The Annona muricata leaf ethanol extract affects mobility and reproduction in mutant strain NB327 Caenorhabditis elegans

Abstract

The C. elegans NB327 mutant strain is characterized for the knockdown of the dic-1 gene. The dic-1 gene is homologous to the dice-1 gene in humans, encoding the protein DICE-1 as a tumor suppressor. Absence or under-regulation of the dice-1 gene can be reflected in lung and prostate cancer [17], [18]. This study evaluated the effect of EEAML on the C. elegans NB327 mutant strain. Phenotypic aspects such as morphology, body length, locomotion, and reproductive behaviour were analyzed. It is important to emphasize that the strain presents a phenotype characteristic with respect to egg laying and hatching. Reported studies showed that Annona muricata extract and its active components evidence anti-cancer and anti-tumor effects, through experimentation in vivo and in vitro models. However, neurotoxicity has been reported as a side effect. The results showed that the mutant strain NB327 was exposed to EEAML (5 mg/ml) concentration, it showed a significant decrease in average locomotion, resulting in 13 undulations in 30 s. This contrasts with the control strain's 17.5 undulations in 30 s. Similarly, the number of progenies was reduced from 188 progenies (control strain) to 114 and 92 progenies at the dose of (1 mg/ml and 5 mg/m) EEAML. The results of this study suggest that EEAML has a possible neurotoxic effect in concentrations equal to or greater than 5 mg/ml. Also, it does not have positive effects on the mutant strain of Caenorhabditis elegans NB327 phenotype.

Keywords: Annona muricata (A. muricata); CLSI, Clinical and Laboratory Standards Institute; Caenorhabtidis elegans (C.elegans); DMSO, Dimethylsulfoxide; EEAML, Ethanol extract of Annona muricata leaves; Gene dic-1; Gene dice-1; LD50, Lethal Dose 50; MHA, Mueller Hinton Agar; MHB, Mueller Hinton Broth; MIC, Minimum Inhibitory Concentration; NGM, Nematode growth medium; Strain NB327.

Fig. 1

Phenotypic characterization of the differences between the mutant strain NB327 and the wild strain N2. A. Strain NB327 larval stages, embryonic stage (egg) is observed, larval L1-L4 stage and adult larvae. B. NB327 eggs and N2 wild strain. The difference in size and shape C. The distribution of eggs in the uterus of the NB327 and N2 wild strain. Stereoscopic pictures taken from the Olympus SZ51 40X, Olympus optical microscope CX31, and Leica DM IL inverted microscope 40X.

Fig. 2

Body length and locomotion of NB327 mutant and N2 wild strain. A. Body length of NB327 mutant strain in reticle microscope, comparison of the NB327 mutant and N2 wild strain body length without extract, exposed EEAML (1 mg/ml and 5 mg/ml) concentrations and control with DMSO 0.2%. Significant effects on length were not observed as being produced by EEAML. B. Undulation photography taken as a reference of the NB327 mutant strain locomotion (*Undulation head) and locomotion comparison between NB327 mutant and N2 wild strain. A significant decrease in locomotion was observed when using EEAML 5 mg/ml concentration. The tests were performed in triplicate and analysis was performed using Graph Pad Prims program version 6 (One-way ANOVA). Significant differences were determined as P<0,05. Photographs taken by the Olympus microscope light CX31 40X and Leica DM IL inverted microscope 40X.

Fig. 3

Reproductive behaviour of the NB327 mutant and N2 wild strain. A. Reproductive behaviour of the NB327 mutant and N2 wild strain expressed in the egg laying; tests performed without extract, using EEAML (1 mg/ml and 5 mg/ml) concentrations, and control with DMSO 0.2%. A significant decrease in the number of eggs is observed using EEAML (1 mg/ml and 5 mg/ml) concentrations. B. Reproduction assay. A reduction in progeny numbers is observed in the strain NB327, exposed to EEAML (1 mg/ml and 5 mg/ml). All assays were performed in triplicate and analysis was performed with the Graphpad Prism program version 6 (One-way ANOVA), showing statistical significance P=0.0682 (P>0.05).