Antibacterial and Antioxidant Potential of Essential Oils of Five Spices

Background: Essential Oils (EOs) of spices may serve as a potential source of antibacterial and antioxidant agents due to the presence of a diverse group of phytochemicals. In the present investigation, an attempt has been made to seek EOs from five commonly used spices that have both strong antibacterial and antioxidant potential to shed some light on these important aspects. Methods: In vitro antibacterial efficacy of black cumin, black pepper, cinnamon, clove, and nutmeg EOs were evaluated against some food-borne bacteria using agar well diffusion, microbroth dilution, and time-kill assay methods. Antioxidant potential was evaluated by 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging, β-carotene linoleic acid bleaching and Fe 2+ ion chelating methods. Chemical characterization of EO components was performed by Gas Chromatography-Mass Spectrometry (GC-MS). Statistical analysis of data was performed using SPSS software, version 18.0. Results: Black pepper, cinnamon, and clove EOs had significantly (p<0.05) higher antibacterial properties comparing to the black cumin oil and nutmeg EOs. Clove and cinnamon EOs showed the highest and lowest antioxidant potential, respectively. GC-MS analysis revealed that eugenol, cinnamaldehyde, and β-caryophyllene were the main constituents of clove, cinnamon, and black pepper oils, respectively. Conclusion: The results provide evidence that EOs of clove, black pepper, and cinnamon may serve as effective natural preservatives in the food industry. Further studies are needed for their plausible applications in the food industry. © 2018, Shahid Sadoughi University of Medical Sciences. This is an open access article under the Creative Commons Attribution 4.0 International License.


Introduction
Food-borne disease is an increasingly major public health problem all over the world (Newell et al., 2010).Microbial contamination and oxidation of food compo-life of food items but they have accumulated evidence that they could be toxic and carcinogenic.Besides, another great concern is the emergence of antimicrobial resistance in food-borne pathogens (Mathew et al., 2007).Natural antimicrobials and antioxidants seem to be the most promising answer to many of the increasing concerns and could yield better results than synthetic food preservatives.Therefore, novel types of effective as well as healthy antimicrobial and antioxidant agents that could protect food against spoilage and also microbial contamination are highly demanded (Lucera et al., 2012).
In this context, Essential Oils (EOs) may serve as a potential source of such compounds due to the presence of diverse groups of phytochemicals and may play a significant role in the development of eco-friendly plantbased food preservatives (Prakash and Kiran, 2016;Raut and Karuppayil, 2014).EOs are effective against a number of microbes due to their hydrophobic nature which allows them to penetrate microbial cells and cause alterations in its structure and function (Burt, 2004).In addition to their antimicrobial properties, EOs may have some antioxidant potential.Therefore, application of EOs as an antimicrobial and also antioxidant agent is a recent growing trend reflecting the interest towards "green consumerism" (Loizzo et al., 2015).
Hence, the diversified use of EOs with broad-spectrum antimicrobial and antioxidant efficacy could play a major role in food preservation both by retarding microbial growth and oxidative deterioration, with enhanced flavour properties.But, knowledge is lacking about EOs that have both strong broad-spectrum antibacterial activity against food-borne bacteria as well as strong antioxidant potential.These evidences are particularly important to facilitate the development of safe and effective natural antimicrobial and antioxidant food preservatives.Although a number of antimicrobial and antioxidant activity study of EOs have been reported by several researchers (Amorati et al., 2013;Mith et al., 2014;Smith-Palmer et al., 1998), but most of the studies were performed using one or two in vitro test models.Evaluation of the antimicrobial as well as antioxidant performance of EOs is, however, a crucial issue, because each of the test models varies in different respects.The lack of standardized methods makes direct comparison of results between studies impossible which subsequently may mislead future research (Amorati et al., 2013;Jennie et al., 2003).Keeping these important points in mind, in the present study, a comprehensive investigation using standard methods has been made to screen safe and effective EOs that have both broad-spectrum antibacterial activity against food-borne bacteria as well as antioxidant potential.

Collection, extraction, and formulation of plant materials
Ethnobotanical description of selected five spices (black cumin, black pepper, cinnamon, clove, and nutmeg) is given in Table 1.These spices were purchased from the local market (Baranagar Bazar Market, Kolkata, India), identified by a botanist and voucher specimens (BC1701, BP1702, CN1703, CL1704, and NT1705) were deposited in Herbarium of Agricultural and Ecological Research Unit, Indian Statistical Institute, Kolkata.The test spices were washed thoroughly in distilled water, dried at 40 ºC and powdered.EOs of powdered test spices were extracted by hydro-distillation in a Clevenger's type apparatus.Yield of test EOs are shown in Table 1.Working solution of test EOs (1000 µg/ml) was prepared by reconstituting them in 0.5% dimethylsulfoxide (DMSO) with Tween 20 (0.02% v/v) as emulsifier.All working solutions were prepared freshly prior to use.

