bader A, Giorgi G, Micheli L In vitro antiproliferative effect of six Salvia species on human tumor cell lines. Phytotherapy Research, 20(8): Javanmardi J, Stushnoff C, Locke E, Vivanco JM Antioxidant activity and total phenolic content of Iranian Ocimum accessions. Food Chemistry, 83: Javidnia K, Miri R, Kamalinejad M, Nasiri A Composition of the essential oil of Salvia mirzayanii Rech. f. & Esfand from Iran, Flavor and Fragrance Journal, 17(6): Kelen M, Tepe B Chemical composition, antioxidant and antimicrobial properties of the essential oils of three Salvia species from Turkish flora. Bioresource Technology, 99(10): Lis-Balchin M, Deans SG Bioactivity of selected plant essential oils against Listeria monocytogenes. Journal of Applied Bacteriology, 82: Löliger J The use of antioxidants in food. Hn O.I. Aruoma & B. Halliwell (Eds.), Free radicals and food additives.(pp ).london: Taylor and Francis. Mirza M, Baher Nik Z, Jamzad Z The extraction and identification in essential oil constituents of Salvia mirzayanii Rech. f. & Esfand, Iranian Journal of Medicinal and Aromatic Plants Research, 19 (2): Nencini C, Cavallo F, Capasso A, Franchi GG, Giorgio G, Micheli L Evaluation of antioxidative properties of Allium species growing wild in Italy. Phytotherapy Research, 21: Newall CA, Anderson LA Herbal medicines. A guide for healthcare professionals. Pharmaceutical Press, London, p Rechinger KH In: Flora Iranica Vol. 152, Akademische Druck- und Verlagsanstalt, Graz. Reynolds JEF Martindale-the Extra Pharmacopeia, 31st ed. Royal Pharmaceutical Society of Great Britain, London. Sadeghi Ghotbabadi F, Alizadeh A, Zadehbagheri M, Kamelmanesh MM, Shaabani M Phytochemical composition of the essential oil, total phenolic content, antioxidant and antimicrobial activity in Iranian Satureja sahendica Bornm. at different ontogenesis conditions, Journal of Medicinal Plants Research, 6(19): Shahidi F, Wanasundara PKJPD Phenolic antioxidants. Crit. Rev. Food Sci. Nut., 32, Singleton VL, Rossi JA Colorimetry of total phenolic with phosphomolybdic- phosphotungestic acid reagents. Am. J. Enol. Vitic., 16: Van Den Dool H, Kratz PD A generalization of the retention index system including linear temperature programmed gas liquid partition chromatography. Journal of Chromatography, 11:463. Wu BW, Pan TL, Leu YL, Chang YK, Tai PJ, Lin KH, Horng JT Antiviral effects of Salvia miltiorrhiza (danshen) against enterovirus 71. American Journal of Chinese Medicine, 35(1): Yamini Y, Khajeh M, Ghasemi E, Mirza M, Javidnia K Comparison of essential oil compositions of Salvia mirzayanii obtained by supercritical carbon dioxide extraction and hydrodistillation methods. Food Chemistry, 108(1): Zargari A Medicinal plants (Vol. 4). Tehran, Iran: Tehran University Press. Zheng W, Wang SY Antioxidant activity and phenolic compounds in selected herbs, Journal of Agricultural and Food Chemistry, 49: No Table 1. Phytochemical composition of the essential oil in wild and cultivated Salvia mirzayanii Components wild S. mirzayanii in natural RI * habitat cultivated S. mirzayanii α-thujene α -Pinene Camphene Sabinene β-pinene Octanone Myrcene n-decane α -Phellandrene α -Terpinene p-cymene Limonene ,8-Cineole (Z)- β -Ocimene (E)- β -Ocimene Terpinene cis-sabinene hydrate trans-linalool oxide Terpinolene Linalool Hotrienol trans-p-mentha-2,8-dien-1-ol trans-pinocarveol Camphor Nerol oxide Pinocarvone Terpineol Terpinene-4-ol p-cymen-8-ol α -Terpineol Myrtenol n-dodecane trans-carveol Nerol Neral

6 Carvacrol methyl ether Linalyl acetate n-decanol Thymol Carvacrol Elemene α -Terpinyl acetate Neryl acetate α -Copaene Geranyl acetate β -Elemene α -Gurjunene (E)-Caryophyllene Aromadendrene α -Guaiene allo-aromadendrene Gurjunene Muurolene Germacrene D β -Selinene α -Selinene Bicyclogermacrene α -Muurolene α -Cuprenene Cadinene epi-α-Selinene Cadinene (Z)-Nerolidol α -Agarofuran Globulol Spathulenol Caryophyllene oxide Viridiflorol epi-α-cadinol β -Eudesmol α -Cadinol neo-Cedranol hydroxy-α-Humulene n-hexadecanol epi-13-manoyl oxide Kaurene Oil Yield (%w/w) Total *RI, Retention Index in elution order from HP-5 column Each value in the table was obtained by calculating the average of three experiments ± standard deviation. Data expressed as percentage of total Table 2. Total phenolic content and antioxidant activity in wild and cultivated S. mirzayanii Rech. & Esfand. No Growth condition Total phenolic content a (mg GAE/g DW) b IC 50 (μg/ml) 1 Wild Salvia mirzayanii b c 2 Cultivated Salvia mirzayanii a b 3 Quercetin (Artificial Antioxidant) a a Data expressed as mg of gallic acid equivalents per g dry weight (DW). b IC 50: Data expressed as μg per millilitre. Lower IC 50 values indicated the highest radical scavenging activity. Each value in the table was obtained by calculating the average of three experiments standard deviation. Means with different letters were significantly different at the level of p<