Eugenol Possesses Colitis Protective Effects: Impacts on the TLR4/MyD88/NF-[Formula: see text]B Pathway, Intestinal Epithelial Barrier, and Macrophage Polarization.

Eugenol (EU) has been shown to ameliorate experimental colitis due to its anti-oxidant and anti-inflammatory bioactivities. In this study, DSS-induced acute colitis was established and applied to clarify the regulation efficacy of EU on intestinal barrier impairment and macrophage polarization imbalance along with the inflammatory response. Besides, the adjusting effect of EU on macrophages was further investigated in vitro. The results confirmed that EU intervention alleviated DSS-induced colitis through methods such as restraining weight loss and colonic shortening and decreasing DAI scores. Microscopic observation manifested that EU maintained the intestinal barrier integrity in line with the mucus barrier and tight junction protection. Furthermore, EU intervention significantly suppressed the activation of TLR4/MyD88/NF-[Formula: see text]B signaling pathways and pro-inflammatory cytokines gene expressions, while enhancing the expressions of anti-inflammatory cytokines. Simultaneously, WB and FCM analyses of the CD86 and CD206 showed that EU could regulate the DSS-induced macrophage polarization imbalance. Overall, our data further elucidated the mechanism of EU's defensive effect on experimental colitis, which is relevant to the protective efficacy of intestinal barriers, inhibition of oxidative stress and excessive inflammatory response, and reprogramming of macrophage polarization. Hence, this study may facilitate a better understanding of the protective action of the EU against UC.

Eugenol Suppresses Platelet Activation and Mitigates Pulmonary Thromboembolism in Humans and Murine Models.

Platelets assume a pivotal role in the pathogenesis of cardiovascular diseases (CVDs), emphasizing their significance in disease progression. Consequently, addressing CVDs necessitates a targeted approach focused on mitigating platelet activation. Eugenol, predominantly derived from clove oil, is recognized for its antibacterial, anticancer, and anti-inflammatory properties, rendering it a valuable medicinal agent. This investigation delves into the intricate mechanisms through which eugenol influences human platelets. At a low concentration of 2 µM, eugenol demonstrates inhibition of collagen and arachidonic acid (AA)-induced platelet aggregation. Notably, thrombin and U46619 remain unaffected by eugenol. Its modulatory effects extend to ATP release, P-selectin expression, and intracellular calcium levels ([Ca2+]i). Eugenol significantly inhibits various signaling cascades, including phospholipase Cγ2 (PLCγ2)/protein kinase C (PKC), phosphoinositide 3-kinase/Akt/glycogen synthase kinase-3ß, mitogen-activated protein kinases, and cytosolic phospholipase A2 (cPLA2)/thromboxane A2 (TxA2) formation induced by collagen. Eugenol selectively inhibited cPLA2/TxA2 phosphorylation induced by AA, not affecting p38 MAPK. In ADP-treated mice, eugenol reduced occluded lung vessels by platelet thrombi without extending bleeding time. In conclusion, eugenol exerts a potent inhibitory effect on platelet activation, achieved through the inhibition of the PLCγ2-PKC and cPLA2-TxA2 cascade, consequently suppressing platelet aggregation. These findings underscore the potential therapeutic applications of eugenol in CVDs.

Multi-Omics Approaches for Liver Reveal the Thromboprophylaxis Mechanism of Aspirin Eugenol Ester in Rat Thrombosis Model.

