Bioactivities and Synergistic Effect of Elsholtzia ciliata Essential Oil and Its Main Components against Lasioderma serricorne.

Investigations have shown that storage bugs seriously harm grains during storage. In the interim, essential oils (EOs) have been proven to be a good botanical pesticide. The anti-Lasioderma serricorne properties of Elsholtzia ciliata essential oil, which was obtained by steam distillation, were evaluated using DL-limonene, carvone, and their two optical isomer components using contact, repelling, and fumigation techniques. Simultaneously, the fumigation, contact, and repellent activities of carvone and its two optical isomers mixed with DL-limonene against L. serruricorne were evaluated. The results showed that E. ciliata, its main components (R-carvone, DL-limonene), and S-carvone exhibited both fumigations (LC50 = 14.47, 4.42, 20.9 and 3.78 mg/L) and contact (LD50 = 7.31, 4.03, 28.62 and 5.63 µg/adult) activity against L.serricorne. A binary mixture (1:1) of R-carvone and DL-limonene displayed an obvious synergistic effect. A binary mixture (1:1) of carvone and its two optical isomers exhibited an obvious synergistic effect, too. Furthermore, the repellent activity of the EO, carvone, and its two optical isomers, DL-limonene, and a combination of them varied. To stop insect damage during storage, E. ciliata and its components can be utilized as bio-insecticides.

Molecular interactions between metformin and D-limonene inhibit proliferation and promote apoptosis in breast and liver cancer cells.

BACKGROUND: Cancer is a fatal disease that severely affects humans. Designing new anticancer strategies and understanding the mechanism of action of anticancer agents is imperative. HYPOTHESIS/PURPOSE: In this study, we evaluated the utility of metformin and D-limonene, alone or in combination, as potential anticancer therapeutics using the human liver and breast cancer cell lines HepG2 and MCF-7. STUDY DESIGN: An integrated systems pharmacology approach is presented for illustrating the molecular interactions between metformin and D-limonene. METHODS: We applied a systems-based analysis to introduce a drug-target-pathway network that clarifies different mechanisms of treatment. The combination treatment of metformin and D-limonene induced apoptosis in both cell lines compared with single drug treatments, as indicated by flow cytometric and gene expression analysis. RESULTS: The mRNA expression of Bax and P53 genes were significantly upregulated while Bcl-2, iNOS, and Cox-2 were significantly downregulated in all treatment groups compared with normal cells. The percentages of late apoptotic HepG2 and MCF-7 cells were higher in all treatment groups, particularly in the combination treatment group. Calculations for the combination index (CI) revealed a synergistic effect between both drugs for HepG2 cells (CI = 0.14) and MCF-7 cells (CI = 0.22). CONCLUSION: Our data show that metformin, D-limonene, and their combinations exerted significant antitumor effects on the cancer cell lines by inducing apoptosis and modulating the expression of apoptotic genes.

Unveiling the Molecular Interactions Between Human Transferrin and Limonene: Natural Compounds in Alzheimer's Disease Therapeutics.

Background: Neurodegeneration is a term describing an irreversible process of neuronal damage. In recent decades, research efforts have been directed towards deepening our knowledge of numerous neurodegenerative disorders, with a particular focus on conditions such as Alzheimer's disease (AD). Human transferrin (htf) is a key player in maintaining iron homeostasis within brain cells. Any disturbance in this equilibrium gives rise to the emergence of neurodegenerative diseases and associated pathologies, particularly AD. Limonene, a natural compound found in citrus fruits and various plants, has shown potential neuroprotective properties. Objective: In this study, our goal was to unravel the binding of limonene with htf, with the intention of comprehending the interaction mechanism of limonene with htf. Methods: Binding was scrutinized using fluorescence quenching and UV-Vis spectroscopic analyses. The binding mechanism of limonene was further investigated at the atomic level through molecular docking and extensive 200 ns molecular dynamic simulation (MD) studies. Results: Molecular docking uncovered that limonene interacted extensively with the deep cavity located within the htf binding pocket. MD results indicated that binding of limonene to htf did not induce substantial structural alterations, ultimately forming stable complex. The findings from fluorescence binding indicated a pronounced interaction between limonene and htf, limonene binds to htf with a binding constant (K) of 0.1×105 M-1. UV spectroscopy also advocated stable htf-limonene complex formation. Conclusions: The study deciphered the binding mechanism of limonene with htf, providing a platform to use limonene in AD therapeutics in context of iron homeostasis.

Inhibition profile of three biological nitrification inhibitors and their response to soil pH modification in two contrasting soils.

