22 minute read
The effect of Artemisia absinthium on Staphylococcus aureus by comparing the zone of inhibition between 10µl and 15µl of Artemisia absinthium
Sherab Pelmo
Armidale Secondary College
Abstract
In this world, antibiotic resistance is one of the most concerning issues. Every year, bacteria are growing resistant towards antibiotics, making it difficult to treat diseases. Given that antibiotics are losing their ability to treat diseases caused by resistant bacteria like Staphylococcus aureus, this leaves medicinal plants such as A.Absinthium with antimicrobial activity as potential solutions in treating those diseases. This research aims to test the effectiveness of A.Absinthium on S. aureus by comparing the potency between 10µl and 15µl of A.Absinthium through its Zone of Inhibition (ZOI)1In this investigation, an active bacterial culture was prepared first before carrying out the disk diffusion assay. During the disk diffusion assay, active S.aureus culture from the broth was added onto 3 nutrient agar plates and was divided into three sections; ethanol, 10µl and 15µl. Following that a disk was placed in the centre of those three sections for all the plates. Then using a microcalibrated pipette, 5µl of ethanol, 10µl and and 15µl of A.Absinthium was added to their respective sections and the agar plates were kept in the incubator under 37℃ for 24hrs. The next day, the radius of the ZOI for all the independent variables was measured and the ZOI 1 was determined. After that, the result was noted in a spreadsheet and the t-test was performed between (ethanol vs 10µl ) and (ethanol vs 15µl) of A.Absinthium using the Analysis tool pack. In addition to that, to further support the findings, Pearson’s correlation coefficient (R^2) was also obtained. The p-value (0.24 ) and R^2 (-0.65) for ethanol vs. 10µl shows that there is no significance between the ethanol and 10µl which denotes that 10µl of A.Absinthium was slightly more effective than ethanol. Contrary to that, the p-value ( 0.029) and R^2 (0.75) of ethanol vs. 15µl implies that there is a significance between ethanol vs. 15µl affirming that 15µl was more successful in destroying the bacteria compared to ethanol. This experiment enlightens us with the knowledge that A.Absinthium holds antimicrobial properties due to its biological constituents such as camphor and thujone whereby its efficiency is directly proportional to the amount of A.Absinthium.
1 Zone of inhibition (ZOI) is the clear circular area around the circular disks where no bacterias are able to grow or are extirpated.
Literature review
Over the last century bacterial resistance against antibiotics has exponentially increased (AK Journals, 2022), becoming a concerning and serious issue in the medical field across the world. Every year, the number of antibiotics produced by pharmaceutical companies decreases, leaving fewer options to treat diseases caused by bacteria such as tuberculosis, pneumonia and gonorrhea (World Health Organisation, 2022). Consequently, synthetic drugs are becoming less effective in inhibiting the growth of bacteria. Research states that almost 25 to 50% of current pharmaceuticals are derived from plants, none of which are used as antimicrobials (National Library of Medicine, 2015). Some medicinal plants are rich in a wide variety of chemical compounds and are found to have antimicrobial activities (National Library of Medicine, 2021), leaving herbal medicines as potential candidates for treating diseases caused by bacteria (National Library of Medicine, n.d). Out of all medicinal plants, A. Absinthium is considered to be highly effective, as research on Artemisia implies that the Artemisia genus exerts significant antimicrobial effects, and constitutes antibacterial compounds (Janz, J, Shayya, N, Bereswill, S and Heimesaat. M, 2022,). This makes A.Absinthium, a promising candidate to treat diseases caused by bacteria that are resistant to antibiotics. Out of all the bacteria, S.aureus is recognised as one of the most consequential bacteria in causing human diseases. S. aureus is a Grampositive 2 bacteria that resides on animal skin and is a leading cause of skin and tissue diseases (APIC, n.d). Studies have shown that an increasing number of people with skin diseases caused by Staphylococcus aureus are resistant to many antibiotics (Minnesota Department of Health, 2022). Given that A. Absinthium holds strong antimicrobial properties, this experiment aims to test the effect of A. Absinthium on the growth of S.aureus.
