Apitoxin was collected from the carniolan honeybee hybrid Apis mellifera carnica, the commercial apiary located in Motobes region Kafr El-Sheikh Governorate, Egypt, during the active period of the summer season 2022. Each colony consisted of five combs (three brood combs, two honey, and stored pollen combs) covered with bees and headed by newly mated queen sisters.

Api collecting electric shock device CJ 401 (Chung-Jin Biotech Ltd., Ansan, Korea) consists of a digital control board, five bee venom collection frames, wire electrodes, battery, Input /Output Voltage: 12 VDC (timer ON: 3 s. and timer-OF: 6 s). Finally, collector Frames: 46 × 28 cm. Honeybees were subjected to a bee venom-collecting electric shock device. There were five parallel wires in the electrode-equipped Api collecting frames of the device. Every frame in a hive was put on top of the combs before being wired to an electro-stimulator. Electrical impulses stimulate bee workers to sting via a latex sheet that has been put on a glass plate of the device frame. Bees that came into touch with the wires were mildly shocked by the electricity and stung the glass surface. The other bees were agitated and mobilized as a result of the alarm odor that was released by the venom, and they began to sting as well. To ensure the safe transmission of the frames containing the fresh Api to the laboratory for 24–48 h, they were meticulously packaged inside a specialized container. The processing of dry Api scraping was implemented by a sharp scraper under laboratory conditions.

Several phases were involved in the extraction of biopolymers of chitin and chitosan from a novel potential source, with dead corniolan honeybee hybrids were collected in front of bee hives during the autumn season of 2022 from the commercial apiary located in Motobes region Kafr El-Sheikh Governorate, Egypt. To extract chitin, the protein (deproteination) and mineral (demineralization) elements of sub-pestilence was first dissolved and removed. The raw honey bee Apis mellifera material was first ground using CM 190 Cemotec TM, Denmark. Demineralization was then performed using the Hackman technique with minor modifications (Haydarova and Ikhtiyarova, 2019), by treating the crushed raw material with 2 M hydrochloric acid for 5 h at 25–27 ⁰С. Then, deproteination was accomplished by treating the pulverized raw materials with a 1N sodium hydroxide solution for 1 h at a temperature of 80−85⁰С. Each step was preceded by a natural wash water reaction (pH = 7) of the raw components. Chitosan, a high molecular weight glucosamine polymer, was synthesized by deacetylation of chitin in the presence of a 35% aqueous NaOH solution at a temperature of 80−85⁰С for 4 h and dried at 60–65⁰С. The resultant mass was decolorized with 3% H₂O₂ and washed with 10% ethanol. The chitin content (WC %) was calculated according to Mol et al., (2018) using the following formula:

Each step was preceded by a natural wash water reaction (pH = 7) of the raw components. Bee chitosan was synthesized by deacetylation of chitin in the presence of a 35% aqueous NaOH solution at a temperature of 80–85°C for 4 h and dried at 60–65°C. The resultant mass was decolorized with 3% H₂O₂ and washed with 10% ethanol.

ChB was diluted in 2% (w/v) acetic acid and filtered to achieve the purification of bee chitosan. The polymer was precipitated by adding 1.0 M NaOH to the chitosan solution while stirring. The precipitated polymer was rinsed with distilled water until the filtrate pH matched the distilled water. The finished product was vacuum-dried in an oven at 60°C after repeated acetone washes. ChB samples were washed with distilled water to a neutral pH according to kaya et al. 2015 (Kaya et al., 2015). The yield (Y) of ChB was estimated by weighing dried chitosan and using the following equation.

Bruker Optics (Tensor, 47IFS), Germany, employed for FT-IR to characterize chitin and chitosan. The following formula was used to calculate the Degree of deacetylation (DD) of chitosan samples:

where the ratio A₁₆₅₅/A₃₄₅₀ for fully N-acetylated chitosan is indicated by the factor of 1.33 (Kaya et al., 2014). The following equations were used to calculate molecular weight (MW), ash (AC), water binding capacity (WBC), fat binding capacity (FBC), and moisture content (MC) of drying honey bee chitosan by vacuum oven for 24 h at 110°C (Sun et al., 2007; Rødde et al., 2008; Marei et al., 2016).

