Geraniol and Carvacrol in Essential Oil Bearing Thymus pulegioides: Distribution in Natural Habitats and Phytotoxic Effect.

Phenolic and non-phenolic chemotypes of Thymus pulegioides L. are common in Europe. Essential oils of these chemotypes, as various compositions of allelochemicals, can have different phytotoxic effects on neighboring plants in natural habitats. The aim of this study was to establish the distribution of carvacrol and geraniol in T. pulegioides, growing wild in Lithuania, and compare phytotoxity of essential oils of carvacrol and geraniol chemotypes on selected plant species. In investigating 131 T. pulegioides habitats, essential oils were isolated by hydrodistillation and analyzed by GC-FID and GC-MS. Phytotoxity of essential oils extracted from carvacrol and geraniol chemotypes transmitted through water and air to selected plants was determined under laboratory conditions. Pharmacologically valuable Hypericum perforatum L. and the important forage grass Phleum pratense L. were respectively selected for experimentation from among 35 medicinal plants and 10 feed Poaceae species, growing in T. pulegioides habitats. Field results showed that carvacrol is common throughout Lithuania's territory, whereas the geraniol is predominantly located under the continental climatic conditions of the eastern region of the country. In the laboratory experiment, it was established that there was stronger inhibition of P. pratense seed germination by the essential oil of the geraniol chemotype than the carvacrol chemotype. None of the H. perforatum seeds germinated after exposure to the essential oil of the geraniol chemotype. In general, this study builds on previous studies by providing further evidence that different T. pulegioides chemotypes have contrasting phytotoxic effects on neighboring plants within their natural habitats.

Effects of essential oil components exposure on biological parameters of Caenorhabditis elegans.

The extensive use of essential oil components in an increasing number of applications can substantially enhance exposure to these compounds, which leads to potential health and environmental hazards. This work aimed to evaluate the toxicity of four widely used essential oil components (carvacrol, eugenol, thymol, vanillin) using the in vivo model Caenorhabditis elegans. For this purpose, the LC50 value of acute exposure to these components was first established; then the effect of sublethal concentrations on nematodes' locomotion behaviour, reproduction, heat and oxidative stress resistance and chemotaxis was evaluated. The results showed that all the components had a concentration-dependent effect on nematode survival at moderate to high concentrations. Carvacrol and thymol were the two most toxic compounds, while vanillin had the mildest toxicological effect. Reproduction resulted in a more sensitive endpoint than lethality to evaluate toxicity. Only pre-exposure to carvacrol and eugenol at the highest tested sublethal concentrations conferred worms oxidative stress resistance. However, at these and lower concentrations, both components induced reproductive toxicity. Our results evidence that these compounds can be toxic at lower doses than those required for their biological action. These findings highlight the need for a specific toxicological assessment of every EOC application.

In vitro toxicological evaluation of mesoporous silica microparticles functionalised with carvacrol and thymol.

The cytotoxicity of carvacrol- and thymol-functionalised mesoporous silica microparticles (MCM-41) was assessed in the human hepatocarcinoma cell line (HepG2). Cell viability, lactate dehydrogenase (LDH) activity, reactive oxygen species (ROS) production, mitochondrial membrane potential (ΔΨm), lipid peroxidation (LPO) and apoptosis/necrosis analyses were used as endpoints. The results showed that both materials induced cytotoxicity in a time- and concentration-dependent manner, and were more cytotoxic than free essential oil components and bare MCM-41. This effect was caused by cell-particle interactions and not by degradation products released to the culture media, as demonstrated in the extract dilution assays. LDH release was a less sensitive endpoint than the MTT (thiazolyl blue tetrazolium bromide) assay, which suggests the impairment of the mitochondrial function as the primary cytotoxic mechanism. In vitro tests on specialised cell functions showed that exposure to sublethal concentrations of these materials did not induce ROS formation during 2 h of exposure, but produced LPO and ΔΨm alterations in a concentration-dependent manner when cells were exposed for 24 h. The obtained results generally support the hypothesis that the carvacrol- and thymol-functionalised MCM-41 microparticles induced toxicity in HepG2 cells by an oxidative stress-related mechanism that resulted in apoptosis through the mitochondrial pathway.

Cytotoxicity screening of Thymus vulgaris L. essential oil in brine shrimp nauplii and cancer cell lines.

