NPC
Natural Product Communications
Composition of Essential Oil from Tagetes minuta and its Cytotoxic, Antioxidant and Antimicrobial Activities
2014 Vol. 9 No. 0 1-4
Nasser A. Awadh Aliab*, Farukh S. Sharopovc, Ali G. Al-kafb, Gabrielle M. Hillb, Norbert Arnoldd, Saeed S. Al-Sokaria, William N. Setzerc and Ludger Wessjohannd a
Pharmacognosy Department, Faculty of Clinical Pharmacy Al-Baha University, KSA Department of Pharmacognosy and Medicinal Chemistry, Faculty of Pharmacy, University of Sana’a, Sana’a, Yemen c Department of Chemistry, University of Alabama in Huntsville, Huntsville, AL 35899, USA b
d
Leibniz Institute of Plant Biochemistry, Department of Bio-organic Chemistry, Weinberg 3, 06120 Halle/Saale, Germany
[email protected] Received: November 5th, 2013; Accepted: December 12th, 2013
The essential oil from the leaves of Tagetes minuta L., growing wild in Yemen, was obtained by hydrodistillation and analyzed by gas chromatography–mass spectrometry. A total of 28 compounds were identified representing 74.2% of total oil composition. Major components of the essential oil were (Z)-ocimenone (15.9%), (E)-ocimenone (34.8%), (Z)-β-ocimene (8.3%), limonene (2.3%), (Z)-tagetone (1.8%), dihydrotagetone (1.4%) and an unidentified dimethylvinylketone derivative (20.6%). The oil showed moderate cytotoxic activity against MCF-7 breast tumor cells, with an IC50 of 54.7 6.2 μg/mL. In the DPPH radical scavenging assay, T. minuta oil showed potent antiradical activity with an IC50 value of 36 µg/mL. Antimicrobial activity was also investigated on several microorganisms, and the essential oil exhibited high activity against methicillin-resistant Staphylococcus aureus (MRSA) with an inhibition zone of 23 mm. It also exhibited remarkable antifungal activity against Candida albicans with an inhibition zone of 26 mm. Keywords: Tagetes minuta, Asteraceae, Essential oil, Cytotoxic, Ocimenone, Antimicrobial, GC-MS.
In recent decades, the essential oils of aromatic plants have been of great interest as sources of bioactive natural products [1]. Antioxidants act as free radical-scavengers and either inhibit or slow down lipid peroxidation and other free radical-mediated processes. Therefore, they have a tendency to protect the body from various disorders which are attributed to free radicals such as cancer, arteriosclerosis, malaria, rheumatoid arthritis, neurodegenerative diseases and aging processes by protecting the organism against oxidative damage [2]. On the other hand, new sources of antimicrobial agents need to be discovered due to the existence and continuous evolution of resistant microorganisms, such as methicillin-resistant Staphylococcus aureus (MRSA). Around 90–95% of S. aureus strains world-wide are resistant to penicillin and in most Asian countries about 75% of the same bacterial strains are methicillin resistant [3,4] Essential oils and their volatile constituents have been widely used for bactericidal, fungicidal, spasmolytic, carminative, hepatoprotective, antiviral, antiparasitic, insecticidal, anticancer, antioxidant, antidiabetic, cardiovascular, and cosmetic and food applications [5-8]. Tagetes is a genus of 56 species [9] of annual and perennial, mostly herbaceous, plants in the Asteraceae. This genus is recognized as a source of carotenoids used as food colorants and feed additives [10], and for possessing anticancer and anti-aging effects [11], T. minuta is traditionally used as an anthelmintic, diuretic, antispasmodic and to treat stomach and intestinal diseases [12]. The essential oils of T. minuta have exhibited biocidal, acaricidal, antifungal, and antimicrobial activities [13-17]. Previous investigations reported the essential oil compositions of T. minuta from different locations, mainly in Argentina, with different
main components. The major components included monoterpene hydrocarbons, oxygenated monoterpenes, and sometimes sesquiterpenes, among which were (Z)- and (E)-β-ocimene isomers, limonene, (Z)- and (E)-ocimenone isomers, dihydrotagetone and spathulenol [13-15,17-21]. As part of our program to evaluate essential oils from Compositous plants [22-24], this work reports, for the first time, the composition, antimicrobial, antiradical and cytotoxic activity of an essential oil from Yemeni T. minuta (EOTM). Leaves of T. minuta yielded an orange-colored essential oil with a percentage yield of 1.5% (v/w), on a dried weight basis. EOTM was subjected to GC/MS analysis. A total of 28 compounds were identified representing 74.2% of the total oil composition, as presented in Table 1. The major components were (Z)-ocimenone (15.9%), (E)-ocimenone (34.8%), (Z)-β-ocimene (8.3%), limonene (2.3%), (Z)-tagetone (1.8%), dihydrotagetone (1.4%) and an unidentified dimethylvinylketone derivative (20.6%). The essential oil contained a large proportion of oxygenated terpenoids (57.9%), represented by oxygenated monoterpenes (57.4%) and oxygenated sesquiterpenes (0.5%) and a lesser proportion of non-oxygenated components (12.1%), represented by non-oxygenated monoterpenes (11.0%) and sesquiterpenes (1.1%). Oxygenated monoterpenes were the most abundant chemical class of compounds in the essential oil. (Z)-Ocimenone and (E)-ocimenone (34.8%) have been reported also as major compounds in T. minuta oils from Argentina [13], Iran [18-20] and India [21]. This is the first report on the cytotoxic effect of the oil, which was assessed against MCF-7 breast tumor cells. The oil showed moderate cytotoxic activity, with an IC50 = 54.7 6.2 µg/mL, compared with the positive control (tingenone) (16.8 1.7 μg/mL).
