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Repelling Aedes aegypti A sustainable plant based solution in Lao PDR
Fredrik Schubert
Arbetsgruppen för Tropisk Ekologi Minor Field Study 188 Committee of Tropical Ecology ISSN 1653-5634 Uppsala University, Sweden
Augusti 2014 Uppsala
Repelling Aedes aegypti A sustainable plant based solution in Lao PDR
Fredrik Schubert
Supervisors: Dr. Hugo de Boer, Systematic Biology, Dept. of Organismal Biology, Uppsala University, Sweden. Prof. Chanda Vongsombath Faculty of Environmental Sciences, National University of Laos, Vientiane, Lao PDR.
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Abstract Background. Vector borne diseases such as dengue fever and malaria are spread through hematophagous insects. Aedes aegypti is a species of mosquito that transmits dengue and chikungunya in Asia. In Lao PDR the estimated direct and indirect cost of dengue fever alone is 5 million USD. Even though research and innovations in the field of vaccines are moving forward there are yet no effective treatments for these diseases. Vector control methods are in place to suppress the Ae. aegypti population but there are still more than 100,000 cases annually. However, insecticide resistance, mosquito behavioral changes, high costs and health issues make todays measures inadequate. An effective measure is to decrease the mosquito-human contact by applying topical repellents. Aims. This study investigates plants used traditionally for repelling hematophagous insects in Laos, with the aim of finding means to empower local communities to create their own repellents. Methods. After interviewing local communities in Laos and reviewing literature, 24 candidate species were compiled. Lemongrass (Cymbopogon citratus) and ginger (Zingiber officinale) were hydro-distilled to extract essential oils. These oils were then analyzed through GS-MS to understand their chemical composition. Finally the essential oils were formulated with soybean oil to pilot a topical repellent that was tested in vivo on Ae. aegypti under controlled conditions. Results. The formulations elicited about 60 minutes of full protection but when combined, a possible additive effect was noted, prolonging the efficacy by nearly 50%. The main constituents of C. citratus are neral (34.77%) and geranial (56.44%) while, in the more complex, Z. officinale the main components are β-Linalool (9.84%), Geranial (14.44%) and Zingiberene (14.43%). Discussion and conclusions. Botanical repellents are a viable, cheap and sustainable solution of repelling hematophagous disease vectors. The mixture of ginger and lemongrass oil can be further improved in formulation by stabilizing it, and thus prolonging the protection. Increasing yield using alternative means of extracting the essential oils would also make these oils more feasible for commercial production.
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Table of contents 1. Introduction .................................................................................................................... 3 1.1 Virus vector ........................................................................................................................... 3 1.1.1 Dengue fever ................................................................................................................................... 4 1.1.2 Chikungunya ................................................................................................................................... 5
1.2 Protection .............................................................................................................................. 5 2. Aims and goals ............................................................................................................... 6 3. Materials and methods ................................................................................................ 7 3.1 Study site ................................................................................................................................ 7 3.2 Candidate plants .................................................................................................................. 8 3.2.1 Literature study ............................................................................................................................ 9 3.2.2 Interviews ........................................................................................................................................ 9
3.3 Hydro-‐distillation ............................................................................................................. 10 3.4 Mosquitoes ......................................................................................................................... 12 3.5 Effective Dose .................................................................................................................... 12 3.6 Complete Protection Time ............................................................................................ 14 3.7 Analysis ................................................................................................................................ 14
4. Results ........................................................................................................................... 16 4.1 Interviews ........................................................................................................................... 16 4.2 Hydro-‐distillation ............................................................................................................. 16 4.3 Effective dose ..................................................................................................................... 17 4.4 Complete Protection Time ............................................................................................ 18 4.5 Analysis ................................................................................................................................ 19
5. Discussion ..................................................................................................................... 23 6. Conclusion .................................................................................................................... 25
7. Acknowledgements ................................................................................................... 26
8. References .................................................................................................................... 27
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1. Introduction Humans have been using plants for medicinal purposes since the birth of civilization. The oldest documentation of this dates back to around 5000 years ago to Nagpur, India. A Sumerian clay piece has been found, upon which 12 different recipes for preparation of drugs are stated and referring to over 250 various plants. Among them poppy, henbane and mandrake (Petrovska 2012). It is likely that medicinal plants have been used for a great deal longer and there are documented cases of apes and monkey using different plants for thought medicinal purposes. Plants of the pepper genus are seen, in Costa Rica, being rubbed on the fur to repel parasites. This is something that is also being used by the human population, especially Piper auritum (Estrada-Reyes et al. 2013). Following the Sumerian clay piece from Nagpur many other recordings follow, up to present day. Subsequently medicinal plants have had and still have a great significance in health care parallel to the synthetic pharmaceutical industry around the world. The Lao People’s Democratic Republic (Lao PDR) is very interesting with regards to ethnobotany. Laos is a multi-ethnical country with a long tradition of using medicinal plants that is very much alive even today (Soejarto et al 2012). The country is relatively unharmed by deforestation consequently having conserved a large portion of its biodiversity. In 2010 Dr. Hugo de Boer and his colleagues conducted interviews in 66 villages in Laos and established the use of 92 different plant species for repelling a variety of hematophagous parasites (De Boer et al 2010). Since then we have conducted further interviews in northwestern Laos acknowledging further species. That, together with further extensive studying of existing literature, a selection of plant candidates for repelling Aedes aegypti emerged (Table 1). 1.1 Virus vector It has long been known that parasitic arthropods may act as vectors for transmitting certain viruses. The Aedes aegypti mosquito is a recognized vector for the dengue fever virus (DENV), chikungunya virus (CHIKV) and yellow fever virus (YFV) (Morrison et al. 2008). These diseases account for 454-900 million cases annually, worldwide (WHO 2014). Yellow fever is not present in Lao People’s Democratic Republic (Laos) and will therefore not be in focus within this report. Ae. aegypti is a small, day active, black mosquito with white stripes on its joints (Fig. 1). As with all mosquitoes, it is only the female mosquitoes that show a haematophagic behavior.