Among natural products, essential oils from aromatic plants have been reported to possess potent anticancer properties. In this work, we aimed to perform the cytotoxic concentration range screening and antiproliferative activity screening of chemically characterized Thymus vulgaris L. essential oil. In vivo bioassay was conducted using the brine shrimp lethality test (BSLT). In vitro evaluation of antiproliferative activity was carried out on three human tumor cell lines: breast adenocarcinoma MCF-7, lung carcinoma H460 and acute lymphoblastic leukemia MOLT-4 using MTT assay. Essential oil components thymol (36.7%), p-cymene (30.0%), γ-terpinene (9.0%) and carvacrol (3.6%) were identified by gas chromatography/mass spectrometry. Analyzed essential oil should be considered as toxic/highly toxic with LC50 60.38 µg/mL in BSLT and moderate/weakly cytotoxic with IC50 range 52.65-228.78 µg/mL in vitro, according to evaluated cytotoxic criteria. Essential oil induced a dose-dependent inhibition of cell proliferation in all tested tumor cell lines and showed different sensitivity. Dose dependent toxicity observed in bioassay as well as the in vitro assay confirmed that brine shrimp lethality test is an adequate method for preliminary toxicity testing of Thymus vulgaris L. essential oil in tumor cell lines.

Limonene-carvacrol: A combination of monoterpenes with enhanced antileishmanial activity.

BACKGROUND: Leishmaniasis is a parasitosis with a wide incidence in developing countries. The drugs which are indicated for the treatment of this infection usually are able to promote high toxicity. PURPOSE: A combination of limonene and carvacrol, monoterpenes present in plants with antiparasitic activity may constitute an alternative for the treatment of these diseases. METHODS: In this study, the antileishmania activity against Leishmania major, cytotoxicity tests, assessment of synergism, parasite membrane damage tests as well as molecular docking and immunomodulatory activity of limonene-carvacrol (Lim-Car) combination were evaluated. RESULTS: The Lim-Car combination (5:0; 1:1; 1:4; 2:3; 3:2; 4:1 and 0:5) showed potential antileishmania activity, with mean inhibitory concentration (IC50) ranging from 5.8 to 19.0 µg.mL-1. They demonstrated mean cytotoxic concentration (CC50) ranging from 94.1 to 176.0 µg.mL-1, and did not show significant hemolytic effect. In the investigation of synergistic interaction, the 4:1 Lim-Car combination showed better fractional inhibitory concentration (FIC) index as well as better activity on amastigotes and IS. The samples caused considerable damage to the parasite membrane this monoterpene activity seems to be more related to Trypanothione Reductase (TryR) enzyme interaction, demonstrated in the molecular docking assay. In addition, the 4:1 Lim-Car combination stimulated macrophage activation, and showed at was the best association, with reduction of infection and infectivity of parasitized macrophages. CONCLUSION: The 4:1 Lim-Car combination appears to be a promising candidate as a monotherapeutic antileishmania agent.

Chicken embryonic toxicity and potential in vitro estrogenic and mutagenic activity of carvacrol and thymol in low dose/concentration.

Thymol and carvacrol are phenolic isomers with the potential developmental toxicity and endocrine disruptions (ED) at low concentrations. However, few reports estimated their toxicity and ED below 10-6 M (150 µg/L) (MW of thymol and carvacrol: 150 g/mol). In this study, both chemicals were determined for the developmental toxicity and potential ED at 500 µg/kg and 50 µg/kg using the chicken embryonic assay, potential estrogenic activity (EA) at 10-12 to 10-7 M (1.5 × 10-4 to 15 µg/L) by the MCF-7 cell proliferation assay, mutagenicity at 10-12 to 10-6 M (1.5 × 10-4 to 150 µg/L) by the Ames test, and an in silico method for ED. Carvacrol showed mutagenic risks at 10-7, 10-8, and 10-11 M (15, 1.5, and 0.0015 µg/L) while thymol at 10-6 and 10-8 M (150 and 1.5 µg/L). Carvacrol negatively impacted embryonic growth at 50 µg/kg, with weak EA at 10-8 M (1.5 µg/L). Carvacrol but not thymol had weak EA at 10-12 M (1.5 × 10-4 µg/L). Molecular docking to 14 types of hormone-related receptors revealed that carvacrol had higher binding affinities to two estrogen receptors and the mineralocorticoid receptor than those to thymol. Carvacrol and thymol varied in toxicities due to a different location of one phenolic hydroxyl group.

Toxic effect and genotoxicity of carvacrol ethers in Drosophila melanogaster.

