Anti-microbial, cytotoxicity, and necrotic ripostes of Pimpinella anisum essential oil

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Inhibitory effect of oil and its preparations were studied using disc-diffusion and Minimum Inhibitory Concentration (MIC) tests, and using corn oil and Streptomycin as controls.

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Nội dung Text: Anti-microbial, cytotoxicity, and necrotic ripostes of Pimpinella anisum essential oil

Annals of Agricultural Science (2015) 60(2), 335–340 H O S T E D BY Faculty of Agriculture, Ain Shams University Annals of Agricultural Science www.elsevier.com/locate/aoas Anti-microbial, cytotoxicity, and necrotic ripostes of Pimpinella anisum essential oil Mohammed A.T. Abdel-Reheem a,*, Mona M. Oraby b a Biochemistry Dept., Faculty of Agric., Ain Shams Univ., Cairo, Egypt b Agric. Microbiology Dept., Faculty of Agric., Ain Shams Univ., Cairo, Egypt Received 4 October 2015; accepted 13 October 2015 Available online 27 November 2015 KEYWORDS Abstract Pimpinella anisum essential oil (EO) residuals and its biotic possessions were deliberated. Anise essential oil; EO was identified using UPLC MS/MS analysis. Inhibitory effect of oil and its preparations were Bio-control; studied using disc-diffusion and Minimum Inhibitory Concentration (MIC) tests, and using corn oil Antimicrobial; and Streptomycin as controls. Tolerant ability of EO treated Hep G2 cells was appraised using Cytotoxicity; MMT assess, and 0.4, 0.8, 1.2 and 1.6% (v/v) of EO for 3 h were used for flow cytometric explo- Hep G2 ration of cell cycle regression. UPLC MS/MS inquiry revealed that EO major ingredients were phe- nyl propanoids followed by monoterpenes (hydrocarbons and phenolics), and trans-anethole followed by cis-anethole, estragole, linalool, a-terpineol, and methyl eugenol were the major EO constituents. EO has high inhibitory effect for Salmonella typhi, Enterococcus faecalis, Staphylo- coccus aureus, Escherichia coli and Micrococcus luteus. Corn oil inhibited E. coli and M. luteus at high concentrations. Streptomycin gave higher inhibition with all bacteria. However both have similar effects with E. faecalis, S. typhi, and M. luteus bacteria. Spraying potato tubers with EO emulsified with soap prior to Ralstonia solanacearum infection and preservation at 4 °C were kept healthy without any rot symptoms. Treating Hep G2 cells with EO for 24 h caused a substantial cytotoxicity as a concentration reliant, and exhibited a significant cytotoxicity at the higher concen- trations. Cell cycle examination of PI-stained did not indicate any changes in the cell cycle regression. There is no substantial escalation in the extension of apoptotic/necrotic Hep G2 cells in sub-G1 segment. Ó 2015 Production and hosting by Elsevier B.V. on behalf of Faculty of Agriculture, Ain Shams University. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/ licenses/by-nc-nd/4.0/). Introduction Most substantial bioactive phytochemical components were * Corresponding author. Tel.: +20 (2)44441172 fax: +20 (2) volatile oil constituents (Edeoga et al., 2005; Al Maofari 44444460. et al., 2013). Using of essential oils in numerous healthiness E-mail address: matbio2050@gmail.com (M.A.T. Abdel-Reheem). arenas; such as makeups, herbal medications, food flavorings, Peer review under responsibility of Faculty of Agriculture, Ain-Shams and preserving agents; is precise ancient due to its safety and University. its antimicrobial and antioxidant activities (Thomas, 2002; http://dx.doi.org/10.1016/j.aoas.2015.10.001 0570-1783 Ó 2015 Production and hosting by Elsevier B.V. on behalf of Faculty of Agriculture, Ain Shams University. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
