RNA-seq analysis reveals narrow differential gene expression in MEP and MVA pathways responsible for phytochemical divergence in extreme genotypes of Thymus daenensis Celak.

BACKGROUND: Here, we investigated the underlying transcriptional-level evidence behind phytochemical differences between two metabolically extreme genotypes of Thymus daenensis. The genotypes 'Zagheh-11' (thymol/carvacrol type, poor in essential oil [EO] [2.9%] but rich in triterpenic acids) and 'Malayer-21' (thymol type and rich in EO [3.8%]) were selected from an ongoing breeding program and then clonally propagated for further experimental use. MATERIALS AND METHODS: GC-MS, GC-FID, and HPLC-PDA were utilized to monitor the fluctuation of secondary metabolites at four phenological stages (vegetative, bud burst, early, and full-flowering stages). The highest phytochemical divergence was observed at early flowering stage. Both genotypes were subjected to mRNA sequencing (approximately 100 million paired reads) at the aforementioned stage. The expression patterns of four key genes involved in the biosynthesis of terpenoids were also validated using qRT-PCR. RESULTS: Carvacrol content in 'Zagheh-11' (26.13%) was approximately 23 times higher than 'Malayer-21' (1.12%). Reciprocally, about 10% higher thymol was found in 'Malayer-21' (62.15%). Moreover, the concentrations of three major triterpenic acids in 'Zagheh-11' were approximately as twice as those found in 'Malayer-21'. Transcriptome analysis revealed a total of 1840 unigenes that were differentially expressed, including terpene synthases, cytochrome P450, and terpenoid backbone genes. Several differentially expressed transcription factors (such as MYB, bZIP, HB-HD-ZIP, and WRKY families) were also identified. These results suggest that an active cytosolic mevalonate (MVA) pathway may be linked to higher levels of sesquiterpenes, triterpenic acids, and carvacrol in 'Zagheh-11'. The chloroplastic pathway of methyl erythritol phosphate (MEP) may have also contributed to a higher accumulation of thymol in Malayer-21. Indeed, 'Zagheh-11' showed higher expression of certain genes (HMGR, CYP71D180, ß-amyrin 28-monooxygenase, and sesquiterpene synthases) in the MVA pathway, while some genes in the MEP pathway (including DXR, ispG, and γ-terpinene synthase) were distinctly expressed in Malayer-21. Future efforts in metabolic engineering of MVA/MEP pathways may benefit from these findings to produce increased levels of desired secondary metabolites at commercial scale.

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.

Application of cuminaldehyde and ciprofloxacin for the effective control of biofilm assembly of Pseudomonas aeruginosa: A combinatorial study.

Pseudomonas aeruginosa is widely associated with biofilm-mediated antibiotic resistant chronic and acute infections which constitute a persistent healthcare challenges. Addressing this threat requires exploration of novel therapeutic strategies involving the combination of natural compounds and conventional antibiotics. Hence, our study has focused on two compounds; cuminaldehyde and ciprofloxacin, which were strategically combined to target the biofilm challenge of P. aeruginosa. The minimum inhibitory concentration (MIC) of cuminaldehyde and ciprofloxacin was found to be 400 µg/mL and 0.4 µg/mL, respectively. Moreover, the fractional inhibitory concentration index (FICI = 0.62) indicated an additive interaction prevailed between cuminaldehyde and ciprofloxacin. Subsequently, sub-MIC doses of cuminaldehyde (25 µg/mL) and ciprofloxacin (0.05 µg/mL) were selected for an array of antibiofilm assays which confirmed their biofilm inhibitory potential without exhibiting any antimicrobial activity. Furthermore, selected doses of the mentioned compounds could manage biofilm on catheter surface by inhibiting and disintegrating existing biofilm. Additionally, the test combination of the mentioned compounds reduced virulence factors secretion, accumulated reactive oxygen species and increased cell-membrane permeability. Thus, the combination of cuminaldehyde and ciprofloxacin demonstrates potential in combating biofilm-associated Pseudomonal threats.

