Industrial-produced lemon nanovesicles ameliorate experimental colitis-associated damages in rats via the activation of anti-inflammatory and antioxidant responses and microbiota modification.

Plant-derived nanovesicles (PDNVs) have recently emerged as natural delivery systems of biofunctional compounds toward mammalian cells. Considering their already described composition, anti-inflammatory properties, stability, and low toxicity, PDNVs offer a promising path for developing new preventive strategies for several inflammatory diseases, among which the inflammatory bowel disease (IBD). In this study, we explore the protective effects of industrially produced lemon vesicles (iLNVs) in a rat model of IBD. Characterization of iLNVs reveals the presence of small particles less than 200 nm in size and a profile of bioactive compounds enriched in flavonoids and organic acids with known beneficial properties. In vitro studies on human macrophages confirm the safety and anti-inflammatory effects of iLNVs, as evidenced by the reduced expression of pro-inflammatory cytokines and increased levels of anti-inflammatory markers. As evidenced by in vivo experiments, pre-treatment with iLNVs significantly alleviates symptoms and histological features in 2,4 dinitrobenzensulfuric acid (DNBS)-induced colitis in rats. Molecular pathway analysis reveals modulation of NF-κB and Nrf2, indicating anti-inflammatory and antioxidant effects. Finally, iLNVs affects gut microbiota composition, improving the consistent colitis-related alterations. Overall, we demonstrated the protective role of industrially produced lemon nanovesicles against colitis and emphasized their potential in managing IBD through multifaceted mechanisms.

Citrus limon Wastes from Part of the Eastern Cape Province in South Africa: Medicinal, Sustainable Agricultural, and Bio-Resource Potential.

The fruits of Citrus limon are often purchased for their vitamin C-rich juice, while the fruit peel and the tree leaves are discarded as wastes. This study obtained the chemical profiles of the essential oils (EOs) of C. limon wastes (the peel and leaves), evaluated their medicinal value as antioxidants, their potential for sustainable use in agriculture as an insecticide for post-harvest preservation of grains, and their potential as a bioresource in livestock feed formulations. The EOs were isolated from C. limon leaves and peel using a hydro-distillation method on a Clevenger apparatus. The oil constituents were identified using the gas chromatography-mass spectrometry (GC-MS) hyphenated technique. The oils were evaluated for their in vitro antioxidant activity using 2,2-diphenyl-1-picrylhydrazyl (DPPH) and ferric-reducing antioxidant power methods. An insecticidal study was conducted using contact toxicity, fumigation, and repellence bioassay methods against Sitophilus zeamais (maize weevils). Finally, the predicted income from using lemon peel as an alternative or substitute ingredient for maize in livestock feed formulations was obtained through a conventional simulation method. Chemically, limonene was found to be present in all the EOs analyzed (12-52%), while α-pinene was only found in the fresh leaf and peel oils (13.3% and 10.6%). Caryophyllene oxide was identified as the major component of the dried leaf oil (17.7%). At 20 µg m, the dry peel oil exhibited the highest inhibitory activity (52.41 ± 0.26%) against the DPPH radical, which was comparable to L-ascorbic acid (a standard antioxidant) at 54.25 ± 3.55%. The insecticidal study revealed that the dry peel oil is a better insect repellent (73.33 ± 6.95% at 10 µL) and fumigant (LC50 = 0.17 µL g-1 after 48 h) natural agent compared to the peel oil. Conversely, the dry peel oil showed a better contact activity (LC50 = 1.69 µL g-1) against the maize weevils compared to the dry leaf oil. The simulation study showed the cost of using dry lemon peel as an alternative to maize in livestock feed formulation to be ZAR 2.8 billion, compared against the higher cost of feed formulation with maize, which currently stands at ZAR 24.9 billion. This study has shown that C. limon wastes (the peel and leaves) contain EOs with unique chemical profiles, valuable medicinal properties as free radical scavengers, and considerable insecticidal properties for agricultural use in post-harvest grain preservation, presenting a cost-effective and promising bioresource for livestock feed production.

Evaluation of morphological traits, biochemical parameters and seeding availability pattern among Citrus limon 'Assam lemon' accessions across Assam.

