Ochoa 1 Medicinal Herbs & Mosquitoes: A Study of Plant Based Insect Repellents Miguel Alejandro Ochoa University of California, Irvine EAP Tropical Ecology and Conservation, Fall 2017 15 December 2017 ABSTRACT Mosquito borne illnesses present a great threat to human health, and result in millions of cases and hundreds of thousands of deaths annually. This study aimed to determine the effectiveness of extracted essential oils from three neotropical plants traditionally us ed in Costa Rica n folk medicine as mosquito repellents. I extracted t he essential oils of Siparuna decipiens (Monimiaceae) , Gliricidia sepium (Fabaceae) , and Neuroloena lobata (Asteraceae) via simple steam distillation. The extracted oils were then examined and compared to a negative control in the field on live volunteers , focusing on the mosquitoes in the Monteverde Zone, Puntarenas Provinc e, Costa Rica. After a total exposure 18 hours and 12 trials over the course of 4 days, I found no significant evidence with relation to the effectiveness of any of the plants . DEET was the only treatment to significantly exhibit a deterrence towards mosquitoes. Plantas Medicinales y Mosquitos: En Busca de Repelentes de Insectos Alternativos RESUMEN La s enfermedades que se transmiten por picadura de z ancudo son de los mÃ¡s problemÃ¡ticas para la salud humana , y resulten e n millones de afectados y muerte s cada aÃ±o. El objetivo del presente estudio fue determinar la eficacia de aceites aislados de tres especies de plantas neotropic ales como repelente de mosquito. Estas plantas han sido utilizadas por la poblaciÃ³n costarricense como remedios tradicionales. Los aceites esenciales de Siparuna decipiens (Monimiaceae) , Gliricidia sepium (Fabaceae) , and Neurol oena lobata (Asteraceae) fueron extraÃdos por medio de destilaciÃ³n sencillo. DespuÃ©s, eran examinadas usando voluntarios en vivo, comparando los aceites a un control negativo con mosquitos silvestres en la Zona Monteverde, Provincia Puntarenas, Costa Rica. DespuÃ©s de 18 horas de exposiciÃ³n y 12 pruebas sobre 4 dÃas, no encontrÃ© evidencia explÃcita que ningÃºn aceite fuera repelente efectivo. Si encontrÃ© que DEET fue el Ãºnico tratamiento con que logrÃ© a ctivamente repele r z ancudos. H umans are notoriously good at driving species to the brink of eradication , so and current mass extinction the Anthropocene (Steffen et al . , 2007) . Nonetheless, an insect that is considered the most prevalent vector of disease known to science has survived the test of time, and continues to be a problem (Gates, 2014) . The mosquito (Family Culicidae) presents social , economic, and scientific challenges contemporary technology and modern medicine have yet to find solutions for. Of the 41 genera of mosquitoes, only a handful are capable of being vectors of diseases such as M alaria, Chikungunya, Dengue
Costa Rican Herbal Remedies as Mosquito Repellents Ochoa 2 F ever, and the newly discovered Zika virus yet these remain among the most prolific diseases in human history , with over 2 billion people currently at risk of exposure (Kweka et al., 2008). Malaria alone kills an estimated 600,000 people annually, and over 200 million cases are reported every year (Gates, 2014). While some treatments look promising, increasing resistance to anti malarial drugs, limited access to healthcare, and drastic environmental changes due to human activity are intensifying the need to approach the problem from a different angl e (Kweka et al., 2008) . In lieu of the difficulties associated with controlling the microorganisms responsible for these ailments, health officials are instead targeting the vector of disease, with a wide array of insecticides and syntheti c repellents at their disposal. Yet, while these compounds are both effective and readily available, they are not cure alls. DEET (diethyltoluamide) is the most common active ingredient in commercial products , but it has also been shown to cause respiratory problems and s kin irritation in humans (Heller et al., 2015) , and researchers were able to breed DEET r esistant strains of mosquitoes in fewer than 5 generations (Stancyzk et al., 2013). Citronella oil, which is marketed as a natural, safer alternative to DEET, was conn ected to decreased body weight in rats with prolonged exposure (Batubara et al., 2015). These findings and other studies