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.

Enhancing the Retention and Oxidative Stability of Volatile Flavors: A Novel Approach Utilizing O/W Pickering Emulsions Based on Agri-Food Byproducts and Spray-Drying.

Spray-drying is a commonly used method for producing powdered flavors, but the high temperatures involved often result in the loss of volatile molecules. To address this issue, our study focused on a novel approach: developing O/W Pickering emulsions with agri-food byproducts to encapsulate and protect D-limonene during spray-drying and storage. Emulsions formulated with lupin hull, lupin-byproduct (a water-insoluble protein-fiber byproduct derived from the production of lupin protein isolate), and camelina press-cake were subjected to spray-drying at 160 °C. The results revealed that these emulsions exhibited good stability against creaming. The characteristics of the dry emulsions (powders) were influenced by the concentration of byproducts. Quantitative analysis revealed that Pickering emulsions enhanced the retention of D-limonene during spray-drying, with the highest retention achieved using 3% lupin hull and 1% camelina press-cake. Notably, lupin-stabilized emulsions yielded powders with enhanced oxidative stability compared to those stabilized with camelina press-cake. Our findings highlight the potential of food-grade Pickering emulsions to improve the stability of volatile flavors during both processing and storage.

Limonene synergistically augments fluconazole susceptibility in clinical Candida isolates from cleft lip and palate patients.

Background: Cleft lip and palate (CLP) patients are prone to Candida infections (oral thrush) mainly due to poor oral hygiene, repetitive surgeries, and orthodontic procedures. Aim: This study was undertaken to evaluate the antifungal efficacy of limonene against clinical Candida isolates from CLP patients. Materials and Methods: The antifungal efficacy of limonene was studied alone and in combination with fluconazole (FLC) against six standards, twenty nine FLC sensitive, and three FLC resistant clinical strains using broth dilution, checkerboard microdilution, agar disk diffusion, growth curves, and spot assays. Results: This nontoxic monoterpene gave low minimum inhibitory concentration (MIC) values of 300-375 µg/mL and 500-520 µg/mL for FLC susceptible and FLC resistant strains, respectively. It showed synergistic interaction with FLC in all clinical and standard Candida strains (fractional inhibitory concentration (FIC) index ≤0.5). Conclusion: Significant chemosensitization of FLC was observed even against resistant clinical isolates. Complete suppression of fungal growth was observed when using combinations. Negligible toxicity, easy availability, and potent antifungal properties suggest that limonene and FLC combinations in appropriate doses can make excellent antifungal mouthwashes during CLP treatment pre and post surgery. Impending in vivo studies are needed to validate the present data.

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.

Efficient production of (S)-limonene and geraniol in Saccharomyces cerevisiae through the utilization of an Erg20 mutant with enhanced GPP accumulation capability.

Monoterpenes and monoterpenoids such as (S)-limonene and geraniol are valuable chemicals with a wide range of applications, including cosmetics, pharmaceuticals, and biofuels. Saccharomyces cerevisiae has proven to be an effective host to produce various terpenes and terpenoids. (S)-limonene and geraniol are produced from geranyl pyrophosphate (GPP) through the enzymatic actions of limonene synthase (LS) and geraniol synthase (GES), respectively. However, a major hurdle in their production arises from the dual functionality of the Erg20, a farnesyl pyrophosphate (FPP) synthase, responsible for generating GPP. Erg20 not only synthesizes GPP by condensing isopentenyl pyrophosphate (IPP) with dimethylallyl pyrophosphate but also catalyzes further condensation of IPP with GPP to produce FPP. In this study, we have tackled this issue by harnessing previously developed Erg20 mutants, Erg20K197G (Erg20G) and Erg20F96W, N127W (Erg20WW), which enhance GPP accumulation. Through a combination of these mutants, we generated a novel Erg20WWG mutant with over four times higher GPP accumulating capability than Erg20WW, as observed through geraniol production levels. The Erg20WWG mutant was fused to the LS from Mentha spicata or the GES from Catharanthus roseus for efficient conversion of GPP to (S)-limonene and geraniol, respectively. Further improvements were achieved by localizing the entire mevalonate pathway and the Erg20WWG-fused enzymes in peroxisomes, while simultaneously downregulating the essential ERG20 gene using the glucose-sensing HXT1 promoter. In the case of (S)-limonene production, additional Erg20WWG-LS was expressed in the cytosol. As a result, the final strains produced 1063 mg/L of (S)-limonene and 1234 mg/L of geraniol by fed-batch biphasic fermentations with ethanol feeding. The newly identified Erg20WWG mutant opens doors for the efficient production of various other GPP-derived chemicals including monoterpene derivatives and cannabinoids.

