In-vitro antimicrobial activity and chemical composition of Sardinian Thymus essential oils

Essential oils and their components are becoming increasingly popular as naturally occurring antimicrobial agents. In this work the chemical composition and the antimicrobial properties of Thymus essential oils and of their main components were determined. Three essential oils obtained from different species of Thymus growing wild in Sardinia and a commercial sample of Thymus capitatus oil were analysed. The essential oil components were identified by GC/MS analysis. The antimicrobial activity of the oils and components was determined against a panel of standard reference strains and multiple strains of food-derived spoilage and pathogenic bacteria, using a broth microdilution method. The GC/MS analysis showed that the major constituents of the oils were monoterpene hydrocarbons and phenolic monoterpenes, but the concentration of these compounds varied greatly among the oils examined. The results of the antimicrobial assay showed that essential oils extracted from Sardinian Thymus species have an antimicrobial activity comparable to the one observed in other thyme oils. It seems also confirmed that the antimicrobial properties of thyme essential oils are mainly related to their high phenolic content. Among the single compounds tested carvacrol and thymol turned out to be the most efficient against both reference strains and food-derived bacteria. The results of this study confirmed the possibility of using thyme essential oils or some of their components in food systems to prevent the growth of foodborne bacteria and extend the shelf-life of processed foods.     

Antimicrobial Activity of Essential Oil Components Against Potential Food Spoilage Microorganisms

The antimicrobial activity of six essential oil components against the potential food spoilage bacteria Aeromonas (A.) hydrophila, Escherichia (E.) coli, Brochothrix (B.) thermosphacta, and Pseudomonas (P.) fragi at single use and in combination with each other was investigated. At single use, the most effective oil components were thymol (bacteriostatic effect starting from 40 ppm, bactericidal effect with 100 ppm) and carvacrol (50 ppm/100 ppm), followed by linalool (180 ppm/720 ppm), α-pinene (400 ppm/no bactericidal effect), 1,8-cineol (1,400 ppm/2,800 ppm), and α-terpineol (600 ppm/no bactericidal effect). Antimicrobial effects occurred only at high, sensorial not acceptable concentrations. The most susceptible bacterium was A. hydrophila, followed by B. thermosphacta and E. coli. Most of the essential oil component combinations tested showed a higher antimicrobial effect than tested at single use. Antagonistic antimicrobial effects were observed particularly against B. thermosphacta, rarely against A. hydrophila. The results show that the concentration of at least one of the components necessary for an antibacterial effect is higher than sensorial acceptable. So the use of herbs with a high content of thymol, carvacrol, linalool, 1,8-cineol, α-pinene or α-terpineol alone or in combination must be weighted against sensorial quality.     

Essential oil production in shoot cultures versus field-grown plants of Thymus caespititius

Thymus caespititius Brot. is an important aromatic species, due to synthesis and production of essential oils for the pharmaceutical and food industries. In the present study, levels of essential oils from two chemotypes, including carvacrol/thymol (CT) and sabinene/carvacrol (SC), were evaluated in proliferating shoot cultures (6–12 subcultures following establishment) and compared to those from field-grown plants. The essential oils were isolated by hydrodistillation and analysed by gas chromatography (GC) and GC–mass spectrometry (GC–MS). Cultures grown under in vitro culture conditions, evaluated over six subcultures, were found to maintain stable composition of essential oils. For the CT chemotype, carvacrol (42 %) and thymol (23 %) were the main essential oil components detected in field-grown plants; in proliferating shoot cultures the levels detected attained 17–25 % in the case of carvacrol and 18–23 % in that of thymol, closely followed by carvacryl acetate (15–23 %) and thymyl acetate (11–15 %). For the SC chemotype, carvacrol (13–28 %), sabinene (18–45 %), and thymol (9–12 %) were the main essential oil components detected in both field-grown and proliferating shoot cultures. Our experiments showed that the essential oil composition in proliferating shoot cultures was not only stable, but also qualitatively similar to that of field-grown plants, notwithstanding minor quantitative differences.     

Chemical Composition and Antimicrobial Activity of Satureja,Thymus, and Thymbra Species Grown in Lebanon

The essential oils were extracted by hydrodistillation from Satureja cuneifolia, Satureja thymbra, Coridothymus capitatus, Thymus syriacus, and Thymbra spicata growing wild in Lebanon. Their phytochemical analysis performed by GC/MS showed that the aforementioned species are characterized either by carvacrol (60.9%) or thymol (54.3%) or by a more or less equal amounts of these two phenols. Assessment of their in vitro antimicrobial activity against Candida albicans and six pathogenic bacteria using the broth dilution method revealed that the tested oils have a broad activity spectrum with minimum inhibitory concentration ranging from 0.1 to 0.8 mg/ml. Among the tested species, S. thymbra EO showed the highest antimicrobial potential whereas T. syriacus showed the lowest inhibitory activity. These results give scientific evidence for the use of those species in the Lebanese folk medicine and lend support to implement them as natural alternatives for synthetic antimicrobials.     

