Print version ISSN 0036-4665
Rev. Inst. Med. trop. S. Paulo vol.48 no.1 São Paulo Jan./Feb. 2006
Atividade de três Piper spp. contra adultos de Stegomyia aegypti (Diptera: Culicidae)
Wej Choochote; Udom Chaithong; Kittichai Kamsuk; Eumporn Rattanachanpichai; Atchariya Jitpakdi; Pongsri Tippawangkosol; Dana Chaiyasit; Daruna Champakaew; Benjawan Tuetun; Benjawan Pitasawat
Department of Parasitology, Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailand
Three Piper species, Piper longum, P. ribesoides and P. sarmentosum, were selected for investigation of adulticidal potential against Stegomyia aegypti, a main vector of dengue and dengue haemorrhagic fever. Successive extraction by maceration with 95% ethanol showed percentage yields of ethanolic extracts, which derived from P. longum, P. ribesoides and P. sarmentosum, of 8.89, 3.21 and 5.30% (w/w), respectively. All Piper extracts illustrated an impressive adulticidal activity when tested against female mosquitoes by topical application. The susceptibility of St. aegypti females to ethanol-extracted Piper was dose dependent and varied among the plant species. The highest adulticidal effect was established from P. sarmentosum, followed by P. ribesoides and P. longum, with LD50 values of 0.14, 0.15 and 0.26 µg/female, respectively. The potential of these Piper species, as possible mosquitocides, established convincing activity for further researches to develop natural substances for combat against adult mosquitoes.
Keywords: Piper; Piper longum; Piper ribesoides; Piper sarmentosum; Stegomyia aegypti; Adulticidal activity; Topical application.
Três espécies de Piper, Piper longum, P. ribesoides e P. sarmentosum, foram selecionadas para investigação da potencialidade contra Stegomyia aegypti adultos, principal vetor de dengue e febre do dengue hemorrágico. Sucessivas extrações por maceração com etanol a 95% mostraram uma porcentagem de extratos etanólicos, derivados de P. longum, P. ribesoides e P. sarmentosum, de 8,89, 3,21 e 5,30% (w/w), respectivamente. Todos os extratos de Piper mostraram atividade adulticida expressiva quando testados contra fêmeas de mosquitos através de aplicação tópica. A suscetibilidade das fêmeas do St. aegypt ao extrato de Piper etanólico foi dose dependente e variou entre as espécies de plantas. O mais elevado efeito adulticida foi demonstrado a partir do P. sarmentosum, seguido pelo P. ribesoides e P. longum, valores LD50 de 0,14, 0,15 e 0,26 µg/fêmea, respectivamente. O potencial destas espécies de Piper, como possíveis mosquiticidas, estabeleceu atividade convincente para futuras pesquisas a fim de desenvolver substâncias naturais para o combate a mosquitos adultos.
Stegomyia aegypti (redirected from Aedes aegypti)18 mosquitoes are the primary carrier of dengue and dengue haemorrhagic fever in many developing countries in the tropical world33. In Thailand, annual dengue incidences are quite high and have been increasing and spreading recently to newer areas10. Like the case of malaria, a vaccine for dengue and dengue haemorrhagic fever is not yet available. Consequently, a reliable way to diminish the incidence of these diseases is to eradicate the mosquito vectors through continued applications of synthetic insecticides. However, the problems of high cost, toxic hazards to humans and other non-target creatures, and development of resistance to conventional insecticides in many mosquito species have revived great interest in exploiting the vector control potential of natural origins. Research and the development of mosquito control alternatives have focused on plant-derived constituents or phytochemicals6,27,30,34.
Botanical insecticides may serve as suitable alternatives to synthetics in future, as they are relatively safe, degradable and readily available in many parts of the world24. Recently, plants in the family, Piperaceae, have been of interest because they contain insecticidal principles2,9. Little work has been carried out on mosquito adulticidal activity, although some Piper spp. and their bioactive constituents are reported to have noteworthy larvicidal potential against various species of mosquitoes such as Culex pipiens pallens, Aedes aegypti, Ae. togoi and Ae. atropalpus8,13,14. In a search for effective and affordable natural substances for use in the control of mosquito-borne diseases, three Piper spp. available in Thailand including Piper longum, P. ribesoides and P. sarmentosum were chosen to evaluate their adulticidal potential against the St. aegypti mosquito.
The Piperaceae family contains approximately 2,000 species, which are widely distributed and commonly used in tropical and subtropical regions as food, spice, traditional medicines and pest control agents11,12,21. P. longum, P. ribesoides and P. sarmentosum, widely grown throughout Thailand, have been locally known as De-plee, Ta-khaan and Cha-plu, respectively. These Piper species have been traditionally used in Thailand for food and medicinal purposes16,19. P. longum is reported as good remedy used for treatment of gonorrhea, menstrual pain, tuberculosis, sleeping problems, respiratory tract infections, chronic gut related pain and arthritic conditions22. Additionally, analgesic and diuretic effects, relaxation of muscle tension and alleviation of anxiety have also been reported23. P. ribesoides and P. sarmentosum are commonly used in folk medicine as a carminative. P. sarmentosum has also been used as an expectorant, and to relieve muscle pain16,19. Methanol extracts of P. longum fruit and P. sarmentosum root have been proved to possess the anti-amoebic effects against Entamoeba histolytica infecting the caecum of mice20. P. longum and its component piperine have accounted as a non-toxic immunomodulator which possess antitumor property also28. The water extract of whole plant of P. sarmentosum has been noted for a hypoglycemic effect in rats15. Brachyamide B and 3',4',5'-trimethoxycinnamoyl pyrrolidine, bioactive compounds derived from P. sarmentosum fruits, have been reported to exert antituberculosis activity and antiplasmodial potential against Plasmodium falciparum parasite17.