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Figure 1. Aedes aegypti mosquito. Male mosquito to the left and female mosquito to the right. Picture taken from Wikimedia Commons 1.1.1 Dengue fever It is estimated that there are about 100.000 cases of dengue fever in Laos alone, annually. Total costs for these episodes every year are believed to be more than USD 5 million (Shepard et al. 2013). In a low-income country, such as Laos, this amounts to a substantial sum of money, which could have been utilized in a more developing manner. DENV comes in four serotypes: 1, 2, 3 and 4. These are very closely related thus serological tests suggest cross-reactivity. Yet, cross-protective immunity does not seem to occur (Gubler 1997). After a mosquito carrying the virus bites a person, the subject undergoes a 3-14 days incubation period. Directly following is the acute onset of fever accompanied by a variety of common influenza symptoms, depending on the serotype. These may include headache, retroorbital pain, joint pains, weakness, nausea and vomiting. The febrile period may last 2-10 days while the virus circulate in the subjects’ blood. This phase is usually followed by a short period of relative feeling of recovery before a second onset of symptoms such as nosebleed, circulatory failure and a characteristic rash (Fig. 2) starting peripherally spreading towards the thorax and back (Gubler 1997 and Buhl & Buhl 2011).
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Figure 2. Characteristic rash attributed to DENV. Permission to use picture given by East Tennessee State University. 1.1.2 Chikungunya As the name implies Chikungunya fever is an acute febrile illness, caused by the virus, CHIKV. The emergence of CHIKV seems to be cyclic (Staples 2009, Institut Pasteur du Laos 2014). It re-emerged in Kenya 2004 and has subsequently spread to novel areas such as Europe, maybe due to an increasingly warmer climate. It has since caused millions of disease cases throughout the globe (Staples 2009). Symptoms include high fever and arthralgia or severe joint pain that occurs in almost all patients. Most infections are resolved within a few weeks but there are reports of cases lasting for many months with recurring episodes of symptoms (Cavrini 2009). Risk of death is about 0.2% and Metz et al. from TIPharma claimed, in 2013, that they have developed a working vaccine against the virus. Refining and distributing such a product is costly and takes time, thus Chikungunya fever may still be regarded as a health issue that may be eluded by botanical repellents. 1.2 Protection Currently there are no effective vaccines against DENV. Nor is there any successful treatment when infected, except relief of symptoms (WHO 2014). The best measure against DENV and CHIKV today is preventive measures. Using repellents that are effective against Aedes aegypti is a successful approach as to avoid bites, which would otherwise lead to risk of infection. According to the World Bank, 2008, 27.6% of the population in Laos lived under the poverty line; meaning one must sustain life on $1.25 or less per day. This generally means that synthetic insect repellents are not prioritized. Synthetic repellents in Laos today are almost exclusively imported from Thailand and based on N,N-Diethyl-meta-toluamide (DEET). A recent study by Corbel and colleagues showed the inhibition of cholinesterases and possible other neurotoxicity in both insect and mammalian nervous system by DEET. This further supports the idea of utilizing an innovative, safe and plant-based solution.
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2. Aims and goals The general aim of this project is to provide the means for local communities in Laos PDR to produce a sustainable, plant based repellent against the Aedes aegypti mosquito, known vector for dengue fever, chikungunya and yellow fever (Morrison et al. 2008). Goals of this project are:
• to build on quantitative ethnobiological data on plant-based repellents gathered in prior studies, and to characterize the active phytochemical constituents or/and fractions containing repellents in species that points towards promising efficacy.
• to develop protocols for local harvesting, extraction and storage of plant-based
repellents in order to empower communities to create their own repellents, independent of ethnicity, location or government programs.
