Platelet Proteomics and Tissue Metabolomics Investigation for the Mechanism of Aspirin Eugenol Ester on Preventive Thrombosis Mechanism in a Rat Thrombosis Model.

Platelet activation is closely related to thrombosis. Aspirin eugenol ester (AEE) is a novel medicinal compound synthesized by esterifying aspirin with eugenol using the pro-drug principle. Pharmacological and pharmacodynamic experiments showed that AEE has excellent anti-inflammatory, antioxidant, and inhibitory platelet activation effects, preventing thrombosis. However, the regulatory network and action target of AEE in inhibiting platelet activation remain unknown. This study aimed to investigate the effects of AEE on platelets of thrombosed rats to reveal its regulatory mechanism via a multi-omics approach. The platelet proteomic results showed that 348 DEPs were identified in the AEE group compared with the model group, of which 87 were up- and 261 down-regulated. The pathways in this result were different from previous results, including mTOR signaling and ADP signaling at P2Y purinoceptor 12. The metabolomics of heart and abdominal aortic tissue results showed that the differential metabolites were mainly involved in steroid biosynthesis, the citric acid cycle, phenylalanine metabolism, phenylalanine, tyrosine, and tryptophan biosynthesis, and glutathione metabolism. Molecular docking results showed that AEE had a better binding force to both the COX-1 and P2Y12 protein. AEE could effectively inhibit platelet activation by inhibiting COX-1 protein and P2Y12 protein activity, thereby inhibiting platelet aggregation. Therefore, AEE can have a positive effect on inhibiting platelet activation.

Multi-omics analyzes of Rosa gigantea illuminate tea scent biosynthesis and release mechanisms.

Rose is an important ornamental crop cultivated globally for perfume production. However, our understanding of the mechanisms underlying scent production and molecular breeding for fragrance is hindered by the lack of a reference genome for tea roses. We present the first complete telomere-to-telomere (T2T) genome of Rosa gigantea, with high quality (QV > 60), including detailed characterization of the structural features of repetitive regions. The expansion of genes associated with phenylpropanoid biosynthesis may account for the unique tea scent. We uncover the release rhythm of aromatic volatile organic compounds and their gene regulatory networks through comparative genomics and time-ordered gene co-expression networks. Analyzes of eugenol homologs demonstrate how plants attract pollinators using specialized phenylpropanoids in specific tissues. This study highlights the conservation and utilization of genetic diversity from wild endangered species through multi-omics approaches, providing a scientific foundation for enhancing rose fragrance via de novo domestication.

Attractiveness, longevity, and release rates of multilure wafers for trapping males of the oriental fruit fly and melon fly (Diptera: Tephritidae).

Invasive fruit flies (Diptera: Tephritidae) pose a serious threat to the production and export of many commercially important fruits and vegetables. Detection of the agricultural pests Bactrocera dorsalis (Hendel) and Zeugodacus cucurbitae (Coquillett) relies heavily on traps baited with male-specific attractants. For B. dorsalis, traps are typically baited with liquid methyl eugenol (ME), and for Z. cucurbitae, traps are baited with liquid cue-lure (CL). Operating large-scale trapping networks is costly, consequently, there is much interest in identifying ways to maintain network sensitivity while reducing costs. One cost-cutting approach is the possibility of combining different male lures in the same dispenser, thus reducing the number of traps requiring servicing. The chief objective of this study was to compare captures of B. dorsalis and Z. cucurbitae males in Jackson traps baited with polymeric wafers impregnated with both ME and raspberry ketone (RK, a hydrolyzed form of CL) versus traps baited with liquid ME or CL freshly applied to cotton wicks. Captures were measured when the ME/RK wafers had been weathered for 12, 18, or 24 wk. Captures of B. dorsalis and Z. cucurbitae males were similar between fresh lure and weathered wafers over all trapping periods, with a single exception apparently due to the lessened potency of the associated killing agent. The residual amount and release rate of ME and RK from the wafers were also measured to examine possible relationships between wafer chemistry and trap catch. The possible implications of the present results to area-wide trapping programs are discussed.

