Larvicidal Activity of the Fruit Mesocarp Extract of Balanites aegyptiaca and its Saponin Fractions against Aedes aegypti Bishnu P . Chapagain and Zeev Wiesman! The Phyto-Lipid Biotechnology Laboratory, Department of Biotechnology Engineering, The Institutes for Applied Research, Ben-Gurion University of the Negev, P .O. Box 653, Beer-Sheva 84105, Israel
Abstract The present study indicates the efficacy of the saponins fraction of the fruit mesocarp extract of Balanites aegyptiaca Del. (Zygophyllace) as a larvicidal agent against the common dengue vector – Aedes aegypti mosquito. A series of concentrations of fruit mesocarp extract of B. aegyptiaca fruit, its crude saponin extract and pure saponin fraction were tested against the laboratory-reared third instars larvae and compared with their LC50 values. The pure saponin fraction was found most active larvicidal, followed by crude saponin extract and mesocarp extract. The LC50 values of pure saponin extract, crude saponin extract and mesocarp extract were 145, 315 and 935 ppm respectively. In separate experiments, concentrations of 35 ppm, 260 ppm and 850 ppm of pure saponin fraction, crude saponin extract and mesocarp extract respectively were found to inhibit the emergence of 50% of the tested larvae population (EC50). The pure saponin fraction also interfered with adult emergence. Keywords: Balanites aegyptiaca, saponins, Aedes aegypti, dengue, larvicidal, adult emergence.
Introduction Dengue is an acute fever which occurs as a dengue fever or dengue haemorrhagic fever (DF/DHF). It is a serious vector-borne disease caused by a virus and transmitted by Aedes aegypti mosquito. This globally prevailing disease has recently grown enormously. The disease is now endemic in more than 100 countries in Africa, the Americas, the Eastern Mediterranean, South-East Asia and the Western Pacific. Every year more than 100 million people residing in these areas are being infected by DF/DHF[1]. There is no vaccine to prevent dengue infection, nor are there drugs to combat the disease in infected persons, so vector control is the most opted solution available so far for !
reducing the morbidity. Most of the widely used vector interruption methods are synthetic insecticides-based. These synthetic insecticides not only affect the non-target population but also increase resistance to the vector[2]. In this regard, the search for natural insecticides which do not have any ill effects on non-target population and are easily degradable is of top priority these days[3]. In an earlier communication we have pointed out saponin-rich extracts from Balanites aegyptiaca and Quillaja saponaria as a possible candidate for natural bioactive agent against Aedes aegypti and Culex pipeins mosquito larvae[4]. However, high concentrations of these preparations were needed for effective control of the mosquito larvae. In continuation of our study we have investigated a saponin fraction,
wiesman@bgu.ac.il; " /Fax: 972-8-6477184 203
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which in much smaller concentration, is found to be active to control the mosquito larvae. Moreover, as adult mosquitoes transmit diseases, the critical concentrations of botanicals, which inhibit 50% of the treated larval population from emerging as adults (EC50), are more meaningful than the LC50[5,6]. Since the determination of effective concentrations that inhibit adult emergence has not received the attention of many researchers, in this communication we report the efficacy of B. aegyptiaca saponin compared with its crude extract against the Ae. aegypti larvae control with concerned LC and EC values.
Materials and Methods Plant materials, extraction and isolation Balanites aegyptiaca Del. (Zygophyllaceae) is a widely grown woody tree of the dryland areas. It is widely grown in the African continent, the Middle-East and South Asia[7]. B. aegyptiaca plant tissues have been used as folk medicines in Africa and Asia. The fruit consists of epicarp (5–9%), mesocarp (28–33%), endocarp (49– 54%) and kernel (8–12%). One estimate shows that every year more than 4 million tons of B. aegyptiaca fruits are produced in Sudan alone[8]. The mesocarp (pulp) was scraped from well-ripened fruits collected from the Aravarift valley of Israel. A voucher specimen (#76816) was deposited at the herbarium of the Hebrew University of Jerusalem. The scraped mesocarp was freeze-dried and then powdered and defatted by petroleum ether 3x1 h at 40 °C. After filtering the petroleum ether, the marc was extracted with methanol 3x1 h with mild heating. The combined methanol extract was concentrated and methanol extract of mesocarp (MCE) was obtained. In order to get the crude saponins extract the MCE was dissolved in methanol and 204
acetone was added (1:5 v/v) to precipitate the saponins as described by Yan et al., 1996[9]. The precipitate was dried under vacuum, turning to a whitish amorphous powder named as crude saponin extract (CSE). To get the pure saponin fraction (PSF), certain amount of CSE was fractionated by applying to Merck silica gel-60 (230-400 mesh) column chromatography and eluted successfully with chloroformmethanol-water (70:30:10) as described by Favel et al., 2005[10]. Five fractions were collected and the solvents were evaporated under reduced temperature; fraction 1 was chosen based on the detection of the total saponin concentration. The total saponin concentration of each fraction was measured by spectrophotometric method as described by Baccou et al., 1977[11] and Uematsu et al., 2000[12] with some modification. The saponin fraction was named as PSF. From the fridgedried fruit mesocarp, a yield of 63.5%, 13.3% and 4.5% (w/w) of each methanol extract (MCE), crude saponin extract (CSE) and pure saponin fraction (PSF) was obtained (Table 1). Table 1. Yield and LC50 of different extract and fraction of B. aegyptiaca fruit mesocarp used in experiment against third instars larvae of Ae. aegypti
Each value is the mean of 3±SE (n=3). Yield represents the % (w/w) of each treatment materials obtained from the fridge-dried fruit mesocarp.