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3. Materials and methods 3.1 Study site Laos is a 236,800km2 oblong country wedged between Thailand and Vietnam. The political scientific idea that geography determines destiny played its role in 1960s and 1970s during the Vietnam War. Laos then became histories most bombed country in the world. According to official figures, the US dropped 2,093,100 tons of bombes over Laos (Bush et al 2010). No one knows the exact figure of victims but this undoubtedly had a great impact on Laos’ socioeconomic climate. According to the Organisation for Economic Co-operation and Developments’ (OECD) DAC list of ODA recipients, Laos is classified as a “least developed country”. Today Laos is a Marxist-Leninist communist state governed by Lao People’s Revolutionary Party since 1975 (Laos National Assembly 2014). This is relevant during research expeditions because proper documentation is required for permission to study and explore certain areas. The 16th-26th December 2013, me and an additional party of eight people; including Dr. Hugo de Boer, Dr. Lars Björk and Dr. Chanda Vongsombath, conducted an expedition (Fig 3). The vegetation in this area is lowland paddies mixed with tropical rainforest mountain terrain. We mainly visited small villages in remote areas established by both Hmong and Lao ethnicities. The objective of this expedition was to conduct interviews with local communities regarding traditional use of plants for repelling hematophagous arthropods and collections of a selection of plants with repelling properties.
Figure 3. A map of Laos with study site route displayed in red marks. Picture remodeled from MapQuest.
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The first expedition took place over a week, setting out and ending from the capital, Vientiane. A number a villages and cities were visited involving different ethnicities and consequently different ethnobotanical data could be gathered. I conducted a second trip, 10/2-20/3 2014, to Vientiane with the objective of gathering selected plants and hydro-distilling them to extract essential oil samples. During this time a supplementary excursion to Kasetsart University and the Health Ministry of Bangkok, Thailand. This was done to organize and execute in vivo tests on mosquitoes in laboratory settings with formulations of extracted essential oils. 3.2 Candidate plants To find and select the appropriate plants for this study several methods were used. Initially an extensive literature study was conducted. By screening various online databases such as the National Center for Biotechnology Information and reading a large number of scientific journals a large number of candidate plants where found that were proven to have repelling effects on Ae. aegypti and other vector species. These were then cross-referenced to a checklist of existing plants in Laos. Since there is no complete and published flora containing all vascular plants of Laos I also deliberated the occurrence of all candidate species with botanist Dr. Vichith Lamxay at NUoL. The interviews from the first expedition that were conducted in several villages in northern Laos were also taken in to account. Eventually, 24 plant species were selected for further reviewing. This selection was based on availability and repellency over 60 minutes. To create a more manageable overview the 24 candidate plants were compiled in a table to ease the comparison of efficiency (Table 1). Table 1. Plant species with repelling effects on Aedes aegypti. Repellency/time is dose dependent thus variations seen on repellency (%) or time (min).
Species Repellency/time Author Caesalpinia pulcherrima 100-73.5%/210min Govindarajan et al. 2011 Capsicum annuum 100-77.5%/1440min* Dadji et al. 2011 Cardiospermum halicacabum 100-84.9%/240min Govindarajan et al. 2012 Citrus aurantifolia 100%/302min Das et al. 2003 Croton roxburhgii 76.3-62.5%/180min Vongsombath et al. 2012 Curcuma longa 100%/138min Tawatsin et al. 2006 Cymbopogon citratus 100%/72min Phasomkusolsil et al. 2011 Cymbopogon nardus 100%/0-120min Trongtokit et al. 2005 Hyptis suaveolens 67-56.8%/180min Vongsombath et al. 2012 Lantana camara 100%/213min Bhargava et al. 2013 Litsea cubeba 76.3-64.1%/180min Vongsombath et al. 2012 Nepeta cataria 100-73%/360min Zhu et al. 2006 Ocimum americanum 100%/180min Tawatsin et al. 2001 Ocimum basilicum 100%/70min Trongtokit et al. 2005 Ocimum gratissimum 100-70.8%120min Oparaocha et al. 2010 Ocimum sanctum 100%/60min Trongtokit et al. 2005 Piper longum 100%/30min Choochote et al. 2007 Piper nigrum 100%/138min Tawatsin et al. 2006 Pogostemon cablin 100%/ 0-120min Trongtokit et al. 2005 Psidium guajava 100%/168min Tawatsin et al. 2006 Sida acuta 100-41%/210min Govindarajan et al. 2010 Syzygium aromaticum 100%/30-120min Trongtokit et al. 2005
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Zanthoxylum limonella 100%/30-120min Trongtokit et al. 2005 Zingiber officinale 100-98.3%/1440min* Dadji et al. 2011
