The mechanism by which MH exerts its cancer-healing properties is multifaceted. In addition to its direct anti-proliferative capacity through the activation of the intrinsic and extrinsic apoptotic pathways19,20, MH regulates immune responses by ameliorating hematological and serological restrictions and activates the intrinsic apoptotic pathway through the modulation of pro-apoptotic protein expression5. The effective inhibitory role of MH at low concentrations on the growth of several cancer cell types, such as melanoma, colorectal cancer, and breast adenocarcinoma, has been reported21. Furthermore, in an implantable melanoma preclinical model, systemic management of MH improved the anti-tumor paclitaxel activity and enhanced the overall survival of the host. The anti-tumor activity of several honey types on cancer cells has also been investigated29. The pro-apoptotic and anti-proliferative features of honey on cancer cells are assumed to be chiefly attributed to its phenolic compounds content, comprising quercetin, luteolin, chrysin, and caffeic acid esters30. It was also established that honey had the ability to induce caspase-mediated apoptosis in diverse cancer cell lines, for instance breast, melanoma, prostate, liver, cervical, and renal cancers31,32. Nevertheless, the mechanism of the initial upstream target in cancer cells influenced by honey treatment needs further clarification.We previously reported that the IC50 value (the concentration that results in 50% inhibition in cell viability) of MH (UMF 10+) against CT26 colon cancer cells was ~ 20 and 10 mg/mL, at 24 and 72 h of culture, respectively21. Despite the fact that we used UMF 20 + MH in this study, the IC50 values against the same cell line were similar, calculated to be 19 mg/ml and 21.5 mg/ml at 24h and 48h, respectively. The equivalent values for pMH were 19.6 mg/ml and 19.8 mg/ml, respectively. Comparing the values for MH with other honey types, the estimated IC50 values of Gelam honey, Nenas honey, and Indian commercial honey were 39–80 mg/mL, 85.5 mg/mL, and 35–40 mg/mL, on human colon cancer HT-29, HCT-15, and HCT-116 cells, respectively, at 24 h32,33. These levels were higher compared to MH in the present work. The observed variations could be mainly attributed to several factors, such as the composition of honey, precisely the diverse varieties of phenolic acids and flavonoids, known as chemo-preventive mediators34. Based on different literature, phenolic compounds, for instance, quercetin, gallic acid, luteolin, kaempferol, and caffeic acid that are also detected in MH, have fundamental roles in the suppression of cancer cell proliferation21,34. In a previous study, we evaluated the early targets of MH and its modulatory properties on the proliferation, invasiveness, and angiogenic potential using two human breast cancer cell lines, the triple-negative MDA-MB-231 cells, and estrogen receptor-positive MCF-7 cells, and the non-neoplastic breast epithelial MCF-10A cell line. The study revealed that while exposure to MH at levels of 0.3–1.25% had no to minimal effect on cell proliferation, significant dose-dependent inhibition of cell migration, colony formation and invasion was observed20. In the same study, we reported that at concentrations higher than 2.5% (or 25 mg/ml), MH was found to be toxic to the proliferation of all three cancer cell lines20.Honey is mainly composed of sugars, water, and other constituents, for instance, proteins, minerals, vitamins, organic acids, volatile compounds, and phenolic compounds, which can be dissolved and stabilized in an aqueous phase; accordingly, honey can also be dissolved in the extraction solvent methanol/water35. It was previously reported that adding formic acid to the extraction solvents resulted in higher responses and sharp peaks in chromatographic separation as it provides the protons to form [M + H]+ ions for analysis under the positive ionization mode36. Another study also revealed that formic acid at low concentrations could improve the stability of the extracted metabolites37. Therefore, a mixture of methanol/water comprising 0.1% formic acid was selected for the extraction of honey metabolites.In this study, UPLC-Q-TOF-MS -based untargeted metabolomics analysis was applied to identify the metabolite characteristics profile of raw MH and pMH. The indicative retention times, mass-to-charge ratios of precursor ions in both positive and negative ion modes, and MS–MS fragment ions, along with the intensities of the identified ions, serve as distinct electro-spray ionization mass spectrometry (ESI–MS) fingerprints for the two MH types. Nevertheless, the classification of all analytical ions proved challenging, as the mass-to-charge values of precursor ions could not be reconciled with MS–MS fragment ions within existing databases. Sun et al.38 analyzed mature and immature honey using LC–MS metabolomics analysis and determined the potential fatty acids marker by using GC–MS. It was shown that a total of nine discriminating metabolites, including bee-originated and plant-originated components, could be supportive to differentiate these two types of honey through metabolomics data analysis.In our study, a thorough analysis confirmed 833 distinct metabolites. Among these, a total of 560 significant metabolites were identified, with 346 showing upregulation and 214 showing downregulation. These findings highlight substantial differential changes between the two types of MH. These metabolites encompass various classes, including carbohydrates, flavonoids, phenols and derivatives, organic acids, and terpenoids. It is interesting to note that while raw MH was enriched in carbohydrates and flavonoids, pMH had increased abundance of phenols and derivatives, organic acids, and terpenoids. All of these metabolite classes contain bioactive compounds that possess anti-cancer properties, either directly or indirectly. For example, carbohydrates, which were the most abundant in our dataset, contain many non-digestible carbohydrates (e.g. Melezitose, Raffinose, Stachyose and Xylobiose) which have shown to effect changes in the gut micrbiota that confer health benefits to the host. These benefits are associated with increased numbers of beneficial microbes like bifidobacteria and lactobacilli in the gut, and increased production of metabolites such as short-chain fatty acids (SCFA) by gut microbes39. The beneficial properties of flavonoid compounds as anticancer agents are well studied and have been extensively reviewed5,40. Many phenols and phenolic acids have anti-inflammatory, anti-metastatic, and anticancer properties41. Additionally, honey polyphenols can improve intestinal inflammation and oxidative stress resistance by modulating gut microbiota42. It is of interest to note that organic acids, including Malonic acid, Propionic acid, Genipinic acid, 2-Methylcitric acid, trans-Aconitic acid, Camphoric acid, Levulinic acid, Oxoglutaric acid, Citric acid, and Malic acid, are more abundant in pMH compared to raw honey. These organic acids help in stabilizing the composition of MH and play a role in its antimicrobial properties43. Terpenoids represent the largest and most diverse group of naturally occurring phytoconstituents, possessing a range of pharmacological activities, including anticancer effects44. Coumarin derivatives, for instance, have been shown to induce apoptosis by up-regulating caspase 3 and caspase 9 expression, while also exhibiting antiproliferative and anti-metastatic effects through PAK1 and PAK2-mediated signaling45.Furthermore, the metabolic pathway analysis showed that the identified metabolites were mostly involved in amino acid metabolism, xenobiotics biodegradation and metabolism, carbohydrate metabolism, terpenoids polyketides metabolism, lipids metabolism, nucleotide metabolism, and energy metabolism. Among the most significant differentially expressed metabolic pathways observed in pMH were tyrosine and purine metabolism which were upregulated and the pentose phosphate pathway and aminoacyl-tRNA biosynthesis which were downregulated. These alterations in metabolite contents between the two types of MH could induce preferential effects on cancer cells. It is important to emphasize that all of these metabolic pathways are intricately involved in the regulation of cancer growth46,47,48,49.