Up to 70% of the nitrogen (N) fertilizer applied to agricultural soils is lost through microbially mediated processes, such as nitrification. This can be counteracted by synthetic and biological compounds that inhibit nitrification. However, for many biological nitrification inhibitors (BNIs), the interaction with soil properties, nitrifier specificity, and effective concentrations are unclear. Here, we investigated three synthetic nitrification inhibitors (SNIs) (DCD, DMPP, and nitrapyrin) and three BNIs [methyl 3(4-hydroxyphenyl) propionate (MHPP), methyl 3(4-hydroxyphenyl) acrylate (MHPA), and limonene] in two agricultural soils differing in pH and nitrifier communities. The efficacies of SNIs and BNIs were resilient to short-term pH changes in the neutral pH soil, whereas the efficacy of some BNIs increased by neutralizing the alkaline soil. Among the BNIs, MHPA showed the highest inhibition and was, together with MHPP, identified as a putative AOB/comammox-selective inhibitor. Additionally, MHPA and limonene effectively inhibited nitrification at concentrations comparable to those used for DCD. Moreover, we identified the effective concentrations at which 50% and 80% of inhibition is observed (EC50 and EC80) for the BNIs, and similar EC80 values were observed in both soils. Overall, our results show that these BNIs could potentially serve as effective alternatives to SNIs currently used.

Synergistic interactions of essential oil components with antibiotics against multidrug-resistant Corynebacterium striatum.

AIM: In this study, it was aimed to examine the antibacterial activity of the essential oil components (EOCs), carvacrol (CAR), cinnamaldehyde (CIN), thymol (TH), alpha pinene (α-PN), eucalyptol (EU), limonene (LIM), and the antibiotics, linezolid (LZD), vancomycin (VAN), gentamicin (GEN), ciprofloxacin (CIP), clindamycin (CLN), and penicillin (PEN) against 50 multidrug resistant Corynebacterium striatum strains, and the synergistic interactions of CAR and CIN with the antibiotics against 10 randomly selected Coryne. striatum strains to explore synergistic interactions to determine if their combined use could enhance antibiotic activity and potentially reduce resistance. METHODS AND RESULTS: The activity of the EOCs and the antibiotics against Coryne. striatum strains isolated from clinical specimens, was examined by broth microdilution method. The synergistic interactions of the EOCs with the antibiotics against 10 randomly selected Coryne. striatum strains were determined by checkerboard method. EOCs, CIN, and CAR and antibiotics, LZD, VAN, GEN, CIP, and CLN were detected to have antibacterial activity against Coryne. striatum strains alone and either synergistic interactions were observed in combinations of the antibiotics with EOCs. CONCLUSIONS: All Coryne. striatum strains were determined to be susceptible to VAN and LZD and resistant to GEN, PEN, CIP, and CLN. Synergistic interactions were observed in all combinations of antibiotics tested with CAR and CIN.

Assessment of the in vitro acaricidal activity of Bravecto® (fluralaner) and a proposed orange oil-based formulation vehicle for the treatment of Sarcoptes scabiei.

BACKGROUND: Sarcoptic mange is a serious animal welfare concern in bare-nosed wombats (Vombatus ursinus). Fluralaner (Bravecto®) is a novel acaricide that has recently been utilised for treating mange in wombats. The topical 'spot-on' formulation of fluralaner can limit treatment delivery options in situ, but dilution to a volume for 'pour-on' delivery is one practicable solution. This study investigated the in vitro acaricidal activity of Bravecto, a proposed essential oil-based diluent (Orange Power®), and two of its active constituents, limonene and citral, against Sarcoptes scabiei. METHODS: Sarcoptes scabiei were sourced from experimentally infested pigs. In vitro assays were performed to determine the lethal concentration (LC50) and survival time of the mites when exposed to varying concentrations of the test solutions. RESULTS: All compounds were highly effective at killing mites in vitro. The LC50 values of Bravecto, Orange Power, limonene and citral at 1 h were 14.61 mg/ml, 4.50%, 26.53% and 0.76%, respectively. The median survival times of mites exposed to undiluted Bravecto, Orange Power and their combination were 15, 5 and 10 min, respectively. A pilot survival assay of mites collected from a mange-affected wombat showed survival times of < 10 min when exposed to Bravecto and Orange Power and 20 min when exposed to moxidectin. CONCLUSIONS: These results confirm the acaricidal properties of Bravecto, demonstrate acaricidal properties of Orange Power and support the potential suitability of Orange Power and its active constituents as a diluent for Bravecto. As well as killing mites via direct exposure, Orange Power could potentially enhance the topical delivery of Bravecto to wombats by increasing drug penetration in hyperkeratotic crusts. Further research evaluating the physiochemical properties and modes of action of Orange Power and its constituents as a formulation vehicle would be of value.

