New insights into changing honey bee (Apis mellifera) immunity molecules pattern and fatty acid esters, in responses to Ascosphaera apis infection.

Monitoring of metabolite changes could provide valuable insights into disturbances caused by an infection and furthermore, could be used to define the status of an organism as healthy or diseased and define what could be defensive elements against the infection. The present investigation conducted a gas chromatography-mass spectrometry (GC/MS) for haemolymph of larval honey bees (Apis mellifera L.) infected with the fungal pathogen Ascosphaera apis in comparison with control haemolymph non-infected insects. Results revealed that the pathogen caused a general disturbance of metabolites detected in the haemolymph of the honey bee. The majority of metabolites identified before and after infection were fatty acid esters. The disease caused an elevation in levels of methyl oleate, methyl palmitate, and methyl stearate, respectively. Further, the disease drove to the disappearance of methyl palmitoleate, and methyl laurate. Conversely, methyl linolelaidate, and ethyl oleate were identified only in infected larvae. A high reduction in diisooctyl phthalate was recorded after the infection. Interestingly, antimicrobial activities were confirmed for haemolymph of infected honey bee larvae. In spite of the presence of some previously known bioactive compounds in healthy larvae there were no antimicrobial activities.

First report on isolation of Mucor bainieri from honeybees, Apis mellifera: Characterization and biological activities.

Fungi are potential biocontrol agents and rich sources of secondary metabolites with demonstrated biological activities. This study aimed to isolate and identify fungi from surface-sterilized honeybees (Apis mellifera), as well as to evaluate their biological activities. One fungal isolate was obtained and identified morphologically and genetically as Mucor bainieri MK-Bee-2. Gas chromatography-mass spectroscopy (GC-MS) analysis of fungus crude extract, showed the existence of six major metabolites representing 92.48% of the total peak area. The crude extract of Mucor bainieri MK-Bee-2 was tested for antimicrobial, antioxidant, and antitumor activities. It demonstrated wide antimicrobial activities against human pathogenic Gram-positive and Gram-negative bacterial strains, as well as Candida albicans, with MIC values ranged from 62.5 to 250 µg/ml. The results revealed that the extract exhibited considerable antioxidant activities indicated by strong inhibition of both DPPH and ABTS free radicals. Additionally, the extract exhibited greater potential anticancer activity against both adenocarcinomic human non-small cell lung cancer cells (A549) [IC50 = 6.45 µg/ml], and immortal cell line hepatoma G2 (HepG2) human liver cancer cells [IC50 = 27.48 µg/ml] and higher selectivity in cancer cells than normal cell lines. Furthermore, the extract showed less cytotoxic activity against normal cells with higher IC50 values of 106.99 and 132.57 µg/ml against human lung fibroblast Wistar-38 (Wi-38) and oral epithelial cells (OEC), respectively. Taken together, the Mucor bainieri MK-Bee-2 extract comprises bioactive compounds as promising potential therapeutic candidates for the treatment of lung cancer. Strikingly, the extract sensitizes the lung cancer cells A549  to the ionizing radiation through the pro-apoptotic pathway as indicated by the annexin V flow cytometry analysis which showed that the extract reduced the apoptosis of lung cancer cells.

Antifungal and antibiofilm activities of bee venom loaded on chitosan nanoparticles: a novel approach for combating fungal human pathogens.

The prevalence of opportunistic human fungal pathogens is increasing worldwide, and antimicrobial resistance is one of the greatest medical challenges the world faces. Therefore, this study aimed to develop a novel agent to control fungal pathogens. The honeybee products (honey, royal jelly, propolis, bee bread, and bee venom) were screened against unicellular fungal (UCF) pathogens (Cryptococcus neoformans, Kodamaea ohmeri, and Candida albicans) and the bee venom was only exhibited an inhibitory effect against them. The protein contents of crude bee venom were separated using the gel filtration technique into eight fractions which were visualized on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to confirm the presence of five bands with molecular weights of 65, 43, 21, 15, and 3 KDa. Bee venom (BV) of Apis mellifera loaded chitosan nanoparticles were prepared by the ionotropic gelation method. The encapsulation efficiency%, average size, zeta potentials, and surface appearance by Transmission electron microscope (TEM) were evaluated for the prepared nanoparticles. The minimum inhibitory concentration (MIC) of crude BV and BV loaded chitosan nanoparticles (BV-CNPs) was evaluated against the offer mentioned UCF where the MIC values of crude BV were 6.25, 3.12 & 6.25 while MIC values in the case of BV-CNPs were decreased to 3.12, 3.12 & 1.56 mg/ml against C. neoformans, K. ohmeri and C. albicans, respectively. Also, the results showed that BV-CNPs suppressed the biofilm formation as well as yeast to hyphal transition formed by the examined UCF. These results revealed that BV-CNPs are a promising natural compound for fungal pathogens treatment.

