Combining Experimental and Computational Methods to Produce Conjugates of Anticholinesterase and Antioxidant Pharmacophores with Linker Chemistries Affecting Biological Activities Related to Treatment of Alzheimer's Disease.

Effective therapeutics for Alzheimer's disease (AD) are in great demand worldwide. In our previous work, we responded to this need by synthesizing novel drug candidates consisting of 4-amino-2,3-polymethylenequinolines conjugated with butylated hydroxytoluene via fixed-length alkylimine or alkylamine linkers (spacers) and studying their bioactivities pertaining to AD treatment. Here, we report significant extensions of these studies, including the use of variable-length spacers and more detailed biological characterizations. Conjugates were potent inhibitors of acetylcholinesterase (AChE, the most active was 17d IC50 15.1 ± 0.2 nM) and butyrylcholinesterase (BChE, the most active was 18d: IC50 5.96 ± 0.58 nM), with weak inhibition of off-target carboxylesterase. Conjugates with alkylamine spacers were more effective cholinesterase inhibitors than alkylimine analogs. Optimal inhibition for AChE was exhibited by cyclohexaquinoline and for BChE by cycloheptaquinoline. Increasing spacer length elevated the potency against both cholinesterases. Structure-activity relationships agreed with docking results. Mixed-type reversible AChE inhibition, dual docking to catalytic and peripheral anionic sites, and propidium iodide displacement suggested the potential of hybrids to block AChE-induced ß-amyloid (Aß) aggregation. Hybrids also exhibited the inhibition of Aß self-aggregation in the thioflavin test; those with a hexaquinoline ring and C8 spacer were the most active. Conjugates demonstrated high antioxidant activity in ABTS and FRAP assays as well as the inhibition of luminol chemiluminescence and lipid peroxidation in mouse brain homogenates. Quantum-chemical calculations explained antioxidant results. Computed ADMET profiles indicated favorable blood-brain barrier permeability, suggesting the CNS activity potential. Thus, the conjugates could be considered promising multifunctional agents for the potential treatment of AD.

BHT - Butylated Hydroxytoluene.

The Expert Panel for Cosmetic Ingredient Safety reviewed updated information that has become available since their original assessment from 2002, along with updated information regarding product types, and frequency and concentrations of use, and reaffirmed their original conclusion that BHT is safe as a cosmetic ingredient in the practices of use and concentration as described in this report.

Antioxidants Amelioration Is Insufficient to Prevent Acrylamide and Alpha-Solanine Synergistic Toxicity in BEAS-2B Cells.

Cells produce free radicals and antioxidants when exposed to toxic compounds during cellular metabolism. However, free radicals are deleterious to lipids, proteins, and nucleic acids. Antioxidants neutralize and eliminate free radicals from cells, preventing cell damage. Therefore, the study aims to determine whether the antioxidants butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) will ameliorate the maximum dose of acrylamide and alpha (α)-solanine synergistic toxic effects in exposed BEAS-2B cells. These toxic compounds are consumed worldwide by eating potato products. BEAS-2B cells were simultaneously treated with BHA 10 µM and BHT 20 µM and incubated in a 5% CO2 humidified incubator for 24 h, followed by individual or combined treatment with acrylamide (3.5 mM) and α-solanine (44 mM) for 48 h, including the controls. Cell morphology, DNA, RNA, and protein were analyzed. The antioxidants did not prevent acrylamide and α-solanine synergistic effects in exposed BEAS-2B cells. However, cell morphology was altered; polymerase chain reaction (PCR) showed reduced RNA constituents but not DNA. In addition, the toxic compounds synergistically inhibited AKT/PKB expression and its downstream genes. The study showed BHA and BHT are not protective against the synergetic toxic effects of acrylamide and α-solanine in exposed BEAS-2B cells.

Ramulus Cinnamomi essential oil exerts an anti-inflammatory effect on RAW264.7 cells through N-acylethanolamine acid amidase inhibition.

