Bacterial inhibition by herbal preparations of Neurolaena lobata Djukic 1 Volatile compounds and microbiota: A ntibacterial properties in traditional preparation methods of medicinal herb Neurolaena lobata ( Asteracea e) Nina Djukic University of California, Berkeley Department of Environmental Science, Policy, and Management EAP Tropical Biology and Conservation Program, Fall 2016 16 December 2016 ABSTRACT I assessed the differential effectiveness of traditional herbal preparation methods of medicinal herb gavilana ( Neurolaena lobata ) on an assay of four bacteria ( E coli, S. aureus, Shigella sp. and Salmonella sp.) known to cause food poisoning symptoms in humans. Medicinal plants are used both traditionally at home in many cultures and in the synthesis of pharmaceuticals. Their medicinal properties often come from their secondary compounds, chemicals that play a role in their ecophysiology and primarily defend them from herbivory. I prepared three types of Neurolaena lobata extracts an oil, an alcohol and a tea and assessed the zones of inhibition of the bacter ia around filter paper disks impregnated with each extract. These assessments were done "blindly" (without knowledge of the inoculum/extract combination on the Petri dish) and I compared the zones of inhibition of the treatments to those surrounding the control. Results showed no significant inhibition of any bacterial inoculum by any Neurolaena lobata treatment ; the majority of disks showed no inhibition and did not differ from the control However, oil seemed to be the least effective preparation method, Shigella sp. and Salmonella sp. appeared to be more resistant to antibacterial activity than E. coli and S. aureus and Neurolaena lobata tea treatments appeared to increase in effectivene ss at higher concentrations. There are still thousands of plants that have yet to be explored for antibacterial or other health related properties, and diverse areas such as tropical forests must be protected in order for this potential to remain alive. Compuestos voltiles y microbiota: Propiedades antibacterianas de mtodos de prepar acin tradicionales de hierba medicinal Neurolaena lobata ( Asteracea e) RESUMEN Evalu la eficacia de los mtodos tradicionales de preparacin de hierbas medicinales de gavilana ( Neurolaena lobata ) en un ensayo de cuatro bacterias ( E. coli, S. aureus, Shigella sp. y Salmonella sp .). Es sabido que estas bacterias causan sntomas de envenenamiento de comida en humanos. Las plantas medicinales se usan tanto tr adicionalmente en el hogar en muchas culturas como en la sntesis de frmacos. Sus propiedades medici nales a menudo provienen de compuestos secundarios, sustancias qumicas que juegan un papel en su ecofisiologa, principalmente como defensa contra herbivo ra. Prepar tres tipos de extractos de Neurolaena lobata aceite, alcohol y t y evalu las zonas de inhibicin de las bacterias alrededor de los discos de papel filtro impregnados con cada extracto. Estas evaluaciones se realizaron "ciegamente" (sin conocimiento de la combinacin inculo / extracto en la placa de Petri) y compar las zonas de inhibicin de los tratamientos con las que rodeaban el control. Los resultados no mostraron una inhibicin significativa de ningn inculo bacteriano por ningn tratamiento de Neurolaena lobata ; La mayora de los discos no mostraron inhibicin y no difirieron del control. Sin
Bacterial inhibition by herbal preparations of Neurolaena lobata Djukic 2 embargo, el aceite pareci ser el mtodo de preparacin menos eficaz. Shigella sp y Salmonella sp. parecieron ser ms resistentes a la acti vidad antibacteriana que E. coli y S. aureus y los tratamientos con t Neurolaena lobata parecieron aumentar la eficacia en concentraciones ms altas. Todava hay miles de plantas en las que an no se ha explorado sus propiedades antibacterianas u otras p ropiedades relacionadas con la salud, y reas diversas como los bosques tropicales deben ser protegidas para que este potencial permanezca vivo. Medicinal plants have been consumed by indigenous societies for thousands of years and continuing into the present. They are used as dietary supplements and spices, as cures for health maladies, and as tonics to relieve a variety of symptoms (Walls, 2009) Beyond direct consumption of the p lant, traditional preparation methods include teas and infusions, as well as de coctions, syrups, and tinctures. Western medicine's increasing interest in herbal medicines has also driven the creation of a variety of commercially available products includin g supplements, capsules, and essential oils (Walls, 2009). The medicinal properties of plants often result from their secondary compounds, synthesized by plants for a variety of ecophysiological interactions including defending themselves against herbivo ry and attracting pollinators and other symbionts (Briskin, 2000). Chemicals used by plants that affect insect herbivore nervous systems may have similar effects on large mammals, due to