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Tea tree oil (TTO), acquired from Melaleuca alternifolia (Maiden & Betche) Cheel, Myrtaceae, is a widely utilized essential oil (EO) due to its bioactive properties. The identification and quantification of TTO ingredients is generally performed by GC-MS, which provides the most accurate results. However, in some instances, the cost and time of analysis may pose a challenge. Thin-layer chromatography (TLC) and high-performance thin-layer chromatography (HPTLC) offer a simpler, faster, cost-effective alternative capable of simultaneously analyzing and quantifying multiple samples. In addition, for more complex oils, two-dimensional (2D) or multigradient development (MGD) TLC provide better separation. Nevertheless, further development is sometimes necessary for the isolation of comigrating components. This study showcases a combined 2D-MGD TLC/HPTLC method for the successful separation of TTO components of interest. While human error, limited separation, and the partial evaporation of volatile components may still present a challenge during the process, considerable recovery of mono- and sesquiterpenes was achieved. This protocol also resulted in the successful isolation of target oxygenated monoterpenes (OMs) producing highly pure terpinen-4-ol (100%) and α-terpineol (≥94%), confirmed by GC-MS. The accurate enantiomeric distribution of these major OMs was verified by GC-FID through the use of a chiral cyclodextrin-based stationary phase. The observed positive enantiomer range (area percent) as well as (+)/(-) ratio for each terpinen-4-ol and α-terpineol were within acceptable ISO criteria.

The aim of this study was to investigate the effects of terpinen-4-ol (TER) supplementation on the intestinal barrier function of pigs. Five groups of fifty 28-day-old piglets with comparable body weights were randomly assigned to the following groups: the control group (CON), the lipopolysaccharide group (LPS), the low TER group (PLT), the middle TER group (PMT), and the high TER group (PHT). The basal diet was given to the CON and LPS groups, and 30, 60, or 90 mg/kg TER was added to the basal diet for the TER groups. After the 21-day trial period, piglets in the LPS and TER groups received an intraperitoneal injection of 100 μg/kg body weight of LPS, whereas the piglets in the CON group received an injection of 0.9% normal saline solution. The results showed that LPS stimulation resulted in a decrease ( p < 0.05) in the depth of colonic crypts in piglets, which was greater ( p < 0.05) in the TER group. Compared with those in the CON group, the number of goblet cells and MUC2 expression were decreased in the colon of piglets in the LPS group, while these parameters were increased in the PMT group ( p < 0.05). The malondialdehyde (MDA) content was greater in the colon of the LPS group than in that of the CON group, while the activities of glutathione peroxidase ( GSH-Px ), superoxide dismutase ( SOD ), and catalase ( CAT ) were lower in the colon of the LPS group; conversely, the MDA content was lower in the colons of the PLT and PMT groups than in those of the LPS group ( p < 0.05). TER also reduced ( p < 0.05) LPS-induced upregulation of IL-1β and TNF-α expression, along with the relative gene expression of NLRP3 , ASC , and caspase-1 in the colon of piglets ( p < 0.05). Compared with those in the CON group, the abundances of Firmicutes and UCG-005 in the LPS group were lower ( p < 0.05), and those in the TER group were significantly greater than those in the LPS group. Compared with those in the CON group, the abundance of Proteobacteria in the LPS group increased ( p < 0.05), while the abundance of Actinobacteria and Phascolarctobacterium increased ( p < 0.05) in the colon of the PHT group compared with that in the LPS group. In conclusion, TER effectively improved the intestinal barrier function of the colon in weaning piglets. Based on the results of this study, the appropriate dose of TER in the diets of weaning piglets was 60 mg/kg.

Following a request from the European Commission, EFSA was asked to deliver a scientific opinion on the safety and efficacy of tea tree oil obtained from leaves and terminal branchlets of Melaleuca alternifolia (Maiden & Betche) Cheel when used as a sensory additive for all animal species. The EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) concluded that tea tree oil was very unlikely to be of safety concern for long-living and reproductive animals and is of no concern for target species for fattening at the following concentrations in complete feed: 1.1 mg/kg for chickens for fattening, 1.5 mg/kg for turkeys for fattening, 1.7 mg/kg for laying hens, 2.0 mg/kg for piglets, 2.4 mg/kg for pigs for fattening, 3.1 mg/kg for sows, 5.0 mg/kg for veal calves (milk replacer) and salmonids, 4.4 mg/kg for cattle for fattening, sheep/goats and horses, 2.9 mg/kg for dairy cows, 1.8 mg/kg for rabbits, 0.9 mg/kg for cats, 5.3 mg/kg for dogs, 6.6 for crustaceans and 15 mg/kg for ornamental fish. These conclusions were extrapolated to other physiologically related species. For any other species, the additive is very unlikely to be of safety concern at 1.1 mg/kg complete feed. No concerns for consumers and the environment were identified following the use of the additive up to the highest safe use level in feed. Regarding user safety, tea tree oil should be considered as an irritant to skin and eyes and as a dermal and respiratory sensitiser. It is classified as a reprotoxic substance (category 1B) following CLP criteria and should be handled accordingly. Since M. alternifolia and its preparations were recognised to flavour food and its function in feed would be essentially the same as that in food, no further demonstration of efficacy was considered necessary., Competing Interests: If you wish to access the declaration of interests of any expert contributing to an EFSA scientific assessment, please contact interestmanagement@efsa.europa.eu., (© 2024 European Food Safety Authority. EFSA Journal published by Wiley‐VCH GmbH on behalf of European Food Safety Authority.)

This study investigated the antistress effects of yuzu synthetic fragrances by employing three experiments on humans and mice using two yuzu synthetic fragrances and five single compounds. We prepared two synthetic fragrances based on the component analysis of two natural yuzu essential oils extracted by cold-pressed and steam-distilled extraction methods. Chromogranin A (CgA) and heart rate (HR) were used as stress indices in human experiments. Immobility time during the forced swim test was used as a stress index in mice experiments. We analyzed brain mechanisms by measuring the expression of neurotrophic factors, brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), and neurotrophin-3 (NT-3) in the mice experiments. Synthetic yuzu fragrance mimicked steam-distilled oil (SD) significantly reduced participants' heart rate in experiment 1. In the forced swim test conducted in experiment 2, SD significantly reduced immobility time, and increased the expression of neurotrophic factors BDNF, NGF, and NT-3 in the hippocampus of mice. In experiment 3, focusing on single compounds, terpinen-4-ol significantly reduced immobility time in the forced swim test. These findings indicate that inhalation of SD and terpinen-4-ol has antistress effects. Terpinen-4-ol is a strong candidate for further investigation as a potential stress-reducing agent.

Owing to their extensive biological potential, essential oils (EOs) and their bioactive phytochemicals have gained attention from the scientific community. Within this domain, Terpinen-4-ol (T-4-ol), a bioactive monoterpene alcohol and the major constituent of tea tree oil (TTO), has made its way into translational research. Recent literature on T-4-ol strongly indicates its diverse pharmacological properties, including but not limited to antimicrobial, antivirulent, anti-oxidant, anti-inflammatory, anti-hypertensive, and anti-cancer effects. Hence, this review is the first to provide a comprehensive overview of the sources, bioavailability, safety, pharmaceutical delivery systems, and multifaceted biological properties of T-4-ol, emphasizing its medicinal potential for widescale application. The antibacterial and antifungal effectiveness of T-4-ol has been discussed, encompassing its role in combating a broad spectrum of bacterial and fungal pathogens. The review delves into the antivirulent prospects of T-4-ol, shedding light on its ability to attenuate virulence and mitigate bacterial pathogenesis. Scientific literature on the anti-oxidant and anti-inflammatory activity of T-4-ol highlighting its role in neutralizing reactive oxygen species and modulating inflammatory pathways has also been collated. Furthermore, the review elaborates on the cardioprotective and anti-hypertensive properties of T-4-ol and augments literature on its anti-cancer mechanism against various cancer cell lines. The review also provides in-depth knowledge of the pharmaceutical formulations of T-4-ol and recent knowledge about its application in clinical/field trials. The exploration of these diverse attributes positions T-4-ol as a promising candidate for further research and therapeutic repurposing in various biomedical applications., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

Terpinen-4-ol (T-4-O) is an important component of tea tree oil and has anti-inflammatory effects. Currently, there are very few studies on the mechanisms by which T-4-O improves lipopolysaccharide (LPS)-induced macrophage inflammation. In this study, LPS-stimulated mouse RAW264.7 macrophages were used as a model to analyze the effects of T-4-O on macrophage inflammatory factors and related metabolic pathways in an inflammatory environment. The results showed that T-4-O significantly decreased the expression levels of inflammatory cytokines induced by LPS. Cellular metabolism results showed that T-4-O significantly decreased the ratio of the extracellular acidification rate and oxygen consumption rate. Non-targeted metabolomics results showed that T-4-O mainly affected glutamine and glutamate metabolism and glycine, serine, and threonine metabolic pathways. qPCR results showed that T-4-O increased the transcript levels of GLS and GDH and promoted glutamine catabolism. Western blotting results showed that T-4-O inhibited the mTOR and IκB, thereby decreasing NF-κB activity. The overall results showed that T-4-O inhibited mTOR phosphorylation to promote glutamine metabolism and increased cell oxidative phosphorylation levels, thereby inhibiting the expression of LPS-induced inflammatory cytokines.