Genome-Wide Analysis and Characterization of the SDR Gene Superfamily in Cinnamomum camphora and Identification of Synthase for Eugenol Biosynthesis.

Short-chain dehydrogenase/reductases (SDRs) are the largest NAD(H)-dependent oxidoreductase superfamilies and are involved in diverse metabolisms. This study presents a comprehensive genomic analysis of the SDR superfamily in Cinnamomum camphora, a species that is one of the most significant woody essential oil plants in southern China. We identify a total of 222 CcSDR proteins and classify them into five types based on their cofactor-binding and active sites: 'atypical', 'classic', 'divergent', 'extended', and 'unknown'. Phylogenetic analysis reveals three evolutionary branches within the CcSDR proteins, and further categorization using the SDR-initiative Hidden Markov model resulted in 46 families, with the CcSDR110C, CcSDR108E, and CcSDR460A families being the most populous. Collinearity analysis identified 34 pairs of CcSDR paralogs in C. camphora, 141 pairs of SDR orthologs between C. camphora and Populus trichocarpa, and 59 pairs between C. camphora and Oryza sativa. Expression profile analysis indicates a preference for the expression of 77 CcSDR genes in specific organs such as flowers, bark, twigs, roots, leaves, or fruits. Moreover, 77 genes exhibit differential expression patterns during the four developmental stages of leaves, while 130 genes show variance across the five developmental stages of fruits. Additionally, to explore the biosynthetic mechanism of methyl eugenol, a key component of the leaf essential oil in the methyl eugenol chemotype, this study also identifies eugenol synthase (EGS) within the CcSDR460A family through an integrated strategy. Real-time quantitative PCR analysis demonstrates that the expression of CcEGS in the leaves of the methyl eugenol chemotype is more than fourfold higher compared to other chemotypes. When heterologously expressed in Escherichia coli, it catalyzes the conversion of coniferyl acetate into a mixture predominantly composed of eugenol (71.44%) and isoeugenol (21.35%). These insights pave the way for future research into the functional diversity of CcSDR genes, with a focus on secondary metabolism.

Design, synthesis, docking studies and bioactivity evaluation of 1,2,3-triazole eugenol derivatives.

Aim: The design, synthesis, docking studies and evaluation of the in vitro antifungal and cytotoxic properties of eugenol (EUG) containing 1,2,3-triazole derivatives are reported. Most of the derivatives have not been reported.Materials & methods: The EUG derivatives were synthesized, molecular docked and tested for their antifungal activity.Results: The compounds showed potent antifungal activity against Trichophyton rubrum, associated with dermatophytosis. Compounds 2a and 2i exhibited promising results, with 2a being four-times more potent than EUG. The binding mode prediction was similar to itraconazole in the lanosterol-14-α-demethylase wild-type and G73E mutant binding sites. Additionally, the pharmacokinetic profile prediction suggests good gastrointestinal absorption and potential oral administration.Conclusion: Compound 2a is a promising antifungal agent against dermatophytosis caused by T. rubrum.

[Box: see text].

Echocardiographic evaluation in koi carp (Cyprinus carpio) under manual restraint compared to anesthesia with isoeugenol.

OBJECTIVE: To establish an echocardiographic technique in koi carp (Cyprinus carpio), compare cardiopulmonary parameters under manual restraint versus anesthesia, and provide a gross anatomical and histologic cardiac description. METHODS: A randomized, crossover echocardiography study was performed in 40 clinically healthy adult, unknown sex, privately owned koi carp on May 10 and 11 through June 26 and 27, 2021. Echocardiography was examined for each koi under manual restraint and isoeugenol at 50 ppm, with 3 measurements per examination performed by a radiologist and cardiologist. Two koi were euthanized for gross anatomic and histologic cardiac evaluation. RESULTS: Mean ejection fraction (EF), stroke volume (SV), and cardiac output (CO) were significantly lower, mean heart rate (HR) was significantly higher, and opercular rate (OPR) was decreased significantly in anesthetized compared to manually restrained koi. Poor reproducibility for EF and SV was observed. CONCLUSIONS: Echocardiography was feasible in both manually restrained and anesthetized koi; however, this technique may best be applied to monitoring trends over time in individual fish due to low reproducibility. Significant differences in multiple cardiopulmonary parameters, including HR, EF, SV, CO, and OPR, were present between manually restrained and anesthetized koi. A gross anatomic and histologic cardiac description is provided for this species to pair with the echocardiographic images. CLINICAL RELEVANCE: This study provides the first description of echocardiography, cardiac gross anatomy, and histology in koi. The results support echocardiography as a safe and practical noninvasive diagnostic for cardiac assessment in koi under both manual restraint and anesthesia.

