Is Grapefruit Essential Oil an Effective Weight Loss Supplement?

By Rachel Layton and Tyler Smart

Abstract

The purpose of our experiment was to test the effectiveness of Grapefruit Essential Oil (GEO) as
a natural weight loss supplement, specifically to see if GEO was more effective if it was taken
preventatively (prior to adipocyte differentiation) or reactively (after adipocyte differentiation). We used
GEO because it has been found to have lipid metabolisers in it which increase the metabolization of fatty
acids in the body reducing the levels of fat. Unfortunately, our cell wells were contaminated, and all of
our cells were killed. This left us with two conclusions: to revise the incubator sterilization procedures so
that contamination doesnt happen again and to be very precise when mixing experimental media so it
doesnt kill the cells.

Introduction

Stem cells are unspecialized cells that are capable of dividing and of turning into specialized cells
such as fat cells (Stem Cell, 2016). Stem cells are great for research because of their ability to reproduce
quickly (Soppler). There are three types of stem cells: embryonic, tissue specific adult, and induced
pluripotent stem cells. Embryonic stem cells are the cells that all cells come from (Watson). They are
pluripotent meaning they can form almost any type of cell (Watson). Tissue specific adult stem cells are
multipotent meaning that they are more limited in their ability to differentiate (Watson). They will
differentiate into a specific type of cell like adipocytes or muscle cells (Watson). Induced pluripotent stem
cells have the qualities of embryonic stem cells but are formed from other bodily cells like skin cells
(Watson). Scientists subject the skin cells to a process that reverts them back to their embryonic stem cell
properties (Watson). This experiment will focus on tissue specific stem cells in the form of fat cells or
adipocytes.
Adipocytes are cells whose primary role is to make and store fat in the body (Medical). These fat
reserves are used as energy and broken down in a process called lipid metabolism (Sanders). Lipid
metabolism weight issues happen when there is a problem with lipid metabolism, preventing the fat to be
metabolized correctly (Sanders). Some weight loss supplements promote lipid metabolism to help the user
lose weight. We will be testing a home remedy type weight loss supplement that is meant to promote lipid
metabolism.
Many people use home remedies for weight loss in place of supplements, as they can be
expensive and hard to obtain (cite). One of the most popular natural supplements is Grapefruit Essential
Oil (GEO) (Babcock 2017). GEO has many uses and benefits, including but not limited to, reducing
depression, cleansing the body, stimulating the immune system, and helping with weight loss (Babcock
2017).
The reason that GEO helps to enhance weight loss is because it contains nootkatone, a compound
that promotes and increases weight loss, partly by activating AMPK (Gamonski 2017). AMP-activated
protein kinase (AMPK) is an enzyme that is present in all mammalian cells (Richter and Ruderman
2009), and serves as the bodys master regulating switch (Robbins 2017). When AMPK is activated it
can reduce storage of body fat and protect against degenerative disorders that are todays leading killers
(Robbins 2017), by triggering the use of stored energy from fats, enhancing removal of fats and sugar
from the blood, increasing production of mitochondria, and reducing inflammation and cellular junk
(Robbins 2017). Calorie restriction and large amounts of exercise can activate AMPK (Robbins 2017), as
well as GEO has been found to activate AMPK, reducing belly fat, cholesterol, blood sugar, and insulin
levels (Robbins 2017).
GEO has a very high concentration, which is why during the experiment we will be using a
diluted version of the oil, so as not to overwhelm our cells. To dilute the oil we will be mixing GEO with
water. However, when water mixes with oil, they naturally separate. When an emulsifier is added to the
system, the droplets remain dispersed, and a stable emulsion is obtained (EFEMA). This is because
emulsifiers contain hydrophilic ends and hydrophobic ends of the compound. The hydrophilic end
connects to water, while the hydrophobic end connects to oil, effectively creating a bond (EFEMA). The
emulsifier being used in this experiment is DMSO. Dimethyl sulfoxide (DMSO) is an organosulfur
compound and a byproduct of the wood industry, and contains both hydrophilic and hydrophobic ends
(Muir 2007).
The purpose of our experiment is to see how Grapefruit Essential Oil (GEO) affects mouse stem
cells that differentiate into adipocytes before and after differentiation. We are specifically looking at how
GEO promotes lipid metabolism in mouse adipocytes suggesting potential weight loss benefits. We
predict that the GEO will be a more effective weight loss supplement if added to adipocytes that have
already been enlarged with fat. We think this because we expect that with more fat in the cells, the more
lipids there will be to metabolize, the more active lipid metabolization will be in the adipocyte cells.

