Bioactivity of Juniperus communis essential oil and post-distillation waste : assessment of selective toxicity against food contaminants

Previously chemically characterized Juniperus communis essential oil (EO) and post-distillation waste (PDW) were tested for cytotoxicity and antimicrobial activity against food contaminants. Microdilution assay showed that PDW induced moderate antifungal (minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) values, ranging between 0.118-0.900 mg mL-1), and an antibacterial effect against Listeria monocytogenes (MIC and minimum bactericidal concentration (MBC) were 0.39 and 0.74 mg mL-1, respectively). Combinations of EO/PDW with selected antibiotics induced synergistic antilisterial activity in the checkerboard assay. The MTT assay determined that cytotoxicity against colon cancer cells was high for the EO but negligible for PDW (IC50 values were 0.087-0.106 and 1.450-6.840 mg mL-1, respectively). The selectivity indices indicated high selectivity of PDW against tested fungi and L. monocytogenes. In the adhesion-inhibition assay, PDW reduced in vitro adhesion of L. monocytogenes to colon cells (29-62% of inhibition). In conclusion, PDW exhibited an antimicrobial effect against important food spoilage and poisoning fungi and L. monocytogenes, and also reduced in vitro adhesion of L. monocytogenes to colon cells. The results indicate that J. communis PDW could be considered as natural preservative against food spoilage and poisonous fungi, and as an adjuvant to conventional therapy of listeriosis.


INTRODUCTION
The search for new antimicrobial agents has become a necessity because growing resistance to existing antibiotics is posing a serious problem to global public health [1].Similarly, the increased use of antifungal agents has resulted in the rapid development of fungal resistance and made it a major clinical problem, due to the limited arsenal of available systemic antifungal agents [2].
Plants are considered as an extremely important source of antimicrobials and many species used in traditional medicine are currently under investigation [3].Junipers (Juniperus spp.), which contain numerous active compounds, are among the most wide-spread species in the Northern Hemisphere.The use of these plants, especially their seed cones ('berries'), in folk medicine and manufacturing is extensive [4].In traditional medicine they are used as diuretics, appetizers, carminatives, stomachics, anticonvulsants and antihypertensive agents, as well as in the treatment of headaches, fever, bronchitis, asthma and some gynecological disorders [5,6].Literature data also indicate remarkable antioxidant, antimicrobial and hypoglycemic activities of these berries [7][8][9][10].Furthermore, juniper extracts have found wide applications in pharmaceutical industry, perfumery and aromatherapy [11].
Juniperus communis L. is well known as the only food spice derived from conifers [12].Juniper berry extracts are used as a flavoring agent in food and alcoholic beverage industries.The most famous alcoholic beverage containing juniper is gin but locally manufactured juniper brandies are also very popular [13,14].In addition, juniper berries are used in European and particularly in Scandinavian cuisine.They are used in meat preparation, especially wild birds and game, and to flavor dishes prepared with pork, cabbage and sauerkraut [15,16].
In recent years, interest in plant-derived food additives is in great expansion, since they possess different health-promoting properties [17].Furthermore, particular adverse effects such as immunologic hypersensitivity have been reported for some synthetic food additives [18], and this has additionally stimulated the search for natural replacements.Among natural compounds that could be used as alternatives to antimicrobial synthetic food additives, essential oils (EOs) are considered as effective candidates [19].In addition, various plant waste materials, including post-distillation waste (PDW), are receiving increased attention since they contain numerous bioactive compounds available for further extraction [20].
The aim of this work was to study the antimicrobial effect of J. communis EO and PDW against selected food-borne pathogenic and spoilage bacteria, as well as food-poisoning and pathogenic fungi.The antibacterial effect was determined for EO and PDW alone or in combination with common antibiotics.To estimate the selective toxicity to microbial strains, the cytotoxic potential against colon cancer cells was also determined and selectivity indices were calculated.Finally, the inhibitory potential of PDW against bacterial adhesion to colon cells was examined in vitro using the most sensitive bacterial strain.

