Natural Products as Antibacterial Agents — Antibacterial Potential and Safety of Post-distillation and Waste Material from Thymus vulgaris L., Lamiaceae

Abstract

Medicinal plants have a long tradition of use in folk and conventional medicine. In recent years numerous studies confirm various bioactivities of natural products, among them antibacterial activity. Natural antibacterial agents such are essential oils and isolated compounds now represent a notable source for pharmaceutical and food industry and are widely used in cosmetology. They meet standards of 'green consumerism' together with excellent antibacterial activity. Aromatic plants such is Thymus vulgaris L. are the major sources of essential oils. Thyme essential oil, as well as dominant compounds thymol and carvacrol are generally recognised as safe and have been registered by European Commission for use as flavouring agents in foodstuffs. However, essential oil is present in very low amount (0,8-2,6%) in thyme leaves. Thus, the majority of plant material remains unused after the isolation. Nowadays, the biological potential of various plant waste materials are in focus of numerous studies. These investigations also include the antimicrobial activity considering the fact that waste material extracts represent the valuable source of different phenolic compounds. Regarding all this, the aim of the present study was to determine antibacterial potential of chemically characterised extracts obtained from waste material remaining after the preparation of drug (stems) and isolation of thyme essential oil (deodorised leaves, postdistillation decoction) on selected bacterial strains. Also, in order to determine safety of waste extracts their cytotoxicity was investigated. All extracts were prepared with maceration using 45% or 75% ethanol (EtOH) for 24 h at room temperature (1:10 w/v). Total phenolic compounds and flavonoids were determined spectrophotometrically. Extracts were chemically characterized by HPLC/DAD analysis. Antibacerial testing was done with broth dilution method against several bacterial strains (Staphylococcus aureus, Bacillus cereus, Salmonella infantis, Escherichia coli and Campylobacter jejuni). Cytotoxicity and cytoprotection studies were performed by XTT assay. Result of HPLC analysis showed that investigated extracts, especially those obtained from deodorised leaves represent a valuable source of rosmarinic acid and luteolin 7-O-glucuronide. Antibacterial testing indicated that all waste material extracts, except the extract T2, possess similar or even stronger bacteriostatic activity than T1. No cytotoxicity nor cytoprotection were determined. In conclusion, results of this study confirmed antibacterial potential investigated thyme extracts. High concentrations of rosmarinic acid and luteolin 7-O-glucuronide, which both have numerous pharmacological activities, were determined. This indicates that thyme postdistillation waste material extracts could be used for isolation of dominant compounds or as addities in pharmaceutical and food industry.

1. Introduction

Plants have been used as food, spice, and medicine since ancient time. Much before the discovery of the existence of microorganisms, plants were an integrated part of the traditional medicine of many communities used for the treatment of various infectious diseases. Likewise, the use of plants as spices, not only affected the organoleptic properties of food, but it was also one of the ways for its preservation [1]. Today, modern consumers demand food that will be minimally processed and contain, as much as possible, additives of natural origin that unlike synthetic ones, will be safe and ecologically acceptable. Also, the approach in raising livestock and production of food of plant origin is shifting towards the use of traditional methods in protection against pests and diseases [2]. In addition, the irrational use of antibiotics not only in human and veterinary medicine, but also to promote growth in agriculture, has led to more frequent occurrence of resistance to conventional antibiotics of some pathogenic microorganisms [3, 4, 5] such are Escherichia coli, Staphylococcus aureus, Enterococcus spp., Salmonella spp., and Campylobacter spp. [6, 7, 8, 9]. Moreover, it is important to bear in mind the potential synergistic action of different natural products and conventional antibiotics in order to affect the emerging resistance [10, 11, 12].

Aromatic plants such as Thymus vulgaris L. (common thyme) and many other representatives of the family Lamiaceae have a long tradition of use in both folk and conventional medicine and in the pharmaceutical industry [13]. Common thyme is indigenous to Europe, especially the Mediterranean region, and is extensively cultivated worldwide. It is a small, bushy herb, with small, elliptical, greenish-grey, and shortly-stalked leaves. Thyme has a characteristic odour of thymol and is used as a culinary herb [14]. The active principle is the essential oil (Thymi aetheroleum), with thymol (25%–50%) and carvacrol (3%–10%) as dominant compounds (Table 1) (Figure 1) [15].

Compounds	Percentage (%)	References
Monoterpene Hydrocarbons
α-Terpinene	1.25–3.17	17
p-Cimene	0.8–16.13	16, 17
γ-Terpinene	7.3–1.87	12, 16, 17
Oxygenated Monoterpenes
1, 8-Cineole	0.8–2.17	16, 17
Linalool	2.03–6.8	12, 16, 17
Menthone	2.2	16
Borneol	2.6–8.9	12, 16
Neomenthol	2.8	16
Terpinen-4-ol	1–2.63	16, 17
Aromatic Oxygenated Monoterpenes
Carvacrol methyl ether	3.9	12
Thymol	35.51–47.9	12, 16, 17
Carvacrol	4.43–12
Sesquiterpene Hydrocarbons
trans-Caryophyllene	1.13–5.1	12, 17
β-Cubebene	2.4	16
Oxygenated Sesquiterpenes
Caryophyllene oxide	0.33–4.6	12, 17
Aliphatic compounds
1-Octen-3-ol	2.8	12

Table 1.

