Whole-cell bioconversion for producing thymoquinone by engineered Saccharomyces cerevisiae.

Thymoquinone, extracted from the black seeds of Nigella sativa, is a natural substance with highly beneficial effects against various human diseases. In this study, we aimed to construct a Saccharomyces cerevisiae strain that, produce thymoquinone from thymol, a relatively inexpensive substrate. To achieve this, cytochrome P450 from Origanum vulgare was expressed in S. cerevisiae for the bioconversion of thymol to thymoquinone, with the co-expression of cytochrome P450 reductase (CPR) from Arabidopsis thaliana, ATR1. Additionally, flexible linkers were used to connect these two enzymes. Furthermore, modifications were performed to expand the endoplasmic reticulum (ER) space, leading to increased thymoquinone production. After integrating the genes into the chromosome and optimizing the media components, a significant improvement in the thymol-to-thymoquinone conversion rate and yield were achieved. This study represents a possibility of the production of thymoquinone, a bioactive ingredient of a plant, using an engineered microbial cell.

Volatile compositions and glandular trichomes of Zataria multiflora in different phenological stages under normal and drought stress conditions.

BACKGROUND: Zataria multiflora Boiss. is a medicinal and aromatic plant from the Lamiaceae family. It is extensively used in Iranian traditional medicine, mostly as a replacement for Thyme species. This study was focused on the analysis of chemical composition and the distribution and types of trichomes of Z. multiflora grown under different conditions. Equilibrium headspace analysis in combination with GC-FID-MS was used to identify volatile compounds released by aerial parts of Z. multiflora in development stages of 50 and 100% flowering under normal and drought-stress conditions. RESULTS: The main constituents were p-cymene (20.06-27.40%), γ-terpinene (12.44-16.93%), and α-pinene (6.91-16.58%) and thymol (8.52-9.99%). The highest content of p-cymene (27.40%) and thymol (9.99%) was observed in the 50% flowering stage at the 90% field capacity, while the maximum γ-terpinene (16.93%) content was recorded in the 100% flowering stage under normal conditions. Using the SEM method, it was found that peltate glandular and non-glandular trichomes are distributed on the surface of the leaf, stem, and outer side of the calyx. However, capitate trichomes only are detected on the stem and calyx in the 100% flowering and beginning of blooming stages, respectively. The type and structure of trichomes do not vary in different development stages, but they differ in density. The highest number of leaf peltate glandular trichomes was observed in the vegetative and beginning of blooming stages at 50% and 90% field capacity, respectively. Non-glandular trichomes of the stem were observed with high density in both normal and stress conditions, which are more densely in 90% field capacity. CONCLUSIONS: Since this plant has strong potential to be used in the food and pharmacological industries, this study provides valuable information for its cultivation and harvesting at specific phenological stages, depending on desired compounds and their concentrations.

Combined in†vitro/in silico approaches, molecular dynamics simulations and safety assessment of the multifunctional properties of thymol and carvacrol: A comparative insight.

Bioactive compounds derived from medicinal plants have acquired immense attentiveness in drug discovery and development. The present study investigated in vitro and predicted in silico the antibacterial, antifungal, and antiviral properties of thymol and carvacrol, and assessed their safety. The performed microbiological assays against Pseudomonas aeruginosa, Escherichia coli, Salmonella enterica Typhimurium revealed that the minimal inhibitory concentration values ranged from (0.078 to 0.312 mg/mL) and the minimal fungicidal concentration against Candida albicans was 0.625 mg/mL. Molecular docking simulations, stipulated that these compounds could inhibit bacterial replication and transcription functions by targeting DNA and RNA polymerases receptors with docking scores varying between (-5.1 to -6.9 kcal/mol). Studied hydroxylated monoterpenes could hinder C. albicans growth by impeding lanosterol 14α-demethylase enzyme and showed a (ΔG=-6.2 and -6.3 kcal/mol). Computational studies revealed that thymol and carvacrol could target the SARS-Cov-2 spike protein of the Omicron variant RBD domain. Molecular dynamics simulations disclosed that these compounds have a stable dynamic behavior over 100 ns as compared to remdesivir. Chemo-computational toxicity prediction using Protox II webserver indicated that thymol and carvacrol could be safely and effectively used as drug candidates to tackle bacterial, fungal, and viral infections as compared to chemical medication.

Protective effect of thymol on glycerol-induced acute kidney injury.

