To what extent does the antimicrobial property of Cinnamon (Cinnamomum verum) work like antibiotics in order to reduce the growth of E.coli bacteria?

Introduction-

Before modern medicine, herbs and spices were frequently used and popularized around the world due to their natural antimicrobial properties used not only as antioxidants and flavorings, but for the treatment of various infectious diseases caused by food spoilage bacteria, such as E.coli. The practice of traditional medicine is highly demanded until now causing an increase in the amount of attention given to the potential of cinnamon (Cinnamomum) antibacterial characteristics to become an alternate source of antimicrobial activity reduction.

According to the World Health Organization, infections are responsible for one-third of all deaths. This indicates to us that the amount of exposure to infection is incredibly high and therefore the consumption of antibiotics has increased dramatically. The high exposure to antibiotics has created for many microorganisms a type of resistance, in which the antibiotic does not respond effectively to drugs. Consequently, the option of developing and analyzing the antimicrobial agents of Cinnamomum, can potentially lead to the use of its natural properties to act against the resistant microorganisms. The aim of this essay is to analyze how the antimicrobial properties of Cinnamon (Cinnamomum) can work like antibiotics in order to reduce the growth of E.coli bacteria.

Figure 1, chemical composition of Cinnamomum verum (Pubchem)

Cinnamon has traditionally been used not only in the culinary world, but in the medical too. It has been known for its oral health benefits, acne and melasma treatments, antioxidant, and anti-inflammatory properties. It’s also a source of tissue regeneration due to how it can increase blood flow. Most importantly, it possesses antibacterial, antifungal, larvicidal, nematicidal, insecticidal properties, which contribute to a greater extent to the idea of treating bacterial and infectious diseases. It’s important to acknowledge how the chemical composition of Cinnamomum allows its antimicrobial properties to carry out. Cinamon most importantly contains cinnamaldehyde.

Figure 2, chemical composition of cinnamaldehyde (Sigmaaldrich)

Cinnamaldehyde is an organic compound that gives cinnamon its flavor and odor, and carries the antimicrobial agent by inhibiting the bacterial growth. Cinnamon contains various essential oils, such as cinnamic acid, and cinnamate. However, cinnamaldehyde is the major component of cinnamon, taking up a concentration of 85%- 98% of the essential oil and its high concentration of 1.25-2.5 mg/ml allows the inhibition to be carried out much greater (International Journal of Pharmaceutical Sciences and Research).

Moreover, in order to test the actual antimicrobial properties of cinnamon as a whole, I decided to test it by inhibiting it with Escherichia coli ATCC (E.coli) which is sensitive to any type of inhibition. This specific type of bacteria is non pathogenic, meaning that it is not specialized enough to enable certain microorganisms to replicate and damage host cells.

Throughout this process it’s crucial to work with this specific type of bacteria as it can’t inhibit the growth and replication of dangerous microorganisms. Furtheron, this bacteria normally lives in the intestines of humans and animals, and it can be transmitted through contaminated water, food, or raw vegetables and meat. Although many E.coli bacteria are non virulent, the inhibition of bacterial growth is necessary to prevent such infection. Through the use of cinnamaldehyde found in cinnamon, E.coli can be inhibited and reduced in the frequency in which it reproduces.

On the other hand, E.coli is typically treated with the use of antibiotics. Antibiotics work by targeting prokaryotic cells, rather than eukaryotic cells. This is due to how they attack characteristics that eukaryotic cells don’t have, such as cell walls, naked strands of DNA, and the way they build proteins and copy DNA. Different antibiotics kill different aspects of the cell, depending on the chemical composition of it that is specific to the bacteria. However, it’s important to take into consideration that the use of antibiotics although it is the most common treatment, it’s not necessarily the most effective way of inhibiting all E.coli infections. For some types of E.coli like O157:H7, the strong chemicals and drugs used in antibiotics can shorten the length of symptoms, but not the actual reduction of the bacteria. The high concentration of

antibiotics, can increase the production of Shiga toxin ¹ , which will worsen the symptoms by

causing severe diarrhea with the presence of blood and mucus in the feces in humans. (WebMd). Therefore, the tendency to opt for less chemically concentrated remedies, such as cinnamon can be more effective in reducing the reproduction of E.coli, and the symptoms that come along with it.

