A study carried out in 2016 showed that there were an estimated 2,927,266 new malaria cases in Ethiopia with 4,782 deaths (Tadele et al., 2019). In addition, malaria is the most widespread disease being number one of the top ten diseases that causes high morbidity in the study site (Fogera District). It is highly prevalent in the rainy season mainly between May and July, and at the end of rainy season in October and December (Bake-Migongo et al., 2012).

Anopheles mosquito is the major public health threat as it is the only vector transmitting malaria which causes mortality, morbidity, economic loss and social disruption (Sakthivadivel and Daniel, 2008). Although adversely contaminating the environment, synthetic insecticides are available for controlling these mosquitoes. However, mosquitoes build up hereditary resistance to synthetic insecticides (Singh et al., 2007). The recent WHO report confirmed that malaria vectors developed resistance to the four commonly used insecticides: pyrethroids, organochlorines, carbamates and organophosphates across the world (WHO, 2018a). Since mosquitoes develop resistance towards chemical insecticides, researchers have been searching for alternative control mechanisms (Markouk et al., 2000).

Traditionally, plant products have been used by the people for many years in insect control. Secondary metabolites present in plants serve as defense mechanism against insect attacks and their derivatives provide alternative source in the control of mosquito (Akhtar et al., 2010). Furthermore, plants possess eco-friendly bioactive chemicals that are alternative to control mosquitoes (Raghavendra et al., 2009). Plant based pesticides are less toxic, easily biodegradable and delay the development of resistance because of its new structure (Kamaraj and Rahuma, 2010). Recent researches have proved effectiveness of plant derived compounds including bioflavonoid, saponin, steroids, alkaloids, essential oils and tannins has potential mosquito larvicidies (Vatandoost and Hanafi-Bojd, 2008; Akhtar et al., 2010).

J. procera has traditional importance for malaria treatment in most rural area of Ethiopia (Desta, 1995). Although researchers have tried to test the plant’s larvicidal activity and its medicinal value in malaria treatment (Kaliyaperumal et al., 2014; Ismat et al., 2018), they have not studied it in in vitro, using different solvents like chloroform, petroleum ether, acetone and ethanol extracts. Therefore, the aim of the study was to evaluate larvicidal activities of J. procera extracts in in vitro against 3rd – 4th instars larvae of A. arabiensis.

Larvicidal bioassay

The existing standard was used to evaluate larvicidal efficacy of J. procera extracts against anopheles mosquito larvae (WHO, 2005). The bioassay was performed at relative humidity of 70-85%, room temperature, and pH 7.0 of distilled water. Twenty 3rd- 4th instar anopheles larvae were taken by pipette and added to each flask containing 1000 mL distilled water. Flasks with mosquito larvae were treated with 200, 400, and 600 mg/L of all solvent extracts of J. procera. Each treatment for experimental group was conducted in three replicates as designed above (Table 1). For control group, twenty 3rd-4th instar larvae were also transferred to 1000 ml distilled water. The larvae of both experimental and control groups were fed an equal amount of sucrose every 24 h. Finally, the effect of the plant extracts on larvae was tested by counting the number of dead larvae. Larvae counting were conducted at 24, 48 and 72 h of treatment and the percentage mortality was calculated. The computed mortality of larvae was adjusted using Abbott’s formula (Abbott, 1925) as shown below: 

Data analysis

Data was analyzed using SPSS version 16. Difference in concentration of plant extracts, type of solvents, and time of exposures were used as independent variables and mortality of larvae was used as dependent variable. One way ANOVA was used to determine the significant difference of the mortality of A. arabiensis larvae between the control and the experimental group and value of 0.05 was taken as significance level.

In the current study, J. procera extracts were tested to have significant secondary metabolites (Table 2). Similar secondary metabolites were screened from J. procera regardless of using different solvents in extraction. Accordingly, from J. procera extracted by chloroform, petroleum ether, ethanol, and acetone solvents, phytochemicals including alkaloids, proteins, carbohydrates, glycosides, phytosterols, saponins, tannins and phenols were identified.

