Bee chitosan nanoparticles loaded with apitoxin as a novel approach to eradication of common human bacterial, fungal pathogens and treating cancer.

Antimicrobial resistance is one of the largest medical challenges because of the rising frequency of opportunistic human microbial infections across the globe. This study aimed to extract chitosan from the exoskeletons of dead bees and load it with bee venom (commercially available as Apitoxin [Api]). Then, the ionotropic gelation method would be used to form nanoparticles that could be a novel drug-delivery system that might eradicate eight common human pathogens (i.e., two fungal and six bacteria strains). It might also be used to treat the human colon cancer cell line (Caco2 ATCC ATP-37) and human liver cancer cell line (HepG2ATCC HB-8065) cancer cell lines. The x-ray diffraction (XRD), Fourier transform infrared (FTIR), and dynamic light scattering (DLS) properties, ζ-potentials, and surface appearances of the nanoparticles were evaluated by transmission electron microscopy (TEM). FTIR and XRD validated that the Api was successfully encapsulated in the chitosan nanoparticles (ChB NPs). According to the TEM, the ChB NPs and the ChB NPs loaded with Apitoxin (Api@ChB NPs) had a spherical shape and uniform size distribution, with non-aggregation, for an average size of approximately 182 and 274 ± 3.8 nm, respectively, and their Zeta potential values were 37.8 ± 1.2 mV and - 10.9 mV, respectively. The Api@ChB NPs had the greatest inhibitory effect against all tested strains compared with the ChB NPs and Api alone. The minimum inhibitory concentrations (MICs) of the Api, ChB NPs, and Api@ChB NPs were evaluated against the offer mentioned colony forming units (CFU/mL), and their lowest MIC values were 30, 25, and 12.5 µg mL-1, respectively, against Enterococcus faecalis. Identifiable morphological features of apoptosis were observed by 3 T3 Phototox software after Api@ChB NPs had been used to treat the normal Vero ATCC CCL-81, Caco2 ATCC ATP-37, and HepG2 ATCC HB-8065 cancer cell lines for 24 h. The morphological changes were clear in a concentration-dependent manner, and the ability of the cells was 250 to 500 µg mL-1. These results revealed that Api@ChB NPs may be a promising natural nanotreatment for common human pathogens.

Bee Venom Toxic Effect on MDA-MB-231 Breast Cancer Cells and Caenorhabditis Elegans.

INTRODUCTION: Bee venom has therapeutics and pharmacological properties. Further toxicological studies on animal models are necessary due to the severe allergic reactions caused by this product. METHOD: Here, Caenorhabditis elegans was used as an in vivo toxicity model, while breast cancer cells were used to evaluate the pharmacological benefits. The bee venom utilized in this research was collected from Apis mellifera species found in Northeast Brazil. The cytotoxicity caused by bee venom was measured by MTT assay on MDA-MB-231 and J774 A.1 cells during 24 - 72 hours of exposure. C. elegans at the L4 larval stage were exposed for three hours to M9 buffer or bee venom. Survival, behavioral parameters, reproduction, DAF-16 transcription factor translocation, the expression of superoxide dismutase (SOD), and metabolomics were analyzed. Bee venom suppressed the growth of MDA-MB-231 cancer cells and exhibited cytotoxic effects on macrophages. Also, decreased C. elegans survival impacted its behaviors by decreasing C. elegans feeding behavior, movement, and reproduction. RESULTS: Bee venom did not increase the expression of SOD-3, but it enhanced DAF-16 translocation from the cytoplasm to the nucleus. C. elegans metabolites differed after bee venom exposure, primarily related to aminoacyl- tRNA biosynthesis, glycine, serine and threonine metabolism, and sphingolipid and purine metabolic pathways. Our findings indicate that exposure to bee venom resulted in harmful effects on the cells and animal models examined. CONCLUSION: Thus, due to its potential toxic effect and induction of allergic reactions, using bee venom as a therapeutic approach has been limited. The development of controlled-release drug strategies to improve this natural product's efficacy and safety should be intensified.

Bee Venom: Composition and Anticancer Properties.

