Qualitative phytochemical analysis and in vitro investigation of the immunomodulatory properties of honeys produced in Kazakhstan

Abstract Honey is known to have antimicrobial, immunomodulatory and wound healing properties. The biological properties of honey have been attributed to phytochemicals derived from their source plants and research has focused on identifying the bioactive phytochemicals with therapeutic potential. In this study, we determined the ability of 5 honeys from Kazakhstan and manuka honey to stimulate TNF-α and TGF-β production by human keratinocytes. TNF-α and TGF-β levels increased over time in honey treated and untreated keratinocytes, whereas cells treated with sugar solutions that matched those of the honeys had reduced levels of both cytokines. This suggests that the non-sugar phytochemical components of the honeys may have prevented this decrease. Analysis by LC-MS confirmed that the honeys contained a diverse range of phytochemicals. Some phytochemicals e.g. pinobanksin and vanillin were present at different levels across the honey types, whereas other components, e.g. dicarboxylic acids and their glycosides, were abundant in all honeys. Graphical Abstract


Introduction
Honey has been recognized for its antimicrobial, immunomodulatory and anticancer properties and medical grade honey is recommended for the clinical management of wound infections (National Health Services, Lothian, Scotland; Pichichero et al. 2010;Majtan 2014;McLoone et al. 2016a). The biological activities of honey are thought to be due, at least partially, to phytochemicals that are derived from plants (Fyfe et al. 2017;McLoone et al. 2021). As bees can collect nectar from different floral sources, the biological activity of different honey types is variable. Recently, scientific attention has focused on understanding the biological activities of honey phytochemical constituents with the aim of developing cosmeceuticals or nutraceuticals suitable for therapeutic use. Some chemical constituents identified in honey have been investigated extensively and have been shown to exert a range of biological effects. For example, chrysin a natural flavonoid found in honey, has been reported to have anticancer, anti-inflammatory, antioxidant, anti-allergic and neuroprotective properties (Naz et al. 2019). Chrysin and its synthetic derivatives are consequently being investigated as prophylactic agents/nutraceuticals to prevent disease. Other examples include pinocembrin which reportedly has anti-inflammatory and antioxidant properties and phenyl lactic acid which has been shown to exert antibacterial activity (Kirkpatrick et al. 2017;Shen et al. 2019). Leptosin, a glycoside of methyl syringate, is another interesting compound found in honeys from the Oceana region including manuka and jelly bush. The quantity of leptosin in manuka honey was found to correlate positively with antibacterial activity (Kato et al. 2012). These are just some examples; there are many other compounds present in honey which have reported biological effects and others which remain unidentified, or their biological properties are not yet fully known. It should be noted, however, that due to synergistic and antagonistic interactions the biological properties of phytochemicals when present in a mixture of compounds, such as in honey, may differ from their individual effects when isolated.
Interestingly, naturally derived compounds/nutraceuticals e.g. from foods, have been reported to have various biological properties that have the potential to improve human health. These include anti-cancer, cardioprotective, anti-diabetic and antidepressive properties ). Nutraceuticals have also been shown to induce immunomodulatory effects that could provide protection against neurodegenerative diseases such as Alzheimer's disease and be beneficial for the management of inflammatory skin conditions such as psoriasis and metabolic disorders such as diabetes mellitus. Alesci et al. 2022a;2022b). Nutraceuticals may exert anti-inflammatory effects, for example, by inhibiting Toll-like Receptors (TLRs) and downregulating pro-inflammatory cytokine production . Some nutraceuticals may exert immunostimulatory effects on the immune system, for example, by modulating cytokine production, stimulating immune cells and promoting anti-viral activity .
In terms of bioactivity of whole honey, it has been reported that some honeys can exert immunomodulatory effects, for example by altering cytokine production by skin and immune cells (McLoone et al. 2016b). This immune modulation is thought to contribute to the wound healing ability of honey. Cytokines such as TGF-b and TNF-a have been shown to play a role in promoting early wound healing (Amento and Beck 1991;Ritsu et al. 2017). Honey has been shown to affect the release of cytokines via TLRs. TLRs are phylogenetically conserved receptors (Lauriano et al. 2021;Alesci et al. 2022c;2022d;2022e;2022f), which play a crucial role in modulating the immune response (Masad et al. 2021).
In Kazakhstan, a wide range of honeys from different floral sources are produced but the regional honeys have not been fully investigated for their biological activities or analyzed for bioactive compounds. In this study, the immunomodulatory activity of 5 honeys produced in Kazakhstan of different floral origins and medical grade manuka honey (Activon) was investigated by measuring the effects of 1% honey concentrations on TNF-a and TGF-b production by human keratinocytes. Furthermore, phytochemicals were putatively identified, and their levels compared in the honeys using Liquid Chromatography-Mass Spectrometry (LC-MS) and components that differed between honeys and were common to all honeys were noted.