ETHNOPHARMACOLOGICAL RELEVANCE: Ramulus Cinnamomi, the dried twig of Cinnamomum cassia (L.) J.Presl., is a traditional Chinese medicine (TCM) with anti-inflammatory effects. The medicinal functions of Ramulus Cinnamomi essential oil (RCEO) have been confirmed, although the potential mechanisms by which RCEO exerts its anti-inflammatory effects have not been fully elucidated. AIM OF THE STUDY: To investigate whether N-acylethanolamine acid amidase (NAAA) mediates the anti-inflammatory effects of RCEO. MATERIALS AND METHODS: RCEO was extracted by steam distillation of Ramulus Cinnamomi, and NAAA activity was detected using HEK293 cells overexpressing NAAA. N-Palmitoylethanolamide (PEA) and N-oleoylethanolamide (OEA), both of which are NAAA endogenous substrates, were detected by liquid chromatography with tandem mass spectrometry (HPLC-MS/MS). The anti-inflammatory effects of RCEO were analyzed in lipopolysaccharide (LPS)-stimulated RAW264.7 cells, and the cell viability was measured with a Cell Counting Kit-8 (CCK-8) kit. The nitric oxide (NO) in the cell supernatant was measured using the Griess method. The level of tumor necrosis factor-α (TNF-α) in the RAW264.7 cell supernatant was determined using an enzyme-linked immunosorbent assay (ELISA) kit. The chemical composition of RCEO was assessed by gas chromatography-mass spectroscopy (GC-MS). The molecular docking study for (E)-cinnamaldehyde and NAAA was performed by using Discovery Studio 2019 software (DS2019). RESULTS: We established a cell model for evaluating NAAA activity, and we found that RCEO inhibited the NAAA activity with an IC50 of 5.64 ± 0.62 µg/mL. RCEO significantly elevated PEA and OEA levels in NAAA-overexpressing HEK293 cells, suggesting that RCEO might prevent the degradation of cellular PEA and OEA by inhibiting the NAAA activity in NAAA-overexpressing HEK293 cells. In addition, RCEO also decreased NO and TNF-α cytokines in lipopolysaccharide (LPS)-stimulated macrophages. Interestingly, the GC-MS assay revealed that more than 93 components were identified in RCEO, of which (E)-cinnamaldehyde accounted for 64.88%. Further experiments showed that (E)-cinnamaldehyde and O-methoxycinnamaldehyde inhibited NAAA activity with an IC50 of 3.21 ± 0.03 and 9.62 ± 0.30 µg/mL, respectively, which may represent key components of RCEO that inhibit NAAA activity. Meanwhile, docking assays revealed that (E)-cinnamaldehyde occupies the catalytic cavity of NAAA and engages in a hydrogen bond interaction with the TRP181 and hydrophobic-related interactions with LEU152 of human NAAA. CONCLUSIONS: RCEO showed anti-inflammatory effects by inhibiting NAAA activity and elevating cellular PEA and OEA levels in NAAA-overexpressing HEK293 cells. (E)-cinnamaldehyde and O-methoxycinnamaldehyde, two components in RCEO, were identified as the main contributors of the anti-inflammatory effects of RCEO by modulating cellular PEA levels through NAAA inhibition.

Protective effects of butylated hydroxytoluene on the initiation of N-nitrosodiethylamine-induced hepatocellular carcinoma in albino rats.