Serine proteases are a class of hydrolytic enzymes involved in various physiological functions like digestion, coagulation, fibrinolysis and immunity. The present study evaluates the serine protease inhibitory potential of phytochemicals liquiritin and terpinen-4-ol present in the herb Glycyrrhiza glabra L. using trypsin as the model enzyme. In silico studies showed that both the compounds have a significant binding affinity towards trypsin with a binding energy of -26.66 kcal/mol and -19.79 kcal/mol for liquiritin and terpinen-4-ol, respectively. Their binding affinity was confirmed through in vitro enzyme inhibition assays. The mode of inhibition was found to be uncompetitive. In order to explain the mode of inhibition, docking of the ligands to the enzyme-substrate complex was also done and binding energy was calculated after MD simulation. The energy values showed that the binding affinities of these compounds towards the enzyme substrate complex are more than that towards the enzyme alone. This explains the uncompetitive mode of inhibition.Communicated by Ramaswamy H. Sarma.

Manuka oil and tea tree oil are essential oils with known antibacterial properties that are believed to be caused by one main component: terpinen-4-ol. Terpinen-4-ol has potent antibacterial activity against caries-related microorganisms. However, few studies have investigated the antimicrobial effects of terpinen-4-ol on bacteria in apical periodontitis. Thus, the objective of the present study was to evaluate the antibacterial and antibiofilm potential of terpinen-4-ol against Enterococcus faecalis, Porphyromonas gingivalis, Prevotella intermedia, and Fusobacterium nucleatum, which have all been detected in apical periodontitis. The minimum inhibitory and minimum bactericidal concentrations of terpinen-4-ol were determined to assess its activity against biofilms. The minimum inhibitory concentration of terpinen-4-ol was 0.25% against E. faecalis and F. nucleatum, 0.05% against P. gingivalis, and 0.1% against P. intermedia. The minimum bactericidal concentration of terpinen-4-ol was 1.0% against E. faecalis, 0.2% against P. gingivalis and P. intermedia, and 0.5% against F. nucleatum. In the biofilm evaluations, all terpinen-4-ol-treated bacteria had significant reductions in biofilm viability compared with controls in experiments assessing attachment inhibitory activity. Furthermore, structural alterations and decreased bacterial cell clumping were observed under scanning electron microscopy, and significantly decreased cell survival was noted using fluorescence microscopy. Together, these results suggest that terpinen-4-ol is a potential antibacterial agent with bactericidal properties, and can also act on established biofilms., (Copyright © 2023 Japanese Society of Chemotherapy, Japanese Association for Infectious Diseases, and Japanese Society for Infection Prevention and Control. Published by Elsevier Ltd. All rights reserved.)

CYP102A1 from Bacillus megaterium is an important enzyme in biotechnology, because engineered CYP102A1 enzymes can react with diverse substrates and produce human cytochrome P450-like metabolites. Therefore, CYP102A1 can be applied to drug metabolite production. Terpinen-4-ol is a cyclic monoterpene and the primary component of essential tea tree oil. Terpinen-4-ol was known for therapeutic effects, including antibacterial, antifungal, antiviral, and anti-inflammatory. Because terpenes are natural compounds, examining novel terpenes and investigating the therapeutic effects of terpenes represent responses to social demands for eco-friendly compounds. In this study, we investigated the catalytic activity of engineered CYP102A1 on terpinen-4-ol. Among CYP102A1 mutants tested here, the R47L/F81I/F87V/E143G/L188Q/N213S/E267V mutant showed the highest activity to terpinen-4-ol. Two major metabolites of terpinen-4-ol were generated by engineered CYP102A1. Characterization of major metabolites was confirmed by liquid chromatography-mass spectrometry (LC-MS), gas chromatography-MS, and nuclear magnetic resonance spectroscopy (NMR). Based on the LC-MS results, the difference in mass-to-charge ratio of an ion (m/z) between terpinen-4-ol and its major metabolites was 16. One major metabolite was defined as 1,4-dihydroxy- p -menth-2-ene by NMR. Given these results, we speculate that another major metabolite is also a mono-hydroxylated product. Taken together, we suggest that CYP102A1 can be applied to make novel terpene derivatives.

Background: SARS-CoV-2's remarkable capacity for genetic mutation enables it to swiftly adapt to environmental changes, influencing critical attributes, such as antigenicity and transmissibility. Thus, multi-target inhibitors capable of effectively combating various viral mutants concurrently are of great interest., Objective: This study aimed to investigate natural compounds that could unitedly inhibit spike glycoproteins of various Omicron mutants. Implementation of various in silico approaches allows us to scan a library of compounds against a variety of mutants in order to find the ones that would inhibit the viral entry disregard of occurred mutations., Methods: An extensive analysis of relevant literature was conducted to compile a library of chemical compounds sourced from citrus essential oils. Ten homology models representing mutants of the Omicron variant were generated, including the latest 23F clade (EG.5.1), and the compound library was screened against them. Subsequently, employing comprehensive molecular docking and molecular dynamics simulations, we successfully identified promising compounds that exhibited sufficient binding efficacy towards the receptor binding domains (RBDs) of the mutant viral strains. The scoring of ligands was based on their average potency against all models generated herein, in addition to a reference Omicron RBD structure. Furthermore, the toxicity profile of the highest-scoring compounds was predicted., Results: Out of ten built homology models, seven were successfully validated and showed to be reliable for In Silico studies. Three models of clades 22C, 22D, and 22E had major deviations in their secondary structure and needed further refinement. Notably, through a 100 nanosecond molecular dynamics simulation, terpinen-4-ol emerged as a potent inhibitor of the Omicron SARS-CoV-2 RBD from the 21K clade (BA.1); however, it did not show high stability in complexes with other mutants. This suggests the need for the utilization of a larger library of chemical compounds as potential inhibitors., Conclusion: The outcomes of this investigation hold significant potential for the utilization of a homology modeling approach for the prediction of RBD's secondary structure based on its sequence when the 3D structure of a mutated protein is not available. This opens the opportunities for further advancing the drug discovery process, offering novel avenues for the development of multifunctional, non-toxic natural medications., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

The chemical composition, antifungal, antibiofilm, and molecular docking studies of the essential oil obtained from Lindera caesia were investigated. A total of thirty-nine components (96.7%) were identified using gas chromatography (GC-FID) and gas chromatography-mass spectrometry (GC-MS). The major components included terpinen-4-ol (26.3%), neo -intermedeol (23.2%), eudesma-4,11-dien-3-one (10.4%), and o -cymene (5.3%). The antifungal activity was tested against Candida albicans and Streptococcus mutans using the broth microdilution assay, whereas the microbial biofilms were determined using a semi-quantitative static biofilm. The essential oil exhibited activity against C. albicans (MIC 125 µg/mL) and S. mutans (MIC 250 µg/mL), and increased the biofilm of C. albicans by 31.25% when treated with 500 µg/mL. The molecular docking study shows neo -intermedeol, eudesma-4,11-dien-3-one, α-selinene, and β-selinene as the good candidate to target Erg11 with a binding energy of -7.3 kcal/mol. These findings demonstrated that the essential oil may have potential in dental application for caries prevention.

Lung cancer is the second most commonly diagnosed cancer and has the highest mortality rate worldwide despite the remarkable advances in its treatment. Origanum majorana Essential Oil (OMEO) has been shown to be effective against non-small cell lung cancer (NSCLC) cells, decreasing their viability and colony growth in vitro, as well as inhibiting tumor growth in chick embryo chorioallantoic membranes (CAM) and nude mice in vivo. OMEO is mainly composed of four monoterpenes, namely terpinen-4-ol, sabinene hydrate, α-terpinene, and γ -terpinene. In this study, we aimed to investigate the potential anticancer effects of these monoterpenes, either alone or in combination, on NSCLC. Our findings indicate that these four monoterpenes significantly decreased NSCLC cell viability in a concentration-dependent manner, reduced their colony growth in vitro, and also downregulated survivin expression in these cells. Moreover, different combined mixtures of these monoterpenes further enhanced their anticancer effects on cellular viability, with a terpinen-4-ol and sabinene hydrate combination being the most potent. We also found that terpinen-4-ol, in combination with sabinene hydrate, markedly enhanced the anticancer effect of the individual monoterpenes on NSCLC viability within a shorter treatment duration through, at least in part, survivin downregulation. Furthermore, this combination enhanced the inhibition of colony growth in vitro and the tumor growth of NSCLC cells xenografted onto chick embryo CAM in vivo. Altogether, our study highlights the potential of these monoterpenes for use in further pre-clinical investigations against various cancer hallmarks.

