Medicinal Plants in Monteverde: Efficacy and Local Use of Neurolaena lobata (Gavilana) and Ageratum conyzoides ( Santa Lucia ) against E. coli and S. aureus Rachel Perera Department of Environmental Science, Policy, and Management University of California Berkeley EAP Tropical Biology and Conservation Program, Spring 2017 June 9, 2017 ABSTRACT I examined the antimicrobial efficacy of both Neurolaena lobata and Ager a tum conyzoides found in Monteverde. These two plants are in the Asteraceae family, and can be used to treat gastr ointestinal discomfort Both have been proven to be effective antimicrobials against E. coli and S. aureus I tested each plant on E. coli and S. aureus usin g a Kirby Bauer assay and an agar well diffusion method. For Neurolaena lobata plated on S. aureus solutions of young leaves exhibited significantly higher inhibition zones than solutions of both old leaves and control disks. I also gathered local knowled ge on medicinal plants using a survey method, in which 31 participants responded to questions regarding their use and knowledge of medicinal plants. Twenty nine out of 31 participants use medicinal plants in some way, and at least 12 different families of medicinal plants are used in Monteverde. Plantas Medicinales en Monteverde: Eficacia y Uso Local de Neurolaena lobata (Gavilana) y Ageratum conyzoides (Santa Lucia) contra E. coli y S. aureus RESUMEN Estudi la eficacia antimicrobiana de Neurolaena lobata y Agertum conyzoides en Monteverde. Estas dos Asteraceae pueden utilizarse para tratar malestares gastrointestinales. Ambas especies han demostrado su eficacia contra E. coli y S. aureus Estudi las bacterias E coli y S. aureus utilizando un mtodo de Kirby Bauer y un mtodo de difusin de pozos de agar. Para soluciones de Neurolaena lobata sobre S. aureus las soluciones de hojas jvenes exhibieron zonas de inhibicin significativamente mayores que las soluciones de hojas viejas y discos de control. N o encontr ninguna otra inhibicin significativa. Compil adems conocimientos locales sobre plantas medicinales mediante una encuesta, en la que 31 participantes respondieron a varias preguntas sobre su uso y conocimiento de plantas medicinales. Veintinu eve de los 31 participantes usan plantas medicinales, y por lo menos 12 familias de plantas medicinales se utilizan en Monteverde. The intersectionality between plants and medicine is often understated in the Western world. With the advent of new compou nds, pills, and treatments, the roots of medicine have become hidden. Yet worldwide, 80% of people rely on traditional medicine as their primar y means of healthcare (Akerele 1992). I n the U.S., despite the lacking use of raw bio material, 118 of the top 150 prescription drugs come from natural sources. Of those 118, 74% come from plants (Roberson, 2008). Thus, medicinal plants hold high importance to human healthcare. Unfortunately, plant biodiversity including medicinal plants, is under threat from hab itat destruction and deforestation. If deforestation continue s at recent rates, 60,000 plant species will
Medicinal Plants in Monteverde Perera 2 disappear by the mid 21 st century (Akerele 1992). The disappearance of plant biodiversity would decrease the abundance of both discov ered and undiscovered medicinal plants, eliciting negative consequ ences for human health (Akerele 1992). Further, the over usage of derived antibiotics has begun an epidemic of antibiotic resistance. Bacteria that could once easily be treated with penicillin, suc h as Staphyloccocus aureus now exhibit high levels of antibiotic resistance. For example, 50% of the S. aureus samples collected in U.S. hospitals in 2003 were Methicillin resistant Staphylococcus aureus (Arias and Murray 2009). The epidemic of antibiotic resistance could be helped by revisiting antimicrobial plants, as they have shown capability of inhibiting antibiotic resistant bacteria (Nascimento 2000). Plant compoun ds can treat certain bacteria for which pharmaceutical compounds are no longer effective Understanding medicinal plants is key to conserving them. Our understanding of medicinal plants is rooted in two main areas: scientific knowledge and traditional knowledge. Notably these two entities are not dichotomous. Local pe ople, regardlss of formal education, retain an immense understanding of the natural histo ries of their own environments using the scientific method. t over the period of a lifetime. S cientific knowledge include s the molecular biology behind an d research on medicinal plants (Martin 1995). Through examining both the efficacy and the local understanding of medicinal plants, with a focus on Neurolaena lobata I hope d to get a holistic and relevance. While in Santa Rosa National Park in Guanacaste many students were afflicted with gastrointestinal discomfort. The knowledgeable Eladio Cruz provided some students with a tea made from Gavilana to combat symptoms including diarrhea. Upon