Plant material, EO and PDW preparation
Plant material (seed cones of Juniperus communis L. var.saxatilis Pall.) was collected on Mt.Stara Planina, Serbia.The voucher specimen (No. 16693) was prepared, identified by Nemanja Rajčević (PhD in botany), and deposited at the Herbarium of the University of Belgrade, Faculty of Biology, Institute of Botany and Botanical Garden "Jevremovac" (BEOU Herbarium).Air-dried and finely ground seed cones were submitted to hydrodistillation in a Clevenger-type apparatus, as previously described [21].The EO was dissolved in dimethyl sulfoxide (DMSO) for all performed bioassays.After distillation of the EO, the residual aqueous solution was evaporated in vacuum at 45°C, resulting in a dry PDW extract.Distilled water (dH 2 O) was used as a solvent for the PDW extract.

Bacterial and fungal strains and human cell cultures
The antimicrobial effect was determined against: (i) Louis, USA, stock solution 0,675 mg mL -1 in sterile dH 2 O) was used as a growth indicator in antibacterial microdilution and checkerboard assays.
The human cells (HT-29 and HCT116) were grown in Dulbecco's modified Eagle's medium (DMEM) with 4.5% glucose and 2 mM L-glutamine, supplemented with 10% fetal bovine serum (FBS) and a penicillin/ streptomycin cocktail.Cells were maintained in an incubator at 37ºC with 5.0% CO 2 in a humidified atmosphere.The cells growing attached to the surface were subcultured at 90% confluence twice a week.Single cell suspensions for subculturing and for experiments were obtained using 0.1% trypsin (from porcine pancreas).Cell viability in suspensions was inspected by the trypan blue dye exclusion method.In the cytotoxicity assay, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Cas.No 298-93-1), 5-fluorouracil (5-FU, Cas No. 51-21-8) and phosphate buffered saline (PBS) were used as indicators of cell viability, positive control and to wash cells, respectively.All media and reagents used to grow and manipulate human cells were purchased from Sigma-Aldrich, St. Louis, USA.

Microdilution assay
The antimicrobial properties of the EO and PDW were determined in microdilution assay performed in 96-well microtiter plates.The serial two-fold dilutions of test substances were made in corresponding medium (BHI for L. monocytogenes and E. faecalis, MHB for other bacteria, and MB for fungi).Suspensions of indicator strains were adjusted to 10 6 CFU mL -1 by preparing an exponential bacterial culture or by washing fungal spores with sterile 0.85% saline containing 0.1% Tween 80 (v/v) from the surface of MA plates.EO was tested in concentration range 0.39-50 mg mL -1 for both bacteria and fungi, while PDW was tested in the ranges 0.195-25 mg mL -1 and 7-900 µg mL -1 for bacteria and fungi, respectively.Assays were performed in triplicate in three individual experiments.
A slightly modified resazurin-incorporated microdilution assay, performed as previously described [22], was used to evaluate the antibacterial properties.Briefly, test substances were serially two-fold diluted along the columns.Bacterial inoculum (10 5 CFU mL -1 ) was added to each well except the sterility control.The growth indicator resazurin (final concentration 0.067 mg mL -1 ) was added to the wells and after 24 h of incubation at 37°C, MICs were determined as the lowest concentrations that did not induce color change.After plating by inoculation loop from each well without visible growth on solid media (BHA/MHA) and incubation (24 h at 37°C), MBCs were determined.As positive controls, conventional antibiotics Str, Amp and Azm were applied in a concentration range of 0.78-100 µg mL -1 .Sterilized solvent (5% DMSO and dH 2 O for EO and PDW, respectively) was used as a negative control.
The microdilution assay, performed as previously described [23], was used to evaluate the antifungal potential.Briefly, the fungal spore suspensions (1.0 × 10 5 CFU mL -1 ) were added to each well containing graded concentrations of test substances.After 72 h of incubation at 28°C, MICs were determined with a binocular microscope as the lowest concentrations without visible growth in seeded wells.The MFCs were determined by the same procedure after serial subcultivation from each well without visible growth into microtiter plates.Standard fungicides Bfz and Kcz, both applied in the concentration range 4-512 µg mL -1 , served as positive controls, while a solvent (sterile 0.85% saline containing 0.1% Tween 80 and dH 2 O for EO and PDW, respectively) was used as a negative control.