t he common ancestry of all multi cellular organisms and their conserved cellular processes and pathways which are affected by similar signaling molecules (Kennedy and Wightman, 2011). Research on pharmaceutical uses for compounds isolated from particular plants has in many cases scientifically affirmed their cultura lly acknowledged medicinal properties. It has also suggested that the preparation method of these plants may be important for bringing out these properties; recent studies on "novel drug delivery systems" (NDDS) for herbal drugs have demonstrated that man y new methods (such as polymeric nanoparticles, nanocapsules, and liposomes) can more effectively harness the desirable compounds of the medicinal plants while protecting consumers from potential toxicity (Ajazuddin, 2010). However, the fact that many phar maceuticals derived from plant compounds cannot be patented decreases the incentive for pharmaceutical companies to invest resources and make these products accessible. Thus, m any people continue to use medicinal plants through traditional preparation meth ods, which may differ in effectiveness as well. A medicinal plant used frequently (and without Western pharmaceutical influence) in traditional Central American medicine is gavilana or Jackass bitters ( Neurolaena lobata ). While digestive problems are one of its primary targets, it is used for everything from fevers and diarrhea to intestinal parasites and skin infections (Bernhardt, 2008). Most scientific studies that have been performed regarding the medicinal properties of Neurolaena lobata have simply i ncluded it in a list of basic tests of medicinal plants rather than studying it in particular (Chariandy et. al., 1999; Cceres et al., 1998; Lentz, 1998). However, studies have shown that it is an "antifeedant" against caterpillars, that it contains sesqu iterpene lactones effective against P lasmodium falciparum (a parasite that carries malaria to humans), as well as other protozoan parasites ( ( Passreitr, 1997; Francois et al. 1996; Berger et al., 2001). Studies done by previous students (Chin, 2015; Burke a nd Sullivan, 2015; Schaller, 2008) have also pointed to differential effectiveness based on concentration and preparation method of Neurolaena lobata and varying methods of other traditional preparations such as drying have been shown to have differentia l effects on the retention of phytochemicals in these plants (Mahanom et al. 1999). T here is also data, including from previous UCEAP students' studies (Chin, 2015; Burke and Sullivan, 2015; Schaller, 20 08) that suggests this plant may have antibacterial properties. The antibacterial potential of Neurolaena lobata is particularly interesting, as bacteria are responsible for a variety of infections, both external and internal, and for much digestive discomfort
Bacterial inhibition by herbal preparations of Neurolaena lobata Djukic 3 (Dyer 2003). A few are known t o be particularly pathogenic to the human gut, producing, depending on their strain, symptoms from food poisoning to dysentery ("Shigella", CDC, 2016). These include Escherichia coli, a gram negative bacillus; Staphylococcus aureus a Gram positive cocci; Shigella sp., a Gram negative, rod shaped bacteria ; and Salmonella sp., a Gram negative, rod shaped bacteria. Traditional remedies from medicinal plants and prepared with traditional, non laboratory methods are still used by many people to treat bacteria l infections. Additionally, they are often used for minor ailments and preventative healthcare, especially where access to clinics and expensive pharmaceutical products is limited. For this reason, it is important in both a social and scientific context t o understand how different methods of consuming herbal remedies affect the effectiveness of the secondary compounds that give them their medical properties, and whether these medical properties are effective due to antibacterial properties or for other phy siological reasons. In this study, I asked the question: how do three different traditional preparation methods of Neurolaena lobata affect its antibacterial properties when tested on an assay of bacteria known to cause food poisoning and stomach ailments in humans? MATERIALS AND METHODS Study Site I conducted research from 20 November 2016 to 2 December 2016. I performed the bacterial experiments and prepared the Neurolaena lobata extracts in the laboratory at the Monteverde Institute. I collected Neurolaena lobata from a single ~4m tree behind the Monteverde Institute between 22 and 23 November 2016. Lab Methods I prepared Neurolaena lobata extracts using three methods that are traditionally used and require materials that can be found in any hom e. I collected all Neurolaena lobata leaves from the same plant and only collected them if they were lacking herbivore damage. I used only adult (full size) leaves that were within my reach (~2m) While I used a whole leaf for the preparation of the tea, as is