Dominant compounds of Thymus vulgaris essential oil

The percentage of the major constituents varies greatly, depending on numerous complex factors, both endogenous and exogenous such are: chemotype, ontogenesis, geographic and climatic conditions, methods used for plant material processing, and essential oil isolation [18]. Also, similar to other medicinal plants, the drug could be adulterated or replaced with other representatives of the genus Thymus, such as T. serpyllum, T. marschallianus, T. pannonicus, etc., that affect the differences in chemical composition and biological activities [19, 20]. Nevertheless, a minimum 40% of the thyme essential oil is required to be aromatic oxygenated monoterpenes [15].

	Thyme essential oil; (µl/mL)	Thymol; (µg/mL)	Carvacrol; (µg/mL)	References
Staphylococcus aureus	0.2–2.5	250–2000	125–280	12, 21, 22
Salmonella sp.	0.45–20	250–3755	125–375	3, 12, 21
Escherichia coli	0.45–1.25	250–2000	125–560	12, 21, 22, 23
Bacillus cereus	1	250–560	125–140	12, 21

Table 2.

Minimal inhibitory concentrations of thyme essential oil, thymol, and carvacrol on selected bacterial strains

Thyme essential oil, thymol, and carvacrol are generally recognised as safe (GRAS status) and have been registered by the European Commission for use as flavouring agents in foods [24]. GRAS is a rigorous process that relies on common knowledge and expert consensus about the safety of the substance for its intended use. Well-conducted toxicology studies are one of the factors that are used to assess the safety of various natural products, for example, plant extracts or essential oils that are composed of a mixture of tens or hundreds of compounds [25].

Figure 1.

Chemical structures of dominant monoterpenes in thyme essential oil

In recent years, numerous studies confirm various bioactivities of thyme essential oil and isolated dominant monoterpenes, among them antibacterial and antifungal activity, together with the inhibition of mycotoxin production (Table 2) [3, 12, 16, 21, 23, 26, 27, 28]. Generally, most of the essential oils used in the pharmaceutical, cosmetic, and food industries possess strong antioxidant activity and represent a notable source of natural additives for different products [12, 29, 30]. What is more important is that they meet standards of 'green consumerism' [21]. However, the essential oil is present in very low amounts (0.8%–2.6%) in thyme leaves. Thus, the majority of plant material remains unused after the isolation.

On the other hand, thyme leaves are found to be a rich source of various phenolic compounds, particularly phenolic acids, flavonoids, and other secondary metabolites that are expected to contribute to its biological activities (Table 3) [14].

Rosmarinic acid is an ester of caffeic acid and 3,4-dihydroxyphenyllactic acid (Figure 5) widely found in the plant kingdom and presumably accumulated as a defense compound. In the Lamiaceae family, the occurrence is mainly restricted to the subfamily Nepetoideae [31] and it is identified as one of the active components of several aromatic medicinal plants (e.g., Salvia officinalis, Mentha x piperita, Lavandula officinalis, Ocimum basilicum, Origanum vulgare, Origanum majorana, Thymus vulgaris, Melissa officinalis, Rosmarinus officinalis) [32]. For rosmarinic acid numerous and notable biological activities are confirmed in different in vitro and in vivo examinations. It is of pharmaceutical importance because of its non-specific complement activation and inhibition of the biosynthesis of leukotriens (leading to an anti-inflammatory effect), as well as its antimicrobial and especially antiviral activity [14, 33]. Additionally, it exhibits a very strong antioxidant effect; it is an inhibitor of acetyl and butyryl cholinesterase and neuroprotector that propose its use in the prevention and symptomatic treatment of Alzheimer's disease. However, it should be considered that bioavailability of rosmarinic acid in the brain is very unlikely. Also, in some test systems rosmarinic acid expresses anticarcinogenic activity [34, 35, 36].

Figure 2.

Chemical structures of flavonoids in investigated thyme extracts (Glu = glucose, Rha = rhamnose)