Field longevity of methyl eugenol and cue-lure plugs and associated insecticidal strips: captures of Bactrocera dorsalis and Zeugodacus cucurbitae (Diptera: Tephritidae) in Hawaii.

Certain species of true fruit flies (Diptera: Tephritidae) cause tremendous damage to commercially important fruits and vegetables, and many countries operate continuous trapping programs which rely on male-specific lures such as trimedlure (TML), methyl eugenol (ME), and cue-lure (CL). Traditionally, these attractants have been applied as liquids to cotton wicks inside traps, although this results in high evaporative loss of the lure. Slow-release, polymeric plugs have been widely adopted for TML, but such devices are not widely used for ME or CL. Recent data, however, suggest that ME and CL plugs may be attractive for as long as 12 wk in the field. The present study investigates whether ME and CL plugs weathered for 18 or 24 wk are effective in capturing males of Bactrocera dorsalis (Hendel) and Zeugodacus cucurbitae (Coquillett), respectively. For B. dorsalis, 6 g ME plugs were as effective as the control treatment (fresh liquid on a wick) after 12 wk of weathering but not after 18 or 24 wk. For Z. cucurbitae, 3 g CL plugs were as effective as the control treatment (fresh CL plugs) after 12 and 18 wk of weathering but not after 24 wk. The residual content and release rate of the 2 lures were also measured over time, but, with the exception of the residual content of ME, we did not find a direct correlation between these parameters and numbers of flies captured.

Suppression of essential oil biosynthesis in sweet basil cotyledons under hypergravity conditions.

The mechanism through which gravity influences the biosynthesis of essential oils in herbs is an important issue for plant and space biology. Sweet basil (Ocimum basilicum L.) seedlings were cultivated under centrifugal hypergravity conditions at 100 g in the light, and the growth of cotyledons, development of glandular hairs, and biosynthesis of essential oils were analyzed. The area and fresh weight of the cotyledons increased by similar amounts irrespective of the gravitational conditions. On the abaxial surface of the cotyledons, glandular hairs, where essential oils are synthesized and stored, developed from those with single-cell heads to those with four-cell heads; however, hypergravity did not affect this development. The main components, methyl eugenol and 1,8-cineole, in the essential oils of cotyledons were lower in cotyledons grown under hypergravity conditions. The gene expression of enzymes in the phenylpropanoid pathway involved in the synthesis of methyl eugenol, such as phenylalanine ammonia lyase (PAL) and eugenol O-methyltransferase (EOMT), was downregulated by hypergravity. Hypergravity also decreased the gene expression of enzymes in the 2C-methyl-d-erythritol 4-phosphate (MEP) pathway involved in the synthesis of 1,8-cineole, such as 1-deoxy-d-xylulose-5-phosphate synthase (DXS) and 1,8-cineole synthase (CINS). These results indicate that hypergravity without affecting the development of glandular hairs, decreases the expression of genes related to the biosynthesis of methyl eugenol and 1,8-cineole, which may cause a decrease in the amounts of both essential oils in sweet basil cotyledons.

The contact toxicity and toxic mechanism of essential oils from Pimenta racemosa and Eugenia caryophyllata against Haemaphysalis longicornis (Acari: Ixodidae).