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 had excellent thromboprophylaxis and inhibition of platelet aggregation. This study aimed to investigate the effect of AEE on the liver of thrombosed rats to reveal its mechanism of thromboprophylaxis. Therefore, a multi-omics approach was used to analyze the liver. Transcriptome results showed 132 differentially expressed genes (DEGs) in the AEE group compared to the model group. Proteome results showed that 159 differentially expressed proteins (DEPs) were identified in the AEE group compared to the model group. Six proteins including fibrinogen alpha chain (Fga), fibrinogen gamma chain (Fgg), fibrinogen beta chain (Fgb), orosomucoid 1 (Orm1), hemopexin (Hpx), and kininogen-2 (Kng2) were selected for parallel reaction monitoring (PRM) analysis. The results showed that the expression of all six proteins was upregulated in the model group compared with the control group. In turn, AEE reversed the upregulation trend of these proteins to some degree. Metabolome results showed that 17 metabolites were upregulated and 38 were downregulated in the model group compared to the control group. AEE could reverse the expression of these metabolites to some degree and make them back to normal levels. The metabolites were mainly involved in metabolic pathways, including linoleic acid metabolism, arachidonic acid metabolism, and the tricarboxylic acid (TCA) cycle. Comprehensive analyses showed that AEE could prevent thrombosis by inhibiting platelet activation, decreasing inflammation, and regulating amino acid and energy metabolism. In conclusion, AEE can have a positive effect on thrombosis-related diseases.

Eugenol alleviates the symptoms of experimental autoimmune encephalomyelitis in mice by suppressing inflammatory responses.

Eugenol is a principal compound in essential clove oil, known for its anti-inflammatory and antioxidant properties. While recent studies have demonstrated its neuroprotective effects on central nervous system (CNS) injuries, such as brain ischemia/reperfusion injuries, but its potential impact on multiple sclerosis (MS), an autoimmune disease of the CNS, has not yet been explored. We evaluated the therapeutic effects of eugenol on experimental autoimmune encephalomyelitis (EAE), an established animal model of MS. EAE was induced in C57BL/6 mice using the myelin oligodendrocyte glycoprotein (MOG)35-55 peptide. Clinical symptoms, including paralysis, were monitored daily, and levels of pro-inflammatory mediators were evaluated using real-time quantitative polymerase chain reaction, Western blot analyses, and immunohistochemistry. Daily oral administration of eugenol to MOG-induced EAE mice led to a notable decline in the severity of clinical symptoms. Eugenol inhibited EAE-related immune cell infiltration and the production of pro-inflammatory mediators. Histological examinations confirmed its ability to mitigate inflammation and demyelination in the spinal cord post-EAE induction. Eugenol alleviates neuroinflammation in the spinal cords of EAE-induced mice, primarily through anti-inflammatory action.

Biofilm formation of C. albicans on occlusal device materials and antibiofilm effects of chitosan and eugenol.

STATEMENT OF PROBLEM: Microbial adhesion on occlusal devices may lead to oral diseases such as candidiasis. Whether chitosan and eugenol provide antibiofilm effects is unclear. PURPOSE: The purpose of this in vitro study was to evaluate the biofilm formation of C. albicans strains on occlusal device materials and the antibiofilm effects of chitosan and eugenol against C. albicans on these surfaces. MATERIAL AND METHODS: A total of 88 specimens (5×10×2 mm) were produced from occlusal device materials with 4 production techniques: vacuum-formed thermoplastic (Group V), head-press (Group H), computer-aided design and computer-aided manufacture (CAD-CAM) (Group C), and 3-dimensionally (3D) printed (Group D) (n=22). After various finishing procedures, the surface properties of the specimens were evaluated by using surface free energy (SFE), surface roughness (SR) measurements, and elemental and topographic analysis. Biofilm formation of C. albicans strain and the antibiofilm effects of chitosan and eugenol against biofilm formation on these surfaces were also examined with a crystal violet assay. The distribution's normality was statistically analyzed with the Kolmogorov-Smirnov test. One-way and two-way analysis of variance with post hoc Tukey tests were used for statistical evaluations (α=.05). RESULTS: Surface roughness values in Groups D and H were significantly higher than in other groups (P<.05). While the highest surface free energy values (except γp) were in Group V, Group C had the highest γp. The lowest biofilm value appeared in Group H. Chitosan exhibited an antibiofilm effect in all groups except Group H, while eugenol was effective in all groups. CONCLUSIONS: The production method affected the susceptibility of occlusal device materials to the adhesion of C. albicans. Eugenol was an effective antibiofilm agent for device materials.