Among the various natural compounds used in alternative and Oriental medicine, toxins isolated from different organisms have had their application for many years, and Apis mellifera venom has been studied the most extensively. Numerous studies dealing with the positive assets of bee venom (BV) indicated its beneficial properties. The usage of bee products to prevent the occurrence of diseases and for their treatment is often referred to as apitherapy and is based mainly on the experience of the traditional system of medical practice in diverse ethnic communities. Today, a large number of studies are focused on the antitumor effects of BV, which are mainly attributed to its basic polypeptide melittin (MEL). Previous studies have indicated that BV and its major constituent MEL cause a strong toxic effect on different cancer cells, such as liver, lung, bladder, kidney, prostate, breast, and leukemia cells, while a less pronounced effect was observed in normal non-target cells. Their proposed mechanisms of action, such as the effect on proliferation and growth inhibition, cell cycle alterations, and induction of cell death through several cancer cell death mechanisms, are associated with the activation of phospholipase A2 (PLA2), caspases, and matrix metalloproteinases that destroy cancer cells. Numerous cellular effects of BV and MEL need to be elucidated on the molecular level, while the key issue has to do with the trigger of the apoptotic cascade. Apoptosis could be either a consequence of the plasmatic membrane fenestration or the result of the direct interaction of the BV components with pro-apoptotic and anti-apoptotic factors. The interaction of BV peptides and enzymes with the plasma membrane is a crucial step in the whole process. However, before its possible application as a remedy, it is crucial to identify the correct route of exposure and dosage of BV and MEL for potential therapeutic use as well as potential side effects on normal cells and tissues to avoid any possible adverse event.

Anaphylactic shock with pulmonary eosinophilic infiltration due to honeybee attack in a donkey: case report / [Choque anafilático com infiltração eosinófila pulmonar devido a ataque de abelhas em um burro: relato de caso]

A case of a donkey attacked by Africanized honeybee is reported here with clinical signs of agitation, dehydration, congestion of the ocular mucous membranes, tongue edema, tachycardia and inspiratory dyspnea, and progression to death. At necropsy, diffuse, severe subcutaneous edema at face and cervical regions and severe diffuse pulmonary hyperemia with abundant edema without parenchymal collapse were observed. Microscopically, marked, diffuse deep dermis and panniculus carnosus edema and marked diffuse alveolar edema, with moderate population of eosinophils predominantly around larger caliber vessels were noted. The final diagnosis of anaphylactic shock was supported by history, clinical signs, and anatomic pathology findings. This is the first report of a honeybee attack with pulmonary eosinophilic infiltration in a mammal.(AU)

Descreve-se um caso de ataque de abelha africanizada em um burro, com sinais clínicos de agitação, desidratação, mucosas oculares congestas, edema de língua, taquicardia e dispneia inspiratória, com progressão e morte. Na necropsia, foram verificados edema subcutâneo difuso grave nas regiões de face e cervical, hiperemia pulmonar difusa grave com edema abundante e sem colapso do parênquima. Microscopicamente, foram observados edema marcado difuso na derme profunda e panículo carnoso e edema alveolar difuso acentuado, com população moderada de eosinófilos predominantemente em torno de vasos de maior calibre. O diagnóstico de choque anafilático foi baseado no histórico, em sinais clínicos e em achados anatomopatológicos. Este é o primeiro relato de ataque de abelhas com infiltração eosinofílica pulmonar em um mamífero.(AU)

Molecular characterization of the anticancer properties associated with bee venom and its components in glioblastoma multiforme.

Glioblastoma multiforme (GBM) is a frequent form of malignant glioma. Strategic therapeutic approaches to treat this type of brain tumor currently involves a combination of surgery, radiotherapy and chemotherapy. Nevertheless, survival of GBM patients remains in the 12-15 months range following diagnosis. Development of novel therapeutic approaches for this malignancy is therefore of utmost importance. Interestingly, bee venom and its components have shown promising anti-cancer activities in various types of cancer even though information pertaining to GBMs have been limited. The current work was thus undertaken to better characterize the anti-cancer properties of bee venom and its components in Hs683, T98G and U373 human glioma cells. MTT-based cell viability assays revealed IC50 values of 7.12, 15.35 and 7.60 µg/mL for cell lines Hs683, T98G and U373 treated with bee venom, respectively. Furthermore, melittin treatment of these cell lines resulted in IC50 values of 7.77, 31.53 and 12.34 µg/mL, respectively. Cell viability assessment by flow cytometry analysis confirmed signs of late apoptosis and necrosis after only 1 h of treatment with either bee venom or melittin in all three cell lines. Immunoblotting-based quantification of apoptotic markers demonstrated increased expression of Bak and Bax, while Caspsase-3 levels were significantly lower when compared to control cells. Quantification by qRT-PCR showed increased expression levels of long non-coding RNAs RP11-838N2.4 and XIST in glioma cells treated with either bee venom or melittin. Overall, this study provides preliminary insight on molecular mechanisms via which bee venom and its main components can impact viability of glioma cells and warrants further investigation of its anticancer potential in gliomas.

