ORIGINAL RESEARCH

Chemical composition, antioxidant, antimicrobial and cytotoxic activities of minuta and Ocimum basilicum essential oils Mohsen Taheri Shirazi1, Hamid Gholami1, Gholamreza Kavoosi1, Vahid Rowshan2 & Asad Tafsiry1

1Institute of Biotechnology, Shiraz University, Shiraz 71441-65186, Iran 2Department of Natural Resources, Fars Research center for Agriculture and Natural Resources, Shiraz 19395-3697, Iran

Keywords Abstract Antimicrobial, cytotoxic, Ocimum basilicum, radical scavenging, Tagetes minuta Chemical composition, antioxidant, antimicrobial and cytotoxic activities of Tagetes minuta (TM) essential oil (TMO) and Ocimum basilicum (OB) essential Correspondence oil (OBO) were examined. The main components for TMO were dihydrotage- Gholamreza Kavoosi, Institute of tone (33.9%), E-ocimene (19.9%), tagetone (16.1%), cis-b-ocimene (7.9%), Biotechnology, Shiraz University, Shiraz Z-ocimene (5.3%), limonene (3.1%) and epoxyocimene (2.03%). The main 71441-65186, Iran. Tel/Fax: +987112272805; components for OBO were methylchavicol (46.9%), geranial (19.1%), neral E-mail: [email protected] (15.15%), geraniol (3.0%), nerol (3.0%), caryophyllene (2.4%). Inhibitory con- Funding Information centrations (IC50) for reactive oxygen species (ROS) and reactive nitrogen spe- This work was supported by the financial cies (RNS) scavenging were 12–17 and 200–250 lg/mL of TMO and OBO, support from Shiraz University (grant no. respectively. Minimal inhibitory concentration (MIC) against Salmonella typhi, 88-GR-AGRST-108). Escherichia coli, Staphylococcus aureus, Bacillus subtilis, Aspergillus niger, and Candida albicans were 150 Æ 8, 165 Æ 9, 67 Æ 8, 75 Æ 7, 135 Æ 15, and Received: 27 July 2013; Revised: 6 October 115 Æ 8 lg/mL of TMO, respectively. MIC for S. typhi, E. coli, S. aureus, 2013; Accepted: 9 October 2013 B. subtilis, A. niger, and C. albicans were 145 Æ 8, 160 Æ 7, 45 Æ 4, 40 Æ 3, Æ Æ l Food Science & Nutrition 2014; 2(2): 146– 80 9, and 95 7 g/mL of OBO, respectively. IC50 for nasopharyngeal can- 155 cer cell line (KB) and liver hepatocellular carcinoma cell line (HepG2) were 75 Æ 5 and 70 Æ 4 lg/mL of TMO, respectively. IC50 for KB and HepG2 were doi: 10.1002/fsn3.85 45 Æ 4 and 40 Æ 3 lg/mL of OBO, respectively. Thus, they could be used as an effective source of natural antioxidant and antibacterial additive to protect foods from oxidative damages and foodborne pathogens. Furthermore, they could be promising candidate for antitumor drug design.

Introduction ment and in a wide variety of fields in its native region and as a popular traditional folk remedies or in the Tagetes minuta (TM) is a marigold in the sunflower complementary and medical therapy. TM has several med- () family. Tagetes species originally has been ical benefits such as remedy for colds, respiratory inflam- used as a source of essential oil for the flavoring in the mations, stomach problem, anti-spasmodic, anti-parasitic, food industries. The powders and extracts of Tagetes are anti-septic, insecticide, and sedative. It is used for chest rich in the orange-yellow carotenoid and are used as a infections, coughs and catarrh, dilating the bronchi, facili- food colorant in foods such as pasta, vegetable oil, marga- tating the flow of mucus and dislodging congestion and rine, mayonnaises, salad dressing, baked goods, confec- can be used in cases of skin infections. It also has a healing tionery, dairy products, ice cream, yogurt, citrus juice, effect on wounds, cuts, calluses and bunions (Gillij et al. mustard and as colorant in poultry feed (Iranian Herbal 2008; Rahimi et al. 2010; Maity et al. 2011). Pharmacopoeia 2002; Zhang et al. 2009; Nerio et al. Ocimium basilicum (OB) is a culinary plant belonging to 2010). TM is also extensively used medicinally as a condi- the Lamiaceae family that is extensively used as a flavoring