The Assam lemon is a highly valued Citrus cultivar known for its unique aroma, flavor, and appearance. This study aimed to investigate the morphological, seeding pattern and biochemical variations within 132 populations of Assam lemon from across 22 districts of Assam along with the control samples, with the objective to offer comprehensive understanding that could facilitate the improvement of breeding programs and further improvement of this important cultivar. Clustering based on UPGMA algorithm for morphological and seeding pattern data were analysed at population level, revealed two major clusters, where all the populations of Upper Assam districts were in the same cluster with the original stock (control population). The populations from Tinsukia and Dhemaji districts displayed more close similarities with the control population in comparison to populations of Upper Assam districts. Another interesting observation was regarding flowering patterns, while populations from Upper Assam districts excluding Golaghat district displayed both bisexual and unisexual flowers with less concentration of unisexual flowers, other remaining districts had bisexual and unisexual flowers of almost equal concentration. Unisexual flowers contained only the male reproductive organs with 40 anthers, while bisexual flowers had 36 anthers. Seeding patterns were examined across the districts, and it was found that populations from Tinsukia, Dhemaji, Lakhimpur, Dibrugarh, Jorhat, and the control population exhibited seedless characteristic while populations from other selected districts displayed a combination of seedless and seeded traits. Interestingly, Golaghat district appears as the linking district and showed availability of both seeded and seedless Assam lemon fruit, connecting the regions of Barak valley, Central, Lower, North and Upper Assam. Biochemical analysis showed significant variations across districts, however, the populations from Dhemaji, Tinsukia, Lakhimpur, Dibrugarh, and Jorhat districts displayed similarity with the control population. The study also investigated variability in soil nutrient content revealing substantial variation among the populations studied. This comprehensive investigation provides valuable insights into the morphological, seeding pattern, and biochemical diversity within the Assam lemon cultivar. These findings can be instrumental in breeding programs to enhance the cultivar, particularly in producing high-quality seedless fruits to meet consumer demands.

Effect of Solanum lycopersicum and Citrus limon-Derived Exosome-Like Vesicles on Chondrogenic Differentiation of Adipose-Derived Stem Cells.

Articular cartilage defect treatment is a very important problem because its therapeutic options are not successful enough. Due to the weak self-repairing capacity of the avascular cartilage, even minor damage can progress and cause joint damage leading to osteoarthritis. Although various treatment strategies have been developed to repair damaged cartilage, cell- and exosome-based therapies are promising. Plant extracts have been used for decades, and their effects on cartilage regeneration have been studied. Exosome-like vesicles, which are secreted by all living cells, are involved in cell-to-cell communication and cell homeostasis. The differentiation potential of exosome-like vesicles isolated from S. lycopersicum and C. limon, which are known to have anti-inflammatory and antioxidant properties, was investigated in the differentiation of human adipose-derived mesenchymal stem cells (hASCs) into chondrocytes. In order to obtain tomato-derived exosome-like vesicles (TELVs) and lemon-derived exosome-like vesicles (LELVs) Aquous Two- Phase system was performed. Characterisation of isolated vesicles based on size, shape were achived via Zetasizer, NTA FAME analysis, and SEM techniques. These results showed that TELVs and LELVs increased cell viability and did not show any toxic effects on stem cells. Although TELVs triggered chondrocyte formation, LELVs downregulated. The expression of ACAN, SOX9, and COMP, known as chondrocyte markers, was increased by TELV treatment. In addition, protein expression of the two most important proteins, COL2 and COLXI, found in the extracellular matrix of cartilage, increased. These findings suggest that TELVs can be used for cartilage regeneration, and may be a novel and promising treatment for osteoarthritis.

Direct relationship between dimeric form and activity in the acidic copper-zinc superoxide dismutase from lemon.

The copper-zinc superoxide dismutase (CuZnSOD) from lemon (SOD_CL) is active in an acidic environment and resists proteolytic degradation. The enzyme occurs as a dimer, which has an indirect effect on the enzyme activity as the monomer retains only ∼35% of the activity. Here, the crystal structure of SOD_CL at 1.86 Šresolution is reported that may explain this peculiarity. The crystal belonged to space group P21, with unit-cell parameters a = 61.11, b = 74.55, c = 61.69 Å, ß = 106.86°, and contained four molecules in the asymmetric unit. The overall structure of SOD_CL resembles that of CuZnSOD from plants. The structure of SOD_CL shows a unique arrangement of surface loop IV that connects the dimer interface and the active site, which is located away from the dimer-interface region. This arrangement allows direct interaction between the residues residing in the dimer interface and those in the active site. The arrangement also includes Leu62 and Gln164, which are conserved in cytoplasmic CuZnSOD. This supports the classification of SOD_CL as a cytoplasmic CuZnSOD despite sharing the highest amino-acid sequence homology with CuZnSODs from spinach and tomato, which are chloroplastic.

