Introduction

Honey is a natural sweetener that is produced by an insect, the bee, from the family Apidae. Since ancient times, it was used among Arabic, Egyptian, and Indian gentility. At that time, honey use was not limited to food, but it was also used as a royal gift or drink, as a sweetener or preservative, and as a home treatment for family members who were suffering from skin rashes, wounds, cough, or sore throat (Molan 1999; Molan 2006; Allsop and Miller 1996). Sweet honey contains phytochemical substances that are obtained when a bee sucks the nectar of flowers or secretions from living parts of plants, such as resin. Resin is found in immature fruit and flower buds. The pollen in the honey is an identifying marker for the botanical origin of the honey. Phytochemical substances are then carried by bees in their honey sacs and stored in honeycombs until the time of harvest. During this process, the natural phytochemical substances are mixed with fluid from the bee’s body, thereby producing bioactive compounds with different structural components that are present in honey. Bioactive compounds possess substantial biological activity (Nile and Park 2014) and display unique abilities to elicit responses (Ramirez-Estrada et al. 2016) in living organisms and to improve health care (Rodriguez-Mateos et al. 2014). Bioactive compounds not only vary from each other with respect to their characteristics and the magnitude of the effects they elicit, but they also differ functionally (Brusotti et al. 2014; Forbes-Hernández et al. 2014). The efficacy of bioactive compounds for the treatment of human diseases has led to extensive profiling studies of the bioactive compounds present in honey or in herbal plants. The aim of this review is to highlight the isolation and characterization of natural bioactive compounds in honey and to explore the applications of polyphenols for the treatment of human diseases.

Diversity of honey

More than 200 polyphenol compounds have been identified in various honey samples (Eteraf-Oskouei and Najafi 2013; Gheldof et al. 2002). For their nutrition, honeybees depend intensely on their ability to forage for aqueous solutions in nearby plants (Ball 2007; Seeley 2009). Most of the plant fluids they consume, such as nectar or resin, are mainly composed of protein, different types of sugars, lipids, minerals, vitamins, and ascorbic acid (Baker and Baker 1983). The composition of such fluids varies depending on the species of plant and natural geographical features (Graham 1992). Thus the composition of a given honey sample depends on its source (Schmidt 1996). Commonly, bees from the genus Apis forage on a single floral source, thus producing monofloral honey. Most honeys are monofloral and are named according to their respective plant species. Manuka honey, the well-known New Zealand and Australian medical-grade honey, is named after the manuka tree, Leptospermum scoparium. Similarly, alfalfa honey, avocado honey, sage honey, and tualang honey are named after their respective source trees. In contrast, bees from the genus Trigona, known as stingless honeybees, forage on diverse floral sources and produce polyfloral or multifloral honey. As such honey cannot be named after a tree species, multifloral honey is named according to the forager bee species, such as Trigona itama honey or Trigona thoracica honey. Nevertheless, the relative efficacy of monofloral and multifloral honeys for the treatment of human disease remains under investigation, as does a conclusion about which type of honey represents a superior therapeutic product.

The most abundant constituents of honey are carbohydrates, which include monosaccharides, disaccharides, and polysaccharides (75%); water (20%); proteins and free amino acids (2%); acids (1%); minerals (1%); and vitamins and enzymes (1%) (Krell 1996). It is a formidable challenge to define a standard honey for human use, due to the great variation in honey compositions and geographical origins (McLoone et al. 2016). For example, the honeys derived from the Apini tribe (non-stingless bee) and the Meliponini tribe (stingless bee) are composed of complex mixtures of natural phytochemical substances; to date, their vital novel compounds that promote human health and consumption remain under investigation.

Honey profiling

Honeys made by the genera Apis and Trigona are both composed of major and minor phytochemical compounds that can be characterized using various analytical methods. The characterization of phytochemical compounds begins with the preparation of honey samples and is followed by extraction and finally by separation or quantification (Khoddami et al. 2013). Prior to each profiling analysis, the preparation of representative samples requires both a sufficient quantity of honey that is free of contaminants and an accurate solution concentration to verify the quantitative and qualitative determinations. Unnecessary analytical repetitions due to errors are time-consuming and can be effectively avoided if sample preparation is precise and optimized. Homogenizing the sample manually or mechanically (Reybroeck et al. 2010; Kurhade et al. 2013), with mild heating to reduce crystallization (Aljadi and Yusoff 2003), is another key procedure for obtaining accurate chromatography results.