signal the need for further research on alternative insect repellent compound s . In Costa Rica, the search for an alternative insect repellent has pointed to the past, as herbal treatments used by native peoples may help in the fight against mosquito borne illnesses. Plant oils are not only biodegradable, they have also been shown to e xpress low toxicity to nontarget organisms an advantage over DEET based products (Dias et al., 2015) . Three plant species in particular have shown historical significance . Madero n egro, or Gliricidia sepium ( Fabaceae ) , is commonly used as a live fence on Costa Rican farms, as its roots are believed to be toxic to rats and field mice, and its leaves have traditionally been used to relieve bug bite itchiness and get rid of body lic e (Bernhardt, 2008). The second , g avilana , or Neuroloena lobata ( Asteraceae), is used in a tea by gardeners as an insecticide, and is also said to have antiparasitic properties (Bernhardt , 2008). The third , l imoncillo , or Siparuna decipiens ( Monimiaceae), is traditionally believed to repel insects with its astringent odor (Bernhard t, 2008 ) , and is a member of the same genus as n egramina (Siparuna guianensis), which has been shown by researchers in Brazil to contain phytochemicals in its essential oil that exhibit toxicity to all developmental stages of Aedes aegypti , the species of mosquito responsible for transmission of Dengue fever (Aguiar et al . , 2015 ) . Could neotropical plants and their essential oils repel mosquitoes with similar potency as DEET? The main aim of this study was to evaluate the efficacy of the ex tracted oils of these plants as mosquito repellents. MATERIALS & METHODS Oil Extraction Using Steam Distillation Using the lab at the Monteverde Institute, I extracted t he essential oils from Siparuna decipiens, Gliricidi a sepium, and Neuroloena lobata using a process of simple steam distillation, and then isolated the fluid using pure acetone, a volatile organic solvent. This process w as done over 4 days, starting 21 November 2017 . I then mixed each oil with ethanol so they would be preserved and readil y mobile . A compiled list of
Costa Rican Herbal Remedies as Mosquito Repellents Ochoa 3 plant species, the geographical coordinates where they were found , and the dates of collection are provided in Table 1. I first broke up 20 g of fresh plant mater ial ( 75% young leaves and 25% small branches by weight ) by hand, and then with a mortar and pestle to increase the exposed surface area during the heating process . I transferred the resulting paste via funnel to a distillation bulb , a nd added 250 mL of deionized water . I covered the top of the bulb with a cork s topper and thermometer, and connected t he side tube of the bulb to a coiled condenser, to prevent the buildup of excess hot steam inside. I set the bulb over a low flame, and allowed the temperature inside to rise to , but not surpass, 100 C , as only the water was to be evaporated and not the oils, which have much higher boiling temperatures (Johnson and Lusas, 1983) . I connected the faucet to rubber tubing and used this to pass cold water continuously through the condenser, quickly cooling the outcoming steam in to water. Once all water was evaporated from inside the distillation bulb, I allowed it to cool to under 56 C (the boiling point o f acetone) and added approximate ly 15 mL of pure acetone to remove the o il from the distillation bulb. I used a colander and colander press to remove the liquid from the leaves, and collected the acetone oil mixture in a 1 L beaker. I left this beaker open to aerate for a minimum of 4 ho urs, a long enough period for the volatile acetone to evaporate out , so that only the oil would be left behind (1983) . I then mixed the remaining oil of the plant with 95% ethanol in a ratio of 1:1 v/v , a preparation method described by other research on essential oils (Alikhani Koupaei et al . , 2014 ) . I placed each oil into covered, opaque vials to slow degradation of phytochemicals by light sources. I repeated this process 3 times, one for each plant, and made sure t o carefully wash the distillation bulb between each extraction. Each oil ethanol mixture was applied directly to the skin during the field trials ( approximately 2 3 mL to cover one arm from knuckle to elbow of volunteer ). Table 1 . Plant Species Collected from Monteverde Zone, Puntarenas, and adjacent provinces. Northwestern region, Costa Rica. Family Plant Species Folk Name Locality Geographical Coordinates Collection Date Monimiaceae Siparuna decipiens Limoncillo San Gerardo Biological Station, Alajuela 27/11/17 Asteraceae Neuroloena lobat a Gavilana EstaciÃ³n BiolÃ³gica, Monteverde, Puntarenas 22/11/17