146 ª 2014 The Authors. Food Science & Nutrition published by Wiley Periodicals, Inc. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. M. T. Shirazi et al. Tagetes minuta and Ocimum basilicum agent in a wide variety of fields in its native region and as a stage. The leaves of the were separated from the popular traditional folk remedies or in complementary and stem and were dried in the shade for 72 h. Seeds of a alternative medical therapy. This plant has several func- native OB plants were directly sowed in experimental tional characteristics including carminative, stimulant, field. During the germination period and first 2 weeks of diaphoretic, diuretic, dyspepsia, antiseptic, anesthetic, flat- plantlet growth, they were irrigated with tap water. The ulence, gastritis, anti-spasmodic, anthelmintic, anti-diar- aerial parts of plants were harvested at the flowering rheal, analgesic and anti-tussive. Other medicinal uses of stage, dissected to leaves and dried at room temperature OB include treatment of some gastrointestinal disorders, for 4–5 days. The shade dried leaves (100 g) were hydro- gastrodynia, diarrhea and vomiting (Mondal et al. 2009; distilled for 3 h using an all-glass Clevenger-type appara- Nerio et al. 2010; Rahimi et al. 2010). Considering the tus (Herbal Exir Co., Mashhad, Iran) according to the aforementioned pharmacological and therapeutic proper- method outlined by the British Pharmacopeia (1998). The ties, OB has played an important role not only in tradi- TMO and OBO were dehydrated over anhydrous sodium tional medicine but also in modern pharmacological and sulfate and stored at À20°C until analyzed by gas chro- clinical investigations. In this perspective, the OB essential matography–mass spectrometry (GC-MS) and then used oil (OBO) has played a crucial role in pharmaceutical as for biological assays. well as food industries. However, such practices are largely based on folklore and train of traditional medicine rather Identification of the essential oils than evidence-based research. components In the present study, reactive oxygen species (ROS) and reactive nitrogen species (RNS) scavenging activities of The essential oils analysis was performed using an Agilent the TMO and OBO were examined using 2, 2′-azino-di gas chromatograph series 7890-A (Agilent, Palo Alto, CA) (3-ethylbenzthiazoline-6-sulfonate) (ABTS) and sodium with a flame ionization detector (FID). The analysis was nitrite scavenging effects, respectively. The TMO and carried out on fused silica capillary HP-5 column OBO were individually tested against two Gram-negative (30 m 9 0.32 mm i.d.; film thickness 0.25 lm). The bacteria (Salmonella typhi and Escherichia coli), two injector and detector temperatures were kept at 250 and Gram-positive bacteria (Staphylococcus aureus and Bacillus 280°C, respectively. Nitrogen was used as carrier gas at a subtilis) and two fungi (Aspergillus niger and Candida flow rate of 1 mL/min; oven temperature program was albicans) caused foodborne outbreaks and illnesses. Cyto- 60–210°C at the rate of 4°C/min and then programmed toxic activity of TMO and OBO against nasopharyngeal to 240°C at the rate of 20°C/min and finally held isother- cancer cell line (KB) and liver hepatocellular carcinoma mally for 8.5 min; split ratio was 1:50. GC–MS analysis cell line (HepG2) was examined using a modified 3-(4, was carried out by use of Agilent gas chromatograph 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide equipped with fused silica capillary HP-5MS column test (MTT) assay. In this study, we offer that TMO and (30 m 9 0.25 mm i.d.; film thickness 0.25 lm) coupled OBO has radical scavenging, antimicrobial and cytotoxic with 5975-C mass spectrometer. Helium was used as car- activities and could be used as safe and effective source of rier gas with ionization voltage of 70 eV. Ion source and natural antioxidant to improve the oxidative stability of interface temperatures were 230 and 280°C, respectively. fatty foods during storage and also can be used as a safe Mass range was from 45 to 550 amu. Oven temperature antimicrobial additive to protect foods from foodborne program was the same given above for the GC. Retention pathogens. Furthermore, they can be used as candidate indices (RI) were determined using retention times (RT) for antitumor drug design. of n-alkanes (C8-C25) that were injected after the essential oil under the same chromatographic conditions. The Materials and Methods retention indices for all components were determined according to the method of using n-alkanes as standard (s). The compounds were identified by comparison of Plant materials and isolation of the retention indices (RI, HP-5) with those reported in the essential oils literature and by comparison of their mass spectra with Seed of TM was obtained from Institute of Medicinal the Wiley GC/MS Library and Mass Finder 2.1 Library Plants, Isfahan, Iran and was grown in green house condi- data published mass spectra data (Adams 2007). tions in Sadra near Shiraz, Iran. The seeds of TM were sown in experimental greenhouse, in September 2011. ROS scavenging assay of essential oils One month later obtained seedlings were transferred to experimental field and distributed homogenously. The To assay ROS scavenging activity, 10 lL of the essential aerial parts of plants were harvested at the flowering oils (0–500 lg/mL in DMSO) was added to 1.0 mL of

ª 2014 The Authors. Food Science & Nutrition published by Wiley Periodicals, Inc. 147 Tagetes minuta and Ocimum basilicum M. T. Shirazi et al. diluted ABTS radical solution (7 mmol/L ABTS and Gram-negative bacteria, Gram-positive bacteria and fungi, 2.54 mmol/L potassium persulfate). After mixing, the respectively. The growth inhibition was estimated by mea- absorbance (A) was read at 734 nm against a blank solu- suring turbidity of the cultured medium at 640 nm using tion containing 1% DMSO using an Ultrospec 2000 spec- a spectrophotometer (Pharmacia, Uppsala, Sweden). The trophotometer (Pharmacia, Uppsala, Sweden). The percentage of growth inhibition was obtained by the equa- percentage of ROS scavenging was calculated as: tion: ([A640blank À A640sample]/A640blank) 9 100. MIC ([A734blank À A734sample]/A734blank) 9 100. The concen- was defined as the lowest concentration of the essential trations that could provide 50% inhibition (IC50) were oils produced >90% growth reduction compared with the calculated from the graph that plotted the inhibition per- growth in the control microtube. MIC was calculated from centage against different essential oil concentrations a graph that plotted inhibition percentage against different (Kavoosi and Rowshan 2013). essential oils concentrations.