Anti-biofilm activities of essential oils rich in carvacrol and thymol against Salmonella Enteritidis

The aim of the present study was to determine the bioactive compounds in four essential oils (EO’s) from Origanum heracleoticum, Origanum vulgare, Thymus vulgaris and Thymus serpyllum and to assess their antimicrobial and anti-biofilm activity against Salmonella Enteritidis. Strains were previously characterized depending on the expression of the extracellular matrix components cellulose and curli fimbriae as rdar (red, dry and rough) and bdar morphotype (brown, dry and rough). This study revealed that the EO’s and EOC’s (carvacrol and thymol) investigated showed inhibition of biofilm formation at sub-minimum inhibitory concentration. Comparing the efficacy of EO’s and EOC’s in the inhibition of biofilm formation between the strains with different morphotype (rdar and bdar) did not show a statistically significant difference. Results related to the effectiveness of EO’s and EOC’s (the essential oil components, carvacrol and thymol) on eradication of preformed 48?h old biofilms indicated that biofilm reduction occurred in a dose-dependent manner over time.     

Antimicrobial activity, essential oil composition and micromorphology of trichomes of Satureja laxiflora C. Koch from Iran

The antimicrobial activity, essential oil composition and micromorphology of trichomes of Satureja laxiflora C. Koch, a native plant from Iran, were studied. The essential oil was obtained from the aerial parts at the flowering stage by hydrodistillation, and analyzed by GC and GC/MS. Thirty-three compounds representing 99.1% of the total oil were characterized. The major compounds were thymol (63.9%) and gamma-terpinene (11.9%) followed by carvacrol (4.8%), p-cymene (3.9%), geraniol (3.2%) and geranyl acetate (3.1%). Furthermore, the essential oil and its three main components were tested against two bacteria and three fungi. The result of the bioassays has been shown that the oil possesses potent antimicrobial property. Chemical studies confirmed that a major portion of this antimicrobial activity is due to thymol present in the oil. Micromorphological analysis by SEM of both vegetative and reproductive organs revealed the presence of abundant sessile capitate and sparse short-stalked glandular trichomes along with retrorse eglandular hairs, giving useful diagnostic characters for identification of this medicinal plant.     

Chemical composition and anti-Helicobacter pylori effect of Satureja bachtiarica Bunge essential oil

Resistance of H. pylori strains to common antibiotics has been developed in different parts of the world and continues to increase. It is important to investigate the novel and efficient anti-H. pylori drugs, among which the plants would be suitable sources.Satureja bachtiarica Bunge is traditionally used as antimicrobial agent. In this study, we evaluated the antibacterial activity of S. bachtiarica Bunge essential oil against 10 clinical isolates of Helicobacter pylori by disc diffusion and agar dilution methods. The chemical composition of essential oil was analyzed by GC and GC–MS. Carvacrol (45.5%) and thymol (27.9%) were the primary constituents of oil, followed by p-cymene (4.4%), and γ-terpinene (4.0%). S. bachtiarica essential oil showed strong antibacterial activity against clinical isolates of H. pylori (17.6 ± 1.1 mm and 0.035 ± 0.13 μl/ml). Carvacrol, as the first main component, had a significant role in this effect, whereas in the presence of thymol, the antibacterial effect of carvacrol was reduced. Therefore, S. bachtiarica essential oil can be applied as an alternative agent for treatment of H. pylori infections. More studies would be required to better clarify its mechanism of action on H. pylori.     

Influence of environmental and genetic factors on the stability of essential oil composition of Thymus pulegioides

Thymus pulegioides plants were collected from various natural habitats of Lithuania and transferred into a new uniform environment. The plants were cloned annually at controlled conditions and their essential oil composition was monitored by capillary GC and GC/MS. The geraniol/geranial/neral (G/G/N), thymol (T), linalool (L), carvacrol/γ-terpinene/p-cymene (C/γT/pC) and thymol/carvacrol/γ-terpinene/p-cymene (T/C/γT/pC) chemotypes of T. pulegioides were studied. It was found that according to the stability of essential oil composition on a sudden change of environmental conditions two types of T. pulegioides can be distinguished: (1) plants which preserve their chemical composition of the essential oils; (2) plants, which considerably change their chemical composition of the essential oils.     

