Blog 4: Correlating the flavonoid and phenol content to the antioxidant activity of various European propolis

Throughout the past year, Dr. Mojica, my faculty advisor and research mentor, have worked with the bee product propolis, or as it otherwise and sometimes more fondly known, bee glue. For the past year, we have worked with many propolis samples from regions throughout the world. The importance of the study of propolis, is its potential to be used as a complementary medicine or as a natural supplement due to its considerable antioxidant content. The problems arise in the effectiveness of different samples of propolis, as there are numerous factors that can alter the compounds present and subsequent antioxidant properties of a particular sample. Such factors include the climate of a region and the botanical sources available to provide bees with bud exudate, a key ingredient in propolis. However, even propolis gathered within the same region at the same time of year can present differently in terms of composition and antioxidant properties. The goal of our study was to use standardized tests for antioxidant activity, phenol content, and flavonoid content on each sample to determine those with the greatest content of bioactive compounds (phenolics and flavonoids) and most antioxidant activity. The DPPH (2,2-diphenyl-1-picryl-hydrazyl)and ABTS (2,2-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid) assays determined the radical scavenging activity of propolis samples, the FRAP (ferric reducing antioxidant power) assay determined the reducing ability, the Folin-Ciocalteu assay determined the total phenol content, and the AlCl3 assay determined total flavonoid content.

It was hoped that following identification of the most and least effective antioxidants, instrumental methods of analysis such as GC-MS could be used to identify the compounds that had contributed to the observed properties, however the present circumstances prevented such data from being collected. The study was however successful in determining antioxidant activity of each sample through three different assays and determining both total phenolic and total flavonoid content for each sample.

Excitingly, the preliminary research Dr. Mojica and I had done last year, prior to this study, is currently being reviewed for publication in an undergraduate research journal, so it was extremely exciting to be enabled to continue this work. This research was also accepted for presentation at the American Chemical Society’s annual meeting, this year’s location being in Philadelphia, prior to its cancellation. This study is also currently serving as the basis for my undergraduate thesis as per the requirements of the Pforzheimer’s Honors College.

Dr. Mojica has been extremely valuable as I am less familiar with writing in the sciences and he has helped me to turn our raw, unformatted data into clear and presentable information. I am extremely grateful to have been a part of this undergraduate research program, as I feel more comfortable, having gone through it, with planning and carrying research in a laboratory setting while collaborating and working with others to continuously improve experiments and our studies as the research progresses.

Blog Post 3: Correlating the flavonoid and phenol content to the antioxidant activity of various European propolis

Since early December, Dr. Mojica and I have focused on collecting and processing as much data as possible and have begun the process of interpreting it such that we can come to a clear conclusion. Over winter break, scheduling was an issue, thought because we were both committed to be this work, we’ve been able to set up out times that we can get work done. Early challenges that we had to work around were troubleshooting when certain protocols that weren’t working. In one instance of an assay not working, we found that the pH of a buffer we were using was too high to facilitate the desired complex formation between reagents and test samples that would produce a measurable color change.  While this was an easy fix, the greater challenges occurred as some results were unexpected in that they did not fit the general trend we were expecting. Specifically, there were countries that displayed high phenolic content and low flavonoid content, though notably variable levels of antioxidant activity and potency. As it is generally expected that high phenol content and high flavonoid content produce strong and potent antioxidant activity, the variability seen in this sample was surprising. Because of this, we’ve considered performing additional tests on the samples to clarify these deviations from the expected trend, though the time constraint is a significant factor in whether or not we will be able to do these tests. Though at times it can be frustrating to get results that don’t align with what was expected, I’ve thoroughly enjoyed the process of finding ways to elaborate our understanding of the natural products we are working with to have a more comprehensive view of the reasons for the surprises in the data, and I look forward to continuing this work.

Blog 2: Correlating the flavonoid and phenol content to the antioxidant activity of various European propolis

Over the past months, the main progress made has been the preparation and testing of the individual samples of propolis from England, Greece, Portugal, Russia, Latvia, Serbia, Hungary, Bulgaria, and Lithuania. The preparation of the samples involved creating an extract through sonication of the propolis in ethanol and subsequent filtration. One unexpected outcome was that some of the samples, after sonication, had hardened into a thick, paste-like mixture. The samples only required additional heat to ultimately be filtered to prepare extracts, though it was interesting that only some samples displayed this feature. We assume that this characteristic of the unique samples is a product of differences in the additional components of propolis such as beeswax, which we were intending on removing as part of preparing the extract.

Once all the samples’ extracts were prepared, we began performing the different assays to measure the antioxidant activity, phenolic content, and flavonoid content. Each assay needs to be performed several times to ensure that the data collected is accurate. For each assay, we tested serial dilutions of each sample extract to more accurately quantify the extent to which the reagents of the assay interact with the compounds in the extracts. Moving forward, more assays will be done to ensure accurate data and then the data obtained can be analyzed to look for trends and relationships.

Blog 1: Correlating the flavonoid and phenol content to the antioxidant activity of various European propolis

The subject of my research is the analysis and correlation of the antioxidant activity, phenolic content, and flavonoid content of bee propolis from several regions of Europe. Bee propolis is a sticky, resinous substance, composed of botanical exudate, pollen, resin, and wax, among other variable substances, each in varying amounts. Known for having many potential health benefits, propolis has been shown to have anti-oxidative, anti-inflammatory, and antimicrobial properties1. The various functionalities of propolis are a result of the wide diversity of chemicals and molecules that are found in it. The phenolics and flavonoids that are present in a given sample of propolis are the compounds that give propolis its anti-oxidative properties, which is why this study will look at how these properties are related to each other. The unique and individual properties of propolis are widely varied as factors such as the region, species of bee, season of collection, and variance of botanical source used by the bee will result in distinctive differences between samples2. The study of these properties will provide insight to the usefulness of propolis as a natural and complementary medicine.

In this study, samples from England, Greece, Portugal, Russia, Latvia, Serbia, Hungary, Bulgaria and Lithuania were used. For each sample, extracts were prepared for analysis. The antioxidant activity and ability will be tested with assays such as the 2,2-diphenyl-1-picryl-hydrazyl (DPPH) free radical scavenging assay, the 2,20-azinobis-(3-ethylbenzothiazoline- 6-sulfonic acid) diammonium salt (ABTS) assay, and Ferric Reducing Antioxidant Power (FRAP) assay. The phenolic content of each sample is measured using Folin-Ciocalteu method and the flavonoid content will be measured by the AlCl3 colorimetric method. Each of these assays involve the interaction of particular compounds in a sample with a reagent that produces a measurable colorimetric change. The results of these assays will be compared within and between different samples to indicate the properties of a particular sample of propolis as well as to allow the comparison of the different samples.

(1)       Kocot, J.; Kiełczykowska, M.; Luchowska-Kocot, D.; Kurzepa, J.; Musik, I. Antioxidant Potential of Propolis, Bee Pollen, and Royal Jelly: Possible Medical Application. Oxid Med Cell Longev2018, 2018. https://doi.org/10.1155/2018/7074209.

(2)       Bankova, V. S.; Castro, S. L. de; Marcucci, M. C. Propolis: Recent Advances in Chemistry and Plant Origin. Apidologie2000, 31(1), 3–15. https://doi.org/10.1051/apido:2000102.