Ligand-based drug design against Herpes Simplex Virus-1 capsid protein by modification of limonene through in silico approaches.

The pharmacological effects of limonene, especially their derivatives, are currently at the forefront of research for drug development and discovery as well and structure-based drug design using huge chemical libraries are already widespread in the early stages of therapeutic and drug development. Here, various limonene derivatives are studied computationally for their potential utilization against the capsid protein of Herpes Simplex Virus-1. Firstly, limonene derivatives were designed by structural modification followed by conducting a molecular docking experiment against the capsid protein of Herpes Simplex Virus-1. In this research, the obtained molecular docking score exhibited better efficiency against the capsid protein of Herpes Simplex Virus-1 and hence we conducted further in silico investigation including molecular dynamic simulation, quantum calculation, and ADMET analysis. Molecular docking experiment has documented that Ligands 02 and 03 had much better binding affinities (- 7.4 kcal/mol and - 7.1 kcal/mol) to capsid protein of Herpes Simplex Virus-1 than Standard Acyclovir (- 6.5 kcal/mol). Upon further investigation, the binding affinities of primary limonene were observed to be slightly poor. But including the various functional groups also increases the affinities and capacity to prevent viral infection of the capsid protein of Herpes Simplex Virus-1. Then, the molecular dynamic simulation confirmed that the mentioned ligands might be stable during the formation of drug-protein complexes. Finally, the analysis of ADMET was essential in establishing them as safe and human-useable prospective chemicals. According to the present findings, limonene derivatives might be a promising candidate against the capsid protein of Herpes Simplex Virus-1 which ultimately inhibits Herpes Simplex Virus-induced encephalitis that causes interventions in brain inflammation. Our findings suggested further experimental screening to determine their practical value and utility.

Pharmacological potential of limonene against opportunistic fungi: Impact on Candida virulence.

The present article aims to evaluate the antifungal and antivirulence effect of the phytoconstituent Limonene against Candida spp. Antifungal assays were performed, where the concentration capable of inhibiting 50 % of fungal growth, the growth inhibition curve, the minimum fungicidal concentration, the evaluation of the modifying effect with fluconazole, the inhibitory effect of the substances on the morphological transition of Candida spp. and the statistical analysis of the results were determined. With this study, it was seen that limonene demonstrated growth inhibition for the strains tested and when associated the natural compound with Fluconazole, there was potentiation of the effect of the drug, since the inhibition of growth by the combination occurred at lower concentrations against all strains tested, when compared to the drug alone, which inhibited growth at the highest concentration. In the test to determine the Minimum Fungicidal Concentration of the products tested alone and in combination, it was found that in the case of Candida strains, growth inhibition by limonene occurred at a concentration of 1024 µg/mL. For Fluconazole, growth impairment ranged from > 1024 µg/mL to 256 µg/mL for the strains. And when combined, limonene potentiated the action of FCZ, making fungal colonization unfeasible at concentrations below 1024 µg/mL. Regarding the morphological transition from yeast to hyphae, limonene was used at concentrations of 1024 µg/mL and 512 µg/mL, and it was found that, for CA and CK, the filaments were reduced in number and size at the highest concentration and against CT, the morphological transition from yeast to hyphae/pseudohyphae was totally inhibited, and if compared to the growth control, limonene was able to reduce fungal growth at concentrations greater than 512 µg/mL. This compound has antimicrobial activity described, due to its ability to interfere in the gene expression of the fungus, the limited therapeutic options and the recent emergence of multidrug-resistant Candida species represent a significant challenge for human medicine and highlight the need for new therapeutic approaches, and in this study a great potential of limonene was revealed in relation to the perspective of increasing the efficiency of commercial drug. This work can bring an important contribution to the scientific database, while emphasizing that in-depth studies and tests on the subject, in order to better investigate its effectiveness and mechanisms by which they exert their effects, are still necessary.

Limonene Exerts Anti-Inflammatory Effect on LPS-Induced Jejunal Injury in Mice by Inhibiting NF-κB/AP-1 Pathway.

