Thymus Vulgaris Essential Oil Research Articles

Modification of the composition of thyme (Thymus vulgaris) essential oil based on the quality of the light

Objective: To identify the changes in the concentration of the main components of thyme (Thymus vulgaris) essential oil in response to five different LED colors. Design/Methodology/Approach: A completely randomized experimental design was used. The design included five treatments (white light; blue light; red light; 75% blue light and 25% red light; and 75% red light and 75% blue light) and 10 repetitions, at a 25 μmol m−2 s−1 luminous intensity, during a 16 h photoperiod. The thyme plants were sown in a pot with a substrate made up of 50% peat, 48% perlite, and 2% vermicompost. Each plant was an experimental unit. The plants were placed in light isolation chambers and subjected to the treatment for 35 days. Results: The concentration of the main molecules in the essential oil recorded considerable changes between treatments: the concentration of thymol (its main component) increased in the white light treatments, as well as in the red light (75%) and blue light (75%) treatments. In addition, the composition of the essential oil resulting from these treatments is different to the composition reported in the references. Study Limitations/Implications: The light intensity used in this experiment was lower than the light intensity required for plant growth; however, it was enough to produce changes in the secondary metabolism. Findings/Conclusions: The changes in the quality of the light modify the composition of the thyme essential oil. Even at a low light intensity (25 µmol m-2 s-1), the changes in the spectrum composition under which the plants grow influence the composition of the essential oil.

Investigating the effects of Thymus vulgaris essential oil, Allium cepa extract, and their active compounds (thymol and quercetin) on expression profile of genes related to Alzheimer's disease in PC12 model cell

Thymus vulgaris and Allium cepa are plants with great medicinal importance. Thymol monoterpene and quercetin, which are present in these plants, have anti-Alzheimer's and antioxidant effects. The objectives of this research were investigating the effects of these compounds on the pathogenesis and progress of Alzheimer's disease in cells modeled by formaldehyde. MTT, flow cytometry, and RT-PCR were used to investigate the toxicity, survival rate and apoptosis of the cells, and the expression level of PP2A, GSK3, NMDAR, BACE1, and APP genes, respectively. Also, the total antioxidant capacity of the modeled cells was measured. The results showed that the two compounds as well as the plants extract and essential oil were able to increase the percentage of cell survival; among them, Thymus vulgaris essential oil had the greatest effect (93.55316 % in 48 h exposure). In addition, quercetin was able to reduce the rate of apoptosis in Alzheimer's cells (4.73 %) which was greater than the effects of other compounds. In general, the essential oil of Thymus vulgaris compared to thymol; and quercetin compared to Allium cepa extract showed more improving effects on the expression of genes involved in the disease. All four compounds increased the antioxidant capacity of the modeled cells compared to the control group, and these effects were almost equal between the compounds. According to the obtained results, both plants, especially Thymus vulgaris can be proposed as candidates to be included in the diet of Alzheimer's patients. In addition, polyphenols thymol and quercetin as derivates from the studied plants can be used in new drugs development for Alzheimer's disease, with greater safety than currently used drugs. These results are significant because most of the drug for Alzheimer's treatments such as cholinesterases (e.g. rivastigmine and donepezil) and memantine are chemically based and have many side effects.

Comparison of Antifungal Activity of Thymus Vulgaris Essential Oil and Triple Antibiotic Paste Against Candida Albicans Isolated from Root Canal (In Vitro Study)

Background The main objective of chemomechanical endodontic therapy is to reduce or eliminate microorganisms from the root canal system, and intracanal medications are used to accelerate the disinfection process. The aim of this study this study was to assess the antifungal activity of thymus vulgaris essential oil against C. albicans compared to that of triple antibiotic paste. Materials and methods C. albicans was isolated from patients who required endodontic treatment. Using the agar well diffusion method, the efficacy of Thymus vulgaris essential oil against C. albicans was determined at 25 mg/ml, 10 mg/ml, 8 mg/ml, 6 mg/ml, 4 mg/ml, and 2 mg/ml and compared to the triple antibiotic paste. The minimum inhibitory and fungicidal concentrations against Candida albicans were determined using the microbroth dilution method. The constituents were identified using a gas chromatography–mass spectrometer. Results Thymus vulgaris essential oil demonstrated antifungal activity against C. albicans, which appeared to increase as the extract concentration increased, and the difference between all concentrations and triple antibiotic paste was significant (p ≤ 0.01). The MIC and MFC for Thymus vulgaris essential oil were 0.015 mg/ml and 0.031 mg/ml, respectively. 53 compounds were identified by (GC-MS) analysis, which represented more than 95% of the oil composition. The main component was thymol (35.48%). Conclusion Thymus vulgaris essential oil is effective against C. albicans, suggesting that it has the potential to be applied as an intracanal medication.