Morphological and molecular characterization of a new root-knot nematode, Meloidogyne limonae n. sp. (Nematoda: Meloidogynidae) parasitizing lemon in China.

Root-knot nematodes of the genus Meloidogyne parasitize the roots of thousands of plants and can cause severe damage and yield loss. Here, we report a new root-knot nematode, Meloidogyne limonae n. sp., parasitizing "lemon" (Citrus limon) in Hainan Province, South China. Lemon trees infected by the root-knot nematode showed poor-quality lemons, chlorosis of foliage, weak growth, and numerous root galls with white females and egg masses protruding outside. Phylogenetic trees of sequences within the ribosomal and mitochondria DNA demonstrated that this species differs clearly from other previously described root-knot nematodes. Morphologically, the new species is characterized by an oval-shaped perineal pattern and the lateral field marked by a ridge of cuticle on one or both sides, the dorsal arch is low with fine to coarse, smooth cuticle striae, vulva slit centrally located at the unstriated area; spicules of males are arcuate, curved ventrally; gubernaculum is distinct and curved; labial disc of second-stage juveniles is prominent and dumbbell-shaped; stylet knobs oval and sloping backwardly; pharyngeal glands not filling the body cavity, overlapping intestine ventrally; conical tail gradually tapering. Phylogenetic trees based on ITS1-5.8S-ITS2, D2-D3 of the 28S rDNA, and the COI and COII-16S rRNA genes of the mtDNA showed that Meloidogyne limonae n. sp. belongs to an undescribed root-knot nematode lineage that is separated from other species with the resemblance in morphology, such as M. floridensis M. hispanica, M. acronea, and M. paranaenis.

Scanning Electron Microscopy Analysis of Smear Layer Removal Ability of Conventional Endodontic Irrigation Regimen, MTAD, and QMix™ Versus a Mixture of Azadirachta indica and Citrus limon: An In Vitro Study.

INTRODUCTION: Smear layer removal from root canals aid in the penetration of both irrigants and endodontic sealer into the dentinal tubules, thereby improving the efficacy of endodontic treatment. The aim of this in vitro study was to compare the smear layer removal ability of a conventional endodontic irrigation regimen, MTAD (mixture of tetracycline, acid, and detergent), and QMix™ (Dentsply Sirona, Charlotte, North Carolina, United States) with that of a mixture of herbal irrigants, namely, aqueous extracts of Azadirachta indica (neem) and Citrus limon (lemon), evaluated using scanning electron microscopy (SEM). MATERIALS AND METHODS: We selected 40 extracted human premolar teeth for the study, which we randomly divided into five groups (eight samples each) according to irrigation solution: (i) Group A (normal saline); (ii) Group B (conventional endodontic regimen, 3% sodium hypochlorite (NaOCl) + 17% ethylenediamine tetraacetic acid (EDTA) + 2% chlorhexidine (CHX)); (iii) Group C (MTAD); (iv) Group D (QMix 2-in-1); and (v) Group E (aqueous extracts of Azadirachta indica and Citrus limon). After we prepared the canals with ProTaper Universal nickel-titanium (Ni-Ti) rotary files (Dentsply Sirona) and the respective irrigants, we split the teeth longitudinally to evaluate the amount of remnant smear layer in the coronal, middle, and apical thirds using SEM photomicrographs. We performed statistical analyses of the data using the Kruskal-Wallis and Mann-Whitney U tests, where the level of significance was set at 0.05. RESULTS: The SEM analysis of the coronal third showed mean values of 3.83 in Group A, 3.67 in Group B, 2.79 in Group C, 3.63 in Group D, and 4.00 in Group E. The SEM analysis of the middle third showed mean values of 4.00 in Group A, 3.88 in Group B, 3.75 in Group C, 3.50 in Group D, and 3.50 in Group E. The SEM analysis of the apical third showed mean values of 3.92 in Group A, 3.63 in Group B, 3.71 in Group C, 3.88 in Group D, and 3.17 in Group E. Therefore, we found that there were significant statistical differences between the groups when an overall comparison was done for the coronal, middle, and apical third, with a p-value of 0.001. On multiple comparisons across the different tooth-section thirds. Groups A and B showed statistically significant differences in the apical third (p-value=0.017). Groups A and C showed statistically significant differences in the coronal third and middle third (p-values=0.001 and 0.010, respectively). Groups A and D showed statistically significant differences in the middle third (p-value=0.001). Groups A and E showed statistically significant differences in all thirds (p-values=0.039, 0.001, and 0.001, respectively) Conclusion: The conventional needle irrigation with MTAD showed the highest level of smear layer removal ability on the root canal surface, followed by QMix 2-in-1, the Azadirachta indica leaf and Citrus limon extract mixture, and the conventional endodontic regimen. Normal saline showed the lowest smear layer removal effect.

