Fatty Acid Composition and Toxicity of Melia azedarach L. Fruits against Malaria Vector Anopheles stephensi

1. Introduction

      The high rate of mortality of malaria disease on one hand and the side effects of chemical insecticide on the other hand has guided the new research fields toward the production of insecticides effective on larvae of the main malaria vector, Anophles stephensi. The extract of Neem or Melia indica (Meliaceae) fruit has recently been shown to have satisfactory larvicidal effects. This plant is native to India and has been studied for its antimalaria [1, 2], as well as spermicidal [3], antifungal [4] and antidiabetes effects [5].

      Meliaceae contains 45 genus and over 750 species in tropical regions [6]. Different parts of the species of this family have been used as medicine and insecticides for pest control [7].

     Melia azedarach L. (Meliaceae) is a short-lived deciduous tree, native in southern Asia and Australia, which reaches a maximum height of 50 feet. It has been cultivated since the sixteenth century, chiefly for ornamental purposes and has become naturalized in most tropical and subtropical countries [8]. The leaves’ extracts of this plant have insecticidal properties [7]. Due to the existence of M. azedarach L. in the north of Iran especially in the coastal parts of Caspian-sea [8], which have never been under research so far, we decided to study the plant considering its toxicity on larvae of the main malaria vector, Anophles stephensi.

Table 1. Chemical composition of the fatty acids methyl ester extracts of the fruits of Melia azedarach from Iran.

^aRI: relative retention indices as determined on a DB-1 column using the homologous series of n-alkanes. ^bThis compound was compared with an authentic sample.

2. Materials and methods

2.1. Plant Materials

     The fruits of M. azedarach L. were collected in September 2003, 8^th km Toskestan, around Gorgan, Golestan, Iran at an altitude of 160 m. Voucher specimen has been deposited at the Herbarium of the Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran (No. 6640 TEH).

     Air-dried and powdered fruits (500 g) were extracted using percolator with 2 liters of n-hexane for 3 times at the room temperature for two weeks. Fatty acids extract was concentrated in a Buchi Rota evaporator Model RE 120 at 50 °C, separately.

     Composition of fatty acids was estimated by gas chromatography. They were separated in the form of methyl ester derivatives. Preparation of the samples was done from 0.15 g of extracted fat, by saponification with 2 ml 0.5 N potassium hydroxide solution in 2 ml of methanol at 75 °C for half an hour, and following esterification with 1 ml of sulfuric acid solution in 5 ml of methanol (2:10 v/v) and 5 ml 0.5 N potassium hydroxide solution at 75 °C. After washing the solution 3 times with 20 ml of n-hexane, the fatty acids methyl ester (FAME) was transferred to the n-hexane layer [9].

2.2. Analytical technique

      Gas chromatography (GC) analysis was carried out using a Thermoquest 2000 GC with capillary column DB-1 (30 m x 0.25 mm x 0.25 mm); carrier gas, He; split ratio, 1:25, and a flame ionization detector. The column temperature was programmed at 50 °C for 1 min. and then heated to 265 °C with a 2.5 °C/min. rate and then kept constant at 265 °C for 20 min. Gas chromatography-Mass spectroscopy (GC-MS) was performed on a Thermoquest 2000 with a quadrupole detector, on capillary column DB-1; carrier gas, He; flow rate, 1.5 ml/min. The column held at 50 °C for 1 min. and programmed up to 265 °C at a rate of 2.5 °C/min and then kept constant at 265 °C for 20 min. The MS operated at 70 eV ionization energy. Retention indices were calculated using retention times of n-alkanes that were injected after the oil at the same chromatographic conditions. The compounds were identified by comparison of retention indices (RI, DB-1) with those reported in the literature and the authentic samples and by comparison of their mass spectra with the Wiley library [10] or with the published mass spectra [11].

2.3. Bioassay test

     The fatty acids extract from M. azedarach fruits were evaluated against late 3^rd and early 4^th instar larvae of An. stephensi under laboratory condition. The mosquito was collected from malarious area of Iran and then maintained at School of Public Health and Institute of Health Research, Tehran University of Medical Sciences. Different concentrations of extract were prepared in a DMSO and then larvae were exposed to different concentrations of the solutions. The minimum concentration was 1 mg/l and maximum 16 mg/l. The method was according to that described by WHO [12]. Mortality was determined after 24 h of exposure. All of the tests were conduced at 25 °C in a laboratory condition. In each concentration at least 100 mosquito larvae were tested. Dosage-mortality regression line was determined by the probit analysis method of Finney [13], using the Probit 79 program. Goodness of fit of the points to a straight line was tested by Chi-square (X²) analysis.

3. Results and discussions

     The constituents of the fatty acids methyl ester (FAME) extraction of M. azedarach are shown in Table 1. The FAME was light green with a distinct sharp coffee odor, in a yield of 0.2% (w/w). Thirteen components were detected in the FAME of M. azedarach, representing 86.84% of the total extract. Some compounds were compared with authentic samples (Table 1). The major constituents were methyl palmitate (18.8%), methyl linolenate (16.1%) and methyl linoleate (9.8%).

     In a previous study on oil of M. indica seeds from forty-two samples from different areas of India, methyl ester of oleic, stearic, palmitic, linoleic, myristic, arachidic (eicosanoic), and behenic (docosanoic) acids were detected. Between 90% and 99% of the total FAME was oleic acid (48.6-69.0%), palmitic acid (14.5-25.0%), and stearic acid (13.4-27.5%), respectively. Other fatty acids methyl ester (myristic, arachidic, linoleic, and behenic) were present in negligible or nondetectable amounts. A wide variation in the contents of different fatty acids is, thus, evident [14].

      Mortality rate of A. stephensi by different concentration of M. azedarach is shown in Table 2. The mortality data were subjected to probit analysis using Finney method [13]. From the regression line between logarithmic dose and probit mortality, all of the parameters including LC₅₀ and 95% confidence interval, LC₉₀ and 95% confidence interval, were determined. Table 3 indicates parameters of regression line. In another study, neemarin an AZ-rich formulation was tested against mosquito larvae both under laboratory and field conditions. LC₅₀ and LC₉₀ values for A. stephensi, the main malaria vector in Iran, was calculated 0.2 and 0.9 mg/l [15].

      Neem products are capable of producing multiple effects on a number of insect species, such as anti-feeding effect, growth regulation, fecundity suppression, and sterilization, oviposition repellency or attractancy and changes in biological fitness [16]. For example neem extracts have been shown to have repellent activity against Mansonia spp., mosquitoes in Ethiopia [17]. In mosquitoes, compounds extracted from neem tree showed mortality for 4^th instar larvae of A. stephensi with LC₅₀ value of 60 ppm [18].

Table 3. Probit regression line parameters of Anopheles stephensi exposed to Melia azedarach

  In conclusion, the extraction of M. azedarach showed biological effects against the larvae of A. stephensi. Similar activities in M. azedarach and other species of Melia also are shown by other researchers around the world [16-19]. Therefore, extracts of this plant offer a valuable candidate for potential development of a botanical insecticide for malaria vectors control.