Chemistry, Biology and Potential Applications of Honeybee Plant-Derived Products

The chemical composition of honey has been widely investigated around the world. Studies have shown how geographical and botanical origin of honey influences its physical-chemical properties, being a useful tool for evaluation of authenticity and differentiation. In this chapter, a review has been performed in order to describe the chemical composition and the most important nutritional properties of honeys from several countries and diverse botanical origins. Reported data of honeys for water, sugars, ash, minerals, color conductivity, aminoacids and quality indicators, such free acidity, enzymes and hydroxymethylfurfural (HMF) are mentioned and also compared to the established limits given by the Codex Alimentarius and the International Honey Commission.

Propolis is a plant derived bee product which serves dual purposes in the honeybee colony: building material and protective substance. Propolis has been used as remedy in the traditional medicine of numerous nations, because of its antimicrobial, antioxidant, and many other beneficial pharmacological actions. In this chapter, the results of the newest (in the last 5 years) chemical studies of propolis from different geographic and plant origin are reviewed, together with the new identified source plants: 152 new constituents of propolis, being 57 new chemical entities, and 12 new chemical types of propolis are listed. The importance of propolis for the bee colony is discussed, with special attention to the activity of propolis and its constituents against bee pathogens and parasites. The review of recent propolis literature demonstrates its potential to serve as a source of new chemical structures and new bioactive compounds due to its chemical diversity. It also reveals the potential of propolis to be used for development of innovative products, mainly in the field of food industries, animal husbandry, and beekeeping. For this to happen, the combined efforts of researchers and technologists from different areas are necessary, in order to make better use of bee glue.

Bee pollen, usually used as an important source of nutrients and micronutrients for the young bees in the hive, is also an important food for humans. This product is very rich in proteins, lipids, free sugars, carbohydrates, and it contains trace amounts of minerals, phenolic acids, flavonoids and a good range of vitamins. A brief look at bee pollen composition, it is easily recognised that it is a balanced food that can be used as a stand-alone food or as a nutritional supplement or even as a medicinal product. Several bioactivities, due to some of these compounds, were studied in bee pollen samples from different floral sources and the results conduce to important properties. The amount and diversity of micronutrients could induce vast benefits if used for health purposes following a complete risk assessment. Nevertheless, the results pointing towards the encouraged use of bee pollen, the risk assessment of some floral species containing toxic compounds has not been fully studied to insure the safety of consumption for all the gathered flowers, so this will also be discussed in this chapter. Admiration for its goodness and medicinal properties, bee pollen has been consumed for centuries, however, currently the efficacy and safety for all consumed products, foods, supplements or medicines is an important tool to guarantee correct quality control and essential to add value to the product.

To summarise, in this chapter we will put the situation of gaps in bee pollen research into some kind of perspective, outlining some important points and discussing in more depth the implications of collecting samples, chemical composition and risk assessment.

Honey, propolis, and pollen are three important components of the beehive produced by honeybees mixing different plant parts (nectar, resin and pollen) with their own secretions, for further usage with different purposes in the hive. The fact that these natural products have been associated with numerous health benefits has attracted the attention of researchers resulting in a significant raise of scientific studies attesting their biological properties. Among the various constituents of honey, propolis and pollen, the phenolic compounds are the ones most frequently related to the beneficial properties of these products and hence, one of the main investigated groups. Their characterization is important to understand individual contribution(s) and synergistic effects of each compound for the overall biological effects of the bee product. To pursuit this goal, spectrophotometric techniques including HPLC, GC and TLC, alongside with the respective detection methods such as DAD, FLD and MS, have been developed and improved in order to offer better and more accurate separative performances.

The aim of this review is to give an approach on the course that the chromatographic techniques have taken until the most recent trends on this field applied to the separation and characterization of the phenolic constituents of honey, propolis and bee pollen as well as an overall perspective of variability in terms of phenolic composition that can be found in the three bee products mentioned.

Over the past fifty years, Nuclear Magnetic Resonance (NMR) spectroscopy proved to be a powerful tool in the structural characterisation of bioactive compounds from natural sources. In this chapter we cover the basic theory behind each NMR technique used to determine the structure of several families of compounds (e.g. carbohydrates, phenolics and sesquiterpenoids) present in honey and propolis. We also provide basic information how 1D and 2D NMR techniques can help in the structure establishment of honeybee constituents. The 1H and 13C NMR data of several of these constituents are compiled and described, being some of them used as botanical and geographical markers. In the case of propolis, a list of compounds identified by NMR is presented. A basic overview in quantitative NMR determinations and in NMR coupled to chemometric methodologies highlights their use to detect honey adulteration and assign their authenticity.

