We are IntechOpen, the world’s leading publisher of Open Access books Built by scientists, for scientists 4,100 116,000 120M Open access books available International authors and editors Downloads Our authors are among the 154 TOP 1% 12.2% Countries delivered to most cited scientists Contributors from top 500 universities Selection of our books indexed in the Book Citation Index in Web of Science™ Core Collection (BKCI) Interested in publishing with us? Contact [email protected] Numbers displayed above are based on latest data collected. For more information visit www.intechopen.com Chapter 6 Natural Products as Antibacterial Agents — Antibacterial Potential and Safety of Post-distillation and Waste Material from Thymus vulgaris L., Lamiaceae Neda Gavarić, Jasna Kovač, Nadine Kretschmer, Nebojša Kladar, Sonja Smole Možina, Franz Bucar, Rudolf Bauer and Biljana Božin Additional information is available at the end of the chapter http://dx.doi.org/10.5772/60869 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 © 2015 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 124 Concepts, Compounds and the Alternatives of Antibacterials (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 cytopro‐ tection 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 deter‐ mined. This indicates that thyme postdistillation waste material extracts could be used for isolation of dominant compounds or as addities in pharmaceutical and food industry. Keywords: aromatic plants, thyme, postdistillation waste, antibacterial potential, cytotoxicity 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 microorgan‐ isms [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, Natural Products as Antibacterial Agents — Antibacterial Potential and Safety of Post-distillation and Waste… 125 http://dx.doi.org/10.5772/60869 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]. Neverthe‐ less, a minimum 40% of the thyme essential oil is required to be aromatic oxygenated mono‐ terpenes [15]. 126 Concepts, Compounds and the Alternatives of Antibacterials Thyme essential oil Thymol Carvacrol References (µl/mL) (µg/mL) (µg/mL) 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].     Thymol  Carvacrol  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 consumer‐ ism' [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 Natural Products as Antibacterial Agents — Antibacterial Potential and Safety of Post-distillation and Waste… 127 http://dx.doi.org/10.5772/60869 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 anticarci‐ nogenic activity [34, 35, 36].   Quercetin  Rutin    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-[37] hydroxyacetophenone derivatives) Methanol, reflux, residue was Acidic polysaccharide [38] extracted with distilled water at 100°C for 2 h Acetone using a Polytron Caffeic acid, Luteolin, Rosmarinic acid, Hispidulin [39] homogenizer for 1 min CHCl soluble fraction 5,4`-Dihydroxy-6,7,8-trimethoxyflavone, 5,3`,4`- [40] 2 2 Trihydroxy-7-methoxyflavone, 5,4`-Dihydroxy-6,7,3`- trimethoxyflavone, 5,4`-Dihydroxy-6,7,8,3`- tetramethoxyflavone β Extracted at 20°C with 1L of EtOH- Roβsmarinic acid, 3`-O-(8``-Z-Caffeoyl) rosmarinic aciβd, [41] HO-HOAc (80:19:1) under N for 5 Eriodictyol, Taxifolin, Luteoline-7-O-glucuronide 2 2 days; further fractionation 128 Concepts, Compounds and the Alternatives of Antibacterials Extraction procedure Identified compounds References Methanol; 1 day at room temperaturebiphenyl compounds (e.g. 3,4,3`,4`-tetrahydroxy-5,5`- [42] diisopropyl-2,2`-dimethylbiphenyl) Deodorised aqueous extracts Total phenolics (mg/g gallic acid equivalents) [43] Methanol extract Monoterpene glucosides (p-Cymenol-9-O-β-glucoside, 2- [44] and 5-O-β-glucosides of thymoquinol, angelicoidenol-O-β- glucoside); Arbutin Ethanol/water (30:70, v/v) with the Rosmarinic acid, Caffeic acid [45] aid of sonication for 10 min methanol extract compounds related to hydroxyjasmone (e.g. 5- [46] hydroxyjasmone-5-O-ß-glucoside) and simple phenol glucosides (3-Hydroxy-4-methoxyphenethyl-3-O-ß- glucoside, eugenol-O-ß-glucoside, syringin) Methanol, 20 min, 60°C in a water Luteolin-glucoside, Rosmarinic acid, Eriodictyol, Luteolin, [47] bath with shaking Apigenin Methanol, 6 h, Soxhlet apparatus Oleanolic acid, Ursolic acid [48] aqueous extracts (boiling water), 30 Luteolin-7-O-glucuronide, Lithospermic acid, Rosmarinic [49] min acid, Caffeic acid Aquous, aq. methanolic, methanolic Arbutin, Hydroquinone, Naringenin, Naringenin-7-O-ß- [50] (1:100 or 1:200); infusion, hot glucoside, Narirutin, Eriodictyol, Eriodictyol-7-O-ß- methanolic, usage of ultrasonic bath glucoside, Eriocitrin, Hesperidin, Luteolin, Luteolin-7-O-ß- for 15 or 30 min glucuronide, Luteolin-7-O-ß-rutinoside, Rosmarinic acid, Methyl rosmarinate, Caffeic acid 95% ethanol at 60°C for 2 h with Total phenolics (mg/g sinapic acid equivalents) [51] stirring Liquid nitrogen powdered samples Caffeic acid, Rosmarinic acid [52] were extracted with acidified methanol 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]. 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 Natural Products as Antibacterial Agents — Antibacterial Potential and Safety of Post-distillation and Waste… 129 http://dx.doi.org/10.5772/60869   Gallic acid  Protocatechuic acid  Syringic acid        Caffeic acid  p‐Coumaric acid  Ferulic acid  Figure 3. Chemical structures of hydroxybenzoic and hydroxycinnamic acids present in thyme extracts 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].     Apigenin  Luteolin      Figure 4. Chemical structures of dominant flavones in investigated thyme extracts Since various plant extracts and isolated natural compounds are widely used and represe nt 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 130 Concepts, Compounds and the Alternatives of Antibacterials   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.   Chlorogenic acid  Rosmarinic acid  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 Natural Products as Antibacterial Agents — Antibacterial Potential and Safety of Post-distillation and Waste… 131 http://dx.doi.org/10.5772/60869 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 spectro‐ photometrically 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,