John Kabasa

John Odda

Paul Waako

Sibylle Kristensen

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J Vector Borne Dis 45, December 2008, pp. 321–324Short Research CommunicationsLarvicidal activity of Combretum collinum Fresen againstAedes aegyptiJohn Oddaa, Sibylle Kristensenb, John Kabasac & Paul WaakoaaDepartment of Pharmacology and Therapeutics, Faculty of Medicine, Makerere University, Kampala, Uganda; bDepartmentof Epidemiology, University of Alabama at Birmingham, U.S.A.; cDepartment of Physiological Sciences, Faculty of VeterinaryMedicine, Makerere University, Kampala, UgandaKey words Aedes  aegypti  – Combretum collinum Fresen – dengue fever – larvicideDespite of the fact that dengue outbreaks in the trop-ics have been worldwide for over 200 years, it is stillthe most important mosquito transmitted viral diseaseaffecting man1. Aedes aegypti (Diptera: Culicidae) isan arbovirus vector responsible for yellow fever incentral and south America and in west Africa. It isalso the vector for dengue hemorrhagic fever (DHF),endemic to south-east Asia, the Pacific Islands, Af-rica and the Americas1.It is estimated that 2.5 billion people are currently atrisk for dengue fever (DF), DHF, and dengue shocksyndrome (DSS)1. The size and spread of the denguepandemic, the unpredictability of the epidemic occur-rences and the circulation of virulent and non-virulentstrains make DHF/DSS a model for emerging infec-tious disease. Despite of this challenge, the develop-ment of dengue virus vaccines is still a long way tobe of any use due to several obstacles2.The threat by DHF/DSS is magnified by the aggres-sive day-time feeding habits of Ae. aegypti making itharder to control compared to Anopheles mosqui-toes3. Current mosquito control strategies include theuse of aerial sprays (toxicants) repellents, larvicides,insecticides as well as mosquito nets for personal pro-tection. However, aerial toxicants against Ae. aegyptiare ineffective because this mosquito is highly do-mesticated and many adults rest indoors in hiddenplaces such as closets. The only effective way of re-ducing mosquito densities to a level where dengueepidemics do not occur is by attacking the larvalbreeding places4. Unfortunately, many of these meth-ods are not readily affordable and available in thedeveloping world setting5. Therefore, in response tothe urgent need for new, affordable, effective andenvironment-friendly mosquito control agents; wescreened a Ugandan medicinal plant of the family—Combretaceae (Combretum collinum Fresen) formosquito larvicidal activity against Ae. aegypti. C.collinum also classified as an aromatic plant is foundgrowing in the wild (western, eastern and northernUganda) with different tribes of Uganda referring toit according to their own dialect—for instance, in theLusoga dialect, it is called Mukoola omukali6.Following is a review of some of the medicinal uses ofC. collinum. In Bulamogi County (eastern Uganda), adecoction from the roots is drunk for the treatment ofdiarrhoea, sterility and pyomyositis. Furthermore, aninfusion from the roots is orally given to deliveringwomen to enhance labour; to children with hydroceleand patients with gonorrhoea to treat the latter condi-tions respectively6. Root powder from C. collinum maybe drunk as ‘tea’; or mixed with bathing water for bathby epileptic patients. Alternatively, the root powderJ  VECTOR  BORNE  DIS  45, DECEMBER 2008 322may be incorporated in petroleum jelly base for topi-cal application in the treatment of epilepsy6.While some medicinal properties of this plant inUganda have previously been studied (isolatedclasses of constituents of Combretaceae family wereshown to include triterpenoids and tannins7) there areno formal reports of its larvicidal activity against Ae.aegypti, hence the need for this study.  To demon-strate the potential of C. collinun as a mosquito lar-vicide against Ae.  aegypti, Neemazal F extract fromAzadirachta indica A. Juss (Neem) from Meliaceaefamily was used as a larvicidal compound8 againstwhich the action of C. collinun was compared.Fresh C. collinum shoot bark was collected fromplants growing naturally in Oculoi village, 15 kmfrom Soroti district (in eastern Uganda) during thesecond week of April 2002. The plant was authenti-cated by a botanist from Makerere University,Kampala, Uganda. The shoot bark of C. collinum waswashed, chopped and air-dried away from direct sun-shine to a constant weight for six months. The driedplant material was pounded into a powder using ametallic mortar and pestle; 500 g of the resultant pow-der was then extracted using 1000 ml of diethyl ether(Fisher Scientific, Jersey, New Jersey, U.S.A.) forfive days at 25°C. The extract was decanted and fil-tered using Whatman No.1 filter paper, followed bysolvent removal using simple distillation