Formulation, characterization and cellular toxicity assessment of a novel bee-venom microsphere in prostate cancer treatment.

Bee venom (B.V.) is a toxin produced naturally by honey bees with several toxic and therapeutic efficacies. It is used in the treatment of different cancer kinds like renal, hepatic, and prostate cancer. Due to its protein nature, it is degraded in the upper gastrointestinal tract. Colon-targeted drug delivery systems represent a useful tool to protect B.V. from degradation and can be administered orally instead of I.V. infusion and traditional bee stinging. In the present study, B.V. loaded enteric-coated cross-linked microspheres were prepared by emulsion cross-linking method. Percentage yield, entrapment efficiency %, swelling degree, and in-vitro release are evaluated for prepared microspheres. Free B.V., optimized microspheres formula (F3), and doxorubicin cytotoxic effects were tested by MTT assay. Results concluded that free B.V. was more effective against the growth of human prostate adenocarcinoma (PC3) cells followed by optimized microspheres than doxorubicin. But both free B.V. and doxorubicin have a cytotoxic effect on normal oral epithelial cells (OEC). According to flow cytometric analysis, the optimized microsphere formula induced apoptosis and reduced necrosis percent at IC50 concentration. Furthermore, microspheres did not affect the viability of OEC. These results revealed that microspheres have a degree of specificity for malignant cells. Therefore, it seems that this targeted formulation could be a good candidate for future clinical trials for cancer therapy.

Mosquitocidal Activity of the Methanolic Extract of Annickiachlorantha and Its Isolated Compounds against Culex pipiens, and Their Impact on the Non-Target Organism Zebrafish, Danio rerio.

In this study, the crude extract and its isolated compounds from the stem bark of Annickia chlorantha were tested for their larvicidal, developmental, and repellent activity against the mosquito vector, Culex pipiens, besides their toxicity to the non-target aquatic organism, the zebrafish (Danio rerio). The acute larvicidal activity of isolated compounds; namely, palmatine, jatrorrhizine, columbamine, ß-sitosterol, and Annickia chlorantha methanolic extract (AC), was observed. Developmentally, the larval duration was significantly prolonged when palmatine and ß-sitosterol were applied, whereas the pupal duration was significantly prolonged for almost all treatments except palmatine and jatrorrhizine, where it shortened from those in the control. Acetylcholinesterase (AChE) enzyme showed different activity patterns, where it significantly increased in columbamine and ß-sitosterol, and decreased in (AC), palmatine, and jatrorrhizine treatments, whereas glutathione S-transferase (GST) enzyme was significantly increased when AC methanolic extract/isolated compounds were applied, compared to the control. The adult emergence percentages were significantly decreased in all treatments, whereas tested compounds revealed non-significant (p > 0.05) changes in the sex ratio percentages, with a slight female-to-male preference presented in the AC-treated group. Additionally, the tested materials revealed repellence action; interestingly, palmatine and jatrorrhizine recorded higher levels of protection, followed by AC, columbamine, and ß-sitosterol for 7 consecutive hours compared to the negative and positive control groups. The non-target assay confirms that the tested materials have very low toxic activity compared to the reported toxicity against mosquito larvae. A docking simulation was employed to better understand the interaction of the isolated compounds with the enzymes, AChE and GST. Additionally, DFT calculations revealed that the reported larvicidal activity may be due to the differing electron distributions among tested compounds. Overall, this study highlights the potential of A. chlorantha extract and its isolated compounds as effective mosquitocidal agents with a very low toxic effect on non-target organisms.