MATERIALS AND METHODS
Plant preparation and extraction: Three Piper species of the Piperaceae family, Piper longum Linn., Piper ribesoides Wall. and Piper sarmentosum Roxb. were obtained from E.A.R. Samunpri, a traditional herb supplier in Chiang Mai province, Thailand. Taxonomic identification of plants was performed by J.F. Maxwell, botanists at the CMU Herbarium, Department of Biology, Faculty of Science, Chiang Mai University, Thailand. The voucher specimens (PARA-PI-001/1, PARA-PI-002/1, and PARA-PI-003/1, respectively) were preserved at the Department of Parasitology, Faculty of Medicine, Chiang Mai University. Dried material of each plant (1 kg) was crushed and successively extracted three times by maceration, with 3 L of 95% ethanol at room temperature for seven days. The ethanolic extracts were separated by suction filtered through a Büchner funnel. Solvent in combined filtrates was removed by rotary evaporator at temperatures below 60 °C until the extract was rather concentrated. The resulting crude extracts were transferred to an open container set on a hot plate and the residual solvent was removed for at least 24 hours. The ethanolic extract of each plant was thus obtained, lyophilized and then refrigerated at -20 °C until testing for adulticidal activity.
Test mosquitoes: Laboratory colonies of Stegomyia aegypti, which originated from larvae collected at various places in Chiang Mai province, northern Thailand, had been reared continuously for several generations in a laboratory free of exposure to pathogens and insecticides. They were maintained at 25-30 °C and 80-90% relative humidity under a photoperiod of 14:10 h (light/dark) in the insectarium of the Department of Parasitology, Faculty of Medicine, Chiang Mai University, Chiang Mai province. Larvae were fed on a ground commercial dog biscuit. The adults were reared in humidified cages and supplied with 10% sucrose and 10% multivitamin syrup. Female mosquitoes were periodically blood-fed on restrained rats to obtain protein used principally for egg production. Under these conditions, the full development from egg to adult lasted about 3-4 weeks. Batches of 2-5 day-old healthy female mosquitoes were used in the adulticidal bioassay.
Adulticidal bioassay: The adulticidal activity of the Piper extracts was determined by topical application of the insecticide to the adult female mosquitoes (Fig. 1), following slightly modified versions of the WHO standard protocols32. Each plant extract was dissolved in acetone yielding a graded series of concentrations. Non-blood fed females were briefly anaesthetized with carbon dioxide (CO2), weighed and placed on a cold plate. Treatment was performed with the aid of a dissecting microscope. A 0.1 µL droplet of plant solution in acetone was applied onto the upper part of the immobilized mosquito's pronotum using a Hamilton's digital syringe (700 series MICROLITERTM, Hamilton Company, USA). Dosages were expressed in the µg of plant material per mg of mosquito body weight. A total of 25 individuals was used at each concentration, with 4-6 concentrations providing a range of 0-100% mortality. Controls were divided into two groups including acetone treated and untreated groups. Both groups were treated in a similar manner to that of Piper treated groups. After application, the females in all groups were maintained at 27 ± 3 ºC and 80% ± 10% RH in plastic cups, with 10% sucrose and 10% multivitamin syrup provided. At the end of a 24-hour recovery period, the mosquitoes were considered dead if they showed no sign of movement such as lying on the bottom of the plastic cup and not responding to mechanical stimulation. Eight replicates were carried out for each plant extract with mosquitoes from different rearing batches, and the results were pooled.
Data management and statistical analysis: It was essential to obtain not less than three mortality counts of between 10% and 90%. Experimental tests that demonstrated more than 20% control mortality were discarded and repeated. However when the control mortality ranged from 5-20%, the observed percentage mortality (%M) was corrected by Abbott's formula1:
The 95% confidence intervals (CI) of the lethal dosage of 50% and 95% (LD50 and LD95, respectively) calculated by a computerized log-probit analysis (Harvard Programming; Hg1, 2) were used to measure differences between test samples.
