Healthy living with bees: Investigating the antioxidant properties of honey and pollen

Reactive oxygen species

Bees rather handily produce a wide range of health-promoting substances, including honey, beeswax, propolis and royal jelly. Even the pollen that bees collect along with the nectar has health benefits. Many of these benefits derive from the fact that these substances are rich in antioxidants such as flavonoids and other polyphenols, which soak up harmful reactive oxygen species (ROS).

Now, chemists from Portugal and Algeria, led by Ana Oliveira-Brett from the University of Coimbra, have come up with a quick and easy analytical process, comprising several comparatively simple analytical techniques, for investigating in detail the antioxidant properties of bee products.

The analytical techniques that make up the process are differential pulse (DP) voltammetry, DPPH (1,1-diphenyl-2-picrylhydrazyl) spectrophotometric assay, high performance liquid chromatography with electrochemical detection (HPLC-ED) and gel electrophoresis. The chemists’ idea was to use both DP voltammetry and the DPPH spectrophotometric assay to determine the antioxidant activity of the bee products. They would then use HPLC-ED to determine the identity and concentrations of the polyphenols present in the products and gel electrophoresis to assess how effectively the products could protect DNA from damage by ROS.

Antioxidant activity

DP voltammetry can determine the antioxidant activity of a substance by recording its oxidation potential at an electrode, as oxidation potential is inversely related to antioxidant activity. In contrast, a DPPH spectrophotometric assay measures antioxidant activity by recording the degree to which a solution of DPPH changes color. DPPH is composed of stable free radicals and forms a purple solution that absorbs light with a wavelength of 516nm.

Antioxidants added to the solution interact with the DPPH, altering its absorbance. This can be seen visually through the loss of the purple colour, but can also be determined more objectively by measuring the absorbance of the 516nm wavelength with a spectrophotometer. The assay works by determining the amount of antioxidant required to reduce the absorbance by 50%: the lower the amount, the greater the antioxidant activity.

The chemists decided to employ ED with HPLC because ED tends to be more sensitive for polyphenols than a standard UV detector. Finally, for testing the protective effects of the bee products, they exposed double-stranded DNA to ROS generated by the Fenton reaction between hydrogen peroxide and iron. The extent of the damage caused by the ROS could be seen from the distribution of the DNA across the electrophoresis gel.

Honey and pollen

They first tested this analytical process on 10 samples of honey from Greece, Portugal, Algeria and Romania, and two samples of pollen from Portugal and Romania. This involved first extracting the polyphenols from the honey and pollen samples by immersing them in a mixture of water, ethanol and methanol, and then exposing them to ultrasound.

DP voltammetry and the DPPH spectrophotometric assay revealed that the extracted polyphenols possessed high levels of antioxidant activity, with the results from both techniques tending to agree with each other.  HPLC-ED revealed that all the honey and pollen samples contained a similar range of polyphenols, including several anthocyanidins and flavanols, although they differed in their concentrations.

These results also indicated that the relationship between polyphenol concentration and antioxidant activity isn’t entirely straightforward. Among the honeys, a honey from Portugal had the highest polyphenol concentrations, even though a Greek honey had the highest antioxidant activity.

Finally, the chemists conducted the gel electrophoresis step with four of the honey samples and one of the pollen samples. This showed that, irrespective of their antioxidant activities or polyphenol concentrations, all the samples helped to protect the DNA from damage by soaking up the ROS.

These findings not only confirm the health benefits of honey and pollen, but also raise the possibility of using this analytical process to explore the health benefits of the various other bee products as well.