Factors Affecting Quality of Honey Bee Venom

Honeybee venom (HBV) with various health benefits is one of the most significant new-generation hive products. Although it has been used in traditional medicine for centuries (Bogdanov, 2012; Cells, 2013; Hwang et al., 2015; Abdela & Jilo, 2016; Eze et al., 2016; Baher & Abo Zeid, 2017; Kader, et al., 2019; Kim et al., 2019; Kurek-Górecka et al., 2020), it has been recently rediscovered by modern medicine for its pharmacological value.

The therapeutic application of HBV in traditional and complementary medicine has been demonstrated to treat such autoimmune and inflammatory diseases as rheumatoid arthritis, osteoarthritis and chronic pain (Abdela & Jilo, 2016). It has also been used for the treatment of such neurological diseases as multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), Parkinson and Alzheimer’s (Hwang et al., 2015). Additionally, antimutagenic, radioprotective, antinociceptive, anti-inflammatory and anticancer effects of HBV have been described by many other studies (Varanda & Tavares, 1998; Kim et al., 2003; Lee et al., 2004; Son et al., 2007; Gajski & Garaj-Vrhovac, 2009; Mahmoodzadeh et al., 2013; Kolayli & Keskin, 2020; Uzuner et al., 2021). The pharmaceutical value of HBV has been referred to for its valuable ingredients, including proteins (phospholipase A₂ (PLA2), phospholipase B, hyaluronidase, phosphatase, alpha glucosidase), peptides (apamin, melittin, MCD peptide, secapine, pamine, minimine, adolapine, procamine A and B, protease inhibitor, tertiapine, cardiopep, melittin F), phospholipids, biogenic amines (histamine, dopamine, noradrenalin), amino acids (aminobutyric acid, alpha-amino acids) in addition to sugars, minerals (P, Ca, Mg), and volatile compounds (pheromones) (Bogdanov, 2012).

Consequently, the production and commercialization of HBV are being developed in Turkey and the world due to a growing interest in its potential as an ingredient for cosmetics and health products. Thus, its production and further processing conditions have become decisive parameters among the producers and the purchasers. According to previous studies, HBV quality is affected by the production method and environmental factors such as humidity, heat, and harvest time (Ghany Zidan, et al., 2018; Hussein et al., 2019). Also, some studies reported the influence of the age of honeybees (Owen et al., 1990; Owen, 1978; Bachmayer et al., 1972; Owen et al., 1974), season (Owen & Bridges, 1982; Owen & Sloley, 1988), and supplementary feeding (Abusabbah et al., 2016; Badaway et al., 2016; Nowar, 2016) on its constituents. Currently, limited researchers, carried out in commercial samples of Anatolian HBV, provide information on its composition (Samancı & Kekeçoğlu, 2019; Kekeçoğlu et al., 2019, Şirin et al., 2016). However, these studies need to be enhanced through focusing on the factors determining its quality, specifically for Anatolian honeybee (Apis mellifera anatoliaca) venom.

High-quality HBV production is essential for the beekeeper, purchaser, and, most importantly, patients who is applied bee venom is concerned. Since HBV is a delicate product, even a little error during its production can affect its quality. Thus, good production techniques and the harvesting season of this unique food became a significant subject for the scientists.

This study aims to investigate how harvesting time, beehive’s site, season and geographic location affect the three main components of Anatolian HBV, apamin, melittin, and PLA2. We expect the study results to stimulate future research focusing on a standard quality of bee venom by guiding good production principles associated with its components.

The results of these groups based on harvesting time (day or night) and collecting site of the beehive (entrance or the inside of the hive) are presented in Tab. 2, and the chromatograms of apamin, melittin and PLA2 for selected samples are provided in Fig. 2. The harvesting time of HBV samples, either at day or nighttime, did not significantly affect the amounts of apamin (2.23%–2.17%), melittin (39.02%–39.88%), or PLA2 (15.79%–13.66%) (Tab. 2). Similarly, the collection site of the HBV samples from the beehives, either during the day or nighttime, resulted in no significant differences in the amounts of apamin (2.33%–2.13%), melittin (39.02%–41.25%), and PLA2 (15.79%–13.66%) as presented in Tab. 2 (P>0.05).

Fig. 2

The selected protein and peptide content of HBV samples produced in different months are presented in Tab. 3. Accordingly, harvesting in four subsequent months from May to August did not significantly influence the amounts of apamin (2.00%–2.31%), melittin (38.64%–44.37%) and PLA2 (12.79%–15.44%) in HBV samples (P>0.05) (Tab. 3).

Tab. 4. Represents the apamin, melittin and PLA2 content of twenty seven HBV samples obtained from beekeepers in Manisa, Muğla, Balikesir, Düzce and Mersin. The statistical analyses revealed significant differences in the amounts of apamin (1.57%–2.56%), melittin (36.71%–51.87%) and PLA2 (11.12%–21.26%), according to the geographic locations (p<0.05). The samples produced in the DAGEM apiary had the highest amounts of apamin and melittin but the second-largest value for PLA2 than other samples (Tab. 4).

