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Enterohemorrhagic Escherichia coli (EHEC) is a subtype of pathogenic E. coli that causes diarrhea or hemorrhagic colitis in humans, which can progresses to hemolytic uremic syndrome (HUS), a leading cause of acute renal failure in children, and morbidity and mortality in adults. Stool samples (n = 273) of patients (1 day-40 years old) suffered from bloody diarrhea and abdominal cramps, were examined bacteriologically and molecularly for the presence and pathogenicity of EHEC with phylogenetic analysis of the obtained stx1, stx2, and eaeA virulence genes' sequences. Overall, 71 (26.01%) E. coli isolates were identified as EHEC with the following serogroupes: O1:H11 (3), O128:H2 (9), O26:H11 (6), O157:H7 (3), O25:H2 (7), O145:H328 (2), O125:H6 (9), O86:H8 (5), O18:H15 (11) and untypable (16). The highest isolation rate were in samples belonged to infants below two years old (42.25%). Antimicrobial susceptibility testing showed that all isolates were highly sensitive to ciprofloxacin, nitrofurantoin, gentamycin, imipenem and vancomycin (100% each), however, they were resistant to ampicillin, cephalexin, penicillin and tetracycline (100% each). In-vitro pathogenicity testing of the isolates revealed that 67 (94.37%) isolates were positive for Congo red test, 47 (66.20%) isolates possessed P fimbriae (MRHA) and 17 (23.94%) possessed type 1 fimbriae (MSHA). Moreover, 46 (64.79%) isolates exhibited hemolysis and 42 (59.15%) isolates showed distinct cytopathic effect to Vero cells. Molecular detection of enterohemorrhagic E. coli (EHEC) pathotype virulence genes, confirmed the presence of stx1 gene in O157:H7 (MA2), O26:H11, O145:H328 and O125:H6 serogroups; stx2 gene in (O157:H7 (MA1), O128:H2 and O25:H2; while all serogroups except (O125:H6) carried the eaeA intimin virulence gene. A phylogenetic tree, based on the nucleotide sequences of toxin-encoding genes, demonstrates that Shiga toxin E. coli (STEC) isolates have considerable genetic variation and belong to various phylogenetic groupings.
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Escherichia coli Enterohemorrágica , Infecciones por Escherichia coli , Proteínas de Escherichia coli , Escherichia coli Shiga-Toxigénica , Adhesinas Bacterianas/genética , Adolescente , Adulto , Animales , Niño , Preescolar , Chlorocebus aethiops , Diarrea , Escherichia coli Enterohemorrágica/genética , Proteínas de Escherichia coli/genética , Humanos , Lactante , Recién Nacido , Filogenia , Toxinas Shiga/genética , Escherichia coli Shiga-Toxigénica/genética , Células Vero , Adulto JovenRESUMEN
PURPOSE: The selective delivery of chemotherapeutic agent to the affected area is mainly dependent on the mode of drug loading within the delivery system. This study aims to compare the physical method to the chemical method on the efficiency of loading DOX.HCl to GNPs and the specific release of the loaded drug at certain tissue. METHOD: Bifunctional polyethylene glycol with two different functionalities, the alkanethiol and the carboxyl group terminals, was synthesized. Then, DOX·HCl was covalently linked via hydrazone bond, a pH sensitive bond, to the carboxyl functional group and the produced polymer was used to prepare drug functionalized nanoparticles. Another group of GNPs was coated with carboxyl containing polymer; loading the drug into this system by the means of electrostatic adsorption. Finally, the prepared system were characterized with respect to size, shape and drug release in acetate buffer pH 5 and PBS pH 7.4 Also, the effect of DOX.HCl loaded systems on cell viability was assessed using MCF-7 breast cancer cell line. RESULTS: The prepared nanoparticles were spherical in shape, small in size and monodisperse. The release rate of the chemically bound drug in the acidic pH was higher than the electrostatically adsorbed one. Moreover, both systems show little release at pH 7.4. Finally, cytotoxicity profiles against human breast adenocarcinoma cell line (MCF-7) exhibited greater cytotoxicity of the chemically bound drug over the electrostatically adsorbed one. CONCLUSION: Chemical binding of DOX·HCl to the carboxyl group of PEG coating GNPs selectively delivers high amount of drug to tumour-affected tissue which leads to reducing the unwanted effects of the drug in the non-affected ones.