Extraction procedure	Identified compounds	References
Butanol-soluble fraction	Acetophenone glycosides (Androsin, Picein, glycosides of 4-hydroxyacetophenone derivatives)	[37]
Methanol, reflux, residue was extracted with distilled water at 100°C for 2 h	Acidic polysaccharide	[38]
Acetone using a Polytron homogenizer for 1 min	Caffeic acid, Luteolin, Rosmarinic acid, Hispidulin	[39]
CH2Cl2 soluble fraction	5,4`-Dihydroxy-6,7,8-trimethoxyflavone, 5,3`,4`-Trihydroxy-7-methoxyflavone, 5,4`-Dihydroxy-6,7,3`-trimethoxyflavone, 5,4`-Dihydroxy-6,7,8,3`-tetramethoxyflavone	[40]
Extracted at 20°C with 1L of EtOH-H2O-HOAc (80:19:1) under N2 for 5 days; further fractionation	Rosmarinic acid, 3`-O-(8``-Z-Caffeoyl) rosmarinic acid, Eriodictyol, Taxifolin, Luteoline-7-O-glucuronide	[41]
Methanol; 1 day at room temperature	biphenyl compounds (e.g. 3,4,3`,4`-tetrahydroxy-5,5`-diisopropyl-2,2`-dimethylbiphenyl)	[42]
Deodorised aqueous extracts	Total phenolics (mg/g gallic acid equivalents)	[43]
Methanol extract	Monoterpene glucosides (p-Cymenol-9-O-β-glucoside, 2- and 5-O-β-glucosides of thymoquinol, angelicoidenol-O-β-glucoside); Arbutin	[44]
Ethanol/water (30:70, v/v) with the aid of sonication for 10 min	Rosmarinic acid, Caffeic acid	[45]
methanol extract	compounds related to hydroxyjasmone (e.g. 5-hydroxyjasmone-5-O-ß-glucoside) and simple phenol glucosides (3-Hydroxy-4-methoxyphenethyl-3-O-ß-glucoside, eugenol-O-ß-glucoside, syringin)	[46]
Methanol, 20 min, 60°C in a water bath with shaking	Luteolin-glucoside, Rosmarinic acid, Eriodictyol, Luteolin, Apigenin	[47]
Methanol, 6 h, Soxhlet apparatus	Oleanolic acid, Ursolic acid	[48]
aqueous extracts (boiling water), 30 min	Luteolin-7-O-glucuronide, Lithospermic acid, Rosmarinic acid, Caffeic acid	[49]
Aquous, aq. methanolic, methanolic (1:100 or 1:200); infusion, hot methanolic, usage of ultrasonic bath for 15 or 30 min	Arbutin, Hydroquinone, Naringenin, Naringenin-7-O-ß-glucoside, Narirutin, Eriodictyol, Eriodictyol-7-O-ß-glucoside, Eriocitrin, Hesperidin, Luteolin, Luteolin-7-O-ß-glucuronide, Luteolin-7-O-ß-rutinoside, Rosmarinic acid, Methyl rosmarinate, Caffeic acid	[50]
95% ethanol at 60°C for 2 h with stirring	Total phenolics (mg/g sinapic acid equivalents)	[51]
Liquid nitrogen powdered samples were extracted with acidified methanol	Caffeic acid, Rosmarinic acid	[52]

Table 3.

Comparison of literature data on identified compounds in Thymus vulgaris leaves

Chlorogenic acid (5-O-caffeoylquinic acid) is a natural antioxidant that is produced in various plants as a response to pathogen microorganisms, different mechanical impairment, or excessive exposure to light (Figure 5) [53]. Due to these characteristics, chlorogenic acid found its use in pharmaceutical and food industries, as well as in cosmetology [54]. Gallic and ferulic acids are also proven antioxidant agents (Figure 3) [45].

Figure 3.

Chemical structures of hydroxybenzoic and hydroxycinnamic acids present in thyme extracts

Furthermore, many beneficial properties have been identified for flavonoids (e.g., quecetin and rutin as the most widely distributed in nature) (Figure 2), and one of the most popularly cited property is their antioxidant activity. Other actions that are proposed to contribute to their biological effects include chelating metal ions, stimulating phase II detoxifying enzyme activity, and inhibiting proliferation and inducing apoptosis. Also, for some of the flavonoids where anti-inflammatory activity has been confirmed, they decrease the vascular cell adhesion and molecule expression, increase endothelial nitric oxide synthase activity, as well as inhibit platelet aggregation [55].

Specifically, it is determined that luteolin (3',4',5,7-tetrahydroxyflavone) exhibit notable antioxidant, antimicrobial, and anti-inflammatory activities [56]. Also, there is some evidence that apigenin and luteolin act as antimutagenic and anticarcinogenic agents (Figure 4) [57].

Figure 4.

Chemical structures of dominant flavones in investigated thyme extracts

Since various plant extracts and isolated natural compounds are widely used and represent one of the major approaches in alternative and complementary medicine that are also accepted by conventional medicine, their quality, efficacy, and safety are of the utmost importance [58]. Safety issues are especially significant due to increasing body of evidence on side effects and interactions between herbal remedies or/and food or conventional drugs. These interactions sometimes can be very serious. They may lead to increased or lack of action of conventional drugs and caution must be taken in the application of plant extracts [59].

Nowadays, the biological potential of various plant waste materials is the focus of numerous studies. This trend includes not only the examination of aromatic plants [43, 60, 61, 62], but also different fruits and plant products such as wine, olives, beetroot, tomato, garlic, and pomegranate [63, 64, 65, 66]. These investigations also include their antimicrobial activity, considering the fact that waste material extracts represent the valuable source of different phenolic compounds. These studies are important, both in terms of economy and ecology, in order to elucidate the way to exploit post-distillation waste material of aromatic plants and other plant waste materials more efficiently.

Figure 5.

Chemical structures of dominant caffeic acid oligomers in thyme extracts

The aim of the present study was to determine the antibacterial potential of chemically well-characterised extracts obtained from waste material that remain after the preparation of the drug Thymi folium (stems) and the isolation of thyme essential oil (deodorised leaves and post-distillation decoction) on selected bacterial strains. Also, in order to determine the safety of the waste extracts, their cytotoxicity was investigated.