This chapter is focused on the application of electrochemical techniques (e.g., sensors and biosensors), as the predominant methodology, to the quantification of individual or total phenolic compounds, either in standard solutions or in real matrices (e.g., plants, fruits and beverages) and their capability for assessing antioxidant activity/capacity. Specially, the potential application to evaluate antioxidant capacity of bee-hives products (e.g., propolis, honey) is addressed. Finally, the voltammetric behavior of Portuguese monofloral honeys is discussed for the first time, taking into account the expected effects of honey color and floral origin.

Also, a possible relation with the expected antioxidant capacity of honeys is discussed, considering their floral origin. Works describing the use of electrochemical detection imbibed on liquid chromatographic or capillary electrophoretic configurations among other analytical methods will not be focused in this review, although their undoubtedly potentials and proved applications.

The importance of honey has been recently promoted due to its nutritional,
pharmaceutical and therapeutical characteristics. In recent years, the combination of
novel and rapid instrumental techniques based on infrared spectroscopy [mid infrared
(MIR), near infrared (NIR)] combined with multivariate data analysis has resulted in
the development of both qualitative and quantitative methods for the analysis of honey
and bee products. The most important applications of these technologies in honey have
been associated with authenticity, discrimination or traceability issues. However, few
reports can be found on the use of both NIR and MIR to quantitatively analyse honey
composition and less information is available for the analysis of other bee products.
This chapter aims to describe and discuss different applications on the use of NIR and
MIR spectroscopies to analyse honey, pollen and bee derived products. A brief
description of some qualitative applications will be also discussed.

The study of antioxidant-antiradical activity of food products has received a rising interest since last decades, parallel to the boom of functional foods and healthy consumption trends, and to the increasing number of scientific evidence linking this physicochemical property to prevention of several degenerative diseases, in particular of cancer. Honey belongs to the category of natural foods showing high antioxidant activity, which depends largely on its botanical/geographical origin. Different studies have been conducted to describe the antioxidant activity of honey by both in vitro and in vivo techniques, which are reported in a vast extension of articles. However, the lack of a standard protocol for measuring the antioxidant activity has been one of the main drawbacks found. Techniques such as 2,2-diphenyl-1-picrylhydrazyl (DPPH), Trolox Equivalent Antioxidant Capacity (TEAC), Ferric Reducing Antioxidant Power (FRAP) and Oxygen Radical Absorbance Capacity (ORAC) are the most common in vitro methods. It has been suggested to use at least two techniques for measuring antioxidant activity, since these are only an approximation to what occurs in the body. It is known that biologically active ingredients which may contribute to the antioxidant effect of honey include vitamins, minerals, organic acids, flavonoids, phenolic compounds and even products derived from Maillard reaction.

Over the last years, the hive products such as propolis and pollen have been highlighted due to their potential health benefits, including antioxidant abilities that have been correlated with their content in phenolic compounds. Regardless of the several factors that may affect propolis and pollen antioxidant activity, these products have been shown to possess, either through the use of in vitro or in vivo models, important features concerning the modulation of cellular oxidative stress caused by environmental factors (e.g. UV-light), metals, pesticides and other xenobiotics. This modulatory effect focus not only on the capture of radicals that these elements might eventually generate, but also by the activation of cellular antioxidant mechanisms such as enzymatic antioxidants or by modifying gene expression patterns. Although the mechanisms behind these responses are not fully known, it has been showed that caffeic acid phenethyl ester, pinocembrin and chrisin are some of the compounds responsible for some of these responses. Taking into account the gathered results, propolis and pollen can be viewed as potential agents in the re-stabilization of cellular oxidative imbalance and in the prevention of oxidative stress related diseases.

This chapter aims to discuss the effects of honeybee plant-derived products in inflammatory processes, with particular focus on honey, pollen and propolis. Honey is mainly composed by fructose and glucose, containing also minerals, proteins, free amino acids, vitamins and polyphenols and has long been used by humans not only for nutritional purposes but also as a medicine. The biological properties of honey can be ascribed to its polyphenolic content which, in turn, is usually associated to its antiinflammatory activity, as well as antioxidant, antiproliferative and antimicrobial benefits. Bee pollen results from the agglutination of flower pollens with nectar and salivary substances of the honeybees. Due to its optimal nutritional balance, it has been considered as a perfect food all around the world and also used as a therapeutical agent. However, there is a lack of scientific support addressing the biological activities of bee pollen. Propolis is produced by bees from secretions of trees, trunks, buds, leaves and pollen, adding wax and substances secreted by bee glands.