336 M.A.T. Abdel-Reheem, M.M. Oraby Blunt, 2003; Aftab et al., 2013; Abdel-Reheem et al., 2014a). Bacteria were assayed on nutrient agar and Nutrient broth, Eternity essential oils and former aromatic plants and spices while C. albicans on Sabouraud Dextrose Agar (Merck, USA). extracts were induced the attention as natural antimicrobial sources due to their prospective usages as other therapies for Disc-diffusion test various contagious illnesses (Buchbauer et al., 1991; Deis, Disc diffusion technique was concluded in duplicate to define 1999; Sokovic´ et al., 2010). Volatile constituents were synthe- the antimicrobial retorts of P. anisum EO according to sized in plants as herbivorous pests, fungi, pathogens, and par- Sokovic´ et al. (2010). Inoculations of 0.1 ml of all tested asites protectors (Walker, 1994; Soliman and Badea, 2002; microbes; grownup in LB medium at 37 °C for 24 h (108–109 Sagdic, 2003). Essential oil of Pimpinella anisum (EO) contents cells/ml); on Mueller–Hinton growth media (Sigma–Aldrich expressively differs with changes of source and farming process Co., USA) were prepared. Sterilized paper discs with absorbed (Al Maofari et al., 2013). Recently P. anisum EO stated as EO (30 ll/disc) positioned against the tested plates at assured replacement of antibiotic in broiler ration, and P. anisum EO spots by soft pressure then incubated at 35 ± 2 °C for 48 h. contains anothole as active ingredient, eugenol, methylchavi- Inhibition regions were measured in millimetres. 1 lL of Strep- col, anisaldehyde and estragole (Mehmet et al., 2005). The tomycin (Sigma P 7794) (from 1 mg/ml stock) was applied to antimicrobial activities of various EO over inclusive series of for each disc while corn oil was used as control. germs have been deliberated comprehensively in vitro and nourishments (Dormont et al., 1998; Ochocka et al., 2002). Minimum Inhibitory Concentration (MIC) test Antimicrobial actions of EO are connected to their chemical assembly, principally the aliphatic and phenolic residuals Oil Minimum Inhibitory Concentration (MIC) was dogged (Macig et al., 2007). Contradictory crude EO was confirmed according to Carson et al. (1995) with modifications. The stock as antimicrobial effectiveness opposing to diverse bacterial seg- solution of oil was dissolved in 8:1 portions of 50% ethanol regates and demonstrated reassuring magnitudes (Morsi, 2000; and Tween 20 in order. Streptomycin was liquefied in 50% Bakathir and Abbas, 2011). P. anisum EO reveals durable inhi- ethanol (positive control); dissolving solvents and corn oil were bitory actions counter to an extensive bacteria and fungi range used also as controls. The nutrient broth media containing and identified as a human and further species pathogen. It two-fold oil dilutions were inoculated with approximately repressed the growing of a diversity of microbes including 5 105 cfu of each active pathogen culture (oil dissolving, Staphylococcus aureus and Escherichia coli; moreover, it is used dilution and inoculation were done in considerateness of as antifungal against several fungi such as Candida albicans achieving the wanted oil concentrations (from 0.02 to (Al-Bayati, 2008). However, no much toxicological studies 15.0 lg/ml)). The cultures were incubated at 30 °C for 24 h were conducted on its essential oil. Therefore, in this work on a rotary shaker (220 rev/min) and examined at 540 nm we probe the constituents, the retorts of P. anisum EO as a nat- for growths. ural, safe and reliable bio-controlling agent against some selected human and plant pathogens, as well as its cytotoxicity Inhibition of potato brown rot during preservation and necrotic activities. Clean and surface sterilized (using 4% sodium hypochlorite for 10 min) market potato tubers (size of about 5 3 cm) were Materials and methods scratched using sterilized scalpel. Five tubers were used for each preparation and controls as follows: (a) Tubers were Extraction of essential oil sprayed with the preparations (oil dilution was prepared by adding 5 ml of oil to 100 ml of distilled water with 3 ml from The air-dried and grained P. anisum ripe seeds were extracted either commercial liquid soap or Tween 80 as emulsifying using water-distillation according to Abdel-Reheem et al. agents), then swapped with R. solanacearum using sterilized (2014a,b). The obtained EO desiccated via anhydrous sodium cotton swap dipped in the bacterial suspension (108 cells/ml). sulphate and stowed at 4 °C. (b) Tubers were swapped with bacteria, then sprayed directly with each preparation, and also sprayed daily for 5 days. Each UPLC MS/MS Inquiry of volatile oil tuber was swapped with constant bacterial inoculum of 1.5 ml; treatments were kept on plastic tray at 4 °C and at room tem- perature (about 30 °C) as a control, and observed daily for rot Ingredients of P. anisum EO extract were achieved via a development till 20 days. Controls were emulsion of corn oil, Waters Alliance system UPLC/USA, auto-sampler and a water with emulsifying agents only and water without emulsi- waters 2996 diode array detector according to Abdel-Reheem fiers or oil. et al. (2014a,b). Antimicrobial activity Culturing of cells Human hepatocellular carcinoma cell line (Hep G2) illuminat- Source of pathogens ing progress without a contact restraint was grown up in Antimicrobial tests were carried out for S. aureus, E. coli, DMEM; complemented with 10% FBS, antibiotic–antimy- Streptococcus pyogenes, Pseudomonas aeruginosa, Salmonella cotic reagent (100, 1 ml/100 ml of medium), and glucose; at typhi, Enterococcus faecalis, Micrococcus luteus, and 37 °C, 5% atmospheric CO2, and 95% humidity. Cell sustain- C. albicans (obtained from Fac. of Medicine, Ain Shams ability was appraised via optical microscopic examination for Univ.). Ralstonia (Pseudomonas) solanacearum was isolated trypan blue segregation. Hep G2 cells consignments revealed from market potato tubers according to Kelman (1954). fewer than 95% cell viability were eliminated.
Pimpinella anisum essential oil 337 Tetrazolium bromide (MTT) scrutiny Table 1 Antimicrobial activity of P. anisum EO in disc- Viability of P. anisum EO Hep G2 treated cells was appraised diffusion method (30 ll/disc). using the MTT assay according to Yen et al. (2012), Saquib Microorganism Inhibition zones in mm et al. (2012a,b), and Abdel-Reheem et al. (2014a, 2014b). The achieved colour was measured at 550 nm via a multi- P. anisum oil Corn oil Streptomycin well microplate reader (Multiskan Ex, Thermo Scientific, Fin- Staphylococcus aureus 15 0 19 land). The solvent (1.6% v/v of EtOH/MeOH) was tracked as Streptococcus pyogenes 12 0 17 a control with alike settings. Escherichia coli 15 2 19 Salmonella typhi 17 1 20 Flow cytometric inquiry for cell cycle regression Pseudomonas aeruginosa 10 0 15 Enterococcus faecalis 16 0 18 Hep G2 cells were cured by 1.6% of EtOH/MeOH as a control, Micrococcus luteus 14 3 20 and 0.4–1.6% (v/v) of P. anisum EO up to 3 h according to Candida albicans 10 1 ND ND = not detected. Table 2 Minimum Inhibitory Concentration (MIC) of Pimpinella anisum EO. Microorganism MIC lg/ml P. anisum oil Corn oil Streptomycin Staphylococcus aureus 3.0 N 1.5 Streptococcus pyogenes 4.0 N 1.0 Escherichia coli 2.5 10.0 1.5 Salmonella typhi 2.0 N 1.5 Pseudomonas aeruginosa 3.0 N 1.5 Enterococcus faecalis 2.0 N 1.0 Micrococcus luteus 2.0 9.0 1.5 Candida albicans 4.0 N ND ND = not detected, N = bacteria not effected at any concentration. Fig. 1 P. anisum EO percentage of various modules of terpene (achieved via UPLC using isocratic mixture of Methanol:Formic Darzynkiewicz et al. (1992), Saquib et al. (2012a,b), and acid (80:20) as a mobile phase and plotted as pie for the minor and Abdel-Reheem et al. (2014a,b). Fluorescence