Mechanism of antimicrobial activity of honeybee (Apis mellifera) venom on Gram-negative bacteria: Escherichia coli and Pseudomonas spp.

Honeybee venom (Apitoxin, BV), a secretion substance expelled from the venom gland of bees, has being reported as antimicrobial against various bacterial species; however, the mechanism of action remains uncharacterized. In this study, the antibacterial activity of BV was investigated on hygiene indicator Escherichia coli and the environmental pathogen and spoilage bacterial species, Pseudomonas putida and Pseudomonas fluorescens. An array of methods was combined to elucidate the mode of action of BV. Viability by culture on media was combined with assessing cell injury with flow cytometry analysis. ATP depletion was monitored as an indicator to metabolic activity of cells, by varying BV concentration (75, 225and 500 µg/mL), temperature (25 [Formula: see text] and 37 [Formula: see text]), and time of exposure (0 to 24 h). Venom presented moderate inhibitory effect on E. coli by viability assay, caused high membrane permeability and significant ATP loss where the effect was increased by increased concentration. The viability of P. putida was reduced to a greater extent than other tested bacteria at comparable venom concentrations and was dictated by exposure time. On the contrary, P. fluorescens appeared less affected by venom based on viability; however, flow cytometry and ATP analysis highlighted concentration- and time-dependent effect of venom. According to Transmission Electron Microscopy results, the deformation of the cell wall was evident for all species. This implies a common mechanism of action of the BV which is as follows: the cell wall destruction, change of membrane permeability, leakage of cell contents, inactivation of metabolic activity and finally cell death.

Emerging therapeutic modality enhancing the efficiency of chemotherapeutic agents against head and neck squamous cell carcinoma cell lines.

The current work aimed to evaluate bee venom (BV) cytotoxic effect and its synergistic action when combined with cisplatin (CIS) against four types of head and neck squamous cell carcinoma (HNSCC) cell lines. 3-(4,5-dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide (MTT) assay for cell viability, reverse transcription-polymerase chain reaction (RT-PCR) for expression of BCL2 associated X (BAX), B-cell lymphoma 2 (BCL2) and epidermal growth factor receptor (EGFR) genes and, flow cytometry for cell cycle analysis were performed. MTT assay revealed that BV caused an approximately 50% cell death for UMSCC12, UMSCC29, UMSCC38 and, UMSCC47 cell lines after 72 hr with 54.809 µg/ml, 61.287 µg/ml, 71.328 µg/ml and, 61.045 µg/ml, respectively. RT-PCR demonstrated a significant up-regulation of BAX gene and a significant down-regulation of BCL2 and EGFR genes among single or combined treatments with CIS and BV as compared to vehicle-treated. The cell lines treated with both BV and CIS showed marked elevation of BAX and a notable drop of BCL2 and EGFR expressions than single-treated groups. Cell cycle analysis via flow cytometry revealed significantly increased cells in the G2/M phase in single or combined-treated cell lines with CIS and BV when compared with vehicle-treated. Moreover, a significant decrease in cells in S phases among all single and combined treatments when matched with vehicle-treated. Briefly, the findings of the present study suggest that BV can exert an anti-cancer effect on HNSCC and may have the potentiality for potentiation of CIS cytotoxic effects and reduction of its adverse effects.

Antimicrobial Activity of Five Apitoxins from Apis mellifera on Two Common Foodborne Pathogens.

Antimicrobial resistance is one of today's major public health challenges. Infections caused by multidrug-resistant bacteria have been responsible for an increasing number of deaths in recent decades. These resistant bacteria are also a concern in the food chain, as bacteria can resist common biocides used in the food industry and reach consumers. As a consequence, the search for alternatives to common antimicrobials by the scientific community has intensified. Substances obtained from nature have shown great potential as new sources of antimicrobial activity. The aim of this study was to evaluate the antimicrobial activity of five bee venoms, also called apitoxins, against two common foodborne pathogens. A total of 50 strains of the Gram-negative pathogen Salmonella enterica and 8 strains of the Gram-positive pathogen Listeria monocytogenes were tested. The results show that the minimum inhibitory concentration (MIC) values were highly influenced by the bacterial genus. The MIC values ranged from 256 to 1024 µg/mL in S. enterica and from 16 to 32 µg/mL in L. monocytogenes. The results of this study demonstrate that apitoxin is a potential alternative agent against common foodborne pathogens, and it can be included in the development of new models to inhibit the growth of pathogenic bacteria in the food chain.