Pharmacodynamic, pharmacokinetic, toxicity, and recent advances in Eugenol's potential benefits against natural and chemical noxious agents: A mechanistic review.

The active biological phytochemicals, crucial compounds employed in creating hundreds of medications, are derived from valuable and medicinally significant plants. These phytochemicals offer excellent protection from various illnesses, including inflammatory disorders and chronic conditions caused by oxidative stress. A phenolic monoterpenoid known as eugenol (EUG), it is typically found in the essential oils of many plant species from the Myristicaceae, Myrtaceae, Lamiaceae, and Lauraceae families. One of the main ingredients of clove oil (Syzygium aromaticum (L.), Myrtaceae), it has several applications in industry, including flavoring food, pharmaceutics, dentistry, agriculture, and cosmeceuticals. Due to its excellent potential for avoiding many chronic illnesses, it has lately attracted attention. EUG has been classified as a nonmutant, generally acknowledged as a safe (GRAS) chemical by the World Health Organization (WHO). According to the existing research, EUG possesses notable anti-inflammatory, antioxidant, analgesic, antibacterial, antispasmodic, and apoptosis-promoting properties, which have lately gained attention for its ability to control chronic inflammation, oxidative stress, and mitochondrial malfunction and dramatically impact human wellness. The purpose of this review is to evaluate the scientific evidence from the most significant research studies that have been published regarding the protective role and detoxifying effects of EUG against a wide range of toxins, including biological and chemical toxins, as well as different drugs and pesticides that produce a variety of toxicities, throughout view of the possible advantages of EUG.

pH-responsive on-demand release of eugenol from metal-organic frameworks for synergistic bacterial killing.

Bacterial infections are a big challenge in clinical treatment, making it urgent to develop innovative antibacterial systems and therapies to combat bacterial infections. In this study, we developed a novel MOF-based synergistic antibacterial system (Eu@B-UiO-66/Zn) by loading a natural antibacterial substance (eugenol) with hierarchically porous MOF B-UiO-66 as a carrier and further complexing it with divalent zinc ions. Results indicate that the system achieved a controlled release of eugenol under pH responsive stimulation, with the complexation ability of eugenol and Zn2+ ions as a switch. Due to the destruction of a coordination bond between eugenol and Zn2+ ions by an acidic medium, the release of eugenol loaded in Eu@B-UiO-66/Zn reached 80% at pH 5.8, which was significantly higher than that under pH 8.0 (51%). Moreover, the inhibitory effect of Eu@B-UiO-66/Zn against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) after 24 h was 96.4% and 99.7%, respectively, owing to the synergistic antibacterial effect of eugenol and Zn2+ ions, which was significantly stronger than free eugenol and Eu@B-UiO-66. We hope that this strategy for constructing responsive MOF-based antibacterial carriers could have potential possibilities for the application of MOF materials in antibacterial fields.

Targeting cardiovascular risk factors with eugenol: an anti-inflammatory perspective.

Inflammation is a multifaceted biological reaction to a wide range of stimuli, and it has been linked to the onset and progression of chronic diseases such as heart disease, cancer, and diabetes. Inflammatory markers found in the blood, including C-reactive protein, serum amyloid A, fibrinogen, plasma viscosity, erythrocyte sedimentation rate, interleukin-6, and soluble adhesion molecules (like intercellular adhesion molecule-1 and vascular cell adhesion molecule-1), are risk factors for cardiovascular diseases such as coronary heart disease, stroke, and peripheral arterial disease. These markers play a crucial role in understanding and assessing cardiovascular health. Due to this complicated relationship between inflammation and cardiovascular disease, anti-inflammatory agents of natural origin have been the subject of many preclinical and clinical studies in recent years. Eugenol is a natural phenolic compound found in clove oil, nutmeg oil, cinnamon oil, and bay leaf oil, as well as other essential oils. Eugenol has been shown to have anti-inflammatory properties in many forms of experimental inflammation. It may scavenge free radicals, which contribute to inflammation and tissue damage. Various studies also suggest that eugenol can limit the production of inflammatory mediators such as prostaglandins, cytokines, and chemokines. Animal models of arthritis, colitis, and lung damage, as well as human clinical studies, have shown that eugenol has phenomenal anti-inflammatory properties. These properties suggest that eugenol may be able to reduce the risk of cardiovascular diseases.