RNS scavenging assay of essential oils Cytotoxic activity of essential oils To assay RNS scavenging activity, 10 lL of the essential The in vitro cytotoxic activity of the essential oils on two oils (0–500 lg/mL in DMSO) was incubated with 0.5 mL human tumor cell lines, viz, nasopharyngeal cancer (KB) of sodium nitrite (10 lg/mL in 100 mmol/L sodium cit- and liver hepatocellular carcinoma (HepG2) cell lines rate pH 5) at 37°C for 2 h. After incubation, 0.5 mL of were examined using a modified MTT assay (Nouri et al. Griess reagent was added and the absorbance (A) was 2000). All cell lines were obtained from the cell bank of read at 540 nm using a spectrophotometer. A solution the Pasteur Institute of Iran. The cells were cultured in a containing 1% DMSO without essential oil was used as humidified atmosphere at 37°C using RPMI-1640 (Gibco, blank. The percentage of RNS scavenging was calculated Karlsruhe, Germany) supplemented with 10% fetal bovine as follows: ([A540blank À A540sample]/A540blank) 9 100. serum (Gibco), 100 U/mL penicillin (Sigma-Aldrich, Saint IC50 was calculated from the graph that plotted the inhi- Louis, MO) and 100 lg/mL streptomycin (Sigma-Aldrich) bition percentage against different essential oil concentra- in a 5% CO2 incubator. The MTT assay was carried out tions (Kavoosi and Rowshan 2013). as follows. Briefly, 180 lL of medium containing cells at a density of 2 9 104 cells/mL were seeded in each well of a flat-bottom 96-well plate (Tissue Culture Plate, Jet Biofil, Antibacterial and antifungal activity assay Kyoto, Japan). Cells were permitted to adhere to the plate of essential oils for 24 h. The adhered cells were treated with various con- All microorganisms were obtained from the Persian type centrations of the essential oils (0–100 lg/mL in DMSO) culture collection (PTCC), Tehran, Iran. The essential oils and incubated for 24 h. Finally, the medium was replaced were individually tested against two Gram-negative bacte- with 200 lL fresh medium containing 0.5 mg/mL of ria (S. typhi PTCC 1609 [Iran isolate] and E. coli PTCC MTT (Sigma-Aldrich). Plates were incubated at 37°C for 1330 [ATCC 8739]), two Gram-positive bacteria (S. aur- another 4 h after which the medium was discarded and eus PTCC 1112 [ATCC 6538] and B. subtilis PTCC 1023 formazan blue, which was formed in the cells, was dis- [ATCC 6633]), and two fungi (A. niger PTCC 5010 solved with 100 lL DMSO at 37°C for 10 min. All tests [ATCC 9142] and C. albicans PTCC 5027 [ATCC and analyses were run in triplicate. DMSO was used as the 10231]). Minimum inhibitory concentration (MIC) was control. The absorbance of each well was determined by determined against serial dilutions of the essential oils spectrophotometer at dual wavelengths of 570 and 630 nm (0-200 lg/mL) using microdilution method recommended on a micro plate ELISA reader (BioTek Elx 808, Winooski, by Clinical and Laboratory Standards Institute (CLSI) VT). Viability percentage was calculated by the following (2006). Bacteria and fungi strains were suspended in Luri- formula: (absorbance of treated cells/absorbance of corre- a-Bertani (LB) media and the densities were adjusted to sponding control) 9 100. The control was essential oil- 0.5 McFarland standards at 640 nm (108 CFU/mL) and untreated cells containing DMSO at the highest concentra- then diluted to 105 CFU/mL with LB. Bacteria and fungi tion used (1%). The concentration providing 50% inhibi- suspensions (0.5 mL) and the essential oils (0.5 mL) were tion (IC50) was calculated from a graph plotting inhibition added to 1.5 mL microtube and incubated with shaking at percentage against different essential oil concentrations. 37°C for 24 h. Medium without bacteria and fungi was used as sterility control. Medium with bacteria but with- Statistical analysis out essential oils was used as growth control (blank). Posi- tive control included Gentamicin, Ampicillin and All data are expressed as the means Æ standard deviations Ketoconazole (all Padtan Teb, Iran, 10 lg/mL) for of at least three independent experiments. The significant