Synthesis of Nanosized γ-Cyclodextrin Metal-Organic Frameworks as Carriers of Limonene for Fresh-Cut Fruit Preservation Based on Polycaprolactone Nanofibers.

To address the issue of bacterial growth on fresh-cut fruits, this paper reports the synthesis of nanosized γ-cyclodextrin metal-organic frameworks (CD-MOFs) using an ultrasound-assisted method and their application as carriers of limonene for antibacterial active packaging. The effects of the processing parameters on the morphology and crystallinity of the CD-MOFs are investigated, and the results prove that the addition of methanol is the key to producing nanosized CD-MOFs. The limonene loading content of the nanosized CD-MOFs can reach approximately 170 mg g-1. The sustained-release behaviors of limonene in the CD-MOFs are evaluated. Molecular docking simulations reveal the distribution and binding sites of limonene in the CD-MOFs. CD-MOFs are deposited on the surfaces of polycaprolactone (PCL) nanofibers via an immersion method, and limonene-loaded CD-MOF@PCL nanofibers are prepared. The morphology, crystallinity, thermal stability, mechanical properties, and antibacterial activity of the nanofibers are also studied. The nanofiber film effectively inhibits bacterial growth and prolongs the shelf life of fresh-cut apples. This study provides a novel strategy for developing antibacterial active packaging materials based on CD-MOFs and PCL nanofibers.

Photopolymerization of Limonene Dioxide and Vegetable Oils as Biobased 3D-Printing Stereolithographic Formulation.

Epoxidized vegetable oils and limonene dioxide, a bis-epoxide derived from the terpene limonene, are photo-copolymerized to yield highly crosslinked networks with high conversion of all epoxide groups at ambient temperature. However, the slow polymerization of such biobased formulation polymerizes is not compatible for a use in a commercial SLA 3D printer. Adding an acrylated epoxidized vegetable oil to the bis-epoxide leads to a decrease of curing time and an increase in LDO conversion to polymer. For example, in a 60:40 wt:wt mixture of LDO and epoxidized soybean oil, the conversions of both exocyclic and endocyclic epoxide groups of LDO are ≥95%. These formulations were successfully used in SLA 3D printers, leading to generation of hard and dry complex objects using biobased formulations.

Investigating How the Properties of Electrospun Poly(lactic acid) Fibres Loaded with the Essential Oil Limonene Evolve over Time under Different Storage Conditions.

Essential oils have been identified as effective natural compounds to prevent bacterial infections and thus are widely proposed as bioactive agents for biomedical applications. Across the literature, various essential oils have been incorporated into electrospun fibres to produce materials with, among others, antibacterial, anti-inflammatory and antioxidant activity. However, limited research has been conducted so far on the effect of these chemical products on the physical characteristics of the resulting composite fibres for extended periods of time. Within this work, electrospun fibres of poly(lactic acid) (PLA) were loaded with the essential oil limonene, and the impact of storage conditions and duration (up to 12 weeks) on the thermal degradation, glass transition temperature and mechanical response of the fibrous mats were investigated. It was found that the concentration of the encapsulated limonene changed over time and thus the properties of the PLA-limonene fibres evolved, particularly in the first two weeks of storage (independently from storage conditions). The amount of limonene retained within the fibres, even 4 weeks after fibre generation, was effective to successfully inhibit the growth of model microorganisms Escherichia coli, Staphylococcus aureus and Bacillus subtilis. The results of this work demonstrate the importance of evaluating physical properties during the ageing of electrospun fibres encapsulating essential oils, in order to predict performance modification when the composite fibres are used as constituents of medical devices.

DFT Studies of the Activity and Reactivity of Limonene in Comparison with Selected Monoterpenes.

Nowadays, the effective processing of natural monoterpenes that constitute renewable biomass found in post-production waste into products that are starting materials for the synthesis of valuable compounds is a way to ensure independence from non-renewable fossil fuels and can contribute to reducing global carbon dioxide emissions. The presented research aims to determine, based on DFT calculations, the activity and reactivity of limonene, an organic substrate used in previous preparative analyses, in comparison to selected monoterpenes such as cymene, pinene, thymol, and menthol. The influence of the solvent model was also checked, and the bonds most susceptible to reaction were determined in the examined compounds. With regard to EHOMO, it was found that limonene reacts more easily than cymene or menthol but with more difficultly than thymol and pienene. The analysis of the global chemical reactivity descriptors "locates" the reactivity of limonene in the middle of the studied monoterpenes. It was observed that, among the tested compounds, the most reactive compound is thymol, while the least reactive is menthol. The demonstrated results can be a reference point for experimental work carried out using the discussed compounds, to focus research on those with the highest reactivity.