Diethylnitrosamine (DEN), a hepatocarcinogen, is found in a variety of smoked and fried foods and was reported to be hepatotoxic in mice. Butylated hydroxytoluene (BHT) is a potent antioxidant used in cosmetic formulations and as a food additive and preservative. As a result, BHT was studied as a potential inhibitor in the early stages of diethylnitrosamine (DEN)-induced HCC. Male Wistar albino rats (n = 24) were equally subdivided. Group 1 was the negative control; Group 2 and 3 administered BHT and DEN, respectively; Group 4 received BHT followed by DEN. Blood samples and rat livers were taken for biochemical and histological investigation. Hepatotoxicity was assessed by increased liver enzymes and HCC indicators, along with reduced antioxidant and pro-apoptotic factors. AFP, AFPL3, GPC3, GSH, SOD, MDA, CASP3 and BAX expression increased significantly after DEN treatment. DEN also reduced GPx, CAT, and CYP2E1 activity, and BCl-2 expression. Moreover, in the hepatic parenchyma, the DEN caused histological alterations. Pretreatment with BHT enhanced antioxidant status while preventing histopathological and most biochemical alterations. BHT pretreatment suppresses DEN-initiated HCC by decreasing oxidative stress, triggering intrinsic mitotic apoptosis, and preventing histopathological changes in liver tissue.

A polypyrrole-cotton pad sorbent as micro-solid phase extractor enclosed in tea bag envelope for determination of synthetic antioxidants in non-alcoholic beverage products.

A polypyrrole (PPy)-cotton pad sorbent enclosed in tea bag envelope was developed and used in micro-solid phase extraction (µ-SPE) for the determination of butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT). After extraction, the extract was qualified and quantified by a gas chromatograph equipped with a flame ionization detector (GC-FID). Parameters influencing this developed method and the efficiency of µ-SPE were studied and optimized. Under the optimal conditions, the developed method provided good linearity in a concentration range of 0.100-100 µg L-1 for BHA and 0.050-50 µg L-1 for BHT, respectively. The limits of detection were 39.27 ± 0.52 ng L-1 for BHA and 16.96 ± 0.17 ng L-1 for BHT. Satisfactory relative recoveries of BHA and BHT were achieved in the range from 86.8 ± 1.9 to 117.1 ± 2.3% with acceptable relative standard deviation (RSD) below 8.1%. Good reproducibility was obtained with RSDs < 3.1%, for n = 6. The developed adsorbent is easy to operate, low cost, eco-friendly, reusable, with high extraction efficiency, and was successfully applied in the simultaneous synthetic antioxidant determination of non-alcoholic beverage samples.

Bacillus cereus WL08 immobilized on tobacco stem charcoal eliminates butylated hydroxytoluene in soils and alleviates the continuous cropping obstacle of Pinellia ternata.

Butylated hydroxytoluene (BHT), as an emerging contaminant in ecosystems, has potential influences on animals, aquatic organisms, and public health, and has been proven to be a major allelochemical of Pinellia ternata. In this study, Bacillus cereus WL08 was used to rapidly degrade BHT in liquid culture. Strain WL08 immobilized on tobacco stem charcoal (TSC) particles notably accelerated BHT removal in contract to its free cells, and exhibited excellent reutilization and storage capacities. The optimal removal parameters of TSC WL08 were ascertained to be pH 7.0, 30 °C, 50 mg L-1 BHT and 0.14 mg L-1 TSC WL08. Moreover, TSC WL08 significantly accelerated the degradation of 50 mg L-1 BHT in sterile and non-sterile soils compared to that of free WL08 or natural dissipation, and notably shortened their half-lives by 2.47- or 362.14- fold, and 2.20- or 14.99- fold, respectively. Simultaneously, TSC WL08 was introduced into the continuous cropping soils of P. ternata, which accelerated the elimination of allelochemical BHT, and notably enhanced the photosynthesis, growth, yield, and quality of P. ternata. This study provides new insights and strategies for the rapid in situ remediation of BHT-polluted soils and effective alleviation of P. ternata cropping obstacles.

Evaluation of the Antioxidant Properties of Carvacrol as a Prospective Replacement for Crude Essential Oils and Synthetic Antioxidants in Food Storage.