Acute kidney injury (AKI) is a syndrome characterized by an accelerating decrease in renal function in a short time. Thymol is one of the main components of thyme species and has a variety of pharmacological effects. Here, we investigated whether thymol could ameliorate rhabdomyolysis (RM)-induced AKI and its related mechanism. Glycerol was used to induce RM-associated AKI in rats. Rats received thymol (20 mg/kg/day or 40 mg/kg/day) gavage 24 h before glycerol injection until 72 h after injection daily. Kidney injury was identified by measuring serum creatinine (Scr) and urea levels and by H&E and PAS staining and immunohistochemistry (the expression of proliferating cell nuclear antigen (PCNA)). Renal superoxide dismutase (SOD), malondialdehyde (MDA), and oxidative stress-related Nrf2/HO-1 signaling pathways were measured. The expression of the inflammatory markers TNF-α, IL-6, MCP-1, and NF-κB was assessed by ELISA and western blotting. Finally, the expression of the PI3K/Akt signaling pathway was detected by western blotting. Glycerol administration induced obvious renal histologic damage and increased Scr, urea, and PCNA expression. Notably, thymol treatment attenuated these structural and functional changes and prevented renal oxidative stress, inflammatory damage and PI3K/Akt pathway downregulation associated with glycerol-induced AKI. In conclusion, thymol might have potential applications in the amelioration of AKI via its antioxidant and anti-inflammatory effects and upregulation of the PI3K/Akt signaling pathway.

Evaluating protective effects of botanicals under inflammation and oxidative stress in chicken apical-out enteroids.

Botanicals (BOTs) are well known for their anti-inflammatory and antioxidant activities. They have been widely used as feed additives to reduce inflammation and improve intestinal functions in agricultural animals. However, the effects of BOTs on chicken intestinal epithelial functions are not fully understood. The 3D apical-out chicken enteroids recapitulate the intestinal tissue, and allow convenient access to the luminal surface, thus serving as a suitable model for investigating gut functions. The aim of this study was to identify the roles of BOTs in protecting the intestinal epithelium in chicken enteroids under challenging conditions. Apical-out enteroids were isolated from the small intestines of 18 days-old chicken embryos. Lipopolysaccharide (LPS, 10 µg/mL) and menadione (400 µM) challenges were performed in the media with or without BOTs. Paracellular Fluorescein isothiocyanate-dextran 4kD (FD4) permeability, inflammatory cytokine gene expression, and reactive oxygen species (ROS) generation were analyzed post-BOTs and challenges treatments. Statistical analysis was performed using one-way ANOVA and post hoc multiple comparisons among treatments. The results showed that the LPS challenge for 24 h induced a 50% increase in FD4 permeability compared with nontreated control; thymol, thyme essential oil, and phenol-rich extract significantly (P < 0.02) reduced FD4 permeability by 25%, 41%, and 48% respectively, in comparison with LPS treatment. Moreover, the gene expression of inflammatory cytokines was upregulated, tight junction proteins and defensins were downregulated (P < 0.05) after 6 h of LPS treatment, while these BOTs treatments significantly restored the LPS-induced gene expression alterations (P < 0.05). Menadione oxidative challenge for 1 h significantly increased the ROS level compared with unchallenged control. Enteroids treated with thymol and thyme essential oils showed 30% reduced ROS levels, while the phenol-rich extract reduced them by 60%, in comparison with the challenged group (P < 0.0001). These data confirmed the role of BOTs in supporting the barrier function and reducing the disruptive effects of inflammation and oxidation in the chicken intestine.

Microencapsulated herbal components in the diet of Lacaune ewes: impacts on physiology and milk production and quality.

This study aimed to determine whether the addition of a microencapsulated herbal blend (MHB) based on thymol, carvacrol, and cinnamaldehyde in dairy sheep feed would improve production efficiency, milk quality, and animal health. Thirty lactating Lacaune ewes were divided into three groups: Control (T0), 150 mg blend/kg of feed (T150), and 250 mg blend/kg of feed (T250). Milk was measured before the beginning of the experiment (d 0), at the end of the adaptation period (d 15), and during the experiment (d 20). In milk samples, was measured the composition, somatic cell count (SCC), reactive oxygen species (ROS), lipoperoxidation (LPO), and total antioxidant capacity. The MHB improved the milk production (only T150 vs. T0 sheep on d 20), productive efficiency and feed efficiency, and reduced the milk SCC (only T250 vs. T0 sheep, on d 20), ROS and tended to reduce the milk levels of LPO (only T250 vs. T0 sheep on d 20). Also, MHB reduced the blood levels of neutrophils and ROS (only T250 vs. T0 sheep on d 20) and increased total protein and globulin levels. Thus, a microencapsulated blend of thymol, carvacrol, and cinnamaldehyde improved the productive performance and milk quality of sheep.