Microorganisms
The bacterial strains [Bacillus cereus (n=6), Listeria monocytogenes (n=8), Micrococcus luteus (n=7), and Salmonella Typhimurium (n=7)] used in the present study were isolated and identified from a total of 50 spoiled and contaminated foods collected from local markets in and around Baranagar, Kolkata, India using standard methods (Kumar et al., 2011).Standard strains [B.cereus (MTCC 1272) and S. Typhimurium (MTCC 3224)] were procured from the Institute of Microbial Technology, Chandigarh, India.Bacterial cultures were maintained on selective agar slants (L.monocytogenes in BHIB; S. Typhimurium in TSB; B. cereus and M. luteus in MHB) following standard guidelines (CLSI, 2005).

Evaluation of antibacterial potential -Determination of Inhibition Zone Diameter (IZD)
IZD of tested EOs against the studied bacteria was determined by a modified agar well diffusion method (Okeke et al., 2001).Briefly, one ml of inoculum (5×10 5 CFU/ml) was spread evenly with a glass rod spreader on selective agar plates (L.monocytogenes in Brain BHIA; S. Typhimurium in TSA; B. cereus and M. luteus in MHA) and six mm diameter wells were created on the surface of agar plates.EO (100 µl) from reconstituted working solution (1000 µg/ml) was pipetted into the wells.Plates were then kept for 2 h at room temperature to allow diffusion of EOs and then incubated at respective temperature (30 ºC/37 ºC) for 24 h.IZD was measured in mm.Ciprofloxacin (10 μg/ml) and 0.5% DMSO were used as experimental positive and negative control, respectively.The threshold limit values for antimicrobial susceptibility testing based on IZD data were classified according to Bauer et al. (1966) as follow: (IZD≥11 mm: sensitive; 8 mm≤IZD<11 mm: intermediate activity; IZD<8 mm: resistant).

-Determination of Minimum Inhibitory Concentration (MIC)
MICs of tested EOs against the studied bacteria were determined according to the guidelines of CLSI ( 2005) with a slight modification.Briefly, test EOs were diluted two-fold serially at varying concentrations (6.25-100 µg/ml) with selective broth from which 100 µl solution was given in each well of 96-well microtiter plates containing 90 µl of selective broth.Ten µl of working inoculum suspension (5×10 5 CFU/ml) was added to each well.A few wells were kept for control of sterility (without giving inoculum), inoculum viability (without giving sample solution) and DMSO inhibitory effect.The plates were then incubated for 24 h at respective temperature (30 ᵒC/37 ᵒC).Then 40 µl of 0.4 mg/ml of INT solution was added to each well and incubated for 6 h.The microtiter plates were examined to determine a colour change.A change in colour from faint yellow to red-purple occurs when viable microorganisms in wells interact with INT solution.The lowest dilution with no colour change was considered as the MIC for that individual oil.The threshold limit values for antimicrobial susceptibility testing based on MIC data were classified according to Chlipala et al. (2010) as follow: (MIC<100 µg/ml: active; MIC≥100 µg/ml: inactive).On the basis of the threshold limit values of antimicrobial susceptibility testing based on IZD and MIC data, EOs that were found active (IZD≥11 mm; MIC<100 µg/ml) against both Gram-positive and Gram-negative bacteria were screened and subjected to further studies.

-Time-kill kinetics assay
Kill-kinetics study of test active EOs was performed following the method of Levinson (2004).Briefly, 90 µl of respective broth, 10 µl of bacterial suspension (5×10 5 CFU/ml) and 100 µl of test active EOs at different concentrations (0.5×MIC, 1×MIC, 2×MIC and 4×MIC) were added in microtiter plate wells and mixed thoroughly.The plates were then incubated at respective temperature (30 ºC/37 ºC) for 24 h.Ten µl sample was removed from wells at 0, 3, 6, and 24 h of incubation, and diluted serially with respective broth.Viable counts were determined by plating 100 µl of diluted aliquots on respective agar plates and incubated for 24 h at respective temperature (30 ºC/37 ºC).Agar plates with 30 to 300 colonies were used for CFU counting.Log 10 CFU/ml was plotted against time for construction of time-kill kinetics curves.Each experiment was repeated thrice.The antibacterial effects of EOs were considered bactericidal when the reduction in colony count was ≥3 log 10 CFU/ml and bacteriostatic when the reduction was <3 log 10 CFU/ml at 24 h with respect to control values (French, 2006).