at 36°C.The mark left on the filter paper after the filtrationprocess was then extracted using 1000 ml of ethanol(PVT, Mumbai, India) at 25°C. The resulting extractwas then decanted and filtered using Whatman No.1filter paper followed by solvent removal using rotaryevaporator in vacuo. The crude extract (41.5 g; 8.3%)was obtained and used in the assay and for phy-tochemical tests. Phytochemical screening gave posi-tive results for tannins and saponins.The C. collinum extract (200 mg) was initially dis-solved in 25 ml of ethanol 95% v/v (PVT, Mumbai,India) to make a stock solution of 8 mg/ml. This wasthen diluted with an appropriate volume of distilledwater (final volume of 20 ml) to make the requiredtest solutions in 250 ml plastic beakers. Therefore, thefinal concentrations ranged from 0.0125–0.200mg/ml. The method of Olwa-Odyek et al9, was thenused for screening of larvicidal activity in all the ex-periments with slight modification —IV instar Ae.aegypti larvae. Laboratory-reared strain maintainedin the Insectary Unit of the Entomology Departmentat the Uganda Virus Research Institute (UVRI),Entebbe, Uganda. The Neemazal F extract from A.indica A. Juss (Neem) from Meliaceae family againstwhich the action of C. collinun was compared and ob-tained from the Entomology Department of UgandaVirus Research Institute (UVRI), Entebbe, Uganda.Ten IV instar larvae were transferred into the testcrude extract solutions (20 ml) in 250 ml plastic bea-kers. The beaker that served as a negative control had0.5 ml of 95% v/v ethanol (PVT, India).Approximately 2% ground mice feed was sprinkledover the contents of each beaker to serve as feed.Another set of nine 250 ml beakers containingNeemazal F, an extract of A. indica A. Juss was usedas positive control8. The concentrations used for thecontrol group ranged from 0.028–0.448 mg/ml. Abeaker containing 0.5 ml of 95% v/v ethanol (PVT,Mumbai, India) served as a negative control in thiscase. The larvae were incubated in the test crudeextract and Neemazal F extract over a 24 h period ofcontinuous exposure at 25°C and 70–80% relativehumidity in the beakers at which larval mortality wasassessed. The larvae were considered dead when theyshowed no signs of movement even after touchingwith a glass rod.Three replicates of each extract and control wereperformed in order to ascertain the consistency ofthe results (Tables 1 and 2). The corrected percentmortalities were analyzed using Abbott’s formula10.The mortality data were analyzed using Prism Ver-sion 3 from which lethal concentration (LC50) values(24 h) and 95% confidence intervals (CI) were deter-ODDA et al:  POTENTIAL LARVICIDE AGAINST DENGUE VECTOR  323mined. The LC50 value of the test extract was com-pared with that of Neemazal F reflecting the poten-cies of the two; the one with a lower LC50 value be-ing more potent of the two.The LC50 of C. collinum Fresen [shown by effectiveconcentration 50 (EC50)] on IV instar larvae was0.051 mg/ml (95% CI of 0.041–0.063 mg/ml) (Table1) compared to 0.078 mg/ml (95% CI of 0.063–0.097mg/ml for A. indica A. Juss (Neemazal F) (Table 2).The crude extract of C. collinun was found to be ac-tive on the IV instar larvae of Ae. aegypti. The larvi-cidal activity varied with the concentration and expo-sure. The larvicidal activity of C. collinun (LC50 =0.051 mg/ml) was comparable to that of A. indica A.Juss with LC50= 0.078 mg/ml. Neemazal F, an extractof A. indica A. Juss (was included in this study forcomparison purposes). The LC50= 0.078 mg/ml valueof A. indica A. Juss in the present study supportsearlier reported LC50 value regarding A. indica A.Juss alcohol extract on IV instar larvae of Ae. aegypti(LC50 = 0.023–0.083 mg/ml)11. The exact active prin-ciple in C. collinum responsible for the larvicidaleffect has not yet been identified, but the Combretumsp of South Africa have been reported to contain suf-ficient amount of tetranortriterpenoids7. The ob-served mosquito larvicidal effects could possibly bedue to these compounds11.Now-a-days, the major strategies for mosquito con-trol are insecticides, mosquito nets, mineral oil larvi-cides, and mosquito repellents5. Unfortunately, mostof these strategies are not sustainable for long-termuse particularly in developing countries because oftheir prohibitive costs5. Mosquito nets are not effec-tive against Ae. aegypti mosquitoes because of theiraggressive day-time feeding habits3.Moreover, there is widespread resistance to availableconventional insecticides5; even conventional aerialspraying with adulticides or sequential fogging givesno prolonged control of the target mosquito popula-tion12.  