Haemaphysalis longicornis, which is widely distributed in China, can transmit various tick-borne diseases such as severe fever with thrombocytopenia syndrome, babesiosis, rickettsia disease and so on, and do great harm to human health and the development of animal husbandry. Chemical acaricides are the most traditional tick control method, but because of its many shortcomings, there is an urgent need to find a substitute with high efficiency, environmental protection and low toxicity. It has been found that some plant essential oils (EOs) have good insecticidal activity and environmental safety. In this study, the components of EOs from Pimenta racemosa and Eugenia caryophyllata were analyzed by gas chromatography-mass spectrometry (GC-MS), and their potential for application in the control of Haemaphysalis longicornis were studied. Gas chromatography-mass spectrometry analysis showed that the main components of P. racemosa EO were eugenol (64.07%), those of E. caryophyllata EO were Hexadecanoic acid, 2-methylpropyl ester (51.84%) and eugenol (39.76%). Larval packet test showed that the EOs of P. racemosa and E. caryophyllata had significant acaricidal activity against unfed larvae of H. longicornis, with LC50 values of 1.20 mg/mL and 0.47 mg/mL and LC90 values of 8.76 mg/mL and 2.91 mg/mL, respectively. The P. racemosa EO, E. caryophyllata EO and eugenol showed significant acaricidal activity against unfed nymph H. longicornis, with LC50 values of 1.65 mg/mL, 2.29 mg/mL and 0.93 mg/mL and LC90 values of 5.03 mg/mL, 11.01 mg/mL and 4.77 mg/mL, respectively. The P. racemosa EO, E. caryophyllata EO and eugenol showed significant acaricidal activity against unfed adults H. longicornis, with LC50 values of 0.51 mg/mL, 2.57 mg/mL and 1.83 mg/mL and LC90 values of 2.44 mg/mL, 11.44 mg/mL and 2.54 mg/mL, respectively. Enzyme assays revealed that the E. caryophyllata EO and eugenol significantly inhibited the activity of carboxylesterase (CarE), eugenol significantly inhibited the activity of catalase (CAT), and two EOs and eugenol had no significant effect on acetylcholinesterase (AchE) (p < 0.05). The above results suggest that the essential oils from P. racemosa and E. caryophyllata have great potential for use as alternatives to synthetic acaricides for tick control.

Vasorelaxant effect of phenylpropanoids: Methyl eugenol and eugenol in human umbilical cord vein.

Methyl-eugenol (ME) and eugenol (EUG) are phenylpropanoids with vasodilatory effects. While EUG's vasorelaxant effect in human umbilical artery (HUA) is known, their action in veins is unclear. This study aimed to evaluate ME and EUG in human umbilical vein (HUV). Isolated HUV underwent tension recordings. ME and EUG caused 100 % relaxation in HUV, with EC50 values corresponding to: 174.3 ± 7.3 and 217.3 ± 6.2 µM for ME and EUG respectively in presence of K+; 362.3 ± 5.4 and 227.7 ± 4.9 µM for ME and EUG respectively and in presence of serotonin (5-HT). It was observed that in presence of BaCl2 and CaCl2 evoked contractions, ME (800 and 1000 µM) and EUG (1000 and 1400 µM) prevent the contractions. In presence of K+ channel blockers it was observed that ME promoted relaxation compared to its control, except in presence of 4-AP, suggesting a possible Ca2+-dependent K+ channel activation for this molecule; EUG increased all EC50 in presence of the K+ blockers except in presence of TEA 1 mM. Greater pharmacological potency was observed for ME. This study highlights natural substances' effects on HUV contractile parameters, suggesting ME and EUG as potential vasodilators in maintaining fetal oxygenation and venous flow during gestational hypertensive syndromes.

Increase of secondary metabolites in sweet basil (Ocimum basilicum L.) leaves by exposure to N2O5 with plasma technology.