the traditional method, I finely chopped leaves with scissors and then ground the m with a mortar and pestle for five minutes for the tinc ture and the infused oil. I made a tea or "infusion" (extracts in water) by steeping one fresh adult leaf (abou t 1.2g) of Neurolaena lobata in 235mL of boiling water just removed from the stove for ten minutes, using methods similar to those used by Chin (2015). This is the most frequent and traditional way the herb treatment is prepared (Balick and Arvigo 1993). Due to lack of observed antibacterial activity after the second trial, I increased the tea concentration for trial three by boiling two leaves (~2g) in 125mL of water for 20 minutes, a method more similar to that used by Schallert (2008). I made a tincture (extract in alcohol), which is traditiona lly made by soaking leaves for two weeks in one part distilled water and three parts 80 proof vodka or other strong alcohol (Walls, 2009). I made my extract, using lab materials, by soaking 5g of fresh Ne urolaena lobata leaf in a 50mL equal parts mixture of 80% methanol and 1% HCL for 48 hours, similar to methods used by Chin (2015). A third herbal preparation method I made for this experiment was an oil infusion. I packed lightly dried and ground plant ma terial, either left out for 12 hours or micro waved between paper towels for one minute and 30 seconds, in with a carrier oil. Traditionally, this would sit in the sun for two weeks, but in the interests of time, I heated 50mL of olive oil with 5g of Neurol aena lobata leaves dried overnight for two hours on low heat (Walls, 2009). I strained the oil and alcohol extractions to remove plant matter after 48 hours. I made the tea fresh the day of the experiment, and stored the other extracts in a cool, dark room in sterilized containers. For controls,
Bacterial inhibition by herbal preparations of Neurolaena lobata Djukic 4 I used boiled tap water, a 1:1 ratio of methanol and HCl with no additives, and olive oil (from the same bottle as used for the infused oil ) with no additives. I plated all bacteria on Mueller Hinton agar, which I prepared and sterilized. As an Agar pouring guideline, I marked all of the plates at approximately 1/4cm from the bottom of the dish. Once cooled, I flipped and refrigerated the plates. Bacterial cultures we re obtained from the company Pre Lab and the Ins tituto Nacional de Aprendizaje in Costa Rica. Due to the high concentration of bacteria upon arrival, I had to isolate individual colonies before using. I diluted bacteria from the original plates received from the lab by using a sterile sy ringe to put 6mL of saline solution in a sterilized test tube. I then swabbed a colony from the original plate and onto a new plate of Mueller Hinton agar. I used another sterile loop to transfer it to another part of the plate, and used a third sterile loop to spread it over the rest of the plate. I then swirled this loop in the s a line solution and used a new sterile swab to plate its contents on a new Petri dish. I incubated these new plates at 37 degrees C for 24 hours and placed them in the fridge to halt growth once I observed the presence of individual colonies. To test antibacterial activity of the Neurolaena lobata extracts, I performed a bioassay using a process similar to Chin (2015), Burke and Sullivan (2015) and the Bauer Kirby disk susceptibility test (Bauer et al., 1966). I plated diluted cultures of Staphylococcus aureu s Escherichia coli Shigella sp., and Salmonella sp. using modified Bauer method. First, I withdrew 3mL of saline solution with a sterilized syringe and filled a small test tube. Then, I used sterile loops to transfer isolated colonies of the bacteria being tested into this solution. Standard Bauer metho d calls for this suspension of bacterial colonies to be compared with a 0.5 McFarland turbidity standard; as I did not have access to materials for this mixture, I instead transferred a standard amount of colonies into the solution. A sterile swab was then used to plate the bacterial suspension. After dipping the swab into the solution and removing excess liquid on the wall of the tube, I streaked all over the surface of the agar with standard side to side motion, and repeated this twice, each after a 60 de gree turn of the Petri dish, for a total of three times. The number of colonies suspended in solution differed slightly by trial For the main trial of my experiment two colonies of E. coli (which had the largest colonies ) and three each of S. aureus, S higella sp., and Salmonella sp. were susp ended in solution. For the follow up t rial, only E. coli and S. aureus were suspended in solution, with one and two colonies, respectively. In the first two trials, nine Petri dishes of each bacteria were plated, an d three plates of each inoculum were used for treatments of each extract oil, alcohol, or t ea I put three treated disks and one control in each plate resulting in nine repetitions for every combination