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Antibióticos Antineoplásicos/farmacocinética , Neoplasias de la Mama/tratamiento farmacológico , Doxorrubicina/farmacocinética , Portadores de Fármacos/química , Liberación de Fármacos , Adsorción , Antibióticos Antineoplásicos/administración & dosificación , Antibióticos Antineoplásicos/química , Neoplasias de la Mama/patología , Doxorrubicina/administración & dosificación , Doxorrubicina/química , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Oro/química , Humanos , Concentración de Iones de Hidrógeno , Células MCF-7 , Nanopartículas del Metal/química , Polietilenglicoles/química , Electricidad EstáticaRESUMEN
Majority of breast cancers originate from epithelial cells in the duct and lobules in the breast. Current systemic treatments for breast cancer are associated with significant systemic side effects, thus warranting localized drug delivery approaches. The aim of this study was to investigate the influence of hydroalcoholic vehicle on topical delivery of 4-hydroxy tamoxifen (4-HT) through the mammary papilla (nipple). The in vitro permeability of 4-HT through porcine mammary papilla was studied using different hydroalcoholic vehicles (0, 33.33, and 66.66% alcohol). Nile red was used as a model lipophilic dye to characterize the drug transport pathway in the mammary papilla. The penetration of 4-HT through the mammary papilla increased with increase in alcohol concentration in the vehicle. The solubility of 4-HT was enhanced by increasing alcohol concentration in the vehicle. On the other hand, the epidermis/vehicle partition coefficient decreased with increase in alcohol concentration. The mammary papilla served as a depot and slowly released 4-HT into the receptor medium. Highest drug penetration was observed with saturated drug solution in 66.66% alcohol, and 4-HT levels were comparable to IC50 value of 4-HT. Results from this study demonstrate the possibility of using mammary papilla as a potential route for direct delivery of 4-HT to the breast.
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Sistemas de Liberación de Medicamentos , Pezones/metabolismo , Tamoxifeno/análogos & derivados , Animales , Neoplasias de la Mama/tratamiento farmacológico , Femenino , Vehículos Farmacéuticos , Solubilidad , Porcinos , Tamoxifeno/administración & dosificación , Tamoxifeno/química , Tamoxifeno/farmacocinéticaRESUMEN
The main focus of this study was on using radiation to make an ultra-absorbent hydrogel (UAH) from sodium alginate (SA) and gelatin (GL) biopolymers. This UAH can effectively handle water and nitrogen in wheat farming during drought stress. The hydrogel was synthesized by gamma irradiation-induced SA/GL/polyacrylamide crosslinking at 10-40 kGy. Varying SA/GL ratios affected swelling and the gel fraction of SA/GL/PAm hydrogels. The (SA/GL 17/83) hydrogel exhibited a 40.03 g/g swelling degree, while increasing SA content resulted in higher swelling, peaking at 75.5 g/g for (SA/GL 83/17). This indicated a synergistic interaction between SA and GL. The gel fraction also increased from 76.8 to 90.3%, with a higher GL content reflecting increased crosslinking. After multiple hydrolysis cycles, the hydrogel achieved 1293 (g/g) swelling and 36 days of water retention. When applied to wheat (Triticuma estivum) under drought stress, it significantly improved shoot length (18%), root length (43%), shoot fresh weight (49%), and shoot dry weight (51%) under extreme drought. The significant increases in protein and carbohydrate content in both shoots (up to 32% and 19%, respectively) and grains (up to 21% and 24%, respectively), along with the reduction in proline content (up to 38%), demonstrate that ultra-absorbent hydrogel (UAH) effectively enhances nitrogen content, photosynthesis, and overall plant health in wheat under varying drought stress levels. This novel SA/GL-based UAH holds promise for addressing water scarcity and agricultural challenges, offering a sustainable solution for water and nitrogen management under drought stress.