The minor phytochemical compounds in honey can be easily isolated by extraction techniques. To increase the concentrations of these minor compounds in a representative sample of honey, sugar and matrix compounds are first extracted by using organic solvents. This procedure may result in uniformly enriched target compounds and less interference from the matrix compound (Tura and Robards 2002). In addition, the use of other solvents, such as ethyl acetate (Kassim et al. 2010a; Kassim et al. 2010b), ethanol (Brudzynski and Miotto 2011; Alvarez-Suarez et al. 2010), methanol (Brudzynski et al. 2012), acetone (Naqvi et al. 2013), water, or mixtures of these solvents (Routray and Orsat 2012; Macdonald et al. 2010) contribute to the optimal extraction of target compounds, especially minor polyphenol substances. Solvents are selected based on the intended analytical technique and on the molecular weight, molecular size, solubility, pH, and volatility of the target compound. An acidic compound is isolated by using a compatible acidic organic solvent. A 70% ethanol solution is suitable for microwave-assisted extraction, ultrasonic extraction, and maceration extraction. On the other hand, ethyl acetate is recommended for liquid-liquid extraction. Multiple extraction methods are used for the isolation of polyphenols; they include supercritical fluid extraction (Junior et al. 2010), pressurized liquid extraction (Blasco et al. 2011), stir-bar sorptive extraction (Huang et al. 2011), and solid-phase micro extraction (Campillo et al. 2012).

Ion-exchange column chromatography is the most common assay for the characterization of minor phytochemical compounds derived from natural honey. Each phytochemical compound is detected based on its ultraviolet absorption peak, its molecular weight, or its retention time. Liquid chromatography-based separation techniques, such as liquid chromatography-mass spectrometry (LC-MS) and high-performance liquid chromatography (HPLC), are the preferred methods for the characterization of polyphenol compounds. Recently, HPLC and GC have been combined with MS to establish HPLC-MS and gas chromatography-mass spectrometry (GC-MS) techniques (Biesaga and Pyrzyńska 2013). Of all these techniques, HPLC and GC-MS are most commonly used (Merken and Beecher 2000; Sun et al. 2012) because of their reliability, efficiency, accuracy, and speed. Generally, GC-MS is suitable for the characterization of phenolic acids and other volatile compounds. Meanwhile, LC-MS and HPLC are useful for the characterization of flavonoids and other major compounds. Separation by LC-MS includes ultraviolet detection with a photodiode detector and diverse subsequent mass-spectrometric methods (Biesaga and Pyrzyńska 2013; Pyrzynska and Biesaga 2009). However, such techniques are not only time-consuming but also tedious and costly. Other techniques, such as ultra-high-performance liquid chromatography (UHPLC) (Kečkeš et al. 2013; Gašić et al. 2014), HPLC with diode-array detection (HPLC-DAD) (Mattonai et al. 2016), reverse-phase HPLC (Can et al. 2015), paper chromatography, thin-layer chromatography (Naczk and Shahidi 2006), and capillary electrophoresis (Asensio-Ramos et al. 2009) are among the analytical choices for minor and major phytochemical profiling. The quantification of bioactive compounds is an optional analysis that is performed by comparing a known commercial standard with each separated compound. Table 1 shows the characterization of polyphenols as verified by different chromatography techniques.

Table 1 Characterization of polyphenols as verified by different chromatography techniques
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Polyphenols

Natural honey is composed of nutritive polyphenols (Manyi-Loh et al. 2011). Polyphenols are primary identifying markers for the botanical origin of honey (Wang and Li 2011), and they display high therapeutic and dietary value (Uthurry et al. 2011). Bioactive polyphenol compounds are responsible for the colors, aromas, and tastes of all plant-based products, including fruits, cereals, and vegetables (Chaturvedula and Prakash 2011). The different polyphenol-derived ranges of honey color, aroma, and taste are directly dependent on the pollen source (Gil et al. 1995). More importantly, polyphenols integrate the physiological functions of a plant and are composed of secondary metabolites of various groups or classes (Di Ferdinando et al. 2014). Specific examples of such metabolites include tocopherol, flavonoids, phenolic acids, alkaloids, chlorophyll substances, amino acids (peptides), amines, carotenoid derivatives, and ascorbic acid (Cavalcanti et al. 2013). The phenolic groups of polyphenols derived from citrus fruits are vital for reducing proinflammatory cytokines in humans (Morand et al. 2011; Bernabé et al. 2013).