Do combinations of fipronil, eugenol and carvacrol have synergistic effects against Rhipicephalus sanguineus?

The tick Rhipicephalus sanguineus is one of the main ectoparasites that affects dogs, causing direct and indirect damage to parasitized animals. Currently, infestation control is mainly carried out by using synthetic acaricidal drugs. However, a decrease in efficacy and an increase in resistance to the main therapeutic protocols against tick infestations have been increasingly reported and confirmed, a factor that has driven research into the potential acaricide activity of natural compounds, including in association with synthetic molecules. The aim of this work was to evaluate whether the combinations of fipronil (FIP) and eugenol (EUG), FIP and carvacrol (CAR), and EUG and CAR would have synergistic effects against immature and unfed adult stages of R. sanguineus through in vitro bioassays. Bioassays were carried out using the larval packet test (FAO 2004) adapted for nymphs and adults. The synergistic activity was explored by combining each solution, based on the estimated LC50, in a 1:1 ratio (FIP: EUG, FIP: CAR and EUG: CAR). CompuSyn software was used to evaluate the various pairwise combinations of FIP, EUG and CAR, checking if there was synergism or antagonism between them. FIP and EUG and FIP and CAR showed combination index (CIn) values above 1.45, indicating antagonism. The synergistic activity between EUG and CAR was verified against all unfed phases of R. sanguineus, since the CIn was below 0.70, a value that indicates synergism. The combination of fipronil with either eugenol or carvacrol presented antagonistic effects against R. sanguineus larvae. On the other hand, carvacrol and eugenol had excellent pharmacological synergism against all tick stages with mortality values in the range of 80 to 100%, including the adult stage, which is less susceptible than immature stages.

Repurposing carvacrol, cinnamaldehyde, and eugenol as potential anti-quorum sensing agents against uropathogenic Escherichia coli isolates in Alexandria, Egypt.

BACKGROUND: Urinary tract infections represent one of the most frequent hospital and community-acquired infections with uropathogenic Escherichia coli (UPEC) being the main causative agent. The global increase in the emergence of multidrug-resistant (MDR) UPEC necessitates exploring novel approaches. Repurposing natural products as anti-quorum sensing (QS) agents to impede bacterial virulence is gaining momentum nowadays. Hence, this study investigates the anti-QS potentials of carvacrol, cinnamaldehyde, and eugenol against E. coli isolated from urine cultures of Egyptian patients. RESULTS: Antibiotic susceptibility testing was performed for 67 E. coli isolates and 94% of the isolates showed MDR phenotype. The usp gene was detected using PCR and accordingly, 45% of the isolates were categorized as UPEC. Phytochemicals, at their sub-inhibitory concentrations, inhibited the swimming and twitching motilities of UPEC isolates, with eugenol showing the highest inhibitory effect. The agents hindered the biofilm-forming ability of the tested isolates, at two temperature sets, 37 and 30 °C, where eugenol succeeded in significantly inhibiting the biofilm formation by > 50% at both investigated temperatures, as compared with untreated controls. The phytochemicals were shown to downregulate the expression of the QS gene (luxS) and critical genes related to motility, asserting their anti-QS potential. Further, the combinatory activity of the phytoproducts with five antibiotics was assessed by checkerboard assay. The addition of the phytoproducts significantly reduced the minimum inhibitory concentrations of the antibiotics and generated several synergistic or partially synergistic combinations, some of which have not been previously explored. CONCLUSIONS: Overall, carvacrol, cinnamaldehyde, and eugenol could be repurposed as potential anti-QS agents, which preferentially reduce the QS-based communication and attenuate the cascades of gene expression, thus decreasing the production of virulence factors in UPEC, and eventually, subsiding their pathogenicity. Furthermore, the synergistic combinations of these agents with antibiotics might provide a new perspective to circumvent the side effects brought about by high antibiotic doses, thereby paving the way for overcoming antibiotic resistance.

