Antimicrobial, Antioxidant, and Cytotoxic Activities of Ocimum forskolei and Teucrium yemense (Lamiaceae) Essential Oils

Background: Ocimum forskolei and Teucrium yemense (Lamiaceae) are used in traditional medicine in Yemen. Methods: The chemical composition, antimicrobial, antioxidant and cytotoxic activities of the essential oils isolated from the leaves of Ocimum forskolei Benth. (EOOF) and two different populations of Teucrium yemense Deflers., one collected from Dhamar province (EOTY-d), and another collected from Taiz (EOTY-t) were investigated. The antimicrobial activities of the oils were evaluated against several microorganisms with the disc diffusion test or the broth microdilution test. The essential oils were screened for in-vitro cytotoxic activity against human tumor cells. EOOF and EOTY-d were screened for free-radical-inhibitory activity using the DPPH radical scavenging assay. Results: Sixty-four compounds were identified in (EOOF) representing 100% of the oil content with endo-fenchol (31.1%), fenchone (12.2%), τ-cadinol (12.2%), and methyl (E)-cinnamate (5.1%) as the major compounds. In EOTY-d, 67 compounds were identified, which made up 91% of the total oil. The most abundant constituents were (E)-caryophyllene (11.2%), α-humulene (4.0.%), γ-selinene (5.5%), 7-epi-α-selinene (20.1%), and caryophyllene oxide (20.1%), while the major compounds in EOTY-t were α-pinene (6.6%), (E)-caryophyllene (19.1%) α-humulene (6.4%), δ-cadinene (6.5%), caryophyllene oxide (4.3%), α-cadinol (9.5%), and shyobunol (4.6%). The most sensitive microorganisms for EOOF were B. subtilis, S. aureus, and C. albicans with inhibition zones of 34, 16, and 24 mm and MIC values of, 4.3 mg/mL, 4.3 mg/mL, and 8.6 mg/mL, respectively. EOTY-t showed antimicrobial activity against S. aureus, B. cereus, A. niger, and B. cinerea with MIC values of 0.156, 0.156, 0.313 and 0.313 mg/mL, respectively. Neither essential oil showed remarkable radical inhibition (IC50 = 31.55 and 31.41 μL/mL). EOTY-d was active against HT-29 human colorectal adenocarcinoma cell lines with IC50 = 43.7 μg/mL. Consistent with this, EOTY-t was active against both MCF-7 and MDA-MB-231 human breast adenocarcinoma cells. Conclusions: The antimicrobial activity of Ocimum forskolei essential oil against B. subtilis and C. albicans is consistent with its traditional use in Yemeni traditional medicine to treat skin infections. Both O. forskolei and T. yemense show wide variations in their respective essential oil compositions; there remains a need to investigate both species botanically, genetically, and phytochemically more comprehensively.