of treatment and bacteria. For the follow up trial, I plated six Petri dishes of each bacteria, and used three dishes of each inoculum for alcohol treatments and three for tea There were still nine repetitions for each combination. This resulted in 36 Petri dishes for the main trial a nd 12 for the f ollow up To plate the extracts on the bacterial inoculum, I dipped a hole punched piece of filter paper into a test tube of each extract. I removed the excess liquid by tapping on the side of the test tube, and I then placed the treated disk in a Petri dish that had been inoculated within the last 30 minutes. I sterilized the tweezers used to plate the disks with a burner between uses. I placed each disk in a separate quadrant at least 20mm from other disks; for each Petri dish, there were three disks of a given treatment as well as a control of the same medium the extract was made from without Neurolaena lobata After I plated the discs, my advisor labeled the plates with a symbolic code un known to me so that measurement could be done "blindly". I allowe d the plates to sit in the incubator at 37 degrees C for 18 hours. In addition to the negative controls included on each Petri dish, I al so plated four positive control plates using a main trial standard inoculum of each bacteria ( E. coli, S. aureus, Sh igella sp., and Salmonella sp.) and a suspension in solution of Amoxicillin, an antibiotic effective against a wide range of bacteria. I placed one disc in the center of each plate of bacterial inoculum using standard plating method. I incubated the plates at 37 degrees C for 18 hours.
Bacterial inhibition by herbal preparations of Neurolaena lobata Djukic 5 Once I removed the Petri dishes from the incubator, I measured their inhibition zones with calipers. Due to small and patchy inhibition zones, this data was classified numerically and visually into three classes of inhibitio n: 0 (no inhibition), 1 (inhibition of 1mm around disk, patchy inhibition, or large (3 4mm )but incomplete inhibition), or 2 (3 4mm of partial inhibition or extremely clear 2mm inhibition).If I was uncertain about the inhibition zone around a disk, I looked at it with light under a dissecting scope. After completion of the experiment, I sterilized my workspace with 10% bleach and passed all contaminated tools through an ethanol burning flame to sterilize them. Bacterial cultures and tools contaminated with inoculum were autoclaved at a the Clnica de Santa Elena with permission from microbiologist Gloriana Barrantes. RESULTS Overall, it did not appear that the Neurolaena lobata extracts had a predictable antibacterial effect. T he majority of all treatments (67/90) resulted in a degree of inhibition of 0 (no inhibition Figure 1). Due to contamination, some repetitions of th e tests were not included for data analysis (nine tests of S. aureus and oil, three of S. aureus and tea, and three of E. coli and tea were contaminated ) There was a statistical difference between the amount of repetitions that showed degrees 0 (none), 1 (slight), and 2 (significant) of inhibition (n=90, X 2 = 17.69 df=6, p<0.05) across bacterial inoculums F rom the graph it would appear this is due to the large number of 0's relative to 1's or 2's (Figure 1) There was no statistical difference between the amount of tests with inhibition by inoculum. Figure 1 Main trial: T otals for each degree of inhibition per treatment and bacterial inoculum. Inhibition was either 0 (no inhibition), 1 (slight inhibition) or 2 (tot al inhibion. E. coli is represented as "E", S. aureus as "St", Shigella sp. as "Sh" and Salmonella sp. as "Sa" Number of repetitions with 0 is majority (67/90) of total. However, a simple totaling method of data representation does not indicate the results of the tests done with controls included in each Petri dish. I determined that a more accurate way to assess inhibition by a treatment was to compare the percent of tests inhibited by the treatment to the percent of tests in hibited by the control. This is because any inh ibition that was observed in bo th th e treatment (with
Bacterial inhibition by herbal preparations of Neurolaena lobata Djukic 6 Neurolaena lobata ) and the control (without Neurolaena lobata ) is not truly inhibition caused by Neurolaena lobata. Data analyzed by this method still show ed no predictable antibacterial effect of Neurolaena lobata Th e results of most repetitions (63/90) were inconclusive, as inhibition from Neurolaena lobata treatment did not differ from the control. Table 1. Main trial : Total inhibitions as compared to co ntrol per bacterial inoculum and Neurolaena lobata treatment. Diminished repetitions due to contamination. Bacterial Inoculum Neurolaena lobata Extraction Method Total Experimental Repetitions # Tests Inhibition of Treatment Greater than Control # Tests Treatment same as Control # Tests Inhibition of Control Greater than of Treatment E. coli Oil 9 0 9 0 Alcohol 9 1 8 0 Tea 6 1 5 0 S. aureus