Abstract
Objective
To evaluate aqueous and ethanol extract of Cassia didymobotrya leaves against immature stages of Culex quinquefasciatus.
Methods
The mortality rate of immature mosquitoes was tested in wide and narrow range concentration of the plant extract based on WHO standard protocol. The wide range concentration tested in the present study was 10 000, 1 000, 100, 10 and 1 mg/L and narrow range concentration was 50, 100, 150, 200 and 250 mg/L.
Results
2nd instar larvae exposed to 100 mg/L and above concentration of ethanol extract showed 100% mortality. Remaining stages such as 3rd, 4th and pupa, 100% mortality was observed at 1 000 mg/L and above concentration after 24 h exposure period. In aqueous extract all the stages 100% mortality was recorded at 1 000 mg/L and above concentration. In narrow range concentration 2nd instar larvae 100% mortality was observed at 150 mg/L and above concentration of ethanol extract. The remaining stages 100% mortality was recorded at 250 mg/L. In aqueous extract all the tested immature stages 100% mortality was observed at 250 mg/L concentration after 24 h exposure period. The results clearly indicate that the rate of mortality was based dose of the plant extract and stage of the mosquitoes.
Conclusions
From this study it is confirmed and concluded that Cassia didymobotrya is having active principle which is responsible for controlling Culex quinquefasciatus. The isolation of bioactive molecules and development of simple formulation technique is important for large scale implementation.
Keywords: Cassia didymobotrya, Culex quinquefasciatus, Larvicide, Pupicide, Aqueous, Ethanol, Extracts
1. Introduction
Cassia didymobotrya (C. didymobotrya) (syn Senna didymobotrya) belonging to the family Fabaceae (Leguminosae) is a widely used medicinal plant in East Africa. It is native plant to tropical Africa found from Congo east to Ethiopia and south to Namibia, Zimbabwe and Mozambigue[1]. In tropical Asia and America these plants were originally introduced as a fodder, green manure and cover crop. However, it is now mainly cultivated as an ornamental plant. These evergreen East African native plants tolerate in full sun even with very little water and blooming with beautiful attractive yellow flowers. In Ethiopia, it is found in dry and moist Kolla and Weyna Dega agroclimatic zones of Arsi, Sidamo, Wolego, Shewa and in the western part of Welo at 1 400-2 400 meter above sea level[2].
C. didymobotrya is a potential medicinal plant and the medicinal values are explored well in many parts of the world by traditional practitioners. In Kenya, traditionally Kipsigis communities were using these plants to control malaria as well as diarrhea. In addition, use to treat skin conditions of humans and livestock infections as well[3]. In Congo, Rwanda, Brundi, Kenya, Uganda, Tanzania, root decoction of these plants was used for the treatment of malaria, other fevers, jaundice and intestinal worm. In addition, root or leaf mixed with water or decoction of fresh parts was used to treat abscess of the skeletal muscle and venereal diseases[4]. The plant is also useful for the treatment of fungal, bacterial infections, hypertension, haemorrhoides, sickle cell anemia, a range of women's diseases such as inflammation of fallopian tubes, fibroids and backache, to stimulate lactation and to induce uterine contraction and abortion[1],[4]. The antibacterial activities of hexane extract against Microsporum gypsum (M. gypsum), dichloromethane extract against Trichophyton mentagrophyte and M. gypsum was reported[5]. According to Joji Reddy et al[6] presence of phenolic compounds, flavonoids and carotenoids in the ethyl acetate extract of leaves are responsible for pronounced antibacterial activities which are comparable with standard antibiotics such as tobramycin, gentamicin sulphate, ofloxacin and ciprofloxacin.