Chemical composition and antimicrobial activity of <Emphasis Type="Italic">Thymus pannonicus</Emphasis> All. (<Emphasis Type="Italic">Lamiaceae</Emphasis>) essential oil

This paper presents the results of a study on chemical composition and antimicrobial activity of Thymus pannonicus All. (Lamiaceae) essential oil from Vojvodina province (north of Serbia). The investigated oil was hydrodistilled from a flowering plant and analysed by GC and GC-MS. Fifty-three constituents were identified (>97% of total oil), with geranial (41.42%, w/w) and neral (29.61%, w/w) as the most prominent. The antimicrobial activity of the oil was evaluated using agar disc diffusion and broth microdilution method against Staphylococcus aureus, Enterococcus faecalis, Pseudomonas aeruginosa, Escherichia coli, two strains of Klebsiella pneumoniae and two strains of Candida albicans. The essential oil exhibited antimicrobial activity to varying degrees against all tested strains. The maximum activity of T. Pannonicus oil was observed against E. coli, S. aureus and both tested strains of C. Albicans (MIC = 50 μ/ml, each). Moderate activity was observed against P. aeruginosa and one of the tested strains of K. Pneumoniae (MIC = 200 μ/ml), while E. faecalis and the other strain of K. Pneumoniae expressed a higher degree of resistance (MIC > 200 μ/ml). This study confirms that essential oil of T. pannonicus possesses remarkable in vitro antimicrobial activity against several medicinally important pathogens. This is attributable to lemon-scented citral, a mixture of geranial and neral, which has well-documented antimicrobial activity against a range of bacteria and fungi.     

Efficacies of Nisin A and Nisin V Semipurified Preparations Alone and in Combination with Plant Essential Oils for Controlling Listeria monocytogenes

The food-borne pathogenic bacterium Listeria is known for relatively low morbidity and high mortality rates, reaching up to 25 to 30%. Listeria is a hardy organism, and its control in foods represents a significant challenge. Many naturally occurring compounds, including the bacteriocin nisin and a number of plant essential oils, have been widely studied and are reported to be effective as antimicrobial agents against spoilage and pathogenic microorganisms. The aim of this study was to investigate the ability of semipurified preparations (SPP) containing either nisin A or an enhanced bioengineered derivative, nisin V, alone and in combination with low concentrations of the essential oils thymol, carvacrol, and trans-cinnamaldehyde, to control Listeria monocytogenes in both laboratory media and model food systems. Combinations of nisin V-containing SPP (25 μg/ml) with thymol (0.02%), carvacrol (0.02%), or cinnamaldehyde (0.02%) produced a significantly longer lag phase than any of the essential oil-nisin A combinations. In addition, the log reduction in cell counts achieved by the nisin V-carvacrol or nisin V-cinnamaldehyde combinations was twice that of the equivalent nisin A-essential oil treatment. Significantly, this enhanced activity was validated in model food systems against L. monocytogenes strains of food origin. We conclude that the fermentate form of nisin V in combination with carvacrol and cinnamaldehyde offers significant advantages as a novel, natural, and effective means to enhance food safety by inhibiting food-borne pathogens such as L. monocytogenes.     

GC-MS analysis and in vitro antioxidant and enzyme inhibitory activities of essential oil from aerial parts of endemic Thymus spathulifolius Hausskn. et Velen

We investigated the antioxidant and enzyme inhibitory activities and chemical composition of the hydro-distilled essential oil (0.35% yield) from aerial parts of Thymus spathulifolius. Antioxidant capacity of the oil was assessed by different methods including free radical scavenging (DPPH and ABTS), reducing power (FRAP and CUPRAC) and phosphomolybdenum assay. Inhibitory activities were analyzed against acetylcholinesterase (AChE), butyrylcholinesterase (BChE), α-amylase, α-glucosidase, and tyrosinase. Twenty-one constituents were identified representing 97.2% of the total oil with thymol (50.5%), borneol (16.7%) and carvacrol (7.7%) as the major components. The essential oil exhibited good antioxidant activity with IC₅₀ values of 3.82 and 0.22?mg/mL determined by free radical scavenging DPPH and ABTS, respectively. EC₅₀ values of FRAP and CUPRAC were found to be 0.12 and 0.34?mg/mL, respectively. The results of the present study support the uses of T. spathulifolius essential oil as a source of natural antioxidants and bioactivities for functional foods and phytomedicines.     

