Top 10 Contributions on Biochemistry Chapter 05 The Biomosquitocides Oreosyce africana (Cucurbitaceae) in the Control of Malaria Vector, Anopheles arabiensis Damtew Bekele* Department of Biology, College of Natural and Computational Sciences, Debre Markos University, Ethiopia *Corresponding Author: Damtew Bekele, Department of Biology, College of Natural and Computational Sciences, Debre Markos University, Ethiopia, Email: damtish99@yahoo.com First Published August 13, 2018 This Book Chapter is an excerpt from an article published by Damtew Bekele, et al. at Biochemistry & Analytical Biochemistry in September 2016. (Bekele D, Tekie H, Asfaw Z, Petros B (2016) Bioefficacy of Solvent Fractions of Oreosyce africana and Piper capense against the Malaria Vector, Anopheles arabiensis with High Performance Liquid Chromatographic and Ultraviolet-Visible Spectroscopic Analysis. Biochem Anal Biochem 5:294. doi:10.4172/2161-1009.1000294) Copyright: © 2018 Damtew Bekele. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source. 2 www.avidscience.com Top 10 Contributions on Biochemistry Abstract The efficacy of synthetic organic insecticides is compromised by increased mosquito resistance to insecticides and use of inorganic insecticides raises environmental toxicity concerns. Therefore, O. africana, plant-based biodegradable insecticides would be used as alternatives to synthetic mosquitocides for the control of mosquito. Malaria remains to be one of the most important diseases in tropical countries including Ethiopia. Attempts to wipe out the mosquito vector, which have been pursued for many years, have so far, yielded no tangible results in the country. Plant product has become an important component in organized mosquito control, due to public and user concerns about synthetic insecticides for their adverse effects to the environment and insecticide resistance. Oreosyce africana was used for many ailments, ranging from anti-malarial to anti-gonorrhea treatment and anthelminthic. The qualitative phytochemical analysis of O. africana crude extracts is known to possess bioactive characteristics. The O. africana purified fractions had the most potent mosquitocidal active components with potency of impregnated nets lasted for two months. The structural elucidation of the active ingredients in first fraction was determined using a combination of 1H- 13C-NMR, DEPT-135 and GC-MS measurements. This revealed the presence of linoleic acid (9, 12-Octadecadienoic acid (Z, Z)- as the major chemical constituent and similar analysis of second fraction showed the presence of dibutyl phthalate as the major chemical constituent. Both compounds have proven insecticidal effects. Use of these botanical derivatives in mosquito control instead of synthetic insecticides could reduce the cost of malaria control in addition to reducing environmental pollution. Keywords Cucurbitaceae; Mosquitocide; Oreosyce Africana; Phytoconstituents www.avidscience.com 3 Top 10 Contributions on Biochemistry Introduction The challenges of malaria control include the complexity of disease control process, the complexity of the vectors and expensive cost of the control program and variations in disease patterns and in the transmission dynamics from place to place. In addition, there is resistance of the parasite to drugs and the increase and spread of insecticide resistance. According to studies conducted in Ethiopia [1-5], An. arabiensis was resistant to an array of insecticides, including dichloro-diphenyltrichloroethane (DDT), permethrin, deltamethrin and malathion. In many countries in the Tropics including Ethiopia, people have little access to modern medicine. The family Cucurbitaceae contributes higher number of medicinal species in Ethiopia [6-9]. The leaf part of O. africana is used as an anthelminthic for intestinal worms and its leaf also used as medicine for a burned part [10]. In southeast Tanzania, traditional practitioners make the boil of