Modulatory effect of thymol on the immune response and susceptibility to Aeromonas hydrophila infection in Nile tilapia fish exposed to zinc oxide nanoparticles.

Zinc oxide nanoparticles (ZnO-NPs) have many exciting properties that make their use in a continuous increase in various biomedical, industrial, and agricultural applications. This is associated with accumulation in the aquatic ecosystems and fish exposure with consequent deleterious effects. To determine the potential of thymol to counteract the immunotoxic effects of ZnO-NPs, Oreochromis niloticus was exposed to ZnO-NPs (⅕ LC50 =1.14 mg/L, for 28 days) with or without feeding a thymol-incorporated diet (1 or 2 g/kg diet). Our data demonstrated a reduction of aquaria water quality, leukopenia, and lymphopenia with a decrease in serum total protein, albumin, and globulin levels in exposed fish. At the same time, the stress indices (cortisol and glucose) were elevated in response to ZnO-NPs exposure. The exposed fish also revealed a decline in serum immunoglobulins, nitric oxide, and the activities of lysozyme and myeloperoxidase, in addition to reduced resistance to the Aeromonas hydrophila challenge. The RT-PCR analysis showed downregulation of antioxidant (SOD) superoxide dismutase and (CAT) catalase gene expression in the liver tissue with overexpression of the immune-related genes (TNF-α and IL-1ß). Importantly, we found that thymol markedly protected against ZnO-NPs-induced immunotoxicity in fish co-supplemented with thymol (1 or 2 g/kg diet) in a dose-dependent manner. Our data confirm the immunoprotective and antibacterial effects of thymol in ZnO-NPs exposed fish, supporting the potential utility of thymol as a possible immunostimulant agent.

The structure of AgamOBP5 in complex with the natural insect repellents Carvacrol and Thymol: Crystallographic, fluorescence and thermodynamic binding studies.

Among several proteins participating in the olfactory perception process of insects, Odorant Binding Proteins (OBPs) are today considered valid targets for the discovery of compounds that interfere with their host-detection behavior. The 3D structures of Anopheles gambiae mosquito AgamOBP1 in complex with the known synthetic repellents DEET and Icaridin have provided valuable information on the structural characteristics that govern their selective binding. However, no structure of a plant-derived repellent bound to an OBP has been available until now. Herein, we present the novel three-dimensional crystal structures of AgamOBP5 in complex with two natural phenolic monoterpenoid repellents, Carvacrol and Thymol, and the MPD molecule. Structural analysis revealed that both monoterpenoids occupy a binding site (Site-1) by adopting two alternative conformations. An additional Carvacrol was also bound to a secondary site (Site-2) near the central cavity entrance. A protein-ligand hydrogen-bond network supplemented by van der Waals interactions spans the entire binding cavity, bridging α4, α6, and α3 helices and stabilizing the overall structure. Fluorescence competition and Differential Scanning Calorimetry experiments verified the presence of two binding sites and the stabilization effect on AgamOBP5. While Carvacrol and Thymol bind to Site-1 with equal affinity in the submicromolar range, they exhibit a significantly lower and distinct binding capacity for Site-2 with Kd's of ~7 µΜ and ~18 µΜ, respectively. Finally, a comparison of AgamOBP5 complexes with the AgamOBP4-Indole structure revealed that variations of ligand-interacting aminoacids such as A109T, I72M, A112L, and A105T cause two structurally similar and homologous proteins to display different binding specificities.

A comparative study on DNA and protein binding properties of thymol and thymoquinone.

Two phytochemicals, thymol and thymoquinone obtained from thymes (Thymus vulgaris L., Lamiaceae etc.) and Nagila Sativa seed, respectively. Both the phytochemicals show several biochemical activities like anticancer, antimicrobial etc. In this paper, we studied the affinities of thymol and thymoquinone towards calf thymus DNA (CT-DNA) and protein (bovine serum albumin). Spectroscopic and molecular modelling studies revealed that both compounds have a high affinity toward both the receptors; DNA and protein. Both phytochemicals binds to the minor grooves of DNA and suitable pockets of protein. Several free energy function and hydrogen bonding play significant role during the binding phenomenon.Communicated by Ramaswamy H. Sarma.