Antimicrobial activity of Thymus longicaulis C. Presl essential oil against respiratory pathogens

Thymus longicaulis C. Presl is a small aromatic perennial herb used as a traditional remedy for cold, flu and cough. Composition of the essential oil of T. longicaulis from Croatia and its in vitro antimicrobial activity against the most common respiratory pathogens were evaluated. The yield of essential oil obtained by hydrodistillation from aerial plant parts was 1.2%. According to the GC-MS analysis, a total of forty one compounds (99%) were identified. Thymol (46.3%), ??-terpinene (16.2%), thymyl methyl ether (11.4%), and p-cymene (9.4%) were the main components. Antimicrobial activity of the essential oil against six clinically isolated bacterial and yeast strains was determined using standard disc agar diffusion method and microdilution broth assay. The essential oil exhibited antimicrobial activity towards all tested respiratory pathogens. The most sensitive strains were Haemophilus influenzae and Streptococcus pneumoniae(MIC=0.78 mg/mL), while Staphylococcus aureus was the most resistant (MIC>25.00 mg/mL). Our results indicate that T. longicaulis essential oil could be effective against clinically relevant respiratory pathogens which have the ability to develop resistance to antimicrobial drugs.     

Assessment of genetic and chemical variability in Thymus caramanicus

Thymus caramanicus is an endemic species grown in Iran with interesting pharmacological and biological properties. In the present work, essential oil compositions and inter-simple sequences repeat (ISSR) markers were used to estimate the relationships among and within seven populations of T. caramanicus, belonging to three provinces in Iran. The studied individuals were distinguished on the basis of ISSR markers and constituents of essential oil. A total of 127 band positions were produced by 12 ISSR primers, of which 105 were found polymorphic with 82.68 % polymorphism. Genetic similarity values among individuals ranged between 0.15 and 0.82 which was indicative of a high level of genetic variation. On the basis of their genetic similarities, ISSR analysis allowed to group the samples into two main clusters. One of these included populations originated from Kerman and Isfahan provinces, and the other cluster consists of populations from Semnan province. Chemical compounds of essential oils were found variable in the various individuals and all samples were principally composed of phenolic constituents (carvacrol and/or thymol). As a consequence, the plants were classified into two major chemotypes including carvacrol and thymol/carvacrol. A relationship between genetic and chemical variability and geographic distribution has been observed in studied populations of T. caramanicus.     

Plant-derived compounds as natural antimicrobials to control paper mill biofilms

Biofilms can cause severe problems in industrial paper mills, particularly of economic and technological types (clogging of filters, sheet breaks or holes in the paper, machine breakdowns, etc.). We present here some promising results on the use of essential oil compounds to control these biofilms. Biofilms were grown on stainless-steel coupons with a microbial white water consortium sampled from an industrial paper mill. Five essential oil compounds were screened initially in the laboratory in terms of their antimicrobial activity against planktonic cells and biofilms. The three most active compounds were selected and then tested in different combinations. The combination finally selected was tested at the pilot scale to confirm its efficiency under realistic conditions. All the compounds tested were as active against biofilms as they were against planktonic cells. The most active compounds were thymol, carvacrol, and eugenol, and the most efficient combination was thymol–carvacrol. At a pilot scale, with six injections a day, 10 mM carvacrol alone prevented biocontamination for at least 10 days, and a 1 mM thymol–carvacrol combination enabled a 67 % reduction in biofilm dry matter after 11 days. The use of green antimicrobials could constitute a very promising alternative or supplement to the treatments currently applied to limit biofilm formation in the environment of paper mill machines.     

Antifungal activities of origanum oil against Candida albicans

The antimicrobial properties of volatile aromatic oils from medicinal as well as other edible plants has been recognized since antiquity. Origanum oil, which is used as a food flavoring agent, possesses a broad spectrum of in vitro antimicrobial activities attributed to the high content of phenolic derivatives such as carvacrol and thymol. In the present study, antifungal properties of origanum oil were examined both in vitro and in vivo. Using Candida albicans in broth cultures and a micro dilution method, comparative efficacy of origanum oil, carvacrol, nystatin and amphotericin B were examined in vitro. Origanum oil at 0.25 mg/ml was found to completely inhibit the growth of C. albicans in culture. Growth inhibitions of 75% and >50% were observed at 0.125 mg/ml and 0.0625 mg/ml level, respectively. In addition, both the germination and the mycelial growth of C. albicans were found to be inhibited by origanum oil and carvacrol in a dose-dependent manner. Furthermore, the therapeutic efficacy of origanum oil was examined in an experimental murine systemic candidiasis model. Groups of mice (n = 6) infected with C. albicans (5 × LD₅₀) were fed varying amounts of origanum oil in a final vol. of 0.1 ml of olive oil (vehicle). The daily administration of 8.6 mg of origanum oil in 100 l of olive oil/kg body weight for 30 days resulted in 80% survivability, with no renal burden of C. albicans as opposed to the group of mice fed olive oil alone, who died within 10 days. Similar results were obtained with carvacrol. However, mice fed origanum oil exhibited cosmetically better clinical appearance compared to those cured with carvacrol. The results from our study encourage examination of the efficacy of origanum oil in other forms of systemic and superficial fungal infections and exploration of its broad spectrum effect against other pathogenic manifestations including malignancy.     