Exposure to N2O5 generated by plasma technology activates immunity in Arabidopsis through tryptophan metabolites. However, little is known about the effects of N2O5 exposure on other plant species. Sweet basil synthesizes many valuable secondary metabolites in its leaves. Therefore, metabolomic analyses were performed at three different exposure levels [9.7 (Ex1), 19.4 (Ex2) and 29.1 (Ex3) µmol] to assess the effects of N2O5 on basil leaves. As a result, cinnamaldehyde and phenolic acids increased with increasing doses. Certain flavonoids, columbianetin, and caryophyllene oxide increased with lower Ex1 exposure, cineole and methyl eugenol increased with moderate Ex2 exposure and L-glutathione GSH also increased with higher Ex3 exposure. Furthermore, gene expression analysis by quantitative RT-PCR showed that certain genes involved in the syntheses of secondary metabolites and jasmonic acid were significantly up-regulated early after N2O5 exposure. These results suggest that N2O5 exposure increases several valuable secondary metabolites in sweet basil leaves via plant defense responses in a controllable system.

Distal mutations enhance efficiency of free and immobilized NOV1 dioxygenase for vanillin synthesis.

Protein engineering is crucial to improve enzymes' efficiency and robustness for industrial biocatalysis. NOV1 is a bacterial dioxygenase that holds biotechnological potential by catalyzing the one-step oxidation of the lignin-derived isoeugenol into vanillin, a popular flavoring agent used in food, cleaning products, cosmetics and pharmaceuticals. This study aims to enhance NOV1 activity and operational stability through the identification of distal hotspots, located at more than 9 Šfrom the active site using Zymspot, a tool that predicts advantageous distant mutations, streamlining protein engineering. A total of 41 variants were constructed using site-directed mutagenesis and the six most active enzyme variants were then recombined. Two variants, with two and three mutations, showed nearly a 10-fold increase in activity and up to 40-fold higher operational stability than the wild-type. Furthermore, these variants show 90-100 % immobilization efficiency in metal affinity resins, compared to approximately 60 % for the wild-type. In bioconversions where 50 mM of isoeugenol was added stepwise over 24-h cycles, the 1D2 variant produced approximately 144 mM of vanillin after six reaction cycles, corresponding to around 22 mg, indicating a 35 % molar conversion yield. This output was around 2.5 times higher than that obtained using the wild-type. Our findings highlight the efficacy of distal protein engineering in enhancing enzyme functions like activity, stability, and metal binding selectivity, thereby fulfilling the criteria for industrial biocatalysts. This study provides a novel approach to enzyme optimization that could have significant implications for various biotechnological applications.

Analysis and Stability Study of Isoeugenol in Aquaculture Products by Headspace Solid-Phase Microextraction Coupled to Gas Chromatography-Mass Spectrometry.

Headspace solid-phase microextraction coupled to gas chromatography-mass spectrometry (HS-SPME-GC-MS) offers an alternative analysis method for isoeugenol (an active ingredient in fish sedatives) that avoids the use of organic solvents, simplifies sample preparation, and can be fully automated. This work focuses on developing and evaluating an HS-SPME-GC-MS method for isoeugenol in aquaculture samples and testing the stability of isoeugenol itself. Because of isoeugenol's relatively low volatility, more polar SPME fiber coatings (polyacrylate and polydimethylsiloxane/divinylbenzene) had better performance and the headspace extractions took over 30 min to reach equilibrium. Additionally, it was found that isoeugenol was relatively unstable compared to a deuterated standard (d3-eugenol) in the presence of water. To address this, after the fish samples were homogenized with water, they were heated at 50 °C for 1 h prior to analysis for equilibration. By using the method developed in this work, isoeugenol's detection limits in multiple aquaculture matrices (shrimp, tilapia, and salmon) were in the low ng/g range (<15 ng/g), well below the target testing level (200 ng/g). Additionally, by adding d3-eugenol as an internal standard, excellent linearity (R2 > 0.98), accuracy (97-99% recoveries), and precision (5-13% RSDs) were all achieved.

Isoeugenol Inhibits Adipogenesis in 3T3-L1 Preadipocytes with Impaired Mitotic Clonal Expansion.

