Controlled in vivo intrinsic detrimental effect of d-Limonene channelized by influential proximal interaction through apoptosis and steatosis in embryonic zebrafish (Danio rerio).

Bioaccumulation of d-Limonene in environment due to the aggrandised usage of their natural sources like citrus food wastes and industrial day to day life products has raised concern to their biotoxicity to environment biotic health. Moreover, their after-usage discharge to aquatic system has enhanced the distress of posing threat and needs attention. This study entails mechanistic and molecular evaluation of in-vivo biotoxicity of d-Limonene in zebrafish embryo models. Experimental analysis excavated the controlled concentration-dependent morphological, physiological and cellular in-vivo impact of d-Limonene in zebrafish embryos through significant changes in oxidative stress, steatosis and apoptosis regulated via 6-fold and 5-fold mRNA expression change in p53 and Sod1 genes. Computational evaluation deduced the cellular mechanism of d-limonene biotoxicity as irregularities in oxidative stress, apoptosis and steatosis due of their intrinsic interaction with metabolic proteins like Zhe1a (-4.8 Kcal/mol), Sod1(-5.3 Kcal/mol), p53, caspase3 and apoa1 leading to influential change in structural and functional integrity of the metabolic proteins. The study unravelled the measured in-vivo biotoxicity of d-Limonene at cellular and molecular level to advocate the controlled usage of d-Limonene related natural and industrial product for a sustainable environmental health.

D-Limonene Inhibits Pichia kluyveri Y-11519 in Sichuan Pickles by Disrupting Metabolism.

The Pichia kluyveri, a proliferation commonly found in Sichuan pickles (SCPs), can accelerate the growth and reproduction of spoilage bacteria, causing off-odor development and decay. Although D-limonene, a common natural preservative, effectively restricts P. kluyveri, its inhibitory mechanism remains unclear. This study aimed to elucidate this molecular mechanism by investigating the impact on basic P. kluyveri metabolism. The findings revealed that D-limonene inhibited P. kluyveri growth and disrupted the transcription of the genes responsible for encoding the enzymes involved in cell wall and membrane synthesis, oxidative phosphorylation, glycolysis, and the tricarboxylic acid (TCA) cycle pathway. The results indicated that these events disrupted crucial metabolism such as cell wall and membrane integrity, adenosine triphosphate (ATP) synthesis, and reactive oxygen species (ROS) balance. These insights provided a comprehensive understanding of the inhibitory effect of D-limonene on the growth and reproduction of P. kluyveri while highlighting its potential application in the SCP industry.

Metabolic profiling and enzyme inhibitory activity of the essential oil of citrus aurantium fruit peel.

BACKGROUND: Bitter orange (Citrus aurantium) is a fruiting shrub native to tropical and subtropical countries around the world and cultivated in many regions due to its nutraceutical value. The current study investigated the metabolic profiling and enzyme inhibitory activities of volatile constituents derived from the C. aurantium peel cultivated in Egypt by three different extraction methods. METHODS: The volatile chemical constituents of the peel of C. aurantium were isolated using three methods; steam distillation (SD), hydrodistillation (HD), and microwave-assisted hydrodistillation (MAHD), and then were investigated by GC-MS. The antioxidant potential was evaluated by different assays such as DPPH, ABTS, FRAP, CUPRAC, and phosphomolybdenum and metal chelating potential. Moreover, the effect of enzyme inhibition of the three essential oils was tested using BChE, AChE, tyrosinase, glucosidase, as well as amylase assays. RESULTS: A total of six compounds were detected by GC/MS analysis. The major constituent obtained by all three extraction methods was limonene (98.86% by SD, 98.68% by HD, and 99.23% by MAHD). Differences in the composition of the compounds of the three oils were observed. The hydrodistillation technique has yielded the highest number of compounds, notably two oxygenated monoterpenes: linalool (0.12%) and α-terpineol acetate (0.1%). CONCLUSION: In our study differences in the extraction methods of C. aurantium peel oils resulted in differences in the oils' chemical composition. Citrus essential oils and their components showed potential antioxidant, anticholinesterase, antimelanogenesis, and antidiabetic activities. The presence of linalool and α-terpineol acetate may explain the superior activity observed for the oil isolated by HD in both radical scavenging and AChE inhibition assays, as well as in the enzyme inhibition assays.