CYP108N14: A Monoterpene Monooxygenase from Rhodococcus globerulus.

Rhodococcus globerulus (R. globerulus) was isolated from the soil beneath a Eucalypt tree. Metabolic growth studies revealed that R. globerulus was capable of living on certain monoterpenes, including 1,8-cineole and p-cymene, as sole sources of carbon and energy. Multiple P450 genes were identified in the R. globerulus genome that shared homology to known bacterial, monoterpene hydroxylating P450s. To date, two of these P450s have been expressed and characterised as 1,8-cineole (CYP176A1) and p-cymene (CYP108N12) monooxygenases that are believed to initiate the biodegradation of these terpenes. In this work, another putative P450 gene (CYP108N14) was identified in R. globerulus genome. Given its amino acid sequence identity to other monoterpene hydroxylating P450s it was hypothesised to catalyse monoterpene hydroxylation. These include CYP108A1 from Pseudomonas sp. (47 % identity, 68 % similarity) which hydroxylates α-terpineol, and CYP108N12 also from R. globerulus (62 % identity, 77 % similarity). Also present in the operon containing CYP108N14 were putative ferredoxin and ferredoxin reductase genes, suggesting a typical Class I P450 system. CYP108N14 was successfully over-expressed heterologously and purified, resulting in a good yield of CYP108N14 holoprotein. However, neither the ferredoxin nor ferredoxin reductase could be produced heterologously. Binding studies with CYP108N14 revealed a preference for the monoterpenes p-cymene, (R)-limonene, (S)-limonene, (S)-α-terpineol and (S)-4-terpineol. An active catalytic system was reconstituted with the non-native redox partners cymredoxin (from the CYP108N12 system) and putidaredoxin reductase (from the CYP101A1 system). CYP108N14 when supported by these redox partners was able to catalyse the hydroxylation of the five aforementioned substrates selectively at the methyl benzylic/allylic positions.

Research into how carvacrol and metformin affect several human proteins in a hyperglycemic condition: A comparative study in silico and in vitro.

Carvacrol (CV) is an organic compound found in the essential oils of many aromatic herbs. It is nearly unfeasible to analyze all the current human proteins for a query ligand using in vitro and in vivo methods. This study aimed to clarify whether CV possesses an anti-diabetic feature via Docking-based inverse docking and molecular dynamic (MD) simulation and in vitro characterization against a set of novel human protein targets. Herein, the best poses of CV docking simulations according to binding energy ranged from -7.9 to -3.5 (kcal/mol). After pathway analysis of the protein list through GeneMANIA and WebGestalt, eight interacting proteins (DPP4, FBP1, GCK, HSD11ß1, INSR, PYGL, PPARA, and PPARG) with CV were determined, and these proteins exhibited stable structures during the MD process with CV. In vitro application, statistically significant results were achieved only in combined doses with CV or metformin. Considering all these findings, PPARG and INSR, among these target proteins of CV, are FDA-approved targets for treating diabetes. Therefore, CV may be on its way to becoming a promising therapeutic compound for treating Diabetes Mellitus (DM). Our outcomes expose formerly unexplored potential target human proteins, whose association with diabetic disorders might guide new potential treatments for DM.

Evidence of the protective role of Carvacrol in a retinal degeneration animal model.