Many species of the plants belongs to the genus Cassia possess potential larvicidial, ovicidal, repellant activities against wide species of immature and adult vector mosquitoes[7]-[10]. Ojewole et al[11] evaluated leaves and stem barks of C. didymobotrya against the malarial vector Anopheles fluviatilis and reported pronounced lethal effect to early stages. The aqueous extract of stem bark at 1%, 0.1% or 0.01% W/V solution showed potential larvicidal activity. In addition, insecticidal properties are also reported but comparatively lesser than nicotine[12]. Therefore, C. didymobotrya is unquestionably potential medicinal plant but there is no much work on their bioactivities against mosquitoes in Gondar area, Amhara region, Ethiopia. The feather likes leaves bearing shrubs or small trees are extensively growing in the study area. Therefore, an attempt was made to evaluate aqueous and ethanol extract of C. didymobotrya leaves against filarial vector, Culex quinquefasciatus (Cx. quinquefasciatus)[13]. It is a vector of lymphatic filariasis, widely distributed in tropical zones with around 120 million people infected worldwide and 44 million people having common chronic manifestation[14].
2. Materials and methods
Larvicidal and pupicidal experiment was conducted in the botany laboratory, Faculty of Natural and Computational Sciences, University of Gondar, Ethiopia from February 2010 to May 2010.
2.1. Plant materials collection and processing
Fully matured dark green leaves of C. didymobotrya were collected in and around the vicinity of Tewodros campus, University of Gondar in the month of February 2010. Plant species was identified by verifying the colour pictures followed by description and identification characters[2]. The plant leaves were thoroughly washed with tap water to avoid dusts and other unwanted materials accumulated on the leaves from their natural environment. The dust free leaves were allowed to dry under shade in the botany laboratory for 20 d. The dried leaves were powdered by using electric blender. Finally, fine powder was collected from the powdered leaves by sieving through the kitchen strainer and used for extraction.
2.2. Extraction procedure
Twenty gram of powdered plant material was kept in 200 mL conical flask and added 100 mL of solvent such as water and ethanol individually. The mouth of the conical flask was covered with aluminum foil and kept in a reciprocating shaker for 24 h for continuous agitation at 150 rev/min for thorough mixing and also complete elucidation of active materials to dissolve in the respective solvent. Then, extract was filtered by using muslin cloth followed by Whatman no 1 filter paper and finally filtered by using vacuum and pressure pump (AP-9925 Auto Science). The solvent from the extract was removed by using rotary vacuum evaporator RE52 with the water bath temperature of 50°C. Finally, the residues were collected and used for the experiment.
2.3. Test concentrations preparation
Stock solution of 10 000 mg/L concentration was prepared by adding 1 g of plant residue with 5 mL of acetone and make up to 100 mL by adding tap water. From the stock solution 0.1% of soap powder was added for emulsification purpose. From the stock solution 1 000, 100, 10 and 1 mg/L concentration was prepared for wide range of tolerance test. After that narrow range concentration such as 50, 100, 150, 200 and 250 mg/L was prepared and tested against immature stages of Cx. quinquefasciatus.
2.4. Maintenance of immature mosquitoes
The immature stages of Cx. quinquefasciatus were collected from the stagnant water with rich organic pollution in and around Tewodros campus and also nearby places around University of Gondar. Mosquito larval collection was done from the breeding site by using large kitchen strainer and transferred to large plastic container and transported to the laboratory. In the laboratory the larvae was kept in enamel try with tap water and provided powdered dog biscuit along with yeast powder (3:1 ratio) as a feed. After acclimatization of the mosquito larvae in the laboratory conditions, subsequent experiments were conducted.
2.5. Larvicidal activity
Larvicidal activity of aqueous and ethanol extract of C. didymobotrya was evaluated by using WHO method[15]. Twenty five 2nd, 3rd, 4th and pupal stage was released in to 250 mL of glass beaker individually. In each beaker, concentration of aqueous and ethanol extract was maintained at 10 000, 1 000, 100, 10 and 1 mg/L concentration with the final water volume of 200 mL for wide range tolerance test. In control experiment except plant materials remaining all added as mentioned in the concentration preparation. The larval mortality rates were recorded at 24 h exposure period[16]. The dead larvae in five replicates were counted individually and converted in to percentage of mortality. Dead larvae were identified when they failed to move when the water was disturbed. Based on immature mortality in wide range test narrow range concentration such as 50, 100, 150, 200 and 250 mg/L was prepared and tested like wide range of concentration. Both the experiment was replicated five times and the percentage mortality was calculated. The corrected percentage of mortality was calculated by using Abbott's formula[17].