Costa Rican Herbal Remedies as Mosquito Repellents Ochoa 4 Fabaceae Gliricidia sepium Madero Negro Life Monteverde Coffee Farm, Guanacaste 24/11/17 Field Testing in the Monteverde Zone I chose the Guindon family farm in Monteverde in an area colloquially called (geographical coordinates 10 , as the main testing site for most of the trials . I conducted 10 of the 12 total trials at this location in the evening from 1600 1800 hr , with each trial lasting a minimum of 1 hour , over three c onsecutive days (from 1 December to 3 December 2017 ). This was repeated over the consecutive days, totaling approximately 18 hours of trials over all days and volunteers. Volunteers (3 on 1 December, 5 on 2 December , 2 on 3 December 2017 ) each had both arms from hand to immediately above elbow exposed to open air (and thus to mosquitoes). One arm (chosen at random) served as the control, and had nothing applied to it. The other arm served as the variable, and I applied either one of the essential oils (approximately 2 3 mL) or 5 spr ays of OFF! b rand DEET (98.25%), and allowed the liquid to dry for 5 minutes. While walking around the farm, I kept track of the number of bites to each arm, also marking how many times a mosquito would land, but not bite. I als o included data from 30 November 2017 con ducted at the Santuario EcolÃ³gico in Monteverde ( geographical coordinates 10 ), but as there were not many mosquitoes found there, it only constituted a small portion of the data. N o tests were conducted at night both the wind and the cold after sunset proved unfeasible for this experiment . Statistical Analysis I ran a Chi Squared test to determine statistic al significance of each treatment category . A p value of 0.1 or less was accepted as a statistically significant difference . RESULTS Aggregate Numbers of Bites & Landings from Field Testing Bites and landings were considered to have equal values, and were summed to create an aggregate. I calculated a control:treatment ratio based on these aggregates. The ratio s for Gliricidia sepium, Neuroloena lobata , and Siparuna decipiens were 29:29, 23:21, & 8:11, resp ectively (Table 2). Meanwhile, DEET showed an aggregate ratio of 9:1 , and was the only treatment I found to be significantly different from the 2 = 6.4, df = 1, p =0.01). I presented each category in Table 2, and showed bites and landings separately, though they were treated as equivalent for the purpose of this experiment and statistical analysis . DEET h ad the highest efficacy at repelling mosquitoes, with only 10% of bites occurring on the treated arm (Table 2) , while the S. decipiens oil application was shown to have the lowest repellent effectiveness, with 57.89% of bites occurring on the treated arm (Figure 1 ). N. lobata had the second lowest percentage of bites and landings on the
Costa Rican Herbal Remedies as Mosquito Repellents Ochoa 5 treated arm (47.73%), while G. sepium fell exactly in the middle, and had a control:treated ratio of 29:29. Table 2. Collected field totals for control and treated arms. Approx. 18 hrs of total exposure over all trials . Treatment Control Bites Control Landings Treatment Bites Treatment Landings Ratio DEET 8 1 1 0 9:1 G. sepium 26 3 23 6 29:29 N. lobata 19 4 19 2 23:21 S. decipiens 7 1 10 1 8:11 Figure 1 . 10% 58% 50% 48% 90% 42% 50% 52% 0.00% 20.00% 40.00% 60.00% 80.00% 100.00% DEET S.decipiens (Limoncillo) G. sepium (Madero Negro) N. lobata (Gavilana) Percent of Bites & Landings on Treated vs. Control Arm Treated Control