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Ali et al.
Table 1: Chemical composition of the essential oil from Tagetes minuta. RI 850 881 975 1008 1028 1038 1048 1053 1078 1100 1133 1142 1145
Compound Ethyl 2-methylbutyrate Isoamyl acetate Sabinene (3Z)-Hexenyl acetate Limonene (Z)-β-Ocimene (E)-β-Ocimene Dihydrotagetone Unidentified Linalool (Z)-Epoxy-ocimene (E)-Epoxy-ocimene (E)-Tagetone
% 0.1 tr 0.3 0.1 2.3 8.3 0.1 1.4 0.6 0.6 0.4 0.1 0.5
RI 1149 1153 1164 1176 1205 1229 1238 1247 1270 1285 1393 1418 1453
Compound Myrcenone (Z)-Tagetone Borneol/Isoborneol Terpinen-4-ol Unidentified (Z)-Ocimenone (E)-Ocimenone Carvotanacetone Unidentified Bornyl/isobornyl acetate Unidentified (E)-Caryophyllene α-Humulene
A possible synergistic effect among the different essential oil components should be taken into account for explaining the cytotoxic effect of the oil. β-Ocimene, (E)-caryophyllene and germacrane D have been reported to exhibit in vitro cytotoxicity to MDA-MB-231 human breast tumor cells [25], and sabinene, a monoterpene hydrocarbon, has been reported to be cytotoxic to breast (MCF7), liver (HEPG2) and colon cancer (HCT116) cells [26]. In the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, the ability of EOTM to act as a donor of hydrogen atoms or electrons for the transformation of DPPH into its reduced form DPPH was investigated. EOTM was able to change the stable violet DPPH radical into yellow-colored DPPH, reaching 50% reduction with an IC50 of 36 µg/mL. The free-radical scavenging activities of essential oils have been associated with phenolic monoterpenoids such as carvacrol and thymol, but other oxygenated monoterpenoids (e.g., 1,8-cineole, linalool, borneol, and terpinene-4-ol) have also shown antioxidant activity [27]. The radical scavenging mechanism involves hydrogen atom abstraction from the substrate molecule by the DPPH radical, and the thermodynamic favorability of this hydrogen abstraction depends on the bond dissociation energy. The bond dissociation enthalpies for carvacrol, thymol, (Z)- and (E)ocimenone have been calculated using density functional theory at the B3LYP/6-311++G**//6-31+G* level of theory in order to provide some insight into the free-radical scavenging activities of these oxygenated monoterpenoids. The bond dissociation energies are calculated to be: carvacrol (BDE = 79.1 kcal/mol), thymol (BDE = 78.4 kcal/mol), (Z)-ocimenone (BDE = 83.2 kcal/mol), and (E)-ocimenone (BDE = 80.4 kcal/mol). Thus, the ocimenones show comparable bond dissociation energies with carvacrol and thymol, and the high concentrations of ocimenones (50.7%) in Yemeni T. minuta essential oil are, therefore, likely to be responsible for the free-radical scavenging activity of this oil. The antimicrobial activity of EOTM was evaluated by the application of the disc diffusion test against some pathogenic bacteria and Candida albicans. The oil exhibited good anticandidal activity against C. albicans with an inhibition zone of 26 mm. It can be seen that the oil possesses strong antifungal potential, as does the commercial drug, nystatin, which was used as a positive control. EOTM showed also a high activity against MRSA with an inhibition zone of 23 mm. There are several clinical studies noting the successful use of essential oils in treating MRSA nasal carriage or MRSA infections. Specifically, Warnke et al. [29] and Sherry et al. [30] reported that topical tea tree oil was as effective as standard therapy for reducing MRSA nasal colonization. At an application of 400 μg, antifungal activity of T. minuta oil was observed using a standardized bioautographic technique, with inhibition zones of 13 mm against the phytopathogenic fungus Cladosporium cucumerinum. There have been few reports on the
% 0.2 1.9 0.7 0.1 0.6 15.9 34.8 0.1 0.6 0.1 1.4 0.3 0.2
RI 1481 1497 1577 1583 1914 2019 2061 2084 2109 2114 2199 2213 2316
Compound Germacrene D Bicyclogermacrene Spathulenol Caryophyllene oxide 5-(3-Buten-1-ynyl)-bithiophene 5-(3-Penten-1-ynyl)-bithiophene cis-Bisocimenone Dehydrojuvibione Unidentified dimethylvinylketone deriv. trans-Bisocimenone Terthiophene Unidentified dimethylvinylketone deriv. 5-Methylterthiophene Total Identified