Antimicrobial-loaded biodegradable nanoemulsions for efficient clearance of intracellular pathogens in bacterial peritonitis.

Intracellular pathogenic bacteria use immune cells as hosts for bacterial replication and reinfection, leading to challenging systemic infections including peritonitis. The spread of multidrug-resistant (MDR) bacteria and the added barrier presented by host cell internalization limit the efficacy of standard antibiotic therapies for treating intracellular infections. We present a non-antibiotic strategy to treat intracellular infections. Antimicrobial phytochemicals were stabilized and delivered by polymer-stabilized biodegradable nanoemulsions (BNEs). BNEs were fabricated using different phytochemicals, with eugenol-loaded BNEs (E-BNEs) affording the best combination of antimicrobial efficacy, macrophage accumulation, and biocompatibility. The positively-charged polymer groups of the E-BNEs bind to the cell surface of macrophages, facilitating the entry of eugenol that then kills the intracellular bacteria without harming the host cells. Confocal imaging and flow cytometry confirmed that this entry occurred mainly via cholesterol-dependent membrane fusion. As eugenol co-localized and interacted with intracellular bacteria, antibacterial efficacy was maintained. E-BNEs reversed the immunosuppressive effects of MRSA on macrophages. Notably, E-BNEs did not elicit resistance selection after multiple exposures of MRSA to sub-therapeutic doses. The E-BNEs were highly effective against a murine model of MRSA-induced peritonitis with better bacterial clearance (99 % bacteria reduction) compared to clinically-employed treatment with vancomycin. Overall, these findings demonstrate the potential of E-BNEs in treating peritonitis and other refractory intracellular infections.

Hyposmia in COVID-19: Temporal Recovery of Smell: A Preliminary Study.

Background and Objectives: Hypo/anosmia is a characteristic symptom of COVID-19 infection. The aim of this study is to investigate the time of smell recovery and to identify a possible order of perception recovery of different odors in COVID-19 patients. Materials and Methods: A prospective observational study was conducted on not hospitalized COVID-19 patients, selected according to eligible criteria. The study was approved by the Ethical Committee. A questionnaire formulated by our team was submitted to patients in order to know the duration of the hypo/anosmia and hypo/ageusia and the order of odor recovery: vanillin (mixed olfactory/gustatory substances), phenyl ethyl alcohol (rosewater) (pure olfactory substances), eucalyptol (mixed olfactory/trigeminal substances), and eugenol (mixed olfactory/trigeminal/gustatory substances). Results: 181 patients were included. Hypo/ageusia and hypo/anosmia lasted on average 10.25 (±8.26) and 12.8 (±8.80) days, respectively. The most frequent odor recovery sequence was: (1) phenyl ethyl alcohol; (2) eucalyptol; (3) vanillin; and (4) eugenol. In COVID-19 patients, hypo/anosmia occurs more often in women and at a young age. Conclusions: This preliminary investigation highlighted novel data: there is a chronological order in perception recovery of different olfactory substances and, therefore, in the restoration of the various sensitive nerve pathways involved in the sense of smell.

Effects of ortho-eugenol on anxiety, working memory and oxidative stress in mice.