Experiment-

The aim of this investigation is to evaluate how different concentrations of Cinnamomum Verum, can act like antibiotics in the reduction of E.coli ACCT growth in a petri dish. Typically, we can assume that higher concentrations of cinnamon will imply higher concentrations of cinnamic acid, cinnamaldehyde and cinnamate which are the most effective inhibitors for the bacteria. Therefore, if there is an increase in the concentration of powdered cinnamon, then the E.coli growth will decrease substantially.

Variables-

As for our independent variable, the concentration is going to increase by 0.01 grams each trial. Creating in total 5 different concentrations (0.01g, 0.02g, 0.03g, 0.04g, 0.05g). Higher concentrations imply more concentrations of cinnamic acid, cinnamaldehyde and cinnamate. A much stronger concentration is able to more efficiently reduce the reproducion, due to how the antimicrobial properties overpower the actual colonie of the bacteria, causing those properties to bind much stronger to them. The reason behind opting to choose small varying quantities is to gradually watch the power of cinnamic acid, cinnamaldehyde and cinnamate on small scaled E.coli colonies.

Moreover, our dependent variable was the actual measurement of the percentage coverage/growth of the E.coli bacteria on a petri dish. The purpose of calculating the percent coverage of the bacteria is to see how the different concentrations impact how quickly the bacteria reproduces and consequently the amount of coverage it has on the petri dish.

However, in order to get as accurate as possible, there are a few variables that should remain constant throughout the experiment. To begin, the same E.coli ACCT should be used for all trials. This specific type of E.coli is sensitive and responsive to any type of inhibition or antimicrobial properties. The use of the same bacteria is meant so that it has equal characteristics, as well as the level of sensitivity and reproductive rate. If we were to use a more aggressive type of E.coli for different trials, for example O157:H7 that produces Shiga toxins, then we wouldn’t have accurate measurements. This is due to how the concentration of the cinnamon might be enough for one type, but not sufficient for the other. Secondly, the same concentration of 1×10 ⁵ should be used by giving an equal chance for the different

concentrations to inhibit the bacteria. Consequently, in order to cultivate the bacteria all should be incubated for 20 minutes ( ± 0.01s) , at a constant temperature of 23 ° C (± 1.5 ° C ) for all

trials. This assured that there was no over concentration and reproduction of the bacteria, we

measured this also through the use of the Oxoid Turbidimeter ² . It’s important also that for

incubating the bacteria, and later on the petri dish should remain a constant temperature of 23 ° C ( ± 1.5 ° C ). This bacteria typically needs a neutrophilic environment ³ If the incubator is overly

hot, the bacteria might not survive, while on the other hand if it’s too cold, the bacteria will solely not reproduce. Moreover, the harvested E.coli should be kept incubated for a period of 24 hours, due to how it’s constantly reproducing as time advances. By setting a time limit for each trial, it allows an approximate equal chance to grow. If the bacteria were placed for longer or shorter amounts of time, it wouldn’t let the cinnamon inhibit properly, as well as it might have spread significantly more or less than expected. Lastly, the same cinnamon powder should be used. A

common market-based product, meaning that is approachable by the normal consumer, may range ingredients. Hence, it’s important to keep the same brand for all trials.

Materials-

• 1 petri dish with multiple colonies of E.coli ACCT

• 7 tests tubes with 1.5 mL of solution

• Powdered cinnamon (natural)

• 25 agar plates (petridish)

• Cotton buds

• Black permanent marker

• Test tube rack

• Incubator at 23 ° C ( ± 5 ° C )

• McFarland solution

• Oxoid turbidimeter

• Pipetes

• Electronic balance ( ± 0005g)

• Aluminium Foil

• Spoon

• T1-84 calculator

• Timer ( ± 001s)

• Gloves

• White light

Procedure for experiment-

1. Gather all materials, make sure that E.coli ATCC is unfreezed and at room temperature ( 23 ° C − 27 ° C )
2. Measure 1.5mL of solution into a test tube

3. With a cotton bud tap it lightly onto the E.coli, picking a small quantity up

4. Place the cotton bud containing the bacteria onto the 1.5mL solution and mix it together

5. Label the test tube according to the trial number

6. Place the test tube into a tube rack, and incubate it for 20 minutes at a constant temperature of 23 ° C ± 1.5 ° C
7. Meanwhile gather an agar plate and label it correspondingly to the trial number

8. Using a molecular balance that is able to measure until 4 decimal places (in grams), measure 0.01g of powder cinnamon
9. After 20 minutes remove the test tubes from incubator

10. Using an Oxoid turbidimeter, place the McFarland solution to calibrate it, the machine should give you a response of “=”
    1. McFarland solution used as a concentration base of 1x10⁵