O. africana with the vegetable gruel for pregnant women to drink which helps makes the birth easy and they also rub themselves with its leaves against trichophytosis [11]. The filtrate obtained from O. africana was reported to be given through hypodermal injection using a syringe to treat gonorrhea [9,12]. The insecticidal properties of plants have been used in Ethiopia, where plant materials are easily available and their use in health practices is a tradition. According to Friedman et al. [13], a study by Bekele et al. [14] showed the people in Akaki district (east-central Ethiopia) traditionally used O. africana’s powder of crushed leaves by sprinkling for mosquito management and control of cattle ticks and other arthropod pests. In another study, O. africana was used for antimalarial treatment [15]. According to World Health Organization report more than 80% of the population in developing countries relies on traditional medicine, and it is now widely accepted that traditional medicines 4 www.avidscience.com Top 10 Contributions on Biochemistry are more affordable, less toxic, and have a wide acceptance around the world [16]. Though several compounds of plant origin have been reported as insecticides-larvicides, there is need for the discovery of more effective plant products particularly in the indigenous flora of lesser studied countries like Ethiopia. Materials and Methods Collection and Identification of Plant Materials The leaves of O. africana Hook. f (Family: Cucurbitaceae) were collected from Yerer Lencho locality of Akaki District (080 50.682’ N, 0380 56.630’ E, altitude 1936 m) in central and western Ethiopia. Plant materials (voucher specimens and materials for extraction and testing) were collected during field trips at the sites. Basic ethnobotanical data focused to the medicinal uses of the target species were collected by interviewing local traditional herbalists and knowledgeable elders. Ethnobotanical data were collected after explaining the purpose of the research and obtaining their consents provided as blessings by those who volunteered to provide information, typical traditional ethical clearance. Voucher specimen no; DB.18 of this species was collected, pressed, dried and authenticity confirmed by taxonomic experts at the Department of Plant Biology and Biodiversity Management and stored at the National Herbarium of Ethiopia, Addis Ababa University for further reference. Fractionation of Oreosyce africana Crude Extracts The dried 80% methanol crude extracts of O. africana was suspended in deionized water and then partitioned with solvents dichloromethane (DCM), ethyl acetate (EtOAc) and deionized water using solvent-solvent extraction at room temperature following the methods of Alkofahi et al. [17]. Portions of 60 g and 65 g of the 80% methanol crude extracts of O. africana was suspended in 600 ml deionized www.avidscience.com 5 Top 10 Contributions on Biochemistry water in separatory funnel and each of them partitioned with 1200 ml dichloromethane. Fractions of each solvent were filtered using Whatman no. 1 filter paper. The mixture was allowed to settle for one day, after which the solvent fraction lower layer was slowly drawn off until only the upper layer remained, and partitions were combined and evaporated at 45oC and labelled as fractions DOF for dichloromethane O. fraction. Portions of 61 g and 60 g of the crude extracts of O. africana was suspended in 600 ml deionized water in a separatory funnel and each of them partitioned with 1200 ml ethyl acetate. The ethyl acetate upper layer filtrates were combined and evaporated to give ethyl acetate O. fraction (EOF). Finally, each of the water residual layer and the solution were evaporated and lyophilized to dryness and labelled as water O. fraction (WOF). Each fraction thus obtained, was filtered and concentrated using rotary vacuum evaporator at 45°C and the dried material was subjected to adulticidal bioassays. These fractions which