HSPiP, Computational, and Thermodynamic Model-Based Optimized Solvents for Subcutaneous Delivery of Tolterodine Tartrate and GastroPlus­Based In Vivo Prediction in Humans: Part II.

In part I, we reported Hansen solubility parameters (HSP, HSPiP program), experimental solubility at varied temperatures for TOTA delivery. Here, we studied dose volume selection, stability, pH, osmolality, dispersion, clarity, and viscosity of the explored combinations (I-VI). Ex vivo permeation and deposition studies were performed to observe relative diffusion rate from the injected site in rat skin. Confocal laser scanning microscopy (CLSM) study was conducted to support ex vivo findings. Moreover, GastroPlus predicted in vivo parameters in humans and the impact of various critical factors on pharmacokinetic parameters (PK). Immediate release product (IR) contained 60% of PEG400 whereas controlled release formulation (CR) contained PEG400 (60%), water (10%) and d-limonene (30%) to deliver 2 mg of TOTA. GastroPlus predicted the plasma drug concentration of weakly basic TOTA as function of pH (from pH 2.0 to 9). The cumulative drug permeation and drug deposition were found to be in the order as B-VI˃ C-VI˃A-VI across rat skin. This finding was further supported with CLSM. Moreover, IR and CR were predicted to achieve Cmax of 0.0038 µg/ mL and 0.00023 µg/mL, respectively, after sub-Q delivery. Added limonene in CR extended the plasma drug concentration over period of 12 h as predicted in GastroPlus. Parameters sensitivity analysis (PSA) assessment predicted that sub-Q blood flow rate is the only factor affecting PK parameters in IR formulation whereas this was insignificant for CR. Thus, sub-Q delivery CR would be promising alternative with ease of delivery to children and aged patient.

Fatty acids and volatile compound of cooked green licuri (Syagrus coronata) and naturally ripe licuri almonds from native flora, popularly consumed in Brazil.

The present study was carried out to investigate the proximate composition, fatty acid (FA) profile and volatile compounds (VC) of cooked green licuri (Syagrus coronata) - an unripe stage that is then cooked - and naturally ripe licuri almonds. The FA profiles were determined by gas chromatography (GC) and the VC composition was evaluated using headspace-solid-phase microextraction coupled with GC-MS. The cooked green licuri presented higher moisture, and lower contents of ashes, proteins and lipids than naturally ripe licuri almonds. The FA profiles of cooked green licuri and naturally ripe licuri almonds showed that saturated FAs were predominant (80%) in both samples, and the concentrations of lauric, palmitic, and oleic acids in naturally ripe licuri almonds were higher than those in cooked green licuri. Limonene was the predominant compound in naturally ripe licuri almonds. The main class of VC in the cooked green licuri were aldehydes, with 3-methyl-butanal and furfural being the main species. Alcohols, such as 3-methyl-butanol and 2-heptanol, were the main class of VC in naturally ripe licuri almonds. Among the volatile compounds, 1-hexanol and 2-nonanone contributed to the aroma of cooked green licuri almonds, whereas 2-heptanone, ethanol, and limonene contributed to the aroma of naturally ripe licuri almonds (almonds not subjected to any cooking process). In a word, cooked green licuri and naturally riped licuri almonds, despite having different proximate compositions, present similar fatty acid profile and distinct aromatic characteristics. Therefore, cooked green licuri and naturally riped licuri almonds are an alternative source of nutrient and could be investigated for the use in the food industry to enhance flavor and aroma to new products.

Variations in the expression of odorant binding and chemosensory proteins in the developmental stages of whitefly Bemisia tabaci Asia II-1.