Flavonoids and phenolic acids

The major polyphenol compounds in honey are flavonoids and phenolic acid (Khalil et al. 2011). Flavonoids and phenolic acid are responsible for inhibiting oxidation and destroying free radicals (Nakajima et al. 2014). The identification and classification of flavonoids and phenolic acid are based on their chemical structures, which consist of one or more hydroxyl groups that are fused with an enclosed ring structure and thereby produce an aromatic ring containing six carbon atoms with hydrogen atoms (Harborne 1989).

The identities of the bioactive flavonoid and phenolic acid compounds in honey are similar across different types of honey but vary in their relative quantities (Can et al. 2015). Table 2 shows the bioactive flavonoid and phenolic acid compounds found in various types of honey. Honey of darker color contains higher quantities of bioactive compounds than lighter-colored honey (Moniruzzaman et al. 2013). This difference is due to the nutritional differences among different sources of pollen (Estevinho et al. 2008; Estevinho et al. 2012).

Table 2 The bioactive flavonoid and phenolic acid compounds found in honey
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Polyphenols in natural honey for the treatment of human diseases

Antimicrobial activity

The presence of flavonoids and phenolic acids confers substantial antimicrobial activity to honey. With respect to the antibacterial effects of honey, previous studies revealed the activity of extracted esters of flavonoids and phenolic acids against various bacterial species, including Bacillus subtilis, Staphylococcus aureus, Staphylococcus lentus, Klebsiella pneumoniae, and Escherichia coli (Estevinho et al. 2008). Specifically, the inhibitory activities of apigenin, quercetin, myricetin, and rutin against E. coli, Vibrio cholerae, Salmonella typhi, and Salmonella typhimurium have been reported (Das et al. 2015). Similarly, the high antibacterial activity of naringenin against streptococci and methicillin-resistant Staphylococcus aureus has also been reported (Tsuchiya and Iinuma 2000). Additionally, myricetin has been described as an inhibitor of DNA synthesis in Proteus vulgaris (Mori et al. 1987; Tenore et al. 2012).

Honey also has shown antiviral effects, as it was found to be effective in the treatment of labial and genital herpes, which are common viral diseases (Onifade et al. 2013; Fingleton et al. 2014). Another study demonstrated the direct effects of several flavonoids, such as chrysin, acacetin, and flavones, as potent inhibitors of HIV replication, which severely infects H9 lymphocytes (Critchfield et al. 1996; Behbahani 2014). Chrysin reportedly inhibits herpes simplex type-1 and type-2 virus in addition to adenovirus-3 (Lyu et al. 2005). Furthermore, apigenin was characterized as an antiviral against hepatitis B surface antigen and hepatitis B e-antigen (Chiang et al. 2005).

Antioxidant activity

Antioxidants are natural chemical substances that are mainly found in plants. Their main role is as a defense mechanism that counteracts free radicals and prevents their damaging oxidative effects (Manyi-Loh et al. 2011; Kumar et al. 2013). Oxidative stress can lead to pathogenesis or to the mutation of cellular DNA. Moreover, free radicals have roles in cardiovascular disease, diabetes, cancer, aging, gastritis, Alzheimer’s disease, and several ulcers and gastrointestinal disorders (Samarakoon et al. 2011; Poorna et al. 2013; Zahin et al. 2010; Kumar et al. 2011). Honey contains antioxidant compounds that are derived from pollen sources (Estevinho et al. 2012) and that can inhibit oxidative reactions (Manyi-Loh et al. 2011; Erejuwa et al. 2012) by increasing total cellular antioxidant capacity and eliminating reactive oxygen species, thereby reducing DNA damage (Erejuwa et al. 2012; Zhou et al. 2012). Quercetin and galangin have shown great antioxidant effects against lipid peroxidation (Lagouri et al. 2014) .