exhibited potent adulticidal activity against An. arabiensis adults were chosen for further bioassay-guided test. Rearing Anopheles Arabiensis Patton Eggs of An. arabiensis for starting a colony were obtained from the Ethiopian Public Health Institute (EPHI) and reared according to the World Health Organization [18] protocol. The colonies were reared and maintained at 25-27oC temperatures and 70-80% relative humidity and 12:12 light and dark photoperiod cycle at the insectary at College of Natural Sciences, Addis Ababa University. Glass Petri dishes (10.5 diameter) lined with wet filter paper were kept inside the cages for oviposition, then eggs laid on the filter paper were transferred to plastic and enamel trays containing 3 liters distilled water and allowed to hatch to first instar larvae and kept until they reach the fourth instar larvae. The larvae were fed on ground Tetramin® fish food pellets (Tetra holding Inc., Blacksburg, VA, USA); the feed was applied on alternate days for normal development. Water of the larval culture was changed every third day to avoid decay. After attaining pupae, they were transferred to cups by disposable pipettes and kept inside the mosquito cages for adult emergence. Anopheles arabiensis 6 www.avidscience.com Top 10 Contributions on Biochemistry adults were kept in cages of 30 cubic cm and they were continuously provided with 10% sucrose solution with cotton wicks by placing on the top of each cage. Adult female An. arabiensis were periodically blood-fed on restrained, shaved back and belly rabbits for egg production. Adulticidal Bioassays Twenty females mosquitoes (2-5 day old glucose fed, blood starved) were collected and gently transferred into a plastic holding tube. The mosquitoes were allowed to acclimatize in the holding tube for 1 hr and then exposed to test paper for 1 hr. At the end of the exposure period, the mosquitoes were transferred back to the holding tube and kept 24 hrs for recovery period. During the test period a pad of cotton soaked with 10% glucose solution was placed on the mesh screen. Mortality of mosquitoes was determined at the end of 24 hrs recovery period. Characterization of Pure Constituents The identity of fraction IV, B2’O and B2”O from O. africana were analyzed using nuclear magnetic resonance (NMR) spectroscopy and gas chromatography-mass spectrometry (GC-MS). Nuclear magnetic resonance spectroscopy 1 H and 13C-Nuclear Magnetic Resonance (NMR) spectra were recorded using Brucker Avance 400 MHz NMR spectrometer at the Department of Chemistry, Addis Ababa University. The NMR analysis for the isolated fraction of O. africana was made and their spectra were recorded at room temperature in deuterated chloroform (CDCl3). The chemical shifts (δ) are reported in parts per million (ppm). For the 13C-NMR spectra, multiplicities were determined by Distortionless Enhancement by Polarization Transfer (DEPT) method. The chemical structures were proposed based on the interpretation of the combined spectra. www.avidscience.com 7 Top 10 Contributions on Biochemistry Gas Chromatography-Mass Spectrometry (GC-MS) Identification of the compounds was carried out by GC-MS analysis at Governors state University, USA. Gas chromatography-mass spectrometry spectra were recorded with Agilent Technologies 7890B GC system, which was combined with Agilent Technologies 6977A MS system. Data Analysis To test variations in An. arabiensis adult mortalities using crude methanol extracts of O. africana, and fractions including DOF, EOF, WOF, the LC50 and LC90 values were determined using probit regression analysis of the statistical package PoloPlus (version 2.0, LeOra Software, Petaluma, California, USA; 2007). Adulticidal Activity of Oreosyce Africana against Anopheles Arabiensis upon Fractionation The adulticidal effect of 80% methanol crude extract of O. africana leaves and fractions DOF, EOF and WOF were evaluated on An. arabiensis at 4, 8, 16 and 32 ppm after 24 hrs exposure on impregnated papers using WHO test tubes (Table 1). 