Checkerboard assay
A slightly modified checkerboard assay was performed as previously described [24].It was used to determine the mode of interactions between the test substances (EO/PDW and antibiotics).One test substance was serially two-fold diluted along the vertical, while the second one was serially two-fold diluted along the horizontal line of 96-well microtiter plates.In combinations prepared with PDW, the concentrations ranged between (1/32)×MIC-4×MIC, while in combinations prepared with EO the concentrations ranged between (1/64)×MIC-2×MIC.The MIC values of combinations were determined by adding resazurin (final concentration 0.067 mg mL -1 ) and inspecting for color changes.Combinations that did not induce color change of resazurin were used for the calculation of the fractional inhibitory concentration index (FICI) for two antimicrobials in combination.The FICI was calculated according to equation ( 1), where substance A was EO or PDW, and substance B was the antibiotic (Str, Amp, Azm).

Cytotoxicity assay
Cytotoxicity was determined by the MTT reduction assay, performed as previously described [23].Briefly, the assay was performed on HT-29 and HCT116 cells inoculated in 96-well plates at a density of 5x10 4 cells/ well and incubated until they formed a monolayer; EO and PDW were serially two-fold diluted in tested concentration ranges (0.016-0.500 and 0.313-20 mg mL -1 , respectively).After 24 h of incubation, the medium was removed and replaced with the MTT solution (final concentration 0.5 mg mL -1 in DMEM); the plates were additionally incubated for 3 h to allow for mitochondrial reduction of MTT into formazan, performed in viable cells.After this step, the medium was carefully removed and the formazan crystals were dissolved in DMSO.Cell viability was determined by measuring the absorbance at 570 nm, using a microplate reading spectrophotometer (Multiskan FC, Thermo Fisher Scientific, Shanghai, China).The cytotoxic activity was evaluated by comparing the absorbance of the wells containing the test substances to that containing the vehicle (DMSO or dH 2 O for EO and PDW, respectively).5-fluorouracil (5-FU) was used as a positive control.For each test substance, two independent experiments with six wells per treatment point were performed.

Selectivity index
In order to estimate the selective toxicity of test substances, a relationship between cytotoxic and antimicrobial effects was determined through the selectivity index (SI).The SI was calculated as previously described [25] using the following equation ( 2): Positive values of SI indicate higher toxicity to bacteria or fungi, while negative values indicate higher toxicity to colon cells.

In vitro adhesion-inhibition assay
The potential to influence bacterial adhesion to colon cells was determined for L. monocytogenes.Both HT-29 and HCT116 cells were used as a model of colon epithelium.The adhesion-inhibition assay was performed using a slightly modified protocol previously described [23].Colon cells were inoculated into 12-well plates at a density 8x10 4 cells/well and incubated at 37°C in 5% CO 2 to form a monolayer.After this step, the medium was removed and the cell monolayers were washed twice with PBS.Two wells were used to enumerate the cell number by trypan blue dye exclusion.L. monocytogenes suspension prepared in DMEM with PDW (concentration equal to ½ MIC) or without it, was added to each well.The final ratio of human and bacterial cell numbers was about 1/10 (the number of bacteria was 10-fold higher than the number of HT-29/ HCT116 cells).To allow adhesion, human cells were co-incubated with bacteria for 1 h at 37°C in 5% CO 2 .The medium was removed and the cell monolayer was washed twice to remove the non-adhering bacterial cells.To enumerate the adhered bacteria, the monolayer was treated with 0.1% trypsin and appropriate dilutions of obtained cell suspensions were plated in triplicate onto BHA and incubated at 37°C for 24 h.The numbers of bacterial cells in suspensions added initially to the cell monolayer were determined in order to calculate the proportion of adhering bacteria.The effect on adhesion was estimated by comparing the percentage of adhering bacteria in the medium with and without the test substance.For each cell line, two independent experiments in triplicate were performed.

Statistical analysis
Experimental data were analyzed by Student's t-Test.The level of statistical significance was defined as p<0.05.