A potential explanation for this phenomenonis that the surviving gravid females that were lessaffected by space spraying will continue their ovipos-iting and are joined by newly emerged mosquitoesfrom unaffected breeding sites12. According to Chunget al12, routine use of chemical adulticide for outdoorpurposes not only needs to be used in combinationwith spraying for indoor control, but also with appli-Table 1.  Larvicidal effects of ethanolic extracts ofC. collinum on IV  instar larvae of Ae.  aegyptiafter a 24 h treatment atroom temperatureConcentration No. of larvae Mortalityof the extract  dead/No.(mg/ml) exposedControl 0/30 00.0125 3/30 100.025 6/30 200.050 12/30 400.100 30/30 1000.200 30/30 100Table 2. Larvicidal effects of Neemazal F after a24 h of treatment at 25°CConcentration No. of larvae Mortalityof the extract  dead/No.(mg/ml) exposedControl 0/30 00.028 6/30 200.056 12/30 400.112 15/30 500.168 24/30 800.224 27/30 900.280 30/30 1000.336 30/30 1000.392 30/30 1000.448 30/30 100J  VECTOR  BORNE  DIS  45, DECEMBER 2008 324cation of mineral oil to kill the larvae. Unfortunately,mineral oil is an environmental pollutant and hencenot environment-friendly. To achieve both, an effec-tive and a sustainable vector control strategy, is nec-essary to come up with an affordable, effective, safeand environment-friendly solution. The most prom-ising strategy meeting these requirements lies inbotanical insecticides hence much interest is nowfocused on plant extracts11. Therefore, we investi-gated the larvicidal activity of C. collinum Fresenshoot bark-ethanol extract by exposing the IV instarlarvae to it and observing the larval mortalities.In conclusion, we found the crude extract of C.collinun effecttive against IV instar larvae of Ae.aegypti and its larvicidal activity is located in theshoot bark which makes it a suitable candidate for thedevelopment of new larvicides, since its exploitationwould not endanger the plant. Moreover, it may bepotentially cheap to produce because its solubility inethanol means that it has the potential to dissolve inwater. Additional biological assays are planned to:(i) carry out toxicological tests on laboratory miceand rats to determine its safety; and (ii) determine ex-actly the constituent responsible for the larvicidalactivity through bioassay directed fractionation.AcknowledgementThe authors are grateful to Professor Ogwal-Okeng ofthe Faculty of Medicine, Gulu University for techni-cal advice; to Ms Namaganda of Department of Botanyat Makerere University for plant identification; andto Dr Mukwaya of the Entomology Department of(UVRI), Entebbe for the provision of mosquito larvaeand Neemazal F extract. Partial financial support forthis study was provided by the University of Alabamaat Birmingham, International Centers of Excellence inResearch (ICER) grant from the Fogarty InternationalCenter, N.I.H. (1 U2R TW06246-04).References1. The Center for Disease Control. The dengue fever factsheet-CDC Division of Vector-Borne Infectious Diseases.[Cited 2007 Dec12 ]. Available at http://www.cdc.gov/ncidod/dvbid/dengue.2. National Institute of Allergy and Infectious Diseases. Den-gue fever-overview. [Cited 2007 Dec12 ] Available at http://www3.niaid.nih.gov/healthscience/healthtopics/dengue/overview.htm.3. Halstead SB. Pathogenesis of dengue: challenges to mo-lecular biology. Science 1998; 239: 476–81.4. Gluber D. Aedes aegypti and Aedes aegypti-borne diseasein the 1990s: top down or bottom up.  Am J Trop Med Hyg1989; 40: 571–8.5. Ogwal-Okeng JW. Studies of the anti malarial activities ofsome Ugandan medicinal plants. [Dissertation]. Kampala:Makerere University 1998.6. Tabuti JRS, Lye KA, Dhillion SS. Traditional herbal drugsof  Bulamogi, Uganda: plants, use and administration. J EthPharm 2003; 88: 19–44.7. Pegel KH, Rogers CB. A review of triterpenoids extractedfrom the leaves of South African Combretum species.Planta Med 1990; 56: 546.8. Pedigo LP. Entomology and pest management. II edn.Lowa State University 1996.9. Olwa-Odyek, Makanga B, Byaruhanga MA. Larvicidal andmolluscidal activities of Solanum aculeastrum Berrymethanol extract. Fitoterapia 1991; 63: 1992.10. Abbott WS. A method of computing the effectiveness ofan insecticide. J Econ Entomol 1925; 18: 265–7.11. Siddiqui BS, Afshan F, Ghiasuddin Faizi S, Naqvi SN,Tariq RM. Two insecticidal tetranortriterpenoids fromAzadirachta indica. J Phytochem 2000; 53: 371–6.12. Chung YK, Lam-phua SG , Chua TY, Yatman R. Evalua-tion of biological and  chemical insecticide mixture againstAedes aegypti larvae and adults by thermal fogging inSingapore. Roesel 2001; 15: 321–7.Corresponding author: Dr Sibylle Kristensen, Department of Epidemiology, University of Alabama at Birmingham, RPHB227C, 1530, 3rd Ave South, Birmingham AL 35294-0022, U.S.A.E-mail: sibylle@uab.eduReceived: 22 May 2008 Accepted in revised form: 25 July 2008

Larvicidal activity of Combretum collinum Fresen against Aedes aegypti

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