Assessment of the Biological Activity of Thyme Essential Oil in the Presence of the Classic Antibiotic Tetracycline.

The present study was purported to assess the activities: (i) antibacterial and synergistic against three types of both Gram-positive and Gram-negative bacteria that are susceptible to drug resistance and (ii) cytotoxic and synergistic on colorectal adenocarcinoma cells, of thyme essential oil (TEo) in combination with tetracycline (Tcyc). Chemical composition of thyme essential oil was evaluated by gas chromatography-mass spectrometry (GC-MS) method, antioxidant capacity by 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) assay, antibacterial and synergistic properties were determined by disk diffusion method and cytotoxic activity by quantifying viable cells by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Thyme essential oil has an elevated antioxidant activity, antibacterial potential against both Gram-positive and Gram-negative bacteria, especially on S. aureus and K. pneumoniae at the highest concentration tested (50µL/mL), also having a synergistic effect when combined with tetracycline (50µL/mL TEo with 10µg/mL Tcyc). Essential oil-treated cells showed a dose-dependent reduction in colorectal adenocarcinoma cell viability, while combination with tetracycline leads to a significantly attenuated decrease in viability.

Introduction

Antibiotics are considered one of the most successful discoveries in the history of medicine [1,2].

For thousands of years, because nothing was known about infections and their prevention, antibiotics or vaccines, people were helpless in the face of huge waves of epidemics, such as cholera, smallpox, plague, typhoid fever, malaria, tuberculosis, leprosy, syphilis.

The period between 1940 and 1960 was the Golden Age of antibiotic discovery [1].

During this period, natural antibiotics isolated from actinomycetes were discovered (aminoglycosides, tetracyclines, amphenicols, macrolides, glycopeptides, ansamycins, lincosamides, streptogramins and cycloserine), from fungal origin (penicillins, cephalosporins) and synthetic antibiotics (e.g., sulfones, nitrofurans, quinolones, azoles, phenazines, ethambutol, thioamides).

Most of these antibiotics are still in clinical use, but due to increased antimicrobial resistance their therapeutic efficacy has decreased [3].

Considering that the development of antibiotic resistance is largely attributed to their overuse, inadequate prescription, and suboptimal dosing, it is necessary to take certain measures to re-evaluate and optimize the current dose of antibiotics. Improving the prescription of antibiotics is directly correlated with well-established criteria, based on the relationship between concentration-dose, beneficial effects-side effects [4].

Natural products offer extraordinary chemical diversity with a wide variety of biological effects, and thus have been the most promising sources for drug discovery and development [5].

Plant extracts, isolated plant compounds and other natural substances are a rich potential source of active molecules that can destroy or attenuate the action of pathogens [6,7].

Medieval societies have used a multitude of natural substances to treat health issues that are currently diagnosed as microbial infections, and there has been various research on the likely effectiveness of these treatments [8].

To meet the challenges of antibiotic resistance, a key approach is to stimulate the discovery of bioactive substances at an early stage.

Complementing the pharmacological actions of classical antibiotics with substances of natural origin is intensively studied to achieve efficiency in the clinic, correlated with a real goal of developing a new generation of chemotherapeutic drugs derived from natural sources, considering their unmatched chemical diversity.

The resistance developed by Gram-positive and Gram-negative bacteria to several drugs made them difficult to treat and/or even untreatable with currently available antibiotics [9].

Moreover, it is of major importance to identify new targets and new classes of antibiotics that can deal with drug-resistant bacterial pathogens.

This requires basic research to discover new gaps and to develop new approaches to antibiotics.

The aim of the present research was to evaluate a combination of thyme essential oil (TEo) and tetracycline (Tcyc) in terms of: (i) antimicrobial activity against Gram-positive (Bacillus cereus, Staphylococcus aureus and Streptococcus pyogenes) and Gram-negative bacteria (Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa) and (ii) cytotoxic activity on human colorectal adenocarcinoma cells (HT-29).

Materials and Methods

Essential Oil Extraction

Aerial parts of thyme were received from Agricultural Technologies Department from Faculty of Agriculture Timisoara.

200g plant material was dried and subjected to hydro distillation for four hours in a Clevenger-type device following methods described in the literature [10].

The final mass resulted was dried and stored at-20˚C until further determinations and evaluations.

Chemical Composition Evaluation by Gas-Chromatography-Mass Spectrometry (GC-MS)

The predominant chemical composition of thyme essential oil was determined by utilizing a GS/MS system (Shimadzu gas-chromatograph couplet with mass spectrometer QP2010 Plus) equipped with a 30m length DB-Wax capillary column (0.32mmx1μm).

Some of the parameters set were: (1) carrier gas helium, (2) flow rate 1mL/min, (3) start temperature 40°C, increased with a rate of 5°C/min until reached 250°C and hold for 5min, (4) injector temperature 250°C, (5) ion source temperature 220°C, (6) injection volume 1μL.