Total sugar content and total phenolic content (TPC) of the honey samples
The percentage total sugar content of the honeys ranged from 76-81% (supplementary data Table S1), which are similar to previous reports (Schneider et al. 2013;Fyfe et al. 2017). Buckwheat honey had the highest TPC content (Table S1).

Phytochemicals identified in the honeys produced in Kazakhstan and medical grade manuka honey (Activon)
Over 100 components were putatively identified across the honey types. Variation between LC-MS profiles of honeys was noted previously (McLoone et al. 2021) and the variation in these samples is illustrated in supplementary data ( Figure S1 & 2). In table S2 (supplementary data), we have noted the variation in honey phytochemicals that have previously been shown to have potential therapeutic interest.
The phytochemicals identified are likely derived from pollen or nectar of plants in the production area. Some of the components were ubiquitous across all samples and some were more characteristic of individual honey types. For example, components such as methyl syringate and leptosperin were only found in manuka honey. Several of the phytochemicals have been reported to have antibacterial, anti-cancer and antiinflammatory properties making them potential bioactive agents for the treatment of disease (e.g. Imran et al. 2020;Mok et al. 2020;Yeo et al. 2020).
It was notable that the different honey types had different phytochemical compositions but decanedioic and decenedioic acids (Sun et al. 2021), and their putative glycosides were consistently major components (see supplementary Fig. S1 & 2). Decanedioic acid (commonly called sebacic acid) and decenedioic acid are well known components of royal jelly and sebacic acid has been shown to have anti-inflammatory effects (Chen et al. 2016). It is possible that these dicarboxylic acid derivatives might synergistically influence cell growth and metabolism.
2.3. Effect of 1% honey concentrations on TGF-b and TNF-a production by adult human keratinocytes (HEKa cells) Figures S3 and S4). The rise in cytokine levels over time may have been due to cell proliferation. Indeed, preliminary experiments measuring HEKa cell proliferation in an MTT assay following treatment with 1% honey suggest that some of the honeys may be promoting cell proliferation (unpublished data). However, this rise in TGF-b and TNF-a levels over time was not observed in the supernatant of cells treated with the sugar only control. As the honeys have a sugar composition comparable to the sugar control, our hypothesis is that other components in the honeys are protecting the cells from the effects of sugar.

TGF-b and TNF-a levels increased over time in the supernatant of both honey treated and untreated cells (see supplementary
As there was no statistically significant difference between cytokine production by the honey treated keratinocytes and the untreated keratinocytes in this study, it was not possible to correlate the immunomodulatory properties of the honeys with the abundance of certain phytochemicals in the honeys.

Conclusions
In conclusion, the honeys from Kazakhstan are rich in phytochemicals with possible therapeutic properties and may exert protective effects on keratinocytes. The findings from this study are important for evaluating the health properties and potential clinical application of the Kazakhstan honeys. In the future, it would be worth investigating further the health benefits of the honeys. In particular, it would be interesting to test the anti-tumorigenic properties of the honeys against skin cancer cells and determine the effects of the honeys on wound healing and other dermatological disorders. Although the biological activity of whole honey was our primary interest, it would be interesting to investigate the biological properties of the honey SPE fractions as these lack the high sugar content but are enriched in the other phytochemical components. For example, it would be interesting if we could obtain a similar protective effect by doping the sugar control with the phytochemicals from an active honey.

Author contributions
PM and GM designed the study, acquired funding, analyzed the data, wrote the original draft and critically revised and reviewed the manuscript. DT, AZ and SY performed experimental procedures and data analysis. SV, JS CA and JWA were involved in data curation, experimental procedures, formal analysis and validation. All authors critically reviewed the manuscript and agreed to the published version of the manuscript.

Disclosure statement
The authors declare no conflict of interest

Data availability statement
The data analysed in this study are available on reasonable request from the corresponding author.