Alpinia zerumbet is a plant from the Zingiberaceae family, popularly used for hypertension treatment. Several studies have demonstrated Alpinia zerumbet vasodilator effect on conductance vessels but not on resistance vessels. Thereby, the aim of this study was to verify the vasodilator effect of the essential oil of Alpinia zerumbet (EOAz) on isolated rat resistance arteries and characterize its mechanism of action. Therefore, the effect of EOAz (3 to 3000 μg/mL) was verified in second-order branches of the mesenteric artery (SOBMA) pre-contracted by KCl and U46619. To study the mechanism of action, the influence of several inhibitors (TEA, 4-AP, Glibenclamide, Atropine, L-NAME, ODQ and indomethacin) on the vasodilator effect of EOAz was evaluated. Some protocols were also performed aiming to study the effect of EOAz on Ca 2+ influx and release from intracellular storage. Furthermore, the binding energy of the main constituents with calcium channels were evaluated by molecular docking. Results showed an endothelium-independent vasorelaxant effect of EOAz on SOBMA, and only ODQ and L-NAME produced significant alteration on its pEC 50 . Regarding the calcium assays, contraction reduction caused by incubation with EOAz was observed in all three protocols. Hence, our results suggest that EOAz has a vasodilator effect mediated by inhibition of Ca 2+ influx and release from intracellular storage, as well as an activation of the NOS/sGC pathway., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interest., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Over the past few decades, complementary and alternative treatments have become increasingly popular worldwide. The purported therapeutic characteristics of natural products have come under increased scrutiny both in vitro and in vivo as part of efforts to legitimize their usage. One such product is tea tree oil (TTO), a volatile essential oil primarily obtained from the native Australian plant, Melaleuca alternifolia , which has diverse traditional and industrial applications such as topical preparations for the treatment of skin infections. Its anti-inflammatory-linked immunomodulatory actions have also been reported. This systematic review focuses on the anti-inflammatory effects of TTO and its main components that have shown strong immunomodulatory potential. An extensive literature search was performed electronically for data curation on worldwide accepted scientific databases, such as Web of Science, Google Scholar, PubMed, ScienceDirect, Scopus, and esteemed publishers such as Elsevier, Springer, Frontiers, and Taylor & Francis. Considering that the majority of pharmacological studies were conducted on crude oils only, the extracted data were critically analyzed to gain further insight into the prospects of TTO being used as a neuroprotective agent by drug formulation or dietary supplement. In addition, the active constituents contributing to the activity of TTO have not been well justified, and the core mechanisms need to be unveiled especially for anti-inflammatory and immunomodulatory effects leading to neuroprotection. Therefore, this review attempts to correlate the anti-inflammatory and immunomodulatory activity of TTO with its neuroprotective mechanisms.

According to previous research, turmeric seeds exhibit anti-inflammatory, anti-malignancy, and anti-aging properties due to an abundance of terpinen-4-ol (T4O). Although it is still unclear how T4O works on glioma cells, limited data exist regarding its specific effects. In order to determine whether or not glioma cell lines U251, U87, and LN229 are viable, CCK8 was used as an assay and a colony formation assay was performed using different concentrations of T4O (0, 1, 2, and 4 μM). The effect of T4O on the proliferation of glioma cell line U251 was detected through the subcutaneous implantation of the tumor model. Through high-throughput sequencing, a bioinformatic analysis, and real-time quantitative polymerase chain reactions, we identified the key signaling pathways and targets of T4O. Finally, for the measurement of the cellular ferroptosis levels, we examined the relationship between T4O, ferroptosis, and JUN and the malignant biological properties of glioma cells. T4O significantly inhibited glioma cell growth and colony formation and induced ferroptosis in the glioma cells. T4O inhibited the subcutaneous tumor proliferation of the glioma cells in vivo. T4O suppressed JUN transcription and significantly reduced its expression in the glioma cells. The T4O treatment inhibited GPX4 transcription through JUN. The overexpression of JUN suppressed ferroptosis in the cells rescued through T4O treatment. Taken together, our data suggest that the natural product T4O exerts its anti-cancer effects by inducing JUN/GPX4-dependent ferroptosis and inhibiting cell proliferation, and T4O will hope-fully serve as a prospective compound for glioma treatment.

This study aimed to observe the effect of terpinen-4-ol(T4O) on the proliferation of vascular smooth muscle cells(VSMCs) exposed to high glucose(HG) and reveal the mechanism via the Krüppel-like factor 4(KLF4)/nuclear factor kappaB(NF-κB) signaling pathway. The VSMCs were first incubated with T4O for 2 h and then cultured with HG for 48 h to establish the model of inflammatory injury. The proliferation, cell cycle, and migration rate of VSMCs were examined by MTT method, flow cytometry, and wound healing assay, respectively. The content of inflammatory cytokines including interleukin(IL)-6 and tumor necrosis factor-alpha(TNF-α) in the supernatant of VSMCs was measured by enzyme-linked immunosorbent assay(ELISA). Western blot was employed to determine the protein levels of proliferating cell nuclear antigen(PCNA), Cyclin D1, KLF4, NF-κB p-p65/NF-κB p65, IL-1β, and IL-18. The KLF4 expression in VSMCs was silenced by the siRNA technology, and then the effects of T4O on the cell cycle and protein expression of the HG-induced VSMCs were observed. The results showed that different doses of T4O inhibited the HG-induced proliferation and migration of VSMCs, increased the percentage of cells in G&#95;1 phase, and decreased the percentage of cells in S phase, and down-regulated the protein levels of PCNA and Cyclin D1. In addition, T4O reduced the HG-induced secretion and release of the inflammatory cytokines IL-6 and TNF-α and down-regulated the expression of KLF4, NF-κB p-p65/NF-κB p65, IL-1β, and IL-18. Compared with si-NC+HG, siKLF4+HG increased the percentage of cells in G&#95;1 phase, decreased the percentage of cells in S phase, down-regulated the expression of PCNA, Cyclin D1, and KLF4, and inhibited the activation of NF-κB signaling pathway. Notably, the combination of silencing KLF4 with T4O treatment further promoted the changes in the above indicators. The results indicate that T4O may inhibit the HG-induced proliferation and migration of VSMCs by down-regulating the level of KLF4 and inhibiting the activation of NF-κB signaling pathway.

Cytochrome P450 proteins (CYPs) in insects can encode various detoxification enzymes and catabolize heterologous substances, conferring tolerance to insecticides. This study describes the identification of a P450 gene ( CYP6BQ8 ) from Tribolium castaneum (Herbst) and investigation of its spatiotemporal expression profile and potential role in the detoxification of terpinen-4-ol, a component of plant essential oils. The developmental expression profile showed that TcCYP6BQ8 expression was relatively higher in early- and late-larval stages of T. castaneum compared with other developmental stages. Tissue expression profiles showed that TcCYP6BQ8 was mainly expressed in the head and integument of both larvae and adults. The expression profiling of TcCYP6BQ8 in developmental stages and tissues is closely related to the detoxification of heterologous substances. TcCYP6BQ8 expression was significantly induced after exposure to terpinen-4-ol, and RNA interference against TcCYP6BQ8 increased terpinen-4-ol-induced larval mortality from 47.78 to 66.67%. This indicates that TcCYP6BQ8 may be involved in T. castaneum 's metabolism of terpinen-4-ol. Correlation investigation between the CYP6BQ8 gene and terpinen-4-ol resistance in T. castaneum revealed that the TcCYP6BQ8 gene was one of the factors behind T. castaneum 's resistance to terpinen-4-ol. This discovery may provide a new theoretical foundation for future regulation of T. castaneum .

Cholesterol is an essential lipid that guarantees several biological processes in eukaryotic cells. Its metabolism is regulated by a complex protein network that could be significantly influenced by numerous exogenous sources, such as essential oils (EOs). For instance, it has been speculated that monoterpenoid and sesquiterpenoid compounds contained in lavender essential oil (LEO) may exert important hypocholesterolemic activities. However, the molecular mechanisms by which LEO influences cholesterol homeostasis are not characterized. In this work, we evaluated the ability of LEO to regulate the protein network that controls cholesterol metabolism in the HepG2 cell line. The main findings indicate that LEO administration increases intracellular cholesterol content. Concurrently, LEO affects the expression of proteins involved in cholesterol uptake, biosynthesis, and trafficking. These effects are partially mediated by terpinene-4-ol, one of the most abundant compounds in LEO. These results demonstrate that LEO modulates cholesterol metabolism in hepatic cells.