1. McFarland solutions needs to be kept in the dark, the darker the environment the more concentrated it is

11. Place the test tube onto the Oxoid turbidimeter to measure the concentration of the coli bacteria. The response should be “=”
    1. If the response is ” ≫ ” , grab some uninfected solution with a pipette, and dilute

it onto the test tube and remeasure it until it gives an equal response

1. If the response is ” ≪ ” grab a small quantity of the E.coli onto a cotton bud a

mix it in until it gives it an equal response

12. Place a cotton bud into the test tube, letting the bacteria soak in it.

13. Cultivate it into all petri dishes by applying a thin equally distributed layer of the bacteria solution over the agar
    1. Label de disk depending on the trial

14. Place the 0.01g of cinnamon onto the center of the petri dish, trying to keep a uniform
    1. It’s important to acknowledge that from the time you placed the bacteria onto the agar, you have an optimum time of 15 minutes to place the cinnamon onto the petri dish
15. Put the lid back on the petri dish, and place the disks onto the incubator once again at a constant temperature of 23 ° C ± 1.5 ° C . Leave it incubated for 24
16. Repeat steps 12-15, 4 more times for 0.01grams of cinnamon

17. Overall repeat steps 2-16, to its correspondent cinnamon concentration

Method for calculating the percentage coverage of the bacteria-

1. Measure the area of the petri dish

2. Once a time span of 24 hours had occurred, take the petri dishes out of the incubator

3. Using a light, place one disk in front of it, where it can clearly outline the bacteria spread

4. Using a whiteboard marker shade the area in which the bacteria has spread

5. Place a transparent grid sheet on top of it and outline the spreading of the bacteria

6. Count how many full squares it covers and estimate the ones that are not fully shaded in

7. Add the total number together

8. Divide it by the area of the petri dish and multiply it by 100

9. Repeat steps 2-8 for each disk

Safety and ethical considerations-

• For this experiment, all IB ethical guidelines for lab studies were followed as stated in the IB curriculum.

It’s important to acknowledge the ethical and safety considerations behind this experiment. Most importantly, the bacteria treated with (E.coli ACCT), should not be virulent ⁴ ,

and should not have a high degree of pathogenicity. However, despite the bacteria being non harmful to an individual, safety precautions should be taken at all costs. Beginning with the use of gloves and an organized and sterilized environment. The use of gloves will protect your skin from contaminating itself from the bacteria, and can prevent any form of spreading it to the body’s most delicate areas, such as the eyes and mouth. The experiment should solely be done inside a lab that has a sterilized environment to prevent the outbreak and spread of the bacteria outside the permitted space. This will prevent it from spreading to unwanted places and objects, and the bacteria can be concentrated into soley one space, which in case of an emergency, it can be treated properly. It’s also important to acknowledge that the bacteria should not be incubated at temperatures higher than 30 ° C , as the cell growth rate of enzyme activity increases in

response to increasing temperature, and overheating will cause the bacteria colonies to become

much more concentrated.  Once the experiment was completed, the petri dishes containing the

E.coli colonies were disposed using an autoclave ⁵ . This device sterilizes materials through the

use of pressure and steam, which is programmed to reach and maintain a temperature that is too high for any microorganisms to live.

Uncertainty of the tools used

Raw Data- All data rounded to 3 significant figures

Table 1

Table 2

Table 3

Table 4

Table 4

Processed Data-

Figure 3

Margin of error =                       n = sample size σ = population standard deviation z = z-score

Σ Standard deviation: 0.2828

95% desired confidence level

Margin of error= 1.96 ×

.2828

√25

=0.05656 x 100 (to find percentage)= 11.01%

Evaluation-

Throughout the experiment we could see a gradual decrease of the E.coli growth and replication as we increased the concentration, which proves our hypothesis correct. The difference from our initial trial of 0.01 grams of cinnamon, compared to trial 5 which was 0.05 grams, decreased by 28.4%. The antimicrobial properties of cinnamomum verum were sufficient to decrease the growth of the bacteria, however it was ineffective in the sense of eliminating it overall and creating a halo of inhibition. Much rather, we saw how the bacteria spread out considerably less from the center of the petri dish where the powdered cinnamon was located. In figure 3, we can observe a decreasing trend in the porcentaje coverage as the cinnamon concentration increases. However, we can also observe how the difference in percentage coverage became smaller when it reached 0.3g. The difference from 0.3g to 0.4g was 1.7%, which started to indicate to us that the E.coli has started to reach its optimum concentration. This refers to the idea that after a certain point the concentration of powdered cinnamon will no longer have an effect in the inhibition of E.coli. There are various weaknesses and strengths that we have to take into consideration about our procedure in order to understand the uncertainty of our results. When taking the percentage coverage of the bacteria, the coloring of the grid spaces was an approximation. Making the percentage coverage has a 95% confidence with 11.01% room for