2. Methods

2.1. Plant material and extract preparation

In July 2013, aboveground parts of thyme (Thymus vulgaris L., Lamiaceae) were collected, just before flowering, in Padej, the Vojvodina province, Republic of Serbia. Voucher specimen of collected plants (no. ThV-15/10) [67] was confirmed and deposited at the Herbarium of the Laboratory of Pharmacognosy, Department of Pharmacy, Faculty of Medicine, University of Novi Sad. Leaves were separated from the previously air-dried stems and stored separately at a cool and dry place for extract preparation. Standard thyme leaves extract [15, 68] was prepared by maceration procedure in 45% ethanol (EtOH) as a solvent for 24 h (1:10 w/v, 10 g dried leaves) at room temperature (T1). Essential oil was isolated using hydrodistillation technique [69]. The waste material, remaining after isolation, was filtered and obtained decoction was used to prepare post-distillation extract (T2). Deodorised leaves were air dried and macerated with 45% (T3) and 75% (T4) EtOH for 24 h. Dried stems were grinded (sieve 0.75) and macerated with 45% EtOH for 24 h to obtain the extract T5.

After the maceration, extracts were collected, filtered, and evaporated to dryness under vacuum. The quantities of dry extracts were determined gravimetrically. Residues were dissolved in water to make 10% (w/v) stock solutions for further investigation. For the high-performance liquid chromatography (HPLC analysis), residues were dissolved in methanol, 1% formic acid mixture (50:50 v/v) to make 2% (w/v) stock solutions.

2.2. Chemical composition

2.2.1. Determination of total phenolic compounds and total flavonoid content

The amount of total phenolic compounds in investigated extracts was determined spectrophotometrically with Folin-Ciocalteu (FC) reagent [70]. The concentration of total phenolic compounds was expressed in mg of gallic acid equivalents (GAE) per g of dry extract (d.e.), using a standard curve of gallic acid (concentration range 0.08–0.24 mg/mL). Total flavonoid content was also determined spectrophotometrically using a method based on the formation of a flavonoid-aluminium complex with an absorptivity maximum at 430 nm [71]. Flavonoid content was expressed in mg of quercetin equivalents (QE) per g of dry extract, using a standard curve of quercetin (concentration range 10–100 μg/mL). All measurements were done in triplicate.

2.2.2. HPLC/DAD analysis

Investigated thyme leaves extracts are rich in a wide range of phenolic compounds [50]. Therefore, a method that allows simultaneous detection of various phenolics in a single run was developed. This decreases the time necessary for analysis and reduces the analysis costs [72]. HPLC analysis was performed using a liquid chromatograph (Agilent 1200 series), equipped with diode array detector (DAD) and Eclipse XDB-C18, 1.8 μm, 4.6×50 mm column, at a flow-rate of 1 mL/min. Solvent gradient was performed by varying the proportion of solvent A (methanol) to solvent B (1% formic acid inwater (v/v)) [73]. The total running time and post-running time were 45 and 10 min, respectively. The column temperature was 30ºC. The injected volume of samples and standards was 5 μL and it was done automatically using an autosampler. The spectra were acquired in the range of 210–400 nm and chromatograms plotted at 280, 330, and 350 nm with a bandwidth of 4 nm, and with reference wavelength/bandwidth of 500/100 nm. Quantification of selected phenolics: gallic acid, protocatechuic acid, caffeic acid, chlorogenic acid, syringic acid, p-coumaric acid, ferulic acid, rutin, rosmarinic acid, quercetin, and apigenin (Figure 6) were done using the calibration curve of standard compounds.

Figure 6.

The chromatogram of the mixture of standards at 280nm used for chemical analysis of investigated thyme extracts. The order of elution of compounds was: 1-gallic acid, 2-protocatechuic acid, 3-p-hydroxybenzoic acid, 4-caffeic acid, 5-vanillic acid, 6-chlorogenic acid, 7-syringic acid, 8-p-coumaric acid, 9-ferulic acid, 10-sinapic acid, 11-rutin, 12-myricetin, 13-rosmarinic acid, 14-cinnamic acid, 15-quercetin, 16-naringenin, 17-luteolin 7-O-glucuronide, 18-kaemferol, 19-apigenin

2.3. Antibacterial testing

2.3.1. Bacterial strains and growth conditions

Selected bacterial strains, namely Bacillus cereus WSBC 10530 (clinical isolate), Staphylococcus aureus ATCC 25923 (clinical isolate), Salmonella Infantis ŽMJ 106 (poultry meat isolate), Escherichia coli O157:H7 ŽM 370 (clinical isolate), and Campylobacter jejuni NCTC 11168) were used for antibacterial testing. All bacterial strains were stored at –80°C. B. cereus, S. aureus, S. Infantis and E. coli were grown on Mueller-Hinton agar (MHA, Oxoid) at 37°C for 24 h. C. jejuni was grown on Columbia blood agar (Oxoid, Basingstoke, UK) at 42°C for 24 h under microaerobic conditions. For the antimicrobial activity assays, cultures were suspended in Mueller-Hinton broth (MHB, Oxoid) to 10⁵–10⁶ CFU/mL.