of the PI was major modules percentages illustration) showing phenyl propa- determined via flow cytometry using Beckman Coulter Flow noids are the foremost constituent. Cytometer (Coulter Epics, USA) using a FL-4 filter (585 nm) and 10,000 events were assimilated. Coulter Epics XL/XL- MCL, System II Software (Version 3.0) was used for data scrutiny. Results and discussion UPLC/SM/MS inquiry P. anisum EO chromatographic data (plotted as logarithmic scale for the minor and major modules percentages illustra- tion) revealed that phenyl propanoids (93.96%) are the major constituent followed by monoterpenoids (5.93%), Hydrocar- bons monoterpenes were 4.52% while Alcohols monoterpenes were 1.41% (Fig. 1). The major constituents of P. anisum EO were trans-anethole (82.1%) followed by cis-anethole (5.8%), estragole (methylchavicol) (2.5%), linalool (2.3%), a- terpineol (1.5%), and methyl eugenol (1.3%) (Fig. 2). In addi- Fig. 2 P. anisum EO percentage of various modules achieved via tion, smaller amounts of other phenyl propanoids and UPLC using isocratic mixture of Methanol:Formic acid (80:20) as monoterpenes were also detected (Fig. 2). Data are in good a mobile phase and plotted as logarithmic scale for the minor and arrangement with what acquired by Arslan et al. (2004), foremost modules percentages illustration. Mehmet et al. (2005), and O¨zcan and Chalchat (2006).
338 M.A.T. Abdel-Reheem, M.M. Oraby Microbiological study Disc-diffusion test Data in Table 1 demonstrate a high effect of P. anisum EO, giving inhibition zones of 17 and 16 mm for S. typhi and E. faecalis, respectively. Corn oil gave inhibition zones of 3 and 2 mm for M. luteus and E. coli, respectively. Streptomycin always gave higher inhibition zones with all bacteria used when compared with P. anisum EO but the differences were not so great with pathogen like E. faecalis (16 and 18 mm zone diameter with P. anisum EO and Streptomycin, respectively). Minimum Inhibitory Concentration (MIC) test Data in Table 2 reveal a high inhibitory effect of P. anisum EO, Fig. 3 The Hep G2 cells cytotoxicity inquiry using MTT assay giving antibacterial activity MIC of 2.0 lg/ml for S. typhi, displaying the cell viability percentage. Cells exposed to P. anisum E. faecalis and M. luteus. Corn oil respectively gave inhibition EO for 24 h. Histograms represent ± S.D. means obtained from at high concentrations; 10 & 9 lg/ml; with E. coli and three independent experiments. **p < 0.01 vs control II. Control I: M. luteus. Streptomycin always gave higher inhibition with untreated control, control II: EtOH:MeOH (1:1). all bacteria with MIC of 1.0–1.5 lg/ml. Also it was found that there are no great differences between oil and Streptomycin infection before or after oil with soap spraying). P. anisum inhibitory effect with S. typhi and M. luteus bacteria. EO antimicrobial action could be related its suppressing ability to the adhesions of microbial cells (Tsuchiya et al., 1996). Inhibition of potato brown rot during preservation Antibiotic activity on the resilient bacteriological isolates was Results in Table 3 prove that spraying potato tubers with enriched when associated with P. anisum EO representing that P. anisum EO and soap prior to R. solanacearum infection the P. anisum EO permitted enhanced diffusion of the medicine and preservation at 4 °C gave the best results, as the 5 used via cell wall external layers, and hindering the enzymes inhibi- replicates were kept healthy without any rot symptoms (with tory influence at microbial cell wall (Darwish and Aburjai, infection before or after oil with soap spraying). Results con- 2010). cluded that potato tubers can be kept safe from brown rot at 4 °C with an interval sprayings of P. anisum EO with soap Cytotoxicity induction in Hep G2 cells via P. anisum EO emulsion. Besides the microbial