Impact of Carbon Fixation, Distribution and Storage on the Production of Farnesene and Limonene in Synechocystis PCC 6803 and Synechococcus PCC 7002.

Terpenes are high-value chemicals which can be produced by engineered cyanobacteria from sustainable resources, solar energy, water and CO2. We previously reported that the euryhaline unicellular cyanobacteria Synechocystis sp. PCC 6803 (S.6803) and Synechococcus sp. PCC 7002 (S.7002) produce farnesene and limonene, respectively, more efficiently than other terpenes. In the present study, we attempted to enhance farnesene production in S.6803 and limonene production in S.7002. Practically, we tested the influence of key cyanobacterial enzymes acting in carbon fixation (RubisCO, PRK, CcmK3 and CcmK4), utilization (CrtE, CrtR and CruF) and storage (PhaA and PhaB) on terpene production in S.6803, and we compared some of the findings with the data obtained in S.7002. We report that the overproduction of RubisCO from S.7002 and PRK from Cyanothece sp. PCC 7425 increased farnesene production in S.6803, but not limonene production in S.7002. The overexpression of the crtE genes (synthesis of terpene precursors) from S.6803 or S.7002 did not increase farnesene production in S.6803. In contrast, the overexpression of the crtE gene from S.6803, but not S.7002, increased farnesene production in S.7002, emphasizing the physiological difference between these two model cyanobacteria. Furthermore, the deletion of the crtR and cruF genes (carotenoid synthesis) and phaAB genes (carbon storage) did not increase the production of farnesene in S.6803. Finally, as a containment strategy of genetically modified strains of S.6803, we report that the deletion of the ccmK3K4 genes (carboxysome for CO2 fixation) did not affect the production of limonene, but decreased the production of farnesene in S.6803.

Characterization of volatile compounds from healthy and citrus black spot-infected Valencia orange juice and essential oil by using gas chromatography-mass spectrometry.

Citrus black spot (Phyllosticta citricarpa, CBS) is an important fungal disease that causes rind blemishes and affects quality of citrus fruits. The response of citrus to CBS in terms of volatiles was evaluated using molecular sensory science approaches. Fifty and twenty-one volatiles were identified in the orange juice and essential oil samples, respectively, via gas chromatography-mass spectrometry (GC-MS). The total volatile content in the samples increased after CBS infection, especially in the severe-infection group (SEG) juice and moderate-infection group (MOG) essential oil, which reached the highest levels. CBS enhanced floral, fruity, and off-flavor aromas and decreased the green aroma in citrus juice. Citrusy, floral, and green aromas increased in the CBS-infected essential oil. Six/five potential markers were screened in citrus juice/essential oil, respectively using the orthogonal partial least-square discriminant analysis (OPLS-DA) model. The changes in aroma profile and the difference in infection levels in citrus were attributed to these odorants.

Smartphone-based digital image colorimetry for the determination of total capsaicinoid content in chili pepper extracts.

A simple smartphone-based digital image colorimetry was proposed for the determination of total capsaicinoid content and the assessment of chili pepper pungency. The biobased solvent D-limonene was used for the first time to isolate analytes. Capsaicinoids were efficiently separated from chili pepper by solid-liquid extraction with D-limonene followed by partitioning of the analytes into the ammonium hydroxide solution to eliminate the matrix interference effect. For colorimetric detection of total capsaicinoid content, a selective chromogenic reaction was performed using Gibbs reagent (2,6-dichloroquinone-4-chloroimide). Measurements were performed using a smartphone-based setup and included image analysis with the program ImageJ. The limit of detection of the proposed procedure was 0.15 mg g-1. The intra-day repeatability did not exceed 10.0 %. The inter-day repeatability was less than 16.5 %. The comparison of the smartphone-based procedure with high-performance liquid chromatography showed satisfactory results.

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.

High perillaldehyde content from essential oils of Ammodaucus leucotrichus subsp. leucotrichus Cosson & Durieu fruits from Aougrout (Algeria).

The chemical composition of the essential oil produced by hydrodistillation from the fruits of Ammodaucus leucotrichus subsp. leucotrichus Cosson & Durieu collected from southern Algeria, precisely in Aougrout District (Adrar Province), was determined by GC-MS analysis. Nineteen (19) compounds, representing 85.71% of the essential oil, were found. The essential oil contained a high percentage of perillaldehyde (80.69%). This composition is compared to numerous reported essential oils of A. leucotrichus subsp. leucotrichus fruits that are found across Algeria and Morocco in various regions. The major component perillaldehyde has drawn a lot of interest in recent years due to its notable antifungal and broader antimicrobial properties, as well as its potential advantages for human health. Thus, the studied plant represents a source of this component that should be used as food additives, perfume ingredients, and in formulations of traditional medicine.