Neurodegenerative pathologies affecting the posterior segment of the eye, are characterized by being devastating and responsible for the majority of visual dysfunctions worldwide. These diseases are primarily degenerative, progressing chronically, and can inflict gradual harm to the optic nerve, retinal ganglion cells (RGC), photoreceptors, and other retinal cells. This retinal damage leads to a progressive loss of vision, marking these conditions as a significant health concern worldwide. The intravitreal administration of the phytochemical Carvacrol (CAR) is expected to demonstrate a neuroprotective and antiapoptotic effect on retinal cells, with a specific focus on RGC. This effect will be observed in a retinal degeneration model (RDM) in rabbits induced by cytotoxic and oxidative agents, namely glutamate (GLUT) and L-buthionine-S, R-sulfoximine (BSO). An in vivo study was conducted using New Zealand rabbits in which retinal damage was created to evaluate the effectiveness of CAR. The effectiveness of CAR on the functionality of retinal neuronal cells in RDM was evaluated using pupillary light reflection (PLR). Furthermore, the phytotherapeutic's influence on cell viability was determined through flow cytometry analysis. Finally, the neuroprotective and antiapoptotic capabilities of CAR were specifically scrutinized in RGC through histological studies, quantifying cell survival, and employing immunohistochemical assays to detect the apoptotic index (%) using the TUNEL technique. Our results demonstrated that CAR promoted the recovery of the pupillary contraction profile over time, maintaining the functionality of retinal cells as healthy controls. Additionally, it showed increased cell viability under oxidative and cytotoxic conditions given by GLUT-BSO agents. Finally, we found that CAR protects the survival of RGC and decreases the percentage of apoptotic cells when compared to RDM. CAR demonstrated to have positive effects on the functionality of photoreceptive nerve cells by restoring pupillary contraction. Likewise, it was shown to have neuroprotective and antiapoptotic effects when evaluated in a general and specific way on retinal nerve cells.

The recent discovery of a promising pharmacological scaffold derived from carvacrol: A review.

Carvacrol, called CA, is a dynamic phytoconstituent characterized by a phenol ring abundantly sourced from various natural reservoirs. This versatile scaffold serves as a pivotal template for the design and synthesis of novel drug molecules, harboring promising biological activities. The active sites positioned at C-4, C-6, and the hydroxyl group (-OH) of CA offer fertile ground for creating potent drug candidates from a pharmacological standpoint. In this comprehensive review, we delve into diverse synthesis pathways and explore the biological activity of CA derivatives. We aim to illuminate the potential of these derivatives in discovering and developing efficacious treatments against a myriad of life-threatening diseases. By scrutinizing the structural modifications and pharmacophore placements that enhance the activity of CA derivatives, we aspire to inspire the innovation of novel therapeutics with heightened potency and effectiveness.

Synergistic interactions of essential oil components with antibiotics against multidrug-resistant Corynebacterium striatum.

AIM: In this study, it was aimed to examine the antibacterial activity of the essential oil components (EOCs), carvacrol (CAR), cinnamaldehyde (CIN), thymol (TH), alpha pinene (α-PN), eucalyptol (EU), limonene (LIM), and the antibiotics, linezolid (LZD), vancomycin (VAN), gentamicin (GEN), ciprofloxacin (CIP), clindamycin (CLN), and penicillin (PEN) against 50 multidrug resistant Corynebacterium striatum strains, and the synergistic interactions of CAR and CIN with the antibiotics against 10 randomly selected Coryne. striatum strains to explore synergistic interactions to determine if their combined use could enhance antibiotic activity and potentially reduce resistance. METHODS AND RESULTS: The activity of the EOCs and the antibiotics against Coryne. striatum strains isolated from clinical specimens, was examined by broth microdilution method. The synergistic interactions of the EOCs with the antibiotics against 10 randomly selected Coryne. striatum strains were determined by checkerboard method. EOCs, CIN, and CAR and antibiotics, LZD, VAN, GEN, CIP, and CLN were detected to have antibacterial activity against Coryne. striatum strains alone and either synergistic interactions were observed in combinations of the antibiotics with EOCs. CONCLUSIONS: All Coryne. striatum strains were determined to be susceptible to VAN and LZD and resistant to GEN, PEN, CIP, and CLN. Synergistic interactions were observed in all combinations of antibiotics tested with CAR and CIN.

Adaptive evolution of Salmonella Typhimurium LT2 exposed to carvacrol lacks a uniform pattern.

