EFFECTS OF GRAPE COMPONENTS ON ORAL BACTERIA AND INFLAMMATION

Summary

Periodontal disease, including gingivitis and more severe periodontitis, is the most common infection-driven inflammatory disease characterized by gingival inflammation. Grapes have been studied for their potential health benefits. Grape and grape products have been reported effective in reducing various inflammatory diseases. Grape components exhibit anti-bacterial capabilities. They are able to inhibit the growth of various bacteria, including some oral bacteria. Grape seed extract (GSE) is also sold as a dietary supplement for its claimed health benefits. Grape extract (GE) is the industrial derivative of whole grapes. Grape extract contains polyphenols and antioxidants such as proanthocyanidin, which are proven to be very powerful in inhibiting the growth of proinflammatory cytokines. The project was based off the question: Do grape components have the ability to inhibit oral bacteria and reduce inflammation? These experiments tested the effects of grape extract, GE (which contains the skin and the seed of the grape, the two most antioxidant rich components of the grape) on bacterial growth and inflammation. To test the inhibition of oral bacteria, a serial dilution and plating of the samples was done. A cell culture and an ELISA experiment was conducted to test for a decrease in proinflammatory cytokines. If there is an increase in the concentration of GE, there will be a decrease in both oral bacteria and proinflammatory cytokines. The results from this project showed that GE is able to inhibit the growth of proinflammatory cytokines and oral bacteria, therefore, grapes may be beneficial to human health and human oral health.

Question / Proposal

Periodontal disease, including gingivitis and more severe periodontitis, is the most common infection-driven inflammatory disease characterized by gingival inflammation (1). Porphyromonas gingivalis (P. gingivalis) is one of the most important etiological pathogens. Grapes have been extensively studied for their potential health benefit, because grapes are high in antioxidants (2). Indeed, grape and grape products have been reported effective in reducing Alzheimer’s disease and some other inflammatory diseases (3). Our previous research showed that grape seed extract can inhibit the growth of oral bacteria. Therefore, it is plausible that grape consumption is beneficial to periodontal disease patients (4). Since grape contains many components, research is needed to illustrate its components on oral health. In my experiment, I will be testing its effects on bacterial growth and inflammation. I formulated the research question:

Are grapes beneficial to human oral health and can they be used to combat Periodontal disease?


I expect that grape extract will inhibit the growth of Porphyromonas gingivalis (P. gingivalis; oral bacteria) and reduce inflammation, therefore making grapes beneficial to oral health.

Research

Grapes have been extensively studied for their potential health benefits and because grapes exhibit antibacterial capabilities, they may be able to inhibit types of oral bacteria, thus, reducing the risk of bacterial diseases. Grape products have also been reported effective in reducing multiple inflammatory diseases.

Individual components of the grape, such as catechin and grape seed extract, have also been proven to exhibit antibacterial capabilities and are found within grape extract (GE). Because grape seed extract is sold as a dietary supplement, and the recommended dosage is 150-300 mg, taking the recommended amounts of grape seed extract over time may prove to be significantly beneficial to human oral health and human health. Studies have shown that because P. gingivalis is implicated in certain forms of periodontal disease, P. gingivalis could be inhibited with GE before the effects of the disease become too harmful, which could be very useful in the treatment and prevention of periodontal disease.

Grape extract contains the catechin, epicatechin, epicatechin gallate, and epigallocatechin found in the stem, fruit, and skin of the grape. Because green tea catechin has the ability to inhibit Porphyromonas gingivalis (P. gingivalis), it is very plausible that grape catechin has the ability to inhibit Porphyromonas gingivalis too, as green tea and grape catechin share similar properties.  

Inflammation is the complex biological response of body tissues to harmful stimuli, such as pathogens, and is also a protective response. Proanthocyanidins (PA) from grapes have an anti-inflammatory effect on experimental inflammation in rats and mice. PA was able to inhibit the production of IL-1 beta and TNF alpha (pro inflammatory cytokines) in the animal model studies (rats and mice). Procyanidin is a potent antioxidant that is found in large quantities in grape extract.

A study conducted by Terra, Montagut, Bustos, Llopiz, Ardèvol, Bladé . . . Blay (2009) found that “rats that were fed a hyperlipidic diet were models of low-grade inflammation as they show an altered cytokine production. In the end, the results suggested a beneficial effect of PE (procyanidin extract) on low-grade inflammatory diseases, which may be associated with the inhibition of the proinflammatory molecules CRP, IL-6 and TNF-α and the enhanced production of the anti-inflammatory cytokine adiponectin.” Procyanidin is found within grape extract, and because this project tests the effects of grape extract (GE) on proinflammatory cytokine TNF-α, then these studies show that there may be a significant amount of proinflammatory cytokine inhibition with the addition of grape extract.  

