Mohamed Derbali1*, Ameur Elaissi2, Imed Cheraief3 and Mahjoub Aouni1
1Laboratory of Transmissible Diseases and Biologically Active Substances, Faculty of Pharmacy, Monastir, Tunisia
2Laboratory of Pharmacognosy, Faculty of Pharmacy, University of Monastir, Tunisia
3Laboratory of Biochemistry, Faculty of Medicine, University of Monastir, Tunisia
Received 31/03/2016; Accepted 06/04/2016; Published 14/04/2016
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Objective: This study is about Allium cepa (A. cepa) seed oil extraction, Fatty acids identification and its antibacterial activity. Methods: Soxhlet extraction method using methanol (MetOH), Fatty acid profile of seed oil using gas-chromatography with flame ionization detector (GC-FID). Disc diffusion and plate micro dilution for the determination of the antibacterial activity. Results: Extraction’s yield was of 24.86 ± 0.98 (% of V/W). Seed extract shows that Linoleic acid (C18:2, ± 70.8%), oleic acid (C18:1, 20.14%) and palmitic acid (C16, ± 4.96%) were the dominant fatty acids in the A. cepa seed oil. Antimicrobial potential was detected against reference bacteria and clinical isolates pathogens. Conclusion: Those results show that A. cepa is a promising oil seed crop with high level of unsaturated fatty acids and antibacterial potential. A bivalent role: nutritive and antibiotic, of A. cepa seed oil prove its importance in food and pharmaceutical applications.
Allium cepa, Seed, Fatty acid, GC-FID, Antibacterial activity, Multi drug resistant bacteria.
Plants seed oils have multiple applications, being used in various fields from cosmetics to cooking. Seeds oils are gaining more importance as studies prove they are a source of tocopherols and polyphenols [1-3], known for their antioixidant [4] and antiinflammatory capacities [5]. Seed oil is known to be rich with fatty acids. Polyunsaturated acids such as linoleic acid are essential for the human metabolism due to the lack of enzymes responsible for their biosynthesis [6]. PUFA play a key role promoting cardiovascular health, enhancing anti-tumoral activity [7,8] and prevent atherosclerosis [9,10].
Onion: Allium cepa L. is known to be an ingredient of food over the world. Researches demonstrated that A. cepa is involved in many biological activities such as antioxidant potential [11], hepatoprotective [12] and antiallergic [13] activities. A. cepa extracts were efficient in reversing organ toxicity [14], treating human leukemia [15]. The seeds of A. cepa L. were used in folk medicine by the Uygur nationality in China and improve the functions of internal organs, treat diarrhea, fervescence, calenture, and puffiness of the face and eyes, and promote blood flood [16]. In this report we focused on A. cepa seed oil extraction with organic solvent, fatty acid profile determination, major compounds identification and testing the antibacterial activity of seed oil against sensitive and multi drug resistant bacteria.
Chemical and materials
Seeds of A. cepa were recolted from our local field in Jelma, Sidi Bouzid, Tunisia. All chemicals used in this study were of analytical grade (Sigma–Aldrich ® 90, St. Louis, Mo., USA) and the reagents used were standardized.
Oil extraction
Seeds were fine powdered using high speed mixer. The oils were extracted from the seeds with soxhlet extractor using MetOH for 4 h. The ratio of solids to solvent used was 1:10. The oil was then recovered by evaporating off the solvent using rotary evaporator.
Fatty acid methyl esters (FAMEs) synthesis and GC-FID
FAMEs were prepared, following the procedure described by AOAC [17]. Aliquot of 10 mg lipid for each solvent extract were esterified after addition of internal standard (0.1 ml of C17:0, 1 mg/ml) and 1 ml of BF3-etherate, the samples were boiled for 30 min at 70°C. The FAMEs were extracted from a salt saturated mixture with hexane (1.0 ml). For drying FAMEs, anhydride Na2SO4 was added and centrifuged (10 min, 3.56 g), then the upper part was poured in specific cell. FAMEs analysis was carried out according to the European Union Commission modified Regulation EEC 2568/91 on a Hewlett-Packard GC (Hewlett-Packard, Palo Alto, CA), fitted with a FID and a split–splitless injector, set at 270°C. The carrier gas was nitrogen (1 ml/min), and elution was performed with a fused silica Agilent DB23 capillary column (60 m length, 0.32 mm inner diameter, and 0.25 m film thicknesses). Conditions were as follows: injector temperature, 270°C; FID, 280°C; injector split ratio, 1:50; the initial column temperature, 130°C; step 1, 6.5°C/min to 170°C; step 2, 2.8°C/min to 215°C and maintained for 12 min; step 3, 40°C/min to 230°C and maintained for 20 min. FAMEs were identified by comparing their relative and absolute retention times to those of authentic cisfatty acid (CFA) and TFA standards [18].