The human gastrointestinal system is a complex ecosystem crucial for well-being. During sepsis-induced gut injury, the integrity of the intestinal barrier can be compromised. Lipopolysaccharide (LPS), an endotoxin from Gram-negative bacteria, disrupts the intestinal barrier, contributing to inflammation and various dysfunctions. The current study explores the protective effects of limonene, a natural compound with diverse biological properties, against LPS-induced jejunal injury in mice. Oral administration of limonene at dosages of 100 and 200 mg/kg was used in the LPS mouse model. The Murine Sepsis Score (MSS) was utilized to evaluate the severity of sepsis, while serum levels of urea and creatinine served as indicators of renal function. Our results indicated that LPS injection induced renal function deterioration, evidenced by elevated serum urea and creatinine levels compared to control mice. However, pretreatment with limonene at doses of 100 and 200 mg/kg mitigated this decline in renal function, evidenced from the reduced levels of serum urea and creatinine. Limonene demonstrated anti-inflammatory effects by reducing pro-inflammatory cytokines (TNF-α, IL-1ß, COX-2), suppressing the TLR4/NF-κB/AP-1 but not IRF3 signaling pathways, and modulating oxidative stress through Nrf2 activation. The results suggest that limonene holds promise as a potential therapeutic agent for mitigating intestinal inflammation and preserving gastrointestinal health.

D-limonene inhibits peritoneal adhesion formation in rats via anti-inflammatory, anti-angiogenic, and antioxidative effects.

The aim of this research was to investigate the effects of D-limonene on decreasing post-operative adhesion in rats and to understand the mechanisms involved. Peritoneal adhesions were induced by creating different incisions and excising a 1 × 1 cm section of the peritoneum. The experimental groups included a sham group, a control group in which peritoneal adhesions were induced without any treatment, and two treatment groups in which animals received D-limonene with dosages of 25 and 50 mg/kg after inducing peritoneal adhesions. Macroscopic examination of adhesions showed that both treatment groups had reduced adhesion bands in comparison to the control group. Immunohistochemical assessment of TGF-ß1, TNF-α, and VEGF on day 14 revealed a significant increment in the level of immunopositive cells for the mentioned markers in the control group, whereas administration of limonene in both doses significantly reduced levels of TGF-ß1, TNF-α, and VEGF (P < 0.05). Induction of peritoneal adhesions in the control group significantly increased TGF-ß1, TNF-α, and VEGF on days 3 and 14 in western blot evaluation, while treatment with limonene significantly reduced TNF-α level on day 14 (P < 0.05). Moreover, VEGF levels in both treatment groups significantly reduced on days 3 and 14. In the control group, a significant increment in the levels of MDA and NO and a notable decline in the levels of GPX, CAT was observed (P < 0.05). Limonene 50 group significantly reduced MDA level and increased GPx and CAT levels on day 14 (P < 0.05). In summary, D-limonene reduced adhesion bands, inflammatory cytokines, angiogenesis, and oxidative stress.

Design and elucidation of an insecticide from natural compounds targeting mitochondrial proteins of Aedes aegypti.

Developing new pesticides poses a significant challenge in designing next-generation natural insecticides that selectively target specific pharmacological sites while ensuring environmental friendliness. In this study, we aimed to address this challenge by formulating novel natural pesticides derived from secondary plant metabolites, which exhibited potent insecticide activity. Additionally, we tested their effect on mitochondrial enzyme activity and the proteomic profile of Ae. aegypti, a mosquito species responsible for transmitting diseases. Initially, 110 key compounds from essential oils were selected that have been reported with insecticidal properties; then, to ensure safety for mammals were performed in silico analyses for toxicity properties, identifying non-toxic candidates for further investigation. Subsequently, in vivo tests were conducted using these non-toxic compounds, focusing on the mosquito's larval stage. Based on the lethal concentration (LC), the most promising compounds as insecticidal were identified as S-limonene (LC50 = 6.4 ppm, LC95 = 17.2 ppm), R-limonene (LC50 = 9.86 ppm, LC95 = 27.7 ppm), citronellal (LC50 = 40.5 ppm, LC95 = 68.6 ppm), R-carvone (LC50 = 61.4 ppm, LC95 = 121 ppm), and S-carvone (LC50 = 62.5 ppm, LC95 = 114 ppm). Furthermore, we formulated a mixture of R-limonene, S-carvone, and citronellal with equal proportions of each compound based on their LC50. This mixture specifically targeted mitochondrial proteins and demonstrated a higher effect that showed by each compound separately, enhancing the insecticidal activity of each compound. Besides, the proteomic profile revealed the alteration in proteins involved in proliferation processes and detoxification mechanisms in Ae. aegypti. In summary, our study presents a formulation strategy for developing next-generation natural insecticides using secondary plant metabolites with the potential for reducing the adverse effects on humans and the development of chemical resistance in insects. Our findings also highlight the proteomic alteration induced by the formulated insecticide, showing insight into the mechanisms of action and potential targets for further exploration in vector control strategies.