Corrected % mortality = (% mortality in test - % mortality in control)/(100-% mortality in control) ×100.
2.6. Pupicidal activity
Freshly emerged pupa was used for pupicidal activity. The concentration of plant extract and methods were followed as that of larvicidal activity. For each concentration 25 numbers of freshly emerged pupae were released individually and the percentage of mortality was recorded after 24 h exposure period. The experiment was replicated five times and percentage of mortality was calculated. The corrected percentage pupal mortality was calculated based on Abbott's formula as mentioned in larvicidal activity.
2.7. Statistical analysis
The experimental data was subjected to statistical analysis to derive mean and standard deviation. The significant difference in concentration of the plant extract and exposure period was confirmed by one way analysis of variance (ANOVA). Further individual mean significant difference was calculated by using post hoc Least Significant Difference test by using SPSS software version 16. The mean percentage larval mortality was subjected to probit analysis for LC50 and LC90, 95% upper and lower confidence (LCL-UCL) limits and Chi-square (X2) significance by using EPA computer probit analysis software program version 1.5[18].
3. Results
Aqueous and ethanol extracts of C. didymobotrya was tested in wide and narrow range of concentrations against immature stages of Cx. quinquefasciatus. According to WHO[13] experimental protocol wide range of tolerance test is important to fix the concentration for narrow range test. In wide range test the mean percentage mortality of immature mosquitoes exposed to ethanol extract after 24 h exposure period was presented in Table 1. Results revealed that 2nd instar larvae exposed to 100mg/L and above concentrations showed 100% mortality. Remaining stages such as 3rd, 4th and pupa, 100% mortality was observed at 1 000 mg/L concentration and above. The immature mosquitoes exposed to aqueous extracts was also presented in Table 1. In aqueous extract all the stages 100% mortality was recorded at 1000mg/L and above concentration. Based on this result narrow range of concentration was prepared and tested.
Table 1. Mean percentage mortality of immature Cx. quinquefasciatus exposed to wide range concentration of ethanol extract of C. didymobotrya after 24 h.
| Concentration tested in mg/L | Ethanol extract |
Aqueous extract |
||||||
| 2nd instar | 3rd instar | 4th instar | Pupa | 2nd instar | 3rd instar | 4th instar | Pupa | |
| 1 | 0.00±0.00 | 0.00±0.00 | 0.00±0.00 | 0.00±0.00 | 0.00±0.00 | 0.00±0.00 | 0.00±0.00 | 0.00±0.00 |
| 10 | 17.60±4.56 | 12.00±2.82 | 11.20±3.34 | 10.40±2.19 | 5.60±2.19 | 3.20±1.78 | 2.40±2.19 | 2.40±2.19 |
| 100 | 100.00±0.00 | 74.40±4.56 | 69.60±6.06 | 64.80±3.34 | 73.60±8.29 | 68.80±6.57 | 63.20±6.57 | 65.60±4.56 |
| 1 000 | 100.00±0.00 | 100.0±0.00 | 100.00±0.00 | 100.00±0.00 | 100.00±0.00 | 100.00±0.00 | 100.00±0.00 | 100.00±0.00 |
| 10 000 | 100.00±0.00 | 100.0±0.00 | 100.00±0.00 | 100.00±0.00 | 100.00±0.00 | 100.00±0.00 | 100.00±0.00 | 100.00±0.00 |
Values are mean percentage±standard deviation of five replications.