Inhibition of Enzymes Involved in Neurodegenerative Disorders and Aß1-40 Aggregation by Citrus limon Peel Polyphenol Extract.

Alzheimer's (AD) and Parkinson's diseases (PD) are multifactorial neurogenerative disorders of the Central Nervous System causing severe cognitive and motor deficits in elderly people. Because treatment of AD and PD by synthetic drugs alleviates the symptoms often inducing side effects, many studies have aimed to find neuroprotective properties of diet polyphenols, compounds known to act on different cell signaling pathways. In this article, we analyzed the effect of polyphenols obtained from the agro-food industry waste of Citrus limon peel (LPE) on key enzymes of cholinergic and aminergic neurotransmission, such as butyryl cholinesterase (BuChE) and monoamine oxidases (MAO)-A/B, on Aß1-40 aggregation and on superoxide dismutase (SOD) 1/2 that affect oxidative stress. In our in vitro assays, LPE acts as an enzyme inhibitor on BuChE (IC50 ~ 73 µM), MAO-A/B (IC50 ~ 80 µM), SOD 1/2 (IC50 ~ 10-20 µM) and interferes with Aß1-40 peptide aggregation (IC50 ~ 170 µM). These results demonstrate that LPE behaves as a multitargeting agent against key factors of AD and PD by inhibiting to various extents BuChE, MAOs, and SODs and reducing Aß-fibril aggregation. Therefore, LPE is a promising candidate for the prevention and management of AD and PD symptoms in combination with pharmacological therapies.

Development of a novel green catalyzed nanostructured Cu(II) macrocyclic complex-based disposable electrochemical sensor for sensitive detection of bisphenol A in environmental samples.

BPA is an endocrine disruptor and the leading environmental pollutant due to its use as raw material in industries. Therefore, the present work reports the sensitive, efficient, and disposable electrochemical paper-based SPE for determining the BPA sensor using an amide-based macrocyclic complex (nanostructured complex of copper acetate with macrocyclic ligand, i.e., CuL (CH3COO)2) synthesized using Citrus limon (lemon) extract via sonication for the first time. The structural, morphological, and electrochemical analyses have been characterized by mass spectroscopy, FTIR, UV-Vis, XRD, FESEM-EDX, elemental mapping and electrochemical techniques. The sensor platform for detecting BPA was fabricated by simple drop-casting on the disposable paper-based SPE using macrocyclic complex, i.e., CuL (CH3COO)2/SPE. After optimizing the conditions, CuL (CH3COO)2/SPE electrode was employed for determining BPA via CV with a wide linear range of 31 × 10-9 µM-0.205 µM, low LOD of 0.027 nM, and high sensitivity of 49.71 µA (log nM)-1 cm-2 having correlation coefficient (R2) of 0.976 which is quite better in compared to other reported SPE sensor for detection of BPA. Further, our sensor also showed good selectivity and reproducibility, in addition to detecting BPA in environmental samples (tube well water, river water and drain water) with acceptable recoveries and RSDs values. In this work, the combination of macrocyclic complex and paper-based SPE has turned out to be a cost-effective electrochemical sensor.

One-pot biogenic synthesis of C. limon/TiO2 with dual applications as an advance photocatalyst and antimicrobial agent.