Oil 0 N/A N/A N/A Alcohol 9 1 8 0 Tea 6 0 6 0 Shigella sp. Oil 9 0 9 0 Alcohol 9 0 8 1 Tea 9 0 8 1 Salmonella sp. Oil 9 0 8 1 Alcohol 9 0 7 2 Tea 9 0 8 1 Although there was no significant difference between inoculums, trends in differe ntial inhibition were observed. These results are based on 24 total repetitions of E. coli 15 total repetitions of S. aureus and 27 total repetitions each of Shigella sp. and Salmonella sp. E. coli showed that treatments were more inhibitory than the cont rol in 2/24 tests; S. aureus showed that treatments were more inhibito ry than controls in 1/15 tests; and Salmonella sp. and Shigella sp. each showed this in 1/27 tests. Additionally, while E. coli and S. aureus did not show that the control was more inhibitory than the treatment in any tests, Shigella sp. showed that the co ntrols were more ef fective than the treatments in 2/27 tests, while for Salmonella sp. this was true in 5/27 t ests. According to this experiment, there appears to be a trend that Shigella sp. and Salmonella sp. are more resistant to Neurolaena lobata treat ments than E. coli and S. aureus
Bacterial inhibition by herbal preparations of Neurolaena lobata Djukic 7 Figure 2. Main trial : Percentage of tests with inhibition of treatment greater than control and inhibition of control greater than treatment, by bacterial inoculum n=24 ( E. coli ), 15 (S aureus ), 27 ( Shigella sp.), 27 ( Salmonella sp.). I also analyzed d ata from the main trial by Neurolaena lobata extraction method. This was based on 27 repetitions with oil, 36 repetitions with alcohol, and 30 repetitions with tea. In tests done with oil treatments, 0/27 of the tests showed Neurolaena lobata oil to be more effective than pure olive oil; however, pure olive oil was more inhibitory than Neurolaena lobata in olive oil in 1/27 tests. Out of repetitions with alcohol extr act (across all bacterial inoculums) 2 /36 tests showed that the treatment was more inhibitory than the control, while 3/36 tests showed that the control was more inhibitory than the treatment. Tea as an extraction method produced similar results; tea was more inhibitory than pure water in 3/36 tests, while pure water was more inhibitory than Neurolaena lobata tea in 3/30 t est s. Tea and alcohol extractions were the only Neurolaena lobata treatments that re sulted in bacterial inhibition.
Bacterial inhibition by herbal preparations of Neurolaena lobata Djukic 8 Figure 3. Main trial: P ercent of tests where treatment was more effective than control and where control more effective than treatment pe r extraction method n= 27 (oil), 36 (alcohol), 30 (tea). Based on which treatments and inoculums resulted in bacterial inhibition i n the main trial, I did a follow up trial with E. coli and S. aureus and only tea and alcohol treatments. The results of this trial were also largely inconclusive with 8/28 tests showing degree 0 inhi bition and the majority ( 18/28) of tests showing treatm ents to have the same degree of inhibition as the control. The sample size was smaller, with 18 tests performed with E. coli, 10 with S. aureus 17 with alcohol treatments, and 11 with tea treatments (these irregularities due to the exclusion of some tests due to contamination). I observed a similar trend as in the main trial in that pure HCl and methanol was more effective than the related Neurolaena lobata treatment in 2/17 of these tests ; a treatment of alcohol and Neurolaena lobata was not more effectiv e than the control in any tests Tea showed the opposite trend, with more inhibition by treatment than control in 3/11 tests and control not more effective than the treat ment in any tests ( 0/10).By inoculum, E. coli was more inhibited by the Neurolaena lobata tre atment than the control in 3/18 tests, while S. aureus was not inhibited by Neurolaena lobata in any trials ( 0/10). E. coli was more inhibited by the con trol than the treatment in 1/18 tests, while S. aureus was more inhibited by the control than the treatment in 1/10 tests. While some of this difference may be explained by sample size, these results may suggest that E. coli was most sensitive to antibacterial effects from Neurolaena lobata overall, as it also showed the gr eatest amou nt of antibacterial effec tive treatments (2/24) in the main trial