In narrow range test mean percentage mortality of immature mosquitoes exposed to ethanol extract after 24 h exposure period was presented in Table 2. Result revealed that in 2nd instar larvae 100% mortality was observed at 150 mg/L and above concentrations. Therefore, results of 50, 100, 150 mg/L concentrations were subjected to calculate LC50, LC90 and values. The LC50, LC90 and values was 52.42 mg/L, 78.17 mg/L and 0.043, respectively. The results of X2 values were not statistically significant (P>0.05) because the calculated value was lower than table value (3.841). However, in general one way ANOVA followed by LSD showed significant difference (F=273.39; P<0.05). The range of 95% confidence limits (LCL-UCL) of LC50 and LC90 values was 48.36-56.26 mg/L and 71.08-90.55 mg/L, respectively. In 3rd, 4th and pupal stages all those values are greater than 2nd instar stage. The LC50 value of 70.27 mg/L and LC90 value of 167.24 mg/L was calculated for 3rd instar stage. The value of 3rd stage was not significantly different (P>0.05) because the calculated value (7.571) was lower than table value (7.815). However, one way ANOVA followed by LSD showed statistically significant results (F=7.571; F=146.29; P<0.05). The LC50, LC90 and X2 value of 4th instar stage was 78.84 mg/L, 205.02 mg/L and 13.315, respectively. In 4th instar stage X2, one way ANOVA and LSD results showed significant difference at 5% level (F=328.37; P<0.05). In pupal stage LC50, LC90 and value was 82.46 mg/L, 217.96 mg/L and 15.249, respectively. The comparison of mean percentage mortality of pupal stage, X2, one way ANOVA and LSD results showed significant difference at 5% level (F=296.28; P<0.05).
Table 2. Mean percentage mortality of immature Cx. quinquefasciatus exposed to narrow range concentration of C. didymobotrya ethanol and aqueous extract after 24 h.
| Concentration tested in mg/L | Ethanol extract |
Aqueous extract |
||||||
| 2nd instar | 3rd instar | 4th instar | Pupa | 2nd instar | 3rd instar | 4th instar | Pupa | |
| 50 | 44.00±6.32a | 32.00±6.32a | 28.00±2.82a | 27.20±1.78a | 41.20±7.69a | 28.80±3.34a | 26.40±2.19a | 26.40±2.19a |
| 100 | 97.60±3.57b | 72.00±5.93b | 67.20±3.34b | 64.00±2.82b | 72.00±8.94b | 69.60±6.06b | 64.80±3.34b | 63.20±1.78b |
| 150 | 100.00±0.00c | 80.00±5.21c | 72.00±4.0c | 68.80±3.34b | 82.00±4.00c | 74.40±4.56c | 69.00±4.56b | 67.20±3.34b |
| 200 | 100.00±0.00c | 94.40±4.56d | 86.40±4.56d | 84.0±6.32c | 94.40±4.56d | 88.00±4.89d | 84.80±5.93c | 82.40±5.36c |
| 250 | 100.00±0.00c | 100.00±0.00e | 100.00±0.00e | 100.00±0.00d | 100.00±0.00e | 100.0±0.00e | 100.00±0.00d | 100.00±0.00d |
| F-value | 272.39 | 146.29 | 328.37 | 296.28 | 69.42 | 196.5 | 264.17 | 388.73 |
| LC50 (LCL-UCL) (mg/L/L) | 52.43 (48.36-56.26) | 70.27 (61.67-78.18) | 78.84 (37.39-110.01) | 82.47 (35.36-117.76) | 62.66 (53.26-71.09) | 75.71 (37.13-104.6) | 82.65 (36.62-117.4) | 84.93 (31.69-124.55) |
| LC90 (LCL-UCL) (mg/L/L) | 78.17 (71.08-90.55) | 167.24 (149.4-192.7) | 205.02 (143.0-572.4) | 217.97 (147.8-789.2) | 164.94 (145.8-193.0) | 193.97 (137.5-480.8) | 213.42 (145.8-724.2) | 227.48 (150.1-1152.2) |
| Chi-square X2 | 0.043 | 7.571 | 13.315 | 15.249 | 6.766 | 12.439 | 15.363 | 17.328 |
Values are mean percentage±standard deviation of five replications. F-values are based on one way ANOVA for individual instars. For 2nd instar the value of 200 and 250 mg/L concentrations of C. didymobotrya ethanol extract are not included for Chi-square analysis. Within the column similar alphabets are not statistically significant by LSD (P>0.05).