Thymus vulgaris essential oil enclosed in β-cyclodextrin as drug delivery system: a promising alternative for antibiotic usage in combating Salmonella

Thymus vulgaris essential oil enclosed in β-cyclodextrin as drug delivery system: a promising alternative for antibiotic usage in combating Salmonella

In vitro Antifungal Activity of Mentha piperita and Thymus vulgaris Essential Oils against Ochratoxigenic Aspergillus carbonarius Isolated from Bozcaada Çavuş Grape

In this study, the antifungal properties of Mentha piperita and Thymus vulgaris essential oils against an isolate of ochratoxin A producer, Aspergillus carbonarius, isolated from Bozcaada Çavuş grape, were evaluated in three steps. By GC-MS of M. piperita and T. vulgaris essential oils, the main components were determined to be menthol (39.911%) and carvacrol (49.042%). Antifungal activity was first evaluated by the agar well diffusion method, and it was determined that the tested essential oils completely inhibited the growth of A. carbonarius and were as effective as fluconazole antifungal. In the second step, the MIC and MFC values of the tested essential oils were determined; both values were 1 µL mL-1. Finally, it was determined that M. piperita and T. vulgaris essential oils completely inhibited the radial growth of A. carbonarius at the MIC value. These results show that M. piperita and T. vulgaris essential oils may be a good strategy to control ochratoxigenic A. carbonarius contamination.

The efficiency of Thymus vulgaris, Lavandula angustifolia, and Cupressus sempervirens essential oils in suppression of Hymenoscyphus fraxineus on Fraxinus angustifolia seedlings

AbstractDue to intense ash dieback, this research tested the bioactivity of Thymus vulgaris, Lavandula angustifolia, and Cupressus sempervirens essential oils (EO) against Hymenoscyphus fraxineus. In vitro testing showed that EOs had a pronounced fungitoxic effect on H. fraxineus. The most effective EO was extracted from T. vulgaris, while EOs obtained from L. angustifolia and C. sempervirens were less effective and showed the same effect. At a concentration of 1%, all tested EOs inhibited mycelial growth, while 0.01% and 0.1% concentrations of EO extracted from T. vulgaris also inhibited the growth of H. fraxineus. The in planta test of EO extracted from T. vulgaris conducted on three‐year‐old Fraxinus angustifolia seedlings showed a reduction in the size of necrotic lesions and a better crown condition compared to untreated seedlings. This research demonstrates, for the first time, the efficiency of EOs in suppressing ash dieback making a path for the development of new biological protection measures against H. fraxineus.

Thymol as Starting Material for the Development of a Biobased Material with Enhanced Antimicrobial Activity: Synthesis, Characterization, and Potential Application.

A biobased material, polythymol (PTF), was prepared using thymol, a monoterpene obtained from the essential oil of Thymus vulgaris (Lamiaceae), as a starting material with the aim of enhancing the antimicrobial properties of this natural product. Initially, different processes were performed in order to optimize the reaction conditions to obtain a macromolecule with a high purity and yield. PTF was characterized using different techniques, such as NMR, infrared, UV-Vis, and thermogravimetric analyses. The antimicrobial activity of both PTF and thymol was evaluated against different microorganisms, including S. aureus, E. coli, P. aeruginosa, and C. albicans. The obtained MIC values showed a higher potential for PTF than the monomer thymol-for example, against S. aureus (500 and 31.5 µg·mL-1 for thymol and PTF, respectively). Therefore, the obtained results show that the polymerization of thymol afforded more active biomaterial than the starting monomeric antimicrobial compound (thymol), suggesting that PTF is an important biomaterial.