Three components of HBV, Melittin, Apamin, and PLA2, are considered important quality indicators for their essential bioactivities suitable for apitherapeutic or cosmetics purposes (Banks & Shipolini 1986). Among these constituents, melittin has been reported to be the major component of bee venom ranging between 40–50%, as reported by many studies (Ali, 2012; Banks & Shipolini, 1986; Bogdanov, 2015; Kye-Sung & Ki-Rok, 2009; Moreno & Giralt, 2015; Zhou et al., 2010; Samancı & Kekeçoğlu, 2019). On the other hand, some studies proclaimed much higher or lower values of melittin. For example, Polish scientists noted a value higher than 70% (Rybak-Chmielewska & Szczęsna, 2004) in their HBV samples. However, Romanian scientists revealed a low level of melittin, about 27%, in their samples harvested from the inside of beehives (Ionete et al., 2013). In our study, the average 40% melittin amount in samples harvested from either inside or at the entrance of beehives during the daytime is consistent with the values reported in the literature.

The protein, PLA2, is the second-largest component of honeybee venom as reported to be in the ranges of 10–12% (Banks & Shipolini, 1986; Bogdanov, 2015; Moreno & Giralt, 2015). However, there are also published scientific data showing its amount to be as low as 7% (Kokot & Matysiak, 2009) or as high as 23% (Ionete et al., 2013). In the present study, the average PLA2 values ranging between 14% and 16% in samples harvested either from both sites of the hives in the daytime (Tab. 2) are higher than these reported ranges for Anatolian HBV.

The apamin content of HBV is much lower than the other two significant components, as reported between 2 and 3% by many scientific studies (Banks & Shipolini, 1986; Bogdanov, 2015; Moreno & Giralt, 2015). In the current literature, the highest apamin value is 5.32% (Ionete et al., 2013), and the lowest is 1.75% (Kokot & Matysiak, 2009). According to our data presented in Tab. 2, the apamin amount averaged from 2.13% to 2.23% for Anatolian HBV is consistent with the available information in the literature, while it reaches a maximum (3.95%) in June (Tab. 3).

Collectors located inside the beehive can be used at the bottom of the hive (Han et al., 2007) or to cover the top of the hive (Robson, 1988). Such devices are capable of collecting higher amounts of venom than hive-entrance collectors, as they may be in contact with larger numbers of bees while operating. The collectors located at the base from the apparatus placed in different parts in the hive are exposed to contamination with bees’ wastes, therefore they are not recommended. On the other hand, those on the hive or in the form of a frame have a lower risk of contamination (Robson, 1988; Serrinha et al., 2019). According to our research, there is no specific study concerning the inside or entrance of the hive site effect on HBV composition. Most of the studies focus on the amount of the production as a result of site differences. In our study, we did not determine any statistical differences between the harvesting site methods through HBV composition. However, we offer the inside hive collection method due to its effect on production amount and sterilization according to the previous studies.

There have been various studies on the seasons’ influence on bee venom protein and peptide components. Owen & Praf (1995) with the dissection method studied HBV melittin content of in connection with seasonal changes and determined that, melittin levels were lower in mid-August than early June. Rybak-Chmielewska & Szczęsna (2004) found that the melittin component differed significantly among HBV samples harvested in different seasons. Ferreira Junior et al. (2010) harvested the bee venom at the entrance of the hives and determined that seasonal changes influence the melittin and PLA2 without a specific association to any climatic parameter.

In contrast with those findings, our results revealed that the seasons had no statistically significant effect on the apamin, melittin and PLA2 components of Anatolian HBV (Tab. 3). However, the highest values for apamin and PLA2 were observed in samples harvested in July and for melittin in August (Tab. 3). Disagreement between previous studies and our results on seasonal effects may be associated with either different harvesting methods or different locations offering different nectar and pollen sources, since venom quality may vary according to the food they consume (Abusabbah et al., 2016; Nowar, 2016).

The study results on locational differences (using the same production method) revealed significant variations in samples collected from five regions in Turkey (Tab. 4). Those locations represent the climate variations as Balıkesir and Manisa are located in the Aegean region, Düzce on the Black Sea and Mersin in the Mediterranean area. In general, the highest mean values for apamin and melittin were observed in samples produced by DAGEM (Düzce University Beekeeping Research and Development Center) Apiary under the controlled conditions of Düzce University. Those sampling locations represent the Black Sea, Aegean and Mediterranean regions with opposing climate parameters and land flora. There may be no direct relationship between the changes in honeybee venom components and climate conditions as described by Ferreira Junior et al. (2010).

Interestingly, the highest reported PLA2 value for Anatolian HBV has been the samples obtained from beekeepers in Muğla have had an average of 28% PLA2 level. Since PLA2 is one of the most effective substances in bee venom content for defense purposes, it may have developed as a self-defense mechanism for the A. m. anatoliaca populations against local enemies. However, these differences might also be related to beekeepers’ improper practices during harvesting and storing, as the most critical parameters for determining HBV quality. In light of all the data obtained from the study, the composition of honey bee venom has been revealed to be unaffected by neither the production time nor harvesting site of the hive, but is affected by geographic locations.

However, there is still a need for more research to clarify the location’s effect on Anatolian HBV quality. This study presented data to be useful for a proposal leading to a standardized production as HBV is a crucial apiculture products due to its pharmaceutical value. As the bee venom market continues to evolve, imitation and adulteration attempts will be a safety problem for authorities worldwide. Therefore, we hope that the presented research data will help the authorities establish the specifications and standards regarding HBV production and its analysis.