However, it is still unclear how grapes might affect human health (Ehrlic, 2015). Few trials have looked to see how grapes (specifically the grape as a whole) affect specific diseases or conditions, and little scientific evidence is available.

Method / Testing and Redesign

The project consists of several parts. In order to measure if there is a decrease in oral bacteria growth, there were serial dilutions and platings of the oral bacteria and grape extract (GE). In order to measure the decrease of pro-inflammatory cytokines, there was an ELISA (enzyme-linked immunosorbent assay) experiment conducted.

The serial dilution and plating experiment for GE (grape extract) was conducted by distributing different dosages of GE (0.05, 0.1, 0.2, and 0.4 mg/ml) into separate centrifuge tubes using a pipette. The next step was distributing 5 × 10^5 CFU/ml of P. gingivalis into each of the tubes using a pipette. Then, the tubes were cultured in an anaerobic incubator for 12 hours at 37 degrees Celsius. Once the tubes finished culturing, the serial dilution experiment was conducted. To conduct the serial dilution, there was 1 ml of a sample pipetted and distributed into 9 ml of PBS (diluent). This created a 10^-1 dilution. 1 ml of the 10^-1 diluted sample was distributed using a clean pipette into another tube that contained 9 ml of PBS. This created a 10^-2 dilution. This process was repeated until each dosage of GE had a 10^-5 dilution.  After the samples had been diluted, the next step was to plate and count the bacteria colonies. Using the L-shaped spreaders, each of the samples from the centrifuge tubes were spread onto separate petri dishes (blood-agar). The plates were then cultured anaerobically for seven days (incubated at 37 °C). Then the bacteria colonies were counted to form results. To count the bacteria colonies, each petri dish was split into four parts. Then the bacteria colonies in one part were counted and the number was multiplied by four.  

Because GE was dissolved in DMSO, there was serial dilution and plating for DMSO as well. This was conducted in order to make sure that the DMSO was not affecting the decrease of oral bacteria colonies and that only the GE was causing the number of colonies to decline. Thus, the same methodology was repeated with DMSO using dosages of 0.05, 0.1, 0.2, and 0.4 mg/ml.

The next step of this project was to conduct an experiment that tested to see if GE has the ability to inhibit proinflammatory cytokines. The first part of this experiment was performing a cell culture. The cell culture was performed for GE by adding 100 ng/ml of P. gingivalis LPS into the 96 wells of a cell culture plate. Then, 2 × 10^5 RAW cells (animal cells from a monocytes cell line) were added into the wells. After that, different dosages of GE were added into each well (0.05, 0.1, 0.2, and 0.4 mg/ml). The plate was then cultured in a cell culture incubator for 12 hours at 37°C. Once the cytokines had secreted from the cells into the medium, the top layer was decanted. The supernatants of each concentration of GE were harvested in order to test for the cytokine concentration by using the ELISA method. 

Results

The Grape Extract vs Number of Bacteria Colonies graph shows that GE (grape extract) was able to inhibit the growth of oral bacteria P. gingivalis. The experiment showed that 10^-5 is the optimal dilution for rendering the best countable colonies (which is the dilution that was plated for both the GE and DMSO samples and then counted to obtain the results found on the graph). Because GE was dissolved in DMSO, DMSO was used as the control. The line indicating DMSO cultured bacterial colonies indicated that the number of bacteria colonies resulting from the samples of DMSO and P. gingivalis stayed relatively the same and relatively high no matter what concentration of DMSO was used. However, the line indicating the number of bacteria colonies that resulted from culturing GE with P. gingivalis showed that higher concentrations of GE produced fewer P. gingivalis colonies. This then shows that the inhibition of P. gingivalis was coming from GE alone, not other substances that could have been involved in the process of obtaining the grape extract, and that DMSO wasn't playing a role in the ability of GE to inhibit P. gingivalis.  

According to the ELISA graphs, grape extract is able to inhibit the cytokine TNF-alpha. With an increased concentration of GE, there is a decrease of TNF alpha cytokine. However, the most significant decrease of TNF alpha occured at 10 micrograms/ml. The ELISA graph conveys how grape extract was able to inhibit the production of TNF alpha cytokine, even with a small concentration of just 10 micrograms/ml. However, the control (DMSO) showed little inhibition of TNF alpha cytokine production at the 10 micrograms/ml concentration. This then shows that the inhibition of TNF alpha was coming from GE alone, not other substances that could have been involved in the process of obtaining the grape extract, and that DMSO wasn't playing a role in the ability of GE to inhibit P. gingivalis.  