8 www.avidscience.com Top 10 Contributions on Biochemistry Table 1: Evaluation of the effect of 80% methanolic crude extract and solvent fractions of O. africana on An. arabiensis adults after 24 hrs exposure (n=60 in each test). Extract LC50 ppm (95% CL) LC90 ppm (95% CL) Slope±SE Tested* Crude extract 13.019 (9.981-16.982) 68.369 (41.861-111.656) 1.779±0.207 DOF 4.267 (1.325-6.451) 14.123 (9.268-49.248) 2.466+0.289 χ2(P) LC50 ratio (95% CL) 5 . 9 0 ND a (0.56) 3.127 3.051(2.3393.980)b (0.47)a EOF 14.562 (13.720-25.292) 82.57 (69.094-98.728) 1.701+0.206 WOF 16.973 (13.456-17.209) 117.877 (88.475-157.372) 1.523+0.204 5 . 0 8 6 (0.53)a 6.65 0.894(0.6921.154) 0.767 (0.5801.1014 (0.48)a Negative control** 0.0 0.0 0.0 0.0 0.0 * The codes used for the fractions was the same as in Table 1; ** DMSO (0.05%) in deionized water; a Good fit of the data to the probit model (P >0.05); b LC50 ratio significant at P <0.05; 95% confidence interval did not comprise the value 1.0; ND-Not determined Among the extracts, dichloromethane fraction of O. africana with LC50 at 4.267 and LC90 at 14.123 ppm showed potent adulticidal effect than crude extract, EOF and WOF (Table 1). The adulticidal effect of 80% methanol crude extract of O. africana leaves and fraction DOF were evaluated on An. arabiensis at 4, 8, 16 and 32 ppm after 24 hrs exposure on impregnated papers using WHO test tubes (Figure 1). www.avidscience.com 9 Top 10 Contributions on Biochemistry Figure 1: Percentage mortality of An. arabiensis after treatment with different concentrations of crude extract and dichloromethane fraction of O. africana. Comparison of DOF with 80% methanol crude extract of O. africana showed dichloromethane fraction of O. africana had more adulticidal effect against An. arabiensis (99% mortality at 32 ppm) than the crude extract, EOF and WOF (Figure 1). The hypothesis test for parallelism was rejected (χ2 = 8.46; P = 0.003) showing that slopes differed significantly. Effects of Purified Potent Fractions of O. Africana against Adult An. Arabiensis Five purified fractions of O. africana exhibited different levels of adulticidal activity as shown in Figure 2. 10 www.avidscience.com Top 10 Contributions on Biochemistry Figure 2: Measure of mortality of Anopheles arabiensis adults following after 24 hrs exposure period on impregnated papers with fractions of O. africana in WHO test tubes (n=45 in each test). With regard to treatment using WHO impregnated papers against adult An. arabiensis with O. africana purified fractions II, III, IV, B2’O and B2’’O at 8 ppm concentration each resulted in 49, 41, 89, 89 And 80% Mortality, Respectively (Figure 2). Chemical Analysis of Oreosyce Africana Fraction The dichloromethane extract of leaves of O. africana were further separated by preparative thin layer chromatography yielded biological active fractions (fractions, B2’O, B2’’O, and IV) and the structure of the compound was determined by spectroscopic means (1H- and 13CNMR, DEPT and GC-MS) and upon comparison with the reported spectral data (Figure 3-5). www.avidscience.com 11 Top 10 Contributions on Biochemistry Figure 3: Chemical structure of a 9, 12-Octadecadienoic acid (Z,Z)- compound in the first fraction (B2’O) isolated from O. africana leaves Figure 4: Chemical structure of dibutyl phthalate in the second fraction (B2’’O) isolated from O. africana leaves The NMR spectral analysis in combination with GC-MS of fraction IV is in agreement with linoleic acid whose structure is depicted in Figure 5. 