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Potential of crude seed extract of celery, Apium graveolens L., against the mosquito Aedes aegypti (L.) (Diptera: Culicidae)
Wej Choochote, Benjawan Tuetun, Duangta Kanjanapothi1, Eumporn Rattanachanpichai, Udom Chaithong, Prasong Chaiwong, Atchariya Jitpakdi, Pongsri Tippawangkosol, Doungrat Riyong, and Benjawan Pitasawat
Department of Parasitology, Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailand 1 Chulabhorn Research Institute, Chiang Mai 50200, Thailand Received 16 April 2004; Accepted 14 May 2004 ABSTRACT: Crude seed extract of celery, Apium graveolens, was investigated for anti-mosquito potential, including larvicidal, adulticidal, and repellent activities against Aedes aegypti, the vector of dengue haemorrhagic fever. The ethanol-extracted A. graveolens possessed larvicidal activity against fourth instar larvae of Ae. aegypti with LD50 and LD95 values of 81.0 and 176.8 mg/L, respectively. The abnormal movement observed in treated larvae indicated that the toxic effect of A. graveolens extract was probably on the nervous system. In testing for adulticidal activity, this plant extract exhibited a slightly adulticidal potency with LD50 and LD95 values of 6.6 and 66.4 mg/cm2, respectively. It showed repellency against Ae. aegypti adult females with ED50 and ED95 values of 2.03 and 28.12 mg/cm2, respectively. It also provided biting protection time of 3 h when applied at a concentration of 25 g%. Topical application of the ethanol-extracted A. graveolens did not induce dermal irritation. No adverse effects on the skin or other parts of the body of human volunteers were observed during 3 mo of the study period or in the following 3 mo, after which time observations ceased. A. graveolens, therefore, can be considered as a probable source of some biologically active compounds used in the development of mosquito control agents, particularly repellent products. Journal of Vector Ecology 29 (2): 340-346. 2004. Keyword Index: Apium graveolens, Aedes aegypti, larvicidal, adulticidal, repellent. INTRODUCTION Aedes aegypti (L.) is generally known as a vector for an arbovirus responsible for dengue fever, which is endemic to Southeast Asia, the Pacific island area, Africa, and the Americas. This mosquito is also the vector of yellow fever in Central and South America and West Africa. Dengue fever has become an important public health problem as the number of reported cases continue to increase, especially with more severe forms of the disease, dengue haemorrhagic fever and dengue shock syndrome, or with unusual manifestations such as central nervous system involvement (Hendarto and Hadinegoro 1992, Pancharoen et al. 2002). About two-fifths of the worlds population are now at risk of catching dengue according to the World Health Organization (WHO 2003). At present, the most successful measures to decrease the incidence of this disease are by personal protection and control of the vector, Ae. aegypti. Although there has been much historical research on the potential of natural plants to protect against mosquitoes and other insect pests (Granett 1940), the interest in herbal-based products was subsequently reduced due to the advent of synthetic chemicals. However, the interest in anti-mosquito products derived from natural origin is being revived because the continued applications of synthetic compounds have some drawbacks, including the widespread development of insecticide resistance. Celery, Apium graveolens (Umbelliferae), commonly known in Thailand as Khuen chaai, is a native of Eurasia and is now grown and consumed all over the world. Leaf stalks and celery seed are used as a popular aromatic herb and spice (Rafikali and Muraleednaran 2001, Kitajima et al. 2003). Certain bioactive compounds derived from A. graveolens seeds have been proven to possess nematocidal activity against Caenorhabditis elegans and Panagrellus redivivus,

