Phenolics in Bee Propolis

Phenolics in Bee Propolis

Research by Josephine Farshi and Professor Elmer Mojica

The primary focus of my Summer 2017 research is the analysis of phenolics in bee propolis. I aim to investigate the properties of this propolis in relation to their use by both bees and humans alike. I will achieve this by studying the antioxidant makeup and nutritional properties of propolis by means of analyzing results gathered from gas-spectrometry mass spectrometry (GC/MS). In this blog post I will describe the purpose of my research, along with how I became interested in the topic.

First, I would like to clarify that bee propolis is a product of bees, but not the commonly presumed product known as honey. The product I am focusing on comes from beehives, just like honey does (Alibino 2014). However, bees make propolis by mixing beeswax with vegetable resin, a brown substance gathered from sap (Castro et al. 2014). Propolis is commonly referred to as bee glue. During the process of GC/MS, the propolis is separated into the chemicals that it consists of during the gas chromatography and further analyzed based on these chemicals during the mass spectrometry overview (Kartal et al. 2002).

Studying the chemical properties, such as flavonoids, will provide insight on how certain chemicals can provide nutritional benefits. The nutritional and food-science aspects behind the bee propolis are what first grabbed my attention about this very topic. For instance, bee propolis is known to contain 300 active compounds, some of which have been used to fight sore throat and stomach ulcers due to the pharmaceutical activities (Rios et al. 2014). I knew that I wanted to work with something related to food-science because I am interested in all aspects of nutrition, including health benefits that certain natural products provide and the effects of socioeconomic status on global nutrient intake. From my research, I aim to see how the chemical composition of bee propolis can be used for benefitting human health.

So far this summer, I have focused on an immense amount of literary exploration. There are several reasons behind why literature is significant in my scientific research. For starters, literature provides me with an understanding of how researchers have previously analyzed the same topic of phenolics in bee propolis. Having a broad understanding of the topic at hand can therefore create a rapport between the audience and myself, as they will be able to trust the authenticity of my studies. By using the Web of Science, I have gathered over 200 abstracts from scientists, published between 2014 and 2017. I now have a broad understanding of the importance of analyzing propolis for both bees and humans alike, along with an established methodology for gathering results.


Quantifying the Number of GFP Expressing Trichomonas Vaginalis Adhering to the Vaginal Epithelial Cells Treated with Amyloid Dyes

Trichomonas vaginalis is a eukaryotic parasite that causes the most common non-viral sexually transmitted infection worldwide. Although it is the most common, it is also the most understudied and poorly comprehended parasite. The research that will be conducted on this organism will help to better understand host-parasite relationship as the trichomonads adhere to vaginal epithelial cells and begin to colonize. The purpose of this research is to successfully quantify the number of GFP expressing Trichomonas vaginalis adhering to the vaginal epithelial cells treated with amyloid dyes. Because there are a lot of trichomonas cells that will be seen under a microscope, the use of GFP or green fluorescent protein will help to magnify and quantify how many cells are present. The goal is to see an increase in fluorescent activity each time the cells are displayed under a microscope.

In order to carryout this research, we first must colonize GFP. To do this, we use GFP that was found in E. coli and smear it onto an agar plate with LB (Luria broth) medium and ampicillin.


Ampicillin is sometimes used on plates because when ampicillin breaks down, it can often lead to more colonies being formed. The plates are then incubated over night to allow for colonization to occur. Once colonization occurs, the plates are moved under a sterile hood and using a sterile pipet and tip, a single colony is extracted and placed into liquid culture made up of the same LB medium with ampicillin and relocated to incubate on a shaker plate over night. The following day, the tubes are removed from incubation and transferred to a microcentrifuge tube and centrifuged out to collect a concise pellet at the bottom of each collection tube. This pellet collected contains bacterial cells that contain GFP, which will eventually be cut out and cloned multiple times. Once the pellet is collected, the DNA is extracted from those cells using a QIA Spin Miniprep kit. The DNA collected after the end of this kit is the unclean DNA, which could possibly contain buffers and residual waste from the kit that was not completely centrifuged out. However, the unclean DNA is tested to make sure that there is indeed DNA present, and to do this, we have to set up a gel electrophoresis.

Gel electrophoresis is a technique used to separate mixtures of DNA, RNA or proteins based on molecular size. Molecules are pushed by an electric field that moves negatively charged particles through the small pores of agarose gel to the positive charge at the other end.


Through this technique, we can compare the sizes of bands to a ladder, or just to simply confirm the presence or absence of a specific molecule. For this purpose, we used the gel to determine the presence of bacterial DNA from the E.coli culture. Below is a picture of the resulting gel after running this technique:

As shown by the picture above, DNA was indeed present in the unclean tubes. From those tubes, a certain amount was then collected and another kit, DNA Clean & Concentrator, was used to further clean the DNA to use for a PCR or polymerase chain reaction.