The present study portrays a facile, cost-effective, and environmentally benign way for the preparation of TiO2 nanoparticles utilizing C. limon extract, which possesses phytochemicals as reducing and stabilizing agents. Structural characterization by XRD reveals that C. limon/TiO2 NPs exhibit anatase-type tetragonal crystallinity. An average crystallite-size is calculated using Debye Scherrer's method (3.79 nm), Williamson-Hall plot (3.60 nm), and Modified Debye Scherrer plot (3.68 nm), which are very closely intercorrelated. The absorption peak at 274 nm (UV-visible spectrum) corresponds to the bandgap (Eg) value of 3.8 eV. The existence of different phytochemicals containing organic groups like N-H, C=O, and O-H, has been elucidated from FTIR along with Ti-O bond stretching at wavenumber 780 cm-1. Micro-structural investigations of TiO2 NPs using FESEM and TEM display different geometrical configurations involving spherical, pentagonal, hexagonal, heptagonal and capsule-like structures. BET and BJH analysis show mesoporous characteristics of synthesized nanoparticles with specific surface-area (97.6 m2 g-1), pore-volume (0.018322 cm3 g-1), and mean pore-diameter (∼7.5 nm) values. In adsorption studies, the influence of reaction parameters, i.e., catalyst dosage and contact-time, for removal of Reactive Green dye is explored along with Langmuir and Freundlich models. The highest adsorption capability is ∼219 mg g-1 for green dye. TiO2 displays an excellent photocatalytic efficiency of ∼96% towards the degradation of reactive green dye within 180 min and excellent reusable performance. C. limon/TiO2 is found to have an outstanding performance with a quantum yield value of 4.68 × 10-5 molecules photon-1 for Reactive Green dye degradation. Additionally, synthesized nanoparticles have exhibited antimicrobial activity against gram-positive Staphylococcus aureus (S. aureus) and gram-negative Pseudomonas aeruginosa (P. aeruginosa) bacteria.

Endophytic Bacillus atrophaeus CHGP13 and salicylic acid inhibit blue mold of lemon by regulating defense enzymes.

Lemons (Citrus limon L.) are one of the most economically important and consumed fruit worldwide. The species is vulnerable to several postharvest decay pathogens, of which Penicillium italicum associated with blue mold disease is the most damaging. This study investigates the use of integrated management for blue mold of lemon using lipopeptides (LPs) extracted from endophytic Bacillus strains and resistance inducers. Two resistance inducers; salicylic acid (SA) and benzoic acid (BA) were tested at 2, 3, 4, and 5 mM concentrations against the development of blue mold on lemon fruit. The 5 mM SA treatment produced the lowest disease incidence (60%) and lesion diameter (1.4 cm) of blue mold on lemon fruit relative to the control. In an in vitro antagonism assay eighteen Bacillus strains were evaluated for their direct antifungal effect against P. italicum; CHGP13 and CHGP17 had the greatest inhibition zones of 2.30 and 2.14 cm. Lipopeptides (LPs) extracted from CHGP13 and CHGP17 also inhibited the colony growth of P. italicum. LPs extracted from CHGP13 and 5 mM SA were tested as single and combined treatments against disease incidence and lesion diameter of blue mold on lemon fruit. SA + CHGP13 + PI had the lowest disease incidence (30%) and lesion diameter (0.4 cm) of P. italicum on lemon fruit relative to the other treatments. Furthermore, the lemon fruit treated with SA + CHGP13 + PI had the highest PPO, POD, and PAL activities. The postharvest quality analysis of the lemon fruit including fruit firmness, total soluble solids, weight loss, titratable acidity, and ascorbic acid content revealed that the treatment SA + CHGP13 + PI had little effect on fruit quality compared to the healthy control. These findings indicate that Bacillus strains and resistance inducers can be used as components of integrated disease management for the blue mold of lemon.

Waste Citrus limon Leaves as Source of Essential Oil Rich in Limonene and Citral: Chemical Characterization, Antimicrobial and Antioxidant Properties, and Effects on Cancer Cell Viability.

This study investigated chemical composition, cytotoxicity in normal and cancer cells, and antimicrobial and antioxidant activity of the essential oil (EO) isolated by hydrodistillation from the discarded leaves of lemon (Citrus limon) plants cultivated in Sardinia (Italy). The volatile chemical composition of lemon leaf EO (LLEO) was analyzed with gas chromatography-mass spectrometry combined with flame ionization detection (GC/MS and GC/FID). The most abundant component of LLEO was limonene (260.7 mg/mL), followed by geranial (102.6 mg/mL) and neral (88.3 mg/mL). The antimicrobial activity of LLEO was tested using eight bacterial strains and two types of yeasts by a microdilution broth test. Candida albicans showed the greatest susceptibility (MIC = 0.625 µL/mL) and Listeria monocytogenes and Staphylococcus aureus were inhibited at low LLEO concentration (MIC values from 2.5 to 5 µL/mL). The C. limon leaf EO displayed radical scavenging ability (IC50 value of 10.24 mg/mL) in the 2,2-diphenyl-1-picryl-hydrazylhydrate (DPPH) assay. Furthermore, the LLEO impact on cell viability was explored by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay in cancer HeLa cells, A375 melanoma cell line, normal fibroblasts (3T3 cells), and keratinocytes (HaCaT cells). LLEO, at 24 h of incubation, significantly reduced viability from 25 µM in Hela cells (33% reduction) and A375 cells (27%), greatly affecting cell morphology, whereas this effect was found from 50 µM on 3T3 fibroblasts and keratinocytes. LLEO's pro-oxidant effect was also established in HeLa cells by 2',7'-dichlorodihydrofluorescein diacetate assay.