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Alginatos , Sequías , Gelatina , Hidrogeles , Nitrógeno , Triticum , Agua , Triticum/crecimiento & desarrollo , Triticum/metabolismo , Triticum/fisiología , Alginatos/química , Gelatina/química , Nitrógeno/metabolismo , Nitrógeno/química , Hidrogeles/química , Agua/química , Estrés Fisiológico , Rayos gammaRESUMEN
Staphylococcus aureus and methicillin-resistant Staphylococcus aureus (MRSA) are classified as high-risk infections that can lead to death, particularly among older individuals. Nowadays, plant nanoparticles such as glycyrrhizic acid are recognized as efficient bactericides against a wide range of bacterial strains. Recently, scientists have shown interest in plant extract nanoparticles, derived from natural sources, which can be synthesized into nanomaterials. Interestingly, glycyrrhizic acid is rich in antioxidants as well as antibacterial agents, and it exhibits no adverse effects on normal cells. In this study, glycyrrhizic acid nanoparticles (GA-NPs) were synthesized using the hydrothermal method and characterized through physicochemical techniques such as UV-visible spectrometry, DLS, zeta potential, and TEM. The antimicrobial activity of GA-NPs was investigated through various methods, including MIC assays, anti-biofilm activity assays, ATPase activity assays, and kill-time assays. The expression levels of mecA, mecR1, blaR1, and blaZ genes were measured by quantitative RT-qPCR. Additionally, the presence of the penicillin-binding protein 2a (PBP2a) protein of S. aureus and MRSA was evaluated by a Western blot assay. The results emphasized the fabrication of GA nanoparticles in spherical shapes with a diameter in the range of 40-50 nm. The data show that GA nanoparticles exhibit great bactericidal effectiveness against S. aureus and MRSA. The treatment with GA-NPs remarkably reduces the expression levels of the mecA, mecR1, blaR1, and blaZ genes. PBP2a expression in MRSA was significantly reduced after treatment with GA-NPs. Overall, this study demonstrates that glycyrrhizic acid nanoparticles have potent antibacterial activity, particularly against MRSA. This research elucidates the inhibition mechanism of glycyrrhizic acid, which involves the suppressing of PBP2a expression. This work emphasizes the importance of utilizing plant nanoparticles as effective antimicrobial agents against a broad spectrum of bacteria.
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Here, the impact of irrigation using untreated wastewater (WW) on carrots (Daucus carota L.) was examined. We hypothesized that the addition of ethylenediaminetetraacetic acid (EDTA), dry algal powder (Spirulina platensis or Chlorella vulgaris), and Salix alba leaves powder would function as chelators for harmful contaminants in wastewater. The findings showed that irrigation of carrot plants with the sampled untreated wastewater led to significant decreases in the shoot lengths, fresh, dry weights of shoots and roots at stage I, the diameter of roots, pigment content, carotenoids, total soluble carbohydrate content, and soluble protein content. Furthermore, a significantly increased level of proline, total phenols, and the activities of polyphenol oxidase (PPO), peroxidase (POX), superoxide dismutase (SOD), and catalase (CAT) was identified in stage I samples. In contrast to the stage I, the length of the roots, the number of leaves on each plant, wet and dry weights of the stage II roots were all greatly enhanced. In spite of the increased yield due to the wastewater irrigation, carrot roots irrigated with wastewater had significantly more cadmium (Cd), nickel (Ni), cobalt (Co), and lead (Pb) than is considered safe. Our data clearly show that the application of Spirulina platensis, Chlorella vulgaris, EDTA, and leaves powder of salix was able to alleviate the toxicity of wastewater on carrot plants. For example, we recorded a significant decrease in the accumulation of carrot's Cd, Ni, Co, and Pb contents. We conclude that the treatments with Spirulina platensis and Chlorella vulgaris can be utilized as eco-friendly tools to lessen the damaging effects of wastewater irrigation on carrot plants.
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Chlorella vulgaris , Daucus carota , Metales Pesados , Contaminantes del Suelo , Spirulina , Cadmio/toxicidad , Aguas Residuales , Ácido Edético/farmacología , Chlorella vulgaris/metabolismo , Plomo/farmacología , Polvos , Metales Pesados/análisis , Contaminantes del Suelo/toxicidadRESUMEN
The present work describes a quinazolinone-based lead optimization for the development of novel purine nucleoside phosphorylase (PNP) inhibitors with quinazolinone scaffold. Nineteen compounds were proposed and docked against PNP, the best 14 compounds with highest docking and affinity scores and low RMSD values were synthesized. Synthesis of new quinazolinone derivatives with variable acetamide substituents on two positions on quinazoline ring was performed. The structures assigned to the products were concordant with the microanalytical and spectral data. In vitro cytotoxicity on human breast cancer cell line (MCF7) was performed and identified compound 6g as the most potent with IC50 (0.99 ± 0.11 µM) which was further tested against five different breast cancer cell lines in addition to normal breast cell to determine the selectivity. Compound 6g was subjected to molecular dynamic simulation study, radiolabelling and biodistribution study to investigate its stability and selectivity toward breast cancers. The in vitro PNP inhibition results were aligned with the in silico, cytotoxicity, and biodistribution results where 6g showed the most potent PNP inhibitory activity with IC50 (0.159 ± 0.007 µM) when compared to Peldesine (BCX-34) IC50 (0.041 ± 0.002 µM).
