Chemical Composition and Antimicrobial Activity of Flower Essential Oil of Jacaranda acutifolia Juss. against Food-Borne Pathogens

Aims: To investigate the chemical composition and antimicrobial activity of the hydrodistilled flower essential oil of Jacaranda acutifolia Juss. (Bignoniaceae) to validate some of its ethnopharmacologial uses such as treatment of wounds and dermatitis. Study Design: Volatile oil isolation, component identification and antimicrobial activity. Place and Duration of Study: Faculty of Pharmacy, Ain Shams University, and the flowers were collected on 15 April 2012 and the study is completed within four months. Methodology: The essential oil of the flowers of Jacaranda acutifolia was extracted by hydrodistillation, analysed by capillary gas chromatography (GC/FID) and gas chromatography– mass spectrometry (GC/MS). Antimicrobial activity was studied in vitro; this included both antibacterial activity against food-borne pathogens and antifungal activity using agar diffusion method. Original Research Article Mostafa et al.; EJMP, 6(2):62-69, 2015; Article no.EJMP.2015.043 63 Results: Forty six components, comprising almost 81.36 of the total peak area, were identified in the analysis. The main components were n-dodecanoic acid (17.48%), n-tetradecanoic acid (15.59%), n-hexadecanoic acid (10.98%), hexahydrofarnesyl acetone (8.2%), n-decanoic acid (7.9%), and nonacosane (7.71%). According to the observed inhibition zone, which ranged from 13 to 26.5 mm, the oil showed significantly high in-vitro antimicrobial activity against Staphylococcus aureus, Escherichia coli and Candida albicans with minimum inhibitory concentration (MIC) values ranging from 0.09 up to 1.09 mg/ml, and moderate antimicrobial activity against Salmonella typhimurium and Shigella flexneri. The potency of the oil was calculated as compared to standard antibiotics, and 1 mg oil was equivalent to 1.05 μg penicillin against S. aureus; 2.72 μg nystatin against C. albicans; 13.55, 1.3 and 272 μg gentamicin against E. coli, Samonella and Shigella species respectively. Conclusion: The essential oil of Jacaranda acutifolia exhibited promising antimicrobial activity, and this makes its local traditional uses rational.


INTRODUCTION
Jacaranda is a member of Bignoniaceae Juss., it contains 49 species around the world [1]. The Bignoniaceae is especially common in the tropics of South America and occurs in habitats consisting of mainly woody trees, shrubs, lianas and rarely herbaceous plants. Many woody representatives of Bignoniaceae as Jacaranda are well known for their use in the timber industry [1]. Chemical constituents recognized in the family are naphthoquinones of the lapachol type, iridoid glucosides, alkaloids, flavones, triterpenes, polyphenols, tannins and seed oils [2].
Chemical studies on the constituents of Jacaranda have only been reported for six species: Jacaranda acutifolia, Jacaranda caucana, Jacaranda copaia, Jacaranda decurrens, Jacaranda filicifolia and Jacaranda mimosifolia. The compounds have been identified as triterpenes, quinones, flavonoids, fatty acids, acetosides and, recently, a novel phenylethanoid dimmer [1]. Members of the genus Jacaranda possess significant pharmacological potential and promising activities of extracts in the context of ethnomedicinal knowledge, especially in the field of tropical diseases, skin problems and venereal illnesses. Jacaranda species might serve as an important source of medicine among people living in tropical regions [1].
Though antimicrobial activity of other species such as Jacaranda cuspidifolia Mart. has been reported, [3] yet, to our knowledge, nothing could be traced regarding the chemical composition and the biological activity of the essential oil obtained from Jacaranda acutifolia. We present here results of GC-MS analysis of the essential oil of flowers of Jacaranda acutifolia Juss. (Bignoniaceae), for the first time, to acquire a comprehensive knowledge on the volatile composition of the leaves. Also, in this study, the antimicrobial activity was evaluated against fungal and food-borne bacteria, to validate some of its ethnopharmacologial uses in the aforementioned disorders.

Isolation of Volatile Components
Fresh plant materials (800 g) were hydrodistillation in a Clevenger-type apparatus [10], using n-hexane as a collecting solvent, until there is no significant increase in volume of oil collected within 4 hours. The yield of the pale yellow oil was 0.01%. The oil was dried over anhydrous sodium sulfate and kept in separated sealed vials at -30ºC for analysis.