Polymerase chain reaction is used to increase a single copy or a few copies of a section of DNA. Below is a table that shows the main ingredients of a PCR and what their role is:

The main goal to achieve from the PCR is to use the primers that have been specialized to highlight the region used to code for GFP in the DNA sequence, amplify it and cut it out. Once the PCR is complete in the set conditions, another gel is run to determine if the primers worked and if the gene was amplified. However, PCR is a very sensitive technique that requires the right conditions, which have yet to be discovered. Below are the results of the gels following a PCR treatment and the conditions that were set:




Using these previous conditions, it will allow for us to easily manipulate the numbers to try and find the most ideal conditions. In the future, these ideal conditions will allow for us to isolate the GFP band with the correct restriction sites and insert it into the parasite.

Moving forward, the research aims to successfully obtain the GFP gene in the PCR using the specialized primers. Once this is obtained, we can perform a gel extraction that will isolate the GFP that contains the specific restriction sites that will eventually be cloned and inserted into the parasite in question. As an individual, I hope to perfect my skills in performing a PCR as well as extracting DNA in the form of a pellet. These skills will eventually aid me in my future career as a forensic scientist where the use of PCR is necessary to amplify the DNA in question to perform further tests on it.

Characterization of the Microbial Community of the Accessory Nidamental Gland of the Longfin inshore squid, Loligo pealei

The title of our experiment is Characterization of the Microbial Community of the Accessory Nidamental Gland of the Longfin inshore squid, Loligo pealei. The research that is being undertaken is focusing in on the symbiotic relationship that is present between marine organisms and the mutualistically beneficial bacteria that grows on these organisms. The foundation of this research was interest in the nidamental gland of the female reproductive organ in squids. The nidamental gland is responsible for coating the eggs with a jelly like substance that is integrated with bacteria to serve as a protective barrier against predators and fowling. If this bacteria is able to be characterized, The properties that allows it to protect the eggs will be understood and potentially be beneficial in other ways. Understanding this bacteria will be attempted through 16S rRNA gene sequencing. Results will assist in understanding of marine symbiotic organisms and how they function. This will be the first study addressing the microbial population of the Longfin inshore Squid nidamental gland as well as their egg cases.


So far, many efforts have been made in reference to lab training for the experimentation itself. LBS agar plates were created in order to perform 3 way streaks and isolate particular bacteria from the marine symbionts. Squid, as well as jellyfish that were collected through wading, were the subjects of bacteria isolation. The process of isolating the bacteria from the jellyfish was extensive, beginning with euthanization of the jellyfish (through freezing),  macerating it, rinsing it with 3x deionized water and then vortexing it. Through performing a three way streak of this sample, particular colonies of bacteria were able to be isolated and examined individually. Colony PCR was performed following this and the samples were then inserted into gel in order to perform gel electrophoresis. PCR clean up was then undertaken using a Qiagen kit. This is done in order to prepare the DNA samples that were amplified to be sequenced. Twelve different bacteria samples were successfully amplified through PCR and able to be sequenced in the lab so further characterization of the bacterial colonies can occur in the future.  


Comparative NMR Spectra of Metronidazole and Ornidazole

We successfully built structures of metronidazole and ornidazole on Gaussian, both of which we calculated the NMR spectra in chloroform. Additionally, we obtained this data using experimental spectroscopic techniques; this was then compared to the theoretical spectra from Gaussian.

Theoretical proton NMR of metronidazole indicated five different types of hydrogens; Gaussian was unable to differentiate between the hydrogens of the methylene group attached to the imidazole ring, both of which were depicted in two different peaks yet with a degeneracy of one. In comparison, our experimental proton spectra depicted these five peaks more downfield. Gaussian predicted six carbon peaks while experimental NMR resulted in five carbon peaks, three of which were shifted more downfield and two of which were very similar in chemical shifts with Gaussian. The missing peak was the carbon with the nitro group on the imidazole ring.

Gaussian provided a more complex proton NMR for ornidazole, in which the hydrogens on the methyl group and the hydrogen on the hydroxide group were reflected in similar peaks. The hydrogens on the methylene group were also reflected in two peaks. Experimental spectra provided one more hydrogen peak. In contrast to theoretical data, the methyl group was more upfield than the hydroxyl group. Seven different carbons were predicted by Gaussian; experimental spectra indicated some similarities in the chemical shifts of three carbons. The carbon with the nitro attachment on the imidazole ring was reflected in a small peak this time.

With these results we were able to adjust our objectives for the future. Gaussian provided a reasonable theoretical analysis of structural information, and in utilization with experimental spectroscopy, we can determine the reaction mechanism of metronidazole and ornidazole. We want to investigate what the activated complex in the interaction between the drug and the microbe looks like, as well as how it forms. This is particularly an area of interest for ornidazole, which has been verified as more potent than metronidazole because of the smaller amount of drug required to kill a larger number of microbes.