Anti-inflammatory activity

Inflammation commonly occurs during the healing process. The opening of a capillary and the movement of plasma to an infected wound results in edema. Mild inflammation is considered normal during the healing process; however, it is harmful (Aljadi and Yusoff 2003), triggering leukocyte activity (Van den Berg et al. 2008), and leading to the production of free radicals. The polyphenols present in honey have an oxidizing ability (Shafin et al. 2014), which confers a corresponding anti-inflammatory effect by inhibiting the production of nitric oxide. Flavones (naringenin), isoflavones (daidzein and genistein), and flavonols (isorhamnetin, kaempferol, and quercetin) are classes of polyphenols that inhibit inducible nitric oxide synthase (iNOS) and nuclear factor-κB (NF-κB). Additionally, genistein, kaempferol, quercetin, and daidzein impaired the activation of the signal transducer and activator of transcription 1 (STAT-1) (Hämäläinen et al. 2007).

Antineoplastic activity

The bioactive compound eugenol has been studied and has shown its antineoplastic efficacy by inhibiting the proliferation of colon cancer cells and prostate cancer cells (Jaganathan et al. 2011; Samarghandian et al. 2011). Similarly chrysin, a flavone derivative in honey, was reported to have antitumor activity after it successfully eliminated a melanoma cell line (Pichichero et al. 2010; Pichichero et al. 2011). The significant effects of honey against radiation-induced mucositis after head and neck treatment (Khanal et al. 2010), in addition to its effect in reducing Walker tumor (Tomasin and Cintra Gomes-Marcondes 2011), have demonstrated its positive impact in diminishing either benign or malignant tumors in both in vitro and in vivo studies (Swellam et al. 2003). Other subclasses of phenolic acids, namely protocatechuic and p-hydroxybenzoic acids, have demonstrated significant inhibition toward the growth of prostate cancer (PC-3) and breast cancer (MCF-7) cells (Spilioti et al. 2014).

Antiulcer activity

Derivatives of flavonoids, such as kaempferol, quercetin, hesperetin, and naringin, possess antiulcer efficacy for the treatment of the gastric mucosa (Ghaffari et al. 2012), duodenal ulcers (Gadkari and Balaraman 2015), and gastritis (Suprijono et al. 2011). Their antiulcer activity manifests as the slowing down or hindrance of lipid peroxidation, the neutralization or reduction of cell proliferation, and an increased susceptibility to apoptosis (Almasaudi et al. 2016).

Cardiovascular disease

Polyphenols play a vital role in the treatment and control of cardiovascular diseases (Habauzit and Morand 2011). Quercetin is beneficial for controlling blood pressure (Sánchez-Moreno et al. 2006) and reduces the risk of stroke and coronary heart disease (Galindo et al. 2012). Kaempferol is of great importance in protection against the accumulation of the low-density lipoprotein cholesterol that can cause cardiac disorders. The effective action of polyphenols against cardiovascular diseases is mainly achieved by preventing the oxidization of low-density lipoprotein cholesterol, controlling coronary vasodilatation and reversing platelet clotting in the blood stream (Alagwu et al. 2011). Thus, the atherosclerosis that leads to arterial hardening and narrowing is effectively counteracted (Kas’ianenko et al. 2010). Generally, polyphenols with activities against either ordinary or chronic diseases are derived from different plant sources (Gadkari and Balaraman 2015) and thus have different therapeutic mechanisms. Table 3 shows the mechanisms by which polyphenols act against several diseases.

Table 3 The polyphenols mechanisms of action against human diseases
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Conclusion

The complex and diverse chemical composition of honey is a prominent key to its broad beneficial activity. However, the polyphenols in honey are not limited to being biomarkers, as they possess direct functions in reducing or terminating the risk of major chronic human diseases, such as cardiovascular disease, cancer, tumors, chronic inflammation, and viral disease. The therapeutic efficacy of honey is mainly derived from the known antioxidant activities of flavonoids or phenolic acids, which naturally exist in small quantities. The isolation and characterization of polyphenols in honey has been a challenging task that has been continuously undertaken by researchers to identify novel bioactive compounds for their future applications in the treatment of human disease.