148 ª 2014 The Authors. Food Science & Nutrition published by Wiley Periodicals, Inc. M. T. Shirazi et al. Tagetes minuta and Ocimum basilicum differences between treatments were analyzed by one-way Table 2. Chemical composition of essential oil from Ocimum analysis of variance (ANOVA) test at P < 0.01 using basilicum. statistical package for the social sciences (SPSS, Abaus %of Concepts, Berkeley, CA) and Prism 5 (Graph Phad, San Compounds RI1 compounds Diego, CA) software. a-Pinene 933 0.3 1-Octen-3-ol 980 0.1 Results 5-Hepten-2-one-6-methyl 991 1.0 p-Cymene 1024 0.1 Chemical composition of TMO and OBO Limonene 1028 0.2 trans-b-Ocimene 1046 0.3 GC-MS analysis of the TMO indicated that the main cis-Sabinene hydrate 1073 0.2 components were dihydrotagetone (33.9%), E-ocimene cis-Verbenol 1142 0.2 (19.9%), tagetone (16.1%), cis-b-ocimene (7.9%), Z- Borneol 1153 0.5 p- 1,5-dien-8 ol 1166 0.4 ocimene (5.3%), limonene (3.1%), and epoxyocimene Methylchavicol 1211 47 (2.0%) (Table 1). GC-MS analysis of the OBO indicated n-Octyl acetate 1215 0.4 that the main components were methylchavicol (46.9%), Nerol 1238 3.2 geranial (19.1%), neral (15.1%), geraniol (3.0%), nerol Neral 1253 15 (2.9%), caryophyllene (2.4%) (Table 2). Geraniol 1265 3.1 Geranial 1284 19 Neryl acetate 1367 0.3 Antioxidant activity of TMO and OBO Geranyl acetate 1387 0.3 Methyl eugenol 1409 0.5 Both TMO and OBO displayed a concentration depen- E-Caryophyllene 1422 2.4 dent ROS and RNS scavenging activity (Table 3). Inhibi- trans-a-Bergamotene 1436 0.5 a tory concentration (IC50) for ROS and RNS scavenging -Humulene 1456 1.1 were 12 Æ 2 and 13 Æ 4 lg/mL of TMO, respectively. Germacrene D 1483 1.2 trans-a-Bisabolene 1543 1.6 IC50 for ROS and RNS scavenging were 200 Æ 11 and Caryophyllene oxide 1589 0.4

1RI, retention indices relative to C8–C25 n-alkanes on the HP-5 Table 1. Chemical composition of essential oil from Tagetes minuta. column.

%of Compounds RI1 compounds Table 3. Radical scavenging activity of essential oils from Tagetes a-Pinene 933 0.33 minuta and Ocimum basilicum. Sabinene 973 0.40 IC (lg/mL) for: T. minuta O. basilicum cis-3-Hexenyl acetate 1005 0.15 50 p-Cymene 1025 0.93 ROS 12 Æ 2 200 Æ 11 Limonene 1029 3.10 RNS 13 Æ 4 210 Æ 16 cis-b-Ocimene 1037 7.90

Dihydrotagetone 1061 33.8 IC50, effective concentration of the test compound which scavenge allo-Ocimene 1135 0.36 the radical by 50%. IC, inhibitory concentration; ROS, reactive oxygen E, Z-Epoxyocimene 1149 2.00 species; RNS, reactive nitrogen species. Tagetone 1160 16.1 cis-Tagetone 1167 0.20 p-Mentha-1,8 dien-3-one 1208 0.18 210 Æ 16 lg/mL of OBO, respectively. TMO had stronger Z-Ocimene 1238 5.30 radical scavenging capacity than OBO. E-Ocimene 1250 19.9 Thymol 1282 0.48 Carvacrol 1298 0.47 Antibacterial activity of TMO and OBO cis-Isoeugenol 1398 0.86 E-caryophyllene 1420 0.32 Both TMO and OBO displayed a concentration-dependent a-Humulene 1455 0.15 antibacterial activity (Table 4). MIC for S. typhi, E. coli, S. Germocrene D 1497 0.43 aureus, and B. subtiliss were 150 Æ 8, 165 Æ 9, 67 Æ 8, Spathulenol 1582 0.42 and 75 Æ 7 lg/mL of TMO, respectively. MIC for S. typhi, 1RI, retention indices relative to C8–C25 n-alkanes on the HP-5 E. coli, S. aureus, and B. subtilis were 145 Æ 8, 160 Æ 7, column. 45 Æ 4, and 40 Æ 3 lg/mL of OBO, respectively. TMO

ª 2014 The Authors. Food Science & Nutrition published by Wiley Periodicals, Inc. 149 Tagetes minuta and Ocimum basilicum M. T. Shirazi et al.

Table 4. Antibacterial activity of Tagetes minuta and Ocimum basili- Table 6. Cytotoxic activity of Tagetes minuta and Ocimum basilicum cum essential oils. essential oils.

