Examining the Efficacy of Hydrosols of Medicinal Plants Endemic to Guam Against Staphylococcus aureus

St. John’s School Guam

Alice Yi

Abstract

Staphylococcus aureus (S. aureus) is a bacterium that is commonly found on our epidermis. However, when one has an open wound, S. aureus can enter the wounds and cause severe infections. Unfortunately, certain strains of S. aureus has become resistant to penicillin, the antibiotic commonly used to treat S. aureus infections. As antibiotic-resistant bacteria increases, scientists have studied the effects of medicinal plant extracts against S. aureus, as an alternative for antibiotics. When these medicinal plants are steam distilled to produce essential oils, they produce hydrosols as a by-product. Many studies have shown essential oils to possess antimicrobial properties; however, scientists are still researching about the antimicrobial properties of hydrosols. The purpose of this research project was to examine the antimicrobial activity of plant hydrosols against S. aureus through its microbiological and chemical properties. Four medicinal plants endemic to Guam were used to produce hydrosols: Azadirachta indica, Cassia alata, Curcuma longa, and Fortunella japonica. Then the Minimum Inhibitory Concentration (MIC) test was performed on these hydrosols to determine the lowest concentrations required to prevent visible bacterial growth after overnight incubation with media in a test-tube. To further assess the accuracy of the MIC, a UV Spectrophotometer was used to measure the light absorbance and indirectly calculate the bacterial density. Cassia alata had the lowest MIC of 20% and the lowest bacterial density at 10%. The Fortunella japonica and Curcuma longa both had the highest MIC of 40% and lowest bacterial density at 10%. Azadirachta indica had an MIC of 30% and lowest bacterial density at 10%. Ergo, Azadirachta indica, Cassia alata, Curcuma longa, and Fortunella japonica all possess antimicrobial activity, but Cassia alata yielded the optimum results for both the MIC and the UV Spectrophotometry, meaning the lowest concentration and bacterial density.

Keywords: S. aureus, Antibiotic-resistant, Essential Oils, Hydrosols, Antimicrobial Properties, Azadirachta indica, Cassia alata, Curcuma longa, Fortunella japonica, Minimum Inhibitory Concentration Test, UV Spectrophotometry

Introduction

Staphylococcus aureus Bacterium

S. aureus is a bacterium that is commonly found on our epidermis and constitute our natural fauna and flora; however, the bacteria can cause severe infections when they enter wounds and areas where the epidermis has been punctured. If the bacteria advance from a break in the skin to the bloodstream, the S. aureus infections can become severe and affect major body parts such as the lungs, the heart, and the central nervous system (Stöppler, Shiel, Staph Infection Symptoms, Causes, Pictures & Treatment. 2017). The designated antibiotic for S. aureus infections is penicillin, a ?-lactam antibiotic (Baorto, Russell, Staphylococcus Aureus Infection Treatment & Management. 2016). But as the prevalence of antibiotic resistance continues to increase, antibiotic-resistant strains of Staphylococcus, such as Methicillin-Resistant-Staphylococcus-aureus (MRSA) and Vancomycin-Resistant-Staphylococcus-aureus (VRSA), have emerged (Fuda, Fisher & Mobashery, Beta-lactam Resistance in Staphylococcus aureus: The Adaptive Resistance of a Plastic Genome. 2005). MRSA is tougher to treat than most strains of aureus, because this pathogen has a universal resistance to beta-lactam antibiotics (Mayo Clinic, MRSA Infection. 2015), as a result of the overuse and misuse of antibiotics in hospitals. As a treatment for MRSA, doctors started to use vancomycin, because this antibiotic was one of the few available treatments against MRSA. However, similar to the situation with MRSA, VRSA emerged. VRSA is resistant to all ?-lactam antibiotics and vancomycin. Consequently, since MRSA and VRSA have become resistant to antibiotics that are commonly used to treat S. aureus infections, these diseases are, generally, worse than the original disease (Stratton, VRSA: The Worst Has Finally Happened. 2002). In many cases, it is difficult to prescribe the appropriate and effective antibiotics as treatments.

Essential Oils

But with that being said, there has been an increasing number of studies regarding the use of essential oils as an alternative treatment against antibiotics. Essential oils are concentrated hydrophobic liquids that have been distilled, commonly by the method of steam distillation, and contain the “essence” of the plant that the oil was distilled from. What this means is that these essential oils are potent plant extracts that hold both the characteristic fragrance and the healing properties of the plants from which they have been distilled from (Jones, Edward, Buck, Shawcross, Dawson, & Dunn, The Effect of Essential Oils on Methicillin-resistant Staphylococcus Aureus Using a Dressing Model. 2004). Essential oils are held in many parts of the plant such as the leaves, stems, flowers, and bark root. When these plants parts are undergo the process of steam distillation, the yield is a two-part aqueous solution. However, only small capacities of essential oils result, and the majority of the yield is an aqueous product called “hydrosols.”