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antifungal activity against Candida albican, C. kruseii and C. parapsilasis, and mosquitocidal effects against Ae. aegypti fourth-instar larvae (Rafikali et al. 2000, Rafikali and Muraleednaran 2001). The literature, however, offers no data about the adulticidal and repellent activities of this plant against mosquito vectors. The aim of this work was to investigate the possible anti-mosquito potential of A. graveolens extract against Ae. aegypti, the major vector of dengue haemorrhagic fever in Thailand (Limrat 1997, Saengtharatip 1997), in the search for an alternative natural product that can be developed and practically used in the control of recurrent dengue epidemics. MATERIALS AND METHODS Plant extract Seeds of celery, Apium graveolens, were obtained from E.A.R. Samunpri, a commercial supplier in Chiang Mai province, Thailand. A voucher named PARA-AP001 was deposited at the Department of Parasitology, Faculty of Medicine, Chiang Mai University, Thailand. Dried and powdered material of this plant (2 kg) was successively extracted three times by maceration, with 3 L of 95% ethanol at room temperature for 2 d. An ethanolic extract was suction filtered through a Buchner funnel and the combined filtrates were concentrated by a rotary evaporator at 60C until the solvent completely evaporated. The residue of ethanol-extracted A. graveolens was thus obtained, lyophilized, and then kept at -20C until testing for larval and adult toxicities and repellent activity. In preparing test concentrations, the lyophilized A. graveolens extract was volumetrically diluted in absolute ethanol and/or Tween 80 at an appropriate test concentration. Test mosquitoes Aedes aegypti larvae, which were derived from various places with clean stagnant water within Chiang Mai province, northern Thailand, were colonized and maintained continuously for several generations since 1997 in a laboratory free of exposure to pathogens, insecticides, or repellents. The laboratory colony was maintained at 25-30C and 80-90% relative humidity under a photoperiod of 14:10 h (light/dark) in the insectary of the Department of Parasitology, Faculty of Medicine, Chiang Mai University, Chiang Mai province. Under these conditions, the full development from egg to adult lasted about 3-4 wk. Larvae were fed on finelyground dog biscuit. The adult colony was provided with 10% sucrose and 10% multivitamin syrup, and it was periodically blood-fed on restrained rats. Two developmental stages, larvae and adult females, were

Human volunteers Healthy volunteers of both sexes (aged 16-50 y; weight 43-65 kg), with no history of allergic reactions to arthropod bites, were recruited from the students and staff of the Department of Parasitology, Faculty of Medicine, Chiang Mai University, and they gave written informed consent before participating in repellent bioassays. The repellent study was reviewed and approved by the Research Ethics Committee of the Faculty of Medicine, Chiang Mai University. Larvicidal bioassay The larvicidal bioassay followed the WHO standard protocols (WHO 1981a) with slight modifications. For experimental treatment, one ml of A. graveolens extract dissolved in absolute ethanol was added to 224 ml of distilled water in a 500-ml enamel bowl, which was shaken lightly to ensure a homogeneous test solution. Then 25 early fourth instar larvae of Ae. aegypti in 25 ml of distilled water were transferred to that bowl. Each experiment was performed in 4 replicates with a final total of 100 larvae for each concentration. The control solution was made with 1 ml of ethanol mixed with 249 ml of distilled water, while the untreated solution contained 250 ml of distilled water only. Symptoms of treated larvae were observed and recorded immediately and at timed intervals, and no food was offered to the larvae. Mortality and survival were registered after 24 h of the exposure period. The moribund and dead larvae in four replicates were combined and expressed as a percentage of larval mortality of each concentration. Dead larvae were identified when they failed to move after probing with a needle in the siphon or cervical region. Moribund larvae were those incapable of rising to the surface (within a reasonable period of time) or showing the characteristic diving reaction when the water was disturbed. They might also show discoloration, unnatural positions, tremors, uncoordination, or rigor. All surviving larvae were separately reared and maintained at 25-30C and 80-90% relative humidity in the insectary. Pupation and adult emergence of these mosquitoes were recorded. The assays were terminated 3 d after the last control mosquito emerged. The experiments were replicated four times. Adulticidal bioassay The adulticidal effect of the ethanol-extracted A. graveolens was assayed following a slightly modified version of the WHO standard method (WHO 1981b) of insecticidal deposits on a paper surface. Aliquots of 1.9 ml of 1:2 preparation of appropriate concentrations of