Method

This experiment will test the effectiveness of GEO at preventing adipocytes from growing and
splitting into other cells (meaning that they are not gaining weight). To test this, we will be mixing the
GEO at two concentrations: 0.0011/mL and 0.0112/mL with the FBS medium that is used to feed the
cells. By mixing the GEO with the medium we are hoping that the cells will take up the GEO, preventing
them from gaining weight. We will use the emulsifier DMSO to mix the GEO oil with the water-based
medium. After mixing the GEO with the medium we will feed the adipocytes with the GEO medium for
the duration of the experiment to ensure maximum exposure. We will be documenting the changes in the
cell confluency every few days to observe any signs of weight gain (or lack of weight gain).

Required Personal Protective Equipment(PPE):

Closed toed shoes
Lab Coat
Goggles
Gloves - Ethanol your gloves

Materials
(1) Micropipette
(1) Pipette
- Pipette tips (lots)
(1) Mouse 3T3-L1 Mouse Embryonic Fibroblasts (adipocytes)
(1) Bottle of Grapefruit Essential Oil (12 oz) Amazon Link
(1) Cell Culture Microscope
- Stem Cell Medium (90% Dulbeccos Modified Eagles Medium, 10% Bovie Calf Serum, 100x Pen
Strep
- Differentiation Medium
(1) Hemocytometer
(1) Ruler (Millimeters)
- Flasks

Independent Variable: Grapefruit Essential Oil

Dependent Variable: How much the adipocyte cells grow/divide while in the GRO
Control Variables: Testing method, amount of supplemental doses, temperature, exposure to fatty acids
Confounding Variable: Not enough DMSO was used, making it difficult to see the cells and potentially
keeping the oil from mixing fully with the cells.

Data Collection:
Quantitative Measure: Number of cells and size of cells
Qualitative Measure: Cell morphology by taking pictures and making observations

We chose these collection measures because we felt that they would accurately show weight gain/loss. By
recording the number of cells and the size of cells we can tell at what rate the cells are dividing and
growing. By collecting photos of our cells we can visually compare stages in growth to see what the
effects of the GEO is. Both methods of data collection will be taken every 2-3 days when we feed the
cells.

Safety:
SDS for Grapefruit Essential Oil - link

Method
1. Serial Dilutions:

Dilution 1:
1. Get two test tubes. Fill one with 4.5 mL of solvent and fill one with 4.328 mL of
solvent
2. Take 0.672 mL of GEO and put it in the 4.328 mL of solvent
3. Take 0.5 mL of the 0.672 mL solute solution and put it into the 4.5 mL solvent
tube. This will give you a solution concentration of 0.0112 ml/ml

Dilution 2:
1. Get three test tubes. Fill two with 4.5 mL of solvent and one with 4.328 mL of
solvent
2. Take 0.672 of GEO and put it in the 4.328 mL of solvent
 3. Take 0.5 mL of the 0.672 mL solute solution and put it into the first 4.5 mL
solvent tube
4. Take 0.5 mL from the diluted solution and put it into the second 4.5 mL solvent
tube. This will give you the desired solution amount of 0.0011