Evaluation of antioxidant potential -DPPH free radical scavenging assay
Radical scavenging activity of test active EOs was determined using DPPH radical scavenging assay method (Wang et al., 1998).For this, test active EOs were diluted two-fold serially (6.25-100 µg/ml) and 100 µl of diluted samples of each of varying concentrations were taken in test tubes containing 3.9 ml of DPPH solution (0.1 mM in methanol), shaken vigorously and kept in dark for 30 min at room temperature.The control was prepared as above without the EO; and methanol was used for zero adjustment.Absorbance of the samples was measured at 517 nm using a spectrophotometer (Thermo Fisher Sci., USA).Free radical scavenging activity of the test active EOs was calculated according to the following formula.%Free radical scavenging=[(A blank -A sample )/A blank ]×100 Where A sample is the absorbance of DPPH solution with EO and A blank is the absorbance of DPPH solution in methanol without EO.BHT was used as reference standard.
-β-Carotene linoleic acid bleaching assay β-carotene linoleic acid bleaching activity of test active EOs was evaluated following the method of Velioglu et al. (1998).Briefly, β-carotene (0.2 mg in 1 ml chloroform), linoleic acid (0.02 ml) and tween 20 (0.2 ml) were taken in round bottom flask.Then 0.2 ml of active EOs or standard BHT at 200 µg/ml (the regulatory limit Downloaded from jfqhc.ssu.ac.ir at 15:09 IRST on Thursday December 27th 2018 [ DOI: 10.29252/jfqhc.5.2.6 ] allowed in food preservation) or 0.2 ml ethanol (control) was added to the tubes.After removing chloroform at room temperature, 50 ml of distilled water was added to the mixture and shaken vigorously to form emulsion.Aliquots (2 ml) of the emulsions were taken into test tubes and placed immediately in a water bath at 50 ᵒ C. Absorbance was measured at 470 nm for every 20 min intervals for 2 h using a spectrophotometer (Thermo Fisher Scientific, USA).Antioxidant activity was expressed as percent of inhibition relative to the control using the following formula: Antioxidant activity%=[(Abs control -Abs sample or standard )/ Abs control ]×100 -Ferrous (Fe 2+ ) ion chelating efficacy The Fe 2+ ion chelating activity of active EOs was estimated by the modified method of Dinis et al. (1994).Briefly, in tubes containing 1 ml of the test EOs at different concentrations, 2.7 ml of deionized water and 0.1 ml of 2 mM ferrous chloride solution were added and mixed thoroughly.After 3 min, the reaction was inhibited by the addition of 5 mM ferrozine (0.2 ml).The mixture was shaken vigorously and kept at room temperature for 10 min.Absorbance of the resulting solution was measured at 562 nm using a spectrophotometer (Thermo Fisher Scientific, USA).A blank was run in the same way by using distilled water instead of test EOs.EDTA and BHT were used as reference standard.Sample control was made for each test sample without adding ferrozine.The percent chelating activity was calculated using the following formula: Iron chelating activity%=[(Abs control -Abs sample or standard )/ Abs control ]×100

Evaluation of cytotoxic potential
The possible cytotoxic potential of test active EOs was evaluated using brine shrimp lethality assay (Meyer et al., 1982).Briefly, artificial sea water was prepared by dissolving 38 g of sea salt in 1 L of distilled water for hatching the shrimp (Artemia salina) eggs.Brine shrimp eggs were incubated in artificial sea water in a specially designed two-compartment plastic tray under a 60 W lamp, providing direct light and warmth (24-26 °C).After that, 48 h were allowed for the shrimp eggs to hatch and mature as nauplii (larva).Then, in each of the nine test tubes containing 4.5 ml of artificial sea water, 10 nauplii were added.
One hundred µl of EOs at different concentrations (7.81-1000 µg/ml) was added to eight tubes.The control tube was devoid of EOs.All the tubes were incubated for 24 h at room temperature.After incubation step, number of nauplli alive was counted with help of a magnifying glass to determine 24 h LC 50 (50% lethal concentration).

Determination of total phenolic content
To determine total phenolic content of test active EOs, Folin-Ciocalteu method (McDonald et al., 2001) was used.Briefly, 0.5 ml of test EOs (100 µg/ml) was mixed with one ml of Folin-Ciocalteu reagent (diluted 1:10 with deionized water) in test tubes, shaken vigorously and kept at room temperature for 3 min.Then, 2% Na 2 CO 3 solution (3 ml) was added to the mixture and kept for 2 h with intermittent shaking for colour development.The absorbance of the resulting blue colour was measured at 760 nm.Gallic acid was used as a reference standard for plotting calibration curve.Total phenolic content was determined from the standard curve prepared with different concentrations of gallic acid.The content of total phenolic compounds was expressed as mg gallic acid equivalent/g of EO.