2.3.2. Broth microdilution method

Antimicrobial test systems should ideally be simple, reproducible, rapid, and inexpensive [74]. Several methods are currently available to determine antimicobial activity of different plant extracts or isolated pure compounds. Since they are not based on the same principle, the obtained results are influenced not only by the selected method, but also by the microorganism used, as well as by the extraction procedure (polar or non-polar extracts, essential oils, etc.) or the solubility of the tested compound [75]. In order to avoid overemphasizing the usefulness of various plant extracts or isolated compounds as antimicrobials, applied methods, both for extraction and antimicrobial testing, should be standardised to obtain comparable and reproducible results, prior to their in vivo application [76]. Broth macro- or microdilution methods for Minimum Inhibitory Concentration (MIC) determination are recommended by the Clinical and Laboratory Standards Institute (CLSI) [77]. In our study, micromethod, as it was less expensive and easier, was used to yield reproducible results. On the other hand, bacterial growth could be assessed visually, spectrophotometrically, or by using bioluminescence of fluorimetry measurements. However, all of the proposed methods have its shortcomings [78] and in this context colorimetric methods could represent an alternative approach, using tetrazolium salts as indicators. Bacteria convert them to coloured formazan derivatives that can be quantified, but difficulties may arise because of autofluorescence, salt reduction, and the antioxidant properties of plant products, especially for XTT (2,3-Bis-(2-Methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide), TTC (2,3,5-triphenyl tetrazolium chloride), and resazurin. This makes them less convenient indicators for MIC assay. Therefore, INT (2-[4-iodophenyl]-3-[4-dinitrophenyl]-5-phenyltetrazolium chloride) was more suitable and was used in our survey. Change of colour occurs due to the reduction process as INT acts as an artificial terminal electron acceptor in respiration. Nevertheless, tetrazolium salts are not appropriate for microaerophilic campylobacters since they indicate the respiratory activity. Therefore, we used broth microdillution method with ATP measurement as rapid and accurate tools [74].

For the broth microdilution test, 50 μL of each bacterial suspension in suitable growth medium was added to the wells of a sterile 96-well microtitre plate already containing 50 μL of two-fold serially diluted standardised (T1) and post-distillation waste thyme extracts (T2, T3, T4, and T5) in proper growth medium. The final volume in each well was 100 μL. Control wells were prepared with culture medium, bacterial suspension only, plant extracts only, and ethanol in amounts corresponding to the highest quantity present. The contents of each well were mixed on a microplate shaker (Eppendorf, Hamburg, Germany) at 800 rpm for 1 min prior to incubation for 24 h in the cultivation conditions described above. The MIC was the lowest concentration where no viability was observed after 24 h on the basis of metabolic activity. To indicate respiratory activity, the presence of colour was determined after adding 10 μL/well of INT (Sigma) dissolved in water (INT 2 mg/mL) and incubated under appropriate cultivation conditions for 30 min in the dark [79]. To determine the ATP activity, the bioluminescence signal was measured by a Microplate Reader (Tecan, Mannedorf/Zurich, Switzerland) after adding 100 μL/well of BacTiter-Glo™ reagent (Promega, Madison, USA) after 5 min of the incubation in the dark [80]. Positive controls were wells with a bacterial suspension in an appropriate growth medium and a bacterial suspension in an appropriate growth medium with ethanol in amounts corresponding to the highest quantity present in the broth microdilution assay. Negative controls were wells with growth medium and plant extract. All measurements of MIC values were repeated in triplicate. Results are expressed in mg of total phenolics (TP).

2.4. Cytotoxicity studies

2.4.1. Cell culture

Human MRC-5 lung fibroblasts were grown in Minimum Essential Medium (MEM, Gibco, Invitrogen, Austria) supplemented with 2 mM of L-glutamine (Sigma, MO, USA), 10% of heat-inactivated fetal bovine serum (FBS, PAA laboratories, Austria), 100 units/mL of Penicillin (PAA laboratories), and 100 µg/mL of Streptomycin (PAA laboratories) (1% Pen/Strep). Cells were kept in a humidified 5% CO₂ atmosphere at 37°C and passaged at 90% confluence. Passages P26 and P27 were used for the assays.

2.4.2. XTT assay for the determination of cytotoxicity

An automated microculture tetrazolium assay (MTA) has long been used for the in vitro assessment of drug effects on cell growth. The use of the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium reagent requires dimethyl sulfoxide solubilization MTT-formazan generated by cellular reduction. This is not only laborious, but also may risk exposure of laboratory personnel to large quantities of potentially hazardous solutions in dimethyl sulfoxide (DMSO). Furthermore, frequent DMSO exposure also produces deleterious effects upon some laboratory equipment. Therefore, to allow the investigation of a simplified MTA and to address potential problems associated with solvent handling, a series of new tetrazolium salts have been developed which, upon metabolic reduction by viable cells, yield aqueous-soluble formazans [81]. One of the possible alternatives was found to be XTT (2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-S-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide), which is metabolically reduced in viable cells to a water soluble formazan product. This allows reproducible estimates of drug sensitivity in a variety of human and other tumor cell lines. However, it is also applicable for the cytotoxicity studies of various natural products, such are plant extracts or isolated natural compounds.