Emergence of genetic variants with increased resistance/tolerance to natural antimicrobials, such as essential oils, has been previously evidenced; however, it is unknown whether mutagenesis follows a general or a specific pattern. For this purpose, we carried out four adaptive laboratory evolutions (ALE) in parallel of Salmonella enterica Typhimurium with carvacrol. After 10 evolution steps, we selected and characterized one colony from each lineage (SeCarA, SeCarB, SeCarC, and SeCarD). Phenotypic characterization of the four evolved strains revealed enhanced survival to lethal treatments; two of them (SeCarA and SeCarB) showed an increase of minimum inhibitory concentration of carvacrol and a better growth fitness in the presence of carvacrol compared to wild-type strain. Whole genome sequencing revealed 10 mutations, of which four (rrsH, sseG, wbaV, and flhA) were present in more than one strain, whereas six (nirC, fliH, lon, rob, upstream yfhP, and upstream argR) were unique to individual strains. Single-mutation genetic constructs in SeWT confirmed lon and rob as responsible for the increased resistance to carvacrol as well as to antibiotics (ampicillin, ciprofloxacin, chloramphenicol, nalidixic acid, rifampicin, tetracycline, and trimethoprim). wbaV played an important role in increased tolerance against carvacrol and chloramphenicol, and flhA in cross-tolerance to heat treatments. As a conclusion, no common phenotypical or genotypical pattern was observed in the isolated resistant variants of Salmonella Typhimurium emerged under carvacrol stress. Furthermore, the demonstration of cross-resistance against heat and antibiotics exhibited by resistant variants raises concerns regarding food safety. KEY POINTS: • Stable resistant variants of Salmonella Typhimurium emerged under carvacrol stress • No common pattern of mutagenesis after cyclic exposures to carvacrol was observed • Resistant variants to carvacrol showed cross-resistance to heat and to antibiotics.

Biocontrol Mechanisms of Three Plant Essential Oils Against Phytophthora infestans Causing Potato Late Blight.

Late blight, caused by the notorious pathogen Phytophthora infestans, poses a significant threat to potato (Solanum tuberosum) crops worldwide, impacting their quality as well as yield. Here, we aimed to investigate the potential use of cinnamaldehyde, carvacrol, and eugenol as control agents against P. infestans and to elucidate their underlying mechanisms of action. To determine the pathogen-inhibiting concentrations of these three plant essential oils (PEOs), a comprehensive evaluation of their effects using gradient dilution, mycelial growth rate, and spore germination methods was carried out. Cinnamaldehyde, carvacrol, and eugenol were capable of significantly inhibiting P. infestans by hindering its mycelial radial growth, zoospore release, and sporangium germination; the median effective inhibitory concentration of the three PEOs was 23.87, 8.66, and 89.65 µl/liter, respectively. Scanning electron microscopy revealed that PEOs caused the irreversible deformation of P. infestans, resulting in hyphal shrinkage, distortion, and breakage. Moreover, propidium iodide staining and extracellular conductivity measurements demonstrated that all three PEOs significantly impaired the integrity and permeability of the pathogen's cell membrane in a time- and dose-dependent manner. In vivo experiments confirmed the dose-dependent efficacy of PEOs in reducing the lesion diameter of potato late blight. Altogether, these findings provide valuable insight into the antifungal mechanisms of PEOs vis-à-vis late blight-causing P. infestans. By utilizing the inherent capabilities of these natural compounds, we could effectively limit the harmful impacts of late blight on potato crops, thereby enhancing agricultural practices and ensuring the resilience of global potato food production.

Synthesis, characterization, and evaluation of the anticancer properties of pH-responsive carvacrol-zinc oxide quantum dots on breast cancer cell line (MDA-MB-231).