Ortho-eugenol is a synthetic derivative from eugenol, the major compound of clove essential oil, which has demonstrated antidepressant and antinociceptive effects in pioneering studies. Additionally, its effects appear to be dependent on the noradrenergic and dopaminergic systems. Depression and anxiety disorders are known to share a great overlap in their pathophysiology, and many drugs are effective in the treatment of both diseases. Furthermore, high levels of anxiety are related to working memory deficits and increased oxidative stress. Thus, in this study we investigated the effects of acute treatment of ortho-eugenol, at 50, 75 and 100 mg/kg, on anxiety, working memory and oxidative stress in male Swiss mice. Our results show that the 100 mg/kg dose increased the number of head-dips and reduced the latency in the hole-board test. The 50 mg/kg dose reduced malondialdehyde levels in the prefrontal cortex and the number of Y-maze entries compared to the MK-801-induced hyperlocomotion group. All doses reduced nitrite levels in the hippocampus. It was also possible to assess a statistical correlation between the reduction of oxidative stress and hyperlocomotion after the administration of ortho-eugenol. However, acute treatment was not able to prevent working memory deficits. Therefore, the present study shows that ortho-eugenol has an anxiolytic and antioxidant effect, and was able to prevent substance-induced hyperlocomotion. Our results contribute to the elucidation of the pharmacological profile of ortho-eugenol, as well as to direct further studies that seek to investigate its possible clinical applications.

Multi-omics reveals aspirin eugenol ester alleviates neurological disease.

BACKGROUND: Exosomes play an essential role in maintaining normal brain function due to their ability to cross the blood-brain barrier. Aspirin eugenol ester (AEE) is a new medicinal compound synthesized by the esterification of aspirin with eugenol using the prodrug principle. Aspirin has been reported to have neuroprotective effects and may be effective against neurodegenerative diseases. PURPOSE: This study wanted to investigate how AEE affected neurological diseases in vivo and in vitro. EXPERIMENTAL APPROACH: A multi-omics approach was used to explore the effects of AEE on the nervous system. Gene and protein expression changes of BDNF and NEFM in SY5Y cells after AEE treatment were detected using RT-qPCR and Western Blot. KEY RESULTS: The multi-omics results showed that AEE could regulate neuronal synapses, neuronal axons, neuronal migration, and neuropeptide signaling by affecting transport, inflammatory response, and regulating apoptosis. Exosomes secreted by AEE-treated Caco-2 cells could promote the growth of neurofilaments in SY5Y cells and increased the expression of BDNF and NEFM proteins in SY5Y cells. miRNAs in the exosomes of AEE-treated Caco-2 cells may play an important role in the activation of SY5Y neuronal cells. CONCLUSIONS: In conclusion, AEE could play positive effects on neurological-related diseases.

Diclofenac and eugenol hybrid with enhanced anti-inflammatory activity through activating HO-1 and inhibiting NF-κB pathway in vitro and in vivo.

A series of diclofenac hybrid molecules were synthesized and evaluated for their NO-inhibitory ability in LPS-induced RAW 264.7 macrophage cells. Among them, compound 1 showed the highest NO-inhibitory ability (approximately 66%) and no significant cytotoxicity. Compound 1 exhibited superior NF-κB-inhibitory ability compared to diclofenac through the activation of Nrf2/HO-1 signaling pathway in RAW 264.7. 20 mg/kg compound 1 resulted in remarkable colitis improvement in dextran sulfate sodium (DSS)-induced mice model by up-regulating HO-1 and down-regulating phosphorylation level of NF-κB p65. Moreover, 50 mg/kg dose of compound 1 showed a lower ulcerogenic potential compared to diclofenac in rats. The diclofenac-eugenol hybrid (compound 1) may serve as a novel anti-inflammatory agent based on its role in inhibiting the NF-κB signaling pathway and activating HO-1 expression with no toxicity in vitro and in vivo.

Therapeutic activity of eugenol towards mitigation of anaemia and oxidative organ damage caused by Plasmodium berghei.