*Effects on Anopheles gambiae. All species in Table 1 are found in Laos with the exception of Nepeta cataria (Checklist of the Vascular Plants of Laos PDR, unpublished book). A further manual filter of selection was made. This was based seasonal availability, low toxicity, resources and effect. Two plants were chosen, namely Cymbopogon citratus and Zingiber officinale. Hyptis suaveolens was initially also worked with but was deemed unfit because the work took place during the dry season and its availability decreased dramatically. The literature study revealed that Capsicum annuum exhibits good repellency on Anopheles gambiae this was viewed to be unsuitable for use as a topical repellent because it was hypothesized that the capsaicins would irritate the skin. Cymbopogon nardus is commonly used today in botanical repellents (Silva et al. 2011) but since C. citratus is commonly used in food, while C. nardus is not, it was expected that the later is more accessible to the general population thus of greater interest in regards to this studies goals. It was also hypothesized that C. nardus and C. citratus would have a similar molecular composition. Z. officinale is also very commonly used as food, thus accessible. Since both plants are consumed on a regular basis it was thought not to be toxic therefore safe for topical use. When collecting the plants for processing, at least three botanists identified and classified the different species. To eliminate the possible difference genotypic expression and molecular composition, material was collected from several sites and mixed. Only fresh samples were collected from gardens, markets or from nature and were processed shortly after. 3.2.1 Literature study Before traveling to Laos an extensive search through literature on the subject was completed. The area of plant based insect repellents is fairly well studied and a large number of scientific articles could be hand picked and studied in depth. Initially an article from 2010 by De Boer et al. was read where him and his colleagues traveled to Laos, conducting interviews to attain traditional ethnobotanical data from villages. This yielded 92 plants traditionally used for repelling hematophagous parasites such as leeches, ticks, mites, lice, bedbugs, mosquitoes and myiasis causing fly larvae. The literature study and interviews performed by me might be considered as a continuation of this work under the supervision of Dr. de Boer. 3.2.2 Interviews Along the route in Figure 1 the team stopped at seven villages and with Dr. Vongsombath as our interpreter we inquired the elders of four villages about their knowledge of plants that have been traditionally used to repel or kill all kinds of hematophagous arthropods, with focus on mosquitoes, flies, ticks, leeches, mites, midges and bedbugs. The interviews were informal and not structured around a prewritten survey. Generally, we stopped at villages around midday (Fig. 4) and asked around for knowledgeable individuals, often village elders. After being invited to their homes (Fig. 5) Dr. Vongsombath started the conversation by explaining who we were
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and what we wanted to achieve. This was always taken well and people were open and very willing to share their traditional knowledge. Partly due to the multiethnic demographics of Laos the information extracted varied greatly. Everything from gunpowder to scientifically evaluated plant species; such as Cymbopogon nardus were suggested.
Figure 4. Hmong village in northern Laos where one interview was conducted.
Figure 5. Hmong villagers sharing traditional knowledge on plant based insect repellents during an interview. 3.3 Hydro-‐distillation When producing topical repellents there are several possible approaches. One thing in common between all methods is to utilize the spread of the active compounds in the
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ambient space; the volatility (Tawatsin et al. 2001, Das et al. 2003, Thomas et al. 2009). To attain the volatile compounds, found in the essential oils, from the candidate plants hydro-distillation was the method of choice since to mobility of equipment and relative low resource requirements (Milojević 2013, Charles and Simon 1990). Terpenes are a diverse class of molecules produced by many plants and many terpenes are volatile (Bowman et al. 1997). Consequently this molecular class is of great interest throughout this study. The setup for hydro-distillation is, as seen in Figure 6-7, a three liter round-bottom flask on an electric heating mantle (Fig. 6). To this an essential oil hydro-distillation column from Humi-glass is connected (Fig. 7). When extracting essential oils from plants through hydro-distillation the general idea is that a mix of plant material and water is heated to its boiling point through which the plant cells are lysed and the oils released. Both hydrophilic and hydrophobic volatiles are then transported with the steam through the still head and condense in the condenser due to the cooling water applied. The hydrophilic distillate is then recycled back into the round flask while the hydrophobic, essential oil is gathered as a supernatant in the distillate receiver. The oils can then be drawn from the collection sprout (Fig. 7). Before each distillation the equipment was cleaned thoroughly with 95% ethanol and acetone. Fresh plant material, leaves for C. citraus and rhizomes for Z. officinale, was washed in water and cut into small pieces (≈1mm3). The amount ranged from about 340g – 450g. Boiling rocks, 1,5 liter water and cut material (Table 2) was placed in the round-bottom flask. The heating mantel was turned on and the distillation process continued for about 2 hours.
Figure 6. Three liter round flask, filled with water and C. citratus, placed in an electric heating mantle.
