Chemotaxonomic Characterization and in-Vitro Antimicrobial and Cytotoxic Activities of the Leaf Essential Oil of Curcuma longa Grown in Southern Nigeria

Curcuma longa (turmeric) has been used in Chinese traditional medicine and Ayurvedic medicine for many years. Methods: The leaf essential oil of C. longa from southern Nigeria was obtained by hydrodistillation and analyzed by gas chromatography–mass spectrometry (GC-MS). The essential oil was screened for in vitro antibacterial, antifungal, and cytotoxic activities. The major components in C. longa leaf oil were ar-turmerone (63.4%), α-turmerone (13.7%), and β-turmerone (12.6%). A cluster analysis has revealed this to be a new essential oil chemotype of C. longa. The leaf oil showed notable antibacterial activity to Bacillus cereus and Staphylococcus aureus, antifungal activity to Aspergillus niger, and cytotoxic activity to Hs 578T (breast tumor) and PC-3 (prostate tumor) cells. The ar-turmerone-rich leaf essential oil of C. longa from Nigeria has shown potent biological activity and therapeutic promise.


Introduction
Curcuma longa L. (syn. C. domestica Vahl.) is a perennial rhizomatous herb of the family Zingiberaceae. The rhizome is the source of turmeric, which has use as a condiment and coloring agent in medicines, confectionery and curry powder [1]. Turmeric has a long history of traditional use in the Chinese and Ayurvedic systems of medicine, particularly as an anti-inflammatory agent [2], and for the treatment of biliary disorders, anorexia, coryza, cough, diabetic wounds, hepatic disorders, rheumatism and sinusitis [1]. The powdered rhizome is used externally as an antiseptic [3] and taken internally to cure gastritis [4]. The rhizome volatiles have been extensively studied (see, for example [5][6][7][8]), and there have been previous reports on the leaf essential oil compositions [9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26]. Research has shown that the quantitative essential oil composition is widely influenced by the genotype, ontogenic development, and environmental and growing conditions [27][28][29]. It also implies the possibility of different medicinal uses of the same plant species grown in different regions [30]. This paper reports the chemical constitution of the leaf essential oil of C. longa grown in the southern part of Nigeria and its antimicrobial effects and anti-neoplastic potential.

Plant Material
Fully grown leaves of C. longa were collected from plants cultivated in the village of Mbaiso, Ikot Ekpene Local Government Area of Akwa Ibom State, Nigeria, in the month of October. Plant materials were authenticated by F. Usang of the Forest Research Institute of Nigeria (FRIN), Ibadan, where voucher specimens were deposited under FHI 106920. The essential oil was obtained by hydrodistillation (4 h) of the air-dried plant leaves using a Clevenger-type apparatus in accordance with the British Pharmacopoeia [31]. The leaf oil was dried over sodium sulfate and kept in refrigeration (4 °C) after estimation of percentage yield.

Gas Chromatographic-Mass Spectral Analysis
The essential oil was subjected to gas chromatography-mass spectrometry (GC-MS) analysis on an Agilent system consisting of a model 6890 gas chromatograph, a model 5973 mass selective detector (MSD), and an Agilent ChemStation data system. The GC column was a Hewlett Packard (HP-5ms) fused silica capillary with a (5% phenyl)-methyl polysiloxane stationary phase (30 m × 0.25 μm film thickness). The carrier gas was helium with a column head pressure of 7.07 psi and flow rate of 1.0 mL/min. Inlet temperature was 200 °C and MSD detector 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 °C/min to 220 °C. The sample was dissolved in CH2Cl2, and 1 μL was injected using a splitless injection technique.
Identification of individual constituents of the essential oils was achieved based on their retention indices (determined with a reference to a homologous series of normal alkanes) and by comparison of their mass spectral fragmentation patterns (National Institute of Standards and Technology, NIST database/ChemStation data system, http://www.agilent.com/chem/nds) and with the literature [32].

Antibacterial Screening
Curcuma longa leaf oil was screened for antibacterial activity against Bacillus cereus (ATCC No. 14579), Staphylococcus aureus (ATCC No. 29213), Pseudomonas aeruginosa (ATCC No. 27853), and Escherichia coli (ATCC No. 10798). Minimum inhibitory concentrations (MICs) were determined using the microbroth dilution technique [33]. Dilutions of the samples were prepared in cation-adjusted Mueller Hinton broth (CAMHB) beginning with 50 μL of 1% w/w solutions of samples in dimethylsulfoxide (DMSO) plus 50 μL CAMHB. The sample solutions were serially diluted (1:1) in CAMHB in 96-well plates to give concentrations of 2500, 1250, 625, 313, 156, 78, 39, and 19.5 μg/mL. 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; the final minimum inhibitory concentration (MIC) was determined as the lowest concentration without turbidity. Gentamicin was used as a positive antibiotic control; DMSO was used as a negative control.