The present study investigated the effect of four carvacrol ethers (propyl-, butyl, octyl- and benzyl) on the viability, level of dominant lethal mutations of Drosophila melanogaster and their influence on the multiplication of the nuclear genome in salivary gland cells. The fertility and viability of fruit flies were assessed after oral administration (0.05 % to culture medium) and inhalation exposure (5 mg per 1 cm2 of polyvinyl alcohol film) of compounds 1‒4 and initial carvacrol. The influence of terpenoid and its ethers on the degree of chromosomes polyteny in salivary gland cells of D. melanogaster larvae has been revealed. Among all tested compounds, carvacrol exhibited the most significant impact on frequency of dominant lethal mutations, fecundity and insect survival when inhaled or adding to the culture medium. Oral administration of ethers 1‒4 was found to decrease the average level of chromosome polyteny degree (366 C-500 C) while pure carvacrol adding to culture medium had the opposite effect (763 C) compared to control (695 C). The possible mechanism of action for carvacrol and its ethers is discussed.

Cuminaldehyde potentiates the antimicrobial actions of ciprofloxacin against Staphylococcus aureus and Escherichia coli.

Escherichia coli and Staphylococcus aureus are important agents of urinary tract infections that can often evolve to severe infections. The rise of antibiotic-resistant strains has driven the search for novel therapies to replace the use or act as adjuvants of antibiotics. In this context, plant-derived compounds have been widely investigated. Cuminaldehyde is suggested as the major antimicrobial compound of the cumin seed essential oil. However, this effect is not fully understood. Herein, we investigated the in silico and in vitro activities of cuminaldehyde, as well as its ability to potentiate ciprofloxacin effects against S. aureus and E. coli. In silico analyses were performed by using different computational tools. The PASS online and SwissADME programmes were used for the prediction of biological activities and oral bioavailability of cuminaldehyde. For analysis of the possible toxic effects and the theoretical pharmacokinetic parameters of the compound, the Osiris, SwissADME and PROTOX programmes were used. Estimations of cuminaldehyde gastrointestinal absorption, blood brain barrier permeability and skin permeation by using SwissADME; and drug likeness and score by using Osiris, were also evaluated The in vitro antimicrobial effects of cuminaldehyde were determined by using microdilution, biofilm formation and time-kill assays. In silico analysis indicated that cuminaldehyde may act as an antimicrobial and as a membrane permeability enhancer. It was suggested to be highly absorbable by the gastrointestinal tract and likely to cross the blood brain barrier. Also, irritative and harmful effects were predicted for cuminaldehyde if swallowed at its LD50. Good oral bioavailability and drug score were also found for this compound. Cuminaldehyde presented antimicrobial and anti-biofilm effects against S. aureus and E. coli.. When co-incubated with ciprofloxacin, it enhanced the antibiotic antimicrobial and anti-biofilm actions. We suggest that cuminaldehyde may be useful as an adjuvant therapy to ciprofloxacin in S. aureus and E. coli-induced infections.

Inhibition of 3D colon cancer stem cell spheroids by cytotoxic RuII-p-cymene complexes of mesalazine derivatives.

The Ru(ii) complex of an imidazole-mesalazine Schiff base is a unique example showing growth inhibition of 3D-colon cancer stem cell spheroids and bulk colon cancer cells at lower dosage than salinomycin or oxaliplatin. Unlike oxaliplatin which increases the expression of stemness genes (SOX2, KLF4, HES1 and Oct4), these complexes maintain a tight regulation.

Antinociceptive and antineuropathic effects of cuminaldehyde, the major constituent of Cuminum cyminum seeds: Possible mechanisms of action.

ETHNOPHARMACOLOGICAL RELEVANCE: In Iranian traditional medicine, Cuminum cyminum is a unique medicinal herb for pain relief. Cuminaldehyde has been distinguished as the major constituent of C. cyminum seeds; even though, the analgesic effect of cuminaldehyde has not yet been examined. AIM OF THE STUDY: The nobility of this study was to assess cuminaldehyde effect on nociceptive and neuropathic pains; furthermore, evaluation of its possible mechanisms of action. MATERIALS AND METHODS: Hot plate, formalin, and acetic acid-induced writhing tests were used to evaluate nociception in mice. Naloxone (opioid receptors antagonist), L-arginine (nitric oxide (NO) precursor), L-NAME (NO synthase inhibitor), sodium nitroprusside (NO donor), methylene blue (guanylyl cyclase inhibitor), sildenafil (phosphodiesterase inhibitor), and glibenclamide (KATP channel blocker) were used to determine the implication of opioid receptors and L-arginine/NO/cGMP/KATP channel pathway. Allodynia and hyperalgesia were investigated in the CCI (chronic constriction injury) model of neuropathic pain in rats. The ELISA method was used to measure the inflammatory cytokines in serum samples of rats. The entire chemicals were intraperitoneally injected. RESULTS: Cuminaldehyde (100 and 200 mg/kg) significantly decreased the latency to nociceptive response in the hot plate test. The outcome of cuminaldehyde was completely antagonized by naloxone (2 mg/kg). Formalin- and acetic acid-induced nociception was significantly inhibited by cuminaldehyde (12.5-50 mg/kg). The antinociceptive effect of cuminaldehyde was reversed in writhing test by L-arginine (200 mg/kg), sodium nitroprusside (0.25 mg/kg), and sildenafil (0.5 mg/kg); however, L-NAME (30 mg/kg) and methylene blue (20 mg/kg) enhanced the effect of cuminaldehyde. Glibenclamide (10 mg/kg) did not alter the antinociceptive effects of cuminaldehyde. In the CCI-induced neuropathy, cuminaldehyde (25-100 mg/kg) significantly alleviated allodynia and hyperalgesia and decreased the serum levels of TNF-α and IL-1ß. CONCLUSION: It was attained magnificently that cuminaldehyde exerts antinociceptive and antineuropathic effects through the involvement of opioid receptors, L-arginine/NO/cGMP pathway, and anti-inflammatory function.