further research of this plant and comparable plants via books and conversations with teachers I found that Neuroloena lobata ( Gavilana ) is well known and used in the Monteverde area. It is used to tre at diarrhea and gastrointestinal discomfort. Additionally, previous studies show that Gavilana is an effective antimicrobial against E. coli Staphylococcus aureus pyogenes and tiphi ( Salmonella enterica subsp. Enterica ) (Madeleno 2010). Previous students examined the efficacy of Gavilana using different preparation methods (including oil, tea, and tonic) and have found relatively low efficacy across all preparations. Notably, there was increas i ng efficacy with increasing concentrations of the tea preparation ( Djukic 2016). Past students observed efficacy of antimicrobial plants such as Siparuna grandiflora (Monimiaceae), Verbena litoralis (Verbenaceae) and Lantana camara (Verbenaceae) by preparing them in boiling water (Schallert 2008 ). Notably, Gavilana is effective due to the presence of secondary metabolites ( Lentz 1998 ). Younger leaves often have higher concentrations of secondary metabolites than older leaves (Coley et al 1996). Based on t hese facts, it is logical to hypothesize tha t the antimicro bial efficacy of younger leaves is higher than antimicrobial efficacy of older leaves. I asked three central questions : How does the antimicrobial efficacy of traditionally prepared Neuroloena lobata change between young, middle aged, and old leaves? Does Ageratum conyzoides collected in Monteverde exhibit antimicrobial efficacy? How do local people in Monteverde interact with and understand medicinal plants, especially with respect to Neuroloena lobata ?
Medicinal Plants in Monteverde Perera 3 MATERIALS AND METHODS I created Mueller Hinton agar by mixing 15 grams of Mueller Hinton powder with 500 mL of water in a glass beaker and then heating the mixture to a boil using a microwave. I sterilized the mixture i n a steam cooker for 15 minutes and allowed it to cool for 20 minute s After cooling I poured the 45 agar plates. The plates were allowed to cool for 15 minutes longer, after which I flipped the plates so that the agar si de was up. Finally, I stored the plates in the refrigerator. To test the efficacy of Neuroloena lobata and Ageratum conyzoides on E. Coli and S. aureus I first cultivated the bacteria. E. coli can be found in abundance in the fresh feces of cattle, and S. aureus is present i n an average of 1/3 human noses (Nowogrodski 2016). Thus, I collected fresh cattle feces at dairy farm using a zip lock bag and gloves. I then diluted a small amount of the feces in deionized water, and plated the solution on a Mueller Hinton agar plate using a small loop. To find S. aureus I swabbed the inside o f three noses at the Monteverde Institute lab I p lated each swab on its own agar plate Both the cow feces plate and the nose swab plates were left to incubate for 48 hours. After 48 hours, there was sufficient growth on the plates. Using known characteristics of E. coli and S. aureus bacterial morphology and coloration, I was able to identify a bacteria that appeared to be E. coli and a bacteria that appeared to be S. aureus After gram staining one potential S. aureus the sample turned out pink, indicating a gram negative species. Since Staphylococcus is gram positive, this sample was not as I supposed ( Bannerman et. a l 2004 ) I thus used a different plate of bacteria that was more likely to be S. aureus I streaked an isolated colony of each type of bacteria for testing using a loop and Mueller Hinton agar They were kept in the incubator at 22 C After sufficient growth, I diluted the bacteria in a water culture. From there I swabbed the bacteria on plates to immediately be used for Kirby Bauer testing. I dipped the swa b i n the culture once, and slid each swab in a horizontal motion across the entire plate. I then rotated the plate twice to repeat the horizontal swabbing motion, and to c reate an even layer of bacteria (Hudzicki 2009). Notably, there are some differences in the dilution levels of the bacteria tested as initial, more concentrated dilutions caused lawn like bacterial growth that diminished any antimicrobial activity. T he following tables outline the different dilutions: Table 1. E. coli dilutions Dilution 1 One loop of E. Coli in 5 mL water Dilution 2 5 mL of dilution 1 mixed with 5 mL of water Dilution 3 5 mL of dilution 2 mixed with 5 mL of water Table 2. S. aureus dilutions Dilution 4 One loop of S. aureus in 5 mL of water Dilution 5 5 mL of dilution 4 mixed with 10 mL of water For the Kirby Bauer assay I used Neuroloena lobata leaves collected from plants in the forest next to the Estacin Biol gica. I collected t he leaves in the morning before testing, and stored them in plastic bags on the journey from the station to the Monteverde I nstitute. There were three separate bags, one for young leaves, one for medium aged leaves, and one for old leaves. Young leaves were classified as being light green, growing at the center meristem of the