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ethanol-extracted A. graveolens in Tween 80 or Tween 80 only (for the control group) and ether mixture were applied to Whatman # 1 filter paper (12x15 cm). The impregnated paper was hung under a shade overnight, by which time all the ether had evaporated and the ethanol-extracted A. graveolens solution had been spread evenly. The blood-deprived 5-7-d-old Ae. aegypti females were then collected and gently transferred to plastic holding tubes at 25 mosquitoes per tube. The mosquitoes were allowed to acclimatize in the holding tube for 1 h before being transferred to a plastic exposure tube lined with the ethanol-extracted A. graveolens impregnated paper, and they were exposed for 1 h. At the end of the exposure period, the mosquitoes were transferred back to the holding tube, which was kept for 24 h. A pad of cotton wool soaked with 10% sucrose and 10% multivitamin syrup was placed on the end of the mesh screen. Four replicates were run for each concentration, yielding a final total of 100 mosquitoes. Percentage mortality was observed 24 h later and mortality was determined when the mosquitoes did not respond to mechanical stimulation. All treatments were replicated four times at 25-30C. Repellent bioassay Two different treatment methods (dose-response study and protection time determination) were used to determine the repellent activity of the ethanol-extracted A. graveolens against laboratory-reared Ae. aegypti after they were applied to human skin. In the dose-response test, the procedure for determining effective dosages of the plant extract against hungry mosquitoes was a modification of the American Society for Testing and Materials Standard ED 951-83 (ASTM 1983). Tests were based on the variable dose-fixed time, free choice method described by Buescher et al. (1982) and were similar to the method described by Coleman et al. (1993, 1994). Because Ae. aegypti is a day biter, the timing of tests was between 08.00 h and 16.00 h. Evaluations were carried out in a 10x10x3 m room, at 25-30C, and relative humidity of 60-80%. Five circles (29 mm in diameter) were outlined on the ventral surface of the volunteers forearm using a plastic template and permanent marker. Applications of 25 ml of the diluent (control) and four serial dilutions of the ethanol-extracted A. graveolens in absolute ethanol were applied randomly on the marked areas. After air drying for 5 min, a plastic cage (4x5x18 cm), divided into 5 compartments, was secured over the area with rubber bands. Each compartment of the plastic cage, with a matching cutout on its floor, contained 10 blood-starved 5-7-d-old Ae. aegypti females. The number of mosquitoes biting on each test site was recorded each minute for 5 min. Tests were carried out three times on

each repellent-treated area and completed within 25 min of repellent application. The experiments were conducted four times on each subject of four human volunteers (2 females, 2 males). Laboratory repellent tests were also conducted to determine the repellent protection time using the humanbait technique of the WHO (1996) standard method, with some modifications. A plastic sleeve was wrapped around each forearm, with a hole cut in alignment with a 3x10 cm area on the inside part of the forearm, and attached with double-sided tape. Thus, only a restricted area of skin was exposed to the mosquitoes, with the hand being protected with a rubber glove. Approximately 0.1 ml of 25 g % ethanol-extracted A. graveolens dissolved in absolute ethanol was spread as evenly as possible on the 30 cm2 test area of one forearm of each volunteer. The other forearm, acting as a control, was treated with absolute ethanol by the same procedure as that for the test repellent. After air drying for 1 min, the test arm was put into a 30x30x30 cm3 cage containing 200 unfed mosquitoes for 3 min. The mosquitoes that landed and attempted to probe and imbibe any blood were recorded. If no mosquito bites occurred in the initial 3 min, the arm was withdrawn from the cage and re-tested every 30 min. The period of repellent protection was then calculated as the time between the extract application and the time when at least two mosquitoes bit in the same 3-min exposure or the time when only one mosquito bit in one exposure period if another bit in the next exposure period (30 min later). When no confirming bites were observed in the period after the initial bite, the treated arm resumed the test until a confirming bite was recorded. During the experiment, successive introductions of the control arm were made in the same manner, prior to inserting the treated arm, in order to confirm the readiness of the mosquitoes to bite. The same test was repeated on each subject of 4 human volunteers (2 females, 2 males). The median complete-protection time was used as a standard measure of the extract repellency against adult female Ae. aegypti in the laboratory. Data management and statistical analysis It was important to obtain not less than 3 mortality (repellency) counts of between 10% and 90%. In cases where the control mortality (larvicidal and adulticidal tests) or control non-biting (repellent test) was between 5-20%, the observed percentage mortality (%M) or repellency (%R) was corrected by Abbotts formula (Abbott 1925):
%M (%R) = % test mortality (non-biting)

% control mortality (non-biting)

100 - % control mortality (non-biting)