The following equation was used to calculate the chitosan crystallinity index (CrI) according to Zhang et al., (2000)

where I_am is the greatest intensity in the corresponding amorphous area at 2θ ≈ 11°, while I₁₁₀ is the maximum intensity at 2θ ≈ 20°.

The ionic gelation process was used to create ChB NPs. Extracted chitosan from dead bees (ChB) solutions were prepared by dissolving 1 g of chitosan in 100 mL of 1.0% aqueous acetic acid and stirring until the liquid became translucent. Following that, NaOH (0.01 N) was used to increase the acidic pH until (pH 5.5) and facilitate the cross-linking reactions between chitosan and tripolyphosphate (TPP) ions. A ChB acidic solution was combined with a basic TPP solution (1.0%) at room temperature and stirred at 150 rmb to create the ChB NPs. TPP was added dropwise to the ChB solution to begin the spontaneous synthesis of ChB NPs. Then, the suspension was stirred under magnetic stirring at room temperature and left to qualify for 30 min. The ChB NPs were then centrifuged at 3000 rmb for 15 min at 3–5°C and freeze-dried with 10% (m/m) trehalose in a Freeze-dryer for 24 h (Arif et al., 2021). The nanoparticles loaded with bee venom were prepared by introducing venom at a concentration of 1 g/20 mL of deionized water and stirring for 10 min. Subsequently, the venom solution was combined with the TPP solution prior to the addition of the chitosan solution (Schematic 1A).

The particle size and ζ-potential were estimated by DLS (Malvern Instruments, UK). 3 mL of bare ChB NPs and Api@ChB NPs were diluted in deionized water and placed in a cell cuvette. The size was estimated four times and then and three ζ-potential charges the mean ± SD (Sharaf et al., 2021). FTIR spectrometers evaluated functional groups in prepared samples. Potassium bromide-treated samples (KBr). A disk was pressurized and measured at 500–4,000 cm⁻¹with a resolution of 4.0 cm⁻¹⁴⁸. To Transmission electron microscopy (TEM) images of ChB and Api@ChB NPs, 1% phosphotungstic acid discolored the samples on copper grids (PTA). After air-drying at ambient temperature, materials were analyzed using (Philips 400; TEM, Netherlands) at 80 kV (Hou et al., 2021).

The One Way ANOVA test was used to compare the arithmetic averages of the inhibition regions to the quantities of the different compounds whose biological inhibitory activity was assessed for bacterial species using Duncan’s polynomial test at *p < 0.05 (Gerber and Voelkl, 2012).

Dead honeybees yielded 25% chitin. The physiochemical analyses of bee chitosan (ChB) showed the yield of chitosan (Y) from the chitin was 45.1 ± 2.5%, possibly due to the source and during deacetylation and precipitation process to remove each of impurities and proteins (Amor et al., 2023). ChB had a DD and WBC values of 79.15 ± 0.7% and 622.7 ± 5.8%, respectively. WBC in ChB was observed to fall within a narrower range (458–805%) than that previously reported by Marei et al. (2016). The FBC value of ChB was 399.5 ± 13.3%. The findings of this study indicate that the FBC of the ChB was comparable to the values reported by Cho et al. (1998) and No et al. (2000), ranging from 217 to 403%.

When chitosan is heated in the presence of air, it totally decomposes, leaving behind the inorganic residue known as ash content (AC). ChB AC concentration indicates calcium carbonate removal and demineralization efficiency. High-quality ChB has less than 1% AC (Nessa et al., 2010). In accordance with Table 1. ChB was found to have the highest AC and MC, coming in at 0.7 ± 0.1% and 9.85 ± 0.3%, respectively. These results indicated that ChB had the highest quality. In addition, the produced ChB has a MW of 20 kDa, which is consistent with a previous study by Sahebzadeh et al. (2024). According to Khan et al. (2000) mentioned that the molecular weight of chitosan samples decreased with increasing alkali treatment times. The alkali treatment resulted in a greater abundance of amine (–NH₂) groups and a reduced abundance of acetyl amide (− NHCOCH₃) groups (Khan et al., 2000). Chitosan’s molecular weight might be reduced by depolymerization in response to the increased alkalinity (Chebotok et al., 2006).