Molecular and Phenotypic Investigation on Antibacterial Activities of Limonene Isomers and Its Oxidation Derivative against Xanthomonas oryzae pv. oryzae.

Xanthomonas oryzae pv. oryzae (Xoo) causes a devastating bacterial leaf blight in rice. Here, the antimicrobial effects of D-limonene, L-limonene, and its oxidative derivative carveol against Xoo were investigated. We revealed that carveol treatment at ≥ 0.1 mM in liquid culture resulted in significant decrease in Xoo growth rate (> 40%) in a concentration-dependent manner, and over 1 mM, no growth was observed. The treatment with D-limonene and L-limonene also inhibited the Xoo growth but to a lesser extent compared to carveol. These results were further elaborated with the assays of motility, biofilm formation and xanthomonadin production. The carveol treatment over 1 mM caused no motilities, basal level of biofilm formation (< 10%), and significantly reduced xanthomonadin production. The biofilm formation after the treatment with two limonene isomers was decreased in a concentration-dependent manner, but the degree of the effect was not comparable to carveol. In addition, there was negligible effect on the xanthomonadin production mediated by the treatment of two limonene isomers. Field emission-scanning electron microscope (FE-SEM) unveiled that all three compounds used in this study cause severe ultrastructural morphological changes in Xoo cells, showing shrinking, shriveling, and holes on their surface. Moreover, quantitative real-time PCR revealed that carveol and D-limonene treatment significantly down-regulated the expression levels of genes involved in virulence and biofilm formation of Xoo, but not with L-limonene. Together, we suggest that limonenes and carveol will be the candidates of interest in the development of biological pesticides.

Essential oil from Citrus depressa peel exhibits antimicrobial, antioxidant and cancer chemopreventive effects.

BACKGROUND: Many diseases may be caused by pathogens and oxidative stress resulting from carcinogens. Earlier studies have highlighted the antimicrobial and antioxidant effects of plant essential oils (EO). It is crucial to effectively utilize agricultural waste to achieve a sustainable agricultural economy and protect the environment. The present study aimed to evaluate the potential benefits of EO extracted from the discarded peels of Citrus depressa Hayata (CD) and Citrus microcarpa Bunge (CM), synonyms of Citrus deliciosa Ten and Citrus japonica Thunb, respectively. RESULTS: Gas chromatography-mass spectrometry analysis revealed that the main compounds in CD-EO were (R)-(+)-limonene (38.97%), γ-terpinene (24.39%) and linalool (6.22%), whereas, in CM-EO, the main compounds were (R)-(+)-limonene (48.00%), ß-pinene (13.60%) and γ-terpinene (12.07%). CD-EO exhibited inhibitory effects on the growth of common microorganisms, including Candida albicans, Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus. However, CM-EO showed only inhibitory effects on E. coli. Furthermore, CD-EO exhibited superior antioxidant potential, as demonstrated by its ability to eliminate 1,1-diphenyl-2-picrylhydrazyl and 2,2'-azinobis-3-ethylbenzthiazoline-6-sulfonate free radicals. Furthermore, CD-EO at a concentration of 100 µg mL-1 significantly inhibited 12-O-tetradecanoylphorbol-13-acetate-induced cancer transformation in mouse epidermal JB6 P+ cells (P < 0.05), possibly by up-regulating protein expression of nuclear factor erythroid 2-related factor 2 and its downstream antioxidant enzymes, such as NAD(P)H:quinone oxidoreductase 1, heme oxygenase-1 and UGT1A. CONCLUSION: These findings suggest that CD-EO exhibits inhibitory effects on pathogenic microorganisms, possesses antioxidant properties and has cancer chemopreventive potential. © 2024 Society of Chemical Industry.

Volatile Compounds Emitted by Plant Growth-Promoting Fungus Tolypocladium inflatum GT22 Alleviate Copper and Pathogen Stress.