12 www.avidscience.com Top 10 Contributions on Biochemistry Figure 5: Chemical structure of linoleic acid for the fraction IV of O. africana Discussion The present study noted that purified fractions of O. africana (leaf extract) possess a very high adulticidal effect against An. arabiensis. It can be considered as effective adulticide based on WHO standards as it produces >60% mortality against the adult stages of the malaria vector An. arabiensis [19]. Data from O. africana leaf extracts against An. arabiensis showed converse relationship between extract efficacy and solvent polarity where efficacy increased as polarity decreases. Overgaard et al. [20] also reported that showed decline in mortality of mosquitoes with increasing solvent polarity of a mosquitocidal plant extract. Moreover, Shaalan et al. [21] described that the bioactivity of phytochemicals against mosquito can vary significantly depending on plant species and solvent used in extraction. Therefore, it is to be expected that since different phytoconstituents dissolve in specific solvents Sukumar et al. [22], the DCM fraction of O. africana would contain constituents with demonstrated adulticidal activity in the present study. Hidayatulfathi et al. [23] indicates that the bioactive components from Acorus calamus (Acoraceae) responsible for the lethal effect on the adults were extracted in greater measures with certain solvents. However, this is not consistent due to differences between the characteristics of active chemicals among plants. From this it is clear that the bioactive components responsible for the lethal effect on mosquitoes were extracted in greater measures with certain solvents only and not with all. www.avidscience.com 13 Top 10 Contributions on Biochemistry The high mosquitocidal activity of DCM fraction of O. africana is consistent with the report of Broussalis et al. [24] for the dichloromethane extracts of Tagetes erecta L. (Fabaceae/Compositae) which showed a significant pesticidal activity against Sitophilus oryzae. In the current study, the higher activity of DCM fraction of O. africana may be due to the presence of bioactive components against adult stage of An. arabiensis. This indicates that the bioactive components in this plant had adulticidal properties against An. arabiensis and were better soluble in DCM than in other solvents. Fractionation of the extracts allowed to minimize the number of compounds in each solvent extract tested for mosquitocidal effect. As the purpose of fractionating crude extracts for bioactivity is to extract as many potentially active constituents as possible, the observed weak to very strong adulticidal effects of the different solvent fractions was an indication that the plant extracts consisted of different phytochemicals with varying adulticidal potencies. The isolates from the leaves extract of O. africana produce a broad spectrum of insecticidal and other bioactive compounds. The larvicidal effect of dichloromethane fraction of O. africana, of which linoleic acid is the major component, is proof to its broad spectrum of bioactivity. Furthermore, the broad spectrum of bioactivity of linoleic acid is evident from its inhibition of parasitemia in mice infected with Plasmodium vinckei and Plasmodium yoelii in a 4-day suppressive test [25]. Ramos-López et al. [26] had reported that linoleic acid isolated from Ricinus communis has insecticidal activity against Spodoptera frugiperda (Noctuidae). The extracts of Annona squamosa and Annona muricata (Annonaceae) contained linoleic acid against adults of Aedes albopictus and Culex quinquefasciatus had significant insecticidal effects compared to mortality induced by deltamethrin [27]. Two purified fractions of O. africana (B2’O and B2’’O), purified using preparative thin-layer chromatography had the potent adulticidal activities against An. arabiensis with an LC50 of 2.206 and LC90 of 7.811 ppm and with an LC50 of 2.62 and LC90 of 11.779 ppm at 24 hrs post-exposure, respectively. The fact that percentage mortality 14 www.avidscience.com Top 10 Contributions on Biochemistry of An. arabiensis adults increased significantly with exposure time in the WHO test tube bioassay of purified fractions of (B2’O and B2’’O), indicates the exposure-time dependence of their lethal effect. Specific evidence for linoleic acid, the chemical compound