Since most solid tumors have a low pH value, a pH-responsive drug delivery system may offer a broad method for tumor-targeting treatment. The present study is used to analyze the anticancer activity of carvacrol-zinc oxide quantum dots (CVC-ZnO QDs) against breast cancer cells (MDA-MB-231). CVC-ZnO QDs demonstrate pH responsive and are specifically released within the acidic pH tumor microenvironment. This property enables targeted drug delivery exclusively to cancer cells while minimizing the impact on normal cells. To the synthesized ZnO QDs, the CVC was loaded and then examined by X-ray diffraction, ultraviolet-visible, Fourier transform infrared spectrophotometer, scanning electron microscopy-energy dispersive X-ray, and transmission electron microscopy. For up to 20 h, CVC release was examined in different pH-buffered solutions. The results showed that carvacrol release was stable in an acidic pH solution. Further, cytotoxicity assay, antioxidant, and lipid peroxidation activity, reactive oxygen species, mitochondrial membrane potential, nuclear damage, and the ability of CVC-ZnO QDs to cause apoptosis were all examined. Apoptosis markers such as Bcl2, Bax, caspase-3, and caspase-9, were also studied. In conclusion, the CVC-ZnO QDs destabilized the MDA-MB-231cells under its acidic tumor microenvironment and regulated apoptosis.

Metabolomic modelling and neuroprotective effects of carvacrol against acrylamide toxicity in rat's brain and sciatic nerve.

The study aimed to investigate the harmful effects of acrylamide (AA), which forms in carbohydrate-rich foods at temperatures above 120°C, on the central and peripheral nervous systems and to evaluate the potential neuroprotective effects of carvacrol (CRV). Male Wistar Albino rats were subjected to AA (40 mg/kg/bw/day) and CRV (50 mg/kg/bw/day) for 15 days. Following the last administration, evaluations revealed disrupted gait, heightened thermal sensitivity and altered paw withdrawal thresholds in AA-exposed rats. Notably, AA reduced glutathione (GSH) and raised malondialdehyde (MDA) levels in both brain and sciatic nerve tissues. AA raised nuclear factor erythroid 2-related factor 2 (Nrf2), caspase 3 and nuclear factor κB (NF-κB) gene expressions while decreasing NR4A2. CRV co-administration mitigated gait abnormalities, elevated GSH levels and lowered MDA levels in both tissues. CRV also modulated gene expression, reducing Nrf2 and NF-κB while increasing NR4A2. Histopathological signs of AA-induced neurodegeneration and elevated glial fibrillary acidic protein levels observed in brain and sciatic nerve tissues were rectified with simultaneous administration of CRV, thereby demonstrating neuroprotective efficacy in both regions. This study is pioneering in demonstrating CRV's neuroprotective potential against AA-induced neurotoxicity in both central and peripheral nervous systems, effectively addressing limitations in the literature. In conclusion, the study revealed AA-induced neurodegeneration in the brain and sciatic nerve, with CRV significantly mitigating this neurotoxicity. This novel research underscores CRV's promise as a neuroprotective agent against AA-induced adverse effects in both the central and peripheral nervous systems.

Determination of in vitro synergy and antibiofilm activities of antimicrobials and essential oil components.

Using existing adrentimicrobials with essential oil components to prevent antimicrobial resistance is an alternative strategy. This study aimed to evaluate the resistance status, synergistic combinations, and in vitro biofilm formation activities of clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA), Stenotrophomonas maltophilia and Candida albicans against antimicrobial agents and cinnamaldehyde, carvacrol, eugenol, limonene and eucalyptol. Antimicrobial activities were evaluated by microdilution, cytotoxicity by XTT, synergy by checkerboard and time-kill, and biofilm inhibition by microplate methods. Cinnamaldehyde and carvacrol showed strong antimicrobial activity. Synergistic effects were observed when using all essential oils with antimicrobials. Only two C. albicans isolates showed antagonism with cinnamaldehyde and fluconazole. The constituents showed cytotoxic effects in the L929 cell line (except limonene). A time-kill analysis revealed a bacteriostatic effect on S. maltophilia and MRSA isolates and a fungicidal effect on C. albicans isolates. These results are important for further research to improve antimicrobial efficacy or to develop new agents.

In vitro and in vivo anthelmintic effect of essential oil obtained from Thymus capitatus flowers against Haemonchus contortus and Heligmosomoides polygyrus.

