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. 2012 Jul 2:12:3.
doi: 10.1186/1472-6769-12-3.

High toxicity and specificity of the saponin 3-GlcA-28-AraRhaxyl-medicagenate, from Medicago truncatula seeds, for Sitophilus oryzae

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High toxicity and specificity of the saponin 3-GlcA-28-AraRhaxyl-medicagenate, from Medicago truncatula seeds, for Sitophilus oryzae

Pedro Da Silva et al. BMC Chem Biol. .

Abstract

Background: Because of the increasingly concern of consumers and public policy about problems for environment and for public health due to chemical pesticides, the search for molecules more safe is currently of great importance. Particularly, plants are able to fight the pathogens as insects, bacteria or fungi; so that plants could represent a valuable source of new molecules.

Results: It was observed that Medicago truncatula seed flour displayed a strong toxic activity towards the adults of the rice weevil Sitophilus oryzae (Coleoptera), a major pest of stored cereals. The molecule responsible for toxicity was purified, by solvent extraction and HPLC, and identified as a saponin, namely 3-GlcA-28-AraRhaxyl-medicagenate. Saponins are detergents, and the CMC of this molecule was found to be 0.65 mg per mL. Neither the worm Caenorhabditis elegans nor the bacteria E. coli were found to be sensitive to this saponin, but growth of the yeast Saccharomyces cerevisiae was inhibited at concentrations higher than 100 μg per mL. The purified molecule is toxic for the adults of the rice weevils at concentrations down to 100 μg per g of food, but this does not apply to the others insects tested, including the coleopteran Tribolium castaneum and the Sf9 insect cultured cells.

Conclusions: This specificity for the weevil led us to investigate this saponin potential for pest control and to propose the hypothesis that this saponin has a specific mode of action, rather than acting via its non-specific detergent properties.

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Figures

Figure 1
Figure 1
Chromatograms ofMedicago truncatulaseed extract.A. Injection into a C18 column (250 × 25 mm, 5 μm, Phenomenex) on an Agilent 1200 HPLC apparatus. The flow was 3.5 mL.min-1. The gradient was H2O + 0.04%TFA (solvent A) / ACN +0.04% TFA (solvent B) 90/10 for 5 min, then 60% solvent B for 25 min. The elution was monitored using a diode array detector at 210 nm. B. The fraction purified in A was injected into the same column with an elution under isocratic conditions (solvent B 30%). Peaks containing entomotoxic activity are indicated.
Figure 2
Figure 2
Negative-ion ESI-mass and tandem mass spectra of 3-O-[β-D-glucuronopyranosyl]-28-O-[β-xylopyranosyl(1 → 4)-α-L-rhamnopyranosyl (1 → 2) - α-L-arabinopyranoside medicagenate (3-GlcA-28-AraRhaxyl-medicagenate).(A) the ESI-mass spectrum of the isotope distribution of the (M-H)- ion at m/z 1087.33, of 3-GlcA-28-AraRhaxyl-medicagenate, (B) MS/MS of 3-GlcA-28-AraRhaxyl-medicagenate, precursor ion is m/z 1087.3, fragment ion at m/z 911.3 correlates to the loss of glucuronic acid (GlcA), (C) MS3 of 3-GlcA-28-AraRhaxyl-medicagenate, precursor ion is m/z 911.3, fragment ion at m/z 501.4 correlates to the sequential loss of sugar substituents arabinose, rhamnose and xylose (Ara-Rha-xyl) and thus the fragment ion at m/z 501.4 corresponds to M-H mass of medicagenic acid.
Figure 3
Figure 3
3-O-[β-D-glucuronopyranosyl]-28-O-[β-xylopyranosyl(1 → 4)-α-L-rhamno-pyranosyl (1 → 2)- α-L-arabinopyranoside medicagenate structure (MW: 1088.5 g/mol). GlcA, Ara, Rha, Xyl indicate respectively glucuronic acid, arabinose, rhamnose and xylose sugar moieties.
Figure 4
Figure 4
DPH fluorescence in increasing 3-GlcA-28-AraRhaxyl-medicagenate concentrations. The calculated CMC was 0.65 mg.ml-1 (0.6 mM).
Figure 5
Figure 5
Effect of 3-GlcA-28-AraRhaxyl-medicagenate on the growth of the yeastSaccharomyces cerevisiae.Yeast growth was followed by the absorbance, at 600 nm, of 1 mL of yeast culture in YNB media. An increasing concentration of saponin from M. truncatula was added to the media; 0 mg.mL-1 (▲); 0.025 mg.mL-1 (⋅); 0.05 mg.mL-1 (x); 0.1 mg.mL-1 (⋅); 0.25 mg.mL-1 (❏); 0.5 mg.mL-1 (;△); 1.5 mg.mL-1 (+).

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