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Figure 7. Distillation column. 1: Still head, 2: Cooling water inlet, 3: Condenser, 4: Cooling water outlet, 5: Vacuum/gas inlet, 6: Distillate receiver, 7: Collection spout, 8: Flow distributor, 9: Water recycler. When calculating the exchange rate (Table 2.) of plant material to essential oil, densities of the oils were obtained from Sigma-Aldrich. 0.887g/mL for lemongrass and 0.871g/mL for ginger (http://www.sigmaaldrich.com/catalog/product/aldrich/w523100?lang=en®ion=SE 2014 and http://www.sigmaaldrich.com/catalog/product/aldrich/w252204?lang=en®ion=SE 2014). The densities of the extracted oils are estimated to be very similar to those of Sigma Aldrich. 3.4 Mosquitoes There were no resources to be able to perform repellency tests on mosquitoes in Laos. I therefore contacted Dr. Hans Jørgen Overgaard, a medical entomologist working at the Entomology department at Kasetsart University (KU), Bangkok. He kindly agreed to facilitate the tests. At KU I was provided with a well-established long-term colony of USDA (U.S. Department of Agriculture) strain of Ae. aegypti. While working at KU I was introduced to Dr. Usavadee Thavara and Dr. Apiwat Tawatsin, professor and associated professor at the Health Ministry in Bangkok. They also kindly agreed to share their extensive knowledge and experience about in vivo mosquito repellency tests. They provided me with a 40-year-old colony of Bangkok (BKK) strain of Ae. aegypti. 3.5 Effective Dose To evaluate what the effective doses are of the formulation, i.e. the concentration of essential oil in dilutant that will repel a percentage of mosquitoes, laboratory studies
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are crucial. The World Health Organization has guidelines (WHO 2009) for these kinds of studies and these were taken into consideration but modified with regards to resources, goals and experience. A serial dilution was made with soybean oil as a dilutant. Soybean oil was used because of it is used in cooking and low cost, therefore availability. Five dilutions were made, volume to volume (v/v): [1%] (v/v), [4%] (v/v), [10%] (v/v), [15%] (v/v) and [20%] (v/v). Preferably dosages should give 10-90% repellency response and 2-3 dosages that give <50% repellency and 2-3 dosages yielding >50% repellency. All volunteers apply doses incrementally so that all dilutions are applied successively during the same day. One test involves the continuous use of the same mosquitoes by the same volunteer. Because of the use of incremental dosages over the same day actual concentration must be calculated cumulatively by adding all dilution strengths together. Initially a 30cm2 patch was outlined and washed with water on both of the volunteers’ forearms. The forearms were then placed in paper bags with a hole of equal size cut into it (Fig. 8).
Figure 8. Outlined 30cm2 area, which will be tested, of volunteer forearm in protective paper bag. Nine tests were performed consecutively as follows. No oil on left arm, pure soybean oil on left arm, 1% formulation on left arm, 4% formulation on left arm, 10% formulation of left arm, 15% formulation on left arm, 20% formulation on left arm, negative control on right arm and positive control on right arm. During the first test 100μl of the respective samples were applied with a pipette. This was deemed to be a too great volume because the skin would not absorb that amount of oil, thus the protocol was optimized and 10μl was used consequently. After the treatment was applied, 20 min had to pass before the test started for the sample to settle and absorb on the skin. The semi-encased and treated forearm was then put in a 30x30x30cm2 textile cage with 250, 4-5 days old, adult, starved, female Ae. aegypti mosquitoes for thirty seconds and number of mosquito probing and feeding was counted (Fig. 9). The mosquitoes were then introduced to the untreated arm to see if the feeding behavior had returned to normal. The same procedure was done for all treatments. All tests were performed indoors at daytime (10:00-18:00) at around 60% humidity and 26°C.
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Protection (P) is then calculated as a proportion of the number of landing and probing mosquitoes on the treated arm (T) in relation to an average of the number of landing and probing mosquitoes on the control (C) of the same individual: 𝑃 = (𝐶 − 𝑇)/𝐶 (i) The dosages yielding P = 1, meaning 100% protection i.e. ED100 were later used to determine Complete Protection Time (CPT) (Table 3).
Figure 9. Test setup for effective dose test. Treated and semi-encased forearm is presented to 250, starved, adult, female Ae. aegypti. 3.6 Complete Protection Time To establish the CPT of the repellents the WHO Guidelines for efficacy testing of mosquito repellents for human skin was modified as written below. Four volunteers; three males and one female, applied a concentration corresponding to ED100. After washing forearms with water, 10μl of sample was applied on 30cm2 on the left forearm as when determining ED (Fig. 8). In the same manner, 100μl of ethanolic Ethyl 3-[acetyl(butyl)amino]propanoate (IR3535) was applied on the right arm as control. IR3535 is classified as a synthetic biochemical substance and is comparable in its efficiency to the world-wide golden standard N,N-diethyl-3-methylbenzamide (DEET) but with a less harmful health profile (Cilek et al. 2004 and Corbel et al. 2009). Every 30 minutes both treated arms were tested separately for three minutes in a 30x30x30cm2 textile cage with 250, 4-5 days old, adult, starved, female Ae. aegypti mosquitoes. This was done until a total of two landings with probing were observed. The tests were performed indoors at daytime (10:00-18:00) but for no more than six hours, at around 60% humidity and 26°C. 3.7 Analysis To analyze the molecular content of the essential oils, produced by hydro-distillation, gas chromatography-mass spectrometry was used. The split-less injection gas chromatographer used is a Hewlett Packard HP 5890 Series GC System that is coupled to the mass spectrometer, which is a Hewlett Packard 5973 Mass Selective Detector. The column used was an Rtx®-5 fused silica column. This is a 30m long, 25mm inner diameter and 0.25μm phase thickness, non-polar column with a diphenyl (5%) dimethyl (95%) polysiloxane stationary phase with a temperature range of -
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60°C to 250°C (http://www.restek.com/catalog/view/134 2014). The pure oils of C. citratus and Z. officinale were diluted 1:10000 in cyclohexane and 1μl of each sample was run on a program starting at 40°C for two minutes, followed by an increase of 4°C/min until 200°C, followed by an increase of 10°C until 250°C is reached. This final temperature is held for 15 minutes. The chromatograms attained from the experiments were investigated by cross-referencing them to a NIST-library database (Fig. 10). This is software containing about 213.000 spectral profiles that will yield a probability of identity with the spectral profile that is investigated (http://www.nist.gov/srd/nist1a.cfm 2014). When a list of probable molecules was identified a further confirmation was performed to establish the true composition of essential oil samples. This was done by running synthetic authentic standards under the same conditions as previous runs and comparing retention indexes (RI) between the authentic standards and investigated molecules (Fig 13-16).