In the present work J. procera extracts were also evaluated to kill Anopheles mosquito larvae. Accordingly, chloroform, ethanol, acetone, and petroleum ether extracts of J. procera applied at a concentration of 200 mg/L, 400 mg/L and 600 mg/L showed significant mean A. arabiensis 3rd- 4th  instars larvae mortality at different time exposure (P=0.00). However, no larval mortality was recorded for the control group within the given time intervals (Table 3). Variations in toxicity of plant extracts against the larvae varied with the solvent used in extraction. Ethanol extracts of J. procera was effective against Anopheles mosquito larvae in which it showed linear increasing of mortality rate for A. arabiensis larvae than others. Although mortality rate increased with concentrations, it decreased with increased time exposure. The most effective time exposure for mortality of larvae was within 24 h. For all solvents used, maximum mean mortality  was  recorded  for  the  highest concentration (600 mg/L).

In the current study, J. procera extracted with chloroform, petroleum ether, ethanol, and acetone solvents had similar phytochemicals including alkaloids, proteins, carbohydrates, glycosides, phytosterols, saponins, tannins and phenols. In agreement with the present finding, other researchers also identified that J. procera leaves extracts are a source of phytochemicals such as tannins, phenols, saponins,  alkaloids and flavonoids in which alkaloids and phenolics have been reported to be insecticides (Shaalan et al., 2005; Ismat et al., 2018). Moreover, studies identified that plants offer great promise as source of phytochemicals with potential as insecticides that can play crucial role in the control of mosquitoes (Ebong, 2008; Pathak et al., 2018). Therefore, screening secondary metabolites from J. procera might have incredible contribution in order to develop new alternative drugs in malaria control.

In the present study J. procera extracted with chloroform, ethanol, acetone, and petroleum ether applied at a concentration of 200, 400 and 600 mg/L killed A. arabiensis larvae. Kaliyaperumal et al. (2014) also evaluated larvicidal activity of J. procera against A. arabiensis and suggested that the plant can be the potential larvicidal agent. In agreement with our study, researchers have shown that plant derived compounds have larvicidal  activities  and  can  be  efficient in  insect control (Vatandoost and Hanafi-Bojd, 2008; Akhtar, 2010; Pathak et al., 2018). Thus, J. procera which has also been identified by the current work to contain significant plant derived compounds such as alkaloids, glycosides, saponins, phytosterols, phenols, and tannins can be an alternative in mosquito control. Such plant derived compounds are more important than other alternatives since they are less toxic and easily biodegradable in the environment (Kamaraj and Rahuman, 2010).

The extraction yield and biological activity of the plant extracts can be affected by solvents used in extraction due to differences of solubility in different solvents (Ajanal et al., 2012; Barchan et al., 2014). As also reported by Ghosh et al. (2012), since insecticidal effects of plant extracts can vary due to the polarity of solvents used during extraction, ethanol extract of J. procera showed linear increase in mortality rate for A. arabiensis larvae than others. In agreement with the current work other study also identified the highest phenolic contents from L. aromatica using ethanol (Quy et al., 2014). The current work also agrees with the report of Sukumar et al. (1991), which indicated variations in toxicity of plant extracts against the larvae that varied with the solvent used in extraction. Although mortality rate increased with concentrations, it decreased with increased time exposure for unknown reason to the current study. The most effective time of exposure and concentration for maximum mean mortality of larvae was within 24 h and 600 mg/L respectively. In agreement to our work, another study done on larvicidal activity of J. procera showed that larval    mortality    rate    is    time   and   dose-dependent (Kaliyaperumal et al., 2014). Ali et al. (2014) also identified that the total mortality is positively correlated with increasing concentrations.

Phytochemical screening of chloroform, petroleum ether, ethanol, and acetone extracts of J. procera showed positive result for alkaloids, carbohydrates, glycosides, phenols, flavonoids, proteins and tannins. J. procera extracted with the above solvents showed significant mean mortality of A. arabiensis larvae within 24, 48 and 72 h at different concentration (200, 400 and 600 mg/L).

The authors have not declared any conflict of interests.

The authors would like to thank Debre Tabor University for financing this study. Bahir Dar University, Department of Chemistry is also appreciated for the laboratory materials and chemicals, as well as Woreta Agriculture College for providing the laboratory room.