GC/FID Analysis
The GC analyses were carried out on a Varian 3400 equipped with an DB-5 fused bonded column (30 m × 0.25 mm × 0.25 μm) (Ohio Valley, Marietta, USA) and FID detector; carrier gas was helium (2 ml/min); the operating conditions were: initial temperature 45ºC, 2 min isothermal, 300ºC, 4ºC / min 300ºC, then 20 min isothermal. Detector and injector temperatures were 300 and 250ºC, respectively. The split ratio was 1:20. PeakSimple 2000 chromatography data system (SRI Instruments, Torrance, USA) was used for recording and integrating of the chromatograms. Average areas under the peaks of three independent chromatographic runs were used for calculating the % composition of each component.

GC/MS Analysis
The analyses were carried out on a Hewlett-Packard gas chromatograph (GC 5890 II; Hewlett-Packard GmbH, Bad Homburg, Germany) equipped with the same column and conditions as for the GC/FID. The capillary column was directly coupled to a quadrupole mass spectrometer (SSQ 7000; Thermo-Finnigan, Bremen, Germany). The injector temperature was 250ºC. Helium carrier gas flow rate was 2 ml/min. All the mass spectra were recorded with the following conditions: filament emission current, 100 mA; electron energy, 70 eV; ion source, 175ºC; diluted samples (0.5% v/v) were injected with split mode (split ratio,

Microbial Strains
The essential oil of Jacaranda acutifolia flowers was tested against Staphylococcus aureus ATCC 2821 (Gram positive bacteria); Escherichia coli ATCC 25922, clinically isolate microorganisms; Shigella flexneri and Salmonella typhimurium (Gram negative bacteria); and Candida albicans ATCC 60193 (fungus). The microorganisms were obtained from the stock cultures of the Department of Microbiology, Faculty of Pharmacy, Ain Shams University.

Screening for Antimicrobial Activity
Antimicrobial activity was assayed via the agar diffusion method [6]. Bacterial and fungal inocula in nutrient agar slants were directly suspended in 10 ml saline. Aliquots (0.25 ml) of bacterial or fungal inocula were spread on the surface of nutrient agar (Lab M, England) using sterile rod to obtain uniform microbial growth on the plates. Small cups (10 mm diameter) were taken out of the inoculated agar surfaces. Each cup was filled accurately with 100 µl solution (30 mg oil was dissolved in 1 ml dimethylsulphoxide (DMSO, Sigma Aldrich, Germany)) as well as DMSO as a control. The plates were incubated overnight at temperature 37ºC for bacteria and 28ºC for fungi. Clear zones of inhibition were developed and diameter of zones of inhibition were measured (in mm) and compared to standard antibiotics [penicillin for Gram (+), gentamicin for Gram (-), nystatin for yeast]. Each test was performed in duplicate and the mean values were calculated.

Determination of the Minimum Inhibitory Concentration
The minimum inhibitory concentration (MIC) was calculated through agar diffusion method [7]. Cups were made in inoculated agar surfaces. Serial two fold dilution of the oil in DMSO was carried out. Each cup is filled with a different concentration of the oil to obtain final concentration range 0.5-3.2 mg oil in each cup. The plates were incubated overnight at temperature 37ºC for bacteria and 28ºC for fungi. Clear zones of inhibition were developed and diameter of zones of inhibition were measured (in mm) and plotted in y-axis against log concentration in x-axis to obtain a standard calibration curve from which log MIC was determined by extrapolation at cup diameter (10 mm). Same method was applied with the above mentioned antibiotics.