error. Although the error still makes the data reliable to some extent reliable, the lack of calculations on the exact area covered when the grid square was not covered complete may have altered the results by overestimating the actual coverage of the bacteria, and hence altering the trend of the graph.

Conclusion-

The behavior of the E.coli bacteria to the exposure of cinnamon allowed us to understand certain key characteristics of the bacteria and cinnamon itself. First, we can deduce that cinnamon contains a type of bacteriostatic agent. This biological agent stops bacteria from reproducing, while not necessarily killing them. On the other hand, the use of cinnamaldehyde extract or oil, has a richer concentration of antioxidants and antimicrobial activities. Since this extract is going to be less processed, and altered for the consumption of it, it will demonstrate higher activity than the powder. Nevertheless, the oil would still act bacteriostatic, causing that if converted into a treatment, the use should be constant and sufficient to allow host defense mechanisms to eradicate the bacteria. Regardless, upon the removal of the treatment the bacteria may typically regrow. By considering the grand scale of the human body, if the individual was affected by the E.coli bacteria, the concentration of such would need to be significantly higher and may cause the immune system to become ill and therefore not produce enough antigens and antibodies to fight off the infection. Thus, the concentration of powdered cinnamon would need to be consumed to extremely large quantities in order to reduce the growth, which would be highly ineffective as a way of treatment.

When we compare this to the use of antibiotics used to treat E.coli contain bactericides agents. This in contrast actually eradicates the bacteria completely by interfering with either the formation of the bacterial cell wall or its cell contents, creating a limited chance for it to regrow again once the treatment is suspended. These bacteriacide agents come in antibiotics due to the amount of different chemicals used to create such. Solely using cinnamon, or the cinnamaldehyde oil, does not contain these agents, and therefore for it to function at the same scale of antibiotics, it should be chemically infused or mixed into a solution with different chemicals to create such an effect. Realistically, in the short run the use of alternative medicine containing cinnamon or cinnamaldehyde extracts to treat E.coli, can be used for early diagnostic of the bacteria. Meaning, that it can be effective in reducing the growth of the bacteria in its initial stages, and it can also be used to treat non virulent bacteria that take a long time to reproduce. In that case, the use of cinnamon is economically beneficial for patients that don’t have the resources available in the short run. However, it’s important to keep in mind that this is a very limited and quick treatment. Inevitably, the use of antibiotics is going to be necessary to not only treat and relieve the symptoms that the bacteria created, but to also prevent it from damaging the immune system in the long run.

Further areas of study and applications-

In either case, the use of technological and microbiological advancements can lead that in the long run the use of cinnamaldehyde extract can be infused with already existing chemicals in the treatment of E.coli. It’s impact is the following but not limited to, reducing the presence of

uneseary strong chemicals and increasing the presence of natural properties. The natural properties are much more gentle to the patient, and may cause less discomfort when it comes to treatment. Most notoriously, the use of a natural extract can reduce the existing antimicrobial resistance. This resistance may come upon the over and misuse of antibiotics, which is caused by the growing number of bacterial infections that are becoming resistant to medications. This causes the bacteria to become less effective as it has had the chance to improve its defenses towards the antibiotic properties. In this case, the concentrated combination of a natural extract in this case cinnamon will be “new” to this resistant bacteria, and it will take a longer time to create a resting agent. Overall, increasing the effectiveness of this new antibiotic.

APPENDIX

¹ A bacterial toxin that can be caused by the presence of E.coli, which may cause bloody

diarrhea and abdominal cramps.

² Oxoid Turbidimeter is a machine used to measure the concentration of bacteria. This uses the McFarland solution as a base concentration.

³ A state in which the environment has a neutral pH (6.5-7.5), and relies upon a temperature of 18 ° C to 38 ° C

⁴ Pathogenicity is defined as the ability of an organism to cause diseases, virulence is referred

to the degree of pathology caused by the organism (tulane)

⁵ The autoclave was made available to me through Laboratorios Zurita&Zurita

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