Purpose: To evaluate the short-term tolerability of five commercially available anti-demodectic eyelid cleansers; OCuSOFT Oust Demodex (OD), I-MED I-Lid'n Lash Plus (ILL+), Labtician BlephaDex (BD), Chrissanthe Eye Cleanse (EC), and Théa Blephademodex (BDdx)., Methods: Thirty healthy non-contact lens wearers (18 female; mean ± SD age, 33 ± 12 years) were enrolled in a prospective randomised crossover study. On separate visits, spaced at least 48 h apart, participants were randomised to receive topical application of one of five eyelid cleansers or saline. Participants rated subjective ocular discomfort during the 10-minute post-application period. Visual acuity, non-invasive tear film stability, conjunctival hyperaemia, and ocular surface staining were assessed at baseline and 10 min., Results: No inter-group differences in ocular parameters were noted at baseline (all p > 0.05). Ocular discomfort scores significantly exceeded baseline scores for 60 s following BD application, 120 s with OD, 135 s with BDdx, 150 s with ILL+, and 195 s with EC (all p < 0.05). Deterioration in non-invasive tear film stability, limbal conjunctival hyperaemia, as well as corneal, conjunctival, and lid margin staining was detected following EC application (all p < 0.05), and increased bulbar conjunctival hyperaemia was observed following both EC and ILL+ treatment (both p < 0.05)., Conclusions: Study outcomes highlight varying tolerability profiles with different anti-demodectic lid cleanser preparations, and the potential to induce tear film instability, conjunctival hyperaemia and ocular surface staining on application. Awareness of possible adverse effects arising from topical application of commercial anti-demodectic lid cleanser formulations may help clinicians set realistic patient expectations and encourage better compliance in their use of lid hygiene therapies., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier Ltd.)

Origanum majorana is a medicinal and aromatic plant that belongs to the Lamiaceae family. It is cultivated in several parts of the world and, due to its splendid aroma and taste, is widely used for culinary purposes and in perfumes. The essential oil of the plant, to which is attributed its aroma, contains many secondary metabolites with valuable biological activity. One of them is the pesticide activity, which has attracted much interest. Given the necessity of replacing synthetic pesticides, essential oils are studied in an attempt to find naturally derived products. Thus, the aim of this review paper is to discuss the chemical profile of O. majorana essential oil and to present data regarding its insecticidal, repellent and fumigant activity. Data were collected from 1992 to 2022. Databases, including PubMed, Google Scholar, ScienceDirect and Scopus, were used for the research, and keywords, including O. majorana , sweet marjoram, essential oil, volatiles, pesticide, insecticide and repellent activity, were used. The results of this review paper indicate that O. majorana essential oil can be an alternative agent to manage pests. However, still, much research should be conducted to evaluate its toxicity against beneficial insects and to ensure its safety for human health.

Antimicrobial resistance is a major public health issue raising growing concern in the face of dwindling response options. It is therefore urgent to find new anti-infective molecules enabling us to fight effectively against ever more numerous bacterial infections caused by ever more antibiotic-resistant bacteria. In this quest for new antibacterials, essential oils (or compounds extracted from essential oils) appear to be a promising therapeutic option. In the present work, we investigate the potential antibacterial synergy between a combination of terpinen-4-ol and α-terpineol (10:1) compared to standard tea tree oil. The minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) were determined. Then, time kill assays, in vitro cytotoxicity and bactericidal activity on latent bacteria (persisters) were investigated. Finally, an in silico study of the pharmacokinetic parameters of α-terpineol was also performed. Altogether, our data demonstrate that the combination of terpinen-4-ol and α-terpineol might be a precious weapon to address ESKAPE pathogens.

Our aim was to verify the modulative TP-4-ol capacity in 4-nitroquinoline-1-oxide induced oral rat cancer. The stereoisomers of TP-4-ol were used against the human tongue squamous cell line and the negative stereoisomer showed lower IC50. Thirty-one Holtzman rats (120-130 g) were cancer-induced by 4-nitroquinoline-1-oxide (4-NQO/8 weeks/25 ppm) and 32 Holtzman rats (120-130 g) were used to healthy and TP-4-ol toxicity experiments. Six groups were used, healthy, 0.1nL/g of TP-4-ol, 8nL/g of TP-4-ol, 4-NQO, 4-NQO + 0.1nL/g of TP-4-ol, and 4-NQO + 8nL/g of TP-4-ol. We performed the toxicity analysis by biochemical and histopathological analysis. The biochemistry analysis includes alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate transaminase (AST), urea, and creatinine and the histopathology analysis includes the liver, kidney, lung, and spleen. Specifically, for malign modulation, we performed a macroscopic and microscopic analysis. The group exposed to 0.1nL/g of TP-4-ol demonstrated a reduced risk of malignancy in dysplasia considering the criteria of architecture and cytology. Similarly, a drop of percentual rats with SCC diagnosis was observed in 4-NQO + 0.1nL/g (41.6%) when compared to 4-NQO (87.5%). Moreover, the 4-NQO group presented a median of 2.62 SCC/rat and the 4-NQO + 0.1nL/g demonstrated a median of 0.75 SCC/rat. For toxicity analysis, 4-NQO + 0.1nL/g showed focal necrosis in the kidney and 4-NQO showed lung hemorrhagic areas. The concentration of 0.1nL/g was more effective in reducing the tongue induction of potentially malignant and malignant lesions by 4-NQO. A kidney toxicity was observed in healthy animals exposed to 0.1nL/g of TP-4-ol. The negative isoform of terpinen-4-ol negatively modulates the development of potentially malignant and malignant lesions in rats (Rattus nonverdicts albinos, Holtzman) exposed to 4-NQO. (-)-Terpinen-4-ol reduced the mice percentual with squamous cell carcinoma, 87.5 to 41.6%, and decreased the cancer/rat ratio of 2.62 in 4-NQO to 0.75 in 4-NQO + 0.1nL/g. This represents 52.4% by group and 71.3% in the cancer/rat ratio., (© 2022. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Numerous plant-based repellents are widely used for personal protection against host-seeking mosquitoes. Vitiveria zizanioides (L.) Nash essential oil and its constituents have demonstrated various mosquito repellent activities. In this study, three chemical actions of vetiver oil and five constituents (terpinen-4-ol, α-terpineol, valencene, vetiverol and vetivone) were characterized against Aedes aegypti, Aedes albopictus and Culex quinquefasciatus by using the high-throughput screening assay system (HITSS). Significant contact escape responses in Ae. aegypti and Ae. albopictus to all test compounds at concentrations between 2.5 and 5% were observed. Spatial repellency responses were also observed in some tested mosquito populations depending upon concentrations. The most significant toxic response on mosquitoes was found at the highest concentration, except for vetivone which had no toxic effect on Ae. aegypti and Ae. albopictus . Results on phototoxic and genotoxic hazard revealed that vetiver oil and their constituents showed no phototoxic potential or any significant genotoxic response. In conclusion, vetiver oil and two constituents, valencene and vetiverol, are potentials as active ingredients for mosquito repellency and present no toxicity., Competing Interests: The authors declare no conflict of interest., (© 2022 The Author(s). Published by Elsevier B.V.)

Background: Harmonia axyridis Pallas (Coleoptera: Coccinellidae) is an important natural enemy of aphids. Plant species and plant health conditions can affect the behavior of H. axyridis. To determine plant effects on this lady beetle, we examined beetle responses to four cover crops: coriander (Coriadrum sativum L., Apiales: Apiaceae), marigold (Tagetes erecta L., Asterales: Asteraceae), sweet alyssum (Lobularia maritima L., Brassicales: Brassicaceae), and alfalfa (Medicago sativa L., Fabales: Fabaceae). Our goal was to better understand this predator's ovipositional behavior in response to different plants and its olfactory response to the aphid-induced volatiles from these plants., Results: We found that this lady beetle did not have any significant oviposition preference among the four plant species, but H. axyridis preferred to lay eggs on the lower surface of leaves, regardless of the plant species. H. axyridis females had a significant preference for aphid-infested marigolds, but were not attracted by any of the other three cover plants or marigolds without aphid damage. Compared to the uninfested marigold plants, the emission of 12 compounds significantly increased on the aphid-infested marigolds, and two of them were attractive to H. axyridis under suitable concentrations., Conclusion: H. axyridis did not show any significant oviposition preference among the four cover crops. Aphid-infested marigolds can attract H. axyridis. Indole and terpinen-4-ol mediated lady beetle attraction. These synomones have potential for manipulating populations of H. axyridis as a component of conservation biological control. © 2022 Society of Chemical Industry., (© 2022 Society of Chemical Industry.)