The phenolic structural analogues of synthetic antioxidants such as butylated hydroxytoluene (BHT) in essential oils have been reported to exhibit antioxidant properties. Additionally, their lipophilicity makes them suitable for use in lipid-rich foods. This study evaluated the antioxidant capacity of carvacrol, a monoterpenoid antioxidant compound in the Monodora myristica (Gaertn.) seed essential oil, compared to the seed essential oil and BHT. In vitro studies (ferric reducing antioxidant power (FRAP), metal chelating activity (MCA), and nitric oxide scavenging activity (NOSA)) were conducted to ascertain if the antioxidant capacity of carvacrol was comparable to that of the seed essential oil. The potential binding affinity and molecular interactions between carvacrol and lipoxygenase (LOX) and its homologous model were investigated in silico. The molecular docking was performed using Autodock Vina, and the best poses were subjected to molecular dynamics simulation. The IC50 for MCA and NOSA were: carvacrol 50.29 µL/mL, seed essential oil (SEO) 71.06 µL/mL; and carvacrol 127.61 µL/mL, SEO 165.18 µL/mL, respectively. The LOX model was Ramachandran favoured (97.75%) and the overall quality factor in the ERRAT plot was 95.392. The results of the molecular docking and molecular dynamics simulations revealed that lipoxygenase has a higher affinity (-22.79 kcal/mol) for carvacrol compared to BHT. In the LOX-BHT and LOX-carvacrol complexes, the root-mean-square deviation (RMSD), root-mean-square fluctuation (RMSF), and the radius of gyration (RoG) were not significantly different, indicating similar molecular interactions. The results obtained from this study suggest that carvacrol exhibits an antioxidant capacity that may be explored as an alternative for crude essential oils and synthetic compounds during the storage of lipid-rich foods.

Integrated the embedding delivery system and targeted oxygen scavenger enhances free radical scavenging capacity.

World trends in oil crop growing area, yield, and production over the last 10 years exhibited an increase of 48 %, 82 %, and 240 %, respectively. Concerning reduced shelf-life of oil-containing food products caused by oil oxidation and the demand for sensory quality of oil, the development of methods the improvement oil quality is urgently required. This critical review presented a concise overview of the recent literature related to the inhibition ways of oil oxidation. The mechanism of different antioxidants and nanoparticle delivery systems on oil oxidation was also explored. The current review provides scientific findings on control strategies: (i) design oxidation quality assessment model; (ii) packaging by antioxidant coatings and eco-friendly film nanocomposite: ameliorate physicochemical properties; (iii) molecular investigations on inhibitory effects of selected antioxidants and underlying mechanisms; (iv) explore the interrelationship between the cysteine/citric acid and lipoxygenase pathway in the progression of oxidative/fragmentation degradation of unsaturated fatty acid chains.

SCCS scientific opinion on Butylated hydroxytoluene (BHT) - SCCS/1636/21.

OPINION TO BE CITED AS: SCCS (Scientific Committee on Consumer Safety), scientific opinion on Butylated hydroxytoluene (BHT), preliminary version of September 27, 2021, final version of December 2, 2021, SCCS/1636/21.

Detection of Synthetic Antioxidants: What Factors Affect the Efficiency in the Chromatographic Analysis and in the Electrochemical Analysis?

Antioxidants are food additives largely employed to inhibit oxidative reactions in foodstuffs rich in oils and fat lipids, extending the shelf life of foodstuffs and inhibiting alterations in color, flavor, smell, and loss of nutritional value. However, various research has demonstrated that the inadequate use of synthetic antioxidants results in environmental and health problems due to the fact that some of these compounds present toxicity, and their presence in the human body, in high concentrations, is related to the development of some cancer types and other diseases. Therefore, the development of analytical methods for identifying and quantifying synthetic antioxidants in foodstuffs is fundamental to quality control and in ensuring consumer food safety. This review describes the recent chromatographic and electrochemical techniques used in the detection of synthetic phenolic antioxidants in foodstuffs, highlighting the main characteristics, advantages and disadvantages of these methods, and specific typical features, which include extraction methods for sample preparation and materials used in the working electrode construction, considering chromatographic and voltammetric methods, since these specific features influence the efficiency in the analysis.