% 0.1 0.5 0.4 0.1 tr tr 0.8 1.8 0.8 0.7 0.2 20.6 tr 74.2
antifungal activity of EOs and their components against C. cucumerinum [30]. In conclusion, EOTM was characterized by a high content of oxygenated monoterpenes with ocimenone as the main active constituent. The use of EOTM is promising as an antioxidant, cytotoxic and antibacterial agent against MRSA, a very significant public health concern. If further studies show safety and efficiency of EOTM, this may represent a valuable weapon against MRSA. Experimental Plant materials: The leaves of Tagetes minuta L. (Asteraceae) were collected during the flowering stage in May 2009, from Dhamar province, Yemen. The plant was identified by Hassan M. Ibrahim of the Botany Department, Faculty of Sciences, Sana’a University. A voucher specimen (YMP-comp-20) has been deposited at the Pharmacognosy Department, Sana’a University, Yemen. Essential oil distillation: Dried leaves from T. minuta were hydrodistilled for 3 h in a Clevenger type apparatus according to the European Pharmacopoeia method [31]. The obtained oil was subsequently dried over anhydrous Na2SO4 and kept at 4C until analysis. Gas chromatographic-mass spectral analysis: EOTM was analyzed by GC-MS using an Agilent 6890 GC with an Agilent 5973 mass selective detector [MSD, operated in the EI mode (electron energy = 70 eV), scan range = 45-400 amu, and scan rate = 3.99 scans/sec], and an Agilent ChemStation data system. The GC column was an HP-5ms fused silica capillary with a (5% phenyl)polymethylsiloxane stationary phase, film thickness of 0.25 μm, a length of 30 m, and an internal diameter of 0.25 mm. The carrier gas was helium with a column head pressure of 48.7 kPa and a flow rate of 1.0 mL/min. Inlet temperature was 200°C and interface temperature was 280°C. The GC oven temperature program was used as follows: 40°C initial temperature, held for 10 min; increased at 3°C/min to 200°C; increased 2°/min to 220°C. A 1%, w/v, solution of the sample in CH2Cl2 was prepared and 1 μL was injected using a splitless injection technique. Identification of the oil components was based on their retention indices determined by reference to a homologous series of n-alkanes (C8-C40), and by comparison of their mass spectral fragmentation patterns with those reported in the literature [32], and stored on the MS library [NIST database (G1036A, revision D.01.00)/ChemStation data system (G1701CA, version C.00.01.080]. The percentages of each component are reported as raw percentages based on total ion current without standardization. Cytotoxicity test: MCF7 cells were grown in a 5% CO2 environment at 37°C in RPMI 1640 medium with L-glutamine and NaHCO3, supplemented with 10% fetal bovine serum, 100,000 units penicillin
Tagetes minuta oil
and 10.0 mg streptomycin per L of medium, pH 7.3. Cells were plated into 96-well cell culture plates at 2.5 ×104 cells per well. The volume in each well was 100 μL. After 48 h, supernatant fluid was removed by suction and replaced with 100 μL growth medium containing 1.0 μL of a DMSO solution of the essential oil, giving final concentrations of either 100 or 50 μg/mL for each well. Solutions were added to wells in 4 replicates. Medium controls and DMSO controls (10 μL DMSO/mL) were used. Tingenone [33] was used as a positive control. After the addition of oils, plates were incubated for 48 h at 37°C in 5% CO2; medium was then removed by suction, and 100 μL of fresh medium was added to each well. In order to establish percent kill rates, the MTT assay for cell viability was carried out [34]. After colorimetric readings were recorded (using a Molecular Devices Spectra MAX Plus microplate reader, 570 nm), average absorbances, standard deviations, and IC50 values (median inhibitory concentrations) were determined using the ReedMuench method [35,36]. Antimicrobial activity: The antimicrobial activity of the essential oil was evaluated by the agar disc-diffusion method, as previously described [23]. The microorganisms used were Escherichia coli ATCC 10536, Pseudomonas aeruginosa