Legionella pneumophila ( Lp ), responsible for a severe pneumonia called Legionnaires' disease, represents an important health burden in Europe. Prevention and control of Lp contamination in warm water systems is still a great challenge often due to the failure in disinfection procedures. The aim of this study was to evaluate the in vitro activity of Terpinen-4-ol (T-4-ol) as potential agent for Lp control, in comparison with the essential oil of Melaleuca alternifolia (tea tree) (TTO. Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of T-4-ol were determined by broth micro-dilution and a micro-atmosphere diffusion method to investigate the anti- Lp effects of T-4-ol and TTO vapors. Scanning Electron Microscopy (SEM) was adopted to highlight the morphological changes and Lp damage following T-4-ol and TTO treatments. The greatest antimicrobial activity against Lp was shown by T-4-ol with a MIC range of 0.06-0.125% v / v and MBC range of 0.25-0.5% v / v . The TTO and T-4-ol MIC and MBC decreased with increasing temperature (36 °C to 45 ± 1 °C), and temperature also significantly influenced the efficacy of TTO and T-4-ol vapors. The time-killing assay showed an exponential trend of T-4-ol bactericidal activity at 0.5% v / v against Lp . SEM observations revealed a concentration- and temperature- dependent effect of T-4-ol and TTO on cell surface morphology with alterations. These findings suggest that T-4-ol is active against Lp and further studies may address the potential effectiveness of T-4-ol for control of water systems.

Tribolium castaneum is an agricultural and stored pest found throughout the world. The cytochrome P450 genes of T. castaneum can encode various detoxification enzymes and catabolize heterologous substances, conferring tolerance to insecticides. Herein, we describe the identification of a P450 gene (CYP9Z6) from T. castaneum and investigated its expression profile and potential role in the detoxification of terpinen-4-ol. TcCYP9Z6 expression was significantly induced after exposure to terpinen-4-ol, and RNA-mediated silencing of TcCYP9Z6 increased terpinen-4-ol-induced larval mortality from 47.75% to 63.92%, showing that TcCYP9Z6 is closely related to the detoxification of terpinen-4-ol. The developmental expression profile revealed that TcCYP9Z6 was mainly expressed in late adults and late larvae. Tissue expression profiling revealed that the highest TcCYP9Z6 expression occurred in the head, in both the adult and the larval tissues, followed by the gut in larvae and the antennae in adults. These developmental stages and tissues with high TcCYP9Z6 expression are closely related to the detoxification of heterologous substances. These results indicated that TcCYP9Z6 may play a pivotal role in the detoxification of terpinen-4-ol, which provides support for using TcCYP9Z6 a potential gene for the RNAi-mediated prevention and control of T. castaneum., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Terpinen-4-ol (T4O), a compound isolated from the seeds of turmeric, has exhibited anti-malignancy, anti-aging, and anti-inflammatory properties in previous studies. However, the specific effects and molecular mechanisms of T4O on pancreatic cancer (PC) cells remain largely unknown. In this study, we demonstrated that T4O markedly suppressed PC cell proliferation and colony formation in vitro and induced apoptosis. Similarly, T4O significantly inhibited the migration and invasion of PC cells in vitro . Through RNA sequencing, 858 differentially expressed genes (DEGs) were identified, which were enriched in the Rhodopsin (RHO)/ Ras homolog family member A (RHOA) signaling pathway. Rho-associated coiled-coil containing protein kinase 2 (ROCK2), a DEG enriched in the RHO/RHOA signaling pathway, was considered as a key target of T4O in PC cells; it was significantly reduced after T4O treatment, highly expressed in PC tissues, and negatively associated with patient outcome. Overexpression of ROCK2 significantly reduced the inhibitory effects of T4O on PC cell proliferation and mobility. Moreover, T4O inhibited cell proliferation in vivo and decreased the Ki-67, cell nuclear antigen, EMT markers, and ROCK2 expression. In conclusion, we consider that T4O can suppress the malignant biological behavior of PC by reducing the expression of ROCK2, thus contributing to PC therapy.

Long noncoding RNAs (lncRNAs) are important regulatory factors in multiple biological processes, and several lncRNAs are known to respond to insecticides. However, the lncRNA functions that are associated with terpinen-4-ol resistance in the red flour beetle ( Tribolium castaneum ) have not yet been identified. In this study, we determined the differentially transcribed lncRNAs between fumigated and control experimental groups. In the six libraries that underwent RNA sequencing, 34,546 transcripts were identified, including 8267 novel lncRNAs, 4155 novel mRNAs, 1151 known lncRNAs, and 20,973 known mRNAs. Among these, we found that the expression of 1858 mRNAs and 1663 lncRNAs was significantly different in the fumigated group compared with the control group. Among the differentially transcribed lncRNAs, 453 were up-regulated and 1210 were down-regulated lncRNAs. In addition, we identified the regulatory function targets of the lncRNAs. Functionally, all lncRNAs and target genes associated with terpinen-4-ol metabolism were enriched in several metabolic pathways, like the ATP-binding cassette transporter, pentose interconversion, and glucuronate interconversion. To the best of our knowledge, this study represents the first global identification of lncRNAs and their potential association with terpinen-4-ol metabolism in the red flour beetle. These results will provide reference information for studies on the resistance to terpinen-4-ol and other essential oil compounds and chemical pesticides, as well as an understanding of other biological processes in T. castaneum .

Purpose/aim of the Study: To evaluate the improvement of ocular signs and symptoms in patients suffering from Demodex blepharitis using a combined treatment approach: use of eyelid wipes impregnated with 2.5% terpinen-4-ol (T4O) and 0.2% hyaluronic acid (HA) in the initial treatment period and investigation of maintenance of the treatment effect with the use of eyelid cleansing wipes., Materials and Methods: Fifty patients with Demodex blepharitis were treated in the initial treatment period with sterile eyelid T4O impregnated wipes for 28 days. In the following four-week maintenance period, 82% patients received sterile eyelid maintenance wipes, while 16% continued treatment with T4O impregnated wipes. Global ocular discomfort, adapted TOSS, SANDE score, and individual blepharitis symptoms were assessed by patients at day 28 and day 56. Ocular signs were evaluated by the investigator at the study visits. Investigator's assessment of the overall treatment performance, patient's assessment of treatment satisfaction, and tolerability were evaluated with questionnaires., Results: All global ocular discomfort symptoms and disease specific symptoms assessed by patients as well as all parameters evaluated by the investigators significantly improved in the initial treatment period with the application of eyelid wipes impregnated with 2.5% terpinen-4-ol until day 28. The therapeutic effect was maintained or even improved during the maintenance period under administration of mainly eyelid maintenance wipes until day 56. Both products were well tolerated. No adverse events and no clinically relevant changes in visual acuity were observed during both periods., Conclusions: Once daily treatment with T4O impregnated eyelid wipes in the initial treatment period significantly improved the ocular symptoms and signs and reduced the mite count in patients with Demodex blepharitis within four-weeks administration. Subsequent maintenance treatment with maintenance wipes for another 4 weeks preserved or further intensified the treatment success. The products were well tolerated and were convenient to use.

Purpose: To investigate in vitro effects of active ingredients in eye cleansing wipes and solutions on the life span of Demodex., Methods: Demodex mites were extracted by eyelash epilation method from volunteers who were found to have chronic demodex blepharitis during an ophthalmological examination. Preparations with three or more motile, live adult Demodex parasites were included in the study. Two drops of the test solution were dropped on the samples: saline, 2% tea tree oil (TTO; Osto®, AKD Medical products, Turkey), 7.5% TTO (Blefaritto®, Jeomed, Turkey), and 0.5% terpinen-4-ol (T4O; Blefastop plus®, NTC, Italy). Under light microscopy at 100× and 400× magnification, samples were examined and followed. The average killing time of Demodex was noted for each preparation., Results: The study was analyzed on 120 patient samples. In the control group with saline, the average killing time of Demodex had an average of 323.5 ± 21.1 min. In other groups, an average mean of 95.9 ± 25.2 min in the 2% TTO group, 67.1 ± 21.8 min in the 7.5% TTO group, and 27.3 ± 6.0 min in the 0.5% T4O group was observed. It was found that the 0.5% T4O group was significantly more effective than the other groups., Conclusion: Chronic Demodex blepharitis treatment is quite difficult in ophthalmology practice. It appears that, even at very low concentrations, T4O has an effective anti-Demodex effect.

Background: This study investigated the effects of terpinen-4-ol on methicillin-resistant Staphylococcus aureus (MRSA) and its biofilm, and the possible mechanisms governing this effect., Results: We observed that terpinen-4-ol has good antibacterial activity and inhibits the formation of MRSA biofilm. The MIC and MBC values for terpinen-4-ol against S. aureus were 0.08% ~ 0.32%. And terpinen-4-ol at 0.32% could kill all bacteria and clear all biofilms. Untargeted metabolomic and transcriptomic analyses showed that terpinen-4-ol strongly inhibited DNA and RNA biosynthesis in MRSA at 2 h after treatment by affecting genes and metabolites related to purine and pyrimidine metabolic pathways. Some differential genes which play important roles in DNA synthesis and the production of eDNA from biofilm exposed to terpinen-4-ol was also significantly decreased compared with that of the control., Conclusions: Terpinen-4-ol has good antibacterial activity and significantly inhibits the formation of MRSA biofilm by inhibiting purine and pyrimidine metabolism., (© 2021. The Author(s).)