Introduction
Ocimum forskolei Benth. (Lamiaceae) is one of about 65 tropical and subtropical species of Ocimum, seven of which are found in Yemen, and these include O. basilicum L., O. tenuiflorum L., O. suave Willd., O. spicatum Deflers, O. gratissimum L., and O. forskolei [1]. Several species of Ocimum are aromatic and used as flavoring agents, insect repellents, and traditional medicines [2]. O. forskolei ranges in East Africa from Egypt, south to Kenya [3], and the southern Arabian Peninsula, including Yemen [4], Oman [5], and the United Arab Emirates (UAE) [6]. O. forskolei is used traditionally in Yemen as a cosmetic, to relieve fever, and to treat skin infections [7]. In UAE, crushed leaves of O. forskolei are used to treat headaches, colds, and ear aches, while the juice is used as eye drops or for insect bites [6]. The plant is used in Eritrea as a mosquito repellent and has demonstrated repellent activity [8]. The dichloromethane extract of O. forskolei has shown weak antibacterial activity against Gram-positive bacteria, while the methanol extract showed minimal radical-scavenging activity [7]. Previous investigations of O. forskolei have shown the essential oil to have weak antioxidant [9] and nematicidal activity [10], and better activities against bacteria and dermatophytes [11].
Teucrium is a large genus belonging to the Lamiaceae which contains about 250 species that are widespread over the world, but are most common in Mediterranean climates [2]. Teucrium species endemic to Yemen are T. yemense Deflers, T. socotranum (Balf. f.) Vierh., and T. balfourii (Balf. f.) Vierh. Plants from this genus are perennial in the form of herbs, shrubs, and sub-shrubs. Teucrium species are widely used in Yemeni folk medicine as antispasmodics and insect repellants [12]. Teucrium species are rich in essential oil and are valued as ornamental plants. Teucrium yemense is mostly found in Djibouti, Ethiopia, Saudi Arabia, Sudan, and Yemen. It is a perennial aromatic plant possessing sessile oblanceolate leaves and dense terminal heads of white flowers [12]. Essential oil studies of various species of Teucrium have shown α-humulene, δ-cadinene, (E)-caryophyllene, α-pinene, β-pinene, linalool, 3-octanol, α-cadinol, caryophyllene oxide, 8-cedren-13-ol, and (E)-β-farnesene to be the major components of the essential oils. A wide range of biological activity has been reported for Teucrium species, some of which are attributed to the high content of essential oil in them. They possess antifungal, antibacterial, larvicidal, antispasmodic, antioxidant, anti-inflammatory, antiulcer, hypoglycemic, antiacetylcholinesterase, and hepatoprotective activities [12].

Plant Materials
The leaves of O. forskolei and T. yemense were collected from several mature plants during the flowering stage in May 2010 from Dhamar province, Yemen. The plants were identified by Hassan M. Ibrahim of the Botany Department, Faculty of Sciences, Sana'a University. Voucher specimens (YMP Lam-33 and YMP Lam-36) have been deposited at the Pharmacognosy Department, Sana'a University, Yemen. Another sample of T. yemense leaves was collected in February 2012 from Taiz town, Yemen. For each sample, the dried leaves (100 g) were hydrodistilled for 3 h in a Clevenger type apparatus according to the European Pharmacopoeia method [13]. The obtained oils were subsequently dried over anhydrous Na 2 SO 4 and kept at 4 • C until analysis.

Gas Chromatography-Mass Spectrometry (GC-MS)
O. forskolei and T. yemense essential oils were 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 = 40-400 amu, and scan rate = 3.99 scans/s), 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 CH 2 Cl 2 was prepared and 1 µL was injected using a splitless injection technique. Identification of the oil components was based on their retention indices as determined by reference to a homologous series of n-alkanes (C 8 -C 40 ), and by comparison of their mass spectral fragmentation patterns with those reported in the literature [14], 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.