The article Antimicrobial Activity of Artemisia Absinthium Against Surgical Wounds Infected by Staphylococcus aureus in a Rat Model (Moselmi, Hoseinzadhe, Badouei, Kafshdouzan, Fard, 2012) focuses on the effectiveness of Artemisia Absinthium, especially against Gram-positive pathogens. The study was carried out in vivo to test the antibacterial components of the plant where it aimed to investigate the antimicrobial effect of A. absinthium against surgical wounds infected by S.aureus in a rat. For the experiment, small circular incisions were made on the rats and 1* 10^4 CFU 3 (Colony forming unit) Staphylococcus aureus was applied to the wound site (Moselmi, 2012). The extract of A.Absinthium was applied twice a day on the site throughout the experiment. The results acquired from the research present that A. Absinthium holds strong antimicrobial activity, especially against Gram-positive bacteria in which the topical application of A. Absinthium extract at the infected wounded site on the rat shows significant antibacterial activity against S. aureus where the bacterial count decreased by 95.7%. The reason for its effectiveness against the bacteria has been suggested to be due to the major components, camphor (14.83%), p-cymene (14.83%) and caryophyllene (6.92%), These composites of the plant have been proven to exhibit potent antibacterial properties against S.aureus where many researchers have reported camphor as the major constituents of a. Absinthium which holds strong antibacterial activity against Gram-positive bacteria (K.Khosravi, 2022).
2 Gram positive bacterias are bacterias that contain a thick cell wall.
3 The Colony Forming Unit (CFU) is a unit which estimates the number of viable microbes in a sample that are able to multiple under stable conditions.
The article, Absinthe against multi-drugresistant bacterial pathogens? (J.Janz, N. Shayya, S.Bereswill, M. Heimesaat, 2022) reinforce the antibacterial effects of specific Aretemsia species; A. Absinthium and Artemisia Annua, implying that biologically active agents like Artemisinin in Artemisia, are potent bacterial agents that enhance the antibiotic susceptibility of resistant bacteria. The article was a secondary research whereby it surveyed the research carried out over the past ten years on the antibacterial effects of distinct Artemisia species. The survey aims to portray the effectiveness of the compounds in Artemisia where the agents can restore the efficiency of the antibiotics against MDR bacteria. The findings obtained from the survey of other literature show that ethanol extracts of A.Absinthium showed promising antibacterial activity, especially against Gram-positive bacteria such as S. aureus whereby the extract of A. absinthium resulted in at least 50% bacterial growth inhibition. The study also discovered that the specific extract of A. Absinthium such as chloroform did not exert any significant antimicrobial effects on E.coli, Enterococcus faecalis and Bacillus cereus (Moselmi, Hoseinzadhe, Badouei, Kashdouzan, Fard, 2012). However, when combined with other chemical components such as berberine, the growth of the bacteria could be successfully inhibited.
Thus, the findings prove that A. Absinthium is a potential candidate that can be used to treat diseases caused by S.ureus due to its potent chemical composition.
Although researchers have investigated the antimicrobial constituents of A. Absinthium and its effectiveness on S.aureus, there is a paucity of research that explores the relationship between the amount of A. Absinthium and its effectiveness against S.aureus. This gap in the research has led to the conducting of this research. Therefore this study aims to explore the effectiveness of A. Absinthium on S.aureus and how its amount influences the effectiveness against the pathogen.
Scientific research question
To test the effectiveness of A.Absinthium on S.aureus by comparing the Zone of inhibition between 10µl and 15µl of A.Absinthiumoil.
Alternate Hypothesis: 15µl A. Absinthium oil will have a greater Zone of Inhibition against S.aureus than 10µl of the A.Absinthium.
Null Hypothesis: There will be no difference between 15µl and 10µl of the A.Absinthium on the Zone Of Inhibition of S.aureus.
Methodology
Contamination and safety
In this investigation, before carrying out the experiment, the whole station was disinfected with ethanol to limit contamination, hands were washed with soap for 20 seconds and gloves were worn at all times. Throughout the experiment, the bunsen burner was turned on to sterilise the equipment and to prevent the contamination of the culture within. In addition to that, in between the experiments, gloves were disinfected with ethanol. Safety glasses, lab coats and gloves were worn at all times.