MIC (lg/mL) for: T. minuta O. basilicum IC50 (lg/mL) for: T. minuta O. basilicum

Salmonella typhi 150 Æ 8 145 Æ 8 KB 75 Æ 545Æ 4 Escherichia coli 165 Æ 9 160 Æ 7 HepG-2 70 Æ 440Æ 3 Staphylococcus aureus 67 Æ 845Æ 4 Bacillus subtilis 75 Æ 740Æ 3 Data are expressed as the means Æ standard deviations for at least three independent experiments. IC50, effective concentration of the Data are expressed as means Æ standard deviation. MIC, minimum test compound which kills 50% of the cells tested; KB, human naso- inhibitory concentration. MIC was defined as the lowest concentration pharyngeal cancer cell line; HepG2, liver hepatocellular carcinoma cell of the compounds produced >90% growth reduction compared with line. the growth in the control well. and OBO had stronger effects on the Gram-positive bacte- Discussions ria than that of Gram-negative bacteria. GC-MS analysis of the essential oil indicated the main com- ponents TMO were dihydrotagetone, E-ocimene, tagetone, Antifungal activity of TMO and OBO cis-b-ocimene, Z-ocimene, limonene and epoxyocimene. TMO and OBO displayed a concentration-dependent Previous study reported the main components of TMO antifungal activity (Table 5). MIC for A. niger and C. were b-ocimene, dihydrotagetone, tagetone, Z-ocimene albicans were 135 Æ 15, and 115 Æ 8 lg/mL of TMO, and E-ocimene (Lopez et al. 2011). Another study reported respectively. MIC for A. niger and C. albicans were thiophenes and polyacetylenic compounds in the Tagetes 80 Æ 9, and 95 Æ 7 lg/mL of OBO, respectively. species and TM had the highest total thiophene yield (Mar- otti et al. 2010). Accordingly, the main components of TMO could be tagetone (cis/trans, ketone/alcohol, alde- Cytotoxic activity of TMO and OBO hyde/alcohol), ocimene (cis/trans, ketone/alcohol, alde- The MTT assay results indicated that at low concentra- hyde/alcohol) and thiophene derivatives (Breme et al. tions (<50 lg/mL), TMO had no effect on KB and 2009; Marotti et al. 2010; Ranilla et al. 2010; Garcia et al. HepG2 viability. However, at higher concentrations (50– 2011; Lopez et al. 2011; Armas et al. 2012). Analysis of the 200 lg/mL), cell viability was significantly reduced in a chemical composition of the OBO by GC-MS indicated concentration-related manner, with the maximum effect that methylchavicol, geranial, neral, geraniol, nerol and at concentrations >200 lg/mL. IC50 for KB and HepG2 caryophyllene were the main constituents. According to was 75 Æ 5 and 70 Æ 4 lg/mL of TMO, respectively previous studies, the most abundant components in OBO (Table 6). The MTT assay results indicated that low con- are phenol-containing monoterpenes (methylchavicol), centrations (1–10 lg/mL), OBO had no effect on KB and alcoholic monoterpenes (linalool, geraniol, nerol), acyclic HepG2 viability. However, at higher concentrations monoterpene aldehyde (geranial, neral), cyclic ether mon- (10–100 lg/mL), cell viability was significantly reduced in oterpenes (1, 8-cineole) and carbure bicyclic sesquiterpene a concentration-related manner, with the maximum effect (caryophyllene) (Rao et al. 2011; Venancio et al. 2011; at 200 lg/mL. IC50 for KB and HepG2 was 45 Æ 4 and Verma et al. 2012). Therefore, the plants analyzed in this 40 Æ 3 lg/mL of OBO, respectively (Table 6). research had roughly same components with other previ- ously analyzed essential oils, however, showed important differences in their quality and quantity of components. The TMO analyzed here possessed potent in vitro ROS Table 5. Antifungal activity of Tagetes minuta and Ocimum basilicum and RNS scavenging activity. The TMO at concentration essential oils. >30 lg/mL had the ability to scavenge all ROS and RNS radicals. ROS are oxygen-derived small molecules, includ- MIC (lg/mL) for: T. minuta O. basilicum ing oxygen radicals such as superoxide, hydroxyl and per- Aspergillus niger 135 Æ 15 80 Æ 9 oxyl and some non-radicals that are easily converted into Candida albicans 115 Æ 895Æ 7 radicals, such as hydrogen peroxide. ROS, once produced,

Data are expressed as means Æ standard deviation. MIC, minimum can interact with various molecules including other small inhibitory concentration. MIC was defined as the lowest concentration inorganic molecules as well as macromolecules such as of the compounds produced >90% growth reduction compared with proteins and lipids. During these interactions, ROS may the growth in the control well. destroy or change the function of the target molecule

150 ª 2014 The Authors. Food Science & Nutrition published by Wiley Periodicals, Inc. M. T. Shirazi et al. Tagetes minuta and Ocimum basilicum