Creation of new germplasm resources, development of SSR markers, and screening of monoterpene synthases in thyme.

BACKGROUND: Thyme derived essential oil and its components have numerous applications in pharmaceutical, food, and cosmetic industries, owing to their antibacterial, antifungal, and antiviral properties. To obtain thyme essential oil with different terpene composition, we developed new germplasm resources using the conventional hybridization approach. RESULTS: Phenotypic characteristics, including essential oil yield and composition, glandular trichome density, plant type, and fertility, of three wild Chinese and seven European thyme species were evaluated. Male-sterile and male-fertile thyme species were crossed in different combinations, and two F1 populations derived from Thymus longicaulis (Tl) × T. vulgaris 'Fragrantissimus' (Tvf) and T. vulgaris 'Elsbeth' (Tve) × T. quinquecostatus (Tq) crosses were selected, with essential oil yield and terpene content as the main breeding goals. Simultaneously, simple sequence repeat (SSR) primers were developed based on the whole-genome sequence of T. quinquecostatus to authenticate the F1 hybrids. A total of 300 primer pairs were selected, and polymerase chain reaction (PCR) was carried out on the parents of the two hybrid populations (Tl, Tvf, Tve, and Tq). Based on the chemotype of the parents and their F1 progenies, we examined the expression of genes encoding two γ-terpinene synthases, one α-terpineol synthase, and maybe one geraniol synthase in all genotypes by quantitative real-time PCR (qRT-PCR). CONCLUSION: We used hybridization to create new germplasm resources of thyme, developed SSR markers based on the whole-genome sequence of T. quinquecostatus, and screened the expression of monoterpene synthase genes in thyme. The results of this study provide a strong foundation for the creation of new germplasm resources, construction of the genetic linkage maps, and identification of quantitative trait loci (QTLs), and help gain insight into the mechanism of monoterpenoids biosynthesis in thyme.

Dietary supplementation with a microencapsulated complex of thymol, carvacrol, and cinnamaldehyde improves intestinal barrier function in weaning piglets.

BACKGROUND: The authors previously prepared a microencapsulated complex of thymol, carvacrol, and cinnamaldehyde (MEEO). This study aimed to evaluate the effect of MEEO on the intestinal mucosal barrier and homeostasis in weaning piglets. A comparison of the effect of MEEO versus chlortetracycline (CTC) was performed in this study. RESULTS: Piglets were divided into three groups - control (Con), MEEO, and CTC groups - and raised for 28 days. The results showed that MEEO significantly elevated the ratio of the villus height and the crypt depth in the jejunum and decreased the crypt depth in the ileum compared with the other groups (P < 0.05); it also upregulated the messenger ribonucleic acid (mRNA) expression of tight junction protein in the small intestine. Compared with the Con group, MEEO increased the concentration of secretory immunoglobulin A (sIgA), cathelicidin antimicrobial peptides (CAMP), and interleukin 10 (IL-10), while decreasing the interleukin 1 beta (IL-1ß) concentration in both jejunal and ileal mucosa (P < 0.05). The mRNA expression of jejunal mucosal MUC1 and ileal mucosal MUC2 was increased in the MEEO group compared with the other groups (P < 0.05). Intestinal microbial analysis showed that dietary treatment had little impact on the ileal microbial structure. A significant rise in the genus Lactobacillus was, however, found in the MEEO group. There is a positive correlation between the Lactobacillus and sIgA, and between the Lactobacillus and CAMP, indicating that an improvement in the mucosal barrier function by the addition of MEEO may be associated with the proliferation of Lactobacillus. CONCLUSION: Dietary supplementation with MEEO improves intestinal barrier function in weaning piglets, the effect of which was superior to CTC. © 2022 Society of Chemical Industry.

Evaluation of Microencapsulated Essential Oils in Broilers Challenged with Salmonella Enteritidis: A Focus on the Body's Antioxidant Status, Gut Microbiology, and Morphology.