Radical Scavenging Assay
For the preliminary test, analytical TLC (thin-layer chromatography) 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 minutes later, active compounds appeared as yellow spots against a purple background. Estimation of a radical scavenging effect was carried out by using a DPPH (2,2-diphenyl-1-picrylhydrazyl) free radical scavenger assay in 96-well microtiter plates (MTPs) according to a modified method [15]. A solution of DPPH (Sigma-Aldrich, Munich, 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 517 nm using a microtiter plate ELISA reader (Thermo Scientific, Helsinki, Finland) and the percentage decolorization was calculated as an indication of the antioxidant activity of a sample. Each experiment was carried out in triplicate and IC 50 (median inhibitory concentration) values were calculated using Origin software (version 7, OriginLab Corp., Northampton, MA, USA). Ascorbic acid (Sigma-Aldrich, Munich, Germany) was used as a positive control.
T. yemense (EOTY-t) essential oil was screened for antibacterial activity against the Gram-positive bacteria Bacillus cereus (ATCC No. 14579) and Staphylococcus aureus (ATCC No. 29213) and the Gram-negative bacteria Pseudomonas aeruginosa (ATCC No. 27853) and Escherichia coli (ATCC No. 10798). The minimum inhibitory concentrations (MICs) were determined using the microbroth dilution technique [17]. Dilutions of the essential oils were prepared in cation-adjusted Mueller Hinton broth (CAMHB) beginning with 50 µL of 1% w/w solutions of oils in DMSO plus 50 µL of CAMHB. The oil solutions were serially diluted (1:1) in CAMHB in 96-well plates. Organisms at a concentration of approximately 1.5 × 10 8 colony-forming units (CFU)/mL were added to each well. Plates were incubated at 37 • C for 24 h, and the final minimum inhibitory concentration (MIC) was determined as the lowest concentration without turbidity. Gentamicin was used as a positive antibiotic control, while DMSO was used as a negative control [17].
The microbroth dilution technique was also applied to determine the antifungal activity of EOTY-t essential oil against Aspergillus niger (ATCC No. 16888), Botrytis cinerea (ATCC No. 126943), and Candida albicans (ATCC No. 90028). For Aspergillus niger and Candida albicans, dilutions of the essential oils were prepared in YM broth beginning with 50 µL of 1% w/w solutions of oils in DMSO plus 50 µL of YM broth. The oil solutions were serially diluted (1:1) in YM in 96-well plates. YM broth inoculated with A. niger hyphal and Candida albicans culture diluted to a McFarland turbidity of 1.0 were added to each well. Plates were incubated at 37 • C for 24 h; amphotericin B was the positive control, while DMSO was used as the negative control. The same process was used for B. cinerea as well, except that the inoculum suspension was prepared in potato dextrose broth (PDB) and the inoculum size was adjusted with respective broth medium between 1.0 × 10 6 and 5.0 × 10 6 spores/mL by maintaining the optical density (OD) between 0.1 and 0.2 at 625 nm. Dilutions were carried out in the fungal growth medium (malt extract broth). Combination antifungal cyprodinil-fludioxonil was used as the positive control and DMSO was used as the negative control. The 96-well plates were incubated at 25 ± 2 • C for 48 h.

Cytotoxicity Assays
EOOF and EOTY-d were screened for cytotoxicity on HT-29 (human colorectal adenocarcinoma) cells using the XTT assay [18]. HT29 cells were grown in a 5% CO 2 environment at 37 • C in RPMI 1640 medium without L-glutamine, supplemented with 10% fetal bovine serum, 1% (200 mM) L-alanyl-L-glutamine, and 1.6% HEPES (1 M). Cells were plated into 96-well cell culture plates at 1.5 × 10 3 cells (HT29) per well. The volume in each well was 100 µL. After 24 h, supernatant fluid was removed by suction and replaced with 100 µL growth medium containing 0.1 µL of a DMSO solution of the essential oil, giving final concentrations of 100, 50, 25, and 12.5 µg/mL for each well. Solutions were added to wells in three replicates. Medium controls without cells and DMSO controls (0.5 µL DMSO/mL) were used. Digitonin (125 µM) was used as a positive control [19]. After the addition of oils, plates were incubated for 72 h at 37 • C in 5% CO 2 ; medium was then removed by suction, and 100 µL of fresh medium was added to each well. In order to establish percent growth inhibition rates, the XTT assay for cell viability was carried out according to Cell Proliferation Kit II protocol, (Roche Diagnostics, Mannheim, Germany). After colorimetric readings were recorded (Molecular Devices SpectraMAX M5 microplate reader, 490 nm), average absorbance, growth inhibition, and standard deviations were determined.
EOTY-t was screened for cytotoxic activity against MCF-7 (human breast adenocarcinoma, estrogen receptor positive) and MDA-MB-231 (human breast adenocarcinoma, estrogen receptor negative) cells using MTT-based cytotoxicity assay [20]. Human MCF-7 (ATCC No. HTB-22) and MDA-MB-231 (ATCC No. HTB-26) breast adenocarcinoma cells were grown in a 3% CO 2 environment at 37 • C in RPMI-1640 medium, supplemented with 10% fetal bovine serum, 100,000 units penicillin, and 10.0 mg streptomycin per liter of medium, 15mM of HEPES, and buffered with 26.7 mM NaHCO 3 , pH 7.35. Cells were plated into 96-well cell culture plates at 2.5 × 10 4 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 DMSO solution of the essential oil (1% w/w in DMSO), giving a final concentration of 100 µg/mL for each well. Solutions were added to wells in four replicates. Medium controls and DMSO controls (10 µL DMSO/mL) were used. Tingenone was used as a positive control [21]. After the addition of compounds, plates were incubated for 48 h at 37 • C in 5% CO 2 ; 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 [22]. After colorimetric readings were recorded (570 nm, using a SpectraMAX Plus microplate reader, Molecular Devices, Sunnyvale, CA, USA), percent kill was calculated.