Effect of Temperature and Water Stress on the Antioxidant and Antidiabetic Activities of Thymus vulgaris Essential Oil

Effect of Temperature and Water Stress on the Antioxidant and Antidiabetic Activities of Thymus vulgaris Essential Oil

Initial Physiological, Biochemical and Elemental Garlic (Allium sativum L.) Clove Responses to T. vulgaris and S. aromaticum Extract Application

Plant extracts are getting attention for their sterilizing, growth-promoting properties in the agricultural field. No exception exists when it comes to the treatment of propagating material with these natural components. Plant extracts may have complex compositions which affect plants or seeds in a positive way. However, they could also cause negative effects, like decreased germination, secondary metabolite contents or biomass gain. As the agricultural field requires sustainable techniques for plant growth and quality assurance in production, it is important to evaluate the potential effects of every alternative natural compound that shows promise for future plant treatment. Garlic (Allium sativum L.) is a popular spice crop grown in various regions of the world. Therefore, our study focused on an investigation of the early physiological and biochemical changes in garlic cloves (cv. ‘Jarus’ and ‘Vasariai’) treated with Thymus vulgaris essential oil and Syzygium aromaticum extract. Experiments were carried out in controlled climate conditions, in which the treated cloves were kept for four weeks. Results show that S. aromaticum extract increased germination in cv. ‘Jarus’ compared to untreated garlic cloves. Meanwhile, T. vulgaris caused lower germination of both cultivars compared to untreated cloves. None of the extracts increased biomass gain in garlic cloves. ABTS antioxidant activity was decreased by both extracts in cv. ‘Jarus’ (~7–47%) and ‘Vasariai’ (~22–32%) compared to untreated garlic cloves. Antioxidant DPPH and FRAP activities were ~6–11% and ~14–15% higher after ‘Vasariai’ treatment with extracts. Meanwhile, in ‘Jarus’ only, S. aromaticum extract caused an increase in DPPH and FRAP antioxidant activities. The elemental content was the highest in garlic cloves treated with T. vulgaris essential oil. Total phenolic compounds (TPC) and flavonoids (TF) were 1.2 times higher after S. aromaticum treatment of ‘Jarus’ compared to untreated cloves, while slightly increased TPC and two times higher TF values were found after treatment with T. vulgaris. Around 10% higher TPC and 1.5–3 times lower TF values were observed after the plant extract treatment of ‘Vasariai’ garlic cloves. Our study demonstrates initial changes in garlic cloves dependent on the plant extract treatment and cultivar. This provides important information for the future utilization of such technology, either alone or in combination with others.

Purification, chemical characterization and evaluation of the antioxidant potential of carvacrol from Thymus vulgaris

This work focuses on the purification, chemical characterization and evaluation of the antioxidant activity of carvacrol in order to determine its contribution in the high antioxidant potential of Thymus vulgaris essential oil (TEO). Firstly, 68% of carvacrol was purified from TEO using chromatography on silica gel column and then chemically characterized using spectroscopic techniques (IR, MS and 1H and 13C NMR). In vitro, the antioxidant activity has been determined using DPPH, ABTS•+ and iron chelating assays. All assays proved the strong radical scavenging and reducing power of carvacrol. In vivo, antioxidant capacity towards stressed Saccharomyces cerevisiae cells was investigated by evaluating cell viability, antioxidant enzymes’ activity, the level of lipid peroxidation (LPO) as well as the activity of succinate dehydrogenase (SDH). Using carvacrol in a dose dependent manner (6.25-25 μg/mL), cell viability was outstandingly improved by 34.5-55% compared to stressed cells. Antioxidant enzymes (CAT, SOD, GR) activities were also brought back to values comparable to control cells along with lower LPO (0.81±0.07 nmol/mg) and SDH (1.15± 0.07 μmol/min/mg of protein) at 25 μg/mL. These findings suggest that the powerful antioxidant properties of TEO found in our previous study were mainly associated to its main component (carvacrol) that showed higher antioxidant activity compared to the other components. Therefore, carvacrol can be of a great use as a pharmacological agent against damages related to oxidative stress.