Bacterial inhibition by herbal preparations of Neurolaena lobata Djukic 9 a) b) Figure 4. Follow up trial Percent of tests treatment more effective than control and control more effective than treatment. a) E. coli and S. aureus inoculums. b) Alcohol and tea treatments. Differences in inhibition and no inhibition between bacterial inoculums were not statistically significant (Figure 4a ). There was, however, a statistical difference between treatments by extraction type ( Figure 4 b; n=28, X 2 =4.94, df=1, p<0.05). Table 2 Follow up trial: There was a significant difference in bacterial inhibition between different Neurolaena lobata treatments (alcohol and tea); (n=28, X 2 =4.94, df =1, p<0.05). Yes No Alcohol 0 17 Tea 3 8 The results of the positive control performed with Amoxicillin showed inhibition zones of 2 3mm around Shigella sp. and Salmonella sp., while the inhibition zone for E. coli was 5 7mm and that for S. aureus was 10mm. DISCUSSION While the majority of tests (main trial: 67/90, follow up trial: 18/28) did not show that Neurolaena lobata treatments produced bacterial inhibition that differed from that of the control, the fact that an antibacterial effect of Neurolaena lobata was obse rved at all means that the potential for continued investigation of the secondary compounds and antibacterial properties of this plant is high. Some treatments used in this experiment appeared to be m ore effective than others, and some bacteria appeared more susceptible; both observations that would benefit from future study. By inoculum, Salmonella sp. and Shigella sp. appeared less vulnerable to Neurolaena lobata treatments overall than E. coli and S. aureus (Figure 2), although the re was no statistical difference. Both Shigella sp. and Salmonella sp. had fewer total tests in the main trial where treatments were more effective than controls than S. aureus and E. coli Additionally, Shigella sp. and Salmonella sp. were the only bacter ia in the main trial to show that the controls were more effective than the treatments and the only tests in which inhibition was greater in the treatment than the control were those tested on inoculums of E. coli and tea S. aureus (Table 1) E. coli had more tests where the inhibition by a Neurolaena lobata treatment was greater than the control than any other bacteria, especially looking at results from the follow up trial (Figure 4b) T he follow up trial also showed greater inhibition by treatment overa ll and did
Bacterial inhibition by herbal preparations of Neurolaena lobata Djukic 10 not include either Shigella sp. or Salmonella sp. (Figure 4b). All of this suggests that Shigella sp. and Salmonella sp. are more resistant to antibacterial properties than E. coli and S. aureus. This hypothesis is also supported by the results of the positive control, which showed that the inhibition zones for the antibiotic Amoxicillin with Shigella sp. and Salmonella sp. were much smaller than those for E. coli and S. aureus Knowledge of the h uman health impacts of these bacteria corroborates a hypothesis of differential aggression as well W hile E. coli and S. aureus both have strains that can be harmful to humans, they also represent large groups of bacteria of which many strains are inert ( E Coli General Information, CDC, 2015 ). S. aureus, for example, is found on the skin and in the nose of about 25% of healthy people and animals (Foster, 1996). Shigella strains, on the other hand, are important causes of disease in the developing world and are capable of causing deadly epidemics (Shigella, CDC, 2016), while Salmonella strains cause one million foodborne illnesses and 380 deaths annually in the United States alone (Salmonella, CDC, 2016) However, t he strains I worked with, as they came from a teaching laboratory, were not pathogenic Extraction methods also showed differential trends in antibacterial effectiveness. For example, oil did not produce greater inhibition from the treatment than the con trol in any of the repetitions where it was used. This supports the hypothesis that antibacterial compounds in Neurolaena lobata are not oil soluble. In the main trial, alcohol and Neurolaena lobata appeared to be more effective in inhibiting bacterial gro wth, producing greater inhibition from the treatment than the control in more tests than tea did. However, I observed the opposite trend in the fo l low up trial, where the difference in inhibition between extraction methods was statistically significant (Ta ble 2; Figure 4a). These results suggest that Neurolaena lobata tea may be more effective at higher concentrations (even higher than those it is traditionally), as I steeped a larger amount of leaves, for longer, in a smaller quantity of water to prepare t he tea for the follow up trial ; however, these results may also be skewed by a smaller sample size Additionally, the main trial showed that pure alcohol (the control) produced greater inhibition than the treatment more often than the treatment produced greater inhibition than the control. This leads me to suggest that alcohol may be more inhibitory to bacteria at higher concentrations, and diminishes in antibacterial effectiveness when mixed with Neurolaena lobata However, further study is necessary to determine the relative effectiveness of these herbal preparations and a nalysis of compounds through a method like gas chromatogrpahy wou ld be beneficial to determine in which substances Neurolaena lobata may be soluble It is interesting to note that previous students' projects such Burke and Sullivan 's (2015 ), showed that Neurolaena lobata inhibit ed bacterial inoculums of E. coli and