Hydrosols

Hydrosols, also known as hydrolats, are the aqueous product that result from steam distillation. Unlike essential oils, hydrosols possess hydrophilic properties, meaning that hydrosols have water-soluble components. Thus, suspended in these hydrosols, are microscopic droplets of essential oils. Depending on the degree of water solubility of the plant components and the distillation parameters, in every liter of hydrosol, there is only about 0.05 to 0.2 milliliters of essential oils, which is less than 1%. Since hydrosols are less concentrated than essential oils, hydrosols are commonly used as fragrances due to their strong aroma. However, studies show that hydrosols do possess antimicrobial properties,  but they are not as concentrated as those in essential oils (Moon, Wilkinson & Cavanagh, Antibacterial Activity of Essential Oils, Hydrosols and Plant Extracts from Australian Grown Lavandula Spp. 2006). Additionally, hydrosols contain carboxylic acids, which may contribute to the observed anti-inflammatory activity. (Harris, R. (2006). Aromatic approaches to wound care. International Journal of Clinical Aromatherapy, Vol 3:2b, 2006). However, scientists are still researching the antimicrobial properties of hydrosols; this research aims to explore that.

The Four Experimental Plants Endemic to Guam

The island of Guam has many indigenous plants that possess medicinal properties. From the time of the ancient Chamorros (native people of Guam) to the present, there have been traditional Chamorro healers, known as “Suruhanas,” that obtain, prepare, and apply these medicinal plants to heal those in need. Thus, this research was conducted on four different types of indigenous medicinal plants that are commonly used by traditional healers.

Azadirachta indica

Azadirachta indica, also known as neem, is an evergreen tree and it is one of the most versatile medicinal plants, due to the rich source of the phytochemical limonoids. The seeds, bark, leaves, and roots of Azadirachta indica are commonly used for extraction. The active constituents of the Azadirachta indica extracts can inhibit the growth of both gram positive and gram negative bacteria (Joshi, Purwar, Wazed Ali S. Rajendran, Antimicrobial Textiles for Health and Hygiene Applications Based on Eco-Friendly Natural Products, 2010). Furthermore, Azadirachta indica has many medicinal properties, such as, antioxidant, anti-inflammatory, and antibacterial (Nagini, Chapter Seven – Neem Limonoids as Anticancer Agents: Modulation of Cancer Hallmarks and Oncogenic Signaling, 2014).

Cassia alata

Cassia alata is a shrubby legume that is commonly used as an antibacterial and antifungal treatment for various skin diseases. Cassia alata has a long history of medical usage. The leaves contain chrysophanic acid, which is a fungicide that can treat fungal infections such as eczema, scabies, and ringworms. Additionally, Cassia alata is used to treat intestinal and respiratory problems, such as, intestinal parasites, bronchitis, and asthma, due to the leaves being sudorific, diuretic, and purgative (Medical Health Guide Staff, 2011).

Curcuma longa

Curcuma longa, also known as turmeric, is a herbaceous perennial plant that has many medicinal uses. Many scientific studies have shown that the active constituents in Curcuma longa can help reverse the effects of certain diseases, such as, arthritis, diabetes, Parkinson’s disease, and cancer. From the rhizome of the Curcuma longa, the phytochemical curcuminoids, primarily curcumin can be obtained. Thus, Curcuma longa can be used as an antioxidant, anti-inflammatory, antibacterial, antiviral, antifungal, and anti-carcinogen. (Abbati, Medicinal Uses and Benefits of Turmeric (Curcumin), 2018)

Fortunella japonica

Fortunella japonica, also known as kumquat, is an evergreen shrub that contains many essential vitamins and minerals for healthy living. The fruit of Fortunella japonica bears phytochemicals like carotene, lutein, and tannins, while the rind of Fortunella japonica is filled with glands of essential oils. Among the essential oils within the Fortunella japonica extraction, the most important one is limonene. Like other citruses, Fortunella japonica, is rich in Vitamin C, which has diuretic and detoxifying properties that help regenerate damaged tissue, boosts immunity, inhibits the growth of cancer cells, treat gallbladder stones, and relieve symptoms of heartburn. Overall, Fortunella japonica is  antioxidant, anti-inflammatory, antibacterial, and anti-carcinogenic (Nair, Value Food Nutrition and Health Information Portal, 2014).