Terpinen-4-ol, the main component of tea tree oil, markedly increases the disease resistance of postharvest strawberry fruit. To understand the mechanism underlying the enhancement of disease resistance, a high-throughput RNA-seq was used to analyze gene transcription in terpinen-4-ol-treated and untreated fruit. The results show that terpinen-4-ol induces the expression of genes in the jasmonic acid (JA) biosynthesis pathway, secondary metabolic pathways such as phenylpropanoid biosynthesis, and pathways involved in plant-pathogen interactions. Terpinen-4-ol treatment reduced disease incidence and lesion diameter in strawberry fruit inoculated with Botrytis cinerea . Terpinen-4-ol treatment enhanced the expression of genes involved in JA synthesis ( FaLOX , FaAOC , and FaOPR3 ) and signaling ( FaCOI1 ), as well as genes related to disease defense ( FaPAL , FaCHI , and FaGLU ). In contrast, treatment with the JA biosynthesis inhibitor salicylhydroxamic acid (SHAM) accelerated disease development and inhibited the induction of gene expressions by terpinen-4-ol. We conclude that the JA pathway participates in the induction of disease resistance by terpinen-4-ol in strawberry fruit. More generally, the results illuminate the mechanisms by which disease resistance is enhanced by essential oils.

Endoplasmic reticulum (ER) stress-mediated phenotypic switching of vascular smooth muscle cells (VSMCs) is key to vascular calcification (VC) in patients with chronic kidney disease (CKD). Studies have shown that activation/upregulation of SIRT1 has a protective effect on CKD-VC. Meanwhile, although terpinen-4-ol has been shown to exert a protective effect against cardiovascular disease, its role and underlying mechanism in VC remain unclear. Herein, we explored whether terpinen-4-ol alleviates ER stress-mediated VC through sirtuin 1 (SIRT1) and elucidated its mechanism to provide evidence for its application in the clinical prevention and treatment of VC. To this end, a CKD-related VC animal model and β-glycerophosphate (β-GP)-induced VSMC calcification model were established to investigate the role of terpinen-4-ol in ER stress-induced VC, in vitro and in vivo. Additionally, to evaluate the involvement of SIRT1, mouse and VSMC Sirt1-knockdown models were established. Results show that terpinen-4-ol inhibits calcium deposition, phenotypic switching, and ER stress in VSMCs in vitro and in vivo. Furthermore, pre-incubation of VSMCs with terpinen-4-ol or a SIRT1 agonist, decreased β-GP-induced calcium salt deposition, increased SIRT1 protein level, and inhibited PERK-eIF2α-ATF4 pathway activation, thus, alleviating VC. Similar results were observed in VSMCs induced to overexpress SIRT1 via lentivirus transcription. Meanwhile, the opposite results were obtained in SIRT1-knockdown models. Further, results suggest that SIRT1 physically interacts with, and deacetylates PERK. Specifically, mass spectrometry analysis identified lysine K889 as the acetylation site of SIRT1, which regulates PERK. Finally, inhibition of SIRT1 reduced the effect of terpinen-4-ol on the deacetylation of PERK in vitro and in vivo and weakened the inhibitory effect of terpinen-4-ol against ER stress-mediated VC. Cumulatively, terpinen-4-ol was found to inhibit post-translational modification of PERK at the K889 acetylation site by upregulating SIRT1 expression, thereby ameliorating VC by regulating ER stress. This study provides insights into the underlying molecular mechanism of terpinen-4-ol, supporting its development as a promising therapeutic agent for CKD-VC., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Purpose: To report a successful treatment of chronic rosacea associated ocular demodicosis with lid scrub containing terpinen-4-ol (T4O)., Observations: A 72-year old woman presented with recurrent and refractory ocular erythema, irritation, dryness, and photophobia despite conventional medical treatment (artificial tears, hypochlorous acid lid hygiene, doxycycline, and erythromycin) for 5 years. Examination revealed facial erythema, telangiectasias on cheeks, nose and lids, and cylindrical dandruff (CD) on bilateral upper and lower lashes. Epilation sampling confirmed demodicosis. After treatment with lid wipe containing T4O (Cliradex, Biotissue, Miami, FL) over face and lids, ocular discomfort, CD, facial and eyelid erythema, telangiectatic vessels were significantly reduced. Complete eradication of demodex mites and resolution of symptoms and signs lasted 8 months of follow-up., Conclusions: This case suggests that T4O is effective in treating chronic rosacea associated ocular demodex blepharitis., Competing Interests: Dr. Scheffer Tseng is the founder and a shareholder of Tissue Tech Inc. that holds patents on use of tea tree oil for the treatment of demodicosis. Sean Tighe is an employee of TissueTech. The following authors have no financial disclosures: (H.Y, A.C.), (© 2021 Published by Elsevier Inc.)

Juniperus excelsa M. Bieb and J. sabina L. contain essential oil (EO), while J. sabina also contains podophyllotoxin, which is used as a precursor for anti-cancer drugs. Two studies were conducted. The first assessed the variability in the EO profile and podophyllotoxin concentration of the two junipers, depending on the location and tree gender. The main EO constituents of J. excelsa were α-cedrol, α-limonene and α-pinene, while the constituents in J. sabina were sabinene, terpinen-4-ol, myrtenyl acetate and α-cadinol. The podophyllotoxin yield of 18 J. sabina accessions was 0.07-0.32% ( w/w ), but this was not found in any of the J. excelsa accessions. The second study assessed the effect of hydrodistillation (Clevenger apparatus) and steam distillation (in a semi-commercial apparatus) on the EO profile and bioactivity. The extraction type did not significantly alter the EO composition. The EO profiles of the two junipers and their accessions were different and may be of interest to the industry utilizing juniper leaf EO. Breeding and selection programs could be developed with the two junipers (protected species) in order to identify chemotypes with (1) a high EO content and desirable composition, and (2) a high concentration of podophyllotoxin in J. sabina . Such chemotypes could be established as agricultural crops for the commercial production of podophyllotoxin and EO.

Purpose: To describe the effectiveness of cyclic treatment with terpinen-4-ol, a tea tree oil component, on Demodex blepharitis., Methods: The presence of Demodex was determined by eyelash rotation as proposed by Mastrota. Schirmer test, tear breakup time, ocular surface disease index, lid margin score, meibomian gland expressibility score, and Oxford grade were performed. Patients were advised to apply heat followed by terpinen-4-ol soaked wipes to eyelids twice a day for 2 weeks. At the end of 2 weeks, treatment was interrupted for 7-10 days. The same treatment was repeated once again. The patients were examined after the first and second cycle of treatment and after 1 year., Results: There was a statistically significant improvement in Schirmer test (10.37 ± 4.73 and 13.13 ± 3.44 mm/5 min), tear breakup time (6.47 ± 3.31 and 7.6 ± 2.89 s), ocular surface disease index (47.94 ± 19.77 and 34.28 ± 13.40), lid margin score (3.2 ± 0.7 and 2.63 ± 0.71), meibomian gland expressibility scores (1.93 ± 0.64 and 1.4 ± 0.67), and Oxford grade (0.9 ± 0.8 and 0.5 ± 0.63) after the first cycle of treatment ( p  < 0.05). The improvement in symptoms and tear function tests of the patients after the second cycle was significantly better than in pre-treatment levels. At 12-month follow-up, symptoms of two patients (93%) relapsed., Conclusion: The administration of terpinen-4-ol to the eyelid margins in a cyclic manner in Demodex blepharitis is effective against adult and hatching Demodex and breaks the vicious cycle.

Various treatments are found to be moderately effective in managing Demodex-related diseases except tea tree oil (TTO) and terpinen-4-ol (T4O), which showed superior miticidal and anti-inflammatory effects in numerous clinical studies. Their possible effects include lowering mite counts, relieving Demodex-related symptoms, and modulating the immune system. This review summarizes the current clinical topical and oral treatments in human demodicosis, their possible mechanisms of action, side-effects and resistance in treating this condition. TTO (especially T4O) is found to be the most effective followed by metronidazole, ivermectin and permethrin in managing the disease. This is because TTO has anti-parasitic, anti-bacterial, anti-fungal, anti-inflammatory and wound-healing effects. Furthermore, nanoTTO can even release its contents into fungus and Pseudomonas biofilms. Combinations of different treatments are occasionally needed for refractory cases, especially for individuals with underlying genetic predisposal or are immuno-compromised. Although the current treatments show efficacy in controlling the Demodex mite population and the related symptoms, further research needs to be focused on the efficacy and drug delivery technology in order to develop alternative treatments with better side-effects profiles, less toxicity, lower risk of resistance and are more cost-effective.