Bioassay test Eggs of the Ae. aegypti mosquitoes were obtained from the Entomology Laboratory of the Israel Ministry of Health, Jerusalem, and Dengue Bulletin – Vol 29, 2005
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necessary larvae were reared in the laboratory of the Institutes for Applied Research, BenGurion University, as described by the standard protocol of WHO, 1973[13]. Twenty larvae of third instars were placed in a 250 ml disposable plastic cup containing 100 ml of treatment solutions. After placing the larvae the plastic cups were kept in the growth room maintained at 27±2 °C with 16 h day (light period) and 40±5 % relative humidity. A multiple 2-fold concentrations were set up to establish a working range (from 25 to 3200 ppm) in triplicate manner and LC50 was calculated. With each experiment, a set of control using just tap water (untreated set) was also run for comparisons. The effects of the treatments were monitored by counting the number of dead larvae each day. For the LC50, the data of 48 h was used because till that time no pupa was observed even in control treatments. During the course of the experiment, a food based on baby food was provided to the larvae. The percentage of mortality was corrected using Abbott’s formula as suggested by Finney, 1971[14]. In another series of experiments, observations on the emergence and larval duration of the larvae that were reared at sublethal doses of the active fractions of the treatments were made and the emergence of the 50% of the test larvae (EC50 values) was determined using the probit programme. Each set of experiment was replicated thrice and the mean and standard error of mean was calculated.
against the third instars larvae of Ae. aegypti. The results showed that all three extracts used in the experiment were found active against the larvae; however, PSF was found to be most active. For PSF to kill 50% of the tested larvae, 145 ppm was needed, which was less than half dose of CSE and one-sixth of MCE. The yield column shows the percentage of yield of each material in relation to the fridgedried B. aegyptiaca fruit mesocarp. This indicates some proportional rate of active ingredients in the basic material. The PSF of the methanol extract of the B. aegyptiaca fruit mesocarp was found very active with regard to its efficacy to inhibit adult emergence. Exposure of the early fourth instars Ae. aegypti larvae to 35 ppm prevented the emergence of 50% of the treated populations (Table 2), whereas a concentration of 260 ppm and 850 ppm of each saponin extract (CSE) and methanolic extract (MCE) respectively was needed to inhibit the emergence of the 50% of the tested population. This shows that in PSF only 21.2% concentration was needed no inhibit 50% emergence whereas more than 80% concentration was needed for CSE and MCE. In separate experiments when the exposure of the larvae to sub-lethal Table 2. EC50 (ppm) and their subsequent % of LC50 value of the different extracts and fraction of the B. aegyptiaca fruit mesocarp used in the experiment against third instars larvae of Ae. aegypti
Results and Discussion Table 1 presents the LC50 (ppm) after 48 h of exposure of the methanolic extract of mesocarp (MCE), crude saponin extract (CSE) and pure saponin fraction (PSF) of the B. aegyptiaca fruit mesocarp treatments used in the experiment Dengue Bulletin – Vol 29, 2005
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concentration of PSE (20 ppm) was carried out, that extended the duration of larval development, pupation and emergence to adult development to 7 to 8 days, compared to the larvae reared only in tap water (control) which required only 11–12 days (data not shown). The extension of the total developmental duration of Ae. aegypti indicated the possible presence of insect growth regulatory activities too in this fraction in sublethal concentration.
Conclusion The results of this study clearly show that saponin fraction of the B. aegyptiaca fruit mesocarp is highly larvicidal against the Ae. aegypti mosquito larvae compared to crude saponin and methanolic extracts. Since the use of a 35 ppm of saponin fraction of B. aegyptiaca fruit mesocarp was found sufficient to inhibit the emergence of 50% of the test larvae population, this will certainly help to reduce the mosquito population drastically. Earlier studies have also indicated the possible use of saponins as a natural larvicidal agent against the mosquito; however, no study so far has reported on the saponin in relation to the inhibition of the emergence of the adult mosquito[4,15,16,17]. This low EC50 value
of the pure saponin fraction of this experiment gives a more reliable evidence for using the saponin as a natural larvicidal agent against mosquito larvae. Since a large proportion of the population living in areas where dengue is a serious problem suffer from varying degrees of poverty, the discovery of plant-derived compounds that could help with the control or eradication of these diseases would of great value, particularly if the concerned plants were readily available to those who needed to use them. In this context, the highly bioactive compound of B. aegyptiaca, which are being grown widely in most of the dengue-infected areas, offer an opportunity for developing alternatives to rather expensive and environmentally hazardous insecticides. Consequently, since these plants are widely grown in rural and remote areas, their commercial exploitation would contribute towards rural economic development.
Acknowledgements The authors thank Dr Zhana Abramovich for his valuable suggestions and technical help. We would also like to thank Kibbutz Samar for maintaining the Balanites aegyptiaca plant. Thanks are also due to the Dibner Foundation (Wilton, Connecticut) for supporting this study.
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