identified as larvicide in this study, as a larvicide exists from Citrullus colocynthis (L.) Schrad (Cucurbitaceae) with an LC50 value of 9.79 ppm and LC90 value of 37.42 ppm against fourth instar larvae of An. stephensi Liston [28]. Another study by Duke [29] also described that Citrullus colocynthis constituted a mixture of fatty acids which had the mosquito larvicidal activity. The structural elucidation of the active ingredients using GCMS analysis showed the presence of 9,12-Octadecadienoic acid (Z,Z) as the major chemical constituent (98.35%) in the first fraction and dibutyl phthalate as the major chemical constituent (97.75%) in the second fraction. The chemical components of the second fraction also have been reported to possess insecticidal bioactivities. When used as a solvent, dibutyl phthalate is in oil-soluble dyes, insecticides, peroxides and other organic compounds (European Chemicals Agency (ECHA), [30]). Dibutyl phthalate and permethrin impregnated clothing provides good protection against chigger mite - the vectors of scrub typhus [31]. Therefore, it is possible that the compounds from O. africana could synergistically or independently cause mosquitocidal effects. Basalah et al. [32] reported one of the main chemical constituent of Citrullus colocynthis Linn. (Cucurbitaceae) is linoleic acid. Sayed et al. [33] also proved the isolation and identification of linoleic acids from Citrullus colocynthis. The adulticidal activity of linoleic acid containing product in the first fraction of the present study, on bioassay test against An. arabiensis with residual activity persisting for up to two months, is a promising discovery in view of the reported decline after two to four months in the residual activity of permethrin, the commonly used synthetic insecticide for mosquito net impregnation [34,35]. Furthermore, the existence of evidence for organic insecticides such as Azadiracthin [36] as a replacement for inorganic/synthetic insecticides, is a good indication for considering the linoleic www.avidscience.com 15 Top 10 Contributions on Biochemistry acid and dibutyl phthalate containing products identified from O. africana in the present study. Today, only a few percent of the population produces the agricultural products needed for our industrialized society. However, recently the Ethiopian Science and Technology Ministry has initiated a preliminary study on indigenous medicinal plants knowledge so as to identify, describe, document and disseminate the existing indigenous knowledge in the country. Therefore, new strategies are constantly being developed to produce high yield and high-quality plant products. Perhaps the brightest future for specializing in plant products will be using O. africana extracts that was found to be effective in the small scale industries and their efficiently control the malaria mosquito by organized vector control agencies. Balandrin et al. [37] described the most economically important of the natural plant compounds used in commercial insect control. Education of the user is also necessary and is best achieved through both the efforts of the producers and county extension agents. The products of O. africana are expected to be patented and it is believed that these results will benefit most agricultural nations. Environmental concern and natural resource development programs have necessitated the utilization of appropriate technological and management techniques in an integrated approach to bring about an effective degree of vector suppression. Therefore, plant derived insecticides will minimize the problem of induction of resistance in the mosquito population and their environmental benefits considerable which will apparently continue to render the extracts effective for a long time as malaria mosquito control agents. Conclusions Substitution of synthetic insecticides with extracts of O. africana is promising adulticidal plants for impregnation into mosquito nets could