Medicinal Plants in Monteverde Perera 4 plant, and being between 0.5 6 cm. Medium leaves were between 7 and 15 cm, and old leaves were growing towards the bottom of the plant and being over 8 cm. If testi ng did not ensue immediately upon arrival, I stored the p lants in the refrigerator in the lower lab of the institute, for a maximum of 4 hours. I measured 1 gram of a given category of plant (young, m edium, or old). I then mixed th e gram of plant material with 50 mL of distilled w ater. I stirre d each respective mixture of leaf material and water by moving each beaker in a circular motion with my hand. Using a pressure cooker, the mixtures were all boiled for 10 minutes. After cooling for 45 minutes, I then produced a Kirby Bauer test. For the Kirby Bauer test, I created disks by pressing coffee filters through a hole puncher. I plated 4 disks per Gavilana agar plate: a young leaf preparation, a medium leaf preparation, an old leaf preparation, and a negative cont rol (which contained only water ). I d ipped t he coffee filter disks into t he solution using tweezers, and removed excess liquid by sliding the dis k across the rim of its given beaker. To test the antimicrobial efficacy of Ageratum conyzoides I used the same technique, except treatments were all done using young leaves. I collected these leaves on the path between the Institute and the road. 1 4 2 3 Figure 1. Disk Type: Neurolaena lobata Figure 2. Disk Type: Ageratum conyzoides I left Kirby Bauer plates to incubate for 24 hours. T he Kirby Bauer S. aureus plates made using dilution 5 were left to incubate for 4 8 hours, due to slower growth. I then measured t he zone of inhibition using calipers. For one trial I used a well method instead of disks, to try to create a stronger zone of inhibition than the disks elicited Before creating the wells I swab bed the plates with either E. coli or S. aureus I created the wells using sterilized micropipette tips, Ageratum conyzoides 1: young leaf solution 2: young leaf solution 3: young leaf solution 4: control (water) Neurolaena lobata 1: young leaf solution 2: medium aged leaf solution 3: old leaf solution 4: control (water)
Medicinal Plants in Monteverde Perera 5 which were a diameter of 9 mm. I sterilized the tips with ethanol, and then washed with deionized water. After creatin g 4 wells in each agar, I micropipette d 150 microliters of each respective type of treatment, in cluding young, medium, and old treatments of Neuroloena lobata as well as a control tha t was 70% ethanol. I left t he treatments to incubate for 24 hours. The tests were done using a blind assay, in which another student marked the plates with a code unknown to me. She marked the agar plates with a number 1, 2, 3, or 4, at each respectiv e disk, and wrote down what treatment corresponded to what number. After taking the measurements of the disks, I would then decode the data, and organize it in Excel. To analyze the Kirb y Bauer test results, I used an ANOVA single factor test. I analyzed significant ANOVA results with JMP. Lastly, to conduct surveys on medicinal plant knowledge, I created a set of questions in Spanish The surveys are anonymous, I handed them out to teachers and homestay parents at the Insti tute and gave them to students to take home to their ho mestay parents. The surveys aimed to determine the relevance of medicinal plants in Monteverde based on multiple choice and short response questions detailing what plants people used (Appendix 1). R ESULTS Antimicrobial Efficacy Test There were no significant differences between young, medium, old, an d control inhibition zone means of Neuolaena preparations on E. Coli at any dilution level (p>0.05) There were also no significant differences between inhibition of E. Coli and inhibition of S. aureus by Neurolaena lobata (p>0.05) Ageratum conyzoides disks and the control disks of water had no significant differences in inhibition (p>0.05) Lastly, there was no significant difference found in any of the well tests (p>0.05) The well tests included: a) Neurolaena lobata with young, old, medium, and control wells (of ethanol) on E. coli b) Neurolaena lobata with young, old, medium, and control wells (of et hanol) on S. aureus c) Ageratum conyzoides with 3 young leaf wells and a control well of ethanol. T he only significant difference lies between the disks of Neurolaena lobata on S. aureus ( Figure 3, F (3,56) = 3.59, p= 0.019). The disks with the solution of new leaves had significantly higher inhibition ( = 7.87 1.04 ) than the old disks ( = 7.44 0.63 ) and the control disks ( = 7.05 0.02). F igure 3. The average inhibitions of new leaves, medium aged leaves, old leaves, and a water control of Neurolaena lobata plated on S. aureus The disk itself has a 7mm radius, so a value of 7 mm indicates no inhibition.