The cotton whitefly, Bemisia tabaci, is considered as a species complex with 46 cryptic species, with Asia II-1 being predominant in Asia. This study addresses a significant knowledge gap in the characterization of odorant-binding proteins (OBPs) and chemosensory proteins (CSPs) in Asia II-1. We explored the expression patterns of OBPs and CSPs throughout their developmental stages and compared the motif patterns of these proteins. Significant differences in expression patterns were observed for the 14 OBPs and 14 CSPs of B. tabaci Asia II-1, with OBP8 and CSP4 showing higher expression across the developmental stages. Phylogenetic analysis reveals that OBP8 and CSP4 form distinct clades, with OBP8 appearing to be an ancestral gene, giving rise to the evolution of other odorant-binding proteins in B. tabaci. The genomic distribution of OBPs and CSPs highlights gene clustering on the chromosomes, suggesting functional conservation and evolutionary events following the birth-and-death model. Molecular docking studies indicate strong binding affinities of OBP8 and CSP4 with various odour compounds like ß-caryophyllene, α-pinene, ß-pinene and limonene, reinforcing their roles in host recognition and reproductive functions. This study elaborates on our understanding of the putative roles of different OBPs and CSPs in B. tabaci Asia II-1, hitherto unexplored. The dynamics of the expression of OBPs and CSPs and their interactions with odour compounds offer scope for developing innovative methods for controlling this global invasive pest.

Therapeutic potential of limonene-based syringic acid nanoemulsion: Enhanced ex-vivo cutaneous deposition and clinical anti-psoriatic efficacy.

Nanoemulsions have carved their position in topical delivery owing to their peculiar features of forming a uniform film on the skin and conquering stratum corneum barrier and hence fostering dermal penetration and retention. The present work developed syringic acid nanoemulsion (SA-NE) by spontaneous emulsification as an anti-psoriatic remedy via the dermal route. SA-NE were prepared with either lauroglycol90, limonene or their combination (oil phase) and tween80 (surfactant) with variable concentrations. The physicochemical characteristics of SA-NE were assessed together with Ex-vivo skin deposition and dermal toxicity. The effectiveness of optimal formula in psoriatic animal model and psoriatic patients was investigated using PASI scoring and dermoscope examination. Results showed that, SA-NE containing mixture of lauroglycol 90, limonene and 10 % tween80 (F5), was selected as the optimal formula presenting stable nanoemulsion for 2-month period, showing droplet size of 177.6 ± 13.23 nm, polydispersity index of 0.16 ± 0.06, zeta potential of -21.23 ± 0.41 mV. High SA% in different skin strata and no dermal irritation was noticed with limonene-based SA-NE also it showed high in-vitro anti- inflammatory potential compared to the blank and control formulations. A preclinical study demonstrated that limonene-based SA-NE is effective in alleviating psoriasis-like skin lesions against imiquimod-induced psoriasis in rats. Clinically, promising anti-psoriatic potential was asserted as all patients receiving F5 experienced better clinical improvement and response to therapy, achieving ≥ 50 % reduction in PASI scores versus only 35 % responders in the Dermovate® cream group. Collectively, the practical feasibility of limonene-based SA-NE topical delivery can boost curative functionality in the treatment of psoriatic lesions.

Molecular-Sensory Decoding of the Citrus latifolia Aroma.

A modified aroma extract dilution approach (AEDA), followed by the determination of flavor dilution (FD) factors, a quantitative analysis and calculation of the relative flavor activity (RFA) and odor activity values (OAVs) as well as recombination experiments were conducted to evaluate the odor- and taste-relevant components of cold-pressed Citrus latifolia peel oil. A 2-fold concentration by distillation and reanalysis, compared with the original oil, revealed relevant components. Partition of the odor-active substances into four reconstitution groups according to their respective FD factors, followed by a recombination, allowed for a better understanding of the contribution of each FD-factor group to the overall aroma. Especially α-pinene, limonene, γ-terpinene, and 7-methoxycoumarin contribute significantly to the distinct aroma profile of C. latifolia. Heptadecanal (CAS 629-90-3) was described for the first time as an odor-active substance in an enriched C. latifolia peel oil. Campherenyl acetate (CAS 18530-07-9) was identified in nature for the first time and described with a herbal, minty and citrus-like odor. The odor profile of the final recombinant mixture, containing 36 components, was similar to cold-pressed C. latifolia peel oil for most descriptors, whereas the taste profile was described as more aldehydic and citral-like.

Effects of aluminum (Al) stress on the isoprenoid metabolism of two Citrus species differing in Al-tolerance.