Antimicrobial properties
The antimicrobial potential of J. communis EO and PDW was determined against selected bacterial and fungal strains.While the antifungal effect of EO was negligible, PDW demonstrated remarkable activity, with MICs and MFCs ranging between 119-900 µg mL -1 and 250-900 µg mL -1 , respectively (Table 1).The highest activity was recorded against A. versicolor.On the other hand, the antibacterial effect of both EO and PDW was weak, with MICs mainly above 3 mg mL -1 (Table 2).The most sensitive bacterium was L. monocytogenes with MIC and MBC values of 3.30 mg mL -1 and 6.25 mg mL -1 for EO, respectively, and 0.39 mg mL -1 and 0.74 mg mL -1 for PDW, respectively.
The effect of EO and PDW against L. monocytogenes was further monitored in the checkerboard assay, where they were combined with conventional antibiotics Str, Amp and Azm.The results showed that certain combinations of EO with Str and Amp, as well as of PDW with all three tested antibiotics, induced a synergistic antimicrobial effect (Table 3).

Cytotoxic potential
Evaluation of cytotoxicity, performed on colon carcinoma HT-29 and HCT116 cells, revealed that both EO and PDW possess a cytotoxic potential (Fig. 1).The estimated IC 50 values (concentrations that decreased cell viability to 50%) pointed at a significantly higher cytotoxicity of EO, comparable to the common cy-tostatic 5-FU (Table 4).PDW induced much lower cytotoxicity, with IC 50 values approximately 20-and 60fold higher than those of 5-FU in HT-29 and HCT116 cells, respectively.The results also showed higher sensitivity of HT-29 cells to all test substances, with the most pronounced discrepancy between cell lines determined for the effect of PDW.Concentrations of test-substances are expressed as mg mL -1 .Student's t-Test used to analyze the experimental data, revealed that a statistically significant difference (p<0.05)corresponding to the control was observed for the following concentrations: A -for EO at 0.125 and 0.0625 mg mL -1 and all lower ones for HT-29 and HCT116 cells, respectively; B -for PDW at 0.625 and 1.25 mg mL -1 and all lower ones for HT-29 and HCT116 cells, respectively; (C) for 5-FU at 0.05 and 0.025 mg mL -1 and all lower ones for HT-29 and HCT116 cells, respectively.

Selective toxicity
Calculation of the SI indicated the absence of selective toxicity of EO to all tested microorganisms, while SI values obtained for PDW were variable.They were almost all positive in respect to HCT116 cells (indicating selectivity against microorganisms), but in respect to HT-29, positive values were determined for L. monocytogenes and almost all fungal strains (Table 5).