Determination of the Antimicrobial Potency
Using agar diffusion method [8], four cups were made in the inoculated agar and removed with the help of sterilized loop. The two opposite wells were filled with the working standard of 1:2 dilutions and marked as S 1 and S 2 , respectively. The remaining two were filled with the sample whose potency was to be determined in the same dilution (1:2) and marked T 1 and T 2 respectively. One hundred micro liters of standards as well as samples were poured with the help of micropipette in the digged holes. The plates were then placed carefully (to avoid spreading of solution due to tilting of the plates) in incubator for 18-24 hours at temperature 37°C for bacteria and 28°C for fungi. Clear zones of inhibition were developed and diameter of zones of inhibition were measured and compared with the known standard.
The % potency of the antimicrobial potency of the tested sample was calculated by the following formula:

RESULTS AND DISCUSSION
The flower oil (0.01% yield) of Jacaranda acutifolia had a pale yellow color, and pleasant odor. Most of their components could be identified unambiguously by direct comparison (mass fragmentation and retention index) with published data as well as computer library search. As shown in Table 1 and Fig. 1, forty six compounds were identified, representing about 81.36 % of the total peak area.
The oil consisted mainly of fatty acids (52.18%) rich in dodecanoic acid (17.48%), tetradecanoic acid (15.59%), hexadecanoic acid (10.98%) and decanoic acid (7.9%); followed by aliphatic hydrocarbons (11.61%), of which nonacosane (7.71%) was identified. While terpene related compounds reached 8.98%, of which Hexahydrofarnesyl acetone (8.2%), represented almost all of terpene related compounds content. The essential oil showed the presence of many methyl and ethyl esters such as methyl linoleate (3.81%), methyl hexadecanoate (1.65%), methyl tetradecanoate (0.24%) and trace amounts of esters of phenylacetate, salicylate and dodecanoate.   Table 4) ranging from 0.9 up to 10.9 mg/ml, while it showed moderate antimicrobial activity against Shigella flexneri and Salmonella typhimurium with inhibition zones of 13 and 14.5 mm, respectively. Only one (E.coli) of the tested strains showed sensitivity to the essential oil in biologically relevant concentrations (MIC of 0.9 mg/ml). Mean inhibition zone diameter (mm) is shown in Table 2 and presented in Fig. 2.
The antimicrobial potency of the oil was calculated as compared to standard antibiotics, and 1mg oil was equivalent to 1.05 µg penicillin against S. aureus; 2.72 µg nystatin against C. albicans; 13.55, 1.3 and 2.72 µg gentamicin against E. coli, Salmonella typhimurium and Shigella Flexner respectively ( Table 3).
The strong antibacterial activity of flower oil of Jacaranda acutifolia may be due to their high content of fatty acids, which constitute 52.18% in content. Fatty acids have been shown to possess antibacterial activities and Gram-negative bacteria are generally more resistant than Grampositive bacteria due to antagonistic effects of fatty acids with their cell wall lipopolysaccharides [9]. This might explain the moderate antibacterial effect seen on Shigella flexneri and Salmonella typhimurium. Dodecanoic acid, decanoic and hexadecanoic acids possess antibacterial activity [9,10]. Also, Hexahydrofarnesyl acetone had proven to demonstrate antimicrobial activity [11].
It must be pointed out, however, that minor compounds may also importantly contribute for the antimicrobial activity of essential oils [12].
Probably, components such as β-linalool and αlinolenic acid detected in our oil could be responsible for this activity as they have shown to be antibacterial [13,14].    An interesting aspect related to the antimicrobial activity of essential oils is that the risk of pathogenic microorganisms developing resistance is very low because these products contain a blend of different antimicrobial substances that have different modes of action [15]. This is a beneficial characteristic of plantderived products as compared to synthetic antimicrobial agents, as their application in food products may provide better food safety and longer shelf life [15].

CONCLUSION
The essential oil of Jacaranda acutifolia flowers contains dodecanoic acid, decanoic acid, tetradecanoic acid, hexahydrofarnesyl acetone, hexadecanoic acid and nonacosane as the main chemical markers. These chemical markers would be a powerful tool for maintaining quality control in the extraction of essential oils for use in medicinal applications, as well as in identification of plant specimens to a taxonomist. The essential oil exhibited potential antimicrobial activity against foodborne bacteria and yeast, and this in-vitro activity makes its local traditional uses rational. Therefore, the essential oil is a potential to be used in food conservation to extend the shelf life and increase the safety of the processed food as alternative to chemical preservatives and can be used as a natural antimicrobial agent in new drugs for therapy of infectious diseases. Further toxicological and clinical studies are required to prove the safety of the oil as a medicine.