X-ray diffraction by a crystalline structure in the sample allows the study of atomic and molecular structure of the crystal. The XRD patterns of honey bee chitin and chitosan samples were located at angles (60 min) at a 2-theta-scale are shown in Figure 1.

Apis mellifera chitin samples showed significant strong peak at 2θ = 9.9°C (020), 2θ =19.8°C (Sangboonruang et al., 2020), 2θ =26.5°C (Elnosary et al., 2023), and minor peaks at 12.1°C, 22.1°C. XRD results of chitin yielded by crab, shrimp, krill, and insects such as Geolycosa vultuosa and Sitophilus granarius are similar to the peak indicated by this study (Yen et al., 2009; Sajomsang, 2010; Liu et al., 2012; Wang et al., 2013; Kaya et al., 2014; Figure 1A). The high crystallinity of pure ChB was 82.2 ± 0.8%. The ordered crystalline structure of ChB is evidenced by the presence of two diffraction peaks at 2θ =10.5⁰ (020) and 2θ =20.2°0 (Sangboonruang et al., 2020), as shown in the XRD pattern of ChB in Figure 1B. Comparatively, the CrI value of chitosan isolated from different insects, such as beetles, cuttlefish, shrimp, and silkworms (B. mori), varied from 36 to 95% (Zhang et al., 2000; Paulino et al., 2006; Kaur et al., 2013; Morsy et al., 2019; Mohan et al., 2020).

Furthermore, FTIR spectra of the functional groups of pure ChB with strong peaks were observed at 3454 cm⁻¹ and referred to –OH group stretching, peaks at 2936 cm⁻¹ indicated carbonyl C–H stretching, a peak at 1087 indicated cm⁻¹ C–O–C stretching, and peaks at 1390 to 1,660 cm⁻¹ were attributed to a protonated amino (–NH₂) group N-H bending vibration and a group C–N bending vibration (see Figure 1F).

One unicellular fungal (Candida auris) and multicellular (Aspergillus nigar) isolates were identified using the VITEK2 system. It showed that these fungi had confidence intervals of 96% (excellent) and 95% (very good). Fluconazole, an antifungal used as a positive control (see Supplementary Figure S2), produced inhibition zones of 13 ± 0.7 mm for A. nigar and 17 ± 1.9 mm for C. auris (see Table 1, Figure 3, and Supplementary Figure S3). The Api, ChB NPs, and Api@ChB NPs were used at a concentration of 100 μg mL⁻¹ against these two fungi. It was found that all three substances had antifungal properties. The Api produced inhibition zones of 7.4 ± 0.8 and 7.1 ± 0.6 mm for C. auris and A. nigar, respectively. The ChB NPs produced inhibition zones of 8.2 ± 1.6 and 15.9 ± 1.3 mm for C. auris and A. nigar, respectively. However, the Api@ChB NPs were the most effective against these fungi, producing inhibition zones of 12.1 ± 0.6 and 22.2 ± 1.6 mm for C. auris and A. nigar, respectively. The results are shown in (Table 1, Figure 3B, and Supplementary Figure S3a). It is interesting that the Api and nanoformulations were effective and could inhibit the growth of A. nigar even after the fungus had been cultured for 5 days. The color of the A. nigar changed from white to black (see Figure 3B and Supplementary Figure S3b). Our results confirmed that the use of bee products in the treatment of infectious diseases caused by Candida is extremely restricted (Lee, 2016). This may be due to the antioxidants in Api, which may regulate the generation of reactive oxygen species (ROSs) in fungi, followed by damage to the fungus’s lipids, proteins, and DNA; cell death may occur when this balance of components is upset inside the fungal cell (Ratajczak et al., 2021). El-Didamony et al. (2022) found that bee venom had a substantial inhibiting impact against C. albicans ATCC 90023, Candida neoformans, and Kodamaea ohmeri, and they attributed this effect to the regulation of ROS generation by oxidative stress. Similar results showed that bee venom had antifungal efficacy against 10 clinical strains of C. albicans, producing MIC values of between 62.5 and 125 μg mL^-1102. Another study showed antifungal activity of bee venom melittin against the fungal yeasts C. albicans, C. parapsilosis, C. tropicalis, and C. krusei, with MIC values of between 30 and 300 μg mL^-1105.

