Honey has been renowned as a natural food supplement and receives much attention because of its high nutritional properties and pharmaceutical activity. It is composed of carbohydrates (96%), and other bioactive natural products including organic acids (e.g., phenolic acids), proteins (certain enzymes), glycoconjugates, carotenoids, vitamins (e.g., ascorbic acid), flavonoids and polyphenols (Sajid and Azim, 2012; Hossen et al., 2017). These minor components are involved in honey's multimodal bioactivities, such as antifungal, antiviral, antinematodal, and antibacterial activity against a broad range of microbes (Sajid and Azim, 2012; Bilal and Azim, 2018; Brudzynski et al., 2012; Brudzynski and Lannigan, 2012). Several minor components of honey are also known to play an important role in antioxidant activity such as polyphenols, phenolic acids, flavonoids, and proteins including major royal jelly proteins (MRJPs) (Schramm et al., 2003; Viuda-Martos et al., 2008). These MRJPs are involved in the production of pro-inflammatory cytokines IL-1β and TNF-α in human monocytic cell lines and significantly boost the innate immune system (Mesaik et al., 2015). The polyphenols in honey are originated from plants (Hossen et al., 2017). Natural honey is well known for containing phenolic acids and flavonoids which are accountable for honey's antibacterial, anti-carcinogenic and anti-mutagenic properties (Jones and Dangle, 2006; Evans et al., 2006; Kroll et al., 2003). In natural honey, these polyphenols non-covalently interact with proteins and oligosaccharides and form multicomponent polymers or glycoconjugates known as melanoidins (Baxter et al., 1997; Brudzynski and Miotto, 2011). The melanoidins are formed by carbohydrates and proteins during the Maillard reaction by the condensation of amino group of proteins and carbonyl group of sugars (Brudzynski and Miotto, 2011).

In our previous studies (Azim and Sajid, 2009; Sajid and Azim, 2012; Bilal and Azim, 2018), we provided evidence demonstrating the antinematodal effects of honey and honey proteins. Specifically, our investigations highlighted the role of honey glycoproteins, particularly major royal jelly proteins derived from natural honey, in exerting anti-C. elegans activity. These proteins were found to induce movement impairments and disrupted egg laying, ultimately leading to the mortality of the worms (Sajid and Azim, 2012; Bilal and Azim, 2018). However, the precise function of small molecules like glycoconjugates in this process has yet to be fully elucidated.

This study presents a comprehensive analysis of the structure and function of honey glycoconjugates found within the 2–5 kDa fraction of honey. Furthermore, we investigate and document their impact on nematodes, specifically their nematicidal effects. For this purpose, the separation of free and peptide-bound glycoconjugates from honey was carried out. Second, structural characterization of honey glycoconjugates was done by MALDI ToF MS/MS. Third, the nematicidal potential of purified low molecular weight (<2 kDa) glycoconjugates was performed using model nematode C. elegans. This nematode species has gained recognition as a user-friendly model system due to its high susceptibility to molecular genetics techniques and its resemblance to other species in the Nematoda phylum. Traditional methods for screening nematicidal drugs are both expensive and labor-intensive, primarily because cultivating and maintaining parasitic nematodes in a laboratory setting is challenging (Holden-Dye and Walker, 2014). The lack of comprehensive molecular and cellular knowledge concerning parasitic nematodes further complicates the examination of their natural habitat (Mathew et al., 2016). Consequently, researchers have turned to C. elegans, which shares similarities with other nematodes, as a means to study antinematodal and anthelminthic compounds (Hunter et al., 2021; Mishra and Agarwal, 2020). It has been observed that compounds that are detrimental to C. elegans also exhibit efficacy against parasitic nematodes (Burns et al., 2015). Hence, to investigate the impact of these honey glycoconjugates on crucial biological pathways in C. elegans, we have undertaken an examination of their influence on the expression patterns of genes associated with various cellular and biochemical pathways. These pathways encompass citric acid metabolism, cytoskeletal organization, body development, embryo development, and meiotic chromosome segregation.