Figure 10. Example of NIST-library search following a GC-MS experiment.
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4. Results 4.1 Interviews The interviews conducted, in Laos, revealed a variety of ethnobotanical data (Fig. 3). We visited one Lao village called Ban Aboum (Fig. 4, Fig. 5) (N 19°00.914’ E 101°29.673’) that revealed ten plant-based remedies for repelling or killing many kinds of hematophagous arthropods, among them Ae. aegypti (Fig. 1), a known vector for dengue fever (Fig 2). Lemongrass was used, in this community, to repel mites from chickens. In another village, a Hmong village (N 18°50.683’ E 101°31.015’) shared five traditional botanical repellents and pesticides. Ginger was used to repel ticks. 4.2 Hydro-‐distillation All hydro-distillation was performed at NUoL according to description in 3.3. A three-liter round-flask was heated on a heat mantel (Fig. 6). A distillation column was used to condense and collect the essential oils (Fig. 7). The data from hydro-distillation experiments are compiled in table 2. Figure 11 and 12 shows a plot of the yielded oil volume over material weight for C. citratus and Z. officinale, respectively. Table 2. Data from hydro-distillation trials of C. citratus and Z. officinale. Shown is the exchange rate of the weight of plant material to volume of essential oil produced. C. citratus Z. officinale Test No. Material
weight (g)
Oil volume (mL)
Exchange rate (%)
Material weight (g)
Oil volume (mL)
Exchange rate (%)
1 405 1.12 0.25% 344 0.25 0.06 2 397 1.36 0.30% 389 1.28 0.29 3 441 1.52 0.31% 366 1.6 0.38 4 451 1.44 0.28% 353 1.0 0.25 5 414 1.2 0.26% 385 1.46 0.33
Figure 11. Plot with essential oil volume over material weight for hydro-distillation experiments of C. citratus.
y = 0.0049x -‐ 0.7336
0 0.2 0.4 0.6 0.8 1
1.2 1.4 1.6
390 400 410 420 430 440 450 460
Oil volume (mL)
Material weight (g)
C. citratus
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Figure 12. Plot with essential oil volume over material weight for hydro-distillation experiments of Z. officinale. 4.3 Effective dose Since during the ED tests dosages are applied successively, on the test area (Fig. 8), concentrations must be added and calculated in cumulative values, as presented in table 3 and table 4. Table 3. Data on number of Aedes aegypti feeding from Effective Dose tests on Z. officinale. Treatment concentrations are shown in cumulative concentrations on left arm. Right arm is only used as control. Z. Officinale Treatment No. Left No. Right Average Protection (%) Control 0% 10 14 12 0
1% 7 N.D. 7 41
5% 5 N.D. 5 58
15% 2 N.D. 2 83
30% 0 N.D. 0 100
50% 0 N.D. 0 100
Table 4. Data on number of Aedes aegypti feeding from Effective Dose tests on C. citratus. Treatment concentrations are shown in cumulative concentrations on left arm. Right arm is only used as control.
C. Citratus Treatment No. Left No. Right Average Protection (%) Control 0% 24 14 19 0
1% 15 N.D. 15 21
5% 8 N.D. 8 58
15% 2 N.D. 2 89
30% 1 N.D. 1 95
50% 0 N.D. 0 100
100% protection is achieved at 30% for Z. officinale and at 50% for C. citratus.
y = 0.0201x -‐ 6.2755
0 0.2 0.4 0.6 0.8 1
1.2 1.4 1.6 1.8
340 350 360 370 380 390 400
Oil volume (mL)
Material weight (g)
Z. ofVicinale
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4.4 Complete Protection Time The experimental set up is shown in figure 9. The complete protection time (CPT) tests end after two landings and probings are observed during the same test period. Both Z. officinale and C. citratus elicit 60 minutes of protection respectively but their combination seems to extend the protection to 90 minutes. Table 5 through 7 shows the data collected from the CPT trials. Table 5. Complete Protection Time tests of Z. officinale [30%] compared to IR3535 as standard on female Aedes aegypti. Z. officinale [30%] Time Male 1 Male 2 Male 3 Female 1 0’ 0 0 0 0 30’ 1 0 1 0 60’ 5 3 3 3 IR3535 [15%] Time Male 1 Male 2 Male 3 Female 1 0’ 0 0 0 0 30’ 1 0 0 0 60’ 0 0 0 0 90’ 0 0 0 0 120’ 0 0 0 0 150’ 1 0 0 0 180’ 0 0 0 210’ 0 0 0 240’ 1 0 1 270’ 1 0 0 300’ 2 2 Table 6. Complete Protection Time tests of C. citratus [50%] compared to IR3535 as standard on female Aedes aegypti. C. citratus [50%] Time Male 1 Male 2 Male 3 Female 1 0’ 0 0 0 0 30’ 0 0 0 0 60’ 4 4 2 4 IR3535 [15%] Time Male 1 Male 2 Male 3 Female 1 0’ 0 0 0 0 30’ 0 0 0 0 60’ 0 0 0 0 90’ 0 0 0 0 120’ 0 0 0 0 150’ 0 0 0 0 180’ 0 0 0 0 210’ 2 1 0 0 240’ 0 0 0 270’ 2 1 2 300’ 3 Table 7. Complete Protection Time tests of mixture of Z. officinale [30%] and C. citratus [50%] on female Aedes aegypti.