Quantification and identification analysis of Ziziphus jujuba Mill. peel pigmentation at different developmental stages.

The fruit peel of a color mutant jujube cultivar, 'Sanbianhong' (SBF), was investigated using an ultra-high performance liquid chromatography quadrupole Orbitrap mass spectrometry (UHPLC-Q-Orbitrap MS) at five ripening stages (S1, Young fruit stage; S2, swelling stage; S3, white-mature stage; S4, pre-mature stage and S5, mature stage). Lutein, ß-carotene, chlorophyll a, chlorophyll b, and 13 anthocyanins were identified. Chlorophyll a and cyanidin 3-O-galactoside were considered key color metabolites in S1 with the content of 1.083 mg/g of fresh weight (FW) and 4.585 mg/g of FW, respectively. Delphinidin (0.488 mg/g FW) and cyanidin (6.259 mg/g FW) were identified as the key pigments in S3. Delphinidin 3-O-glucoside (0.256 mg/g FW) was identified as the key anthocyanin in maturity S5. Herein, the identification and quantitation of pigment-related metabolites of SBF were studied for the first time, and the results provide a theoretical basis for understanding the pigment changes of jujube fruit during ripening.

Effect of a polymerization inhibitor on the chemomechanical properties and consistency of experimental resin composites

Abstract This study investigated the effect of butylated hydroxytoluene (BHT) inhibitor on degree of conversion (DC), flexural strength (FS), flexural modulus (FM), Knoop microhardness (KH), microhardness reduction (HR), and consistency of experimental resin composites at different BHT concentrations: C0 (control-0%); C0.01 (0.01%); C0.025 (0.025%); C0.05 (0.05%); C0.1 (0.1%); and C0.5 (0.5%). For the consistency, the composites were tested immediately after being exposed to a dental chair headlight (0, 20, 40 and 60 s). Data concerning DC, FS, FM, KH, and HR were submitted to one-way ANOVA, while the consistency data was submitted to 2-way ANOVA; mean values were then compared (Tukey's test; α=0.05). The KH, FS and FM analyses showed no significant difference among the composites tested. For DC, C0 showed the highest mean value (74.2%) and differed only from C0.5 (67.2%). For HR, C0.5 showed the lowest mean (13.09%) value and differed from C0 (26.4%) and C0.01 (24.87). The consistency analysis showed no difference among C0.05, C0.1 and C0.5, considering 0 and 20 s of light exposure, while C0 (14.07 mm), C0.01 (13.97 mm), and C0.025 (14.18 mm) showed higher mean values at 0 s when compared to 20 s (12.67, 12.77 and 13.05 mm, respectivelly). Polymerization occurred within 40 s of light exposure for C0, C0.01, C0.025, and C0.05 and within 60 s for C0.1. In conclusion, the BHT concentrations had no significant influence on FS, FM and KH. The higher the BHT concentration, the longer was its handling time under light, with a significant improvement in the HR, but a decrease in DC. Therefore, BHT at 0.1% showed the best outcomes concerning all the BHT concentrations tested.