Isoprenoid metabolism and its derivatives took part in photosynthesis, growth regulation, signal transduction, and plant defense to biotic and abiotic stresses. However, how aluminum (Al) stress affects the isoprenoid metabolism and whether isoprenoid metabolism plays a vital role in the Citrus plants in coping with Al stress remain unclear. In this study, we reported that Al-treatment-induced alternation in the volatilization rate of monoterpenes (α-pinene, ß-pinene, limonene, α-terpinene, γ-terpinene and 3-carene) and isoprene were different between Citrus sinensis (Al-tolerant) and C. grandis (Al-sensitive) leaves. The Al-induced decrease of CO2 assimilation, maximum quantum yield of primary PSII photochemistry (Fv/Fm), the lower contents of glucose and starch, and the lowered activities of enzymes involved in the mevalonic acid (MVA) pathway and 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway might account for the different volatilization rate of isoprenoids. Furthermore, the altered transcript levels of genes related to isoprenoid precursors and/or derivatives metabolism, such as geranyl diphosphate (GPP) synthase (GPPS) in GPP biosynthesis, geranylgeranyl diphosphate synthase (GGPPS), chlorophyll synthase (CHS) and GGPP reductase (GGPPR) in chlorophyll biosynthesis, limonene synthase (LS) and α-pinene synthase (APS) in limonene and α-pinene synthesis, respectively, might be responsible for the different contents of corresponding products in C. grandis and C. sinensis. Our data suggested that isoprenoid metabolism was involved in Al tolerance response in Citrus, and the alternation of some branches of isoprenoid metabolism could confer different Al-tolerance to Citrus species.

Dual-Regulation in Peroxisome and Cytoplasm toward Efficient Limonene Biosynthesis with Rhodotorula toruloides.

Rhodotorula toruloides is a potential workhorse for production of various value-added chemicals including terpenoids, oleo-chemicals, and enzymes from low-cost feedstocks. However, the limited genetic toolbox is hindering its metabolic engineering. In the present study, four type I and one novel type II peroxisomal targeting signal (PTS1/PTS2) were characterized and employed for limonene production for the first time in R. toruloides. The implant of the biosynthesis pathway into the peroxisome led to 111.5 mg/L limonene in a shake flask culture. The limonene titer was further boosted to 1.05 g/L upon dual-metabolic regulation in the cytoplasm and peroxisome, which included employing the acetoacetyl-CoA synthase NphT7, adding an additional copy of native ATP-dependent citrate lyase, etc. The final yield was 0.053 g/g glucose, which was the highest ever reported. The newly characterized PTSs should contribute to the expansion of genetic toolboxes forR. toruloides. The results demonstrated that R. toruloides could be explored for efficient production of terpenoids.

D-Limonene reduces depression-like behaviour and enhances learning and memory through an anti-neuroinflammatory mechanism in male rats subjected to chronic restraint stress.

D-limonene is a widely used flavouring additive in foods, beverages and fragrances due to its pleasant lemon-like odour. This study aimed to investigate the effects of D-limonene on the central nervous system when subjected to chronic restraint stress in rats for 21 days. Forty rats were randomly divided into five groups: i) control, ii) D-limonene, iii) restraint stress, iv) restraint stress+D-limonene and v) restraint stress+fluoxetine. Following the induction of restraint stress, the sucrose preference test, the open field test, the novel object recognition test and the forced swimming test were performed. The levels of BDNF, IL-1ß, IL-6 and caspase-1 were measured from hippocampal tissue using the ELISA method. Sucrose preference test results showed an increase in consumption rate in the stress+D-limonene and a decrease in the stress group. The stress+D-limonene group reversed the increased defensive behaviour observed in the open-field test compared to the stress group. In the novel object recognition test, the discrimination index of the stress+D-limonene group increased compared to the stress group. BDNF levels increased in the stress+limonene group compared to the stress group. In contrast, IL-1ß and caspase-1 levels increased in the stress group compared to the control and decreased in the stress+limonene group compared to the stress group. In this study, D-limonene has been found to have antidepressant-like properties, reducing anhedonic and defensive behaviours and the impairing effects of stress on learning and memory tests. It was observed that D-limonene showed these effects by alleviating neuroinflammation induced by chronic restraint stress in rats.

Bioactivities of essential oil extracted from Elsholtzia densa Benth. And its main components against Tribolium castaneum eggs and pupae.