Antibacterial activity of the essential oil of Thymus vulgaris and thymol in the control of Curtobacterium flaccumfaciens

Curtobacterium flaccumfaciens é um dos principais patógenos que acometem sementes de feijão e soja, causando diversos prejuízos às culturas, já que não existe nenhum produto registrado para seu controle. Dentre os métodos de controle que possuem eficiência comprovada frente à diversos patógenos, destacam-se os óleos essenciais e seus derivados. Sendo assim, este trabalho buscou avaliar o efeito do óleo essencial (OE) de Thymus vulgaris (tomilho) e timol contra a bactéria Curtobacterium flaccumfaciens (cff). A cepa foi inoculada em meio BHI (Brain Heart Infusion) e incubada a 28ºC/96h. Os testes com OE e timol foram realizados sob duas perspectivas: prevenção e remediação. As microplacas foram incubadas por 24h e 48h em shaker orbital e posteriormente adicionou-se as soluções em contato com a cepa. As análises antibiofilme foram realizadas após 24h visando a prevenção da formação dessas comunidades. Para verificar o efeito de remediação sobre biofilmes maduros, as análises foram realizadas após 48h. Foram realizadas análises para quantificação de unidades formadoras de colônias e biomassa. Os resultados da análise antibiofilme demonstraram que ambos os antimicrobianos foram efetivos, sendo que, o timol foi mais eficaz, pois apresentou melhores resultados em todas as concentrações testadas. Dessa forma, pode-se concluir que a utilização de ambas as substâncias é uma alternativa viável para o controle da murcha vascular causada pelo patógeno.

Antibacterial Activity of Thymus vulgaris against Dental Caries

Introduction: Essential oils possess antibacterial properties that can successfully combat disorders caused by microorganisms, such as bacteria, viruses, and protozoa. The aim of this study was to assess the effectiveness of Thymus vulgaris essential oils against bacterial strains associated with dental caries.
 Methods: Saliva specimens were obtained from individuals with dental caries at the Karanganyar Community Health Center during January–February 2023. The essential oil extracted from Thymus vulgaris was diluted to various concentrations ranging from 0.20 mg/mL to 100 mg/mL. Each concentration was made in 25–30 ml containers, with ten containers for each selective medium.
 Results: The different amounts of essential oil stopped all the isolates from growing. In summary, the oil exhibits the least inhibitory effect at a concentration range of 16 μL/mL. At a concentration of 128 μl/mL, Thymus vulgaris EO effectively inhibited all isolates, except for Streptococcus mutans-1 and Escherichia coli.
 Conclusion: Essential oil from Thymus vulgaris showed strong activity against clinical strains belonging to Staphylococcus sp., Pseudomonas sp., Escherichia sp., Bacillus sp., Klebsiella sp., Streptococcus sp., and Lactobacillus sp isolated from the oral cavity at a dose of 64 and 128 μl/mL.

Eugenia caryophyllata, Cinnamomum sp., Mentha piperita ve Thymus vulgaris esansiyel yağların Aspergillus niger suşuna karşı antifungal aktivitesi

Aspergillus species are pollutants found in both food and air. The increase in the metabolic activity of Aspergillus leads to spoilage of foodstuffs and large economic losses. In addition, some Aspergillus species have the ability to produce aflatoxins and ochratoxins, secondary metabolites called, namely, mycotoxins. Especially mycotoxins are very important in terms of food safety and human health. Since the protection of human and animal health and the prevention of economic losses is a very important issue, our study aimed to determine the antifungal activity of Eugenia caryophyllata Thunb., Cinnamomum sp., Mentha piperita L. and Thymus vulgaris L. essential oils (EO) against Aspergillus niger Tiegh. NRRL 321 strain. In the second step, MIC and MFC values of EOs were determined. It was determined that the MIC value of Cinnamomum sp, M. piperita and T. vulgaris EOs was 0.01 µL/mL, and E. caryophyllata EO was 0.5 µL/mL. It was determined that E. caryophyllata, Mentha piperita and Thymus vulgaris EOs completely inhibited radial colony growth at MIC, 2xMIC and 4xMIC values. It was determined that the inhibition of radial growth of Cinnamomum sp. EO varies depending on the concentration, and the inhibition rate increases as the concentration increases. As a result, evaluations should be made considering the in vivo conditions that the tested EOs showed strong antifungal activity against the A. niger strain.