Cell proliferation kit II (XTT) (Cat. No. 11 465 015 001) was obtained from Roche Diagnostics (Mannheim, Germany). Aliquots (100 µL) of 1x10⁵ cells/mL were seeded into 96 well plates (flat bottom) and grown for 24 h before the extracts were added. Afterwards, cells were incubated for 4 h or 24 h with investigated thyme extracts (20 µg/mL) before XTT solution was added. The XTT solution consisted of a XTT labeling reagent and an electron-coupling reagent and was always freshly prepared. XTT is a yellow tetrazolium salt and cleaved by metabolic active cells into an orange, water-soluble formazan dye. Since these color changes only occur in viable cells, it can be directly quantified using a scanning multiwell spectrophotometer [81]. Numbers of viable cells were determined with the following formula and expressed as percentage of control [82]:

Absorbance of treated cells/Absorbance of untreated cells x 100

Control cells represent vehicle-treated cells. Vehicle was 0.5% of DMSO (final concentration), which did not affect the cells as shown by control experiments.

2.4.3. Cytoprotection assay

Aliquots (100 µL) of 1x10⁵ cells/mL were seeded into 96 well plates (flat bottom) and grown for 24 h before the extracts (20 µg/mL) were added for 4 h or 24 h. Afterwards, the medium with the extracts was removed and replaced by 100 µl of a H₂O₂ solution (50 µM, 100 µM, 200 µM or 500 µM) in MEM medium and further incubated for 2 h or 24 h. To finally determine the cell viability, XTT solution was added and analyzed as described above.

2.5. Statistical analysis

The data were reported as mean values ± standard deviation (SD) and processed using Microsoft Office Excel for Windows v. 2007.

3. Results

3.1. The amount of dry extract

The amounts of dry extracts obtained by maceration of thyme leaves (T1), deodorised thyme leaves (T3 and T4), and thyme stems (T5) and post-distillation extract remaining after the evaporation of thyme decoction (T2) are shown in Table 4. Generally, the amount of dry extract T1 is in accordance with previously published data, considering the fact that numerous factors could influence the extraction yield [47, 83]. When the thyme waste extracts were analyzed, the highest amount was obtained in T2 [43]. The difference in the extraction yield between T1 and T2 may be due to the mere extraction process since T2 is obtained after hydrodistillation of thyme leaves that favours extraction of polar substances, but also present carbohydrates genuine to the leaves or generated after hydrolysis of present glycosides. Deodorised leaves and stems yield a smaller amount of dry extracts when compared to T1.

3.2. Total phenolic compounds and flavonoid content

Spectrophotometric methods used are simple and reproducible for rough screening of total phenolic compounds and total flavonoids before detailed chemical analysis. The results are presented in Table 4. Total phenolic compounds in examined thyme leaves and stem extracts varied from 35.69 (T2) to 62.45 (T4) mg GAE/g d.e., while total flavonoids were in the 11.31 (T5)–52.89 (T3) mg QE/g d.e. range. Interestingly, deodorised leaves extracts (T3 and T4) have a relatively high content of total phenolic compounds and total flavonoids when compared to T1. The extract T2, for which the highest amount of dry extract was determined, is poor in total phenolic compounds and total flavonoids when compared to T1, but also to T3 and T4 [43, 51]. However, when absolute amounts (in plant material) are considered, T2 may also represent a valuable source of phenolic compounds and flavonoids. Stem extract (T5) have relatively high total phenolics, while are poor in total flavonoids.

Type of extract	% dry extract; (g d.e./100 g of plant material)	Total phenolic compounds; (mg GAE/g d.e.)	Total flavonoid content; (µg QE/g d.e.)
T1	9.31±0.05	49.71±0.02	33.88±0.014
T2	37.19±0.1	35.69±0.019	22.79±0.005
T3	3.45±0.03	58.23±0.019	52.89±0.012
T4	3.85±0.04	62.45±0.006	50.09±0.006
T5	5.39±0.05	45.95±0.03	11.31±0.005

Table 4.

The amount of dry extract, total phenolic compounds, and total flavonoid content

3.3. HPLC analysis

The qualitative-quantitative data from the chromatographic analyses are presented in Table 5, with representative chromatograms for T1 and T3 (Figure 7). The following components: gallic, protocatechuic, caffeic, chlorogenic, syringic, p-coumaric, ferulic, and rosmarinic acid, as well as rutin, quercetin, luteolin 7-O-glucuronide andapigenin were identified and quantified within the investigated samples.

Type of extract	T1	T2	T3	T4	T5
c (mg/g d.e.)
Gallic acid	2.46	2.58	1.76	0.77	4.99
Protocatechuic acid	0.23	0.22	0.27	0.37	0.22
Caffeic acid	0.35	0.23	0.13	0.33	0.53
Chlorogenic acid	2.06	2.02	3.77	4.75	7.75
Syringic acid	0.23	0.42	0.3	0.36	0.25
p-Coumaric acid	0.06	0.04	0.05	0.06	0.16
Ferulic acid	1.05	0.89	1.56	1.9	5.77
Rutin	0.4	0.12	2.4	3.68	0.32
Rosmarinic acid	62.22	60.49	86.01	105.08	52.36
Quercetin	0.35	0.04	2.2	3.98	0.36
Luteolin 7-O-glucuronide	42.20	34.44	81.24	87.11	11.01
Apigenin	1.81	0.79	3.36	4.45	0.27

Table 5.