The broth dilution technique was used to determine the bacteriostatic effects of Api, ChB NPs, and Api@ChB NPs against various harmful bacteria (Selim et al., 2020). As shown in Table 2, ChB NPs outperformed Api in antimicrobial assays against Gram-negative and Gram-positive bacteria. At low concentrations, ChB NPs were highly antimicrobial. However, adding Api increased the activity significantly. The visual turbidity test showed that Api@ChB NPs inhibited E. faecalis at 12.5 μg mL⁻¹; S. hominis, A. baumannii, and E. coli at 25 μg mL⁻¹; and S. aureus and K. pneumoniae at 50 μg mL⁻¹. For the comparable bacterial strain, control inhibition effectiveness varied from 8 μg mL^-1107.

The second-order derivative of ChB NPs can show how a double concentration hinders these microorganisms’ microbial growth; it ranged from 25 μg mL⁻¹ for E. faecalis to 50 μg mL⁻¹ for S. hominis, A. baumannii, and E. coli, to 100 μg mL⁻¹ for S. aureus and K. pneumoniae. Like previous studies, we proved that the killing efficiency of Api@ChB NPs with these bacterial strains was mostly less than that of the standard antibiotic gentamicin. Interestingly, ChB NPs had a statistically higher inhibition efficiency against Gram-positive bacteria at low concentrations than the other bacterial strains. In contrast, all the tested bacteria were strongly resistant to Api at concentrations of 100 μg mL⁻¹ or more. These findings were similar to those of Issam et al. (2015), who reported that bee venom and melittin exhibited a broad spectrum of antibacterial activity against 51 strains of Gram-positive and Gram-negative bacteria. They had strong activity against even MRSA and vancomycin-resistant enterococci (VRE), with MIC values of between 6 and 800 μg mL⁻¹. Thus, the MIC results revealed that Api@ChB NPs are more potent against Gram-negative and Gram-positive bacteria than other nanosubstances. Generally, the MIC values for Api@ChB NPs were lower than those for ChB NPs, suggesting that the entrapment process impacted the effectiveness of bee venom. This finding may be connected to the following theory. Because the examined microorganisms were only exposed to the experimental ChB NP concentrations once at the beginning of the experiment, some of these concentrations may have become tolerated by the time the experiment ended, resulting in relatively high MIC values. However, the Api@ChB NPs were available in moderate numbers throughout the experiment, and this may have resulted in decreased MIC values because the microbes may have developed tolerance to them. Prior studies have corroborated this theory (Sahoo et al., 2007; El Rabey et al., 2019).

Api, ChB NPs, and Api@ChB NPs were tested against Vero ATCC CCL-81 Cercopithecus aethiops normal kidney cells, Caco2 ATCC ATP-37 human colon cancer cells, and HepG2 ATCC HB human liver cancer cells at concentrations of 31.25–1,000 μg mL⁻¹ to determine their ability to inhibit cancer cell growth. The test was conducted for 24 h at 37°C. There were three replicates for each concentration and the untreated control sample (Figures 4A–C). The severity of the toxicological impact was calculated according to the growth inhibition rate for the Api, ChB NPs, and Api@ChB NPs in relation to the control group, which grew at 100%.

The normal Vero ATCC CCL-81 cells were selected in order to evaluate the potential of Api, ChB NPs, and Api@ChB NPs as cytotoxic agents. Overall, all the tested concentrations showed cytocompatibility at concentrations of up to at least 62.5 μg mL^˗1 (51.74%) of Api, 500 μg mL^˗1 (94.60%) of ChB NPs, and 500 μg mL^˗1 (>74.89%) of Api@ChB NPs after 24 h of incubation and IC50 values were 72.17, 800.53, and 676.28 μg mL⁻¹ for the Api, ChB NPs, and Api@ChB NPs, respectively (Figure 4A).