The result of immature stage mosquitoes exposed to aqueous extract was presented in Table 2. Result revealed that in 2nd instar larvae 100% mortality was observed at 250 mg/L concentration. The LC50, LC90 and X2 values was 62.66 mg/L, 164.93 mg/L and 6.766, respectively. The results of X2 value was not statistically significant (P>0.05) because calculated value was lower than table value (7.815). However, in general one way ANOVA followed by LSD showed significant difference (F=69.415; P<0.05). The range of 95% confidence limits (LCL-UCL) of LC50 and LC90 values was 53.26-71.09 mg/L and 145.80-193.03 mg/L, respectively. In 3rd, 4th and pupal stages all those values are greater than 2nd instar stage. The LC50 value of 75.70 mg/L and LC90 value of 193.97 mg/L was recorded for 3rd instar stage. The value showed statistically significant difference at 5% level (X2=12.439; P<0.05). In addition, one way ANOVA followed by LSD was also confirmed statistically significant results (F=196.50; P<0.05). The LC50, LC90 and value of 4th instar stage was 82.65 mg/L, 213.42 mg/L and 15.363 mg/L, respectively. The statistical comparison of mean percentage mortality of 4th instar stage, X2, one way ANOVA and LSD results showed significant difference at 5% level (X2=15.363; F=264.17; P<0.05). In pupal stage LC50, LC90 and X2 value was 84.93 mg/L, 227.48 mg/L and 17.328, respectively. The comparison of mean percentage mortality of pupal stage, X2, one way ANOVA and LSD results showed significant difference at 5% level. ( X2=17.328; F=388.73; P<0.05).
4. Discussion
Exploring bioactive medicinal plants in vector and insect pest management program is one of the eco-friendly approaches because they are easily biodegradable in nature. Naturally plants are rich store houses for potential bioactive compounds which are gaining appreciation in recent times among the scientific communities. Any plant species showing promising bioactivity in aqueous extract is a valuable source for the wealth of resource poor communities. According to Berenbaum[19] crude extracts of the plants may have mixtures of active compounds which act synergistically and their overall bioactivity was also greater than individual compounds[20]. In the present study aqueous and ethanol extracts of C. didymobotrya was showed promising bioactive potential against immature stages of Cx. quinquefasciatus.
Bioactivity of C. didymobotrya extract was significantly varied based the solvents used for extraction, concentration of the extract and stage of the immature mosquitoes tested. In this present study IInd instar larva was highly susceptible than late stages. The percentage of mortality was also significantly greater in higher concentration. Irrespective of solvent used for extraction 100% mortality was recorded at 150 mg/L concentration of ethanol extract in 2nd instar larva and the remaining stages 100% mortality was recorded at 250 mg/L concentration. In general, ethanol extract was showed promising result in all the immature mosquitoes. However, aqueous extract was also showed significant result but the concentration of the extract was little greater. When compare to cost effect aqueous extract was far better than ethanol extract. Present findings are in agreement with the report of Ojewole et al[11] reported that pronounced larvicidal activity in early stages of Anopheles fluviatilis and the larvicidal activity was dependent on concentration and incubation time. In their studies 100% mortality of the larvae was observed at 1% solution of root bark, stem bark and leaf extracts. However, present study 100% mortality was recorded at 250 mg/L concentration of leaves extract. The variation may be due to presence of active principle varied in geographical distribution of plant species and another possible reason Anopheles fluviatilis may be highly resistant than Cx. quinquefasciatus[21]. Obviously it is true in our study area chemical pesticide utilization in mosquito control program was very meager. Therefore, present study Cx. quinquefasiatus may be highly sensitive to exposed plant extracts that may be one of the reasons for higher mortality in less concentration.