Procedure
1) Add dose of 0.0112 mL/mL of GEO using a micropipette (one serial dilution
from 0.672 mL concentration) to adipocyte cells that have not differentiated yet;
2) Count cells before, after 24 hours, after 48 hours; measure the size before, after
24 hours, after 48 hours, etc.
3) Photograph before, after 24 hours, after 48 hours; etc.
4) Add dose of 0.0112 mL/mL (one serial dilution from 0.672 mL concentration) to
adipocyte cells that have differentiated
5) Repeat steps 2-3 for the differentiated cells
6) Repeat steps 1-5 for the 0.0011 solution

2. Splitting The Cells:

Safety
Biohazard Level 1- All procedures involving open cell containers must be done under fume hood.
Bleach bottle must be tightly closed before putting Ethanol into fume hood. The reaction between
Ethanol and Bleach creates poisonous Chlorine Gas. Immediately close bleach container and
dispose of all pipette tips and chemically contaminated materials such as kimwipes and paper
towels in the biohazard bin.

Required Personal Protective Equipment (PPE)

Closed toed shoes
Lab Coat
Goggles
Gloves - Ethanol your gloves

Protocol
1. Remove and discard all media
2. Add 2.0-3.0 mL of Trypsin- EDTA solution to flask and observe cells under an
inverted microscope until the cell layer is dispersed (5-10 minutes).
NOTE: To avoid clumping do not agitate the cells by hitting or shaking the flask while
waiting for the cells to detach. Cells that are difficult to detach may be placed at 37 C to facilitate
dispersal.
3. Add 5 mL of complete growth medium to deactivate the trypsin and aspirate cells
by gently pipetting.
4. Transfer media and free cells to centrifuge tube.
5. Spin @ 1000 rpm for 6 minutes.
 6. Remove supernatant, taking care not to disturb the cells at the bottom of the
plate.
7. Add new media to centrifuge tube, resuspending cells to allow them to mix
appropriately.
8. Add appropriate aliquots of the cell suspension to new culture vessels(diagram
shown below).
9. Incubate cultures at 37 C.

Do not prematurely add media to incubator because it cannot be in the incubator for a long time.

Figure 1. Rendering of well plate.

3. Cell Feeding:

Safety
Biohazard Level 1- All procedures involving open cell containers must be done under fume hood.
Bleach bottle must be tightly closed before putting Ethanol into fume hood. The reaction between
Ethanol and Bleach creates poisonous Chlorine Gas. Immediately close bleach container and
dispose of all pipette tips and chemically contaminated materials such as kimwipes and paper
towels in the biohazard bin.

Required Personal Protective Equipment (PPE)

Closed toed shoes
Lab Coat
Goggles
Gloves

Protocol
Feed cells by replacing media every 2-3 days or when media in flasks comes tinted yellow in
color.

Wash hands and don PPEs.

You are required to ethanol gloves, micropipette & tips, hood, microscope, your
workspace and anything else you will be using.
 Place bottle of media in incubator for 18 minutes
Ethanol bottle of media before placing inside of the hood.
When opening the micropipette tip, make sure the side with the measurements is
facing you so you can read it. Make sure to not touch the tip or anywhere where the tip
will touch the substance.

Figure 2. Example of feeding cells.

Carefully tilt wells so media gathers at the bottom. Extract as much media as
possible until there is a thin layer of media in the well and make sure not to suck up any
cells.

Figure 3. Pipetting into a flask.

Open the 70% bleach and pour in the old media. Make sure not to touch the pipette tip
with anything and to close the cap when youre done.
Slide the pipette tip back into its package and dispose it into the biohazard bin.
Attach a new pipette tip to the pipette.
Open the 0.0112/mL media tube. Extract 2 mL from the media and insert it into the cells
by gently dropping the media back in. Minimize shaking up the cells.
Slide the pipette tip back into its package and throw it away into the biohazard bin.
 Repeat media extraction and 0.0112/mL media feeding for the appropriate well.
Once done with the 0.0112/mL media, repeat the same feeding steps for the 0.0011/mL
media
Slide the pipette tip back into its package and throw it away into the biohazard bin.
Check to see if the cells are still there by putting the wells under the microscope.
Put the cells back into the incubator.
Return media bottle to fridge
Ethanol gloves, hood, microscope, your workspace and anything else you used
Take off PPEs & be mindful of removing gloves without touching them.
Wash hands