(Circu and Aw 2010). The ROS reducing activity of TMO antibacterial activity of both water extracts (the water observed in our study implies the beneficial role of this remaining after hydro-distillation) and essential oils of product for reducing damages in biological tissues. The TM against Paenibacillus larvae. The water remaining after radical scavenging activity of compounds is mainly due to hydro-distillation showed the highest antibacterial activi- their oxidation-reduction potential, which can play an ties while essential oils had less activity for the inhibition important role in neutralizing free radicals. This activity of P. larvae (Gonzalez and Marioli 2010). The total extract is related to phenolic hydroxyl groups (Katalinic et al. and fractions with different solvents, obtained from leaves 2006). TMO mainly contains dihydrotagetone, ocimene, of TM showed several degrees of antimicrobial activity tagetone and limonene which all are monoterpenes. against Gram-positive and Gram-negative bacteria. The Despite polyphenol absence TMO exhibited significant same fractions were inactive against Lactobacillus (Gram- radical scavenging activity. This antioxidant activity was positive), Zymomonas (Gram-negative) and Saccharomyces confirmed by previous researches with IC50 between 35 (fungi kingdom) species (Tereschuk et al. 1997). and 344 lg/mL (Gong et al. 2012; Gupta et al. 2012; OBO analyzed in this research also showed potent anti- Perez-Cruz et al. 2013). Thus, TMO analyzed in this bacterial activities. OBO is a good source of phenol-con- research showed stronger antioxidant activity compared taining monoterpenes, with a significant antibacterial to previously analyzed TMOs. activity against S. aureus, Salmonella enteritidis, and E. coli The OBO analyzed in this research showed potent in and antiseptic against Proteus vulgaris, B. subtilis and Sal- vitro ROS and RNS scavenging activity. The OBO at con- monella parathypha. The essential oil of sweet oil centrations more than 500 lg/mL had the ability to scav- displayed a great potential for antibacterial activity with enge all ROS and RNS radicals which indicates its their MIC values of 62.5–500 lg/mL, which is similar to moderate radical scavenging activity. The OBO also pos- our results (Hossain et al. 2010; Rattanachaikunsopon sessed antioxidant activity as measured by the 2, 2-diphe- and Phumkhachorn 2010). Previously identified major nyl-1-picrylhydrazyl (DPPH) and ABTS assay (Tarchoune compounds of OBO are methylchavicol, gitoxigenin, et al. 2010; Kaurinovic et al. 2011; Sgherri et al. 2011). trimethoquinol, b-guaiene, aciphyllene, alizarin, naphtha- OBO mainly contains methylchavicol, geranial/geraniol line, caryophyllene and mequinol (Hossain et al. 2010). and neral/nerol. The radical scavenging activity of meth- Considering high concentration of methylchavicol (78%) ylchavicol and essential oil bearing methylchavicol to in essential oil of O. basilicum and showing broad spec- some extent was confirmed (Tominaga et al. 2005; Mo- trum antibacterial and antifungal activities (Rao et al. hamad et al. 2011). Previous studies on the radical scav- 2011), our results, as we expected, exhibited high antimi- enging activity and antioxidant capacity of geraniol and crobial activities. geraniol bearing essential oil using tsigniertiary-butyl The antibacterial activity which is recognized in the hydroperoxide stressed rat alveolar macrophages clearly essential oils of several medicinal plants established that showed that geraniol increased the cell viability and the antibacterial activity of essential oils are related to the showed 45% increase in superoxide dismutase (antioxi- attack on the phospholipids present in the cell mem- dant enzyme) activity and 120% increase in glutathione branes, which causes increased permeability and leakage content (antioxidant). Geraniol was found to have radical of cytoplasm, or to their interaction with enzymes located scavenging and significantly decreased lipid peroxidation on the cell wall (Paparella et al. 2008). Thus, the resis- and inhibited nitric oxide (NO) release and ROS genera- tance of Gram-negative bacteria to the essential oils likely tion in pretreated cells as compared to stressed cells. laid in the protective role of their cell wall lipopolysac- These results indicated the antioxidant activity of geraniol charide or outer membranes proteins, which restricts by scavenging ROS and RNS and induction of antioxi- diffusion of hydrophobic compounds through its lipo- dant defense systems such as superoxide dismutase and polysaccharide layer (Oussalah et al. 2007; Garcia-Garcia glutathione (Choi et al. 2000; Tiwari et al. 2010). Thus, et al. 2011). Essential oils have the ability to disrupt lipid the radical scavenging activity of OBO could be related to structure of the cell wall of bacteria, leading to destruc- geraniol/geranial followed by methylchavicol. The ROS tion of cell membrane, cytoplasmic leakage, cell lysis and and RNS reducing activities of OBO observed in our ultimately cell death. The decrease in pH that occurs due study imply the beneficial role of this product for reduc- to cell membrane disruption results in a loss of control of ing damages in biological tissues. cellular process such as ATP biosynthesis, DNA transcrip- TMO analyzed in this research also showed potent tion and protein synthesis (Xu et al. 2008). antibacterial activities. TMO has a significant antibacterial TMO analyzed in this research also showed antifungal activity against both gram-positive and gram-negative activities. Several studies have also described antifungal bacteria (Hethelyi et al. 1986; Tereschuk et al. 1997; activities of TMO against Candida, Penicillium and Asper- Cespedes et al. 2006). Gonzalez and Marioli studied gillus species (Dunkel et al. 2010; Thembo et al. 2010). The