Antimicrobial activity of the interaction between essential oils against resistant Staphylococcus aureus isolates from bovine mastitis

The control of bovine mastitis is crucial for the sustainability of dairy production. Phytotherapy emerges as a potential alternative for controlling resistant bacteria and potentially replacing conventional therapies. This study aimed to assess the in vitro antimicrobial effect of the Pelargonium graveolens (GEO) and Thymus vulgaris (TEO) essential oils, as well as the interaction between them against strains of Staphylococcus aureus isolated from bovine mastitis. Eight strains of S. aureus isolated from bovine mastitis and two standard strains of human origin were used. The Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) were determined using the microdilution method. The interaction between the oils was assessed using the checkerboard method. It was observed that both essential oils exhibited inhibitory effects when evaluated separately; however, TEO demonstrated superior bactericidal efficacy. The combination of both oils resulted in a bactericidal complex with a 99% reduction in the effective concentration of GEO. In conclusion, the TEO/GEO complex may contribute to improved therapeutic efficacy, reducing adverse effects, costs, and bacterial resistance development.

Preparation and properties of films loaded with cellulose nanocrystals stabilized Thymus vulgaris essential oil Pickering emulsion based on modified tapioca starch/polyvinyl alcohol

Pickering emulsions were prepared by stabilizing thymus vulgaris essential oil (TEVO) with cellulose nanocrystals (CNCs), which formed composite films by loading the emulsions into modified tapioca/polyvinyl alcohol (PVA)-based films. The results showed that the 1.0 % CNCs-15 % TEVO emulsion had optimal stability and smaller particle size. The emulsion increased the thickness of the composite film in the form of solid material additions (thickness, 0.062–0.099 mm), which opacity given the laminating film's superior UV-blocking ability compared to blank film. The emulsion plasticizing effect enhanced the film's elongation at break (EAB, 123–159 %). In addition, due to the hydrophobicity and influencing the diffusion path of water molecules in the emulsion, the denser microstructure composite film had a lower water vapor transmission coefficient (WVP, 6.22 × 10-11-5.35 × 10-11g∙cm/cm2∙s∙Pa) to impede moisture penetration. Meanwhile, the composite film can effectively maintain the color and inhibit the growth of microorganisms to extend the storage time of fish fillets.

New insights into the cytotoxic effects of Thymus vulgaris essential oil on the human triple-negative breast cancer cell line MDA-MB-231

Essential oils (EOs) are natural products that have gained wide interest due to their biological activities and anticancer properties through various mechanisms. The present study aimed to test the cytotoxicity of Thymus vulgaris L. (thyme) EO of Italian origin, rich in thymol (49.6%) and p-cymene (18.8%), towards the triple-negative breast cancer cell line MDA-MB-231 and to investigate the biochemical mechanisms underlying its antitumor activity. Thyme EO reduced cancer cell viability in a dose-dependent manner after 24 h treatment, with an IC50 value equal to 75.1 ± 15.2 μg/ml; simultaneously, the inhibition of cancer cell migration and colony formation capacity was evidenced. Thyme EO antiproliferative effects were related to the induction of apoptosis as demonstrated by the increased expression of the pro-apoptotic proteins Bax, cleaved caspase-3, phospho-p53, and SMAC/Diablo and by the reduction of the anti-apoptotic proteins Bcl-2, cIAP-1, cIAP-2, HIF-1α, survivin, and XIAP. Thyme EO administration led to the early formation of intracellular ROS, followed by the increment of MDA as an index of lipid peroxidation and by the decreased expression of the antioxidant enzymes catalase and PON2. The upregulation of Nrf2 mRNA expression and the strong induction of HO-1 sustained the activation of the Nrf2 pathway by thyme EO. These data showed that the EO from Thymus vulgaris L. might inhibit the malignant phenotype of MDA-MB-231, thus suggesting potential benefits against human triple-negative breast cancer.