Vaporized D-limonene selectively mitigates the acute anxiogenic effects of Δ9-tetrahydrocannabinol in healthy adults who intermittently use cannabis.

BACKGROUND: Cannabis contains hundreds of chemical constituents beyond delta-9-tetrahydrocannabinol (THC), which is believed to drive most of its acute pharmacodynamic effects. The entourage effect theory asserts that non-THC constituents can impact acute cannabis effects, but few empirical studies have systematically evaluated this theory in humans. This study assessed whether the cannabis terpenoid d-limonene mitigates the acute anxiogenic effects of THC. METHODS: Twenty healthy adults completed nine, double-blind outpatient sessions in which they inhaled vaporized THC alone (15mg or 30mg), d-limonene alone (1mg or 5mg), the same doses of THC and d-limonene together, or placebo; a subset of participants (n=12) completed a tenth session in which 30mg THC+15mg d-limonene was administered. Outcomes included subjective drug effects, cognitive/psychomotor performance, vital signs, and plasma THC and d-limonene concentrations. RESULTS: When d-limonene was administered alone, pharmacodynamic outcomes did not differ from placebo. Administration of 15mg and 30mg THC alone produced subjective, cognitive, and physiological effects typical of acute cannabis exposure. Ratings of anxiety-like subjective effects qualitatively decreased as d-limonene dose increased and concurrent administration of 30mg THC+15mg d-limonene significantly reduced ratings of "anxious/nervous" and "paranoid" compared with 30mg THC alone. Other pharmacodynamic effects were unchanged by d-limonene. D-limonene plasma concentrations were dose orderly, and concurrent administration of d-limonene did not alter THC pharmacokinetics. CONCLUSIONS: D-limonene selectively attenuated THC-induced anxiogenic effects, suggesting this terpenoid could increase the therapeutic index of THC. Future research should determine whether this effect extends to oral dose formulations and evaluate the interactions between other cannabis terpenoids or cannabinoids and THC.

Deformable Vesicles with Edge Activators for the Transdermal Delivery of Non-Psychoactive Cannabinoids.

BACKGROUND: Transdermal delivery of highly lipophilic molecules is challenging due to the strong barrier function of the skin. Vesicles with penetration enhancers are safe and efficient systems that could improve the transdermal delivery of non-psychoactive cannabinoids such as cannabidiol and desoxy-cannabidiol. In the last decades, research interest in desoxy-cannabidiol as a potent drug with anti-nociceptive properties has risen. Still, its scarce market availability poses a limit for both research and clinical applications. Therefore, it is necessary to improve the synthesis to produce sufficient amounts of desoxy-cannabidiol. Moreover, also the formulation aspects for this drug are challenging and require to be addressed to meet an efficient delivery to the patients. OBJECTIVE: This work aimed to develop innovative phospholipid-based vesicles with propylene glycol (PG), oleic acid (OA), or limonene as edge activators, for the transdermal delivery of highly lipophilic drugs such as non-psychoactive cannabinoids. In particular, desoxy-cannabidiol was selected thanks to its anti-nociceptive activity, and its synthesis was improved enhancing the stereoselectivity of its synthon's production. METHODS: Desoxy-cannabidiol was synthesized by Lewis acid-mediated condensation of p-mentha-2,8-dien- 1-ol and m-pentylphenol, improving the stereoselectivity of the first synthon's production. Transethosomes containing 20-50% w/w PG, 0.4-0.8% w/w OA, or 0.1-1% w/w limonene were optimized and loaded with cannabidiol or desoxy-cannabidiol (0.07-0.8% w/w, 0.6-7.0 mg/mL). Ex-vivo studies were performed to assess both the skin permeation and accumulation of the cannabinoids, as well as the penetration depth of fluorescein- loaded systems used as models. RESULTS: An enantioselective bromination was added to the pathway, thus raising the production yield of pmentha- 2,8-dien-1-ol to 81% against 35%, and the overall yield of desoxy-cannabidiol synthesis from 12% to 48%. Optimized transethosomes containing 0.6 mg/mL cannabinoids were prepared with 1:10 PG:lipid weight ratio, 0.54 OA:lipid molar ratio, and 0.3 limonene:lipid molar ratio, showing good nanometric size (208 ± 20.8 nm - 321 ± 26.3 nm) and entrapment efficiency (> 80%). Ex-vivo tests showed both improved skin permeation rates of cannabinoids (up to 21.32 ± 4.27 µg/cm2 cannabidiol), and skin penetration (depth of fluorescein up to 240 µm, with PG). CONCLUSION: Desoxy-cannabidiol was successfully produced at high yields, and formulated into transethosomes optimized for transdermal delivery. Loaded vesicles showed improved skin penetration of desoxy-cannabidiol, cannabidiol and a lipophilic probe. These results suggest the potential of these carriers for the transdermal delivery of highly lipophilic drugs.