Costa Rican Herbal Remedies as Mosquito Repellents Ochoa 6 DISCUSSION I found that none of the plant based mosquito repellents deterred mosquitoes. Though previous literature led me to predict that the extracted oils would work as ef fective repellents, below I descri be some reasoning as to why my study did not support these claims. Researchers in South America found that Siparuna camporum (in the same genus as the S. decipiens I tested) exhibited larvicidal toxicity on Aedes aegypti (Dias et al., 2015) , and was primarily composed of sesquiterpene hydrocarbons compounds that have been identified to have insect repellent properties in concentrations of up to 60% ( Williams et al., 2006). This same group of terpene molecules was prese nt in a species of Australian frog ( Litoria caerulea ) which exhibited mosquito repellent properties in its skin secretions though the terpen es were not found in laboratory reared individuals, signaling that the repellent compounds originated from dietary sources, which could be traced back to terpene producing plants down the trophic line (Williams et al., 2006). Sesquiterpene lactones were also found in Neuroloena lobata , and have traditional uses in the Panamanian province of Darien as an insect repellent (Borges del Castillo et al . , 1982). Isolated compounds from N. lobata neurolenin A and neurolenin B , are extremely bitter substances present in the pl above properties (Manchand & Blount, 1978). Gliricidia sepium is a medium sized tree, common on many farms in Costa Rica as both a food source for rumen animals and a live fence to border a property, often operating under the belief that its toxic roots will ward off rodents (Bernhardt, 2008). G. sepium has also been shown to achieve 100% larvae mortality for A. aegpyti and Culex quinquefasciatus though at high concentrati ons of 16,000 ppm and has demonstrated ovicidal, larvicidal, and pupicidal toxicity against Anopheles stephensi , a malarial vector (Sharma et al., 2008; Krishnappa et al., 2012). A known toxic compound , coumarin , has been found in leaf oil (1 8.2%) and flower oil (43.1%), prompting Kaniampady et al. to warn against the use of the plant extract as a mosquito repellent (2007). Therefore, even if a significant difference had been found between the G. sepium extract and the control, it would be wis e to warn against the use of it in living spaces. Possible explanations for the inability of each of the extracted oils to effectively phytochemicals and the tested mosquitoes. In reference to S. decipiens , members of the same genus in Brazil have been found to actively attract mosquitoes , as mosquitoes are capable of pollinating the flowers a characteristic the researchers argue may be found across the entire g enus (Valentini et al., 2010). Another critical supplement that must be considered is that many of the studies use d lab reared mosquito populations, and Culicidae that have been raised as such have been shown to be more susceptible to pesticides, stemming from a lack of environmental pressures and/or low genetic variation (Dias et al., 2015).
Costa Rican Herbal Remedies as Mosquito Repellents Ochoa 7 An important addition match my prediction is that plant specimens of different ages, health, genetic s , and environ mental stressors may create fluctuating levels of alkaloids and terpenes among populations and even individuals (Dias et al., 2015). Natural variation in populations of N. lobata in distinct regions can cause significant differences to the amount of neurolenins present, which i s a possible explanation for why no evidence of mosquito repellent compounds were found in this experiment (Passreiter et al., 1998). There were also difficulties in field testing associated with the unpredicatble variability in day to day weather, as the Monteverde region is well documented to have wind gusts of up to 24 kph during the month of December, when the rainy season transitions into the dry season (accuweather.com). Researchers in West Africa have found that host seeki ng females of certain species of Anopheles mosquitoes have a maximum flight speed around 6.5 kph (1.8 m/s) (Gillies and Wilkes, 2009), and the northeasterly trade winds in the TilarÃ¡n mountain easily exceed those speeds, possibly explaining the low density of mosquitoes in the tested area s . Still, the abovementioned plant extracts show some promise as alternatives to industrially synthesized insect repellents currently in use. Developed by the U.S. Army after encountering jungle warfare during World War II, DEET has now been found to cause respiratory problems and skin irritation in humans for mild cases of sensitivity, and seizures, coma, and death in severe ones (Heller et al., 2015). Evidence has also been found that excessive use of DEET may actively sel ect for resistant populations