Previous studies on the intricate interactions between plants and microorganisms have revealed that fungal volatile compounds (VCs) can affect plant growth and development. However, the precise mechanisms underlying these actions remain to be delineated. In this study, we discovered that VCs from the soilborne fungus Tolypocladium inflatum GT22 enhance the growth of Arabidopsis. Remarkably, priming Arabidopsis with GT22 VCs caused the plant to display an enhanced immune response and mitigated the detrimental effects of both pathogenic infections and copper stress. Transcriptomic analyses of Arabidopsis seedlings treated with GT22 VCs for 3, 24 and 48 h revealed that 90, 83 and 137 genes were differentially expressed, respectively. The responsive genes are known to be involved in growth, hormone regulation, defense mechanisms and signaling pathways. Furthermore, we observed the induction of genes related to innate immunity, hypoxia, salicylic acid biosynthesis and camalexin biosynthesis by GT22 VCs. Among the VCs emitted by GT22, exposure of Arabidopsis seedlings to limonene promoted plant growth and attenuated copper stress. Thus, limonene appears to be a key mediator of the interaction between GT22 and plants. Overall, our findings provide evidence that fungal VCs can promote plant growth and enhance both biotic and abiotic tolerance. As such, our study suggests that exposure of seedlings to T. inflatum GT22 VCs may be a means of improving crop productivity. This study describes a beneficial interaction between T. inflatun GT22 and Arabidopsis. Our investigation of microorganism function in terms of VC activities allowed us to overcome the limitations of traditional microbial application methods. The importance of this study lies in the discovery of T. inflatun GT22 as a beneficial microorganism. This soilborne fungus emits VCs with plant growth-promoting effects and the ability to alleviate both copper and pathogenic stress. Furthermore, our study offers a valuable approach to tracking the activities of fungal VC components via transcriptomic analysis and sheds light on the mechanisms through which VCs promote plant growth and induce resistance. This research significantly advances our knowledge of VC applications and provides an example for further investigations within this field.

In vitro antiproliferative and apoptotic effects of thiosemicarbazones based on (-)-camphene and R-(+)-limonene in human melanoma cells.

A series of 38 thiosemicarbazone derivatives based on camphene and limonene were evaluated for their antiproliferative activity. Among them, 19 were synthesized and characterized using proton and carbon-13 nuclear magnetic resonance (1H and 13C NMR). For initial compound selection, human melanoma cells (SK-MEL-37) were exposed to a single concentration of a compound (100 µM) for 24, 48, and 72 hours, and cell detachment was visually observed. Cell viability was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. Nineteen compounds (4, 6, 8, 11, 13, 14, 15, 16, 17, 18, 20, 22, 25, 26, 31, 3', 4', 6', and 9') yielded cell viability below 20%. Subsequently, IC50 values for these compounds were determined, ranging from 11.56 to 55.38 µM, after 72 hours of treatment. Compound 17 (o-hydroxybenzaldehyde (-)-camphene-based thiosemicarbazone) demonstrated the lowest IC50 value, followed by compound 4 (benzaldehyde (-) camphene-based thiosemicarbazone) at 12.84 µM. Regarding compound 4, we observed the induction of a characteristic ladder pattern of DNA fragmentation through gel electrophoresis. Furthermore, fluorescence, flow cytometry and scanning microscopy assays revealed morphological changes consistent with apoptosis induction. Additionally, the measurement of caspase 6 and 8 activity in cellular extracts after treatment for 2, 4, 6, and 24 hours suggested the potential involvement of the extrinsic apoptosis pathway in the mechanism of action of compound 4. Further investigations, including molecular docking studies, are required to fully explore the potential of compound 4 and the other selected compounds, highlighting their promising role in future melanoma therapy research.

Combined effects of Limonene and Ivermectin on P-glycoprotein-9 gene expression of lambs Infected with Haemonchus contortus.

Although ivermectin (IVM) has a wide spectrum and long half-life, its frequent use as an anthelmintic for the last 42 years led to its worldwide tolerance by Haemonchus contortus. We evaluated the combination of limonene (LIM), a P-glycoprotein (Pgp) modulator, with IVM in lambs infected with a multidrug-resistant H. contortus. Twenty-four male Dorper lambs were artificially infected with two doses (seven days apart) of 8000 infective larvae of a multidrug-resistant isolate of H. contortus. The infection was patent 25 days later. Fifteen days before treatment with IVM (DAY -15), animals were divided into 4 groups: Infected-untreated control (CTL), IVM, LIM, and LIM+IVM. From DAY -15 to DAY + 14, groups LIM and LIM+IVM received 200 mg/kg of body weight/day of LIM via oral. On DAY 0, a single dose of IVM at 200 µg/kg of body weight was administered orally to groups IVM and LIM+IVM. On DAY + 7 and DAY + 14, fecal egg counts (FEC) were performed and on DAY + 14 animals were euthanized for total worm count (TWC), worm length, fecundity of females, and Pgp-9 gene expression. On DAY + 7, group LIM+IVM had 96.29% efficacy based on Fecal Egg Count Reduction TEST (FECRT) and a highly significant reduction in FEC (P = 0.0005) when compared to CTL. On DAY + 14, the efficacy of LIM+IVM was 82.87% on FECRT, although no differences were found among groups for FEC, TWC, worm length, or Pgp-9 gene expression. Female worms from the CTL group had higher egg counts in their uterus when compared to LIM. No differences were found for hematological or biochemical parameters, body weight, or weight gain among groups. Thus, LIM given daily at 200 mg/kg was safe for animals and, when combined with IVM, decreased egg shedding and could reduce pasture contamination, although it was unable to kill multidrug-resistant H. contortus.