X 100

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Data for the anti-mosquito potential (larvicidal, adulticidal, and repellent effects) were analyzed by means of computerized probit analysis (Harvard Programming; Hg1, 2), yielding a level of effectiveness at 50% and 95% mortality or repellency, and 95% confidence intervals (95% C.I.). RESULTS AND DISCUSSION Ethanolic extract of A. graveolens, with a yield of 1.14% (w/w), was semi-solid, light brownish, and slightly aromatic. Larvicidal activity of this plant extract against fourth instar larvae of Ae. aegypti is shown in Table 1. The susceptibility of Ae. aegypti to serial dilutions of the ethanol-extracted A. graveolens was dose dependent. Increasing the plant extract level from 40 to 120 mg/L increased the larval mortality range from 3 to 100%. High mortality (> 50% mortality) values were observed at 80 to 120 mg/L. No mortality was observed in control or untreated groups. Mortality of 93-100% was observed in the highest concentration, 120 ppm of the ethanolextracted A. graveolens. At the lowest concentration, 40 mg/L, there was very low mortality (3-5%). However, in contrast to the control and untreated groups (pupation rate = 98-100%, emergence rate = 96-98%), many surviving larvae derived from the concentration of 40 mg/L failed to pupate (6.2-11.3%) and emerge as adults (11.6-13.2%). The ethanol-extracted A. graveolens showed promising larvicidal activity with LD50 and LD95 values of 81.0 and 176.8 mg/L, respectively. Observations carried out through the exposure period at room temperature revealed that immediately after exposure to ethanol-extract A. graveolens solution, all larvae were still active and exhibited a normal appearance with the siphon pointed up and head hung down. The process of larval feeding, both collectingfiltering in the water column and collecting-gathering at submerged surfaces, were clearly seen. Between 5 and 10 min after treatment, some of the larvae became restless and frequently sank down and floated up quickly. At 15 min, the restlessness persisted, and tremor and convulsion at the bottom of the container were observed in approximately 2-3 larvae. Similar evidence of restlessness, tremors, and convulsions followed by paralysis was clearly seen at 45 min in approximately 45 larvae. At 1 h, approximately 1-2 moribund and dead larvae were found. For as long as 4 h after treatment, approximately one-third of the larvae were paralyzed and sank to the bottom of the bowl. More and more larvae exhibited toxic symptoms during 5 to 6 h. Subsequently, all of them died within 7 h in the 120 mg/L treatment. The ethanol-extracted A. graveolens did not cause rapid mortality, suggesting a delayed type of larval killing

property. The symptoms observed in treated larvae were similar to those caused by nerve poisons, i.e. excitation, convulsions, paralysis, and death. The ethanol-extracted A. graveolens showed adulticidal activity against Ae. aegypti with LD50 and LD95 values of 6.6 and 66.4 mg/cm2, respectively. The extract was found to cause a mosquito knockdown (the rapidly and normally reversible paralysis) after exposure to varying concentrations of A. graveolens impregnated paper. Following exposure to 3.5 to 10.6 mg/cm2 for 515 min, almost all mosquitoes showed signs of paralysis, i.e. unable to walk and lay at the bottom of the exposure tube. At the end of a one h exposure period, all mosquitoes became inactive. Nevertheless, when transferred from the exposure tube to the holding tube, approximately one-third of the adults recovered within 1 h. The subsequent record at the end of a 24 h holding period revealed mortality values ranging from 16 to 76%. It was also apparent that mortality in adults was dose dependent. Although the insecticidal potency of the crude seed extract of A. graveolens against both larva and adult mosquitoes was lower than that of other plant extracts against Ae. aegypti and other mosquito species obtained in comparable laboratory conditions (Pitasawat et al. 1998, Choochote et al. 1999, Mansour et al. 2000, Yang et al. 2002, 2003), its probable nerve poison to mosquitoes was certainly not negligible. Rafikali et al. (2000) isolated 4 compounds from the hexane extract of celery seed. The first three compounds, b-selinene, 3-nbutyl-4, 5-dihydrophthalide, and 5-allyl-2methoxyphenol were reported to have a pronounced larvicidal potential with 100% mortality of fourth instar Ae. aegypti at 50, 25, and 200 g/ml, respectively, while the last one, 1,3-Di [(cis)-9-octadecenoyl]-2-[(cis,cis)9-12-octadecadienoyl] glycerol was not biologically active. Additionally, the knockdown effect on adult mosquitoes obtained from the present adulticidal bioassay was very interesting for further repellent study. In the repellent study, the ethanol-extracted A. graveolens possessed significant repellent effect against Ae. aegypti adults on human volunteers, as shown in Table 3. The ED50 and ED95 values were 2.03 and 28.12 mg/cm 2, respectively. It also provided a median complete-protection time of 3.0 (2.5-3.0) h against Ae. aegypti bites when applied at a concentration of 25 g%. No skin irritation, hot sensations, or rashes were observed during the 3 mo of the study period or in the following 3 mo, after which time observations ceased, although dermatitis and phototoxic skin reactions from celery in some farm or grocery store workers were reported (Palumbo and Lynn 1953, Seligman et al. 1987). However, the toxicity of this substance under long-term