have good potential for malaria vector control program at the domestic level. Therefore, it is desirable to use this bioactive plant as 16 www.avidscience.com Top 10 Contributions on Biochemistry alternative agents for the control of mosquitoes as they are locally available, potentially less expensive, they can be propagated easily and sustainable, biodegradable and have a low environmental impact. Moreover, in order to accelerate the acceptability of this plant-based vector control agents, it will be necessary to develop formulations suitable for field applications and to promote large-scale cultivation required to provide the raw material base. References 1. Abose T, Yeebiyo Y, Olana D, Alamirew D, Beyene Y, et al. Reorientation and definition of the role of malaria vector control in Ethiopia. WHO/MAL. 1998; 1085: 31. 2. Balkew M, Gebre-Michael T, Hailu A. Insecticide susceptibility level of Anopheles arabiensis in two agro-development localities in Eastern Ethiopia. Parasitologia. 2003; 45: 1-3. 3. Yewhalaw D, Van Bortel W, Denis L, Coosemans M, Duchateau L, et al. First evidence of high knockdown resistance frequency in Anopheles arabiensis (Diptera: Culicidae) from Ethiopia. Am J Trop Med Hyg. 2010; 83: 122-125. 4. Abate A, Hadis M. Susceptibility of Anopheles gambiae s.l. to DDT, malathion, permethrin and deltamethrin in Ethiopia. Trop Med Int Hlth. 2011; 16: 486-491. 5. Yewhalaw D, Wassie F, Steurbaut W, Spanoghe P, Bortel WV, et al. Multiple insecticide resistance: an impediment to insecticide-based malaria vector control programme. Plos One. 2011; 6: e16066. 6. Giday M, Ameni G. An ethnobotanical survey on plants of veterinary importance in two woredas of Southern Tigray, Northern Ethiopia. SINET: Ethiop J Sci. 2003; 26: 123-136. 7. Balemie K, Kelbessa E, Asfaw Z. Indigenous medicinal plant utilization, management and threats in Fentalle area, Eastern Shewa, Ethiopia. Ethiop J Biol Sci. 2004; 3: 37–58. www.avidscience.com 17 Top 10 Contributions on Biochemistry 8. Hunde D, Asfaw Z, Kelbessa E. Use and management of ethnoveterinary medicinal plants by indigenous people in ‘Boosat’, Wolenchiti area. Ethiop J Biol Sci. 2004; 3: 113–132. 9. Yineger H, Yewhalaw D. Traditional medicinal plant knowledge and use by local healers in Sekoru district, Jimma Zone, south-west Ethiopia. J Ethnobiol Ethnomed. 2007; 3: 24. 10. Yamada T. A report on the ethnobotany of the Nyindu in the eastern part of the former Zaire. African Study Monographs. 1999; 20: 1-72. 11. Neuwinger H, Porter A. African Ethnobotany: Poisons and Drugs: Chemistry, Pharmacology, Toxicology. London: Chapman and Hall. 1996; 1996: 941. 12. Yineger H, Yewhalaw D, Teketay D. Ethnomedicinal plant knowledge and practice of the Oromo ethnic group in southwestern Ethiopia. J Ethnobiol Ethnomed. 2008; 4: 11. 13. Friedman J, Yaniv Z, Dafni A, Palewitch D. A preliminary classification of the healing potential of medicinal plants, based on a rational analysis of an ethnopharmacological field survey among Bedouins in the Negev Desert, Israel. J. Ethnopharmacol. 1986; 16: 275–287. 14. Bekele D, Asfaw Z, Petros B, Tekie H. Ethnobotanical study of plants used for protection against insect bite and for the treatment of livestock health problems in rural areas of Akaki district, Eastern Shewa, Ethiopia. Topclass J Herbal Med. 2012; 1: 40-52. 15. Chifundera K. Contribution to the inventory of medicinal plants from the Bushi area, South Kivu Province, Democratic Republic of Congo. Fitoterapia. 2001;72: 351-368. 18 www.avidscience.com Top 10 Contributions on Biochemistry 16. WHO. Traditional Medicine: Growing Needs and Potential. WHO Policy Perspectives on Medicines. Geneva: World Health Organization. 2002; 1-6. 17. Alkofahi A, Rupprecht JK, Anderson JE, Mclaughlin JL, Mikolajczak KL et al. Search for new pesticides from higher plants. In: Arnason JT, Philogens BJR, Morand P, editors. Insecticides of plant origin. ACS Symp Ser 387. Washington: American Chemical Society. 