(In case the slides doesn't work: https://docs.google.com/presentation/d/1f6Ui04uFMDr-O37I3CidkRvyfkRQaVeKFMxiN6-68M8/edit?usp=sharing)

 

 

Conclusion

From this project, I was able to find that grape extract inhibits oral bacterial growth, as well as inflammation, and is therefore beneficial to human oral health. The results shown from my project convey that there was a significant decrease in proinflammatory cytokine TNF-alpha with the addition of grape extract. The results also show a significant decrease in oral bacteria P. gingivalis with the addition of grape extract. Because many of the phytochemicals found within grape are common within other fruits or vegetables, I'd assume that the beneficial properties of grape can also be found with fruits or other plants that have a similar chemical composition as grapes (ex: green tea leaves). My project was relatively successful; due to time constraints, I wasn't able to test all of the variables that I wanted to. If I were to change some of my methodology, I'd consider performing the cell culture simultaneously during the oral bacteria culture. By doing this, I could have saved a lot more time and tested more variables in order to further support my hypothesis. Throughout this process, I became interested in future routes that I could take this project in the future. I'm interested in testing the various phytochemicals found within the grape extract and researching which of the chemicals is most prominent in the inhibition of proinflammatory cytokines and oral bacteria. In order to further test my current hypothesis, I'm also interested in testing grape extract on the human oral bacteria microbiome, not just P. gingivalis. This would give me an idea of how grape extract reacts with the various microbes found within the oral cavity, and not just one specific strain of bacteria. I may also want to expand my current project to encompass the entire human body and test how grape extract reacts with the gut microbiome, as well as the oral. If I were to continue my project in this direction, I would be more focused on how grape extract can improve overall human health, not just human oral health. 

About me

I am a sophomore at duPont Manual High School in Kentucky. My decision to focus my project on the treatment of Peridontal disease by the usage of grapes (more specifically grape extract) was prompted by research showing that Peridontal disease is an extremely prevalent and severe problem in developing countries. People in developing countries are often unable to recieve, the treatment needed to mitigate the effects of Peridontal disease. Peridontal disease, if left untreated, leads to oral cancer, the eighth most common cancer in the world. Around 60-90% of schoolchildren in developing countries are affected by Periodontal disease and if left untreated, will be at risk for severe oral cancer. Winning the Google Science fair is a dream of mine and would allow me to inform others about the impacts of Peridontal disease and make a long lasting impact on global oral health. I would have the opportunity to work with experts in the STEM field and meet and interact with some of the brightest teenagers in the world. 

Outside of school I volunteer with many organizations, including the Red Cross. I am very involved in the STEM activities that my school offers and participate in my school's science olympiad and math teams. My continuous work in the field of oral microbiology inspired my dream to become a oral surgeon when I grow up. One of my many inspirations is Antje Boetius, an amazing female microbiologist whose dedication to her work encouraged me to pursue my own projects in microbiology.

Health & Safety

The experiment was conducted in a BSL-1 lab at the University of Louisville in order to use the bacteria and lab equipment that they have there. Biosafety Level 1 is suitable for work involving well-characterized agents not known to consistently cause disease in immune competent adult humans, and present minimal potential hazard to laboratory personnel and the environment. Work was conducted on open bench tops using standard microbiological practices. Special containment equipment or facility design was not required for this project. Laboratory personnel has specific training in the procedures conducted in the laboratory and was supervised by a scientist with training in microbiology and immunology. Any contaminated materials were disposed of into a biohazard waste basket.

The lab supervisor and mentor was Dr. Huizhi Wang, who can be contacted at huizhi01wang@gmail.com. The lab protocol that was followed during experimentation was based off of the general laboratory guidelines provided by the University of Louisville. They can be accessed here: http://louisville.edu/dehs/ohs/lab-manual-general-safety#General%20Rules%20for%20Laboratory%20Safety. 

Bibliography, references, and acknowledgements

References 

I would like to acknowledge my parents for their constant support throughout my project, as well as my science teachers for always be willing to lend their resources whenever it was required. I would also like to acknowledge the University of Louisville School of Dentistry for providing me with the tools needed to complete my project. Mentor Dr. Huizhi Wang helped me greatly by teaching me the proper protocol for performing an ELISA experiement, as well as serial dilution and plating tecniques. The University of Louisville provided me with a sterile hood, as well as various other equipment including pipettes and blood-agar plates. 

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