The parasite responsible for causing malaria infection, Plasmodium, is known to exhibit resistance to a number of already available treatments. This has prompted the continue search for new antimalarial drugs ranging from medicinal plant parts to synthetic compounds. In lieu of this, the mitigative action of the bioactive compound, eugenol towards P. berghei-induced anaemia and oxidative organ damage was investigated following a demonstration of in vitro and in vivo antiplasmodial effects. Mice were infected with chloroquine-sensitive strain of P. berghei and thereafter treated with eugenol at doses of 10 and 20 mg/kg body weight (BW) for seven days. The packed cell volume and redox sensitive biomarkers in the liver, brain and spleen were measured. Our result demonstrated that eugenol significantly (p < 0.05) ameliorated the P. berghei-associated anaemia at a dose of 10 mg/kg BW. In addition, the compound, at a dose of 10 mg/kg BW, significantly (p < 0.05) alleviated the P. berghei-induced organ damage. This evidently confirmed that eugenol plays an ameliorative role towards P. berghei-related pathological alterations. Hence, the study opens up a new therapeutic use of eugenol against plasmodium parasite.

Eugenol relieves the pathological manifestations of Alzheimer's disease in 5×FAD mice.

BACKGROUND: Alzheimer's disease (AD) is a progressive neurodegenerative disease characterized by the accumulation of amyloid-ß (Aß) and excessive neuroinflammation, resulting in neuronal cell death and cognitive impairments. Eugenol, a phenylpropene, is the main component of Syzygium aromaticum L. (Myrtaceae) and has multiple therapeutic effects, including neuroprotective and anti-inflammatory effects, through multimodal mechanisms. PURPOSE: We aimed to investigate the effect of eugenol on AD pathologies using a 5× familiar AD (5×FAD) mouse model. METHODS: Eight-month-old 5×FAD and wild-type mice were administered with eugenol (10 or 30 mg/kg/day, p.o) for 2 months. Y-maze and Morris water maze tests were performed to assess the cognitive function of mice. After the behavioral test, molecular analysis was conducted to investigate the therapeutic mechanism of eugenol. RESULTS: Our findings indicate that eugenol treatment effectively mitigated cognitive impairments in 5×FAD mice. This beneficial effect was associated with a decrease in AD pathologies, including neuronal cell loss and Aß deposition. Specifically, eugenol inhibited necroptosis activation and increased microglial phagocytosis, which were the underlying mechanisms for the observed reductions in neuronal cell loss and Aß deposition, respectively. CONCLUSION: Overall, our data suggest that eugenol would be a potential therapeutic candidate for AD.

The Antileishmanial Activity of Eugenol Associated with Lipid Storage Reduction Rather Than Membrane Properties Alterations.

Leishmaniasis is a neglected tropical disease that still infects thousands of people per year throughout the world. The occurrence of resistance against major treatments for this disease causes a healthcare burden in low-income countries. Eugenol is a phenylpropanoid that has shown in vitro antileishmanial activity against Leishmania mexicana mexicana (Lmm) promastigotes with an IC50 of 2.72 µg/mL and a high selectivity index. Its specific mechanism of action has yet to be studied. We prepared large unilamellar vesicles (LUVs), mimicking Lmm membranes, and observed that eugenol induced an increase in membrane permeability and a decrease in membrane fluidity at concentrations much higher than IC50. The effect of eugenol was similar to the current therapeutic antibiotic, amphotericin B, although the latter was effective at lower concentrations than eugenol. However, unlike amphotericin B, eugenol also affected the permeability of LUVs without sterol. Its effect on the membrane fluidity of Lmm showed that at high concentrations (≥22.5× IC50), eugenol increased membrane fluidity by 20-30%, while no effect was observed at lower concentrations. Furthermore, at concentrations below 10× IC50, a decrease in metabolic activity associated with the maintenance of membrane integrity revealed a leishmaniostatic effect after 24 h of incubation with Lmm promastigotes. While acidocalcisomes distribution and abundance revealed by Trypanosoma brucei vacuolar H+ pyrophosphatase (TbVP1) immunolabeling was not modified by eugenol, a dose-dependent decrease of lipid droplets assessed by the Nile Red assay was observed. We hereby demonstrate that the antileishmanial activity of eugenol might not directly involve plasma membrane sterols such as ergosterol, but rather target the lipid storage of Lmm.