Hypothesis

The medicinal plant Azadirachta indica is known to inhibit the growth of both gram positive and gram negative bacteria, due to its rich source of the phytochemical limonoid. However, among Azadirachta indica, Cassia alata, Curcuma longa, and Fortunella japonica, it is unknown which of the four medicinal plants is the most efficacious against S. aureus. Thus, in this research, it was hypothesized that if the hydrosol of Azadirachta indica is used to eliminate S. aureus, then it will yield the greatest antimicrobial activity against S. aureus compared to the hydrosols of Cassia alata, Curcuma longa, and Fortunella japonica. Therefore, this research was conducted to evaluate and assess the extent of antimicrobial potential of the hydrosols of Azadirachta indica, Cassia alata, Curcuma longa, and Fortunella japonica against S. aureus, as well as, to determine the most efficacious hydrosol against S. aureus among the four medicinal plants endemic to Guam that were experimented on.

 

Materials and Methods

Steam Distillation

The hydrosols of Azadirachta indica, Cassia alata, Curcuma longa, and Fortunella japonica were obtained from the process of steam distillation. The glassware equipments used in order to set up the steam distillation apparatus were obtained from the University of Guam. It is very important to place at least seven boiling chips in the distillation pot, being the round bottom flask (indicated with “A”), that holds the prepared medicinal plants; failing to do this step may cause an explosion. All of the glassware of the steam distillation apparatus was clamped together. The first half of the steam distillation apparatus was first wrapped with cotton and then aluminum foil to create an insulation coat (indicated with “B”) that ensures that the maximum heat is kept within the steam distillation apparatus. After having set up the steam distillation apparatus, the four medicinal plants were prepared for efficient distillation. The leaves of the Azadirachta indica and Cassia alata were ripped into smaller pieces in order for the leaves to fit into the distillation pot. The Curcuma longa was chopped into smaller pieces to fit into the distillation pot. The rinds of the Fortunella japonica were blended to break into small pieces to put into the distillation pot.

To begin the steam distillation, clamp the distillation pot with the experimental plant and start with 100 ml of distilled water in the top most tube (indicated with “C”) and add more distilled water when needed to replenish the distillation pot. The heat was produced by the Variable Autotransformer (indicated with “D”) on a setting of 40, which produced a steady temperature of 100°C. Each plant was distilled for two hours. The yield of steam distillation is cooled in the condenser tube (indicated with “E”), as the two mini tubes attached to the condenser tube are connected to a running faucet of cold water. Then the yield is captured in the graduated cylinder (indicated with “F”). Multiple graduated cylinders or beakers may be needed to capture all the yield from the two hour steam distillation period. The top 5 ml of the yield was separated as this portion may contain microscopic droplets of essential oils. The remaining aqueous product is the hydrosol component.

Minimum Inhibitory Concentration Test (MIC)

To prepare for the MIC, 48 test tubes were divided into groups of four and were labelled according to their essential oil type, essential oil concentration percentage, and trial letter. In another test tube, half the tube was filled with the trypticase soy broth (TSB) growth medium. Using the S. aureus grown in the University of Guam microbiology laboratory, a single colony was mixed in this test tube with the growth medium until the colony was hardly visible. Then in the 48 experimental test tubes, the respective capacity of the TSB with S. aureus mixture was pipetted first and then 0.5 ml of hydrosol was pipetted second. Below are the ratios of hydrosol capacities to TSB with S. aureus mixture capacities to create a concentration from 10% to 40%.

• 10%: 0.5 ml of hydrosol: 5 ml of TSB with S. aureus mixture
• 20%: 0.5 ml of hydrosol: 2.5 ml of TSB with S. aureus mixture
• 30%: 0.5 ml of hydrosol: 1.7 ml of TSB with S. aureus mixture
• 40%: 0.5 ml of hydrosol: 1.25 ml of TSB with S. aureus mixture

The 48 experimental test tubes were stored in the incubator set to 37°C for 24 hours. After 24 hours the turbidity of each test tube was qualitatively assessed.

UV Spectrophotometry

The model of the UV Spectrophotometer available at the University of Guam was the Varian Cary 50 Bio UV-Visible Spectrophotometer, which can scan the entire wavelength range of 190-1100 nm. Using the UV Spectrophotometer, the antimicrobial activity of different concentrations of hydrosol and TSB with S. aureus mixtures from the MIC was qualitatively assessed. To begin, 1 ml of the hydrosol and TSB with S. aureus mixture from the MIC test tubes was pipetted into the plastic disposable cuvettes. After sliding the lid of the UV Spectrophotometer open, the cuvettes were inserted into the slot, then the lid was closed, and the light absorbance was measured. The light absorbance indirectly indicates the bacterial densities of the samples.