S. aureus, potentially to a greater extent than the results of my experiment showed. There are a number of possible interpretations to explain a lack of observed bacterial inhibition in this study First of all, it is possible that the use of pure cultures ordered from labs meant that I was using bacteria that were either different strains than those used by previous students more resistant to antibacterial effects, or simply in too high of a concentration. Additional factors, such as the seasonality, age of the leaves, and location of the plant, also could play into the antibacterial potency of Neurolaena lobata leaves. F actors like weather, soil, and environmental conditions (e.g. amount of light) could have affected the growth and resources available for secondary compound production (Coley and Barone, 1996) in the Neurolaena lobata plant I used. Additionally, I used exclusively adult (full size) leaves to make my extractions; however, some research has shown that new leaves generally have higher concentrations of the secondary compounds that give them antibacterial properties (Coley and Barone, 1996). While the effectiveness of Amoxicillin, the antibiotic used in the positive control, to cause bacterial inhibition sh ould not be overlooked, there are still many reasons to continue investigating the potential of traditional medicinal plant remedies. A ntibiotics like Amoxicillin have proven to be effective against a wide range of bacteria, including those that cause tons illitis, bronchitis, pneumonia, and various infections of the ear, nose, throat, skin, or urinary tract ("Amoxicillin Uses, Side Effects & Dosage
Bacterial inhibition by herbal preparations of Neurolaena lobata Djukic 11 Guide", Drugs.com, 2016) ; however, these can also be expensive, difficult to access, and ineffective or causing unpleasant side effects. Traditional medical remedies prepared with traditional, non laboratory methods are still used by many people to treat bacterial infections, and continue to be used for used for minor ailments (such as digestive problems) and preventative healthcare, especially where access to clinics and expensive pharmaceutical products is limited. The continuing social and medical relevance of medicinal plants such as Ne urolaena lobata means that continuing investigation of the mechanism for their medicinal properties (and potential future applications) is both an important and immediate goal. Further study could investigate the secondary compounds present in Neurolaena lobata through methods like gas chromatography; this would help determine in which solvents these compounds are soluble to verify the ideal preparation method. Additionally, further research with methods of concentration, such as distillation, would be use ful in determining the physiological impacts of Neurolaena lobata. Additional assays performed with more concentrated treatments would be helpful in assessing the antibacterial activity of its compounds and determining whether Neurolaena lobata is more inh ibitive to certain kinds of bacteria than others. Finally, the conduction of clinical trials with safe and concentrated Neurolaena lobata treatments would be necessary to make conclusions about its effects on the human body. Sources of Error Working in a lab without certain tools or standard items available meant that many things used in my experiment I made myself, which may have reduced the accuracy of my experiments. Agar was boiled and "autoclaved" in a pressure cooker by me, and then plated using just my assessment and a line on the Petri dish to make a certain thickness, which was likely not standard. It was hard to grab exactly one colony to transfer into the suspension, and swabbing was difficult to standardize as well, as it was hard to make it a s mooth "mat" of bacteria and not streaky. The sterilization methods I used for the tools in my experiment may not have removed all bacteria, or e xtracts may have become contaminated through storage, as some of the Petri dishes were contaminated. Finally, w ith the disk plating method, hot tweezers were sometimes used to plate filter disks, which could either promote or destroy bacterial growth, and it was hard to standardize the amount of extract on the filter disk. Conclusion The efficacy of medicinal pla nt preparations to bring a bout beneficial health impacts for the human body is a question of high relevance for a number of people in the world. Though there may be little data on the specific impacts of Neurolaena lobata the study of chemical compounds synthesized by plants and their effects on human physiology is a growing field that is being actively contributed to, even in small ways such as this study. While plant phytochemicals affect plant physiology, the common ancestry of all multi cellular organ isms means that many have conserved cellular processes and pathways that are affected by similar chemicals (Kennedy and Wightman, 2011). Thus, the potential for compounds