Enhancing collagen based nanoemulgel for effective topical delivery of Aceclofenac and Citronellol oil: Formulation, optimization, in-vitro evaluation, and in-vivo osteoarthritis study with a focus on HMGB-1/RAGE/NF-κB pathway, Klotho, and miR-499a.

The majority of conventional osteoarthritis (OA) treatments are based on molecular adjustment of certain signaling pathways associated with osteoarthritis (OA) pathogenesis, however there is a significant need to search for more effective and safe treatments. This study centers around formulating Aceclofenac (ACF) with high bioavailability in combination with Citronellol oil and collagen. The optimal concentrations of Citronellol oil/D-Limonene oil, Tween 80, and Transcutol HP were determined using a pseudoternary phase diagram. The formulated nanoemulsions were studied for thermophysical stability. Thermodynamically stable formula were analyzed for droplet size, zeta potential, and in-vitro permeation. Then, collagen based nanoemulsion were prepared to capitalize on its efficacy in reducing osteoarthritis side effects and characterized for nano size properties. Formulae F10 and F10C were chosen as optimum nanosize formula. Hense, they were prepared and characterized as nanoemulgel dosage form. The nanoemulgel formulae F10NEG1 and F10CNEG1 showed reasonable viscosity and spreadability, with complete drug release after 4 h. These formulae were chosen for further In vivo anti-OA study. Collagen based ACF/citronellol emugel were able to modulate HMGB-1/RAGE/NF-κB pathway, mitigating the production of inflammatory cytokine TNF-α. They were also able to modulate Klotho and miR-499, reducing serum CTXII and COMP, by reducing the cartilage destruction. Histological investigations validated the efficacy, safety, and superiority of Aceclofenac in combination with Citronellol oil and collagen (F10CNEG1) over solo the treated group (F10NEG1 and blank). Hence, the findings of the current work encourage the use of this promising combined formula in treatment of OA patients.

Characterization and investigation of cytotoxicity and antimicrobial properties of coencapsulated limonene and thymol into the Ferula assafoetida gum microparticles.

Thymol (Th) and d-limonene (L) exhibit low stability and are prone to oxidation when exposed to air, light, humidity, and high temperatures. This study examined the coencapsulation of Th and L into Ferula assafoetida gum (AFG) microparticles. Scanning electron microscope (SEM), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), and thermogravimetric analyzer (TGA) were done to characterize the obtained complexes. Furthermore, the encapsulation efficiency, antibacterial properties, cytotoxicity, and anticancer properties of both the free and encapsulated forms of L and Th were measured. For all samples, by increasing the percentage of bioactive compound (L, Th, and L-Th) from 2.5 to 5 % w/w, the EE was increased. FTIR and XRD analysis results demonstrated that Th and L were successfully incorporated into the AFG. Additionally, thermogravimetric analysis showed that in the thermal graphs of all samples, the first weight loss occurred between 30 °C and 160 °C, which was due to the evaporation of water. In the free L and Th graph, a sharp reduction peak was observed in which 80 % of compounds were lost. These reduction peaks disappeared in the thermal graphs of L: AFG and Th: AFG revealing that the thermal stability of Th and L was significantly increased upon their incorporation into the AFG. The inclusion of Th into the AFG also led to an increase in its antibacterial activity, while L exhibited acceptable antibacterial activity, albeit not as high as Th. Additionally, according to the MIC results, Th: AFG had the best antibacterial activity among all compounds, especially on gram-positive bacteria. According to the result of the MTT assay, there was a significant difference between the IC50 of free Th (123.4 µg/ml) and Th: AFG (2312 µg/ml), and free L (1762 µg/ml) and L: AFG (2480 µg/ml) showing that encapsulated Th and L into the AFG has decreased the cytotoxicity of free compounds against L929 cell line. Also, Th: AFG had the best anticancer activity against Hella and CT26 cell lines among all compounds. Finally, the flow cytometry analysis demonstrated that the encapsulated particles effectively eliminated cancer cells. The outcomes imply that AFG can be employed as a suitable delivery system to enhance the use of Th and L into the food and pharmaceutical industries.