Results
Our experiment used the Adipocyte cell line, undifferentiated with the hopes that the cells would
differentiate over time. We tested the effects of grapefruit essential oil on fat cells to see if it would have
any effect on their size. Our independent variable was the grapefruit essential oil, our dependent variable
was originally the size of the cells but evolved into the confluency of the cells. Our control variables
include our testing method, the amount of supplemental doses, temperature, and exposure to fatty acids.
Our confounding variable was that our wells became contaminated with mold, which could have been
caused by a few different things. We were only able to do one round of inconclusive trials over the course
of one week because all of our cells died due to contamination. Our data is inconclusive because our cells
died.

Our graphs show the change in confluency over the week that we observed our cells before they
were killed. This was the only kind of data that we were able to gather because we had only just added
our experimental media to our wells before they were killed. As you can see, the cells were becoming
more confluent just before they were killed. Also, all graphs look the same because they grew at the same
rate and died at the same time.

Figure 4. Average confluency Figure 5. Average confluency Figure 6. Average confluency

graph for one control group. graph for one control group. graph for well with 0.0112 mL
concentration.
 Figure 7. Average confluency Figure 8. Average confluency Figure 9. Average confluency
graph for well with 0.0112 mL graph for well with 0.0011 mL graph for well with 0.0011 mL
concentration. concentration. concentration.

Photographs

Figure 10. Control before differentiation. In the background are dead

cells, and in the foreground is contamination (mold).

Figure 11. Photo of one of our wells after we have fed it our experiential
media. It is obvious that the radius of DMSO to GEO is uneven, which is
why there are large bubbles in another plane from the cells.

Because all six of our wells grew at the same rate and then died at the
same time, all of our graphs look identical. We were unable to compare data because of this. We were
also unable to analyze our data very much because of the contamination. This could have been caused by
a few different things. For one, it could have been that our media containing the grapefruit essential oil
killed the cells, however this seems unlikely because our control groups also died (as a disclaimer, none
of our cells differentiated, all of the cells we tested on were undifferentiated). We believe that the
contamination occurred because our well plate was left open in the incubator for an unknown extended
amount of time. We were unable to gather any conclusive data regarding our experimental question due to
this contamination.

Conclusion
 As weve stated before, the purpose of our experiment was to test the effectiveness of GEO as a
natural weightloss supplement when used as a preventative and reactive supplement. We had expected the
reactive supplement to be the most effective because it would increase lipid metabolism. Unfortunately,
our results were inclusive. We found this because our wells experienced contamination in the second
week of data collection. The contamination came in the form of mold, however there were also dead cells
in the surrounding wells. There are a few different things that could have caused the contamination. For
one,the grapefruit essential oil could have killed our cells. If this was the case, it could have been because
the ratio of GEO to DMSO was not equal. The contamination also could have been caused by the well
plate being open inside the incubator. As mentioned before, we are unsure where the contaminating
organisms came from and how they were introduced to our experimental wells. We were told that our
wells were left uncovered in the incubator for a period of time. There is a good chance that this is when
the contaminants were able to get into the wells. However, there is a possibility that the contaminants
were in the GEO/DMSO mixture and were introduced to the cells when the GEO/DMSO media was
added to the wells. The contamination happened directly after both of these events (the GEO/DMSO
media was added just before the well was left exposed) so were unsure which one caused the
contamination. We suggest for future experiments that extra caution needs to be taken with regards to
sterilization practices so that cells arent contaminated. We also suggest that when measuring mediums
the concentrations are correct. Our results matter because they show that contamination is very possible in
a lab setting even when you follow sterile procedures. It shows that for experiments to be successful the
protocol needs to be strictly followed. Also, extra sterilization procedures probably need to be added to
the incubator maintenance protocol because there is a chance that our wells were contaminated in the
incubator.

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