Restrictions on antibiotic use encourage researchers to seek natural substitutes with the same effects without adverse end effects resulting from antibiotic use. Savory and black pepper have been challenged against Salmonella enteritidis (S. enteritidis) bacterium using the spray dryer method to evaluate growth performance, antioxidant status, immune response, and intestinal health parameters in broilers. In this study, thyme essential oil (50%), savory (25%), peppermint (12.5%), and black pepper seeds (12.5%) were mixed to form essential oil-loaded spherical microcapsules with the particle size of 323 nm and encapsulation efficiency of 96.2%. The main bioactive compounds used in the core of microcapsules included thymol, carvacrol, p-cymene, γ-terpinene, and menthol. Moreover, modified starch (25%) and maltodextrin (55%) were used for the preparation of spherical microcapsules for the enclosed wall with 20% whey protein concentrate. The dietary addition of microcapsules containing essential oil significantly reduced the S. enteritidis population in both ileum and cecum (P<0.05). The results revealed that the dietary inclusion of essential oil-loaded microcapsules significantly (P<0.05) increased the villus height, villus width, V: C ratio, and the number of goblet cells and decreased the crypt depth. Microcapsules have antioxidant and antibacterial activity and their dietary use as feed additive at 0.5, 1, and 2 kg/t concentrations in broilers has been challenged and showed that the final weight, total feed intake, and FCR improved the body's antioxidant status, structure, and inflammation in the ileum tissue.

Therapeutic potential of second degree's skin burns by topical dressing of Teucrium ramosissimum that promotes re-epithelialization.

The aim of the report is to assess the protective effect of powder aerial part of Teucrium ramosissimum (TS) on the in vivo wound-healing of second-degree burn injuries. Teucrium phytocompounds were characterized by FTIR, HPLC, and GC/MS spectra. Burn wound models were employed to evaluate the in vivo wound-healing activity. Thirty six wistar rats with burn wounds were divided into six groups and treated daily with TS, the mixture of Teucrium and honey (TS-HY), thymol and Dermosalic® (0.05%) (DS) creams. Skin epithelialization was monitored on the 4th, 13th, and 21st days. Proteins and the level of malondialdehyde in the burned skin were assessed. Microscopic and macroscopic investigations of skin wound tissues showed significant wound closure rate via complete epidermal reepithelization and regeneration, higher protein content, collagen synthesis and deposition, hair follicles growth post wounding that were promoted in TS-, thymol-, TS-HY- and DS-treated wound tissues compared to the untreated burned wound tissues that was characterized by the absence of the epithelialization, vascularization and the formation of the epidermis layer. Additionally, the skin healing potential of TS and TS-HY was validated by markedly decreased of lipid peroxidation. Overall, TS was found to possess complete wound closure and improves the healing process.

Thymol improves salinity tolerance of tobacco by increasing the sodium ion efflux and enhancing the content of nitric oxide and glutathione.

BACKGROUND AND OBJECTIVE: Salt stress is one of the most important abiotic stresses affecting the yield and quality of tobacco (Nicotiana tabacum). Thymol (a natural medicine) has been widely used in medical research because of its antibacterial and anti-inflammatory activities. However, the influence of thymol on the root growth of tobacco is not fully elucidated. In this study, the regulatory effects of different concentrations of thymol were investigated. METHODOLOGY: Here, histochemical staining and biochemical methods, non-invasive micro-test technology (NMT), and qPCR assay were performed to investigate the effect of thymol and mechanism of it improving salinity tolerance in tobacco seedlings. RESULTS: In this study, our results showed that thymol rescued root growth from salt stress by ameliorating ROS accumulation, lipid peroxidation, and cell death. Furthermore, thymol enhanced contents of NO and GSH to repress ROS accumulation, further protecting the stability of the cell membrane. And, thymol improved Na+ efflux and the expression of SOS1, HKT1, and NHX1, thus protecting the stability of Na+ and K+. CONCLUSION: Our study confirmed the protecting effect of thymol in tobacco under salt stress, and we also identified the mechanism of it, involving dynamic regulation of antioxidant system and the maintenance of Na+ homeostasis. It can be a new method to improve salinity tolerance in plants.

Carcass characteristics and meat evaluation of cattle finished in temperate pasture and supplemented with natural additive containing clove, cashew oil, castor oils, and a microencapsulated blend of eugenol, thymol, and vanillin.