Effect of vitamin E, D-limonene, and their combination on nulliparous rabbit reproductive performance.

Oxidative status is important in reproductive performance and using two natural antioxidants is more beneficial than one in nulliparous rabbits. The goal, effect of vitamin E (VitE), D-limonene (DL), and VitE+DL on maternal LBW (MLBW), conception (CR), pregnancy (PR), and kindling rates (KR), gestation length (GL), total litter size at birth (TLSB) and weaning (TLSW), live kits at birth (LKB) and weaning (LKW), dead kits at birth (DKB) and weaning (DKW), kits mortality rate at weaning (KMRW), Kit weight at birth (KWB) and weaning (KWW), total kit weight at birth (TKWB) and weaning (TKWW), and concentrations of progesterone (P4) and Malondialdehyde (MDA), during first two pregnancies. A total of 24 healthy female WNZ rabbits were randomly selected and assigned into four groups (6/each). Control (animals injected with 1.0 mL propylene glycol), VitE (60 mg IM injection/animal, 2X/week pre-mating and 3X post-mating until mid-pregnancy, DL (20 mg IM injection/animal, 2X/week pre-mating and 1X at mating, and VitE+DL (IM injection/animal with the same doses and times applied in VitE and DL groups. All animals were treated during 1st pregnancy only. The results confirmed that animals treated with VitE+DL gained significant maternal LBW in 1st pregnancy, reduced dead kit number at birth and kit mortality rate at weaning, increased live kits and total kit weight at birth and weaning in the two pregnancies, and also increased significantly kit weight at birth and weaning in the treatments than controls in the two pregnancies, and DL was greater in 1st pregnancy. Progesterone concentrations in mid-pregnancy rose significantly in VitE+DL during 1st pregnancy and DL in 2nd pregnancy. Malondialdehyde concentrations dropped significantly in VitE and VitE+DL in mid-pregnancy in the two pregnancies. Eventually, the integration of VitE and DL displayed their unique properties for improving productive and reproductive performance in nulliparous rabbits.

Expediting Multiple Biological Properties of Limonene and α-Pinene: Main Bioactive Compounds of Pistacia lentiscus L., Essential Oils.

BACKGROUND: Screening new natural molecules with pharmacological and/or cosmetic properties remains a highly sought-after area of research. Moreover, essential oils and volatile compounds have recently garnered significant interest as natural substance candidates. In this study, the volatile components of Pistacia lentiscus L. essential oils (PLEOs) isolated from the fruit and its main compounds, alpha-pinene, and limonene, are investigated for antioxidant, antidiabetic, and dermatoprotective activities. METHODS: In vitro antioxidant activity was investigated using 2,2'-diphenyl-1-picrylhydrazyl (DPPH), fluorescence recovery after photobleaching (FRAP), and 2,2-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) methods. The antidiabetic and dermatoprotective effects were studied using enzyme inhibitory activities. RESULTS: Antioxidant tests showed that PLEO has the best activity (ranging from 29.64 ± 3.04 to 73.80 ± 3.96 µg/mL) compared to its main selected molecules (ranging from 74 ± 3.72 to 107.23 ± 5.03 µg/mL). The α-glucosidase and α-amylase assays demonstrated that the elements tested have a promising antidiabetic potential with IC50values ranging from 78.03 ± 2.31 to 116.03 ± 7.42 µg/mL and 74.39 ± 3.08 to 112.35 ± 4.92 µg/mL for the α-glucosidase and α-amylase assays, respectively, compared to the standard drug. For the tyrosinase test, we found that the EOs (IC50 = 57.72 ± 2.86 µg/mL) followed by limonene (IC50 = 74.24 ± 2.06 µg/mL) and α-pinene (IC50 = 97.45 ± 5.22 µg/mL) all exhibited greater inhibitory effects than quercetin (IC50 = 246.90 ± 2.54 µg/mL). CONCLUSIONS: Our results suggest that the biological activities of PLEO, as well as its main compounds, make them promising candidates for the development of new strategies aimed at improving dermatoprotection and treating diseases associated with diabetes mellitus and oxidative stress.