Preparation of the active bacterial culture
To grow the active bacterial culture, S.aureus culture, an inoculating loop and luria broth were used. First of all, the inoculating loop was sterilised by being heated in the bunsen for 3 minutes. After that the colonies from the S.aureus culture in the Petri dish were collected using the inoculating loop. Then the luria broth container was tilted and placed near the bunsen flame where the colonies collected were rubbed on the side of the container so that once it was upright the colonies get soaked into the broth. This process was carried out near the heated bunsen burner to limit the contamination of broth and the materials. Once the colony was rubbed, the inoculating loop was heated again before placing it down. Then the luria broth container was enclosed tightly and was placed inside an incubator for 24 hrs ready to be used in the disk diffusion assay.
Disk Diffusion Assay
1ml of the active S.aureus culture from the broth was added onto the petri plate using a 1ml pipette and was carefully spread onto a solid nutrient agar petri plate with a sterile spreader. Following that, the active bacterial culture was left to dry for 5 minutes. Once the bacterial culture had dried, the plates were divided into 3 sections; ethanol (control) and 10 µl and 15µl of A. Absinthium. Each section was marked using a permanent marker. Then, for all of the petri plates, three sterile filter paper disks were placed at the centre of ethanol, on the 10 µl and 15µl of A. Absinthium section using a tweezer. After placing each disk, the tweezer was sterilised by placing it on the Bunsen burner. 5µl of ethanol, 10 µl and 15µl of A.Absinthium were added onto their respective paper disks using calibrated micropipettes and were left to dry for 5 minutes. This procedure was repeated two more times before incubating the Petri plates for 24 hours under 37℃
Measuring the Zone of Inhibition (ZOI)
After 24 hours, the effectiveness of A. Absinthium was carefully examined through the Zone of Inhibition test. Before calculating the ZOI, it was carefully examined and marked with a marker pen. Then the diameter of the inhibition zone for each disk was measured in millimetres using a ruler. After that, the area of the inhibition zone was calculated using the circle formula. (A=πr^2). Once the area has been measured the findings were recorded in a table.
Data analysis – t-test
After the data was collected, the t-test was conducted. Before doing the t-test, 4 the XLMiner Analysis ToolPak was downloaded from the add-on in the extension operation. After that, all the results collected were put into a table in a spreadsheet (Google spreadsheet was mainly used for this research). In the first column, the independent variables (ethanol and 10 and 15 µl of A. Absinthium) were noted and the ZOI (mm^2) in the second column. Two more tables comparing the ZOI of ethanol to 10 µl and 15 µl of A. Absinthium was included. Following, the extension operation was opened and the XLMiner Analysis ToolPak was chosen. In the XLMiner Analysis ToolPak, the ‘t-test: Two Samples Assuming unequal variance’ was picked. For the variable 1 range, the column of the ethanol’s ZOI and for the variable 2 range, the ZOI of 10µl was highlighted. As for the hypothesised mean, 0 was noted and the labels section was ticked off. Lastly, an empty tab on the sheet was chosen for the output range to generate the graph. Finally, the table was generated. The same methods were followed to generate the t-test for Ethanol’s ZOI and 15µl of A. Absinthium oil. Following that using the same tables, comparison column charts were generated. Once the column graphs were created, the customise section was chosen and the series section was selected. After that the trendline box and the Pearson’s correlation coefficient (R^2)^5 box was ticked and the Pearson’s correlation coefficient was obtained.
4 From the t-test, p-values are determined which help show whether there is significance between the independent variables and the dependent variable. It is also used if the results rejects or accept the null or the alternate hypothesis.
5 The Pearson’s Correlation Coefficient value that tells how strong the relationship is between the variables and helps compare them.
Results
Discussion
In this investigation, A.Absinthium has exhibited antimicrobial activity towards S.aureus and this is substantiated when both 10µl and15µl of A. Absinthium oil displayed Zones of inhibition (ZOI) which can be seen in Tables 1 and 2 where the average ZOI for 10µl and 15µl of A. Absinthium is 60.0mm2(3 significant figs) and 31.7mm2(3 significant figs). The antimicrobial activity of A. Absinthium is due to its predominant constituents such as thujone and camphor. These findings are consistent with the earlier research conducted (Moselmi, Hoseinzadhe, Badouei, Kafshdouzan, Fard, 2012).
