Most mosquito repellents today include harmful, synthetic chemicals that potentially

Transcription

1 Lit Review Jack Mao Analysis of Essential Oil Effectiveness at Repelling Mosquitoes Introduction Most mosquito repellents today include harmful, synthetic chemicals that potentially have side effects after prolonged use. DEET, the active ingredient in the most commonly used insect repellent, has no known side-effects aside from occasional skin rashes, blisters, and skin and mucus membrane irritation (CDC, n. d.). Another common chemical used to make insect repellent clothing is permethrin. However, this active ingredient is known to be carcinogenic, can cause thyroid damage, and negatively affects pets such as cats or fish. (Bremmer, 2006) Because imperfections exist in the current mosquito repellents, new repellents made from natural ingredients are needed. While some mosquito repellents made from natural plant oils exist on the market, they are not effective as DEET or other synthesized repellents. Also, the natural oils are highly volatile, thus reducing the duration of effectiveness of the repellent. Researchers interested in developing natural mosquito repellents have therefore been comparing the effectiveness of different essential oils.

2 What is an Essential Oil? Essential oils, also known as essences, volatile oils, etheric oils, or aetheroleum, are defined as extracts of plant raw material that have been obtained through physical means. Such extraction techniques include hydrodistillation, steam distillation, or dry distillation. For Citrus fruits, common extraction techniques include cold pressing. Proper essential oils exclude aromatic and volatile compounds extracted through solvent extraction, supercritical fluid extraction, and microwave assisted extractions. (ISO 9235: 2013) Essential oils are usually a mixture of volatile or semi-volatile compounds that are odorant and lipophilic. Many the major ingredients of many essential oils include terpenes ((C5H8)n), which are derived from the chemical isoprene (C5H8). The most dominant terpenes in essential oils are monoterpenes (C10H16) and sesquiterpenes (C15H24). (ISO 9235: 2013) Ways of Extracting Oils Extracting Essential Oil Using Steam Distillation Steam distillation is used to extract the essential oil from plants. There are three main processes that happen during steam distillation: hydrodiffusion, hydrolysis, and decomposition by heat. (Rao, 2016) In hydrodiffusion, warm water and oil are diffused through the plant membrane. When the hot steam soaks the plant, the plant cells swell due to osmosis. Although the cell walls are impermeable to the volatile essential oils, they are permeable to water vapor. Small amounts of the volatile essential oils dissolve in the hot water that can escape the plant membranes. Because hydrodiffusion dissolves the essential oils in the solution to free them from the plant membranes,

3 the rate of oil extraction is dependent upon the solubility of the oil in water. The temperature of the water and the volatility of the oil does not affect the rate of diffusion. (Rao, 2016) In the hydrolysis stage, components of essential oils react with water to produce an acid and an alcohol. Plant essential oils contain esters, which are functional groups made by replacing the hydrogen of an acid by an alkyl or other organic group. The following equation describes the esterification reaction. (Rao, 2016) Figure 1: Esterification Reaction (source bc this is a picture not something you drew) According to Le Châtelier's principle, when stress is applied to a system at equilibrium, the system will tend to shift its equilibrium to counteract the effect of the stress. The hydrolysis stage applies Le Châtelier's principle by adding warm water to esters. In the presence of warm water, the reverse reaction occurs because stress is placed on the products of the reaction, causing partial reaction in both directions. The equilibrium constant equation is shown in the following equation. (Rao, 2016) K = [alcohol]a [acid] B [ester] C [water] D Figure 2: Equilibrium constant reaction acting in steam distillation In steam distillation, plant materials are harvested and dried. After macerating the plant materials into powder, the powdered materials are placed in a round bottom flask with water. A double walled tube filled with cold water will be attached to the lip of the round bottom flask.

4 The round bottom flask with the mixture will be placed above a hot plate at about 150 C and allowed to boil for three hours. Water is added periodically when the mixture does not have enough water to produce more vapor. The result are two immiscible liquids containing the essential oil and hydrosol (water mixed with some plant matter). Because the oil is less dense than the hydrosol, the oil is the liquid above the hydrosol. To obtain the oil from the hydrosol, the entire solution is placed in a funnel and allowed to settle. After the oil and the hydrosol are clearly settled, the hydrosol can be released from the funnel, leaving only the oil. (Rao, 2016) Essential Oils Lavender Oil Lavender oil is a common active ingredient used in natural mosquito repellents. The essential oil obtained from lavender consists primarily of linalyl acetate, linalool, 1,8-cineol, lavandulyl acetate and camphor, as determined by GC-MS. (Lis-Balchin and Hart, 1999) Lemon Eucalyptus Oil Lemon Eucalyptus oil is another common active ingredient used in natural mosquito repellents. The chemical compounds present in the lemon eucalyptus oil consist mainly of piperitone (40.5%), α-phellandrene (17.4%), p-cymene (8.5%) and terpin-4-ol (4.7%). (Gilles, 2010) Geranium Oil