The Effect of Combined Treatment of Psilocybin and Eugenol on Lipopolysaccharide-Induced Brain Inflammation in Mice.

Inflammation is an organism's biological defense mechanism. Acute and chronic inflammation of the body triggers the production of pro- and anti-inflammatory pathways that can affect the content of cytokines in the brain and thus cause brain inflammation. Disorders such as depression and posttraumatic stress disorder (PTSD) are often associated with elevated inflammation. Recently, positive and promising clinical results of psilocybin for the treatment of depression and PTSD were reported. Thus, we decided to test whether psilocybin alone or in combination with eugenol, an anti-inflammatory and antioxidant agent, would prevent the increase in or decrease the content of cytokines in the brain of C57BL/6J mice injected with lipopolysaccharides (LPS). Two experiments were performed, one with pre-treatment of mice through gavage with psilocybin (0.88 mg/kg), eugenol (17.6 mg/kg), or combinations of psilocybin and eugenol (1:10, 1:20, or 1:50), followed by intraperitoneal injection of LPS, and the second, post-treatment, with initial injection with LPS, followed by treatment with psilocybin, eugenol, or their combination. Brain tissues were collected, and cytokines were analyzed by qRT-PCR, Western blot, and ELISA. Data were analyzed with a one-way ANOVA followed by Tukey's post hoc test or with multiple unpaired t-tests. LPS upregulated mRNA expression of COX-2, TNF-α, IL-1ß, and IL-6. All pre-treatments decreased the expression of COX-2 and TNF-α, with psilocybin alone and in 1:50 combination, with eugenol being the most effective. In the post-treatment, all combinations of psilocybin and eugenol were effective in reducing inflammation, with the 1:50 ratio displaying the most prominent results in reducing the mRNA content of tested cytokines. Western blot analysis confirmed the effect on COX-2 and IL-1ß proteins. Finally, the ELISA showed that post-treatment with psilocybin + eugenol (1:50) demonstrated the best results, decreasing the expression of multiple markers including IL-6 and IL-8. This demonstrates the anti-inflammatory effects of a combination of psilocybin and eugenol in the brain of animals with systemically induced inflammation.

Eugenol modulates the NOD1-NF-κB signaling pathway via targeting NF-κB protein in triple-negative breast cancer cells.

Background: The most aggressive subtype of breast cancer, triple-negative breast cancer (TNBC), has a worse prognosis and a higher probability of relapse since there is a narrow range of treatment options. Identifying and testing potential therapeutic targets for the treatment of TNBC is of high priority. Methods: Using a transcriptional signature of triple-negative breast cancer collected from Gene Expression Omnibus (GEO), CMap was utilized to reposition compounds for the treatment of TNBC. CCK8 and colony formation experiments were performed to detect the effect of the candidate drug on the proliferation of TNBC cells. Meanwhile, transwell and wound healing assay were implemented to detect cell metastasis change caused by the candidate drug. Moreover, the proteomic approach was presently ongoing to evaluate the underlying mechanism of the candidate drug in TNBC. Furthermore, drug affinity responsive target stability (DARTS) coupled with LC-MS/MS was carried out to explore the potential drug target candidate in TNBC cells. Results: We found that the most widely used medication, eugenol, reduced the growth and metastasis of TNBC cells. According to the underlying mechanism revealed by proteomics, eugenol could inhibit TNBC cell proliferation and metastasis via the NOD1-NF-κB signaling pathway. DARTS experiment further revealed that eugenol may bind to NF-κB in TNBC cells. Concludes: Our findings pointed out that eugenol was a potential candidate drug for the treatment of TNBC.