Sheep haemonchosis is a disease that causes serious losses in livestock production, particularly with the increase of cases of anthelmintic resistance around the world. This justifies the urgent need of alternative solutions. The aim of this study was to determine the chemical profile, in vitro, and, in vivo, anthelmintic properties of Thymus capitatus essential oil. To evaluate the, in vitro, anthelmintic activity of the T. capitatus EO on Haemonchus contortus, two tests were used: egg hatch assay (EHA) and adult worm motility (AWM) assay. The nematicidal effect of this oil was evaluated, in vivo, in mice infected artificially with Heligmosomoides polygyrus using faecal egg count reduction (FECR) and total worm count reduction (TWCR). Chromatographic characterization of T.capitatus composition using gas chromatography coupled to mass spectrometry (GC-MS) demonstrated the presence of carvacrol (81.16%), as the major constituents. The IC50 values obtained was 1.9 mg/mL in the EHT. In the AWM assay; T. capitatus essential oil achieved 70.8% inhibition at 1 mg/mL after 8 h incubation. The in vivo, evaluation on H. polygyrus revealed a significant nematicidal effect 7 days post-treatment by inducing 49.5% FECR and 64.5% TWCR, using the highest dose (1600 mg/kg). The results of present study, demonstrate that T.capitatus EO possess a significant anthelmintic properties. Furthermore, it could be an alternative source of anthelmintic agents against gastrointestinal infections caused by H. contortus.

Carvacrol-Loaded Nanoemulsion Promotes Tocolytic and Anti-Dysmenorrhea Effects in Rodents.

INTRODUCTION: Carvacrol is a phenolic constituent of essential oils that has antinociceptive, anti-inflammatory, and antioxidant activities. METHOD: This study aimed to evaluate the in vitro spasmolytic and in vivo anti-dysmenorrhea potential of a nanoemulsion-containing carvacrol (nanoCARV). RESULTS: In isolated rat uterus, nanoCARV reduced spontaneous contractions (pEC50 = 3.91 ± 0.25) and relaxed preparations pre-contracted with oxytocin (pEC50 = 3.78 ± 0.2), carbachol (pEC50 = 4.15 ± 0.4), prostaglandin F2α (pEC50 = 3.00 ± 0.36), and KCl (pEC50 = 3.98 ± 0.32). The investigation of the mechanism of action revealed significant differences (p < 0.05) between the pEC50 values of nanoCARV in the absence or presence of aminophylline or tetraethylammonium. In a primary dysmenorrhea model, treatment with nanoCARV reduced the number of oxytocin-induced abdominal writhes. CONCLUSIONS: These data indicate that the anti-dysmenorrhea effect of nanoCARV may be related to the relaxation of uterine smooth muscle, with participation of the cAMP signaling pathway and potassium channels.

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.

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.

Biofilm inhibition of denture cleaning tablets and carvacrol on denture bases produced with different techniques.

OBJECTIVES: This study compares the biofilm inhibition effects of denture cleaning tablets, carvacrol, and their combined use against Candida albicans on denture bases produced with different techniques. Additionally, the surface roughness and contact angles of these denture bases were evaluated. MATERIALS AND METHODS: Test samples were prepared from four different denture base materials (cold-polymerized, heat-polymerized, CAD/CAM milling, and 3D-printed). The surface roughness and contact angles of the test samples were measured using a profilometer and goniometer, respectively. For the evaluation of biofilm inhibition, samples were divided into 5 subgroups: Corega and carvacrol, separately and combined treatments, positive (inoculated with C. albicans) and negative control (non-inoculated with C. albicans, only medium). Biofilm mass was determined using the crystal violet method. An additional prepared test sample for each subgroup was examined under scanning electron microscopy (SEM). RESULTS: The surface roughness values of the 3D-printed test samples were found to be statistically higher than the other groups (P < .001). The water contact angle of all test materials was not statistically different from each other (P > .001). Corega and carvacrol, separately and combined, significantly decreased the amount of biofilm on all surfaces (P < .0001). Treatment of corega alone and in combination with carvacrol to the 3D-printed material caused less C. albicans inhibition than the other groups (P < .001; P < .05). CONCLUSIONS: The surface roughness values of all test groups were within the clinically acceptable threshold. Although Corega and carvacrol inhibited C. albicans biofilms, their combined use did not show a synergistic effect. CLINICAL RELEVANCE: Carvacrol may be used as one of the disinfectant agents for denture cleaning due to its biofilm inhibition property.