Successive extraction by maceration with 95% ethanol showed the percentage yield of ethanolic extracts, which derived from P. longum, P. ribesoides and P. sarmentosum in relation to the starting dry material that averaged 8.89, 3.21 and 5.30% (w/w), respectively. These alcoholic extracts showed various strengths of odor and a high solubility in absolute ethanol, DMSO or acetone (Table 1). Investigating against female St. aegypti by topical application in this study revealed a pronounced toxic effect in all Piper extracts (Table 2). The susceptibility of St. aegypti females to a graded series of concentrations of each ethanol-extracted Piper was dose dependent. Following treatment with increasing concentrations of P. longum, P. ribesoides and P. sarmentosum from 0.10 - 0.50, 0.025-0.40 and 0.05-0.30 µg/mg female, respectively, the mortality values increased from 6.5-84.5, 6.0-97.5 and 3.0-89.5%, respectively. Comparison of adulticidal activity of these ethanolic-extracted peppers demonstrated that the highest potential was established from P. sarmentosum, followed by P. ribesoides and P. longum with LD50 values of 0.14, 0.15 and 0.26 µg/mg female, respectively. Although P. sarmentosum provided slightly better activity than P. ribesoides, no statistically significant difference was found, based on the overlapping of 95% confidence interval at LD50 values. No mortality was detected in any acetone treated or untreated groups.
Piperaceae plants are widely used in tropical and subtropical regions throughout the world as medicines, spice, food flavoring and pest control substances21,25. Recently, there has been a growing interest in plants belonging to the family, Piperaceae, as potential sources of bioactive chemical compounds against insect pests. While excellent effects on mosquito vectors from larvicidal constituents that derived from some Piper species have been reported8,13,14,35, little work has been carried out on Piper toxicity against adult mosquitoes. The potential of three Piper species, P. longum, P. ribesoides and P. sarmentosum against St. aegypti, as observed at 24 hours following treatment in this investigation, was strong and found to have various degrees of adulticides. St. aegypti adults were most susceptible to P. sarmentosum followed by P. ribesoides and P. longum (LD50: 0.14, 0.15 and 0.26 µg/mg female, respectively). However, the adulticidal activity of the two former species, P. sarmentosum and P. ribesoides, showed no statistically significant difference, and was considered to be approximately equal, and higher than that of P. longum. The variety in adulticidal activity of these extracts is probably due to variation in the types and levels of active ingredients that depend on not only the genetic characteristics of the plant species, but also the conditions under which they were grown and harvested29,31.
The literature offers no publication on the toxicity of natural plant products against adult St. aegypti or other mosquito species, measured by topical application. KHAN & AHMED7 revealed the toxicity of crude neem leaf extract against the adult of housefly Musca domestica, investigated by topical application, with LD50 and LD95 values of 8.4 and 169.8 µg/fly, respectively. The LD50 values of male and female M. domestica treated with commercial eucalyptol were 118 and 177 µg/fly, respectively, whereas those of male and female Chrysomya megacephala were 197 and 221 µg/fly, respectively26. The St. aegypti mosquito is actually smaller in size and lower in weight (» 10-fold) than M. domestica and C. megacephala. With regard to the size and weight of insect and lethal dosage parameter, although insect susceptibility to each plant product generally demonstrates an interspecies variability, it could be expected that the toxic effect of Piper products was greater than that of eucalyptol and neem, the popular plant-based insecticide. The adulticidal potential of P. ribesoides, P. sarmentosum and P. longum investigated in this study is encouraging, but not as much as that of conventional synthetic insecticides. CORBEL et al.5 reported the excellent intrinsic toxicity of some chemical insecticides measured by topical application against many mosquito species. The LD50 of bifenthrin, permethrin and temephos were 0.077, 0.24 and 195 ng/mg female, respectively, when tested against Ae. aegypti adults. The adulticidal activity of the Piper spp. seemed much less toxic against female St. aegypti (Ae. aegypti) than these commonly used insecticides by more than 10-fold at the LD50 level. Pyrethroids and organophosphates resistance, however, is now widely developed in mosquitoes of medical importance5.
Aerial toxicants are partially effective in eradicating Ae. aegypti, which are highly domesticated, and many adults hide indoors4. Insecticide space-spraying against adult mosquitoes is still necessary during epidemics of dengue. However, the use of aerosolized pyrethroid insecticides in public health programs can cause allergies in the human population3. There have also been questions raised regarding the long-term persistence and other possible hazards to human health from conventional applications. The results from this research are encouraging and clearly demonstrate the potential of these Piper species as possible mosquitocides against St. aegypti. The pepper plants, P. longum, P. ribesoides and P. sarmentosum may be used as alternatives for managing adult flying mosquitoes. Further work on Piper-derived constituents is needed to develop effective formulations for controlling adult mosquitoes. Moreover, further research to identify the biologically active compounds in Piper extracts, which showed potent adulticidal activity, is already in progress.
The authors acknowledge the staff members of the Department of Parasitology, Faculty of Medicine, Chiang Mai University for their kind cooperation and great assistance in this research project. Acknowledgment is extended to the Faculty of Medicine Endowment Fund for its financial support of this research project and the Faculty of Medicine Endowment Fund for Research Publication for its financial support in publishing this paper.
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Department of Parasitology, Faculty of Medicine, Chiang Mai University
Chiang Mai 50200, Thailand
Phone +66-53-945343, Fax +66-53-217144
Received: 4 February 2005
Accepted: 4 October 2005