Medicinal Plants in Monteverde Perera 6 Survey Results A total of 31 peo ple participated in the survey Together they use 21 different species of plants from 12 different families as natural medicine. Of these, four plants were in the Asteraceae family Twenty four out of 31 of respondents prepare medicinal plants at home rather than buying ready made medicine Further, 21/31 respondents use both medicinal plants and pharmaceuticals as means of treatment. Five out of 31 particip ants correctly identified the Gavilana based on photographic recogni tion of a black and white image (A ppendix 1 ). Table 3 The reasons people cited for choosing to use a medicinal plant instead of a pharmaceutical. Each respondent was allowed to select more than one response Reason Number of Responses C heaper 14 For a specific ailment 12 Easier to obtain 9 Other Reason 9 Works better than pharma ceuticals 6 Habitual 4 Table 4 The means by which people learned how to prepare any given medicinal plant. Each respondent was allowed to select more than one response Method of Learning Number of Responses F amily 19 Another person 8 Another medium (tv, books, etc.) 2 Other 1 0 2 4 6 8 10 12 14 all diluted Zone of Inhibition (mm) Treatment Type new medium old control Figure 4. Solutions of Neurolaena lobata on all E. coli dilutions (1, 2, 3) as compared to dilutions 2 and 3 alone
Medicinal Plants in Monteverde Perera 7 Table 5 Categorization of medicinal plant obtainment from people who use medicinal plants as typically being home prepared, bought, or both. Method of Obtainment Number of Responses P repared 24 Store bought 2 B oth 2 Table 6 Medicinal usage of respondents categorized as either medicinal plants exclusively, a mixture of plants and pharmeceuticals, or exclusively pharmeceuticals. Plants and pharmaceuticals 21 Plants 6 Pharmeceuticals 1 Table 7 Gavilana photo recognition by age group. People were able to either correctly identify Gavilana Gavilana Neurolaena lobata, Age groups are divided into 10 year segments, with bet ween 2 11 people per age group Age Respondents in age group Number of Frequency successful ID 50+ 4 2 0.50 40 to 50 9 1 0.11 30 to 40 11 2 0.18 20 to 30 4 0 0.00 10 to 20 2 0 0.00 DISCUSSION The only antimicrobial test that elicited a significant difference in mean inhibition zones between young, medium, old, and control disks was Neurolaena lobata on S. aureus T he significance lies between new leaf treatments and old leaf treatments, as well as between new leaf tre atments a nd control treatments (Figure 3 ). This supports the idea that young leaves have higher secondary co mpounds than older leaves. A possible reason for this difference could be because leaf toughness and thickness are unavailable as defenses when a leaf is young, so chemicals are the primary means of defense (Coley et al 1997). Additionally, s econdary metabolite difference could be ontogenic in the early life stages of a leaf, and could be induced by environmental fac tors later in life (Liu et al 1998). In Neurolaena lobata the secondary metabolites tha t hold antimicrobial properties are sesquiterpene lactones (Passreiter et al 1995). Because of the observed difference in antimicrobial efficacy of new and old Neurolaena lobata versus S. aureus the young leaves likely had higher concentrations of sesquiterpen e lactones than the old leaves. Though the E. coli tests did not reveal any significant differences between young, medium, old, and control treatments, there is an increase in ihibition whe n dilution 1 (the most concentrated solution) is separated from the data. Figure 4 shows an increase in variability betwee n average inhibition zone of treatments when the E. coli becomes more diluted. Dilution 1
Medicinal Plants in Monteverde Perera 8 plates were thick with E. coli to a point where 0 inhibition was observed on all but one of the disks. As for Ageratum conyzoides, the antimicrobial activity has been well studied in Bangladesh, South Africa, Brazil, and other locations (Durodola 1977; Hossan et al 2010; Nogueira et al 2010) but information on testing from plants located in Monteverde, or even Costa Rica, is less available In Costa Rica, Santa Lucia is typically mixed with Myrtus communis to create eye drops (Morton 1977). In other areas of the world, Santa Lucia is used as an anti inflammatory, an analgesic, and an anti diarrheal (Okunade 2002). Though Neurolaena lobata on E. coli and Ageratum conyzoides did not exhibit significant inhibition differences between trials and control, it is important to recognize t he already proven antimicrobial activity of these substances, as well as the potential areas for error in my study. The ratio of medicinal compounds to bacteria is likely much higher upon ingestion of prepared tea as compared to disk solutions on a petri p late. This could lead to a ratio of secondary compounds to bac teria that is too low to elicit antimicrobial activity. Based on these results, future studies ought to begin testing with a dilution of E. coli and S. aureus that is roughly 1 loop per 30 mL, to avoid lawn like growth that stifles antimicrobial inhibition. Further, uniform