Isoeugenol (IEG), a natural component of clove oil, possesses antioxidant, anti-inflammatory, and antibacterial properties. However, the effects of IEG on adipogenesis have not yet been elucidated. Here, we showed that IEG blocks adipogenesis in 3T3-L1 cells at an early stage. IEG inhibits lipid accumulation in adipocytes in a concentration-dependent manner and reduces the expression of mature adipocyte-related factors including PPARγ, C/EBPα, and FABP4. IEG treatment at different stages of adipogenesis showed that IEG inhibited adipocyte differentiation by suppressing the early stage, as confirmed by lipid accumulation and adipocyte-related biomarkers. The early stage stimulates growth-arrested preadipocytes to enter mitotic clonal expansion (MCE) and initiates their differentiation into adipocytes by regulating cell cycle-related factors. IEG arrested 3T3-L1 preadipocytes in the G0/G1 phase of the cell cycle and attenuated cell cycle-related factors including cyclinD1, CDK6, CDK2, and cyclinB1 during the MCE stage. Furthermore, IEG suppresses reactive oxygen species (ROS) production during MCE and inhibits ROS-related antioxidant enzymes, including superoxide dismutase1 (SOD1) and catalase. The expression of cell proliferation-related biomarkers, including pAKT and pERK1/2, was attenuated by the IEG treatment of 3T3-L1 preadipocytes. These findings suggest that it is a potential therapeutic agent for the treatment of obesity.

A weight of evidence evaluation of the mode of action of isoeugenol.

Isoeugenol is one of several phenylpropenoid compounds that is used as a fragrance, food flavoring agent and in aquaculture as a fish anesthetic. Carcinogenicity testing in rats and mice by NTP resulted in clear evidence of carcinogenicity (hepatic adenomas/carcinomas) in male mice only. A nongenotoxic threshold mode of action (MOA) is postulated for isoeugenol and is discussed considering the IPCS MOA and Human Relevance Framework. The weight of evidence indicates that isoeugenol is not genotoxic and that the carcinogenic outcome in male mice relates directly to the metabolism of individual compounds. Benchmark Dose (BMD) modeling was conducted to determine a Point of Departure (POD) and potential threshold of carcinogenicity. The results of the BMD evaluation for isoeugenol resulted in an estimated POD for carcinogenicity in the male mouse of 8 mg/kg with a lower limit of 4 mg/kg, representing a POD for the determination of an acceptable daily intake. With application of uncertainty factors, an ADI of 40 µg/kg is calculated. This daily dose in humans would be protective of human health, including carcinogenicity. A corresponding maximum residual level (MRL) of 3200 µg/kg fish is also estimated based on this POD that considers the threshold MOA.

[Isolation and functional characterization of (iso)eugenol O-methyltransferase (IEMT) gene in Asarum sieboldii].

Methyleugenol is one of the main active constituents in the volatile oil of the traditional Chinese medicine Asari Radix et Rhizoma. It possesses various pharmacological effects such as analgesic, anesthetic, and anti-inflammatory properties. In biosynthesis, the initial precursor phenylalanine is finally converted into methyleugenol through a series of intermediate compounds including coniferyl acid, courmaryl acid, caffeic acid, ferulic acid/ferulic-CoA, coniferyl aldehyde, conferyl alcohol, cnfiferyl acetate, and eugenol/isoeugenol, which are produced through catalysis of a large number of enzymes. Eugenol O-methyltransferase(EOMT) is one of the key enzymes in the biosynthesis pathway, capable of methylating eugenol on the para-site hydroxyl group of the benzene ring, thereby generating methyleugenol. Here, an(iso)eugenol O-methyltransferase(IEMT) gene was cloned for the first time from Asarum siebo-ldii, holding an open reading frame that consisted of 1 113 bp and encoded a protein containing 370 amino acid residues. Bioinformatics analysis results showed that this protein was equipped with the characteristic structural domains of methyltransferases such as S-adenosylmethionine(SAM) binding sites and dimerization domains. The prokaryotic expression recombinant plasmid pET28a(+)-AsIEMT was constructed, and the candidate protein was induced and purified. In vitro enzyme assays confirmed that AsIEMT had dual functions. The enzyme could catalyze the production either of methyleugenol from eugenol or of methylisoeugenol from isoeugenol, although the latter was more prevalent. When isoeugenol was used as the substrate, the kinetics parameters K_m and V_(max) of catalytic reaction were(0.90±0.06) mmol·L~(-1) and(1.32±0.04)nmol·s~(-1)·mg~(-1), respectively. This study expanded our understandings of critical enzyme genes involved in phenylpropanoid metabolic pathways, and would facilitate the elucidation of quality formation mechanisms of the TCM Asari Radix et Rhizoma.