Results

Minimum Inhibitory Concentration

The tested percentage concentrations of hydrosols were 10%, 20%, 30%, and 40%, because 10% to 40% was the effective concentration range for plant extracts against S. aureus according to my previous research comparing the potency of commercial essential oils against S. aureus. The MIC of Azadirachta indica hydrosol was 30%. The MIC of Cassia alata hydrosol was 20%. The MIC of Curcuma longa hydrosol was 40%. And, the MIC of Fortunella japonica hydrosol was 40%. Thus, the Cassia alata hydrosol required the lowest concentration of 20% in order to prevent visible bacterial growth after overnight incubation with media in a test-tube.

UV Spectrophotometry

The bacterial densities of each concentration were calculated by averaging the bacterial densities yielded from three trials. The average bacterial densities of each concentration were then plotted on a line graph to model the calibration curve of absorbance versus concentration. The values of the standard error of the mean (SEM) were used to calculate the error bars.

(10% concentration) The bacterial density of the Azadirachta indica hydrosol was 0.686 AU with a SEM value of 0.0444.

(20% concentration) The bacterial density of the Azadirachta indica hydrosol was 01.16 AU with a SEM value of 0.007.

(30% concentration) The bacterial density of the Azadirachta indica hydrosol was 1.60 AU with a SEM value of 0.152.

(40% concentration) The bacterial density of the Azadirachta indica hydrosol was 2.13 AU with a SEM value of 0.340.

Thus, the Azadirachta indica hydrosol of 10%  had the lowest bacterial density of 0.0444 AU. But the Azadirachta indica of 20% had the smallest SEM value of 0.007, meaning the average bacterial density at 20% was the most accurate reflection of the average bacterial density within the parameters of the error.

The bacterial densities of each concentration were calculated by averaging the bacterial densities yielded from three trials. The average bacterial densities of each concentration were then plotted on a line graph to model the calibration curve of absorbance versus concentration. The values of the standard error of the mean (SEM) were used to calculate the error bars.

(10% concentration) The bacterial density of the Cassia alata hydrosol was 0.566 AU with a SEM value of 0.00445.

(20% concentration) The bacterial density of the Cassia alata hydrosol was 0.660 AU with a SEM value of 0.0417.

(30% concentration) The bacterial density of the Cassia alata hydrosol was 1.50 AU with a SEM value of 0.484.

(40% concentration) The bacterial density of the Cassia alata hydrosol was 2.09AU with a SEM value of 0.548.

Thus, the Cassia alata hydrosol of 10%  had the lowest bacterial density of 0.566 AU. But the Cassia alata hydrosol of 20% had the smallest SEM value of 0.0417, meaning the average bacterial density at 20% was the most accurate reflection of the average bacterial density within the parameters of the error.

 

The bacterial densities of each concentration were calculated by averaging the bacterial densities yielded from three trials. The average bacterial densities of each concentration were then plotted on a line graph to model the calibration curve of absorbance versus concentration. The values of the standard error of the mean (SEM) were used to calculate the error bars.

(10% concentration) The bacterial density of the Curcuma longa hydrosol was 0.590 AU with a SEM value of 0.1.

(20% concentration) The bacterial density of the Curcuma longa hydrosol was 2.27 AU with a SEM value of 0.392.

(30% concentration) The bacterial density of the Curcuma longa hydrosol was 1.45 AU with a SEM value of 0.0385.

(40% concentration) The bacterial density of the Curcuma longa hydrosol was 2.74 AU with a SEM value of 0.0746.

Thus, the Curcuma longa hydrosol of 10%  had the lowest bacterial density of 0.590 AU.

But the Curcuma longa hydrosol of 30% had the smallest SEM value of 0.0385, meaning the average bacterial density at 30% was the most accurate reflection of the average bacterial density within the parameters of the error.

The bacterial densities of each concentration were calculated by averaging the bacterial densities yielded from three trials. The average bacterial densities of each concentration were then plotted on a line graph to model the calibration curve of absorbance versus concentration. The values of the standard error of the mean (SEM) were used to calculate the error bars.

(10% concentration) The bacterial density of the Fortunella japonica hydrosol was 0.490 AU with a SEM value of 0.0636.

(20% concentration) The bacterial density of the Fortunella japonica hydrosol was 1.03 AU with a SEM value of 0.038.