1989; 25-43. 18. WHO. Guidelines for laboratory and field testing of mosquito larvicides. Geneva: WHO/CDS/WHOPES/GCDPP/2005. 2005; 41. 19. Maharaj R, Maharaj V, Crouch NR, Bhagwandin N, Folb PI, et al. Screening for adulticidal bioactivity of South African plants against Anopheles arabiensis. Malaria. J. 2011; 10: 233. 20. Overgaard HJ, Sirisopa P, Mikolo B, Malterud KE, Wangensteen H, et al. Insecticidal activities of bark, leaf and seed extracts of Zanthoxylum heitzii against the African malaria vector Anopheles gambiae. Molecules. 2014; 19: 2127621290. 21. Shaalan EA, Canyon D, Younes MWF, Abdel-Wahab H, Mansour A. A review of botanical phytochemicals with mosquitocidal potential. Environ Int. 2005; 31: 1149-1166. 22. Sukumar K, Perich MJ, Boobar LR. Botanical derivative in mosquito control: a review. J Am Mosq Control Assoc. 1991; 7: 210-237. 23. Hidayatulfathi O, Sallehuddin S, Ibrahim J. Adulticidal activity of some Malaysian plant extracts against Aedes aegypti L. Trop Biomed. 2004; 21: 61-67. www.avidscience.com 19 Top 10 Contributions on Biochemistry 24. Broussalis AM, Ferraro GE, Martino VS, Pinzo´n R, Coussio JD. Argentine plants as potential source of insecticidal compounds. J Ethnopharmacol. 1989; 67: 219–223. 25. Krugliak M, Deharo E, Shalmiev G, Sauvain M, Moretti C, et al. Antimalarial effects of C18 fatty acids on Plasmodium falciparum in culture and on Plasmodium vinckei petteri and Plasmodium yoelii nigeriensis in vivo. Exp Parasitol. 1995; 81: 97–105. 26. Ramos-López MA, González-Chávez MM, Cárdenas-Ortega NC, Zavala-Sánchez MA, Pérez GS. Activity of the main fatty acid components of the hexane leaf extract of Ricinus communis against Spodoptera frugiperda. AJB. 2012; 11: 4274-4278. 27. Ravaomanarivo LHR, Razafindraleva HA, Raharimalala FN, Rasoahantaveloniaina B, Ravelonandro PH, et al. Efficacy of seed extracts of Annona squamosa and Annona muricata (Annonaceae) for the control of Aedes albopictus and Culex quinquefasciatus (Culicidae). Asian Pac J Trop Biomed. 2014; 4: 798-806. 28. Rahuman AA, Venkatesan P, Gopalakrishnan G. Mosquito larvicidal activity of oleic and linoleic acids isolated from Citrullus colocynthis (Linn.) Schrad. Parasitol Res. 2008; 103: 1383-1390. 29. Duke JA. Promising phytomedicinals. In: J Janick, JE Simon, editors. Advances in new crops. Portland: Timber Press. 1990; 491–498. 30. ECHA. Background document for dibutyl phthalate, developed in the context of ECHA first Recommendation for the inclusion of substances in Annex XIV, 1 June 2009. 2009. http://echa.europa.eu/doc/authorisation/annex_xiv_rec/ subs_spec_background_docs/d 20 www.avidscience.com Top 10 Contributions on Biochemistry 31. Frances SP, Yeo AE, Brooke EW, Sweeney AW. Clothing impregnations of dibutyl phthalate and permethrin as protectants against a chigger mite, Eutrombicula hirsti (Acari: Trombiculidae). J Med Entomol. 1992; 29: 907- 910. 32. Basalah MO, Ali Whaibi MH, Sher M. Comparative study of some metabolities of Citrullus colocynthis Schrad and Cucumis prophetarum L. J Biol Sci Res. 1985; 16: 105-123. 33. Sayed MD, Balbaa S, Afifi MS. Lipid content of the seeds of Citrullus Colocynthus. Planta Med. 1973; 24: 41–45. 34. Darriet F, Robert V, Tho Vien N, Carvenale P. Evaluation of the efficacy of permethrin-impregnated intact and perforated mosquito nets against vectors of malaria. WHO mimeographed document WHO/VBC. 1984; 84-899. 35. Lines JD, Myamba J, Curtis CF. Experimental hut trials of permethrin impregnated nets anddeve curtains against malaria vectors in Tanzania. Med Vet Ento. 1987; 1: 37-51. 36. Shivakumar MS, Purohit H, Annasamundram S, Patel PV. Efficacy of azadirachtin treated nets on adults of Aedes aegypti and Culex quinquefasciatus ( Diptera: Culicidae). J Ecobiotechno. 2010; 2: 76-79. 37. Balandrin MF, Klocke JA, Wurtele ES, Bollinger WH. Natural plant chemicals: Sources of industrial and medical materials. Science, new series. 1985; 228: 1154-1160. www.avidscience.com 21