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Table 1. Larvicidal activity of the ethanol-extracted A. graveolens against fourth instar Ae. aegypti. A. graveolens (mg/L) 40 50 60 80 100 120 Control Untreated % Mortality (MeanSE) 3.51.0 13.02.2 27.81.5 44.012.8 71.27.4 96.22.4 0 0 Larvicidal activity (95% C.I., mg/L) LD50 81.0 (71.3-98.2) LD95 176.8 (129.4-443.7)

Table 2. Adulticidal activity of the ethanol-extracted A. graveolens against Ae. aegypti. A. graveolens (mg/cm2) 1.8 3.5 7.0 10.6 Control % Mortality (MeanSE) 17.21.3 32.02.9 42.84.0 71.53.7 0 Adulticidal activity (95% C.I., mg/cm2) LD50 6.6 (5.6-8.1) LD95 66.4 (38.0-173.0)

Table 3. Initial repellency of the ethanol-extracted A. graveolens against adult female Ae. aegypti. A. graveolens (mg/cm 2) 1 2 4 6 Control % Mosquitoes repelled (MeanSE) 31.891.70 48.713.25 66.382.46 78.663.89 0 Repellency (mg/cm2) (95% C.I.) ED50 2.03 (1.63-2.50) ED95 28.12 (16.05-75.53)

Table 4. Repellency (median complete-protection time) of the ethanol-extracted A. graveolens against adult female Ae. aegypti. Treatment Ethanol-extracted A. graveolens (25 g%) Control (ethanol) Median complete-protection time (Range, h) 3.0 (2.5-3.0) 0

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usage has not yet been investigated and characterized in humans. Because two h is the minimum protection time allowed for the sale of mosquito repellents in Thailand, ethanol-extracted A. graveolens, which provides a protection time of 2.5-3.0 h, is considered a satisfactory candidate for improving the practicality and extension of marketable formulations of repellent. This plant is cheap and widely cultivated in rural areas, consequently its commercial exploitation would contribute toward rural economic development. In conclusion, A. graveolens offers potential against Ae. aegypti, particularly in its markedly repellent effect. Further studies of the active principles involved and their mode of action, formulated preparations for enhancing potency and stability, toxicity and effects on non-target organisms and the environment, and field trials are needed to recommend A. graveolens as an anti-mosquito product used to combat and protect from mosquitoes in a control program. Acknowledgments The authors are very grateful to the individuals who served as subject volunteers and also the staff members of the Department of Parasitology, Faculty of Medicine, Chiang Mai University for their cooperation. Acknowledgment is extended to the Faculty of Medicine Endowment Fund for its financial support and the Faculty of Medicine Endowment Fund for Research Publication for helping to defray the publication cost. REFERENCES CITED Abbott, W. S. 1925. A method of computing the effectiveness of an insecticide. J. Econ. Entomol. 18: 265-266. ASTM. 1983. Standard test methods for laboratory testing of non-commercial mosquito repellent formulations of the skin. Standard E951-83, Annual Book of ASTM Standards. American Society for Testing and Materials, Philadelphia, PA. Buescher, M. D., L. C. Rutledge, R. A. Wirtz, K. B. Glackin, and M. A. Moussa. 1982. Laboratory tests of repellents against Lutzomyia longipalpis (Diptera : Psychodidae). J. Med. Entomol. 19: 176-180. Choochote, W., D. Kanjanapothi, A. Panthong, T. Taesotikul, A. Jitpakdi, U. Chaithong, and B. Pitasawat. 1999. Larvicidal, adulticidal and repellent effects of Kaempferia galanga. Southeast Asian J. Trop. Med. Publ. Hlth. 30: 470-476. Coleman, R. E., L. L. Robert, L. W. Roberts, J. A. Glass, D. C. Seeley, A. Laughinghouse, P. V. Perkins, and R. A. Wirtz. 1993. Laboratory evaluation of