dilutions and comparisons would be more reliable evidence of the difference in Neurolaena lobata inhibition of E. coli and S. aureus It is known that E. coli is less sensitive to the antimicrobial effects of Neurolaena lobata leaves than S. aureus (Smith et al 2000) and future testing of this phenomenon would be interesting The survey responses revealed that a variety of medicinal plants are used and relevant in Monteverde. The fact that 5/31 people were able to recognize Gavilana simply based on the pic res communicated in a black and white image shows a high understanding and knowledge of the plant within some respondents. Of the five able to recognize Gavilana two were older than 50 years old, one between 40 and 50 years and two were between 30 and 40 years Notably, the two data points in the 30 t o 40 category were likely biased by participants with whom I had discussed my project. There is some positive corre lation between age and plant recognition, perhaps indicating a higher knowledge base with older age on this subject. However, mo re thorough testing is needed. Further, the 12 different families identified shows that a variety of medicinal plants are used i n Monteverde, and that the protection of plant biodiversity directly affects human heal thcare. Future tests of an in person plant recognition would be useful to gauge a more general familiarity of Neurolaena lobata CONCLUSION Neurolaena lobata elicited significantly different zones of inhibition on S. aureus between new disks and old disks, as well as between new disks and control disks. This supports the fact that plants have higher levels of secondary compounds,some of which are antimicrobial, in younger leaves than in older leaves. It also supports evidence that Neurolaena lobata is antimicrobial against S. aureus Other plants and bacteria tested did not rev eal significant results, perhaps due to bacteria that w as too concentrated Differences in inhibition of S. aureus and E. coli alluded to known evidence that Neurolaena lobata is more effective against S. aureus than E. coli The su rveys revealed that Monteverde residents hold a plethora of knowledge regarding medicinal plants in their own environments. This information is likely retained through teachings from family members and the practice of preparing the medicine from scratch, as elicited by the survey responses. Further, older age may correlate with ability to identify medici nal plants.
Medicinal Plants in Monteverde Perera 9 Future studies could be improved by proper dilutions of the bacteria, more uniform and comparable dilutions, and conducting interviews one by one and in person. ACKNOWLEDGEMENTS Thank you to Sofa Arce Flores for guiding me as my point advisor, and for encouraging me to be flexible and willing during unexpected changes and developments. Thank you as well to Frank Joyce as my secondary advisor, for comments and questions that consistently shaped and refined my thoughts and study. I would like to thank all of my other teachers and advisors, who helped and encouraged me though each step of this proces s, including Federico Chincilla, Emilia Triana, Andrs Camacho, F lix David Huertas Salazar, and Paola Rojas. I would also like to thank Lorenzo Vargas for helping me locate Santa Lucia to Ellyen for helping me locate Gavilana and to Jessie Ziga for modifying and editing the survey. Thank you to Annie Gorges and Julian Cassano for allowing me to swab p lates of the S. aureus from your noses and to Liana Warren for implementing the blind code for my agar plates. Lastly, thank you to everyone who took the time and patienc e to participate in the survey, and to my fellow students for your constant support. WORKS CITED Akerele, O. (1992). WHO guidelines for the assessment of herbal medicines. Fitoterapia 63 99 104. Arias, C. A., & Murray, B. E. (2009). Antibiotic resistant bugs in the 21st century a clinical super challenge. New England Journal of Medicine 360 (5), 439 443. Bannerman, D. D., Paape, M. J., Lee, J. W., Zhao, X., Hope, J. C., & Rainard, P. (2004). Escherichia coli and Staphylococcus aureus elicit differential innate immune responses following intramammary infection. Clinical and diagnostic laboratory immunology 11 (3), 463 472. Coley, Phyllis D., and J. A. Barone. "Herbivory and plant defenses in tropical forests." Annual review of ecology and systematics 27.1 (1996): 305 335. Djukic N. (2016). Bacterial inhibition by herbal preparations of Neurolaena lobata. EAP Retrieved May 1, 2017. Durodola, J. I. (1977). ANTIBACTERIAL PROPERTY OF CRUDE EXTRACTS FROM A HERBAL WOUND HEALING REMEDY AGERATUM CONYZOIDES, L. Planta medica 32 (08), 388 390. Hossan, S., Agarwala, B., Sarwar, S., Karim, M., Jahan, R., & Rahmatullah, M. (2010). Traditional use of medicinal plants in Bangladesh to treat urinary tract infections and sexually transmitted diseases. Ethnobotany Research and Applications 8 061 074. Hudzicki, J. (2009). Kirby Bauer disk diffusion susceptibility test protocol. Jeffrey, C. 1982. An introduction to plant taxonomy. Cambridge University Press: Cambridge, UK.
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