Effect of dietary aspirin eugenol ester on the growth performance, antioxidant capacity, intestinal inflammation, and cecal microbiota of broilers under high stocking density.

This study was designed to examine the impact of aspirin eugenol ester (AEE) on the growth performance, serum antioxidant capacity, jejunal barrier function, and cecal microbiota of broilers raised under stressful high density (HD) stocking conditions compared with normal density broilers (ND). A total of 432 one-day-old AA+ male broilers were randomly divided into 4 groups: normal density (ND, 14 broilers /m2), high density (HD, 22 broilers /m2), ND + AEE, and HD + AEE. The results of the study revealed a significant decrease in the growth performance of broiler chickens as a result of HD stress (P < 0.05). The total antioxidant capacity (T-AOC) in serum demonstrated a significant decrease (P < 0.05) at both 28 and 35 d. Conversely, the serum level of malondialdehyde (MDA) exhibited a significant increase (P < 0.05). Dietary supplementation of AEE resulted in a significant elevation (P < 0.05) of serum GSH-PX, SOD and T-AOC activity at both 28 and 35 d. Moreover, exposure to HD stress resulted in a considerable reduction in the height of intestinal villi and mRNA expression of tight junction proteins in the jejunum, along with, a significant elevation in the mRNA expression of inflammatory cytokines (P < 0.05). However, the administration of AEE reversed the adverse effects of HD-induced stress on villus height and suppressed the mRNA expression of the pro-inflammatory genes, COX-2 and mPGES-1. Additionally, the exposure to HD stress resulted in a substantial reduction in the α-diversity of cecal microbiota and disruption in the equilibrium of intestinal microbial composition, with a notable decrease in the relative abundance of Bacteroides and Faecalibacterium (P < 0.05). In contrast, the addition of AEE to the feed resulted in a notable increase in the relative abundance of Phascolarctobacterium and enhanced microbial diversity (P < 0.05). The inclusion of AEE in the diet has been demonstrated to enhance intestinal integrity and growth performance of broilers by effectively mitigating disruptions in gut microbiota induced by HD stress.

Dehydrodieugenol isolated from Ocotea cymbarum induces cell death in human breast cancer cell lines by dysregulation of intracellular copper concentration.

In this work, two neolignans - dehydrodieugenol (1) and dehydrodieugenol B (2) - were isolated from leaves of Ocotea cymbarum (H. B. K.) Ness. (Lauraceae). When tested against two human breast cancer cell lines (MCF7 and MDA-MB-231), compound 1 was inactive (IC50 > 500 µM) whereas compound 2 displayed IC50 values of 169 and 174 µM, respectively. To evaluate, for the first time in the literature, the synergic cytotoxic effects of compounds 1 and 2 with ion Cu2+, both cell lines were incubated with equimolar solutions of these neolignans and Cu(ClO4)2·6H2O. Obtained results revealed no differences in cytotoxicity upon the co-administration of compound 2 and Cu2+. However, the combination of compound 1 and Cu2+ increases the cytotoxicity against MCF7 and MDA-MB-231 cells, with IC50 values of 165 and 204 µM, respectively. The activity of compound 1 and Cu2+ in MCF7 spheroids regarding the causes/effects considering the tumoral microenvironment were accessed using fluorescence staining and imaging by fluorescence microscopy. This analysis enabled the observation of a higher red filter fluorescence intensity in the quiescence zone and the necrotic core, indicating a greater presence of dead cells, suggesting that the combination permeates the spheroid. Finally, using ICP-MS analysis, the intracellular copper disbalance caused by mixing compound 1 and Cu2+ was determined quantitatively. The findings showcased a 50-fold surge in the concentration of Cu2+ compared with untreated cells (p > 0.0001) - 18.7 ng of Cu2+/mg of proteins and 0.37 ng of Cu2+/mg of protein, respectively. Conversely, the concentration of Cu2+ in cells treated with compound 1 was similar to values of the negative control group (0.29 ng of Cu2+/mg of protein). This alteration allowed us to infer that compound 1 combined with Cu2+ induces cell death through copper homeostasis dysregulation.