The large and diverse number of chemicals in propolis may justify their biological activities, namely anti-inflammatory properties. Herein we emphasize the antiinflammatory potential of the honeybee plant-derived products propolis, honey and pollen. Whenever possible we also disclose the action mechanisms and the principal compounds responsible for the biological activity. The intracellular signaling targets of propolis, honey and pollen are highlighted and summarized in Fig. (1). Overall, the production of inflammatory mediators, i.e. nitric oxide (NO) and prostaglandins, are inhibited by the three products partially due to the inhibition of nuclear factor kappa B (NF-κB) and mitogen activated protein kinases (MAPKs) signaling pathways.

The majority of cancers have no curable treatment and the main available therapies have serious side effects, justifying the need for development of new antitumor agents. Several efforts have been made to identify natural products useful in the cancer setting. This area has emerged as an important research field, providing the possibility to both identify novel potentially useful agents and to study the mechanisms of antitumor action. Honeybee plant-derived products have shown anti-cancer activity in a series of experimental and clinical studies with cell lines, animals and humans. Honey, the viscous, golden and sweet liquid produced by bees from the nectar of flowering plants has proven to display antiproliferative and apoptotic effects, along with other activities that contribute for its antitumor properties. Propolis, a special substance made by honeybees through mixing tree saps with salivary secretions, is used to seal fissures and openings in the hive, strength combs, seal brood cells and protect the hive from infections. Propolis contains phytonutrients that may be useful in different pathological conditions, including cancer.

Bee pollen, the bees´ primary food source, is plant pollen collected from a variety of plants and processed by honeybees. Demand for this natural product is rising since it has effects on a variety of biological functions, which contribute to the fight and prevention of cancer. This review focuses on the antitumor properties of honey, propolis and bee pollen as well as on the potential use of these honeybee plant-derived products to develop new therapeutic approaches for patients with different types of tumors.

Bee products have always been used as foods and/or as therapeutic agents against a number of diseases in alternative medicine since almost immemorial times. The millennial track record of the health and nutritional benefits of such natural bee products is being supported more recently by scientific research. The antimicrobial activity of bee products is considered one of its widespread and most important bioactivities, and highlights the potential of these natural products as promising antimicrobial agents for clinical and biotechnological applications. Honey and propolis fulfill all the criteria of ideal candidates for treatment of non-healing wounds and other diseases caused by microorganisms. These natural products find application against resistant pathogenic microorganisms without the risk of antimicrobial resistance acquisition and prevent the formation or distortion of biofilms. Honey is regarded as a pure natural and functional product of high nutritional value. Honey factors that contribute to its antimicrobial activity are diverse but researchers consider both the high sugar concentration and low pH as well as the presence of hydrogen peroxide, methylglyoxal and bee defensin-1 (an antimicrobial peptide) as the most relevant in different honeys.

Propolis is a sticky resin produced by bees, especially from coniferous, having plantderived and bee-released compounds, but with a chemical composition difficult to standardize due to its dependence on vegetation, season and environmental conditions at the collection site. The mechanism of propolis antimicrobial activity is complex, probably relying in a synergistic activity between different phenolic compounds such as flavonoids and phenolic acids along with other components. Pollen is a source of phytochemicals and nutrients, extremely rich in carotenoids, flavonoids and phytosterols. Although less studied for this bioactivity, bee pollen has also been proven to possess antimicrobial activity.

Although honey extraction is an ancient practice, its use as a source of income is more recent. Over the years several techniques have been developed to achieve greater amounts of honey and also to ensure the quality of the product. Furthermore, bees during their life cycle produce wax, royal jelly, pollen and propolis besides honey. Pollen and propolis are such as honey, plant derivatives, while the wax and royal jelly are products secreted by glands of the bees. This chapter aims to present ways to add value to bee products plant-derived, namely: honey, pollen and propolis. Uses and new applications are presented. Moreover, the production of honey-fermented products such as mead, honey beer and honey vinegar are also discussed.