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Neoplasias de la Mama , Purina-Nucleósido Fosforilasa , Humanos , Femenino , Purina-Nucleósido Fosforilasa/metabolismo , Neoplasias de la Mama/tratamiento farmacológico , Quinazolinonas/farmacología , Distribución Tisular , Inhibidores Enzimáticos/farmacología , Acetamidas , Relación Estructura-ActividadRESUMEN
The study addresses the potential negative impacts of climate change on water resources, specifically irrigation water for crops. The radiation technique produces the biomaterial hydrogel as a soil conditioner by polymerizing hydroxy ethyl cellulose/acrylamide (HEC/AAm) at various irradiation doses and copolymer concentrations. A maximum swelling of 23.4 g/g is attained by (HEC/PAAm) hydrogel at 1/7.5 ratio, prepared by 10 kGy gamma irradiation. The study introduces a new class of ultra-absorbent hydrogel (UAH) to address the low swelling limitation for soil conditioner applications. The alkaline hydrolysis treatments with NaOH, LiOH, and KOH enhance the water absorbency of (HEC/PAAm) hydrogel, with the highest capacity of 1220 g/g achieved by the KOH treatment, surpassing NaOH (622 g/g) and LiOH (540 g/g). The cumulative release of fertilizers from the UAH sample shows a slow and controlled release behavior. Urea takes 22 days to reach 100 % release. The UAH demonstrates water retention for 28 days, improving the growth of Zea mays L. at drought stress levels of 0 %, 25 %, 50 %, and 100 %, revealing an increase in shoot length by 16 %, 19 %, 24 %, and 20 %, respectively. Also, UAH increased the contents of chlorophyll a, b, a + b, and carotenoid on maize plant leaves compared to the control sample.
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Fertilizantes , Hidrogeles , Fertilizantes/análisis , Zea mays , Sequías , Clorofila A , Hidróxido de Sodio , Suelo , AguaRESUMEN
Herein, two seaweed extracts (Sargassum latifolium and Corallina elongate), and two commercial seaweed products (Canada power and Oligo-X) with a concentration of 5% were used to alleviate the drought stress on wheat plants. The extract of C. elongate had the highest capacity to ameliorate the deleterious effects of water scarcity followed by S. latifolium and the commercial products. The drought stress reduced wheat shoots length and the contents of pigments (chlorophyll and carotenoids), carbohydrates, and proteins. While the highest increment in the total carbohydrates and protein contents of the wheat shoot after two stages, 37-and 67-days-old, were noted in drought-stressed plants treated with C. elongate extract with values of (34.6% and 22.8%) and (51.9% and 39.5%), respectively, compared to unstressed plants. Decreasing the activity of antioxidant enzymes, peroxidase, superoxidase dismutase, and polyphenol oxidase in drought-stressed plants treated with algal extracts indicated amelioration of the response actions. Analysis of phytohormones in wheat plants exhibited increasing GA3 and IAA contents with percentages of (20.3-13.8%) and (72.7-25%), respectively. Interestingly, all morphological and metabolic characteristics of yield were improved due to the algal treatments compared with untreated drought-stressed plants. Overall, the algal extracts, especially those from seaweed of C. elongate, could represent a sustainable candidate to overcome the damage effects of water deficiency in the wheat plant.