inhibitory effect of P. anisum Disclosure of Hep G2 cells to P. anisum EO for 24 h leads to a EO, the presence of soap can help in increasing this effect significant cytotoxicity as application reliant. From MTT and gave an efficient method in protecting potatoes from assay, the exposure of Hep G2 cells to 0.4%, 0.8%, 1.2% infection. and 1.6% of P. anisum EO has exhibited a significant cytotox- Results of the present study demonstrated a high effect of icity only at the higher concentrations of 1.2% and 1.6%. P. anisum EO, giving inhibition zones of 17 and 16 mm for Compared to the solvent control exposure, P. anisum EO at S. typhi and E. faecalis, respectively. Also P. anisum EO 1.2% and 1.6% has resulted in 34% and 58.2% reduction in revealed a high inhibitory effect giving antibacterial activity cell viability (Fig. 3). MIC of 2.0 lg/ml for S. typhi, E. faecalis and M. luteus. It was found that there are no great differences between oil and Apoptosis/necrosis in Hep G2 cells via P. anisum EO Streptomycin inhibitory effect with S. typhi and M. luteus bacteria. Results also proved that spraying potato tubers with Cell cycle examination of PI-stained revealed that no devia- P. anisum EO and soap prior to R. solanacearum infection and tions in the cell cycle regression were found in P. anisum EO preservation at 4 °C gave the best results, as the 5 used treated cells. There was no significant increase in the character- replicates were kept healthy without any rot symptoms (with istic peak of apoptosis/necrosis (SubG1) (Figs. 4 and 5). Table 3 Potato brown rot inhibition using P. anisum EO emulsions. Treatment Condition Number of tubers showing rot symptoms* P. anisum & tween P. anisum & soap Corn oil & tween Corn oil & soap Water & tween Water & soap Water Preserved at A 2 0 4 4 5 3 5 4 °C B 2 0 4 3 5 4 5 Preserved at RT A 3 1 4 3 5 4 5 B 3 2 5 4 5 4 5 A: Spraying prior to infection, B: Infection before spraying. * Five potato (P) tubers were reserved at 4 °C or at room temperature (RT).
Pimpinella anisum essential oil 339 Fig. 4 Illustrates flow cytometric metaphors unveiling deviations in the regression of normal cell cycle in Hep G2 cells after 3 h of P. anisum EO treatment. Subsections are symbolized as (A): untreated control; (B): solvent control (1.6%) EtOH:MeOH (1:1); (C): 0.4%; (D): 0.8%; (E): 1.2% and (F): 1.6%. Conclusion The foremost ingredients of the P. anisum EO were phenyl pro- panoids followed by monoterpenes (hydrocarbons and pheno- lics), and trans-anethole followed by cis-anethole, estragole, linalool, a-terpineol, and methyl eugenol were the main EO modules. P. anisum EO retains obvious inhibitory influence contrary to numerous vegetal and animal pathogens and non- pathogenic germs and there are no great differences between oil and Streptomycin inhibitory effect. Antimicrobial activity of P. anisum EO is mainly related to its phenolic contents. Treatment of Hep G2 cells to P. anisum EO for 24 h caused a significant cytotoxicity as a concentration dependent, and exhibited a significant cytotoxicity at the higher concentra- tions. No changes in the cell cycle regression were designated. Proportion upsurges of apoptotic/necrotic Hep G2 cells in sub- Fig. 5 Deviations in the regression of normal cell cycle in Hep G1 stage were insignificant. G2 cells after 3 h of P. anisum EO treatment. Histograms represent ± S.D. means obtained from three independent exper- Acknowledgements iments. G1, S, and G2/M are the proportions of cells existing in cell cycle typical phases; SubG1 signifies proportion of apoptosis/ Gratitude to King Saud Univ., Deanship of Scientific necrosis cells. Control I: untreated control, control II: EtOH: Research, and College of Sciences’ Research Center for sup- MeOH (1:1). porting this work.
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