(30% concentration) The bacterial density of the Fortunella japonica hydrosol was 1.21 AU with a SEM value of 0.291.

(40% concentration) The bacterial density of the Fortunella japonica hydrosol was 0.951 AU with a SEM value of 0.259.  

Thus, the Fortunella japonica hydrosol of 10%  had the lowest bacterial density of 0.490 AU.

But the Fortunella japonica of 20% had the smallest SEM value of 0.038, meaning the average bacterial density at 20% was the most accurate reflection of the average bacterial density within the parameters of the error.

Discussion and Conclusion

This research was aimed to evaluate the efficacy of the four medicinal plants endemic to Guam (Azadirachta indica, Cassia alata, Curcuma longa, and Fortunella japonica) against S. aureus and to determine the medicinal plant with the greatest antimicrobial activity against S. aureus among the four plants. The results from the MIC and UV Spectrophotometry indicate that hydrosols of the medicinal plants endemic to Guam possess antimicrobial properties against S. aureus, by inhibiting the growth of S. aureus. From the qualitative data produced by the MIC, Cassia alata hydrosol had the lowest MIC of 20%, Azadirachta indica hydrosol had an MIC of 30%, and Fortunella japonica and Curcuma longa both had the highest MIC of 40%. However from the quantitative data produced by the UV Spectrophotometry, all of the tested hydrosols had the lowest bacterial densities at 10%. But with that being said, three out of four (Azadirachta indica, Cassia alata, and Fortunella japonica) of the tested hydrosols yielded the smallest SEM values at 20% concentration, which indicate that the average bacterial densities of the hydrosols at 20% yielded the most accurate and reliable results. Interestingly, the higher concentrations of hydrosols of Azadirachta indica, Cassia alata, Curcuma longa resulted with higher bacterial densities. Generally, higher concentrations result with more effective results, because the more concentrated the substance is the more potent the substance is. However, in this research, there seems to exist a range of effectiveness for the medicinal plants, being less than 10%, while the higher portion of the concentrations (30% and 40%) had more bacterial growth. Unfortunately this research does not include research on the efficacy of hydrosols against S. aureus with concentrations less than 10%. Oddly, the bacterial density chart of Curcuma longa hydrosol had a high bacterial density at 20% then low bacterial density at 30% and then high again at 40%. This may also give insight into the range of effectiveness of Curcuma longa or it may have been human error. Therefore, the results in its entirety add to the research of the antimicrobial efficacy of hydrosols; however, more research is needed to confirm the antimicrobial activity of hydrosols.

To conclude this research, the hypothesis was rejected, because both the results from the MIC and UV Spectrophotometry indicate that the hydrosol of Azadirachta indica was not the most effective in eliminating S. aureus compared to the other three medicinal plants endemic to Guam (Cassia alata, Curcuma longa, and Fortunella japonica).

The results indicate that hydrosols of medicinal plants endemic to Guam possess antimicrobial properties against S. aureus, as all of the tested hydrosols had the lowest bacterial densities at 10%. Furthermore, among the tested hydrosols, Cassia alata hydrosol yielded the greatest antimicrobial ability against S. aureus, as Cassia alata hydrosol resulted with both the lowest MIC concentration of 20% and the lowest bacterial density at 10%.

Further Research

If this experiment were to be conducted again, the changes that would be made would be to use a gas chromatograph and determine the constituents of the hydrosols that contribute the most to the antimicrobial activity. A gas chromatography can be performed on essential oils to determine the constituents of the essential oils that contribute the most to the antimicrobial activity and then analyze the functional groups in essential oils and hydrosols to determine whether there is a correlation between the two groups of plant extracts. Another improvement would be to develop a more conventional way to distill essential oils. Finally, more trials would have been better when analyzing results.

Acknowledgments

I would like to thank Karla Wang, the bio-laboratory technician, for giving me permission to use the biology laboratory at the University of Guam (UOG), providing the laboratory materials and S. aureus cultures, and supervising my laboratory work. I would also like to thank Dr. Vuki, Ms. Precy, and Dr. Suleman, the Chemistry professors at UOG, for giving me permission to use the chemistry laboratory at UOG and the chemistry equipment to set up the steam distillation apparatus. Additionally, I would thank Dr. Yang, the Food Science professor at UOG for letting me use his laboratory to perform the UV Spectrophotometry. Lastly, I would like to thank Ms. Gilmore and Ms. Afaisen, my chemistry and science teachers, respectively, for guiding and assisting me throughout my research, as well as, for reviewing my manuscript.

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