used by plants for their predators and symbionts to be harnessed to perform similar f unctions in humans, whether they be antibacterial or stimulative to the nervous system, is high (Briskin, 2000). This study was conducted in Costa Rica, a place where only a quarter of the original forest cover is still standing (Blasak, 2011). Tropical f orests such as those found in Costa Rica differ in a variety of ways from temperate forests, meaning that threats posed to them have different consequences. Although tropical forests such as these cover only about 7 percent of the land surface of the earth they harbor close to half of all species (Lindsey 2007). Additionally, as a consequence of plant herbivore interactions, tropical plants possess an assortment of protective measures including a wide variety of secondary metabolites, many of which are mo re common in tropical than temperate forests (Coley and Barone, 1996). As one of many examples, Neurolaena lobata is found only between southern Mexico and
Bacterial inhibition by herbal preparations of Neurolaena lobata Djukic 12 northern South America; many species of tropical forests are so specialized to microhabitats that th ey can only be found in small areas, making them vulnerable to extinction ("Neurolaena lobata", Lindsey 2007). As the beneficial medicinal effects of plants largely result from the combinations of their secondary compounds, and the diversity and abundanc e of plant secondary metabolites is greater in tropical than temperate forests, it follows logically that the conservation of tropical forests is the only way that these compound combinations and their potential for human health may be kept alive (Briskin, 2000; Coley and Barone, 1996). The potential benefits for human bodies are one of the anthropocentric reasons to protect tropical fo rests, extending beyond even the ecological impacts on other species or the atmospheric composition of the earth. Already, people rely on a range of products derived from plants for nutritive and medical needs, both in traditional and pharmaceutical settings; in recent years, compounds from plants have been shown to do everything from enhance brain function to attack cells tha t cause cancer (Briskin, 2000; Kennedy and Wightman, 2011). However, many traditional medicinal plants have not been well studied, nor have the majority of plants with secondary compounds been researched with regard to the composition of these compounds o r their impacts on human physiology. The potential for discovery with these plants is enormous; and if the remaining tropical forest is cut down, they will never be found. ACKNOWLEDGMENTS First, I would like to thank my advisor, Sofa Arce Flores, who is intelligent, witty, and wonderful, for spending hours tampering with potentially pathogenic Petri dishes in the lab with me, helping me ascend a huge learning curve in microbiology, and encouraging me even when I was discouraged. I would also like to than k Andrs Camacho, for obtaining my long list of materials and providing comic relief; microbiologist Gloriana Barrantes, for taking time after work to inspect my ameteur plating techniques; and the staff at the Monteverde Institute and the Estac in Biologica for their resources and their indefatigable helpfulness ; and my host family, for giving me the opportunity to be a part of their family for a little while. As with most things in life, what has stood at the basis of all this learning has been my emotional well being and I would like to thank my peers (and all EAP staff) for their accumulated wisdom, knowledge and curiosity and for hiking 12 miles in rubber boots, swimming with sharks, eating rice and beans, and laughing, agonizing, and living with me T hese memories will be cherished Finally, I would like to thank Frank Joyce, who gave me the confi dence to undertake this project, maintained the spirit of the course as one of humor, support, and freedom, is patient, thoughtful, and inspiring a nd gen uinely, actually really good at his job To be present in so many novel, challenging, and stimulating situations surrounded by mentors and friends has made this such a n enjoyable and educational time in my life and whichever path I go down I will trace some root of it to this I could not have done any of this without you. Thanks to all. LITERATURE CITED Ajazuddin, S. S. 2010. Applications of novel drug delivery system for herbal formations. Fitoterapia 81(2010): 680 689. Amoxicillin Uses, Side Effects & Dosage Guide. Uses, Side Effects & Dosage Guide Drugs.com. Drugs.com .22 July 2016. Web. 05 Dec. 2016. Balick, M. J., and Arvigo, R. 1993. Messages from the gods: a guide to the useful plants of Belize Oxford University Press, USA. Bauer, A. W., Kirby, W. M. M., Sherries, J. C., and Turck, M. 1966. Antibiotic Susceptibility Testing by a Standardized Single Disk Method. TURCK American Journal of Clinical Pathology 45: 493 496.
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