5 Geranium oil is a common active ingredient used in natural mosquito repellents. The oil consists mainly of monoterpenoids (58.4%), sesquiterpenoids (19.4%), and phenyl propanoids (10.1%). Compounds found in large amounts in geranium essential oil include fenchone (10.7%), methyl eugenol (9.9%), α-pinene (9.4%), α-thujene (7.6%), limonene (6.4%), spathulenol (4.7%), sabinene (4.3%), linalool (4.2%), (E)-caryophyllene (4.2%), terpinen-4-ol (3.2%), β- pinene (2.9%), caryophyllene oxide (2.2%) and bicyclogermacrene (2.1%). (Verma, 2013) Rosemary Oil Rosemary oil is a common active ingredient used in natural mosquito repellents. The major compounds in rosemary oil consist of 1,8-cineole (26.54%), α-pinene (20.14%), camphor (12.88%), and camphene (11.38). (Jiang, 2011) Essential Oil Additives Vanillin Vanillin is a phenolic aldehyde, which means that it contains a phenol group and an aldehyde. Vanillin contains three functional groups: aldehyde, hydroxyl, and ether. (Vanillin, n.d.)

6 Figure 3: Vanillin (National Center for Biotechnology Information CID = 1183, n. d.) Vanillin is mostly used in the food industry as flavoring. It is also used in the fragrance industries in perfumes to mask unpleasant odors or tastes in medicines, livestock fodder, and cleaning products. Other uses include chemical intermediates in producing pharmaceuticals and other fine chemicals. (Vanillin, n.d.) Natural vanilla extract, which is extracted from vanilla plants, is a mixture of hundreds of different compounds in addition to vanillin. Because the process of isolating vanillin from the vanilla plant is difficult and costly, most commercially available vanillin is synthetic. (Vanillin, n.d.) In addition to uses in the food industry, vanillin can extend the protection time of mosquito repellents. One study have shown that when DEET is mixed with vanillin in 1:1 ratio, the protection time increased by 95% (Khan, 1975). Another study has shown that by adding 5% vanillin into other essential oils, the protection time of the essential oil (carvacryl oil) solution was increased by 7 hrs. (Yang, 2005)

7 Soybean Oil Soybean oil is a vegetable oil extracted from soybean seeds. Normally, soybean oils are used in cooking and can provide an important source of protein for the human body. (Silva, 2009) Soybean oil contains many compounds. It has low concentrations of polyunsaturated (hydrocarbons having two or more double or triple bonds between its carbon atoms) linolenic acid and linolenic fatty acids as well as high oleic acid content, thus making the soybean oil less volatile. Due to the double bond of the unsaturated linolenic acid, the linoleic acid could be oxidized when subjected to heat, light, and oxygen. (Silva, 2009) Fig 4: Alpha-linolenic fatty acid, (National Center for Biotechnology Information CID = , n. d.)

8 Fig 5: Oleic fatty acid (National Center for Biotechnology Information CID = , n. d.) Ways of Testing Efficiency of Mosquito Repellents Cage Test The cage test is the most common way of testing the effectiveness of a mosquito repellent. A cage with a slot for inserting an arm is used. According to World Health Organization (WHO) guidelines, the cage should have the dimension of 35 cm to 40 cm per side. The cage should be able to contain the mosquitoes without allowing them to escape. (WHO, 2009) For testing purposes, the cage should be supplied with a minimum of 200 mosquitoes that were only fed with sucrose solution during breeding (WHO, 2006). The mosquitoes should not be fed 24 hours prior to experimentation. However, more updated cage tests use fewer

9 mosquitoes (about 30) to simulate lower densities, hence providing a more realistic biting environment (WHO, 2009). To conduct the experiment, volunteers should refrain from smoking or using scented products for 12 hours before testing. Prior to testing, volunteers must wash their hands with unscented soap. The volunteers right arms will be covered with treated materials and their left arms will be covered with untreated materials. After inserting both arms into different cages, the data on the number of landings on both hands will be collected. If within three minutes, less than two mosquitoes have landed on the untreated side, then the experiment will stop and the volunteers will remove their hands from the cages due to the fact that the volunteer might be naturally repellent to mosquitoes, therefore causing the results to be inconclusive. (Anuar, 2016) To calculate the percent protection against mosquitoes, the following formula is used. U T p = [ U ] 100% Figure 6: Formula for the protection of mosquito repellents for cage test (Anuar, 2016) In this formula, p is protection, U is the number of mosquito landings in the untreated sample, and T is the number of mosquito landings in the treated sample. (WHO, 2009) Advantages of a cage test include direct observation of mosquitoes landing and probing, thus providing realistic data. A disadvantage of cage test is that direct human involvement is required. Therefore, mosquitoes without disease must be provided and stringent procedure must be observed. (Anuar, 2016) Cone Test