ª 2014 The Authors. Food Science & Nutrition published by Wiley Periodicals, Inc. 151 Tagetes minuta and Ocimum basilicum M. T. Shirazi et al. antifungal activity of aqueous and organic extracts of T. (Gupta et al. 2012), which are lower rather than TMO minuta, Lippia javanica, Amaranthus spinosus and Vigna analyzed in this research. The results of MTT assay unguiculata against Fusarium verticillioides, F. proliferatum, indicated that OBO had no effects on the viability of KB Aspergillus flavus and A. parasiticus were investigated. All and HepG2 cells at low concentrations (<10 lg/mL). extracts except for the water extracts showed growth inhibi- However, at higher concentrations (10–100 lg/mL) cell tory activity against most isolates of the Fusarium spp. The viability was significantly reduced in a concentration most active were the methanol and hexane extracts of V. dependent manner, the maximum effect was 100% at unguiculata and A. spinosus with MIC values of 500 lg/mL 200 lg/mL. The in vitro anticancer activity of the essential against Fusarium spp. (Thembo et al. 2010). These antifun- oil from O. basilicum was examined using methyl thiazol gal activities may be attributed, at least in part, to the pres- tetrazolium assay against the human cervical cancer cell line ence of thiophenes and flavonoids in the extracts (Gonzalez (HeLa) and human laryngeal epithelial carcinoma cell and Marioli 2010; Thembo et al. 2010). line (Hep-2). The IC50 values obtained were 90.5 and OBO analyzed in this research also showed antifungal 96.3 lg/mL, respectively. In other studies the major constit- activities. Several studies have also described antifungal uents were found to be methyl cinnamate (70.1%), linalool activities of OBO against C. albicans, Penicillium natum, (17.5%), b-elemene (2.6%) and camphor (1.52%) (Kathir- A. niger and Microsporum gyseum (Siddiqui et al. 2012). vel and Ravi 2012). The major oil constituents of O. basili- Essential oils from O. basilicum (methylchavicol 78%), O. cum were linalool (30–40%) and eugenol (8–30%) had no gratissimum (eugenol 84%), O. tenuiflorum (methyeuge- cytotoxic activity to mammalian cells (Zheljazkov et al. nol 72%) and O. kilimandscharicum (camphor 51.7%) 2008). were exhibited broad spectrum antifungal activities (Rao Although the constituents of essential oils can act as et al. 2011). The effects of essential oils from sweet O. antioxidants, they may also act as prooxidants and affect basilicum (linalool 65%) on fungal decay and quality inner cell membranes and organelles such as mitochon- parameters of the Thompson seedless table grape were dria in eukaryotic cells. Depending on the type and con- evaluated. Results showed that the essential oils have a centration, this effect may result in cellular cytotoxicity. good inhibitory effect on the development of fungal decay The essential oils that showing different levels of cytotox- in Thompson table grapes (Abdollahi et al. 2012). The icity also exhibited different antioxidative capacities essential oil of the aerial parts of O. basilicum mainly con- depending on the composition of the oil especially on stituted from linalool (29.68%), (Z)-cinnamic acid methyl their phenolic content (Bakkali et al. 2008). Essential oils ester (21.49%), cyclohexene (4.41%) and showed signifi- by penetrating through the cytoplasmic and organelles cant antifungal activity against some plant pathogenic membranes disrupt and permeabilize them and especially fungi (Zhang et al. 2009). damage mitochondrial membranes. The mitochondria by Essential oils and their components generally displayed changes in electron flow through the electron transport potent fungicidal activity and flow cytometry confirmed chain produce free radicals which oxidize and damage lip- the occurrence of damage to the plasma membrane and ids, proteins and DNA. Phenolic components of essential cell death (Vale-Silva et al. 2012). In addition, fungicidal oils are oxidized by contact with ROS producing very activity of essential oil apparently originates from the reactive phenoxyl radicals. These types of radical reactions inhibition of ergosterol biosynthesis and the disruption of are enhanced by the presence of transition metal ions membrane integrity (Ahmad et al. 2011). Furthermore, such as Fe2+ and Cu2+ (Shamim et al. 2008). Phenolic fungicidal activity of essential oils might be due to the compounds oxidation appears to take place in the cytosol induction of calcium stress and up-regulation of genes by contact with ROS to form phenoxyl radical. In the 2+ 2+ involved in metabolic and energy pathways, stress presence O2, transition Fe and Cu metal ions catalyze response, autophagy, and drug efflux (Rao et al. 2010). the oxidation of phenol ring forming phenoxyl radical The results of MTT assay indicated that TMO had no leading to the formation of ROS and hydroxyl radical effects on the viability of KB and HepG2 cells at low then damage mitochondria (Kyselova 2011). Lipophilic concentrations (<50 lg/mL). However, at higher concen- phenolic compounds themselves permeabilize the mito- trations (50–200 lg/mL) cell viability was significantly chondrial membranes where transition metal ions Fe2+ reduced in a concentration dependent manner, the and Cu2+ are sequestered in the inter membrane space maximum effect was 100% at concentrations >200 lg/mL. and provoke a leakage of these ions and ROS from mito- Cytotoxic activity of the ethanol extract of T. eracta roots chondria (Mehta et al. 2006). Thus, phenolic compounds against prostate (PC-3) and HeLa cancer cell lines was are oxidized during permeabilization and leakage at the investigated by Gupta et al. using MTT assay. The extract mitochondrial level giving rise to phenoxyl radicals which conferred noticeable cytotoxicity against both PC-3 and continue prooxidants chain reactions with proteins and

HeLa cell lines with IC50 of 407 and 164 lg/mL, respectively DNA and generate new ROS (Hansen et al. 2006).