Free Radical Scavenging
O. forskolei and T. yemense leaf oils from Dhamar (2010) were screened for free-radical-inhibitory activity using the DPPH· radical scavenging assay. Neither essential oil showed remarkable radical inhibition (IC 50 = 31.55 and 31.41 µL/mL, respectively). This is not surprising; neither essential oil has high concentrations of phenolic compounds such a carvacrol, thymol, or eugenol, which are known to be excellent radical scavenging agents [47]. endo-Fenchol, the major component in O. forskolei oil, (E)-caryophyllene and α-humulene, major components in T. yemense oil, have shown only weak antioxidant activities [48].

Antimicrobial Activity
O. forskolei and T. yemense essential oils were screened for antibacterial and antifungal activity ( Table 3). The disc diffusion assay showed O. forskolei to have weak antibacterial activity against Staphylococcus aureus, but stronger activity against Bacillus subtilis with an inhibition zone of 34 mm, and antifungal activity against Candida albicans with an inhibition zone of 24 mm. EOTY-d was virtually devoid of antimicrobial activity. Using the broth microdilution assay, EOTY-t showed antibacterial activity against S. aureus and B. cereus (MIC = 156 µg/mL), as well as antifungal activity against Aspergillus niger and Botrytis cinerea with MIC of 313 µg/mL. The antibacterial activity of O. forskolei essential oil against B. subtilis and S. aureus is likely due to methyl (E)-cinnamate and τ-cadinol. Methyl cinnamate has shown antibacterial activity against S. aureus [49] while τ-cadinol has shown activity against B. cereus and S. aureus [50]. Apparently neither fenchone nor fenchol are antibacterial [51] and fenchone is also not antifungal [52].

Cytotoxic Activity
O. forskolei leaf oil was screened for cytotoxic activity against HT-29 human colorectal adenocarcinoma cells, but was inactive (Table 4). EOTY-d was active against this cell line with IC 50 = 43.7 µg/mL. Consistent with this, EOTY-t was active against both MCF-7 and MDA-MB-231 human breast adenocarcinoma cells. a "na" = not active (0% kill at 100 µg/mL); b "nt" = not tested.

Conclusions
In summary, this preliminary phytochemical and bioactivity investigation has described the composition of Ocimum forskolei leaf essential oil from Yemen for the first time, and has shown the leaf oil to present moderate antimicrobial properties against B. subtilis and C. albicans. This activity is consistent with its uses in Yemeni traditional medicine for some skin infections. The variation in the chemical compositions and biological activities of T. yemense leaf essential oils can be due to the different environmental factors such as altitude, latitude, or time of collection. Both populations of T. yemense showed good cytotoxic activity. There remains the need to follow up this preliminary study and investigate both species more comprehensively in terms of botanical, genetic, phytochemical, and biological properties. This is particularly important because much primary health care in Yemen still relies on traditional herbal practices.