Antimicrobial activity
Microorganisms
The microorganism strains employed in the biological assays are listed in Table 1. Different American Type Culture Collection (ATCC) reference bacteria were used as well as multi-drug resistant strains from clinical isolates.
| Strain | Zone inhibition (mm) | MIC (µg/ml) | Gentamycin |
|---|---|---|---|
| GRAM negative | |||
| Enterobactercloacae MDR | 9,33 ± 0.57 | 500 | R |
| Escherichia coli ATCC25922 | 12.66 ± 0.57 | 250 | 24 |
| Escherichia coli MDR | 12.33 ± 0.57 | 250 | 24 |
| Klebsiellapneumoniae MDR | 14 ± 1 | 250 | R |
| Proteus mirabilis MDR | 10,33 ± 0.57 | 500 | 19 |
| Pseudomonasaeruginosa ATCC 27853 | 10.66 ± 0.57 | 500 | 20 |
| Pseudomonas aeruginosa MDR | 11 ± 1 | 500 | 20 |
| GRAM positive | |||
| Staphylococcus aureus ATCC 25923 | 18,33 ± 0.57 | 125 | 26 |
| Staphylococcus aureus MDR | 18 ± 1 | 125 | 26 |
| Staphylococcus epidermidis ATCC12228 | 14,33 ± 0.57 | 250 | 25 |
| Streptococcuspseudopneumonaie MDR | 14 ± 1 | 250 | 24 |
| Bacillus subtilus ATCC 6633 | 20.66 ± 0.57 | 125 | 28 |
| Enterococcusfaecalis ATCC 29212 | 16 ± 1 | 125 | 17 |
Table 1: Zone inhibition and MIC of Allium cepa seed extract.
Disk diffusion assay
Antimicrobial activity was carried out using disc-diffusion method [19]. Petri plates were prepared with 20 ml of sterile Mueller–Hinton Agar [20] for bacteria. The test cultures were swabbed on the top of the solidified media and allowed to dry for 10 min. A. cepa seed oil was diluted in 10% dimethyl sulfoxide (DMSO) and 20 μl was loaded on discs (1 mg per disc). Discs of Gentamycin (30 μg) were used as positive control. The loaded discs were placed on the surface of the medium and left for 30 min at room temperature for compound diffusion. The plates were incubated at 37°C. After 24 h, zones of inhibition were recorded in millimeters.
Determination of minimum inhibitory concentration
The minimal inhibitory concentration (MIC) preventing visible bacterial growth was measured by the broth dilution method (microdilution using 96-well microplates) [21]. Extract stock solution were prepared by dissolution in 10% DMSO. The MIC of each extract was defined as the lowest concentration which inhibited bacterial growth, after incubation at 37°C between 18 and 24 h.
Statistical analysis
All the experiments were performed in triplicate, and statistical significance was calculated by one-way analysis of variance (ANOVA). A value of p<0.05 indicated statistical significance using Duncan’s multiple range tests.
Fatty acid profile
Results shown in Table 2 shows that A. cepa is rich of MUFA and PUFA responsable of lipid regulation [22] and their protective effects against seizures, cognitive impairment and hippocampal oxidative DNA damage [23] especially oleic acid and linoleic acid. MUFA and PUFA represent more than 92% of total fatty acid profile, providing omega-3 and omega-6 fatty acids. Those essential fatty acids should be provided by food intake since humans have lack of enzymes responsible of their synthesis [24,25]. In fact, Linoleic acid is an essential fatty acid from which the whole omega-6 fatty acid family is derived. These fatty acids are important components of the cell membranes and are precursors of other substances involved in many physiological responses. By contrast, palmitic acid is considered as an atherogenic compound, when consumed in high amount [26].