BACKGROUND: Forty crossbred steers were supplemented with different doses (from 0 control to 6000 mg/animal/day) of natural additive blend containing clove essential oil, cashew oil, castor oil, and a microencapsulated blend of eugenol, thymol, and vanillin for 80 days. Carcass characteristics, drip loss, and antioxidant activity were evaluated 24 h post mortem on longissimus thoracis, and the effects of aging (until 14 days) were evaluated for water losses (thawing/aging and cooking), texture, color, and lipid oxidation. RESULTS: The use of the natural additive blend did not modify (P > 0.05) carcass characteristics but did, however, modify body composition (P < 0.05). Drip losses were unaffected by the treatments tested (P > 0.05). There was an observed quadratic effect (P < 0.05) on losses from thawing/aging on the first day of storage. Regarding the effects of natural additives on cooking losses, there was a quadratic effect (P < 0.05) among the treatments on day 7 of aging. Differences between days of aging were only observed with control treatment. Shear force was similar among treatments on days 1 and 7 of aging. On day 14 a linear effect (P < 0.05) was observed. Also, a linear effect (P < 0.05) appeared on meat lightness, meat from the control group being clearer on day 1. No changes were observed in redness among treatments or days of storage (P > 0.05). Yellowness was not modified by the treatments (P > 0.05)but only by the days of storage in control and the lowest dosage used. CONCLUSION: The blend of natural additives has potential use in pasture feeding and could improve meat quality. However, doses should be adjusted. © 2021 Society of Chemical Industry.

The biosynthesis of thymol, carvacrol, and thymohydroquinone in Lamiaceae proceeds via cytochrome P450s and a short-chain dehydrogenase.

Thymol and carvacrol are phenolic monoterpenes found in thyme, oregano, and several other species of the Lamiaceae. Long valued for their smell and taste, these substances also have antibacterial and anti-spasmolytic properties. They are also suggested to be precursors of thymohydroquinone and thymoquinone, monoterpenes with anti-inflammatory, antioxidant, and antitumor activities. Thymol and carvacrol biosynthesis has been proposed to proceed by the cyclization of geranyl diphosphate to γ-terpinene, followed by a series of oxidations via p-cymene. Here, we show that γ-terpinene is oxidized by cytochrome P450 monooxygenases (P450s) of the CYP71D subfamily to produce unstable cyclohexadienol intermediates, which are then dehydrogenated by a short-chain dehydrogenase/reductase (SDR) to the corresponding ketones. The subsequent formation of the aromatic compounds occurs via keto-enol tautomerisms. Combining these enzymes with γ-terpinene in in vitro assays or in vivo in Nicotiana benthamiana yielded thymol and carvacrol as products. In the absence of the SDRs, only p-cymene was formed by rearrangement of the cyclohexadienol intermediates. The nature of these unstable intermediates was inferred from reactions with the γ-terpinene isomer limonene and by analogy to reactions catalyzed by related enzymes. We also identified and characterized two P450s of the CYP76S and CYP736A subfamilies that catalyze the hydroxylation of thymol and carvacrol to thymohydroquinone when heterologously expressed in yeast and N. benthamiana Our findings alter previous views of thymol and carvacrol formation, identify the enzymes involved in the biosynthesis of these phenolic monoterpenes and thymohydroquinone in the Lamiaceae, and provide targets for metabolic engineering of high-value terpenes in plants.

Effect of Methyl Jasmonate on Thymol, Carvacrol, Phytochemical Accumulation, and Expression of Key Genes Involved in Thymol/Carvacrol Biosynthetic Pathway in Some Iranian Thyme Species.

Thyme species are a good source of thymol and carvacrol, which play a key role in controlling diseases. For the first time, the expression patterns of γ-terpinene synthase (TPS2), CYP71D178, and CYP71D180 genes and the amount of phenolics compounds were evaluated in T. migricus and T. daenensis after different methyl jasmonate (MeJA) treatments. The highest thymol and carvacrol contents were observed in T. migricus (86.27%) and T. daenensis (17.87%) at MeJA 100 µM, which was consistent with the expression patterns of the three investigated genes. All species treated showed high total phenolic and flavonoid content compared to control plants for which the highest amounts were observed in T. vulgaris treated with 100 µM and 10 µM MeJA. Furthermore, in the 100 µM MeJA treatment, the relative expression of TPS2 and CYP71D178 in T. migricus increased 7.47 and 9.86-fold compared with the control, respectively. The highest level of CYP71D180 transcripts (5.15-fold) was also observed for T. daenensis treated. This finding highlights the notion that thymol was known as the dominant component of the essential oil rather than carvacrol in diffident thyme species. This implies that MeJA at different concentrations influenced metabolic pathways and induced expression changes, resulting in a rise in essential oil levels.