Expression of VEGF and CD-31 in traumatic ulcer of diabetic Wistar rats after application of Citrus limon peel essential oil.

Objective: Diabetes mellitus (DM) is a metabolic disease that delays the healing process, including the interruption of the processes of angiogenesis and vasculogenesis. The etiology of most angiogenic-related diseases, such as diabetes complications, includes the presence of hypoxia caused by declined vascular endothelium growth factor (VEGF) and CD-31. d-limonene, one of the main constituents of Citrus limon, is considered to have angiogenic, antioxidant, hypoglycemic, and anti-inflammatory activities. However, the exact mechanism of this process remains unclear. This study aimed to determine the potential of C. limon as a medication for diabetic ulceration. Methods: A total of 30 Wistar rats (Rattus novergicus) induced with DM and traumatic ulcers on the lower lip mucosa were divided into six groups-three each for control and treatment groups. Control groups were treated with CMC 5% gel, and treatment groups were administered with C. limon peel essential oil gel. The expression of VEGF and CD-31 was observed on days 5, 7, and 9. Immunohistochemical examinations were performed with the monoclonal antibodies anti-VEGF and anti-CD-31. ANOVA was conducted to analyze the differences between the groups (p < 0.05). Result: An increase in VEGF and CD-31 expression in the treatment group was observed compared with that of the control group (p < 0.05). Conclusion: Citrus limon peel essential oil gel increased VEGF and CD-31 expression during the healing process of traumatic ulcers in diabetes-afflicted Wistar rats.

Synthesis and Characterization of Sulfur Nanoparticles of Citrus limon Extract Embedded in Nanohydrogel Formulation: In Vitro and In Vivo Studies.

The study aimed to synthesize non-noxious, clean, reliable, and green sulfur nanoparticles (SNPs) from Citrus limon leaves. The synthesized SNPs were used to analyze particle size, zeta potential, UV-visible spectroscopy, SEM, and ATR-FTIR. The prepared SNPs exhibited a globule size of 55.32 ± 2.15 nm, PDI value of 0.365 ± 0.06, and zeta potential of -12.32 ± 0.23 mV. The presence of SNPs was confirmed by UV-visible spectroscopy in the range of 290 nm. The SEM image showed that the particles were spherical with a size of 40 nm. The ATR-FTIR study showed no interaction, and all the major peaks were preserved in the formulations. An antimicrobial and antifungal study of SNPs was carried out against Gram-positive bacteria (Staph. aureus, Bacillus), Gram-negative bacteria (E. coli and Bordetella), and fungal strains (Candida albicans). The study showed that Citrus limon extract SNPs exhibited better antimicrobial and antifungal activities against Staph. aureus, Bacillus, E. coli, Bordetella, and Candida albicans at a minimal inhibitory concentration of 50 µg/mL. Different antibiotics were used alone and in combination with SNPs of Citrus limon extract to evaluate their activity against various strains of bacteria and fungal strains. The study showed that using SNPs of Citrus limon extract with antibiotics has a synergistic effect against Staph.aureus, Bacillus, E. coli, Bordetella, and Candida albicans. SNPs were embedded in nanohydrogel formulations for in vivo (wound healing) studies. In preclinical studies, SNPs of Citrus limon extract embedded within a nanohydrogel formulation (NHGF4) have shown promising results. To be widely used in clinical settings, further studies are needed to evaluate their safety and efficacy in human volunteers.

Disease alleviating effects following prophylactic lemon and coriander essential oil treatment in mice with acute campylobacteriosis.