Anti-adhesive properties
Taking into account that SI values for PDW indicated higher toxicity to L. monocytogenes than to both human colon cells, we performed the in vitro adhesioninhibition assay and monitored the potential of PDW to reduce the ability of L. monocytogenes to adhere to the colon cells.The obtained results indicated that PDW significantly decreased adhesion of L. monocytogenes to both HT-29 and HCT116 cells (Table 6).The inhibitory potential was more prominent in the case of HCT116 cells: 62% inhibition vs 29% inhibition in HT-29 cells.
Concerning the antifungal properties tested in this work, a general observation was that EO induced  only a weak, almost negligible effect, while the effect of PDW was remarkably higher.Literature data also indicates a weak antifungal effect of EO prepared from J. communis berries [28] and a slightly higher effect of oil prepared from needles [29].To the best of our knowledge, the antifungal effect of PDW has not been previously tested, but literature data show the antifungal effect of the hydroalcoholic extract of J. communis [30].According to Holetz et al. [31] who classified the antimicrobial agents according to MIC values into groups with high (MIC<0.1 mg mL -1 ), moderate (0.1<MIC<0.5 mg mL -1 ) and weak (0.5<MIC<1 mg mL -1 ) antimicrobial activities, the antifungal effect of PDW could be considered as moderate.Bearing in mind that toxigenic fungi associated with food spoilage and poisoning belong mainly to three genera, Aspergillus, Fusarium and Penicillium, and that Trichoderma species could also be important food contaminants [32,33], the antifungal activity of PDW obtained in this work could be of special interest for food preservation.According to SI values, the cytotoxicity of PDW against human colon cells was markedly lower than its antifungal effect, especially in the case of the more resistant HCT116 cells.The different sensitivities of the two cell lines could be attributed to their intrinsic differences [34], and they show the importance of using more than one cell line for SI assessment.The generally high SI values determined for almost all tested fungi are particularly important because biocontrol of toxigenic fungi present in foods has to make it safe for human use.
The antibacterial effect of J. communis EO and PDW was generally weak.Similar to our results, Glišić et al. [28] revealed low antibacterial activity of J. communis EO, but higher activity of different preparations of EO fractions.However, good antibacterial properties of some J. communis EOs were also previously reported [10,35], indicating that the origin and distilling procedure significantly influences the chemical composition and consequently the biological activities of the oils.The low antimicrobial activity of our EO could be due to the unusually high content of hydrocarbon terpenes, which commonly possess a lower antimicrobial potential than oxygenated terpenoids [36].On the other hand, the higher antimicrobial activity of PDW, especially against micromycetes and L. monocytogenes, could be attributed to the antimicrobial properties of polyphenolics which are well established [37].
L. monocytogenes is an invasive food-borne pathogen that commonly enters the host by consumption of contaminated food.In our experiments, L. monocytogenes was the most sensitive bacterium to J. communis EO and especially PDW.Even more important is the finding that EO and PDW could act synergistically with the tested antibiotics against L. monocytogenes, lowering their MICs.While EO decreased the MICs of Str and Amp 4-and 8-fold, respectively, PDW decreased the MICs of all three antibiotics 32-fold.Considering the growing problem of antibiotic resistance, the use of natural compounds as adjuvants capable of increasing the efficacy of conventional antibiotics could be an important strategy to combat infections [38].Amplified antibiotic activity could consequently decrease their therapeutic doses, which is additionally important in terms of their registered side effects [39].
J. communis PDW showed selective toxicity against L. monocytogenes and reduction of adhesion of this bacterium to intestinal cells in vitro.This is another important finding since L. monocytogenes and many other food-borne pathogens need to cross the epithelial barrier of the intestine to cause a systemic disease.The first step in this process is attachment to host intestinal cells, thus the search for natural products with anti-adhesive properties is encouraged [23,40].The ability of J. communis products to inhibit adhesion of Campylobacter jejuni to polystyrene has been recently reported [41].Moreover, in the same study, anti-adhesive properties were even increased in cocultured C. jejuni and L. monocytogenes.Yet, to the best of our knowledge this is the first report indicating the potential of J. communis PDW to reduce adhesion of L. monocytogenes to intestinal cell lines.
In conclusion, the moderate antimicrobial potential of PDW of Juniperus communis against Aspergillus, Penicillium and Trichoderma species, as well as against Listeria monocytogenes, were demonstrated.Both EO and PDW synergistically potentiated the effect of conventional antibiotics against L. monocytogenes.Regarding the colon cell lines used, high selective toxicity of PDW to tested micromycetes and L. monocytogenes was detected.PDW also significantly reduced in vitro adhesion of L. monocytogenes to HT-29 and HCT116 colon cells.The obtained results indicate that material remaining after the distillation of J. communis EO contains bioactive compounds and endorse it for further study as a potential natural antimicrobial preservative, as well as an adjuvant in conventional therapy of listeriosis.

Fig. 1 .
Fig. 1.Cytotoxicity of EO (A), PDW (B) and positive control 5-FU (C) against colon carcinoma cell lines HT-29 (O) and HCT116 (■).Concentrations of test-substances are expressed as mg mL -1 .Student's t-Test used to analyze the experimental data, revealed that a statistically significant difference (p<0.05)corresponding to the control was observed for the following concentrations: A -for EO at 0.125 and 0.0625 mg mL -1 and all lower ones for HT-29 and HCT116 cells, respectively; B -for PDW at 0.625 and 1.25 mg mL -1 and all lower ones for HT-29 and HCT116 cells, respectively; (C) for 5-FU at 0.05 and 0.025 mg mL -1 and all lower ones for HT-29 and HCT116 cells, respectively.

Table 1 .
Antifungal activity of Juniperus communis EO, PDW and conventional fungicides.

Table 2 .
Antibacterial activity of Juniperus communis EO, PDW and conventional antibiotics.

Table 3 .
Antilisterial effects of combinations of J. communis EO/ PDW with different antibiotics in the checkerboard assay.

Table 4 .
IC50 concentrations (mg mL -1 ) of J. communis EO and PDW and the positive control, 5-FU against colon carcinoma cell lines HT-29 and HCT116.

Table 6 .
Anti-adhesive properties of PDW against L. monocytogenes on HT-29 and HCT 116 cells.