Individual compounds were calculated as percentage based on GC peaks and LRI (linear retention indices) were determined as described in the literature [11].

Antioxidant Potential

The DPPH (2,2-diphenyl-1-picrylhydrazyl) assay was used to estimate the radical-scavenging ability of the different dilutions of thyme essential oil. Shortly: (1) 0.5mL of sample was diluted with 2.5mL ethanol 50%, (2) 0.5mL of DPPH 1mM was added, (3) absorbance was measured at 516nm, (4) ascorbic acid was used as positive control while negative control was distilled water.

Antioxidant activity expressed as percentage was calculated according to the formula presented in literature [12].

Antimicrobial Activity Analysis

Essential oil of thyme in combination with tetracycline was evaluated for antimicrobial potential against Gram-positive and Gram-negative bacteria (strains detailed in Table 1) acquired from American Type Culture Collection (Manassas, USA).

Table 1

Bacterial strains utilized in the present study in order to evaluate the activity of thyme essential oil combined with tetracycline

Type	Strain	American Type Culture Collection Code
+	B. cereus	11778
+	S. aureus	25923
+	S. pyogenes	19615
-	E. coli	25922
-	K. pneumoniae	700603
-	P. aeruginosa	27853

Experiments were conducted in specific Petri plates by using a 0.001 dilution of the fresh bacterial strains culture and an inoculum equivalent to a 0.5 McFarland standard, incubation at 37°C for 24-48h.

Water was used as negative control and gentamicin disks as positive control.

+

+

+

-

-

-

Cell Culture and Viability Assessment

The cell line used in this study was human colorectal adenocarcinoma (HT-29) obtained from American Type cell Collection (ATCC) as frozen sample, and preserved in liquid nitrogen until the cultivation for conducting experiments.

McCoy's 5a modified medium purchased from ATCC was necessary for cell culture (on standard conditions, 37°C and 5% CO2), supplemented with 10% FBS (fetal bovine serum) and 1% of antibiotic mixture.

In brief, 10,000 cells were seeded in 96-well plates and after a~90% confluence was reached, cells were treated with five concentrations of TEo (5, 7.5, 10, 25 and 50µL/mL) or Tcyc (1, 2.5, 5, 7.5 and 10µg/mL) and three concentrations of TEo and Tcyc mixture (10, 25, or 50µL/mL with 10µg/mL).

After 48 hours of stimulation, 10µL/well of MTT solution was added, followed by three hours of incubation.

Number of viable cells was determined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) test according to the protocol presented in the literature [13].

Results

Following the GC-MS analysis, 16 known chemical constituents were identified in the essential oil of thyme and are presented in Table 2.

Table 2

Chemical composition quantification by GC-MS of thyme essential oil

No.	Compounds	LRI	Molecular formula	Concentration (%)
1	Cyclohexane	729	C6H12	0.694
2	α-Pinene	1010	C10H16	2.358
3	Camphene	1046	C10H16	0.725
4	(+)-4-Carene	1149	C10H16	3.062
5	β-Myrcene	1155	C10H16	1.634
6	γ-Terpinene	1223	C10H16	16.869
7	p-Cymene	1283	C10H14	28.792
8	o-Cymene	1298	C10H14	1.117
9	Caryophyllene	1562	C15H24	2.304
10	Terpinen-4-ol	1606	C10H18O	0.921
11	Humulene	1649	C15H24	0.491
12	Borneol	1690	C10H18O	2.546
13	α-Terpineol	1718	C10H18O	0.696
14	Caryophyllene oxide	1975	C15H24O	1.225
15	(−)-Spathulenol	2121	C15H24O	0.345
16	Thymol	2153	C10H14O	35.420
				99.199

The highest percentages were as follows: thymol (~35%), p-cymene (~29%), γ-terpinene (~17%), (+)-4-Carene (~3%), α-pinene (~2%), borneol (~3%), and caryophyllene (~2%).

-Cymene

In the Figure 1 is presented the antioxidant activity of thyme essential oil tested at three concentrations.

Figure 1

Antioxidant activity of thyme essential oil evaluated by DPPH method and expressed as percentage

Samples showed a moderate antioxidant activity compared to that of ascorbic acid, the values being directly proportional to the concentration.

Therefore, at the highest concentration the average antioxidant activity is around 71%, at the first dilution it decreases to the value of 41% and at the lowest concentration it is around 30% (Figure 1).

Bacteria susceptibility to the thyme essential oil and its combination with tetracycline, determined by disk diffusion method, pointed out that TEo shows increased dose-dependent activity on all strains tested except P. aeruginosa, with the highest inhibitory effects produced inhibition zones of 41 mm diameter.