10 The cone test is used to evaluate the toxicity of insecticide-treated bed nets against mosquitoes. This test is especially useful for studying treated materials that are designed to immobilize or kill the mosquito upon landing. One advantage of the cone is that the cone test does not require human participation as a part of the experiment. Instead, artificial blood is used as bait to attract mosquitoes. (Anuar, 2016) In a cone test, a transparent plastic cone with a cork is placed on top of a treated surface. Masking tape is placed on the cone and on the treated surface to secure the sample. Five to ten females are blown into the cone using an aspirator and thus exposed to the surface. The mosquitoes are exposed to the treated surface for three minutes. After the exposure, the mosquitoes are transferred into the plastic cups free from insecticides for further observation. The number of mosquitoes that are immobilized or dead is recorded after twelve hours. To calculate the percent mosquito repellency, the following formula is used: % Mosquito mortality = MR MC MC 100 Figure 7: Formula for calculating the percent mosquito repellency in cone test (WHO, 2009) In this formula, MR stands for the mortality from treated samples and MC stands for the mortality from untreated samples. (Anuar, 2016) Excito Chamber The excito chamber is a customized method for testing mosquito behavior change towards treated material. In the excito chamber test, a modified mosquito cage is used. The

11 mosquito cage will be made from glass panels and contain an entrance and an exit portal. The floor of the cage will be covered by treated materials. (Anuar, 2016) The mosquitoes used in the experiment are starved for four to six hours prior to the experiment. Once the mosquitoes are inputted through the entrance, the entrance is closed off. Observation is recorded ten to thirty minutes after the mosquitoes have been placed in the excito chamber. The percentage of mosquito repellency is modelled by the following formula: % Mosquito repellency = NES + NDE NEX 100 Figure 8: Formula for determining the percent mosquito repellency for excito chamber test (Anuar, 2016) In this equation, NES represents the number of mosquitoes escaped, NDE represents the number of mosquitoes exposed, and NEX represent the total number of mosquitoes exposed to the treated materials. (Anuar, 2016) Field Trials Field trials are conducted after laboratory testing. The objective of field trials is to estimate the optimum dosage, persistence, and efficacy of a repellent material. The recommended field trial procedure by the WHO requires volunteers adept at using aspirators or collecting tubes to catch mosquitoes landing on an exposed limb before the mosquitoes commence to probe. The protection of the repellent is calculated by the formula: p = [1 ( T )] 100% C

12 Figure 9: Formula for determining the percent protection of the repellent (WHO, 2009) In the equation, p is protection, T is the number of times the mosquitoes landed on the attractor in a chamber with the repellents, and C is the number of times the mosquitoes landed on the attractor in a chamber without the repellents. (WHO, 2009)

13 References Khan, A. A., Maibach, H. I., & Skidmore, D. L. (1975). Addition of vanillin to mosquito repellents to increase protection time. Mosq. News, 35, Anuar, A., & Yusof, N. (2016). Methods of imparting mosquito repellent agents and the assessing mosquito repellency on textile. Fashion and Textiles, 3(1), doi: /s y Bremmer, H. J., Blom, W. M., van Hoeven-Arentzen, P. H., Prud'Homme de Lodder, L. C. H., Van Raaij, M. T. M., Straetmans, E. H. F. M.,... & Van Engelen, J. G. M. (2006). Pest Control Products Fact Sheet. To assess the risks for the consumer. Updated version for ConsExpo 4. Brenes, M., García, A., García, P., Rios, J. J., & Garrido, A. (1999). Phenolic compounds in Spanish olive oils. Journal of Agricultural and Food Chemistry, 47(9), doi: /jf990009o Centers for Disease Control and Prevention. (n. d.) Fight the Bite for Protection from Malaria Guidelines for DEET Insect Repellent Use. Christophers, S. R. 1. (1947). Mosquito repellents; being a report of the work of the mosquito repellent inquiry, cambridge England: G. N. SAPKALE, S. M. PATIL, U. S. SURWASE, & P. K. BHATBHAGE. (2010). Supercritical fluid extraction. Int. J. Chem. Sci, Gilles, M., Zhao, J., An, M., & Agboola, S. (2010). Chemical composition and antimicrobial properties of essential oils of three australian eucalyptus species. Food Chemistry, 119(2), doi: /j.foodchem Jiang, Y., Wu, N., Fu, Y., Wang, W., Luo, M., Zhao, C.,... Liu, X. (2011). Chemical composition and antimicrobial activity of the essential oil of rosemary. Environmental Toxicology and Pharmacology, 32(1), doi: /j.etap Lis-Balchin, M., & Hart, S. (1999). Studies on the mode of action of the essential oil of lavender (lavandula angustifolia P. miller). Phytotherapy Research : PTR, 13(6), 540. Retrieved from Masetti, A., & Maini, S. (2006). Arm in cage tests to compare skin repellents against bites of aedes albopictus. Bulletin of Insectology, 59(2), National Center for Biotechnology Information. PubChem Compound Database; CID = 1183, (accessed Dec 7, 2016). National Center for Biotechnology Information. PubChem Compound Database; CID = , (accessed Dec 7, 2016). National Center for Biotechnology Information. PubChem Compound Database; CID = , (accessed Dec 7, 2016).

