Grinded seeds of A. squamosa were percolated with methanol. Solvent was evaporated to produce a crude MeOH extract which was then partitioned between CHCl₃ and H₂O. The residue produced by further evaporation of the chloroformic phase was chromatographed on a silica gel column by using chloroform, ethyl and finally methanol as eluents. The isolation of ACG from the subextract was guided by thin layer chromatography (TLC) with the Kedde’s reagent. Chromatographic fractions were processed on high-performance liquid chromatography (HPLC) by using RI detection, a Beckman C 18 column (5 mm particle size, 25 cm × 1 cm i.d.) and mixtures of methanol and water (90:10) as the mobile phase to yield pure ACG. Characterization of tested natural ACG was assessed by spectroscopic techniques (IR, ¹H-NMR, ¹³C-NMR, and MS) as well as α_D determination, in comparison to previously reported data.

Acetylated analogues, squamocin (3 OAc) (11), molvizarin (3 OAc) (12) and motrilin (3 OAc) (13) were obtained by chemical acetylation with acetic anhydride and addition of triethylamine (25˚C, N₂ atmosphere) followed by solvent evaporation and purification on silica gel column. The compounds of interest were identified by high resolution spectroscopic methods (¹H and ¹³C-NMR) [20] .

Methoxy methylated derivatives, squamocin (MOM) (14) and motrilin (MOM) (15) were obtained by adding N,N-diisopropylethylamine and methoxymethyl chloride to a dichloromethanic solution of natural ACG under a nitrogen atmosphere. The reaction was followed by TLC until the formation of products. MOM derivatives were purified by flash column chromatography. The identification of the bis-THF ACG methoxy methylated, were achieved by comparison of spectroscopic data ¹H-NMR and ¹³C-NMR with the sample of the original ACG.

Spodoptera frugiperda’s natural life cycle is between 26 and 31 days. Females lay between 200 and 300 eggs for 5 to 6 days. Larvae emerge between 48 and 72 h and remain in this state between 12 and 15 days. Then, they pass to pupal stage where they remain for 7 days, after which the adults emerge. The larval diet of S. frugiperda consists in a mixture of yeast (3 g), boiled and milled bean (250 g), wheat germ (12.5 g), ascorbic acid (1.5 g), agar agar (12.5 g), methyl p-hydroxybenzoate (1.5 g), formaldehyde (4 mL of a 38 % water solution), and water (500 mL).

The results are reported as mean values ± SD. The differences between the mean values were evaluated by analysis of variance (one way ANOVA). The Tukey test was used for all pair wise multiple comparisons of groups. P > 0.05 were considered not significant difference between treatments [22] .

Chromatographic processes permitted the separation of 10 known ACG: squamocin (1) [23] , molvizarin (2) [24] , motrilin (3) [24] , rolliniastatin-2 (4) [25] , almuñequin (5) [26] , cherimolin-1 (6) [4] , cherimolin-2 (7) [4] , annonacin (8) [27] , squamocin D (9) [28] and asiminecin (10) [29] (Figure 1), that were exhaustively purified by reversed phase HPLC to be employed in the bioassays. The ACGs 4, 6, 7 and 10 had not been previously isolated from A. squamosa.Acetogenins represent around 0.07% of the seed weight.

ACG acetylated (OAc) derivatives: squamocin (3 OAc) (11), molvizarin (3 OAc) (12) and motrilin (3 OAc) (13), and their methoxy methylated (MOM) derivatives: squamocin (MOM) (14) and motrilin (MOM) (15) (Figure 2) were synthesized and purified, and then characterized by spectroscopic techniques (Table 1 and Table 2).

As indicated in Table 1, the incorporation of 100 µg/g of diet of ACG squamocin into the artificial diet of S. frugiperda displayed the highest antifeedant effects (FR₅₀) followed by squamocin (3 OAc) (0.66 ± 0.17 and 0.77 ± 0.14; respectively), and caused a marked decrease in larval growth under the conditions of the experiment. The ACGs motrilin, motrilin (3 OAc), molvizarin and molvizarin (OAc) did not present a significant antifeedant action under the conditions of the experiment (Table 3).

The results obtained in the tests of toxicity for each ACG show that all natural ACG, possesses a lethal effect on the S. frugiperda larvae in the early stages (Table 3) with

^aMethyl: CH₃; ^bMethylene: CH₂; ^cCarbonyl: C=O.

^aMean ± SD.

high mortality effect at 100 µg/g of diet over than 80%. The ACGs structurally transformed by acetylation showed less toxicity than natural ACGs (Table 3). The pupal mortality of squamocin (OAc), molvizarin (OAc) and motrilin were 30%, 30% and 15%, respectively. It is apparently related to the observed malformations as shown in Figure 3(A) and Figure 3(B). The surviving adults showed malformations and decrease in size relative to the control (Figure 4(A) and Figure 4(B)), that led them to death before laying eggs. Significant increments in the duration of the larval stage where observed for all ACGs tested.

According to results obtained on food consumption and its utilization, it has been observed that motrilin, squamocin (OAc) and molvizarin (OAc) at 100 µg/g of diet, influenced GR and ECI of instar larvae. A drop in ECI indicated that more food has been metabolized for energy and less has been converted to insect biomass (i.e., growth). These, resulted in an important larval growth decrease and subsequent significant larval mortality at very early stages in their life cycle and would reveal the presence of toxic compounds when compared with control (P < 0.05, Tukey multiple range test). Motrilin (3 OAc) had the lowest consumption percentage and the lowest conversion of absorbed nutrient (Table 4). Although, the addition of 100 µg/g of MOM derivatives produced no significant toxic effects (Table 3) and changes in nutritional index values (Table 4) all emerged adults died within 24 h.

The most important toxic action was observed with natural ACG against S. frugi- perda larvae at the dose tested, following acetylated and MOM derivatives. These results are in agreement with a previous report from our team for other ACGs isolated from different species of Annonaceae [14] .

^aNumbers in columns represent mean ± SD. ^bMeans within a column followed by the same letter are not significantly different (P > 0.05, Tukey multiple range test). N/D: Not determined (Mortality at early instars).

Since hydroxyls groups flanking the THF have been blocked in MOM and acetylated derivatives a decrease in insecticidal activity is observed. This highlights the importance of the hydroxyl group to the insecticidal activity of natural ACG.