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A rapid and accurate high-performance liquid chromatographic method was developed for the determination of both abamectin and closantel in the veterinary formulation. The chromatographic separation was conducted on an Agilent 1200 with a UV detector using Waters C18 (4.6 mm × 50 mm; 2.7 µm). The mobile phase consisted of acetonitrile:water (80:20 v/v) adjusts pH 3.0 using diluted phosphoric acid. The flow rate of 1.5 mL min-1 was used. An injection volume of 10 µL was used The calibration curve of abamectin B1b was linear with a correlation coefficient (r2) = 0.9996; over a concentration range of 2.0-8.0 µg/mL, abamectin B1a was linear with a correlation coefficient (r2) = 0.9997; over a concentration range of 8.0-32.0 µg/mL; with a retention time of 2.18 and 3.72 minutes for avermectin B1b and avermectin B1a, respectively. While the calibration curve of closantel was linear with a correlation coefficient (r2) = 0.99929; over a concentration range of 250.0-1,000.0 µg/mL for; with a retention time of 5.84 minutes. Correlation coefficient was r2 ≥ 0.999. The relative standard deviation was found to be ≤ 2. The proposed method was validated and successfully applied for the simultaneous determination of abamectin and closantel in the veterinary formulation.
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Cromatografía Líquida de Alta Presión/métodos , Ivermectina/análogos & derivados , Salicilanilidas/análisis , Drogas Veterinarias/análisis , Ivermectina/análisis , Límite de Detección , Modelos Lineales , Reproducibilidad de los ResultadosRESUMEN
Khellin was successfully extracted from Ammi visnaga fruits with a recovery percent of 96.15%. Next radio-iodination of Khellin was successfully achieved with a high yield. The biodistribution study of [131I]iodo-khellin in tumour bearing mice revealed that khellin preferentially localization at tumour tissue. Target prediction study for [131I]iodo-khellin revealed that PI3K and VEGFR are potential targets for iodo-khellin with good affinity. The results of this study potentiate [131I]iodo-khellin as a good theranostic agent for tumour imaging and therapy.
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Radioisótopos de Yodo/administración & dosificación , Khellin/metabolismo , Neoplasias/terapia , Medicina de Precisión , Animales , Cromatografía Líquida de Alta Presión/métodos , Humanos , Radioisótopos de Yodo/química , Khellin/química , Khellin/aislamiento & purificación , Masculino , Ratones , Simulación del Acoplamiento Molecular , Neoplasias/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Receptores de Factores de Crecimiento Endotelial Vascular/metabolismo , Distribución TisularRESUMEN
The main goal of any data storage model on the cloud is accessing data in an easy way without risking its security. A security consideration is a major aspect in any cloud data storage model to provide safety and efficiency. In this paper, we propose a secure data protection model over the cloud. The proposed model presents a solution to some security issues of cloud such as data protection from any violations and protection from a fake authorized identity user, which adversely affects the security of the cloud. This paper includes multiple issues and challenges with cloud computing that impairs security and privacy of data. It presents the threats and attacks that affect data residing in the cloud. Our proposed model provides the benefits and effectiveness of security in cloud computing such as enhancement of the encryption of data in the cloud. It provides security and scalability of data sharing for users on the cloud computing. Our model achieves the security functions over cloud computing such as identification and authentication, authorization, and encryption. Also, this model protects the system from any fake data owner who enters malicious information that may destroy the main goal of cloud services. We develop the one-time password (OTP) as a logging technique and uploading technique to protect users and data owners from any fake unauthorized access to the cloud. We implement our model using a simulation of the model called Next Generation Secure Cloud Server (NG-Cloud). These results increase the security protection techniques for end user and data owner from fake user and fake data owner in the cloud.
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Nube Computacional , Confidencialidad , Seguridad Computacional , Almacenamiento y Recuperación de la InformaciónRESUMEN
Microbial endophytes organize symbiotic relationships with the host plant, and their excretions contain diverse plant beneficial matter such as phytohormones and bioactive compounds. In the present investigation, six bacterial and four fungal strains were isolated from the common bean (Phaseolus vulgaris L.) root plant, identified using molecular techniques, and their growth-promoting properties were reviewed. All microbial isolates showed varying activities to produce indole-3-acetic acid (IAA) and different hydrolytic enzymes such as amylase, cellulase, protease, pectinase, and xylanase. Six bacterial endophytic isolates displayed phosphate-solubilizing capacity and ammonia production. We conducted a field experiment to evaluate the promotion activity of the metabolites of the most potent endophytic bacterial (Bacillus thuringiensis PB2 and Brevibacillus agri PB5) and fungal (Alternaria sorghi PF2 and, Penicillium commune PF3) strains in comparison to two exogenously applied hormone, IAA, and benzyl adenine (BA), on the growth and biochemical characteristics of the P. vulgaris L. Interestingly, our investigations showed that bacterial and fungal endophytic metabolites surpassed the exogenously applied hormones in increasing the plant biomass, photosynthetic pigments, carbohydrate and protein contents, antioxidant enzyme activity, endogenous hormones and yield traits. Our findings illustrate that the endophyte Brevibacillus agri (PB5) provides high potential as a stimulator for the growth and productivity of common bean plants.