Results of HPLC-DAD analysis of thyme leaves and post-distillation waste extracts

Regarding the hydroxybenzoic, hydroxycinnamic acids, and caffeic acid oligomers, rosmarinic acid was identified as the major component in all the investigated thyme extracts and was present within the range of 52.36 (T5) to 105.08 (T4) mg/g d.e, which is similar to the previously published data for officially prepared thyme extracts (T1) [50]. Also, gallic, ferulic, and chlorogenic acids were present in considerable amounts. In general, 75% EtOH extract of the deodorised leaves (T4) abundant in rosmarinic, chlorogenic, and ferulic acid, whilst 45% EtOH contained higher amounts of gallic acid. In the flavonoid fraction, luteolin 7-O-glucuronide was the dominant and the second major identified component in all investigated extracts (from 11.01 (T5) to 87.11 (T4) mg/g d.e.). Similarly to the total phenolic content, the highest amounts of almost all quantified components, especially the dominant ones were determined in deodorised leaves extracts (T3 and T4). Also, although the stem is confirmed to be relatively poor in phenolic compounds, chlorogenic and ferulic acids are present in considerable amounts.

Figure 7.

Chromatograms of examined thyme leaves extracts (T1 and T3) at 280 nm: 1-gallic acid, 2-protocatechuic acid, 3-caffeic acid, 4- chlorogenic acid, 5-syringic acid, 6-p-coumaric acid, 7- ferulic acid, 8-rutin, 9-rosmarinic acid, 10-quecetin, 11-luteolin 7-O-glucuronide, 12-apigenin

3.4. Antibacterial testing

Numerous studies have confirmed the use of herbs and spices as anti-infective agents [84]. Among them, different essential oils and phenolic compounds, or plant extracts rich in phenolic compounds stand out for their antibacterial activity [85, 86]. This is particularly important, given that phenolic compounds possess a strong antioxidant potential. This joint action is significant for their usage in pharmaceutical and food industries, but also in all other fields where it is important to prevent both oxidation and contamination of certain products [87].

All investigated post-distillation thyme waste extracts exhibited relatively strong antimicrobial potential, against all tested bacterial strains, especially deodorised leaves extracts (T3 and T4) (Table 6) [88]. Also, thyme stem extract (T5) expressed notable antibacterial action. What is more important, by comparing these results with the data obtained for butylhydroxytoluene (BHT), often used as a synthetic additive, antibacterial potential of thyme post-distillation waste material extracts becomes even more evident. Also, interestingly, all investigated thyme extracts showed lower minimal inhibitory concentration on all investigated bacterial strains when compared to isolated rosmarinic acid. This may indicate that phenolic compounds possess a synergistic antibacterial effect, which is important in terms of emerging occurrence of resistance.

	Staphylococcus aureus; MIC (mg TP/mL)	Salmonella liverpool; MIC (mg TP/mL)	Escherichia coli; MIC (mg TP/mL)	Bacillus cereus; MIC (mg TP/mL)
T1	0.09	0.28	0.14	0.14
T2	0.14	0.39	0.56	1.55
T3	0.04	0.12	0.12	0.03
T4	0.06	0.12	0.12	0.03
T5	0.09	0.13	0.19	0.06
BHT (mg/mL)	0.55	0.55	0.55	0.28
Rosmarinic acid^*	5	2.5	2.5	5

Table 6.

Minimal inhibitory concentrations of investigated thyme extracts on selected bacterial strains

^* reference [74]

Despite many recent technological advances in the food industry, which have contributed to increased safety in the food supply chain, we are still facing high incidence of foodborne illnesses. In 2011 alone, there were 69,553 human cases originating from foodborne zoonotic outbreaks in the EU [89]. Ninety-three of them were fatal. In the same year, Campylobacter, the most prevalent foodborne zoonotic agent, which is especially a problem in the production and processing of meat, caused 220 of the 209 registered, mostly sporadic human illnesses. It is assumed that the real burden is even much higher [4].

Two selected extracts, T3 prepared from deodorised leaves and T1 as an officially prepared one, were tested against Campylobacter jejuni. Both T1 and T3 expressed relatively strong antibacterial potential. The activity of deodorised extract T3 in gram negative C. jejuni was comparable to that in gram positive S. aureus and B. cereus. On the other hand, standard thyme extract T1 expressed substantially stronger activity in C. jejuni compared to all other tested species, suggesting that the phenolic compounds present in standard extract T1 may contribute more to the antimicrobial activity in C. jejuni than in other species. Furthermore, obtained MIC values of extracts were lower in comparison with rosmarinic acid, indicating the importance of synergistic effects of different compounds present in investigated extracts that can contribute to the antimicrobial activity through different mechanisms. They can interfere with bacterial membrane and thereby increase the cell leakage or act indirectly antimicrobial by facilitating the influx of antimicrobial phenolic compounds [21]. In this, as well as the economical aspect, the use of plant extracts might be more sensible than the use of pure compounds. However, obtained MIC values were much higher than those of selected antibiotics (Table 7).

	Campylobacter jejuni	References
T1	0.05	[88]
T3	0.06	[88]
Rosmarinic acid	1.25	[74]
Ciprofloxacin; Erythromycin	0.25	[4]
0.25	[4]

Table 7.