152 ª 2014 The Authors. Food Science & Nutrition published by Wiley Periodicals, Inc. M. T. Shirazi et al. Tagetes minuta and Ocimum basilicum

Components of natural products especially phenolic Adams, R. P. 2007. P. 456 in Identification of essential oil components which show antioxidant activity, can be oxi- components by gas chromatography/mass spectrometery. dized by ROS and thus generate additional radical species 4th ed. Allured Publishing Corporation, IL. like panoxyl, hydroxyl and superoxide radicals and hydro- Ahmad, A., A. Khan, F. Akhtar, S. Yousuf, I. Xess, L. A. Khan, gen peroxide (El-Agamey et al. 2004). Indeed, antioxi- et al. 2011. Fungicidal activity of thymol and carvacrol by dants by interacting with ROS are converted into disrupting ergosterol biosynthesis and membrane integrity prooxidants which are able to oxidize lipids, proteins and against Candida. Eur. J. Clin. Microbiol. Infect. Dis. 30:41– DNA (Atsumi et al. 2006). Volatile terpenic and phenolic 50. components of essential oils can function as prooxidants Armas, K., J. Rojas, L. Rojas, and A. Morales. 2012. by affecting the cellular redox status. This may lead to Comparative study of the chemical composition of essential late apoptosis and/or necrosis including damage to pro- oils of five Tagetes species collected in Venezuela. Nat. Prod. – teins and DNA and overall cytotoxic effects (Bakkali et al. Commun. 7:1225 12226. 2008). If the antioxidant concentrations is too weak to Atsumi, T., K. Tonosaki, and S. Fujisawa. 2006. Induction of permeabilize mitochondrial membranes, conversion into early apoptosis and ROS-generation activity in human prooxidant may not occur, and the antioxidant would gingival fibroblasts (HGF) and human submandibular gland keep its activity. Thus, at low concentrations, antioxidant carcinoma (HSG) cells treated with curcumin. Arch. Oral – was not oxidized and could not damage mitochondria. In Biol. 51:913 921. contrast, at high concentration antioxidant could damage Aydin, S., A. A. Basaran, and N. Basaran. 2005. Modulating effects of and its major ingredients on oxidative DNA and permeabilize mitochondria, oxidized to prooxidant damage in human lymphocytes. J. Agric. Food Chem. and could react as prooxidant damaging DNA and 53:1299–1305. proteins (Aydin et al. 2005). Bakkali, F., S. Averbeck, D. Averbeck, and M. Idaomar. 2008. Biological effects of essential oils-a review. Food Chem. Conclusions Toxicol. 46:446–475. Breme, K., P. Tournayre, X. Fernandez, U. J. Meierhenrich, H. Considering all of these results, TMO and OBO had ROS Brevard, D. Joulain, et al. 2009. Identification of odor and RNS scavenging activities. Radical scavenging activity impact compounds of Tagetes minuta L. essential oil: of TMO was higher than that of OBO. Thus, they can be comparison of two GC-olfactometry methods. J. Agric. Food used as a safe, effective and easily accessible source of natu- Chem. 57:8572–8580. ral antioxidants to improve the oxidative stability of fatty British Pharmacopeia. 1998. Pp. 137–138 in British foods during storage. In addition, TMO and OBO had anti- pharmacopeia. HMSO, London, U.K. bacterial and antifungal activities. Antibacterial and anti- Cespedes, C. L., J. G. Avila, A. Martınez, B. Serrato, J. C. fungal activities of OBO were higher than that of TMO. Calderon-Mugica, and R. Salgado-Garciglia. 2006. Thus, they could be used as safe antimicrobial agents Antifungal and antibacterial activities of Mexican . against food burn pathogens to preserve foods staff against J. Agric. Food Chem. 54:3521–3527. these pathogens. Furthermore, TMO and OBO had cyto- Choi, H. S., H. S. Song, H. Ukeda, and M. Sawamura. 2000. toxic activity against KB and HepG2 cell lines. Hence, they Radical-scavenging activities of citrus essential oils and their can be used as candidate for antitumor drug design. components: detection using 1, 1-diphenyl-2-picrylhydrazyl. J. Agric. Food Chem. 48:4156–4161. Acknowledgments Circu, M. L., and T. Y. Aw. 2010. Reactive oxygen species, cellular redox systems, and apoptosis. Free Radical Biol. This work was supported by the financial support from Med. 48:749–762. Shiraz University (grant no. 88-GR-AGRST-108). Clinical and Laboratory Standards Institute (CLSI). 2006. Methods for dilution antimicrobial susceptibility tests for Conflict of Interest bacteria that grow aerobically, approved standard. 7th ed. Clinical and Laboratory Standards Institute, CLSIM7-A7, None declared. Wayne, PA. Dunkel, F. V., S. T. Jaronski, C. W. Sedlak, S. U. Meiler, and K. D. Veo. 2010. Effects of steam-distilled shoot extract of References Tagetes minuta (: Asteraceae) and Abdollahi, A., A. Hassani, Y. Ghosta, I. Bernousi, M. H. entomopathogenic fungi on larval Tetanops myopaeformis. Meshkatalsadat, R. Shabani, et al. 2012. Evaluation of Environ. Entomol. 39:979–988. essential oils for maintaining postharvest quality of El-Agamey, A., G. M. Lowe, D. J. McGarvey, A. Mortensen, Thompson seedless table grape. Nat. Prod. Res. 26:77–83. D. M. Phillip, T. G. Truscott, et al. 2004. Carotenoid radical

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