Gold nanocluster-based ratiometric fluorescence immunoassay for broad-spectrum screening of five eugenols.

BACKGROUND: Eugenol compounds (EUGs), which share chemical similarities with eugenol, belong to a group of phenolic compounds primarily found in clove oil. They are highly valued by fish dealers due to their exceptional anesthetic properties, playing a crucial role in reducing disease incidence and mortality during the transportation of live fish. Despite their widespread use, the safety of EUGs remains a contentious topic, raising concerns about the safety of aquatic products. This underscores the need for efficient and sensitive analytical methods for detecting EUGs. RESULTS: Nanomaterial-based ratiometric fluorescence immunoassay has gained increasing attention due to its integration of the immunoassay's excellent specificity and compatibility for high-throughput analysis, coupled with the exceptional sensitivity and anti-interference capabilities of ratiometric fluorescence assays. In this study, we developed a sensitive ratiometric fluorescence immunoassay for screening five EUGs. This method employs a broad-specificity monoclonal antibody (mAb) as a recognition reagent, selective for five EUGs. It leverages the horseradish peroxidase (HRP)-triggered formation of fluorescent 2,3-diaminophenazine (DAP) and the quenching of fluorescent gold clusters (Au NCs) for detection. The assay's detection limits for eugenol, isoeugenol, eugenol methyl eugenol, methyl isoeugenol, and acetyl isoeugenol in tilapia fish and shrimp were found to be 9.8/19.5 µg/kg, 0.11/0.22 µg/kg, 19/36 Tilapia ng/kg, 8/16 ng/kg, and 3.0/6.1 µg/kg, respectively. Furthermore, when testing spiked Tilapia fish and shrimp samples, recoveries ranging from 84.1 to 111.9 %, with the coefficients of variation staying below 7.1 % was achieved. SIGNIFICANCE: This work introduces an easy-to-use, broad-specificity, and highly sensitive method for the screening of five EUGs at a pg/mL level, which not only provides a high-throughput strategy for screening eugenol-type fish anesthetics in aquatic products, but also can serve as a benchmark for developing immunoassays for other small molecular pollutants, rendering potent technological support for guarding food safety and human health.

Curry-Odorants and Their Metabolites Transfer into Human Milk and Urine.

SCOPE: The excretion of dietary odorants into urine and milk is evaluated and the impact of possible influencing factors determined. Furthermore, the metabolic relevance of conjugates for the excretion into milk is investigated. METHODS AND RESULTS: Lactating mothers (n = 20) are given a standardized curry dish and donated one milk and urine sample each before and 1, 2, 3, 4.5, 6, and 8 h after the intervention. The concentrations of nine target odorants in these samples are determined. A significant transition is observed for linalool into milk, as well as for linalool, cuminaldehyde, cinnamaldehyde, and eugenol into urine. Maximum concentrations are reached within 1 h after the intervention in the case of milk and within 2-3 h in the case of urine. In addition, the impact of glucuronidase treatment on odorant concentrations is evaluated in a sample subset of twelve mothers. Linalool, eugenol, and vanillin concentrations increased 3-77-fold in milk samples after treatment with ß-glucuronidase. CONCLUSION: The transfer profiles of odorants into milk and urine differ qualitatively, quantitatively, and in temporal aspects. More substances are transferred into urine and the transfer needs a longer period compared with milk. Phase II metabolites are transferred into urine and milk.