百里香提取物抑菌特性的研究

以百里香水和酒精提取物及百里香芳香油作为抑菌剂进行抑菌试验 ,结果表明 ,所用抑菌物对供试菌金黄色葡萄球菌 (Staphalococcusaureus)、枯草芽孢杆菌 (Bacillussubtilis)和大肠杆菌 (Escherichiacoli)均有不同程度的抑制作用 ,可望将其用于食品工业作为防腐和抑菌剂。     

Essential oil variability in Thymbra capitata (L.) Cav. growing wild in Southern Apulia (Italy)

Essential oils of Thymbra capitata (Thymus capitatus) collected from Southern Apulia (Italy) were analysed using gas chromatography and gas chromatography–mass spectrometry techniques, to check for chemical variability. The study showed that among the 75 components of the oils the most recurrent ones were thymol and carvacrol, which always constituted more than 50% of the oils, as well as γ-terpinene, borneol and p-cymene. Cluster analysis led to the identification of three chemotypes: thymol, carvacrol and thymol/carvacrol; this was presumably a crossbreed between the other two chemotypes. Principal component analysis showed the direct correlation among myrcene, α-terpinene and γ-terpinene; anti-correlation between thymol and carvacrol, and the inverse correlation between linalool and myrcene. Moreover, lower thymol concentrations were accompanied by an increase in myrcene, α-terpinene and γ-terpinene.     

玫瑰精油的化学成分及其抗菌活性

通过水蒸汽同步蒸馏法提取玫瑰精油,采用GC-MS方法分析了玫瑰精油的化学组成,共鉴定出其中14个化学成分并测定其相对含量,占总含量的95.25%。香茅醇为玫瑰精油的主要成分,相对含量为90.37%。体外抑菌实验表明,玫瑰精油除对黑曲霉没有抗菌活性外,对其它7种供试菌均具有不同程度的抑制作用,其中对表皮葡萄球菌、金黄色葡萄球菌和大肠杆菌的最小抑菌浓度(MIC)为0.063%(v/v),对枯草芽孢杆菌、变形杆菌和白色念珠菌的最小抑菌浓度（MIC）为0.125%(v/v),而对绿脓杆菌(Pseudomonas aeruginosa)的抗菌活性相对较弱,MIC为0.5%(v/v)。抑菌直径结果也表明了玫瑰精油除对黑曲霉、绿脓杆菌的抗菌活性较弱外,对其它6种菌株的抑菌直径都大于8.5 mm。考察了玫瑰精油对3种敏感菌株包括金黄色葡萄球菌(革兰氏阳性菌)、大肠杆菌(革兰氏阴性菌)和白色念珠菌(真菌)的杀菌动态过程,为玫瑰精油的应用提供了理论依据。     

Comparative analysis of essential oil compositions in seven populations of Bakhtiarian savory in natural and field conditions

Bakhtiarian savory (Satureja bachtiarica Bunge) is an endemic herb growing wild in Iran with interesting pharmacological and biological properties. In this research, the variability of essential oil content and phytochemical compositions among seven populations of this species was studied in both natural and field conditions. There was a wide interpopulation variability in phytochemical variation of studied populations under both natural and field conditions, indicating the existence of different chemotypes. Six chemical compositions showed high value in studied plants so that carvacrol and thymol were known as major compounds and confirmed by principal component analysis. Ilam population had the highest thymol in both field (77.10 %) and natural (42.32 %) conditions and was known as a thymol chemotype. Beside, Yazd and Shahrekord populations, respectively, showed the highest carvacrol percentages in both field (84.83 and 63.81 %) and natural (72.50 and 58.05 %) conditions and were known as carvacrol chemotypes. In other populations, carvacrol and thymol were the main components and showed partly equal percentage and these populations were known as carvacrol/thymol chemotypes. According to Pearson correlation analysis, carvacrol was negatively correlated with p-cymene, γ-terpinene and thymol (r = ?0.61, ?0.70 and ?0.98, respectively), indicating very high reverse correlation between carvacrol and thymol. In conclusion, the high value of the essential oil compositions in studied S. bachtiarica populations could provide useful information for conservation and selection of cross parents in breeding programs to develop improved cultivars.