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Preliminary molecular characterization of a proinflammatory and nociceptive molecule from the Echinometra lucunter spines extracts.
Sciani JM
,
Zychar B
,
Gonçalves LR
,
Giorgi R
,
Nogueira T
,
Pimenta DC
.
Abstract
BACKGROUND: Sea urchins are animals commonly found on the Brazilian shoreline, being Echinometra lucunter the most abundant species. Accidents caused by E. lucunter have been reported as one of the most frequent in Brazil, and are characterized by intense pain and inflammation, consequence of spine puncture in the skin. In order to characterize such toxic effects, we isolated one molecule that caused inflammatory and nociceptive effects.
METHODS: E. lucunter specimens were collected without gender distinction. Spines were removed and molecules were extracted, fractionated by RP-HPLC and assayed for inflammatory and nociceptive activity, in a biological-driven fractionation way, until the obtainment of one active molecule and its subsequent analysis by mass spectrometry (MS and MS/MS). For inflammation, intravital microscopy was performed on the mouse cremaster muscle, in order to evaluate rolled, adherent and migrating leukocytes. Paw edema was also evaluated. For the nociceptive activity, the paw pressure test was performed in rats.
RESULTS: One molecule could be isolated and related to the inflammatory and nociceptive activity. Regarding inflammation, increase in adherent and migrating cells was observed in the cremaster muscle after the administration of the molecule. Corroborating the inflammatory response, paw edema was also observed, although only in 20% of controls and 20 min after injection. Additionally, this molecule was able to decrease significantly the pain threshold, characterizing hyperalgesia. This molecule was analyzed by mass spectrometry, and according to the exact molecular mass, isotopic distribution and fragmentation profile, it was possible to propose the molecular formula C29H48N3O10.
CONCLUSIONS: One isolated molecule from the spine extract of E. lucunter is able to elicit inflammation and hypernociception in animal models, which is in agreement with the effects observed in sea urchin accidents.
Fig. 1. Leukocyte count on the cremaster muscle microcirculation 2 h after 25 and 50% SPE fractions injection. * p < 0.05
Fig. 2. Fractionation of SPE 25% and biological assay of its fractions. a RP-HPLC purification of 25% SPE fraction, in a C18 column, elution by 0 to 80% acetonitrile containing 0.1% TFA in water. In the arrow, the proinflammatory peak, named p3. b Leukocytes count on the cremaster muscle microcirculation 2 h after p3 injection. * p < 0.05
Fig. 3. Re-purification of p3 and its inflammatory effects. a RP-HPLC purification of p3, in a C18 carbon column, in an isocratic elution with 0.1% TFA in water. The arrow indicates the proinflammatory peak, named p3E. b Leukocyte count on the cremaster muscle microcirculation 2 h after p3E injection. c Paw edema percentage (control-related) after p3E injection. d Pain threshold evaluation after p3E injection. * p < 0.05
Fig. 4. Mass spectrometry analysis of p3E and its characterization. a MS profile. b Fragmentation of 599 m/z ion. c Software analysis for prediction of molecular formula
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