over subsequent generations, as a team of researchers at the University of Nottingham was able to successfully breed DEET resistant populations of mosquitoes, where up to 50% of the females exhibited some degree of insensitivi ty to the repellent (Stanczyk et al., 2013). This same study found that the offspring of these mosquitoes also demonstrated some insensitivity, signaling that this gene may be dominant. Mosquitoes of the genus Culex a transmitter of the West Nile virus were even shown to exhibit Pavlovian learning when prompted with olfactory conditional stimuli, and could learn to associate an arbitrary odor with a food source (Tomberlin et al., 2006). Synthesis of findings of both aforementioned studies could mean tha t not only are certain strains of mosquitoes DEET resistant, but that genetically resistant colonies may learn to associate the smell of DEET with a food source (humans) and actively seek it out, changing their host preferences to suit available alimentati on, though this phenomenon requires more in depth study to be confirmed (Stanczyk et al., 2013; Tomberlin et al., 2006). Another popular repellent, Citronella oil, is often marketed as a natural, safer alternative to DEET based insect repellents. An extra ct from the Indonesian Lemongrass Cymbopogon narbus L. , this oil is found in everything from candles to lotions, and is marked as safe for human use by the FDA and EPA ( Shurren 2006). This widespread notion that its origin dictates its safety is misinforme d, as the toxicity of the G. sepium illustrates that simply because a product comes from a plant it does not necessarily mean it is safe for everyday human use (Kaniampady et al., 2007). Over 2,000 plants have been identified to contain insect repellent compounds , and as more species are discovered and more chemical assays are conducted this number is likely to increase (Krishnappa et al., 2012). In this investigation, I found no significant evidence that any of the extracted oils of Siparuna decipiens, G liricidia
Costa Rican Herbal Remedies as Mosquito Repellents Ochoa 8 sepium, and Neuroloena lobata were effective as mosquito repellents, though studies with contrasting results indicate a need for further investigation. Future research should focus on isolating the chemical compounds that compose sential oil, and examining them individually for mosquito repellent properties. There also may be many more plants that exhibit these properties, either in the same genera of the species I studied, or in different families and habitats altogether. Lastly, the toxicity of G. sepium should be further investigated, as its use as a popular use as a food source for cattle and other domesticated animals make it of prime interest to toxicology. ACKNOWLEDGEMENTS I would like to thank my advisors, Sofia Arce Flores and Federico Chinchilla , for their unconditional support towards this project even as seemingly impossible obstacles presented themselves as well as Frank Joyce, Emilia Triana, Andres Camacho, Felix Salazar and elp along the way. I would also like to thank Eladio and Anais Cruz for helping with everything from identification of species to making me feel right at home in Monteverde. Thank you also to all the volunteers who helped my data collection by getting bit by mosquitoes Brianne, Kara, Nicole, and Georgia. A final thanks to the staff of La EstaciÃ³n BiolÃ³gica, Life Monteverde Coffee Farm, t allowing me to conduct my research on their premises, and for always satisfying my curiosity whenever a question came to mind. REFERENCES Aguiar, R.W.S., Dos Santos, S.F., Morgado, F.d.S., Ascencio, S.D. 2013. Repellent Activity of Siparuna guianensis Aubl. (Negramina) against Aedes aegypti and Culex quinquefasciatus PLoS One. 2013. Web. Alikhani Koupaei, M., Mazlumzadeh, M., Sharifani, M., Adibian, M. 2014. Stability of Essential Oils by Microencapsulation for Preservation of Button Food Science & Nutrition 2(5): 526 533. 2014. Batubara, I. , Citronella Oil and Related Compounds on Rat Body Weight and Brown Adipose Nutrients 7(3):1859 70. 2015. Web. Bernhardt, E. 2008. Medicinal Plants of Costa Rica . Zona Tropical S.A. 2008. Borges del Castillo, J., M anresa Ferrero, M.T., Rodriguez Lius, F., Vazquez Bueno, P., Gupta, M.P. 1982. New Sesquiterpene Lactones from N eurolaena
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