Apitoxin and Its Components against Cancer, Neurodegeneration and Rheumatoid Arthritis: Limitations and Possibilities.

Natural products represent important sources for the discovery and design of novel drugs. Bee venom and its isolated components have been intensively studied with respect to their potential to counteract or ameliorate diverse human diseases. Despite extensive research and significant advances in recent years, multifactorial diseases such as cancer, rheumatoid arthritis and neurodegenerative diseases remain major healthcare issues at present. Although pure bee venom, apitoxin, is mostly described to mediate anti-inflammatory, anti-arthritic and neuroprotective effects, its primary component melittin may represent an anticancer therapeutic. In this review, we approach the possibilities and limitations of apitoxin and its components in the treatment of these multifactorial diseases. We further discuss the observed unspecific cytotoxicity of melittin that strongly restricts its therapeutic use and review interesting possibilities of a beneficial use by selectively targeting melittin to cancer cells.

Antimicrobial activity of apitoxin from Apis mellifera in Salmonella enterica strains isolated from poultry and its effects on motility, biofilm formation and gene expression.

Salmonella is a major global food-borne pathogen. One of the main concerns related to Salmonella and other food-borne pathogens is their capacity to acquire antimicrobial resistance and produce biofilms. Due to the increased resistance to common antimicrobials used to treat livestock animals and human infections, the discovery of new antimicrobial substances is one of the main challenges in microbiological research. An additional challenge is the development of new methods and substances to inhibit and destruct biofilms. We determined the antimicrobial and antibiofilm activities of apitoxin in 16 Salmonella strains isolated from poultry. In addition, the effect of apitoxin on Salmonella motility and the expression of biofilm- and virulence-related genes was evaluated. The minimum inhibitory concentrations (MIC) of apitoxin ranged from 1,024-256 µg/mL, with 512 µg/mL being the most common. Sub-inhibitory concentrations of apitoxin significantly reduced biofilm formation in 14 of the 16 Salmonella strains tested, with significant increases in motility. MIC concentrations of apitoxin destroyed the pre-formed biofilm by 27.66-68.22% (47.00% ±â€¯10.91). The expression of biofilm- and virulence-related genes and small RNAs was differentially regulated according to the strain and the presence of apitoxin. The transcription of the small RNAs dsrA and csrB, related to antimicrobial resistance, was upregulated in the presence of apitoxin. We suggest that apitoxin is a potential antimicrobial substance that could be used in combination with other substances to develop new drugs and sanitizers against food-borne pathogens.

Bee Venom: Overview of Main Compounds and Bioactivities for Therapeutic Interests.

Apitherapy is an alternate therapy that relies on the usage of honeybee products, most importantly bee venom for the treatment of many human diseases. The venom can be introduced into the human body by manual injection or by direct bee stings. Bee venom contains several active molecules such as peptides and enzymes that have advantageous potential in treating inflammation and central nervous system diseases, such as Parkinson's disease, Alzheimer's disease, and amyotrophic lateral sclerosis. Moreover, bee venom has shown promising benefits against different types of cancer as well as anti-viral activity, even against the challenging human immunodeficiency virus (HIV). Many studies described biological activities of bee venom components and launched preclinical trials to improve the potential use of apitoxin and its constituents as the next generation of drugs. The aim of this review is to summarize the main compounds of bee venom, their primary biological properties, mechanisms of action, and their therapeutic values in alternative therapy strategies.

Efecto antiinflamatorio de apitoxina de Apis mellifera sobre prostaglandina E2 del fluido crevicular gingival de pacientes con y sin enfermedad periodontal, sometidos a apiterapia: ensayo preliminar / Anti-inflammatory effect of apitoxin and Apis mellifera on prostaglandin E2 in gingival crevicular fluid of patients with and without periodontal disease, submitted to apitherapy: preliminary test