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Mixture of Z. officinale [30%] and C. citratus [50%] Time Male 1 Male 2 Male 3 Female 1 0’ 0 0 0 0 30’ 0 0 0 0 60’ 0 3 0 1 90’ 5 2 4
4.5 Analysis All analysis was performed with GS-MS hardware and Xcalibur™ software (Fig. 10). Initially a sample of cyclohexane was run to determine the column background and condition (Fig. 13). This was deemed clean enough to not having to change the column.
Figure 13. Cyclohexane run on Rtx®-5 30m, 25mm id, 0.25μm – 40(2); 4°C/min-200°C; 10°C/min-250°C (15). Authentic hydrocarbon standards (C9-C28) were run to be able to properly calculate RI (Fig. 14)
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Figure 14. Authentic hydrocarbon standards, C9-C28, 5ng in cyclohexane run on Rtx®-5 30m, 25mm id, 0.25μm – 40(2); 4°C/min-200°C; 10°C/min-250°C (15). The two major peaks of the C. citratus are α-Citral and β-Citral (Fig. 15 and Table 8). This is consistent with other findings (Moreira et al. 2010)
Figure 15. C. citratus essential oil in cyclohexane diluted 1:10000 run on Rtx®-5 30m, 25mm id, 0.25μm – 40(2); 4°C/min-200°C; 10°C/min-250°C (15). The results from the Z. officinale (Fig. 16 and Table 9) is more complex that the C. citratus. Ali showed, in 2007, that there might be quite a big variation in the relative molecular composition in Z. officinale. These results still show that this sample contains what is to expect, such as zingiberene, borneol, geraniol, α-Citral and β-Citral.
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Figure 16. Z. officinale essential oil in cyclohexane diluted 1:10000 run on Rtx®-5 30m, 25mm id, 0.25μm – 40(2); 4°C/min-200°C; 10°C/min-250°C (15). Table 8. List of compounds identified in C. citratus through GC-MS experiments.
RI Compound RT %Area C. citratus 1041 β-Pinene a 12.18 1.11 1089 β-Ocimene a, b 13.9 0.47 1098 3-Carene a 14.28 0.25 1156 β-Linalool a, b 16.23 0.57 1288 β-Citronellol a 20.89 0.67 1301 β-Citral a, b 21.38 34.77 1316 Geraniol a 21.83 1.93 1334 α-Citral a, b 22.41 56.44 1698 Eudesmol a 33.42 3.47 N.A. Unidentified N.A. 0.32 Tot: 100
a, confirmed through retention index and mass spectra cross-referenced with NIST-library b, confirmed through GC-MS with authentic standards. Table 9. List of compounds identified in Z. officinale through GC-MS experiments. RI Compound RT %Area Z. officinale 980 α-Pinene a, b 10.13 0.34 995 Camphene a, b 10.65 0.94 1026 β-Pinene a 11.66 1.02 1080 β-Phellandrene a 13.56 3.1 1098 β-Ocimene a, b 14.28 1.09 1155 β-Linalool a, b 16.2 9.84 1224 Borneol a, b 18.69 1.88 1302 β-Citral a, b 21.39 6.86
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1316 Geraniol a 21.83 1.17 1334 α-Citral a, b 22.41 14.44 1553 1-(1,5-dimethyl-4-hexenyl)-4-methyl-benzene a 29.26 6.35 1565 Zingiberene a 29.62 14.43 1577 Bisabola-4,7(11),10(15)-trienea 29.99 4.89 1594 Cedrene a 30.5 5.15 1633 Azulene a 31.6 1.79 N.A. Unidentified N.A. 26.7 Tot: 100 a, confirmed through retention index and mass spectra cross-referenced with NIST-library b, confirmed through GC-MS with authentic standards.