Bioactivity of some Apiaceae essential oils and their constituents against Sitophilus zeamais (Coleoptera: Curculionidae).

Sitophilus zeamais is a key pest of stored grains. Its control is made, usually, using synthetic insecticides, despite their negative impacts. Botanical insecticides with fumigant/repellent properties may offer an alternative solution. This work describes the effects of Anethum graveolens, Petroselinum crispum, Foeniculum vulgare and Cuminum cyminum essential oils (EOs) and (S)-carvone, cuminaldehyde, estragole and (+)-fenchone towards adults of S. zeamais. Acute toxicity was assessed by fumigation and topical application. Repellence was evaluated by an area preference bioassay and two-choice test, using maize grains. LC50 determined by fumigation ranged from 51.8 to 535.8 mg L-1 air, with (S)-carvone being the most active. LD50 values for topical applications varied from 23 to 128 µg per adult for (S)-carvone > cuminaldehyde > A. graveolens > C. cyminum > P. crispum. All EOs/standard compounds reduced significantly the percentage of insects attracted to maize grains (65-80%) in the two-choice repellence test, whereas in the area preference bioassay RD50 varied from 1.4 to 45.2 µg cm-2, with cuminaldehyde, (S)-carvone and estragole being strongly repellents. Petroselinum crispum EO and cuminaldehyde affected the nutritional parameters relative growth rate, efficiency conversion index of ingested food and antifeeding effect, displaying antinutritional effects toward S. zeamais. In addition, P. crispum and C. cyminum EOs, as well as cuminaldehyde, showed the highest acetylcholinesterase inhibitory activity in vitro (IC50 = 185, 235 and 214.5 µg mL-1, respectively). EOs/standard compounds exhibited acute toxicity, and some treatments showed antinutritional effects towards S. zeamais. Therefore, the tested plant products might be good candidates to be considered to prevent damages caused by this pest.

Design, synthesis and pharmacological evaluation of CVIB, a codrug of carvacrol and ibuprofen as a novel anti-inflammatory agent.

The search for new drugs with anti-inflammatory properties remains a challenge for modern medicine. Among the various strategies for drug discovery, deriving new chemical entities from known bioactive natural and/or synthetic compounds remains a promising approach. Here, we designed and synthesized CVIB, a codrug developed by association of carvacrol (a phenolic monoterpene) with ibuprofen (a non-steroidal anti-inflammatory drug). In silico pharmacokinetic and physicochemical properties evaluation indicated low aqueous solubility (LogP ≥5.0). Nevertheless, the hybrid presented excellent oral bioavailability, gastrointestinal tract absorption, and low toxicity. CVIB did not present cytotoxicity in peripheral blood mononuclear cells (PBMCs), and promoted a significant reduction in IL-2, IL-10, IL-17, and IFN-γ cytokine levels in vitro. The LD50 was estimated to be approximately 5000 mg/kg. CVIB was stable and detectable in human plasma after 24 h. In vivo anti-inflammatory evaluations revealed that CVIB at 10 and 50 mg/kg i.p. caused a significant decrease in total leukocyte count (p < 0.01) and provoked a significant reduction in IL-1ß (p < 0.01). CVIB at 10 mg/kg i.p. efficiently decreased inflammatory parameters better than the physical mixture (carvacrol + ibuprofen 10 mg/kg i.p.). The results suggest that the codrug approach is a good option for drug design and development, creating the possibility of combining NSAIDs with natural products in order to obtain new hybrid drugs may be useful for treatment of inflammatory diseases.