Introducción: La apitoxina que es producida por la Apis mellifera posee efecto antiinflamatorio sobre una serie de marcadores biológicos. La prostaglandina E2 forma parte de ellos, estando presente en el fluido gingival crevicular (FGC). La prostaglandina E2 es evidenciada en la enfermedad periodontal. Objetivo: En este estudio se evaluó el efecto antiinflamatorio de la apitoxina sobre la concentración de prostaglandina E2 del FGC de un paciente sin enfermedad periodontal (SEP) y otro con enfermedad periodontal (CEP). Materiales y Método: Se seleccionó un paciente SEP y otro CEP, que sometidos a apiterapia durante 28 días, se registraron 5 muestras por paciente de FGC, siendo almacenadas, centrifugadas y refrigeradas para su conservación. Posteriormente se midió la concentración de prostaglandina E2 crevicular mediante inmunodifusión radial simple en placas petri con concentración de anticuerpo anti prostaglandina E2 de 1:1000. Selladas a 4°C, se esperó 72 horas para permitir su difusión, tiñéndose con Azul brillante de Coomasie, determinándose la concentración de cada placa. Resultados: Paciente SEP inmediatamente antes de apiterapia presentó una concentración de 0.9636 ± 0.0055 (ug/uL), finalizando con una concentración de 0.9196+/-0.0733 (ug/uL) al completar 28 días de tratamiento. El paciente CEP antes de recibir apiterapia presento una concentración de 1.1866 +/- 0.0867 (ug/uL), finalizando con una concentración de 0.9858 +/- 0.0074 (ug/uL) al completar 28 días de tratamiento. Discusión: Los hallazgos de este estudio demuestran una disminución de la concentración de PGE2 del FGC tanto para el paciente CEP y SEP sometidos a apiterapia durante 28 días, siendo esta disminución 3.7 veces mayor en el paciente CEP.

Introduction: Apitoxin, which is produced by Apis mellifera, has anti-inflammatory effect on a number of biomarkers. Prostaglandin E2 is one of them, being present in gingival crevicular fluid (GCF). Prostaglandin E2 is evidenced in periodontal disease. Objective: This study evaluated the antiinflammatory effect of apitoxin on concentration of prostaglandin E2 FGC in a patient with no periodontal disease (SEP) and other with periodontal disease (CEP). Materials and Methods: We selected both a SEP and CEP patient who were subjected to apitherapy for 28 days. There were 5 samples per patient of FGC, being stored, centrifuged and refrigerated for their preservation. Subsequently, the concentrations of crevicular prostaglandin E2 were measured by simple radial immunodiffusion in petri dishes with antibody concentration of prostaglandin E2 of 1:1000. Sealed at 4 °C, after 72 hours to allow diffusion, they were stained with Coomassie Brilliant Blue, determining the concentration of each plate. Results: SEP patient immediately before apitherapy presented a concentration of 0.9636 +/- 0.0055 (g / mL), ending with a concentration of 0.9196 +/- 0.0733 (g / mL) upon completion of 28 days of treatment. CEP patient before receiving apitherapy showed a concentration of 1.1866 +/- 0.0867 (g/mL), ending with a concentration of 0.9858 +/- 0.0074 (g/mL) upon completion of 28 days of treatment. Discussion: The findings of this study show a decrease in the concentration of PGE2 of FGC both for the CEP and SEP patient subjected to apitherapy for 28 days, being this decrease 3.7 times higher in the CEP patient.

Foreign Body Granuloma Following Dried Honey Bee Venom (Apitoxin Inj) Injection / 대한피부과학회지

Bee sting therapy is sometimes used for the treatment of chronic recalcitrant neuralgia and arthralgia in traditional Korean herbal medicine, but retained sting materials at the treatment site may induce granulomatous inflammation. Recently, dried honey bee venom (Apitoxin Inj, Guju Pharma. Co., Seoul, Korea) has been approved by the Korea Food and Drug Administration (KFDA) as an anti-inflammatory drug. The adverse events associated with dried honey bee venom injection include itching, edema, pain, headache, fever and myalgia, but foreign body granuloma caused by drug injection has not been previously reported. We herein report two interesting cases of foreign body granuloma induced by dried honey bee venom injection.

Minimal Change Nephrotic Syndrome After Apitoxin Therapy: A Case Report / 대한신장학회지

Bee stings have previously been implicated in the development of nephrotic syndrome, but the reported cases in the literature are rare. Furthermore, there has been no case of nephrotic syndrome after bee venom (apitoxin) therapy. We experienced a 28-year-old female who developed generalized edema 6 days after an intramuscular injection of apitoxin. The physical examination and laboratory findings were relevant with nephrotic syndrome and the renal biopsy revealed minimal change nephrotic syndrome. The corticosteroid treatment induced prompt remission with resolution of edema and normalization of the laboratory findings. There was no relapse of the disease during the 6-month follow-up. We report this case together with brief review of literatures.