The aim of this study was to analyze the 4-carvomenthenol (carvo) oral treatment on the experimental model of the combined allergic rhinitis and asthma syndrome (CARAS). BALB/c mice were OVA-sensitized on day zero and 7th (50 μg/mL OVA in 10 mg/mL Al (OH)3) and OVA-challenged (5 mg/mL, 20 μL/animal) for three weeks. In the last week, the animals were dally challenged with aerosol of OVA and the carvo treatment (12.5, 25 or 50 mg/kg) occurred one hour before each OVA-challenge. Data were analyzed and p < 0.05 was considered significant. Carvo (12.5-50 mg/kg) decreased significantly the eosinophil migration into the nasal (NALF) and bronchoalveolar (BALF) cavities as well as on the nasal and lung tissues of sick animals. The treatment also decreased mucus production on both tissue sections stained with PAS (periodic acid-Schiff satin). In addition, the histological analyzes demonstrated that sick mice presented hyperplasia and hypertrophy of the lung smooth muscle layer followed by increasing of extracellular matrix and carvo (50 mg/kg) inhibited these asthmatic parameters. We analyzed the allergic rhinitis signals as nasal frictions and sneezing and observed that carvo decreased these two signals as well as serum OVA-specific IgE titer, type 2 cytokine synthesis, mainly IL-13, with increasing of IL-10 production. Decreasing of IL-13 production corroborated with decreasing of mucus production and these effects were dependent on p38MAPK/NF-κB(p65) signaling pathway inhibition. Therefore, these data demonstrated that a monoterpene of essential oils presents anti-allergic property on an experimental model of CARAS suggesting a new drug prototype to treat this allergic syndrome., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Staphylococcus aureus is able to rapidly develop mechanisms of resistance to various drugs and to form strong biofilms, which makes it necessary to develop new antibacterial drugs. The essential oil of Melaleuca alternifolia is used as an antibacterial, a property believed to be mainly due to the presence of terpinen-4-ol. Based on this, the objective of this study was to evaluate the antibacterial and antibiofilm potential of terpinen-4-ol against S. aureus . The Minimal Inhibitory and Minimal Bactericidal Concentrations (MIC and MBC) of terpinen-4-ol were determined, and the effect of its combination with antibacterial drugs as well as its activity against S. aureus biofilms were evaluated. In addition, an in silico analysis of its pharmacokinetic parameters and a molecular docking analysis were performed. Terpinen-4-ol presented a MIC of 0.25% ( v/v ) and an MBC of 0.5% ( v/v ) (bactericidal action); its association with antibacterials was also effective. Terpinen-4-ol has good antibiofilm activity, and the in silico results indicated adequate absorption and distribution of the molecule in vivo. Molecular docking indicated that penicillin-binding protein 2a is a possible target of terpinen-4-ol in S. aureus . This work highlights the good potential of terpinen-4-ol as an antibacterial product and provides support for future pharmacological studies of this molecule, aiming at its therapeutic application.

This study aimed to reveal the effects and possible mechanism of terpinen-4-ol, the main component of tea tree oil (TTO), on the disease resistance of strawberry fruit. When the effects of TTO and its components were compared on the decay development in fruit inoculated with Botrytis cinerea after treatment, strawberry treated with terpinen-4-ol showed the lowest disease incidence (44.4%) after 48 h and also the smallest lesion diameter during the whole storage. This indicates that terpinen-4-ol induces the highest disease resistance in strawberry compared with TTO and other components. Untargeted metabolomic analysis showed that terpinen-4-ol treatment strongly activated phenylpropanoid biosynthesis and flavonoid metabolism pathway by increasing the accumulation of cinnamaldehyde, coniferyl aldehyde, naringenin, taxifolin, quercetin, and quercitrin in fruit at 12 h after treatment. In addition, terpinen-4-ol treatment also caused the accumulation of total phenolics and lignin by enhancing activities and relative gene expression of key enzymes in the phenylpropanoid metabolism pathway. These results suggest that terpinen-4-ol, as the key component of TTO, is the most important contributor to the effectiveness of TTO in improving disease resistance of strawberry fruit through activating the phenylpropanoid metabolism pathway.

The Mediterranean fruit fly, Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) poses a major threat to fruit and vegetable production in the United States and throughout the world. New attractants and detection methods could improve control strategies for this invasive pest. In this study, we developed a method that combined thin-layer chromatography (TLC) of tea tree essential oil (TTO) ( Melaleuca alternifolia ) with short-range bioassays to isolate attractive kairomones for male C. capitata . After development, the TLC chromatogram indicated that TTO separated into five major spots, designated as zones 1 to 5. When the TLC plate was exposed to flies, zones 1 and 3 were strongly attractive to male C. capitata . To confirm activity, the developed TLC plate was cut into five zones which were then tested in short-range bioassays. Again, flies were observed to aggregate around zones 1 and 3, which corresponded with R f values of 0.93 and 0.59. In addition, zones 1 to 5 were separated using preparative-TLC, and olfactory responses to volatile emissions from the five fractions were quantified by electroantennography (EAG). Highest amplitude EAG responses were recorded with fractions 1 and 3, further supporting the bioactivity of these samples. In conclusion, a TLC-based bioassay system can provide an effective, rapid screening protocol for initial isolation of insect kairomones from complex mixtures such as essential oils or plant extracts. Further analysis of TTO fractions 1 and 3 is needed to identify the specific constituents attractive to male C. capitata .

Terpinen-4-ol has high fumigating activity to stored-grain pests including Tribolium confusum. To understand the detoxification of terpinen-4-ol in insects, proteomic analysis was performed to identify related proteins and pathways in response to terpinen-4-ol fumigation in T. confusum. By using isobaric tags for relative and absolute quantitation (iTRAQ)-based strategy, 4,618 proteins were obtained from T. confusum adults in the present study. Comparative proteomic analysis showed that 148 proteins were upregulated and 137 proteins were downregulated in beetles under the LC 50 of terpinen-4-ol treatment for 24 hr. According to functional classifications, differentially expressed proteins (DEPs) were enriched in xenobiotic metabolism pathways. In the detoxification pathway, the levels of 25 cytochrome P450s, 5 glutathione S-transferases, and 2 uridine diphosphate (UDP)-glucuronosyltransferases were changed, most of which were upregulated in T. confusum exposed to terpinen-4-ol. The results indicated that terpinen-4-ol was potentially metabolized and detoxified by enzymes like P450s in T. confusum., (© 2019 Wiley Periodicals, Inc.)

Tea tree oil (TTO) and its two characteristic components (terpinen-4-ol and 1,8-cineole) have been shown to inhibit Botrytis cinerea growth. In this study, we conducted a transcriptome analysis to determine the effects of TTO and its characteristic components, alone and in combination, against B. cinerea. Most differentially expressed genes (DEGs) from B. cinerea cells treated with terpinen-4-ol participated in the biosynthesis of secondary metabolites, and the metabolism of amino acids, carbohydrates, and lipids. All treatments containing terpinen-4-ol potentially induced mitochondrial dysfunction and oxidative stress. These were further confirmed by the decreased activities of several enzymes (e.g., succinate dehydrogenase (SDH), malate dehydrogenase (MDH), α-ketoglutarate dehydrogenase (α-KGDH), isocitrate dehydrogenase (ICDH)), the increased activities of certain enzymes (e.g., catalase (CAT), peroxidase (POD), superoxide dismutase (SOD)), and increased content of hydrogen peroxide (H 2 O 2 ). 1,8-Cineole mainly affected DEGs involved in genetic information processing, resulting in cell death. This study provides insight into the molecular mechanism of B. cinerea inhibition by TTO, and explains the synergistic effect of terpinen-4-ol and 1,8-cineole on B. cinerea.