Lactoferrin-thymol complex for the disinfection of gram-positive Staphylococcus aureus and Gram-negative Escherichia coli.

Seeking all-nature derived antibacterial agents with effective disinfection function, high human safety as well as environment-friendly characteristics are highly required in the food industry. Herein, we report the lactoferrin-thymol (LF-Thy) complex as an effective killing agent against Gram-negative Escherichia coli (E. coli) and Gram-positive Staphylococcus aureus (S. aureus). The multi-spectroscopy results clearly demonstrate the combination of LF and Thy to form the LF-Thy complex, accompanied with LF conformation variations including the increase in the hydrophobicity of amino acid residues and changes in the types of secondary conformation distribution in LF. Molecular docking results show that LF exhibits three possible binding sites and five predicted stable binding modes for Thy with the help of hydrogen bonding and hydrophobic interactions. Moreover, LF-Thy demonstrated a significantly higher antibacterial ability compared to LF and displays effective disinfection function against E. coli and S. aureus. The minimum inhibitory concentration (MIC) of LF toward E. coli and S. aureus is >40 mg mL-1 and 40 mg mL-1, which decreases to 10 mg mL-1 and 5 mg mL-1 after combination with Thy, respectively. This work demonstrates the promising antibacterial activities of the LF-Thy complex and provides an alternative agent for combating bacterial infection in the food industry, which holds great potential for promoting the development of the all-natural healthcare food complex.

Bacteria-specific pro-photosensitizer kills multidrug-resistant Staphylococcus aureus and Pseudomonas aeruginosa.

The emergence of multidrug-resistant bacteria has become a real threat and we are fast running out of treatment options. A combinatory strategy is explored here to eradicate multidrug-resistant Staphlococcus aureus and Pseudomonas aeruginosa including planktonic cells, established biofilms, and persisters as high as 7.5 log bacteria in less than 30 min. Blue-laser and thymol together rapidly sterilized acute infected or biofilm-associated wounds and successfully prevented systematic dissemination in mice. Mechanistically, blue-laser and thymol instigated oxidative bursts exclusively in bacteria owing to abundant proporphyrin-like compounds produced in bacteria over mammalian cells, which transformed harmless thymol into blue-laser sensitizers, thymoquinone and thymohydroquinone. Photo-excitations of thymoquinone and thymohydroquinone augmented reactive oxygen species production and initiated a torrent of cytotoxic events in bacteria while completely sparing the host tissue. The investigation unravels a previously unappreciated property of thymol as a pro-photosensitizer analogous to a prodrug that is activated only in bacteria.

Natural Antibiotic Oregano in Hydroxyapatite-Coated Titanium Reduces Osteoclastic Bone Resorption for Orthopedic and Dental Applications.

Traditional infection prevention and treatment methods include synthetic antibiotics, which can cause severe adverse side effects. Carvacrol and thymol are biologically active monoterpenoid extractants from oregano leaves with antibiotic capabilities; however, little is known regarding their effects on bone tissue engineering. The objective of this work is to understand their effects on osteogenesis, specifically with osteoblast and osteoclast cells, from surface-modified Ti6Al4V with plasma sprayed hydroxyapatite (HA) coatings. This system is an alternative to cemented implants to aid in bone healing. Results reveal that full carvacrol release from the HA matrix is successful in aqueous environments and modulation of release kinetics can also be made using polycaprolactone (PCL) and polyethylene glycol (PEG) polymers. From HA-pressed disc samples in physiological pH, full carvacrol release is achieved in 10 days using PCL/PEG, about 95% release in 50 days using no polymer, and 60% in 50 days when using a PCL coating. Without polymer, full carvacrol release is achieved after 3 days from HA coatings in both physiological pH and acidic pH, mimicking the post-surgery environment. The release is assessed as a diffusion-based mechanism in phosphate-buffered saline but degradation-based mechanism in acetate buffer solution. Carvacrol and thymol show bacterial inhibition of Staphylococcus epidermidis and no cytotoxic effects on osteoblast proliferation in vitro. Carvacrol and thymol also induce a significant 7% reduction in osteoclast tartrate-resistant acid phosphatase (TRAP) activity, caused by poorly attached cellular morphologies, leading to an approximately 65% reduction in osteoclast resorption pit formation. Our goal is to demonstrate a natural medicinal system that can support bone healing while providing infection prevention and reducing costly revision surgeries for orthopedic and dental applications.