Resumo O objetivo foi investigar o efeito do inibidor de polimerização BHT no grau de conversão (GC), resistência à flexão (RF), módulo de flexão (MF), microdureza Knoop (KH) e redução da microdureza (RKH), e consistência de um compósito experimental contendo diferentes concentrações de BHT (% em peso): C0 - controle (0%); C0,01 (0,01%); C0,025 (0,025%); C0,05 (0,05%); C0,1 (0,1%); e C0,5 (0,5%). Para o teste de consistência, os compósitos foram testados imediatamente após serem expostos à luz de um refletor odontológico por 0, 20, 40 e 60 s. Os dados relativos a GC, RF, MF, KH e RKH foram submetidos a one-way ANOVA, enquanto os dados de consistência foram submetidos a two-way ANOVA; os valores médios foram comparados (teste de Tukey; α = 0,05). As análises de KH, RF e MF não mostraram diferença significativa entre os compósitos testados. Para GC, C0 apresentou o maior valor médio e diferiu apenas de C0,5. Para a RKH, C0,5 apresentou o menor valor médio e diferiu de C0 e C0,01. A análise de consistência não mostrou diferença entre C0,05, C0,1 e C0,5, considerando-se 0 e 20 s de exposição à luz, enquanto C0, C0,01 e C0,025 apresentaram maiores valores médios a 0 s quando comparados a 20 s. A polimerização ocorreu dentro de 40 s de exposição à luz para C0, C0.01, C0.025 e C0.05 e dentro de 60 s para C0.1. Em conclusão, as concentrações de BHT não tiveram influência significativa sobre RF, MF e KH. Quanto maior a concentração de BHT, maior o tempo de manuseio sob luz, com melhora significativa da RKH, mas diminuição da GC. Portanto, o compósito contendo 0,1 % de BHT apresentou os melhores resultados entre as demais concentrações testadas.

Safety and efficacy of a feed additive consisting of butylated hydroxytoluene (BHT) for all animal species (Lanxess Deutschland GmbH).

Following a request from the European Commission, the Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) was asked to deliver a scientific opinion on the safety and efficacy of butylated hydroxytoluene (BHT) as a feed additive for all animal species. The additive BHT is considered safe for chickens for fattening and weaned piglets at the maximum proposed concentration of 150 mg/kg complete feed. This conclusion is extended to chickens reared for laying and extrapolated to pigs for fattening. In the absence of data, no conclusion on the safety for the other target species could be drawn. The exposure of the consumer to BHT from tissues and products of animals fed the additive ranged from 1% to 3% of the acceptable daily intake (ADI). The FEEDAP Panel concluded that the use of BHT as a feed additive at the proposed conditions of use is of no concern for the safety of the consumers. Exposure of the user to BHT via inhalation is likely; however, the Panel is not in the position to conclude on the potential inhalation toxicity of the additive. BHT is a skin and eye irritant, no conclusions can be drawn on the potential of the additive to be a skin sensitiser. In the absence of data, the FEEDAP Panel cannot conclude on the safety of BHT for the environment. The additive BHT is considered an efficacious antioxidant in feedingstuffs for all animal species.

Safety and efficacy of a feed additive consisting of butylated hydroxytoluene (BHT) for all animal species (Katyon Technologies Limited).

Following a request from the European Commission, the Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) was asked to deliver a scientific opinion on the safety and efficacy of butylated hydroxytoluene (BHT) as a feed additive for all animal species. The additive BHT is considered safe for chickens for fattening and weaned piglets at the maximum proposed concentration of 150 mg/kg complete feed. This conclusion is extended to chickens reared for laying and extrapolated to pigs for fattening. In the absence of data, no conclusion on the safety for the other target species could be drawn. The exposure of the consumer to BHT from tissues and products of animals fed the additive ranged from 1% to 3% of the acceptable daily intake (ADI). The FEEDAP Panel concluded that the use of BHT as a feed additive at the proposed conditions of use is of no concern for the safety of the consumers. Exposure of the user to BHT via inhalation is likely; however, the Panel is not in the position to conclude on the potential inhalation toxicity of the additive. BHT is a skin and eye irritant, no conclusions can be drawn on the potential of the additive to be a skin sensitiser. In the absence of data, the FEEDAP Panel cannot conclude on the safety of BHT for the environment. The additive BHT is considered an efficacious antioxidant in feedingstuffs for all animal species.

[Study of Rapid HPLC Method for the Determination of Antioxidants in Foods, and Standard Solution Stability during Storage].