These essential constituents are complex mixtures of volatile secondary metabolites and are hydrophobic in nature (MDPI, 2021). The characteristic of these constituents enables them to target the cell wall of S.aureus allowing them to kill the pathogen (MDPI, 2021). S. aureus has a soft permeable outer layer cell wall made up of 90% - 95% peptidoglycan (MDPI, 2013). The structure of Gram-positive bacteria can be seen in the figure (3.1) below. In S.aureus their cell wall plays an important role in enhancing their survivability rate. Its cell wall is significant in; maintaining its cell shape (which is important in determining how well the cell will grow), reproduction and also its ability to obtain nutrients (LibreTexts, n.d). This indicates that destroying its cell wall will reduce the survivability rate of the pathogen and ultimately extricate it. Although the cell wall performs several functions that are essential for the bacteria to function successfully, its permeable cell wall is the limitation to its survivability. The permeability of the cell wall allows the hydrophobic molecules to easily penetrate the cells, enabling them to act on both the cell wall and within the cytoplasm (National Library of Medicine, 2013). Considering that the constituents of A. Absinthium are hydrophobic, this allows them to enter the cell and disrupt the cell wall and other organelles.
This implies that it is due to the hydrophobic characteristic of A.Absinthium and the cell wall of S.aureus that enables the biological constituents of A.Absinthium disrupts the bacteria’s cell wall and membrane. The lack of a hard protective outer layer, the ability of the peptidoglycan layer to absorb biomolecules and its high porosity that allows most substances to pass through the cell wall with little difficulty permits these constituents to enter the bacteria easily and destroy them (AAT Bioquest, 2023). Once these biomolecules enter the pathogen, they are reported to interfere with the fluidity of the membrane (MDPI, 2020). The effect of A. Absinthium on S. aureus depends on the amount of the constituents ( thujone and camphor) present. At lower concentrations, they can just interact with the enzymes of the bacteria inhibiting their functions but at higher concentrations, they can denature the bacteria’s proteins (National Library of Medicine, 2013). As the proteins become denatured, the constituents also suppress the biosynthesis of other proteins in the bacteria. Given that proteins are the building block of life (Medline Plus, 2023) if the proteins are denatured or suppressed, the cell won’t function effectively, leading to more cell deaths. In addition to that, higher amounts of A.Absinthium can highly interfere with the fluidity of the membrane which causes the cells to become biologically impaired, preventing them from carrying out their cellular processes. Moreover, at higher concentrations more constituents of A.Absinthium is able to enter S.aureus in comparison to lower amounts of A.Absinthium As more amounts of A.Absinthium is absorbed by S.aureus, and the hydrocarbons ( the constituents) accumulate in the pathogen, causing it to swell and leading to cellular content leakage (MDPI, 2021) and therefore death.
The scientific understanding between the effectiveness and concentration is supported by the findings of this investigation where 15µl of A. Absinthium displayed more effectiveness than 10µl. This is supported by the p-values in T-test tables 1 and 2. In the ttest table 1, the p-value is 0.24 and considering that the p-value is ＞0.05 this shows no significance between ethanol and 10µl of A.Absinthium. It also denotes the lower difference in the ZOI between these two variables whereupon the 10µl A.Absinthium was just slightly more effective in destroying the bacteria compared to the ethanol. Thus the p-value of 10µl A.Absinthium rejects the alternate hypothesis and accepts the null hypothesis, emphasising that there is no difference between the 10µl and 15µl in destroying S.aureus. Contrary to that, in the ttest table 2, the p-value for ethanol and 15µl of A.Absinthium is 0.029. Given that the pvalue is <0.05, this proves the significance between ethanol and 10µl of A.Absinthium, therefore rejecting the null hypothesis and affirming the alternate hypothesis. This affirms the larger difference in the ZOI for ethanol and 15µl of A. Absinthium, showing that the 15µl of A. Absinthium was more successful in extirpating more bacteria than ethanol. Similarly, in graphs 1 and 2, Pearson’s correlation coefficients ( R^2) also confirm that 15µl has a greater effect against S.aureus than 10µl. This is evident when the R^2 = 0.75 for 15µl in graph 1 and in graph 2 R^2= 0.65 for 10µl. Even though Pearson’s correlation for both the independent variables is not strong, given that the R^2 for 15µl is greater than 10µl, this suggests that 15µl exhibits greater antimicrobial activity than 10µl of A. Absinthium. Thus, the results acquired underline that higher amounts of A. Absinthium has a higher content of antimicrobial properties, like camphor and thujone that denature the proteins and interfere with the fluidity of S. aureus. This contributes to it presenting a greater ZOI against S. aureus which indicates its higher effectiveness against S.aureus compared to the 10µl A.Absinthium.