Antifungal Screening
Antifungal activity was determined, as described above for bacteria, using Candida albicans (ATCC No.10231) in a yeast-nitrogen base growth medium with approximately 7.5 × 10 7 CFU/mL. Amphotericin B was used as the positive control. An additional test for antifungal activity against Aspergillus niger (ATCC No. 16888) was determined as above using yeast mold (YM) broth inoculated with A. niger hyphal culture diluted to a McFarland turbidity of 1.0. Amphotericin B was the positive control.

Cell Culture
Human Hs578T breast ductal carcinoma cells (ATCC No. HTB-129) [34] were grown in a 3% CO2 environment at 37 °C in DMEM with 4500 mg glucose per liter of medium, supplemented with 10% fetal bovine serum, 10 μg bovine insulin, 100,000 units penicillin and 10.0 mg streptomycin per liter of medium, and buffered with 44 mM NaHCO3, pH 7.35.

Cytotoxicity Screening
Hs578T cells were plated into 96-well cell culture plates at 1.0 × 10 5 cells per well and PC-3 cells at 1.9 × 10 4 cells per well. The volume in each well was 100 μL for both cell types. After 48 h, supernatant fluid was removed by suction and replaced with100 μL growth medium containing either 2.5 or 1.0 μL of DMSO solution of oils (1% w/w in DMSO), giving a final concentration of 250 or 100 μg/mL, respectively, for each oil. Hs578T cells were tested with final concentrations at 250 μg/mL and PC-3 at a final concentration of 100 μg/mL. Solutions were added to wells in four replicates. Medium controls and DMSO controls (25 or 10 μL DMSO/mL) were used. Tingenone (250 or 100 μg/mL) was used as a positive control [36]. After the addition of the sample, plates were incubated for 48 h at 37 °C; 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 Cell Titer 96 ® AQueous Non-Radioactive Cell Proliferation assay was performed [37]. After colorimetric readings were recorded (using a Molecular Devices SpectraMAX Plus microplate reader, Sunnyvale, CA, USA, 490 nm), average absorbances, standard deviations and percent kill ratios (% killoil/% killDMSO) were calculated.
The results from the antimicrobial screening and the cytotoxicity screening of the leaf oil of C. longa are summarized in Table 2. The leaf oil demonstrated particularly strong antibacterial activity against the Gram-positive organisms B. cereus and S. aureus (MIC = 78 μg/mL) and antifungal activity against A. niger (MIC = 19.5 μg/mL). The antimicrobial activities observed in C. longa leaf oil can be attributed to the major component ar-turmerone. Consistent with these results, ar-turmerone had previously shown strong antibacterial activity against Clostridium perfringens and weak inhibitory activity against Escherichia coli [38]. The antimicrobial results in this study showed that Gram-positive bacteria were more sensitive to the leaf oil of C. longa (Table 2). This result is consistent with those reported by other workers [39][40][41][42]. According to Alzoreky and Nakahara [42], Gram-negative bacteria are less susceptible to plant extracts due to lipopolysaccharides in their outer membrane. Similarly, turmerone-rich essential oils have shown antifungal activity [43,44], and ar-turmerone itself has shown antifungal activity against Aspergillus flavus [45]. The observed in vitro cytotoxicity against breast tumor (Hs578T) and prostate tumor (PC-3) cells is also consistent with the previously observed cytotoxicity of ar-turmerone. Thus, ar-turmerone has shown cytotoxic activity to HL-60, K-562, L-1210 [46], HeLa [47], U-937, and RBL-2H3 cells [48]. Furthermore, ar-turmerone has been shown to induce apoptosis coupled with DNA fragmentation in several cell lines [48][49][50][51].

Conclusions
High concentrations of turmerones were identified in the leaf essential oil of Curcuma longa from Mbaise, Nigeria. The composition of the leaf oil represents a new chemotype of this plant. The significant antibacterial, antifungal, and cytotoxic activities of the leaf oil may be attributed to the turmerone content. Due to the turmerone-rich essential oil, this chemotype of C. longa from southern Nigeria has promising therapeutic potential.