In this study, we simultaneously determined three antioxidants, butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), and tert-butylhydroquinone (TBHQ), using HPLC equipped both with a photodiode array detector and a fluorescence detector in 25 minutes per sample. Due to the combined use of the two detectors, we could achieve improved target selectivity. Further, quantification at the specific wavelengths for each target substance particularly increased BHT detection sensitivity. This approach enabled us to avoid repeated measurements during daily inspections. Furthermore, detections were performed using LC-MS/MS instead of GC-MS to overcome the problem of helium gas shortage.In addition, we investigated antioxidant stability in standard solutions during storage. Although TBHQ was stable in methanol with ascorbic acid at -20℃, ascorbic acid storage has possibility to lead to decrease in BHT and BHA concentrations. We recognized that the mixture of BHT and BHA dissolved in methanol at 4℃ and that of BHT, BHA and TBHQ dissolved in methanol with ascorbic acid at -20℃ were suitable for about one year.

Comparative study of the antioxidant activity of the essential oils of five plants against the H2O2 induced stress in Saccharomyces cerevisiae.

The purpose of this work was to investigate the protective effect of five essential oils (EOs); Rosmarinus officinalis, Thymus vulgaris, Origanum compactum Benth., Eucalyptus globulus Labill. and Ocimum basilicum L.; against oxidative stress induced by hydrogen peroxide in Saccharomyces cerevisiae. The chemical composition of the EOs was analyzed by gas chromatography (GC) and gas chromatography-mass spectrometry (GC/MS). The in vitro antioxidant activity was evaluated and the protective effect of EOs was investigated. Yeast cells were pretreated with different concentrations of EOs (6.25-25 µg/ml) for an hour then incubated with H2O2 (2 mM) for an additional hour. Cell viability, antioxidants (Catalase, Superoxide dismutase and Glutathione reductase) and metabolic (Succinate dehydrogenase) enzymes, as well as the level of lipid peroxidation (LPO) and protein carbonyl content (PCO) were evaluated. The chemical composition of EOs has shown the difference qualitatively and quantitatively. Indeed, O. compactum mainly contained Carvacrol, O. basilicum was mainly composed of Linalool, T. vulgaris was rich in thymol, R. officinalis had high α-Pinene amount and for E. globulus, eucalyptol was the major compound. The EOs of basil, oregano and thyme were found to possess the highest amount of total phenolic compounds. Moreover, they have shown the best protective effect on yeast cells against oxidative stress induced by H2O2. In addition, in a dose dependent manner of EOs in yeast medium, treated cells had lower levels of LPO, lower antioxidant and metabolic enzymes activity than cells exposed to H2O2 only. The cell viability was also improved. It seems that the studied EOs are efficient natural antioxidants, which can be exploited to protect against damages and serious diseases related to oxidative stress.

A comparison of cell death mechanisms of antioxidants, butylated hydroxyanisole and butylated hydroxytoluene.

Butylated hydroxyanisole (BHA) and the chemically similar butylated hydroxytoluene (BHT) are widely used as antioxidants. Toxicity of BHA and BHT has been reported under in vitro and in vivo experimental conditions. However, the mechanism of BHA-induced toxic effects in cells is unclear. In this study, the cytotoxic effects of BHA and differences in cell death mechanism for BHA and BHT were investigated in rat thymocytes by flow cytometric analysis using a fluorescent probe. We observed a significant increase in propidium iodide fluorescence in the population of cells treated with 100 µM and 300 µM BHA (dead cells). Thymocytes treated with 100 µM BHA showed increased intracellular Ca2+ and Zn2+ levels and depolarized cell membranes. BHA (30-100 µM) decreased non-protein thiol content of cells, indicating decreased glutathione content. Co-stimulation with 100 µM BHA and 300 µM H2O2 acted synergistically to increase cell lethality. Moreover, BHA significantly increased caspase-3 activity and the number of annexin-V-positive cells in a concentration-dependent manner, indicating apoptosis. However, BHT reduced caspase-3 activity and increased the number of annexin-V-negative dead cells, indicating non-apoptotic cell death. Our results reveal the toxicity of BHA could be attributed to increased levels of intracellular Ca2+ and Zn2+, resulting in an increased vulnerability of rat thymocytes to oxidative stress. In addition, we demonstrate that whereas BHA induced apoptosis, BHT induced non-apoptotic cell death in rat thymocytes. Therefore, these results may support the safety of BHA, but also demonstrate the importance of performing toxicity evaluation at the cellular level besides the tissue level.