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5. Discussion The topic of producing botanical repellents is entering the spotlight of ethnobotanical research. With a greater understanding and concern for health issues regarding synthetic chemicals as pesticides, insecticides and repellents, the interest of investigating plant-based solutions is growing fast. A popular plant used is Corymbia citriodora, or lemon eucalyptus, which main ingredient is citronellal (Maia and Moore 2011). Using eucalyptus is not as viable as using annual herbs because of its slow growth rate and limited cultivation zone. Lemongrass is also used as an ingredient in commercially available repellents today, especially Cymbopogon nardus, because of its similar composition. Citronellal is known from previous studies to be present in C. citratus (Lu et al. 2014). In the lemongrass tested in this study, C. citratus, no citronellal was found through GC-MS studies but β-citronellol was found. This can be synthesized by hydrogenating citronellal, a quite simple metabolic process (Weiyong et al. 2000). It is well known that the composition of plant constituents vary greatly and the lack of citronellal in the C. citratus studied in this thesis might be due to several reasons such as genotype, time of the day when collected, season, extraction method etc. (Tawatsin et al. 2006). The ethnobotanical interviews reaffirmed the selection of the plants tested in this project but also gave rise to new interesting species to test. This goes to show the scientific potential in this area with many species, methods of formulation and application as of yet untested. While collecting the essential oils during the hydro-distillation experiments it is worth noticing a few things. The amount of plant material used is quite low since using large amount creates problems with over boiling. This is more prominent when distilling Z. officinale because a large amount of foam is created suggesting the presence of saponins (Liu et al 2010). Also notable is the linear gradient on the plot of C. citratus (Fig. 11). It is assessed to be a linear ratio because the standard deviation is below 0.03%. The value of k according to y=kx+m is low meaning that the gain of processing large amounts of plant material will not yield significantly more oils. This might be due to the properties of the column used. Z. officinale does not seem to behave in the same manner. This might also be due to the presence of saponins (Liu et al 2010). It is also notable that more data is needed for viable statistics. The overall exchange rate of essential oils is quite low (<0.4%). The collection and distilling occurred during a dry season and the exchange rate may well increase during a wet season (Rolim de Almeida et al 2014). α-citral or geranial and β-citral or neral has also been proven to be effective repellents (Oyedele et al. 2002, Makhail et al. 2004, Stotz et al. 2008). In our studied selection of C. citratus geranial and neral constitutes 91.21% of the essential oil. The final product tested in vivo did, as hypothesized, demonstrate complete protection until 60 minutes passed (Table 5). This is slightly lower than what Phasomkusolsil found in 2011 (Table 1). It might be explained by a difference in the applied concentration. The formulation used in this study is equivalent to 0.15mg/cm2, while Phasomkusolsil used 0.21mg/cm2.
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Since Z. officinale is more complex in its composition, the results are slightly more challenging to interpret. One way of rooting out which components elicit effects on insects is to perform Electroantennography (EAG). Campbell and colleagues performed a study in 2010 where they coupled an EAG with a GC-MS. They proved that, zingiberene, geraniol, linalool, β-pinene, β-phellandrene and borneol stimulate antennal responses of female Ae. aegypti, which are all present in the essential oil of the studied Z. officinale. To fully evaluate which specific compounds elicit repellent effects and not only antennal stimuli, which only acknowledges presence of specific receptors, further studies are needed. Repeating the in vivo experiments and testing all compounds individually would yield a definitive answer. Complete protection was held for 30-60 minutes (Table 4). This, by far, recedes the expected efficacy (Table 1). The expected value was taken from a study performed on Anopheles gambiae and it must be noted that Ae. aegypti seem to be less sensitive to a great variety of repellents (Tawatsin et al. 2001, Tawatsin et al. 2006, Govindarajan 2010, Kongkaew et al. 2011, Phasomkusolsil et al. 2011). Interestingly, the mixture of C. citratus and Z. officinale seem to have an additive effect upon one another, thus increasing the CPT from 60 minutes to 90 minutes (Table 6). To further investigate this hypothesis more data is needed for reliable statistics. Many of today’s commercially available botanical repellents seem to contain several plants even though their recipes are well guarded. It appears as if the more complex a repellent is the less likely it is for mosquitoes to overcome the volatile repellent barrier.
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6. Conclusion As a concluding remark it has been shown that diluting the essential oils of lemongrass, Cympobogon citratus and ginger, Zingiber officinale in soybean oil has a repelling effect on Aedes aegypti. The hypothesis was thus correct. The plants are abundant in Laos and the methods used to produce the repellent are simple therefore this might very well be implemented as a viable mean of repelling Aedes aegypti. Teaching and installing a hydro-distillation laboratory is of marginal cost when comparing to the direct costs of allowing dengue fever to spread among communities. One issue regarding the product is the protection time. Even though it enables complete protection for one hour, the product would be user-friendlier if this time could be increased. More work is needed in order to potentially stabilize the product thus increasing its protection time. I suggest that focus should be on oxidizing the terpenes, making them less volatile and adding additional plant species, making the formulation even more complex in composition.
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7. Acknowledgements I would like to direct grateful and most sincere gratitude to all the people that have helped me complete this thesis. Without Dr. Hugo de Boer, Dr. Lars Björk, Prof. Anna-Karin Borg-Karlson, Assoc. Prof. Raimondas Mozūraitis, Assoc. Prof. Chanda Vongsombath, Prof. Theeraphap Chareonviriyaphap, Dr. Hans Jørgen Overgaard, Dr. Apiwat Tawatsin and Dr. Usavadee Thavara, among others, this would not have been possible. I am very thankful to the Swedish International Development Cooperation Agency for partly funding this project. Thank you to all PhD students and technicians helping me facilitating the work progress. I would also like to take this opportunity to thank my family for moral support.
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