14 Pin Yang, & Yajun Ma. (2005). Repellent effect of plant essential oils against aedes albopictus. Journal of Vector Ecology : Journal of the Society for Vector Ecology, 30(2), Retrieved from Rao, V. P. (2006). Extraction of essential oil and its applications (Doctoral dissertation, National Institute of Technology Rourkela). Silva, J. B. d., Carrão-Panizzi, M. C., & Prudêncio, S. H. (2009). Chemical and physical composition of grain-type and food-type soybean for food processing. Pesquisa Agropecuária Brasileira, 44(7), doi: /s x Turek, C., & Stintzing, F. C. (2013). Stability of essential oils: a review. Comprehensive Reviews in Food Science and Food Safety, 12(1), VANILIN, O. S. FOREWORD INTRODUCTION. bli, 1, 2. Verma, R. S., Padalia, R. C., & Chauhan, A. (2013). Fragrant volatile oil composition of nutmeg geranium (pelargonium fragrans willd.) from india. Natural Product Research, 27(8), doi: / World Health Organization. Dept. of Control of Neglected Tropical Diseases, & WHO Pesticide Evaluation Scheme. (2006). Guidelines for testing mosquito adulticides for indoor residual spraying and treatment of mosquito nets Geneva : World Health Organization. Retrieved from World Health Organization. Dept. of Control of Neglected Tropical Diseases, & WHO Pesticide Evaluation Scheme. (2009). Guidelines for efficacy testing of mosquito repellents for human skin Geneva : World Health Organization. Retrieved from

15 Research Plan A. The Question The question guiding this science fair experiment is: Will the mosquito repellency of essential oils be more effective when used together? B. The Hypothesis The hypothesis is: The mixture of the natural essential oils lemon eucalyptus and lavender with the additive vanillin will form an effective mosquito repellent as demonstrated through choice chamber experiments. C. Why is this research important? Mosquitoes are common annoyances to people all over the world. In addition, they are vectors for many deadly diseases, including but not limited to dengue, yellow fever, West Nile, and Zika. Many insect repellents were developed to deter mosquitos from approaching humans. However, many of these mosquito repellents contain dangerous chemicals such as DEET, which is carcinogenic and can cause skin rashes, and permethrin, which is toxic to humans and animals, so many new repellents use natural essential oils. Because pure essential oils are less potent and more volatile than DEET, a new essential oil solution is needed. D. Customized Mosquito Chamber Mosquito chambers will be made from clear acrylic boxes. An opening will be made for releasing the mosquitoes inside the chamber. On the bottom of the box, a removable drawer will be inserted. The removable drawer will include an attractor (heat source) to lure the mosquitoes in. Adjacent to the attractor will be the mosquito repellent, which will serve as the deterrent

16 against the mosquitoes. Thirty mosquitoes will be released into the chamber per trial. The effectiveness of the mosquito repellent will be modeled by the following equation: p = [1 ( T )] 100% C In this equation, p is protection, T is the number of times the mosquitoes landed on the attractor in a chamber with the repellents, and C is the number of times the mosquitoes landed on the attractor in a chamber without the repellents. If the protection is greater than 90%, the repellent will be considered very effective. If the protection is greater than 80%, the repellent will be considered effective. If protection is greater than 70%, the repellent will be considered moderately effective. If the protection is greater than 60%, the repellent will be considered slightly effective. If the protection is less than 60%, the repellent will be considered ineffective.