Naringin prevents cyclophosphamide-induced erythrocytotoxicity in rats by abrogating oxidative stress.

Earlier reports have shown that Cyclophosphamide (CYCP), an anti-malignant drug, elicited cytotoxicity; and that naringin has several beneficial potentials against oxidative stress and dyslipidaemias. We investigated the influence of naringin on free radical scavenging, cellular integrity, cellular ATP, antioxidants, oxidative stress, and lipid profiles in the CYCP-induced erythrocytotoxicity rat model. Rats were pretreated orally by gavage for fourteen consecutive days with three doses (50, 100, and 200 mg/kg) naringin before single CYCP (200 mg/kg, i.p.) administration. Afterwards, the rats were sacrificed. Naringin concentrations required for 50 % scavenging hydrogen peroxide and nitric oxide radical were 0.27 mg/mL and 0.28 mg/mL, respectively. Naringin pretreatment significantly (p < 0.05) protected erythrocytes plasma membrane architecture and integrity by abolishing CYCP-induced decrease in the activity of erythrocyte LDH (a marker of ATP). Pretreatment with naringin remarkably (p < 0.05) reversed CYCP-induced decreases in the erythrocytes glutathione levels, activities of glutathione-S-transferase, catalase, glutathione peroxidase, and glutathione reductase; attenuated CYCP-mediated increases in erythrocytes levels of malondialdehyde, nitric oxide, and major lipids (cholesterol, triacylglycerol, phospholipids, and non-esterified fatty acids). Taken together, different acute pretreatment doses of naringin might avert CYCP-mediated erythrocytes dysfunctions via its antioxidant, free-radical scavenging, and anti-dyslipidaemia properties.

The Role of Cell Proliferation and Extracellular Matrix Accumulation Induced by Food Additive Butylated Hydroxytoluene in Uterine Leiomyoma.

Leiomyoma is the most common benign uterine tumor in reproductive-age women. Increasing numbers of studies are focusing on the effects of environmental exposure on the incidence and progression of tumors. One major step taken in the food industry is the addition of food preservatives to maintain freshness. Butylated hydroxytoluene (BHT) is a synthetic phenolic antioxidant, which is widely used as an additive to develop fat-soluble characteristics, as well as in cosmetics and rubber. Previous studies also highlighted that BHT may be related to increased fibrosis capacity and carcinogenic effects. In this study, we explored the effects of the commonly used food additive BHT on leiomyoma progression, and the related mechanism. The exposure of the ELT-3 leiomyoma cell line to BHT for 48 h increased the proliferative effect. Since leiomyoma progression is related to increases in extracellular matrix (ECM) accumulation and matrix metalloproteinase (MMP), BHT could effectively increase ECM-related protein expression, as well as MMP-2 and MMP-9 protein expression. This increase in ECM, in response to BHT, may be linked to the activation of the phosphoinositide 3-kinase (PI3K)/Akt and mitogen-activated protein kinase (MAPK) signaling pathway. Through PI3K inhibition, BHT's effect on leiomyoma progression could be partially modulated. These results suggest the harmful effect of BHT exposure on leiomyoma progression may relate to PI3K modulation. However, an in vivo study is necessary to confirm these findings.