The cancer Caco2 ATCC ATP-37 and HepG2 ATCC HB-8065 cells were selected in order to evaluate the potential of Api, ChB NPs, and Api@ChB NPs as anticancer agents. For Caco2 ATCC ATP-37 cells after 24 h incubation, all the tested concentrations showed cytocompatibility at concentrations of up to at least 62.5 μg mL^˗1 (70.35%) of Api, 31.25 μg mL^˗1 (83.93%) of ChB NPs, and 250 μg mL⁻¹ (69.87%) of Api@ChBNPs and IC₅₀ values were 59.87, 34.19, and 197.99 μg mL⁻¹ for the Api, ChB NPs, and Api@ChB NPs, respectively (Figure 4B). For HepG2 ATCC HB-8065 cells after 24 h incubation, all the tested concentrations showed cytocompatibility at concentrations of up to at least 125 μg mL⁻¹ (79.56%) of Api, 62.5 μg mL⁻¹ (75.40%) of ChB NPs, and further evaluation of the Api@ChB NPs showed the lowest level of cytotoxic activity against at a concentration of less than 250 μg mL⁻¹ (67.98%) and IC₅₀ values were 74.59, 48.7, and 206.75 μg mL⁻¹ for the Api, ChB NPs, and Api@ChB NPs, respectively (Figure 4C).

This finding corresponded well with the findings of Taher et al. (2017), who reported that the size of bee venom–loaded nanoparticles prepared with a concentration of chitosan of 1 mg/ml had good anticancer properties. Previous studies have also reported that most insect venoms in human stings contain various chemical combinations. They include amino acids, peptides, proteins, enzymes, carbohydrates, biogenic amines, volatile compounds, phospholipids, and pheromones (Carpena et al., 2020; Sangboonruang et al., 2020).

Furthermore, the morphological features of normal Vero ATCC CCL-81 cell lines and Caco2 ATCC ATP-37 and HepG2 ATCC HB-8065 cancer cell lines treated with different concentrations of Api, ChB NPs, and Api@ChB NPs were reported (Supplementary Figures S4–S6) and compared with those of the untreated cells. The 3T3 Phototox software estimated the concentrations of prepared samples in different cell lines using the absorbance values disclosed following the capture of the red dye and the relevant amounts of the Api, ChB NPs, and Api@ChB NPs employed in the viability studies. After the entry of chitosan nanoparticle-loaded Api into the cellular environment, a direct impact on the nucleus was seen, leading to the activation of certain genes involved in the increased generation of apoptosis. The pyridine-based chitosan thiosemicarbazones and their copper (II) complexes have shown a favorable structure for inhibiting the growth of tumorigenic MDCK and MCF-7 cancer cell lines (Adhikari et al., 2022). A more recent in vitro study showed that the combined use of isatin and 5-chloroisatin-based chitosan thiosemicarbazones displayed anticancer activity against MDCK and MCF-7 cancer cell lines (Adhikari et al., 2023). The sustained induction of apoptosis may lead to a significant reduction in the population of cancer cells, which can be achieved by the use of chitosan nanoparticles (Adhikari et al., 2022). The overall outcome was a reduction in cell viability, accompanied by the inactivation of cell division, growth promoter genes, and other virulence genes. In a previous study conducted by Rajivgandhi et al. (2020) and Wang et al. (2021), it was shown that chitosan nanoparticles exhibit anticancer activities against a range of cancer cells. Furthermore, the inhibitory effects were found to be dependent on the concentration of the nanoparticles.

Some limitations in this work need additional investigation. Firstly, it is necessary to examine the in vitro releasing profile of Api from Api@ChB NPs. Furthermore, due to limited financial resources, we were unable to conduct tunnel or flow cytometry experiments with annexin V must be performed anticancer results to demonstrate that apoptosis is occurring. Additionally, we recommend in vivo experiments in order to validate the effectiveness of the formulations we have developed.