In the present study LC50 and LC90 values of C. didymobotrya showed great variation in 2nd instar larvae in ethanol and water extract compared to other immatures. The LC50 value of 2nd instar larvae exposed to ethanol extract was 52.42 mg/L and aqueous extract it was 62.66 mg/L. This variation was attributed with dissolving nature of the active ingredients in respective solvent or the polarity of the solvent was also another important factor to determine dissolving nature of active principles. Many earlier reports confirmed the variation in the percentage of larval mortality in different group of mosquito species. Rajkumar and Jebanesan[8] reported ethanol extract of C. obtusifolia was showed LC50 value of 52.2 mg/L against 3rd late instar larvae of Anopheles stephensi. Govindarajan[7] reported LC50 value of methanol, benzene and acetone extract of Cassia fistula was 8.45 mg/L, 18.27 mg/L and 23.95 mg/L, respectively against Aedes aegypti. However, similar solvent extracts of the same plant LC50 value of Culex tritaeniorhynchus and Anopheles subpictus was 45.57 mg/L, 48.55 mg/L, 52.17 mg/L and 33.76 mg/L, 36.43 mg/L, 39.01 mg/L, respectively after 24 h exposure period. These earlier findings and current reports clearly indicate the important of solvent used for extraction. In conclusion, present study clearly revealed that leaf extracts of C. didymobotrya possessing bioactive compound to control immature stages of Cx. quinquefasciatus. However, hypothesis such does the plant extract are effective in natural mosquitoes breeding places need to be addressed. Further isolation of bioactive compound in a cost effective way and to develop simple formulation techniques is very much useful in large scale implementation of these plant extracts in mosquito control program.
Acknowledgments
Author is thankful to University of Gondar for financial assistant (UOG/Budget/no.6215) for the successful completion of research work. The acknowledgement extended to Solomon Tesfaye, Head, Department of Biology, Faculty of Natural and Computational Sciences for providing laboratory facilities. The continuous cooperation of my undergraduate student Angisom Tesfaye in mosquito larval collection and assisting in experiment is greatly appreciated.
Footnotes
Foundation project: Supported by University of Gondar (UOG/Budget/no.6215).
Conflict of interest statement: We declare that we have no conflict of interest.
References
- 1.Tabuti JRS. Senna didymobotrya (Fresen.) H.S. Irwin & Barneby. In: Schmelzer, GH, Gurib-Fakim A. Prota II(1): Medicinal plants/Plantes medicinales 1. (CD-Rom). PROTA, Wageningen, Netherlands. 2007.
- 2.Bekele-Tesemma A. Useful trees of Ethiopia: Identification, propagation and management in 17 agroecological zones. 2007. p. 552. Nairobi: RELMA in ICRAF Project.
- 3.Njoroge GN, Bussmann RW. Ethnotherapeutic management of skin diseases among the Kikuyu of central Kenya. J Ethanopharmacol. 2007;111:303–307. doi: 10.1016/j.jep.2006.11.025. [DOI] [PubMed] [Google Scholar]
- 4.Kamatenesi-Mugisha M. Medicinal plants used in reproductive health care in Western Uganda: documentation, phytochemical and bioactivity evaluation. Ph.D. thesis. 2004. p. p.228. Makerere University, Kampala, Uganda.