Carvacrol Encapsulation in Chitosan-Carboxymethylcellulose-Alginate Nanocarriers for Postharvest Tomato Protection.

Advancements in polymer science and nanotechnology hold significant potential for addressing the increasing demands of food security, by enhancing the shelf life, barrier properties, and nutritional quality of harvested fruits and vegetables. In this context, biopolymer-based delivery systems present themselves as a promising strategy for encapsulating bioactive compounds, improving their absorption, stability, and functionality. This study provides an exploration of the synthesis, characterization, and postharvest protection applications of nanocarriers formed through the complexation of chitosan oligomers, carboxymethylcellulose, and alginate in a 2:2:1 molar ratio. This complexation process was facilitated by methacrylic anhydride and sodium tripolyphosphate as cross-linking agents. Characterization techniques employed include transmission electron microscopy, energy-dispersive X-ray spectroscopy, infrared spectroscopy, thermal analysis, and X-ray powder diffraction. The resulting hollow nanospheres, characterized by a monodisperse distribution and a mean diameter of 114 nm, exhibited efficient encapsulation of carvacrol, with a loading capacity of approximately 20%. Their suitability for phytopathogen control was assessed in vitro against three phytopathogens-Botrytis cinerea, Penicillium expansum, and Colletotrichum coccodes-revealing minimum inhibitory concentrations ranging from 23.3 to 31.3 µg·mL-1. This indicates a higher activity compared to non-encapsulated conventional fungicides. In ex situ tests for tomato (cv. 'Daniela') protection, higher doses (50-100 µg·mL-1, depending on the pathogen) were necessary to achieve high protection. Nevertheless, these doses remained practical for real-world applicability. The advantages of safety, coupled with the potential for a multi-target mode of action, further enhance the appeal of these nanocarriers.

Carvacrol as a Stimulant of the Expression of Key Genes of the Ginsenoside Biosynthesis Pathway and Its Effect on the Production of Ginseng Saponins in Panax quinquefolium Hairy Root Cultures.

The accumulation of ginsenosides (triterpenic saponins) was determined in Panax quinquefolium hairy root cultures subjected to an elicitation process using carvacrol at 5, 10, 25, 50, 100, 250, and 500 µM concentrations during 24 and 72 h exposure. This study was the first one in which carvacrol was applied as an elicitor. The content of eight ginsenosides, Rb1, Rb2, Rb3, Rc, Rd, Rg1, Rg2, and Re, was determined using HPLC analysis. Moreover, the quantitative RT-PCR method was applied to assess the relative expression level of farnesyl diphosphate synthase, squalene synthase, and dammarenediol synthase genes in the studied cultures. The addition of carvacrol (100 µM) was an effective approach to increase the production of ginsenosides. The highest content and productivity of all detected saponins were, respectively, 20.01 mg∙g-1 d.w. and 5.74 mg∙L-1∙day-1 after 72 h elicitation. The production profile of individual metabolites in P. quinquefolium cultures changed under the influence of carvacrol. The biosynthesis of most examined protopanaxadiol derivatives was reduced under carvacrol treatment. In contrast, the levels of ginsenosides belonging to the Rg group increased. The strongest effect of carvacrol was noticed for Re metabolites, achieving a 7.72-fold increase in comparison to the control. Saponin Rg2, not detected in untreated samples, was accumulated after carvacrol stimulation, reaching its maximum concentration after 72 h exposure to 10 µM elicitor.