Gas Chromatography-Mass Spectrometry (GC-MS) analysis
The test active EOs were subjected to GC-MS analysis to identify the major chemical constituents present in EOs.The sample was subjected to GC and MS (JEOL GCMATE II, USA) equipped with a secondary electron multiplier.The column (HP5) was fused silica 50 m×0.25 mm I.D.The experimental conditions were 20 min at 100 °C, column temperature: 235 °C for 3 min; injector temperature: 240 °C; carrier gas: helium; and split ratio: 5:4.One μl of the sample was evaporated in a splitless injector at 300 °C and the run time was 40 min.The components were identified by GC coupled with MS.GC-MS analysis was performed at the SAIF, IIT-Madras, Tamil Nadu, India.

Statistical analysis
All the experiments were performed in triplicate.Statistical analysis of data (mean±SD) was performed using SPSS software, version 18.0.One-way ANOVA test followed by Tukey's range test was applied to analyze the data with level of significance set at p<0.05.

Results
The IZD of test EOs against the studied food-borne bacteria are shown in Table 2 showing significant difference (p<0.05).Black pepper, cinnamon, and clove oils were found to be effective (IZD≥11 mm) against all the both Gram-positive and Gram-negative bacteria; whereas black cumin and nutmeg oils showed intermediate activity (11 mm>IZD≥8 mm) against Gram-positive bacteria and were found to be inactive (IZD<8 mm) against the studied Gram-negative bacteria.On the basis of MIC values, clove oil (14.58±4.79 to 27.38±7.51µg/ml) Downloaded from jfqhc.ssu.ac.ir at 15:09 IRST on Thursday December 27th 2018 [ DOI: 10.29252/jfqhc.5.2.6 ] showed highest antibacterial activity against all the studied bacteria, followed by black pepper oil (16.66±6.01 to 32.14±11.57µg/ml) and cinnamon oil (19.44±6.39 to 34.52±12.44µg/ml).Based on MIC results, black cumin oil and nutmeg oil had significantly (p<0.05)lower antibacterial properties than the other EOs (Table 3).3).As seen in Figure 4, all the three test active EOs exhibited concentration-dependent Fe 2+ ion chelating activity.Clove EO showed the highest chelating activity (9.56±0.08 to 72.68±0.10%),followed by black pepper EO (6.64±0.01 to 62.48±0.01%),and cinnamon EO (2.13±0.04 to 43.86±0.07%).The Fe 2+ ion chelating activity of test active oils and reference standards were found to be in the following decreasing order: EDTA>clove oil>BHT>black pepper oil>cinnamon oil.
There was a close association (R 2 =0.95) between total phenolic content and antioxidant activity.Also, a linear relationship was found (R 2 =0.75-0.90) between total phenolic content and antibacterial activity (data not shown).According to the cytotoxicity experiment, none of the three test active EOs showed cytotoxic effects at recommended dosage level; and their 24 h LC 50 values were found to be >1000 μg/ml (Table 4).
GC-MS analysis of EOs revealed that eugenol, cinnamaldehyde, and β-caryophyllene were the main constituents of clove, cinnamon, and black pepper oils, respectively (Table 5).[ DOI: 10.29252/jfqhc.5.2.6 ] caryophyllene were found to be the major constituents of test active EOs which probably had the most important role for antibacterial and antioxidant activities.It is well documented in the literature that the biological properties of the EOs are generally determined by their major components, including two groups of distinct bio-synthetical origin.Terpenes and terpenoids comprise the main groups whereas aromatic and aliphatic constituents comprise the other group, all characterized by low molecular weight (Pichersky et al., 2006).

Conclusion
The present study provides some evidence about considerable antibacterial and antioxidant potential of clove, cinnamon, and black pepper EOs.These EOs might indeed be used both as natural broad-spectrum antibacterial and antioxidant agents for preventing food spoilage and contamination as well as oxidation of food components which subsequently may help in developing safe and effective natural food preservatives.

Figure 1 :Figure 2 :Figure 4 :
Figure 1: Time-kill curves of black pepper, cinnamon and clove essential oils against food-borne bacteria Bacillus cereus and Salmonella Typhimurium

Table 1 :
Ethnobotanical description and yield of essential oils from spices used in the present study

Table 4 :
Cytotoxic potential (mortality%) of black pepper, cinnamon, and clove oils in brine shrimp lethality assay * * The LC50 for all treatments is >1000 µg/ml.

Table 5 :
Major constituents of black pepper, cinnamon, and clove oil obtained by GC-MS analysis