- 5.Korir RK, Mataj C, Kiiyukia C, Bii C. Antibacterial activity and safety of two medicinal plants traditionally used in Bomet district of Kenya. Res J Med Plant. 2012;6:370–387. [Google Scholar]
- 6.Joji Reddy L, Beena Jose Anjana JC, Ruveena TN. Evaluation of antibacterial activity of Trichosanthes cucumerina, L. and Cassia didymobotrya Fres. Leaves. Int J Pharm Pharmaceu Sci. 2010;2(4):153–155. [Google Scholar]
- 7.Govindarajan M. Bioefficacy of Cassia fistula Linn. (Leguminosae) leaf extract against chikungunya vector, Aedes aegypti (Diptera: Culicidae) Eur Rev Med Pharmacol Sci. 2009;13:99–103. [PubMed] [Google Scholar]
- 8.Rajkumar S, Jebanesan A. Larvicidal and oviposition activity of Cassia obtusifolia Linn (Family: Leguminosae) leaf extract against malarial vector, Anopheles stephensi Liston (Diptera: Culicidae) Parasitol Res. 2009;104:337–340. doi: 10.1007/s00436-008-1197-8. [DOI] [PubMed] [Google Scholar]
- 9.Govindarajan M, Sivakumar R, Rajeswari M. Larvicidal efficacy of Cassia fistula Linn. leaf extract against Culex tritaeniorhynchus Giles and Anopheles subpictus Grassi (Diptera: Culicidae) Asian Pac J Trop Dis. 2011a:295–298. doi: 10.1016/S1995-7645(11)60135-1. [DOI] [PubMed] [Google Scholar]
- 10.Govindarajan M, Mathivanan T, Elumalai K, Krishnappa K, Anandan A. Ovicidal and repellent activities of botanical extracts against Culex quinquefasciatus, Aedes aegypti and Anopheles stephensi (Diptera: Culicidae) Asian Pac J Trop Biomed. 2011b:43–48. doi: 10.1016/S2221-1691(11)60066-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Ojewole JAO, Rahim S, Shode FO. Mosquito larvicidal properties of aqueous extract of Senna didymobotrya. Nigerian J Nat Prod Med. 2000;4:46–47. [Google Scholar]
- 12.Worsley RR. The insecticidal properties of some East African plants. Ann Appl Biol. 2008;2(4):649–669. [Google Scholar]
- 13.Paramanik Manas, Chandra Goutam. Studies on seasonal fluctuation of different indices related to filarial vector, Culex quinquefasciatus around foothills of Susunia of West Bengal, India. Asian Pac J Med. 2010;3(9):727–730. [Google Scholar]
- 14.Bernhard L, Bernhard P, Magnussen P. Management of patients with lymphoedema caused by filariasis in North-eastern Tanzania: alternative approaches. Physiotherapy. 2003;89:743–749. [Google Scholar]
- 15.WHO. World Health Organization. Guidelines for laboratory and field testing of mosquito larvicides. WHO/CDS/WHOPES/GCPP/2005
- 16.Madhu SK, Vijayan VA, Shaukath AK. Bioactivity guided isolation of mosquito larvicide from Piper longum. Asian Pac J Med. 2011;4(2):112–116. doi: 10.1016/S1995-7645(11)60048-5. [DOI] [PubMed] [Google Scholar]
- 17.Abbott WS. A method of computing the effectiveness of an insecticide. J Econ Entomol. 1925;18:265–267. [Google Scholar]
- 18.EPA probit analysis, version 1.5. [Online] Available from: http://www.epa.gov/nerleerd/stat2.htm.
- 19.Berenbaum MR. Brementoun revisited: allochemical interations among in plants. Rec Adv Phytochem. 1985;19:139–169. [Google Scholar]
- 20.Chen W, Isman MS, Chiu SF. Antifeedant and growth inhibitory effects of limonoid toosendanin and Melia toosendanin extracts on the variegated cutworm Prodroma saucia (Lep., Noctuidae) J Appl Entomol. 1995;119:367–370. [Google Scholar]
- 21.Rana Inder Singh, Rana Aarti Singh. Efficacy of essential oils of aromatic plants as larvicide for the management of filarial vector Culex quinquefasciatus Say (Diptera: Culicidae) with special reference to Foeniculum vulgare. Asian Pac J Dis. 2012;2(3):184–189. [Google Scholar]