Concerning antimicrobial activity, the diameter inhibition zone expressed in mm (as the average of two determinations in report to the effectiveness of the positive control) are presented in Figures 2 and 3.

Figure 2

Antimicrobial activity of thyme essential oil and its association with tetracycline expressed as inhibition zones (mm) on Gram-positive bacterial strains

Figure 3

Antimicrobial activity of thyme essential oil and its association with tetracyc line expressed as inhibition zones (mm) on Gram-negative bacterial strains

The maximum zone of inhibition revealed by thyme essential oil was at the maximum concentration tested (50µL/mL) against S. aureus (~35mm, Figure 2) and K. pneumoniae (~40mm, Figure 3) while the combination with tetracycline led to a slight increase in diameter, S. aureus (~38mm) and K. pneumoniae (~41mm).

To determine the cytotoxic effect of thyme essential oil and tetracycline, were tested different concentrations on human colorectal adenocarcinoma cell line-HT-29.

Cell viability was assessed by using the MTT assay at 48-hour intervals.

In the case of tetracycline, a slight decrease in cell viability was observed only at the highest concentration tested (10µg/mL) as can be seen in the Figure 4.

Figure 4

Percentage of human colorectal adenocarcinoma viable cells (HT-29) assessed in vitro after stimulation with thyme essential oil, tetracycline and their combination at different concentrations, by MTT assay. One-way ANOVA analysis, followed by Tukey’s post-test were employed in order to establish statistical differences (*p<0.1, **p<0.01, ***p<0.001 and ****p<0.0001).

In contrast, thyme essential oil exerts a decrease in cell viability by more than 30 percent at the highest concentration tested (50µL/mL).

Regarding the evaluation of the highest concentration of tetracycline and three different concentration of thyme essential oil on the behaviour of cells the following values were obtained: ~98% (Tcyc 10µg/mL+TEo 10µL/mL), ~92% (Tcyc 10µg/mL+TEo 25µL/mL) and ~84% (Tcyc 10µg/mL TEo 50µL/mL).

Discussions

Thymus vulgaris is a plant commonly found in the Balkans and known for several biological properties, including a very pronounced antimicrobial activity [14].

In the present study, the major compounds identified in thyme essential oil were thymol, p-cymene, and γ-terpinene.

Different studies have described the majority composition of volatile T. vulgaris oil based on p-cymene, γ-terpinene, and thymol our data being in accordance with those described in the literature regarding predominant compounds [15,16,17,18].

Certainly, these compounds show percentage variations depending on the geographical area and climatic conditions [19].

A viable strategy to control antibiotic-resistant bacteria is to use natural antimicrobial compounds and/or their association with clinically used antibiotics.

It is well known that thyme essential oil rich in carvacrol and thymol possesses strong antimicrobial effects [20].

Its antibacterial activity but also the synergistic effect in combination with classical antibiotics was evaluated with very promising results and have been described in several studies [21,22,23].

The association between essential oil and tetracycline, in the present study, had positive effects in Gram-positive bacteria (especially S. aureus) but also in Gram-negative ones (especially K. pneumoniae).

The study conducted by Miladinović et al highlighted the predominantly synergistic and additive effects after using the combination of T. glabrescens oil and tetracycline against K. pneumoniae and E. coli [24].

Inhibition of protective enzymes, sequential inhibition of common biochemical pathways, combination of active compounds on the membrane and use of membranotropic compounds are some of the possible mechanisms associated with the diffusion of antimicrobials by synergism [25].

The combined therapy of classical antibiotics with bioactive agents can be a real way to address the various problems caused both by the development of bacterial resistance mechanisms and by the registered side effects.

Stimulation of cells with different concentrations of thyme essential oil has reduced cell viability in a dose-dependent manner, as described in the literature [26,27,28].

The HT-29 cell line was selected due to its use mainly in studies related to the transport of drugs and food compounds but also to study the intestinal immune response to bacteria (survival, adhesion, invasion of microorganisms) [29].

These cells can be considered a viable model because it expresses the characteristics of enterocytes and are essential for observing attachment but also for evaluating certain mechanisms.

Secretion of mucus is important because the mucus layer plays an important role in modulating adhesion [29].

The association between TEo and tetracycline attenuated the decrease in cell viability exerted by thyme essential oil but also inhibited antibiotic-induced cell proliferation.

In the future, more detailed studies are needed to elucidate the mechanisms involved.

Conclusion

The current research study revealed that thyme essential oil manifests a series of biological properties quantified by an increased antioxidant activity, a broad-spectrum antibacterial activity against both Gram-positive and Gram-negative bacteria and a good cytotoxic potential.

The association between the biological product and the classic antibiotic tetracycline has shown the preservation of its cytotoxic properties and the increase of antimicrobial activity against S. aureus, E. coli and K. pneumoniae.

Conflict of interests

None to declare.