Introduction: Given the worldwide increasing prevalence of human Campylobacter jejuni infections and the emergence of multi-drug resistant enteropathogenic strains, antibiotic-independent approaches applying non-toxic natural compounds for the treatment and prophylaxis of campylobacteriosis appear utmost desirable. In our placebo-controlled intervention study, we surveyed potential disease-alleviating including anti-pathogenic and immune-modulatory effects upon prophylactic oral application of lemon-essential oil (LEM-EO) and coriander-essential oil (COR-EO) in acute experimental campylobacteriosis. Methods: Therefore, secondary abiotic IL-10-/- mice were orally challenged with either LEM-EO or COR-EO starting seven days prior to peroral C. jejuni infection. Results and discussion: Six days post-infection, slightly lower pathogen loads were assessed in the colon of mice from the LEM-EO as opposed to the COR-EO cohort if compared to placebo counterparts. Prophylactic application of both EOs improved the clinical outcome of acute campylobacteriosis which was paralleled by less distinct pathogen-induced colonic epithelial cell apoptosis. Moreover, mice subjected to LEM-EO and COR-EO prophylaxis displayed lower colonic numbers of macrophages/monocytes and of T lymphocytes, respectively, whereas in both verum groups, basal IL-6 and IFN-γ concentrations were measured in mesenteric lymph nodes on day 6 post-infection. The oral challenge with either EOs resulted in diminished secretion of distinct pro-inflammatory mediators in the kidney as well as serum samples derived from the infected mice. In conclusion, the results from our preclinical in vivo study provide evidence that LEM-EO and COR-EO constitute promising prophylactic measures to prevent severe campylobacteriosis which may help to reduce the risk for development of post-infectious sequelae in C. jejuni infected individuals.

Presence of potent inhibitors of bacterial biofilm associated proteins is the key to Citrus limon's antibiofilm activity against pathogenic Escherichia coli.

In an era of antibiotic resistance where natural antibiotic substitutes are considered essential, the antimicrobial and antibiofilm activities of Citrus limon extract on strains of pathogenic Escherichia coli isolated from pork were evaluated. The strains which form biofilms were more resistant (MIC50 = 2.5 mgml-1) compared to non-biofilm forming strains (MIC50 = 1.25 mgml-1). Use of C. limon extract at 20 mgml-1 concentration has resulted in inhibition of biofilm formation by 53.96%. Cyclobarbital, 5, 8-dimethoxycumarin, orotic acid and 3-methylsalicylhydrazide were the major phytochemicals in C. limon extract with highest docking affinities against the biofilm associated proteins in E. coli. The results of simulation studies have clearly illustrated the energy stability of the protein-ligand complexes. Absorption, distribution, metabolism, excretion and toxicity (ADMET) profiles revealed that the phytochemicals in C. limon could be used in the drug design studies to preferentially target the specific receptors to combat biofilms associated with E. coli.

Citrus limon phytocompounds decorated nanoparticles control poultry pathogens.

Antibiotic resistance poses a significant threat to the global health, food security, and environment. In poultry and livestock, antibiotics are beneficial since they improve poultry performance and are economically effective. Therefore, it is crucial to search for alternatives that can be environmentally safe and successful in treating these infections. In this study, we employed molecular docking to evaluate lemon peel phytochemical's protein binding capability against various poultry pathogens. The nanoparticles (LP AgNPs) obtained from the lemon peel were characterized and tested for their antibacterial activity against more poultry pathogens. LP AgNPs were characterized by using UV-Visible absorption spectra, which revealed an absorption peak at a wavelength of 420-440 nm. The FT-IR analysis demonstrated that flavonoids and phenolic acids acted as capping, reducing, and stabilizing agents during the biosynthesis of AgNPs. EDAX showed a strong peak was observed at 3 keV which revealed the absorption of metallic silver nanoparticles. The mean diameter was from 2 to 20 nm through HRTEM. Zeta potential of the LP AgNps at - 17.2 mV showed the high stability of the green synthesized AgNps. Maximum inhibitory concentrations of LP AgNps against the isolated poultry pathogens were 50 µg/ml concentration. The toxicity tests were performed in the Vigna radiata seedlings and Artemia nauplii, which showed less toxic effects and eco-friendly nature of the LP AgNps. LP AgNps have the potential to treat antibiotic resistant poultry pathogens, thereby paving the way for the development of value-added novel products incorporated with nanoparticles for treating various infection caused by antibiotic-resistant poultry pathogens.

Effects of Essential Oils and Hydrolates on the Infectivity of Murine Norovirus.