In this investigation, the experiment was carried out more than once for all the variables. This is supported when the experiment was carried out 3 times. Considering that the experiment was conducted 3 times, the results were averaged to reduce the spread of the data, lessening the inaccuracy of the results. This can be seen in Tables 1 and 2 of this study. Although the findings of the experiment show less precision, this study is reliable which enhances the accuracy of the experiment. In addition to that, the use of detailed standardised procedures in the experiment reduces/ eliminates systematic errors as it allows for precise control of the variables. This is evident when the amount of ethanol added and the S.aureus added to the agar plates were all kept the same. The standardised procedure of the experiment also makes the results accurate by removing and reducing systematic errors.
Although this experiment was carried out successfully, there are some limitations. One of the main limitations of this investigation was ethanol being used instead of distilled water. In this study, 5µl ethanol was used when comparing against 10µl and 15µl of A. Absinthium. Considering that the amount of ethanol is not the same as the different amounts of A. Absinthium, we cannot accurately make a comparison between its potency. This makes the comparison between the 10µl and 15µl A.Absinthium challenging and less accurate which affects the validity and accuracy of the experiment. Even if the amounts of ethanol were used the same as A. absinthium, this would make the comparison invalid. If 10µl of ethanol were to be used against 10 µl of A.Absinthium and 15µl of ethanol against the 15 µl, this makes the 10µl and 15µl incomparable. This is because since ethanol is antimicrobial, the higher concentration will display greater ZOI compared to the lower concentration which will exhibit a lower ZOI. The different ZOI exhibited by 10µl and 15µl amounts of ethanol makes it almost impossible to draw a comparison between the potency of 10µl and 15µl of A. Absinthium on S.aureus. Therefore, to improve the research, distilled water should be used instead of ethanol as it will make the comparison between the two amounts easier and more accurate.
The other limitation was the inconsistent amount of the 10µl. In the first plate for the 10µl, an extra amount of A.Absinthium was accidentally added using a wrong-calibrated pipette. This caused the high ZOI to be displayed in the first nutrient plate and this is evident as the ZOI is 112mm2 which is 70% higher than the other disks with the 10µl of A.Absinthium. The 113mm2 ZOI is an outlier to the data causing the average for the 10µl of A.Absinthium to be higher compared to the 15µl of A.Absinthium. Due to the outlier, this lowers the precision and accuracy of the findings obtained in the t-test and also the Pearson’s correlation. Although the outlier could be removed from the data, it was decided not to, given that it would alter the pvalues and Pearson’s correlation which would affect the whole findings. If the ZOI were to be removed, the p-values would have been smaller and the Pearson’s correlation would have been close to 1 and positive instead of negative. Thus, in the future, more results should be replicated to limit/ remove the outliers. If more experiments were performed, the outliers (noise) could be easily removed from the data to make the results more precise and accurate.
One of the potential future studies could investigate the relationship between temperature and effectiveness of A.Absinthium on S.aureus. This research will help determine the optimal temperature needed for the A.Absinthium to work effectively and successfully against S.aureus.
Conclusion
This study aimed to investigate the effectiveness of A.Absinthium on S.aureus by comparing the ZOI between 10µl and 15µl of A. Absinthium oil. The findings indicated that constituents such as camphor and thujone are the biological compounds that make A.Absinthium antimicrobial. The soft permeability of the bacterial cell wall allows these biological compounds to easily enter the cell. Once these molecules enter the cell, they interfere with the fluidity of the bacteria, interact with the enzymes and even denature the proteins which impairs the bacteria’s function and reduces its survivability rate. The p-values and Pearson’s correlation coefficient of this experiment accentuate the effectiveness of A. Absinthium is directly proportional to its amount whereby a higher concentration of the oil allows more proteins to be denatured which causes the S.aureus to extirpate after it enters the cell. Contrary to that, at lower concentrations, it implies that the biological constituents of A.Absinthium only interferes with the enzymes, killing less S.aureus. The overall results of this experiment support and prove the alternate hypothesis which claims that 15µl A. Absinthium oil has a greater Zone of Inhibition against S.aureus compared to 10µl of the A. Absinthium.
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