repellents against four anopheline mosquitoes (Diptera: Culicidae) and two phebotomine sand flies (Diptera: Psychodidae). J. Med. Entomol. 30: 499502. Coleman, R.E., A.L. Richards, and G.J. Magaon. 1994. Laboratory and field trials of four repellents with Culex pipiens (Diptera: Culicidae). J. Med. Entomol. 31: 17-22. Granett, P. 1940. Studies of mosquito repellents. II Relative performance of certain chemicals and commercially available mixtures as mosquito repellent. J. Econ. Entomol. 33: 566-572. Hendarto S.K. and S.R. Hadinegoro. 1992. Dengue encephalopathy. Acta. Paediatr. Jpn. 34: 350-357. Kitajima, J., T. Ishikawa, and M. Satoh. 2003. Polar constituents of celery seed. Phytochemistry. 64: 1003-1011. Limrat, D. 1997. Distribution of Aedes aegypti in rural and urban areas and correlation between the index of this mosquito and reported cases of DHF in Ayuthaya province. J. Malar. 32: 112-122. Mansour, S.A., S.S. Messeha, and S.E. El-Gengaihi. 2000. Botanical biocides. 4. Mosquitocidal activity of certain Thymus capitatus constituents. J. Nat. Toxins. 9: 49-62. Palumbo, J. F and E.V. Lynn. 1953. Dermatitis from celery. J. Am. Pharm. Assn. 42: 57. Pancharoen, C., W. Kulwichit, T. Tantawichien, U. Thisyakorn, and C. Thisyakorn. 2002. Dengue infection: a global concern. J. Med. Assoc. Thai. 85: S25-33. Pitasawat, B., W. Choochote, D. Kanjanapothi, A. Panthong, A. Jitpakdi, and U. Chaithong. 1998. Screening for larvicidal activity of ten carminative plants. Southeast Asian J. Trop. Med. Publ. Hlth. 29: 660-662. Rafikali, A.M., S.R. Russel, and G. N. Muraleednaran. 2000. Bioactive compounds and 1,3-Di [(cis)-9octadecenoyl]-2-[(cis,cis)-9-12-octadecadienoyl] glycerol from Apium graveolens L. seeds. J. Agric. Food Chem. 48: 3785-3788. Rafikali, A.M. and G. N. Muraleednaran. 2001. Mosquitocidal, nematicidal and antifungal compounds from Apium graveolens L. seeds. J. Agric. Food Chem. 49: 142-145. Saengtharatip, S. 1997. Prevalence of tiger mosquitoes (Aedes aegypti) and reported cases of Dengue hemorrhagic. J. Malar. 32: 17-33. Seligman, P.J., C.G. Mathias, M.A. OMalley, R.C. Beier, L.J. Fehrs, W.S. Serrill, and W.E. Halperin. 1987. Phytophotodermatitis from celery among grocery store workers. Arch. Dermatol. 123: 1478-1482. World Health Organization. 1981a. Instructions for

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determining the susceptibility or resistance of mosquito larvae to insecticides. WHO/VBC/81.807. World Health Organization. 1981b. Instructions for determining the susceptibility or resistance of adult mosquitoes to organocholrine, organophosphate and carbamate insecticides: diagnostic test. WHO/ VBC/81.806. World Health Organization.1996. Report of the WHO informal consultation on the evaluation and testing of insecticides. CTD/WHOPES/IC/96.1, Control of Tropical Diseases Division. Geneva: WHO.

World Health Organization. 2003. Dengue (online, access in 03/06/2003). Available at http:// w.w.w.who.int/inf-fs/en/fact117.html. Yang, Y.C., S.G. Lee, H.K. Lee, M.K. Kim, S.H. Lee, and H.S. Lee. 2002. A piperidine amide extracted from Piper longrum L. fruit shows activity against Aedes aegypti mosquito larvae. J. Agric. Food Chem. 50: 3765-3767. Yang, Y.C., M.Y. Lim, and H.S. Lee. 2003. Emodin isolated from Cassia obtusifolia (Leguminosae) seed show larvicidal activity against three mosquito species. J. Agric. Food Chem. 51: 7629-7631.