The use of natural substances with antiviral properties might reduce foodborne viral diseases. In this study, we evaluated the virucidal effect of Citrus limon and Thymus serpyllum essential oils (EOs) and of Citrus Limon, Thymus serpyllum and Thymus vulgaris hydrolates on murine norovirus (MNV), a human norovirus surrogate. To assess the virucidal effect of these natural substances, the reduction in viral infectivity was estimated by comparing the TCID50/mL of untreated viral suspension and the viral suspension treated with hydrolates and EOs at different concentrations. The results showed a natural loss of infectivity of the untreated virus after 24 h of approx. 1 log. The EO (1%) of T. serpyllum, and hydrolates (1% and 2%) of T. serpyllum and T. vulgaris immediately caused a reduction in MNV infectivity of about 2 log but did not provide a further significant decrease after 24 h. Instead, the EO (1%) and hydrolate (1% and 2%) of C. limon exerted an immediate reduction in the viral infectivity of about 1.3 log and 1 log, respectively, followed by a further reduction in infectivity of 1 log after 24 h for the hydrolate. These results will allow for the implementation of a depuration treatment based on the use of these natural compounds.

Gas Chromatography-Mass Spectrometry (GC-MS) Metabolite Profiling of Citrus limon (L.) Osbeck Juice Extract Evaluated for its Antimicrobial Activity Against Streptococcus mutans.

Purpose The present study aimed to determine the antimicrobial nature of Citrus limon juice extract against Streptococcus mutans and to identify its metabolic profile by gas chromatography-mass spectrometry (GC-MS) technique. Materials and methods The cariogenic bacteria S. mutans were procured from Microbial Type Culture Collection and Gene Bank (MTCC), Chandigarh, India, and revived on brain heart infusion (BHI) agar. The C. limon (L.) Osbeck fruits were authenticated from the University of Trans-Disciplinary Health Sciences and Technology (TDU), Bengaluru, India. The antibacterial property of lyophilized lemon juice extract (LJE) dissolved in methanol was evaluated against S. mutans (MTCC 497) by the agar well diffusion assay. GC-MS technique was employed to identify the volatile metabolite profile of the methanolic LJE sample. The metabolite masses of the respective compounds were identified using the National Institute of Standards and Technology (NIST) library. Results The methanolic LJE sample concentration from 5 to 25 mg/ml did not demonstrate antimicrobial activity, while 30 to 100 mg/ml displayed antibacterial activity against S. mutans. Chlorohexidine (100 µg/ml) was used as the positive control, while methanol was used as the negative control. Significant antimicrobial metabolites were detected in the methanolic LJE sample by GC-MS analysis. Maleic anhydride, succinic anhydride, 6-Oxa-bicyclo[3.1.0] hexan-3-one, and 3-methyl-2,5-Furandione were the key metabolites identified in the methanolic LJE sample.  Conclusion The present study reports that C. limon is a potential candidate for the pharmaceutical industry as it possesses bioactive compounds demonstrating therapeutic properties. Further investigations are warranted to determine the individual and synergetic effects of identified metabolites in the methanolic LJE sample for its antimicrobial property. Special attention needs to be focussed on translational research for the development of anti-caries products from C. limon.

Evaluation of Cryogen-Free Thermal Modulation-Based Enantioselective Comprehensive Two-Dimensional Gas Chromatography for Stereo-Differentiation of Monoterpenes in Citrus spp. Leaf Oils.

This study evaluates the applicability of enantioselective gas chromatography (eGC) and enantioselective comprehensive two-dimensional gas chromatography (eGC×GC) coupled with flame ionization detection for the stereospecific analysis of designated chiral monoterpenes within essential oils distilled from the leaves of Citrus hystrix (CH), C. limon (CL), C. pyriformis (CP), and C. microcarpa (CM). A cryogen-free solid-state modulator with a combination of enantioselective first-dimension and polar second-dimension column arrangements was used to resolve potential interferences in Citrus spp. leaf oils that can complicate the accurate determination of enantiomeric compositions. Interestingly, considerable variations were observed for the enantiomeric fractions (EFs) of the chiral terpenes. (+)-limonene was identified as the predominant enantiomer (60.3-98.9%) in all Citrus oils, (+)-linalool was the major enantiomer in CM (95.9%), (-)-terpenin-4-ol was the major isomer in CM (66.4%) and CP (61.1%), (-)-α-pinene was the dominant antipode in CL (55.5%) and CM (92.1%). CH contained (-)-citronellal (100%) as the pure enantiomer, while CL and CP have lower proportions (9.0-34.6%), and citronellal is absent in CM. The obtained enantiomeric compositions were compared and discussed with results from eGC using the same enantioselective column. To our knowledge, this work encapsulates the first report that details the EFs of these chiral monoterpenes in Citrus spp. leaf oil.