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Producción de Cultivos/métodos , Endófitos/metabolismo , Phaseolus/efectos de los fármacos , Reguladores del Crecimiento de las Plantas/farmacología , Adenina/análogos & derivados , Adenina/farmacología , Biomasa , Brevibacillus/metabolismo , Ácidos Indolacéticos/farmacología , Phaseolus/crecimiento & desarrollo , Phaseolus/microbiología , FotosíntesisRESUMEN
A microbial fuel cell (MFC) integrated with adsorption system (MFC-AHS) is tested under various operating temperatures with palm oil mill effluent as the substrate. The optimum operating temperature for such system is found to be at â¼35°C with current, power density, internal resistance (Rin), Coulombic efficiency (CE) and maximum chemical oxygen demand (COD) removal of 2.51 ± 0.2â mA, 74 ± 6â mWâ m-3, 25.4â Ω, 10.65 ± 0.5% and 93.57 ± 1.2%, respectively. Maximum current density increases linearly with temperature at a rate of 0.1772â mAâ m-2â °C-1, whereas maximum power density was in a polynomial function. The temperature coefficient (Q10) is found to be 1.20 between 15°C and 35°C. Present studies have demonstrated better CE performance when compared to other MFC-AHSs. Generally, MFC-AHS has demonstrated higher COD removals when compared to standalone MFC regardless of operating temperatures. ABBREVIATIONS: ACFF: activated carbon fiber felt; APHA: American Public Health Association; CE: Coulombic efficiency; COD: chemical oxygen demand; ECG: electrocardiogram; GAC: granular activated carbon; GFB: graphite fiber brush; MFC: microbial fuel cell; MFC-AHS: microbial fuel cell integrated with adsorption hybrid system; MFC-GG: microbial fuel cell integrated with graphite granules; POME: palm oil mill effluent; PTFE: polytetrafluoroethylene; SEM: scanning electron microscope.
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Fuentes de Energía Bioeléctrica , Adsorción , Análisis de la Demanda Biológica de Oxígeno , Electricidad , Electrodos , Aceite de Palma , TemperaturaRESUMEN
Diabetes is one of the leading causes of impaired wound healing. The objective of this study was to develop a bee venom-loaded wound dressing with an enhanced healing and anti-inflammatory effects to be examined in diabetic rats. Different preparations of polyvinyl alcohol (PVA), chitosan (Chit) hydrogel matrix-based wound dressing containing bee venom (BV) were developed using freeze-thawing method. The mechanical properties such as gel fraction, swelling ratio, tensile strength, percentage of elongation and surface pH were determined. The pharmacological activities including wound healing and anti-inflammatory effects in addition to primary skin irritation and microbial penetration tests were evaluated. Moreover, hydroxyproline, glutathione and IL-6 levels were measured in the wound tissues of diabetic rats. The bee venom-loaded wound dressing composed of 10 % PVA, 0.6 % Chit and 4 % BV was more swellable, flexible and elastic than other formulations. Pharmacologically, the bee venom-loaded wound dressing that has the same previous composition showed accelerated healing of wounds made in diabetic rats compared to the control. Moreover, this bee venom-loaded wound dressing exhibited anti-inflammatory effect that is comparable to that of diclofenac gel, the standard anti-inflammatory drug. Simultaneously, wound tissues covered with this preparation displayed higher hydroxyproline and glutathione levels and lower IL-6 levels compared to control. Thus, the bee venom-loaded hydrogel composed of 10 % PVA, 0.6 % Chit and 4 % BV is a promising wound dressing with excellent forming and enhanced wound healing as well as anti-inflammatory activities.