Minimal inhibitory concentrations of selected thyme extracts (mg TP/mL), rosmarinic acid (mg/mL) and selected antibiotics (µg/mL) on Camphylobacter jejuni

3.5. Cytotoxicity studies

Cancer is a leading cause of death worldwide and sustained focus is on the discovery and development of newer and better-tolerated anti-cancer drugs, especially from plants. About 50% of all anti-cancer drugs approved internationally are either natural products or natural product mimics. They were developed on the basis of the knowledge obtained from small or macromolecules existing in nature [90].

The major challenges associated with currently available anti-cancer agents include selectivity, toxicity, resistance, and development of a secondary malignancy. These drawbacks have motivated the search for newer, more efficacious, and better tolerated anti-tumor drugs, with natural products, especially plants, offering an inexhaustible reservoir for new drug discovery and development. Also, molecular targeted agents are currently being studied in all treatment settings including that of chemoprevention, which is defined as the use of natural or synthetic nonessential dietary agents to interrupt the process of carcinogenesis and to prevent or delay tumor growth [91].

In vitro cytotoxicity studies are the first step in selecting from the vast number of still unexamined plant extracts or isolated compounds. Also, these studies can provide a valuable insight in the safety of different natural products. This is especially important for waste plant material, in our case for pos-tdistillation waste material of thyme leaves, if their possible future application in pharmaceutical or food industry should be considered.

Figure 8.

Metabolically active cells (% of control) in XTT assay after post-distillation thyme extracts application

Figure 9.

Absorbance values (490–650 nm) of extracts mixed with XTT solution in culture medium

The effects of investigated post-distillation thyme waste extracts against human MRC-5 lung fibroblasts are presented in Figures 8–15. Cytotoxicity of the extracts, a possible reaction with the XTT solvent and a potential cytoprotective activity were evaluated. As shown in Figure 8, none of the investigated extracts at concentrations of 20 µg/ml exhibited cytotoxicity after 4 h or 24 h of incubation. To exclude a direct reaction of the extracts with the XTT solvent and, therefore, false-negative results, extracts and XTT solution were mixed in culture medium and incubated for 4 h or 24 h. There were no significant differences in the absorbance values of medium and XTT alone compared to DMSO (control) or extracts (20 µg/ml) in medium (Figure 9) that reveals that the extracts did not interact with the XTT solution.

Finally, a potential cytoprotective effect of the extracts was investigated. Cells were incubated with the extracts for 4 h or 24 h and cytoprotection was evaluated by adding different concentrations of H₂O_2, which is a well-known cell damaging agent causing oxidative stress.

Figure 10.

Cells incubated with the extracts for 24 h, medium replaced by 50 µM H₂O₂ and further incubated for 4 h (XTT added after 2h and read out after 2h)

Figure 11.

Cells incubated with the extracts for 24 h, medium replaced by 100 µM H₂O₂ and further incubated for 4h (XTT added after 2 h and read out after 2 h)

Figure 12.

Cells incubated with the extracts for 24 h, medium replaced by 200 µM H₂O₂ and further incubated for 4h (XTT added after 2 h and read out after 2 h)

Figure 13.

Cells incubated with the extracts for 24 h, medium replaced by 500 µM H₂O₂ and further incubated for 4 h (XTT added after 2 h and read out after 2 h)

Figure 14.

Cells incubated with the extracts for 4 h, medium replaced by 500 µM H₂O₂ and further incubated for 4 h (XTT added after 2 h and read out after 2 h)

Figure 15.

Cells incubated with the extracts for 4 h, medium replaced by 500 µM H₂O₂ and further incubated for 24 h (XTT added after 24 h and read out after 2 h)

The effects were compared to vehicle-treated (DMSO) cells (control) and to H₂O₂ treated cells (H₂O₂ control). As shown in Figures 10–15, none of the investigated extracts exhibited notable cytoprotective activity.

4. Further research

Results of microbiological tests indicate the possibility to use thyme postdistillation waste extracts primarily in the food or pharmaceutical industry as natural additives, regarding the relatively strong antibacterial activity on investigated bacterial strains.

Also, examined extracts represent a valuable source of phenolcarboxylic acids and flavonoids, which have numerous biological effects. Therefore thyme post-distillation waste material could be used for the isolation of rosmarinic acid and luteolin 7-O-glucuronide, as dominant compounds.

However, only limited data are presently available on the antimicrobial activity of plant extract in food systems and this indicates continuation of our study and the need for further work. Additionally, there is growing evidence on synergistic action of different plant products (extracts, essential oils, isolated compounds) with each other or with conventional antibiotics used in human and veterinary medicine [92]. Besides, it would be very interesting to collect more data on the efficiency of both thyme essential oil and investigated thyme extracts as a dietary supplementation in animal feed on protective or prebiotic effects on the gut microflora. Also, it is important to test their influence on antioxidant and antimicrobial stability and shelf life of meat and meat products [93, 94, 95].

Furthermore, the results of our study recorded neither cytotoxicity, nor cytoprotection of examined extracts. Yet, detailed in vivo toxicology analyses are required in order to confirm the safety of usage of thyme postdistillation waste material.

Acknowledgments

The authors are thankful to the research agencies of Slovenia, Serbia, and Austria for financing the bilateral projects BI-034-SLO-SR-2010/2011 and WTZ SI 03/2011 and to the The Provincial Secretariat for Science and Technological Development of Vojvodina (grant number: 114-451-2056/2011-01).