The monoterpenoid terpinen-4-ol (4TERP) is known to inhibit cell excitability, has low toxicity and important pharmacological activities, which are likely related to neural excitability, such as anti-inflammatory, antiepileptic and antinociceptive effects. However, the pharmacological characteristics and mechanisms underlying the effects of 4TERP on blockade of neural action potential are not completely elucidated. Since Na + current (I Na ) through voltage-dependent Na + channels (Na V ) is a major mechanism for excitability, the present study investigated the pharmacological characteristics and mechanisms of the action of 4TERP on I Na through Na V . For this aim, dissociated small neurons of dorsal root ganglia of adult rats were used for whole cell patch-clamp recordings. 4TERP concentration-dependently inhibits I Na (IC 50 0.8 ± 0.3 mM; pharmacological efficacy 42.89 ± 5.54%). 4TERP interfered with I Na through a mechanism with various components, which includes predominantly channel pore block and sensitivity to frequency of use. In presence of 4TERP (3 mM), decreasing stimulation from 5 Hz to very low frequency (75 s of quiescence previously to stimulation) induced I Na decrease to 65.17 ± 5.86% of control. 4TERP also altered (left shift) voltage sensitivity of the steady state activation of Na V . Data are discussed aiming to interpret the importance of blockade of I Na through Na V as participant of 4TERP-induced inhibition of membrane excitability., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Terpinen-4-ol showed highly insecticidal activity to stored-grain pest Sitophilus zeamais, and cytochrome P450s were strongly induced in response to terpinen-4-ol fumigation. Understanding of the function of P450 enzyme system in the susceptibility to terpinen-4-ol in S. zeamais will benefit the potential application of terpinen-4-ol in controlling stored-grain pests. In the present study, the synergist piperonyl butoxide increased the toxicity of terpinen-4-ol to S. zeamais, with a synergism ratio of 3.5-fold. Two isoforms of NADPH-cytochrome P450 reductase (SzCPR) were identified, with the difference at the N-terminal. SzCPR contained an N-terminal membrane anchor, FMN, FAD, and NADP binding domains. Expression levels of SzCPR were upregulated by tea tree oil (TTO) and its main constituent terpinen-4-ol under different concentrations and time periods. RNAi was generated for S. zeamais by feeding adults dsRNA and the knockdown of SzCPR increased the susceptibility of S. zeamais to terpinen-4-ol, with higher mortality of adults than control under terpinen-4-ol fumigation. Further RNAi analysis showed that P450 gene CYP6MS1 mediated the susceptibility of S. zeamais to terpinen-4-ol. These results revealed that cytochrome P450 enzyme system, especially CYP6MS1 participated in the susceptibility of S. zeamais to terpinen-4-ol. The findings provided a foundation to clarify the metabolic mechanisms of terpinen-4-ol in stored-grain pests., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Aim: To investigate the effects of Terpinen-4-ol on quorum sensing (QS)-regulated biofilm formation and virulence factors production in Pseudomonas aeruginosa . Materials & methods: QS inhibition, molecular docking analysis and gene expression studies were performed to check attenuation effect of Terpinen-4-ol on virulence of P. aeruginosa . Production of various virulence factors and biofilm formation were studied at sub-MIC of Terpinen-4-ol alone and in combination with ciprofloxacin. Results: Terpinen-4-ol at sub-MIC exhibited QS inhibition and downregulated all key QS genes. Molecular docking analysis showed high binding affinities of Terpinen-4-ol with QS receptors. Terpinen-4-ol exhibited synergistic interaction with ciprofloxacin and further reduced production of all the virulence factors and biofilms formation. Conclusion: Terpinen-4-ol could be developed into antivirulence drug after its in vivo evaluation for treatment strategies.

Aim: This study investigated the effect of terpinen-4-ol against Sporothrix schenckii complex and its interactions with antifungals. Materials & methods: The antifungal activity of terpinen-4-ol was evaluated by broth microdilution. The potential effect on cellular ergosterol concentration was evaluated by spectrophotometry. The antibiofilm activity was evaluated by violet crystal staining and XTT reduction assay. The potential pharmacological interactions with antifungals were evaluated by the checkerboard assay. Results: terpinen-4-ol (T-OH) showed minimal inhibitory concentrations ranging from 4 to 32 mg/l decreasing cellular ergosterol content and presented a SMIC ranging from 64 to 1024 mg/l for Sporothrix spp. The combinations of T-OH with itraconazole or terbinafine were synergistic. Conclusion: T-OH has antifungal activity against Sporothrix spp. and acts synergistically with standard antifungals.

Purpose: Demodex folliculorum is a ubiquitous mite that can infest the eyelash follicles. Two commercial lid hygiene products have asserted their effectiveness in killing Demodex mites, yet there has never been a comparative trial between these two products. This study evaluated the demodicidal activity of 0.01% hypochlorous acid (HOCl) solution (Avenova; NovaBay Pharmaceuticals; Emeryville, CA) and 4% terpinen-4-ol (T4O) solution (Cliradex, Bio-Tissue; Miami, FL) in comparison to mineral oil (MO), a negative control., Methods: Live Demodex mites were obtained from volunteers. Samples were immersed in 1-2 drops of test solution: 0.01% HOCl, 4% T4O, or 100% MO. Samples were observed under the microscope every 10 mins for up to 90 mins. Kill time was defined as the elapsed time between the addition of test solution and all cessation of movement of the body, legs, mouth and pedipalps for a minimum of 60 seconds., Results: T4O demonstrated a mean kill time of 40±0.0 mins. HOCl had a mean kill time of 87.86±4.23 mins, with 79% of samples surviving the full 90 mins. In the MO group, all samples survived through the 90 min mark. Kill time was statistically significant in favor of T4O as compared to HOCl ( p =0.0005). There was no statistically significant difference in kill time between HOCl and MO ( p =0.25)., Conclusion: 4% T4O effectively killed all adult mite samples within 40 mins of exposure. In contrast, the demodicidal activity of 0.01% HOCl was minimal, and comparatively similar to 100% MO., Competing Interests: Dr Alan G Kabat is a consultant for Bio-Tissue, Inc. Dr Alan G Kabat reports non-financial support from TissueTech, Inc., during the conduct of the study; personal fees from Avellino Labs, Bio-Tissue, Inc., Bruder Healthcare Company, Inc., Lacrivera, OCuSOFT, Inc., Shire, Sight Sciences, Sun Pharmaceuticals, Vmax Vision, Kala Pharmaceuticals, Inc., National Vision Administrators, LLC, Quidel, TearScience and EyeGate Pharma, outside the submitted work. The author reports no other conflicts of interest in this work.

Purpose: This study investigated the anti-Candida activity and the Shore A hardness of a tissue conditioner (Softone™) modified by incorporation of terpinen-4-ol and cinnamaldehyde., Material and Methods: Agar diffusion, microdilution, and mechanism of action methods were performed to determine to evaluate the antifungal activity of phytoconstituents. Then, phytoconstituents in varying concentrations were incorporated into the tissue conditioner. The anti-Candida effect of the modified conditioner was evaluated through agar punch well and biofilm formation methods. Shore A hardness of the experimental liners was evaluated after baseline, 24 h, 48 h, 4 days, and 7 days immersion on artificial saliva., Results: The phytoconstituents incorporated into Softone showed completely inhibited fungal growth in concentrations of 20-40% and did not present significant antifungal activity until their concentrations where higher than 5%. There were differences between non-modified Softone and M5, M10, C10, and T10% (p < 0.05). The groups containing 10-40% of cinnamaldehyde incorporated into Softone were able to completely inhibit the biofilm. Concentrations below 40% of terpinen-4-ol showed unsatisfactory biofilm inhibition. The T40% and C40% groups presented the lowest Shore A hardness values. Hardness values from groups T40% at 7 days (p = 0.476); C40% at 4 days (p = 0.058); and T20% (p = 0.058), C20% (p = 0.205), T30% (p = 0.154), and C30% (p = 0.874) after 48 h did not differ from the control group., Conclusions: Cinnamaldehyde incorporated into Softone inhibited Candida biofilm formation at concentrations of 10-40%, being more effective than terpinen-4-ol modification despite of halo inhibition observed by both products., Clinical Relevance: All modifications showed a very similar pattern of hardness being useful for clinical practice.

Purpose: To evaluate the clinical improvement in ocular symptoms and signs in patients suffering from Demodex anterior blepharitis after using a novel cleansing wipe impregnated with 2.5% terpinen-4-ol and 0.2% hyaluronic acid. Study design: This was an exploratory, multicenter, open, randomized, two-parallel group comparative study. Methods: Forty-eight patients with Demodex anterior blepharitis were randomly assigned to apply the sterile wipe T1172 (Blephademodex ® ), either once daily (n=24, Group 1) or twice daily (n=24, Group 2) for 29 days. Overall ocular discomfort and other individual symptoms were measured using a 0-10 numeric rating scale at Day 8 and Day 29. Ocular signs, including eyelid margin hyperemia and number of cylindrical dandruff, were examined at slit lamp. Overall treatment performance by investigator, patient satisfaction and tolerance were evaluated with questionnaires. Results: Overall ocular discomfort was significantly reduced from baseline ( p <0.0001) in both groups at Day 8 (-3.6±0.3 in Group 1 and -4.0±0.4 in Group 2) and Day 29 (-5.7±0.4 and -6.8±0.7, respectively), with no difference between groups (D8: p =0.718; D29: p =0.505). Each ocular symptom associated with Demodex blepharitis was improved in both groups. Eyelid margin hyperemia was significantly reduced at Day 8 (-0.7±0.7; p <0.001) and Day 29 (-1.1±0.7; p <0.0001) in Group 1. Similar results were observed in Group 2 (Day 8: p <0.001; Day 29: p <0.0001). Total disappearance of cylindrical dandruff was reported in 30.4% of patients in Group 1 and 43.5% in Group 2. Improvements in other ocular signs were observed in both groups. The product was well tolerated. All patients were satisfied or very satisfied and would continue using it. Conclusion: Daily eyelid hygiene using this cleansing wipe impregnated with 2.5% terpinen-4-ol and 0.2% hyaluronic acid during a 4-week period led to a rapid and marked reduction in ocular symptoms and signs associated with Demodex anterior blepharitis and was well tolerated., Competing Interests: MK and SD are consultants for Laboratoires Théa. SD reports personal fees from Alcon-Novartis, Bausch&Lomb, Horus, Santen, and Théa, outside the submitted work. The authors report no other conflicts of interest in this work.