Limonene anti-TMV activity and its mode of action.

The main component of orange peel essential oil is limonene. Limonene is a natural active monoterpene with multiple functions, such as antibacterial, antiseptic and antitumor activity, and has important development value in agriculture. This study found that limonene exhibited excellent anti-tobacco mosaic virus (TMV) bioactivity, with results showing that its protection activity, inactivation activity, and curative activity at 800 µg/mL were 84.93%, 59.28%, and 58.89%, respectively-significantly higher than those of chito-oligosaccharides. A direct effect of limonene on TMV particles was not observed, but limonene triggered the hypersensitive response (HR) in tobacco. Further determination of the induction activity of limonene against TMV demonstrated that it displayed good induction activity at 800 µg/mL, with a value of 60.59%. The results of physiological and biochemical experiments showed that at different treatment days, 800 µg/mL limonene induced the enhancement of defense enzymes activity in tobacco, including of SOD, CAT, POD, and PAL, which respectively increased by 3.2, 4.67, 4.12, and 2.33 times compared with the control (POD and SOD activities reached highest on the seventh day, and PAL and CAT activities reached highest on the fifth day). Limonene also enhanced the relative expression levels of pathogenesis related (PR) genes, including NPR1, PR1, and PR5, which were upregulated 3.84-fold, 1.86-fold and 1.71-fold, respectively. Limonene induced the accumulation of salicylic acid (SA), and increased the relative expression levels of genes related to SA biosynthesis (PAL) and reactive oxygen species (ROS) burst (RBOHB), which respectively increased by 2.76 times and 4.23 times higher than the control. Systemic acquired resistance (SAR) is an important plant immune defense against pathogen infection. The observed accumulation of SA, the enhancement of defense enzymes activity and the high-level expression of defense-related genes suggested that limonene may induce resistance to TMV in tobacco by activating SAR mediated by the SA signaling pathway. Furthermore, the experimental results demonstrated that the expression level of the chlorophyll biosynthesis gene POR1 was increased 1.72-fold compared to the control in tobacco treated with 800 µg/mL limonene, indicating that limonene treatment may increase chlorophyll content in tobacco. The results of pot experiment showed that 800 µg/mL limonene induced plant resistance against Sclerotinia sclerotiorum (33.33%), Phytophthora capsici (54.55%), Botrytis cinerea (50.00%). The bioassay results indicated that limonene provided broad-spectrum and long-lasting resistance to pathogen infection. Therefore, limonene has good development and utilization value, and is expected to be developed into a new botanical-derived anti-virus agent and plant immunity activator in addition to insecticides and fungicides.

In Silico Prediction of Selected Bioactive Compounds Present in Alpinia elegans (C.Presl) K.Schum Seed Oil as Potential Drug Candidates Against Human Cancer Cell Lines.

OBJECTIVE: Alpinia elegans (Zingiberaceae) is a Philippine endemic plant reported to have various folkloric uses. The seed oil of A. elegans has been shown to contain a majority of the following bioactive compounds: D-limonene, α-pinene, and caryophyllene oxide. The study sought to determine if the bioactive compounds found in A. elegans seed oil would be a good natural, inexpensive, and less-detrimental alternative for cancer treatment. METHODS: The study utilized in silico (Way2Drug predictive services, SwissADME, AutoDock 4) experiment to examine the aforementioned compounds as viable therapeutic candidates against human cancer cell lines. RESULT: Results determined that the compounds D-limonene, α-pinene, and caryophyllene oxide were most potent against thyroid gland carcinoma (8505C) cells, brain glaucoma (Hs 683) cells, and promyeloblast leukemia (HL-60) cells, respectively. Additionally, D-limonene was the only compound to show arrhythmia as an adverse effect. Predictions showed that the compounds could inhibit cellular growth factors and serine/threonine-protein kinase activity. The compounds generated a bioavailability score of 0.55 and exhibited blood-brain barrier (BBB) penetration. D-limonene, α-pinene, and caryophyllene oxide had binding energy of -4.59, -5.43, and -6.92, respectively. CONCLUSION: The binding energy indicated that the ligands could securely dock to the receptors, thus suggesting that interaction between the ligands and receptors was stable. Results have shown that the compounds are promising candidates against human cancer cell lines by inhibiting cell proliferation and inducing apoptosis.