| S. No. | Fatty acids Yield (%) | |
|---|---|---|
| SFA | ||
| 1. | Butyricacid C4 :0 | 0.004 |
| 2. | Caproicacid C6 :0 | 0.003 |
| 3. | Capricacid C10 :0 | 0.006 |
| 4. | Lauricacid C12 :0 | 0.058 |
| 5. | Tridecylicacid C13 :0 | 0.002 |
| 6. | Myristicacid C14 :0 | 0.052 |
| 7. | Pentadecylicacid C15 :0 | 0.001 |
| 8. | Palmiticacid C16 :0 | 4.968 |
| 9. | Margaricacid C17 :0 | 0.040 |
| 10. | Stearicacid C18 :0 | 1.772 |
| 11. | Arachidicacid C20 :0 | 0.213 |
| 12. | Heneicosylicacid C21 :0 | 0.033 |
| 13. | Behenicacid C 22 :0 | 0.135 |
| 14. | Tricosylicacid C23 :0 | 0.001 |
| MUFA | ||
| 15. | Myristoleicacid C14 :1 | 0.022 |
| 16. | Pentadecenoicacid C15 :1 | 0.005 |
| 18. | Palmitoleicacid C16 :1 | 0.049 |
| 19. | Heptadecenoicacid C17 :1 | 0.038 |
| 20. | Oleicacid C18 :1 | 20.14 |
| 21. | Vaccenicacid C18 :1 | 0.914 |
| 22. | Icosenoicacid C20 :1 | 0.313 |
| 23. | Erucicacid C22 :1 | 0.008 |
| PUFA | ||
| 24. | Linoleicacid C18 :2 | 70.80 |
| 25. | Alpha-linolenicacid C18 :3 | 0.080 |
| 26. | Gamma-linolenicacid C18 :3 | 0.002 |
| 27. | Eicosadienoicacid C20 :2 | 0.054 |
| 28. | Dihomo-gamma-linolenic acid C20:3 | 0.007 |
| 29. | Eicosatrienoicacid C20 :3 | 0.016 |
| 30. | Arachidonicacid C20 :4 | 0.001 |
| 31. | Docosadienoicacid C22 :2 | 0.008 |
| 32. | Docosatrienoicacid C22 :3 | 0.051 |
| 33. | Docosatetraenoicacid C22 :4 | 0.003 |
| 34. | Docosapentaenoicacid C22 :5 | 0.042 |
| Total SFA | 7.29 | |
| Total MUFA | 21.50 | |
| Total PUFA | 71.21 | |
Table 2: Fatty acid composition of Allium cepa seed extract as % of total fatty acid profile.
Antibacterial activity
The in vitro antimicrobial activity of the metOH seed extract from A. cepa against tested microorganisms including multidrug resistant pathogens was qualitatively and quantitatively assessed by the inhibition zones and the minimum inhibitory concentration were determined using serial microdilutions technique.
According to the results shown in Table 1, the seed oil exhibited a potent inhibitory effect with diameter of inhibition zones ranging from 9 to 20 mm. MIC values vary from 125 to 500 μg/ml for ATTCC microorganisms and multidrug resistant pathogens. The MetOH extract were more effective against GRAM negative strains. Staphylococcus aureus ATCC 25923, Staphylococcus aureus MDR, Bacillus subtilus ATCC 6633 and Enterococcus faecalis ATCC 29212 were the most affected GRAM negative bacteria by the MetOH seed extract with an MIC value of 125 μg/ml. Escherichia coli ATCC25922, Escherichia coli MDR and Klebsiella pneumoniae MDR were the most affected GRAM positive tested strains to the MetOH seed extract with MIC value of 250 μg/ml.
Those findings are original concerning MetOH seed extract of A. cepa and they are in accordance with antimicrobial activity of aerial part extracts and essential oil of A. cepa [27-30]. Those results took a highly importance regarding the inhibitory effects especially against tested multidrug resistant bacteria from clinical isolates from Tunisian University Hospitals concerning their potential danger and their threat to the human health.
Those findings are original concerning MetOH seed extract of A. cepa and they are in accordance with antimicrobial activity of aerial part extracts and essential oil of A. cepa [27-30]. Those results took a highly importance regarding the inhibitory effects especially against tested multidrug resistant bacteria from clinical isolates from Tunisian University Hospitals concerning their potential danger and their threat to the human health.
This report revealed that A. cepa seed oil is well rich with MUFA and PUFA and it possess an important antibacterial activity against ATTCC strains and multi drug resistant pathogens. Taken together A. cepa seed oil combines the nutritive and the pharmaceutical aspects wich testifies it’s highly utilities as food additive and food preservative from bacterial pathogens (Figures 1 and 2).
Figure 1: Fatty acids chromatogramm of Allium cepa seed extract.
Figure 2: Chemical structure of major fatty acids of Allium cepa seed extract.
The authors are grateful to the Laboratory of Microbiology, University Hospital Fattouma Bourguiba, and TU-5000 Monastir, for providing multi drug resistant clinical isolates pathogens.
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