ATCC 25619, Staphylococcus aureus ATCC 29737, Bacillus subtilis ATCC 6633, methicillin-resistant Staphylococcus aureus MRSA and Candida albicans ATCC 2091. Müller-Hinton Agar (MHA) (Merck, Darmstadt, Germany) was used for bacterial culture at 37ºC. Sabouraud dextrose agar (Merck, Darmstadt, Germany) was used to cultivate Candida albicans. Determination of antioxidant activity: For the preliminary test, analytical TLC on silica gel plates was developed under appropriate conditions after application of 5 μL of oil solution, dried and sprayed with DPPH solution (0.2%, MeOH). Five min later, active compounds appeared as yellow spots against a purple background. Estimation of a radical scavenging effect was carried out by using a DPPH free radical scavenger assay in 96 well microtiter plates (MTP) according to the modified method [37]. A solution of DPPH (Sigma-Aldrich, Germany) was prepared by dissolving 5 mg DPPH in 2 mL of methanol, and the solution was kept in the dark at 4°C until use. Stock solutions of the samples were prepared at 2 mg/mL and diluted to different concentrations. Methanolic DPPH solution (5 μL) was added to each well. The plate was shaken for 2 min to ensure thorough mixing before being wrapped in aluminum foil and stored in the dark. A methanolic solution of DPPH served as control. After 30 min, the optical density (OD) of the solution was measured at a wavelength of 517nm using a microtiter plate ELISA
Natural Product Communications Vol. 9 (0) 2014 3
reader (Thermo, Finland) and the percentage decolorization calculated as an indication of the antioxidant activity of a sample. Each experiment was made at least in triplicate and IC50 values were calculated using Origin software. Ascorbic acid (SigmaAldrich, Germany) was used as a positive control. DPPH scavenging activity is usually presented by an IC50 value, defined as the concentration of the antioxidant needed to scavenge 50% of DPPH present in the test solution. Antifungal assay: Initial tests of fungicidal activity were carried out by the method of Gottstein et al. [38, 39]. This semi-quantitative test allows a relative estimation of the activity of compounds with similar diffusion characteristics. The phytopathogenic fungus Cladosporium cucumerinum Ell. et Arth. was used as test organism. Antifungal tests were performed on TLC plates (glass plates, 20 x 20 cm, silica gel 60 HF254, thickness 0.5 mm (Merck). The essential oil was applied to the TLC plate using microsyringes at concentrations of 50 μg, 100 μg, 200 μg and 400μg as individual spots (diameter 1 cm, corresponding to a surface of 78 mm2). Subsequently, the plates were dried in a warm air stream in order to evaporate remaining solvent. Each plate was covered with approx. 10 mL spore suspension of C. cucumerinum (approx. 2.5 × 106 spores/mL). Afterwards the plates were dried at room temperature for some mins, placed into a TLC chamber lined with water-soaked filter paper and covered. After 48 h incubation at 25°C in an incubator a dark grey mycelium had developed. Benomyl (Riedelde- Haen, Germany) was used as positive control. The evaluation of the antifungal effect was based on the area of the white spots corresponding to fungus growth inhibition. Three independent tests were performed and an average of the observations was calculated (n = 3). Bond dissociation enthalpies: All calculations were performed using the Spartan ’14 program package [40]. In this study, bond dissociation energies for R–H → R• + H• were calculated using the hybrid B3LPY functional [41]. The 6-31+G* basis set [42] was used for geometry optimization. Single-point energy calculations and computation of harmonic vibrational frequencies using the 631+G* optimized geometries were performed with the 6-311++G** basis set for both parent molecules and their corresponding radicals in order to characterize all of their conformations as minima and to evaluate the zero-point energy (ZPE) corrections. The total enthalpy at 298 K consisted of the thermal correction to the enthalpy and the B3LYP calculated ZPE values.
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