Synergistic effects of Thymus vulgaris essential oil in combination with antifungal agents and inhibition of virulence factors of Candida albicans

BackgroundCombination antifungal therapy has become a prominent approach in medical practice as it takes advantage of synergistic interactions by interfering with multiple targets of the pathogen, broadening the spectrum of activity, reducing the development of resistance, and improving treatment outcomes. Therefore, combining conventional antifungal drugs with natural products can increase antifungal activity, reduce side effects, and optimize therapeutic effects. PurposeThe present study aimed to evaluate the antifungal activity of thyme essential oil (ThyEO) in combination with amphotericin B, caspofungin, fluconazole, itraconazole, and posaconazole (PSZ), as well as its inhibitory effect on Candida albicans virulence factors. Materials and MethodsThe microbroth dilution assay was employed to determine the minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC). Interactions were assessed using the microdilution checkerboard assay. Ergosterol and methylene blue assays were used to detect effects on fungal membrane, while the sorbitol assay was used to detect effects on fungal cell wall. Inhibition of yeast virulence factors (adherence to epithelial cells, germ tube and pseudomycelium formation, secretion of hydrolytic enzymes, and biofilm formation) was assessed with previously reported methods. ResultsAlmost all combinations showed additivism against C. albicans, except ThyEO/PSZ (31.25/0.0039 µg/ml), which showed partial synergism. Furthermore, all mixtures were fungicidal against C. albicans strains. ThyEO/PSZ, its components alone, and thymol were shown to disrupt the fungal cytoplasmic membrane, increasing its permeability. ThyEO/PSZ, at sub-inhibitory concentrations, significantly decreased the ability of C. albicans to adhere to buccal epithelial cells. ThyEO/PSZ, ThyEO, and PSZ were able to reduce the pseudomycelium production of C. albicans while thymol completely inhibited its formation. ThyEO/PSZ, each combination component on its own, and thymol inhibited biofilm formation and preformed biofilm of C. albicans. Notably, ThyEO/PSZ showed synergistic and fungicidal activity against a resistant strain of C. albicans, reducing the PSZ dose by 4-fold. ConclusionThese findings make ThyEO and ThyEO/PSZ mixture valuable candidates for the development of alternative antifungals with a lower incidence of adverse effects.

Antibacterial and antifungal activity of Lavandula angustifolia and Thymus vulgaris essential oils grown in the Algerian west coast

ABSTRACT The screening of natural products in the quest for new antimicrobial and antifungal agents has become the need of the hour in order to combat drug-resistant pathogenic microbes.The aim was to evaluate the antibacterial and antifungal activities of Lavandula angustifolia and Thymus vulgaris essential oils (EOs).The antimicrobial activity was tested on Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus using agar disc diffusion and the antifungal screening was done in the same way against Candida albicans. The Thymus Vulgaris EO collected from Oran was very active against Staphylococcus aureus and Candida albicans. Lavandula angustifolia EO collected from Oran was extremely active against Pseudomonas aeruginosa and Candida albicans, Candida albicans was the most sensitive to the essential oils. Essential oils of L. angustifolia and T. vulgaris are highly effective natural antibacterial and antifungal agents that can be used to treat infections and food poisoning.

Cuticular property affects the insecticidal synergy of major constituents in thyme oil against houseflies, Musca domestica

Plant essential oils are intricate blends comprising predominantly of monoterpenes and some sesquiterpenes. These oils display diverse bioactivities against targeted organisms, often arising from complex interactions among their constituents, which may demonstrate synergistic or antagonistic effects. Despite their wide use as botanical insecticides, the mechanisms behind these interactions and their effects on bioactivity are poorly understood. This study investigated the synergistic interaction of thymol and p-cymene, two major constituents of Thymus vulgaris essential oil, on the larvae and adults of the housefly, Musca domestica. The results showed that p-cymene synergized the insecticidal activity of thymol in adult houseflies, but not in larvae. GC–MS analyses and bioassays indicated the increased cuticular penetration of thymol by p-cymene was the mechanism of synergy, which was observed only in the adults. Two potential routes were proposed: the expansion of the wetting area, or the disruption of cuticular integrity through dissolving the wax layer. The sequential application and large-volume treatment bioassay results suggested that the former was the more likely mechanism. Also, the hydrophobicity of the cuticle seemed critical for this stage-specific synergy. Wax-devoid adults failed to show synergistic toxicity, whereas artificially wax-coated larvae gained a synergistic effect. Overall, the findings provide insights into the synergistic mechanism of insecticidal activity of plant essential oils and suggest potential applications in developing effective strategies using penetration-enhancing synergists.

Postharvest control of green and blue molds on Mexican lime fruit caused by Penicillium species using Thymus vulgaris essential oil and carboxy methyl cellulose

Postharvest control of green and blue molds on Mexican lime fruit caused by Penicillium species using Thymus vulgaris essential oil and carboxy methyl cellulose