This study aimed to develop a relatively natural and safe botanical insecticide for controlling the storage pest Tribolium castaneum in the egg and pupal stages. It examined how Elsholtzia densa Benth. essential oil (EO) and its primary components, ß-caryophyllene and limonene, affected T. castaneum eggs and pupae through contact and fumigation. Among th, the contact activities of ß-caryophyllene against T. castaneum eggs and pupae are LD50 (median lethal dose, 50%) = 0.156 mg/cm2 and ED50 (median effective dose, 50%) = 16.35 mg/pupa respectively. The study also investigated the effect of ß-caryophyllene and limonene on T. castaneum eggs and pupae through synergistic contact and fumigation. When the mixing ratio of ß-caryophyllene and limonene was 7:1, the LD50 value of contact activity against T. castaneum eggs was reduced to 0.100 mg/cm2, displaying an obvious synergistic effect. Experiments were conducted to investigate the antitoxic effect of ß-caryophyllene on T. castaneum eggs and pupae, as well as its effects on the enzymatic activity of acetylcholinesterase, succinate dehydrogenase, glutathione S-transferase and carboxylesterase in T. castaneum pupae. Finally, the molecular docking techniques were employed to confirm the aforementioned effects on enzyme function. The findings of this study might help improve storage pest control with T. castaneum and create eco-friendly insecticides using E. densa EO, ß-caryophyllene, and limonene.

Optical discrimination of terpenes in citrus peels with a host:guest sensing array.

A simple aqueous host:guest sensing array can selectively discriminate between different types of citrus varietal from peel extract samples. It can also distinguish between identical citrus samples at varying stages of ripening. The discrimination effects stem from detection of changes in the terpenoid composition of the peel extracts by the host:guest array, despite the overwhelming excess of a single component, limonene, in each sample. The hosts are insensitive to limonene but bind other monoterpenes strongly, even though they are similar in structure to the major limonene component. This work demonstrates the capability of host:guest arrays in sensing target molecules in environments with the competing agents present at high abundances in the sample matrix.

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.

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.

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.

Chemical Characterization of the Essential Oil Compositions of Mentha spicata and M. longifolia ssp. cyprica from the Mediterranean Basin and Multivariate Statistical Analyses.

This present study aims to characterize the essential oil compositions of the aerial parts of M. spicata L. and endemic M. longifolia ssp. cyprica (Heinr. Braun) Harley by using GC-FID and GC/MS analyses simultaneously. In addition, it aims to perform multivariate statistical analysis by comparing with the existing literature, emphasizing the literature published within the last two decades, conducted on both species growing within the Mediterranean Basin. The major essential oil components of M. spicata were determined as carvone (67.8%) and limonene (10.6%), while the major compounds of M. longifolia ssp. cyprica essential oil were pulegone (64.8%) and 1,8-cineole (10.0%). As a result of statistical analysis, three clades were determined for M. spicata: a carvone-rich chemotype, a carvone/trans-carveol chemotype, and a pulegone/menthone chemotype, with the present study result belonging to the carvone-rich chemotype. Carvone was a primary determinant of chemotype, along with menthone, pulegone, and trans-carveol. In M. longifolia, the primary determinants of chemotype were identified as pulegone and menthone, with three chemotype clades being pulegone-rich, combined menthone/pulegone, and combined menthone/pulegone with caryophyllene enrichment. The primary determinants of chemotype were menthone, pulegone, and caryophyllene. The present study result belongs to pulegone-rich chemotype.

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.

Degradation of limonene and trans-cinnamaldehyde in soil, and detection of their metabolites by UHPLC and GC-HRMS.

Two commercial biopesticides were studied to determine their persistence in two soil types, such as sandy clay loam and clay loam soils. For this purpose, an orange oil-based biopesticide was used, being limonene its main ingredient. The other biopesticide was based on cinnamon extract and trans-cinnamaldehyde as its main component. Degradation of these compounds was monitored, and transformation products or metabolites were detected. Limonene and its metabolites were analyzed by gas chromatography (GC) and trans-cinnamaldehyde by ultra-high-performance liquid chromatography (UHPLC). Both techniques were coupled to a high-resolution mass (HRMS) analyzer, such as quadrupole (Q)-Orbitrap. Limonene and trans-cinnamaldehyde were rapidly degraded as result of first-order kinetics. Possible metabolites such as thymol, cymene, isoterpinolene and cymenene for limonene, and hydroxycinnamic acid for trans-cinnamaldehyde were tentatively identified. Moreover, four other metabolites of trans-cinnamaldehyde, some of them not previously described, were also detected.