Extrato hexânico da entrecasca de Calophyllum brasiliense : avaliação da atividade antipreneoplásica, isolamento e avaliação de toxicidade e atividade anti-Helicobacter pylori de suas cromanonas

Abstract

Calophyllum brasiliense (Calophyllaceae) is a tree used in folk Medicine to treat various diseases, among which are gastrointestinal pathologies. Previous studies have shown that the hexane extract of Calophyllum brasiliense stem bark (HECb) and its chromanone-rich fractions protect against acute and chronic gastric ulceration in murine models. The objective of this work was: to isolate, for the first time, the principal chromanones of HECb in their natural acidic form, as they occur in the plant, in addition to investigating their effects on Helicobacter pylori activity and on gastric preneoplasic, in mouse model of Helicobacter felis infection. The brasiliensic (BRA), and isobrasiliensic (ISO) acids were isolated using a sequence of liquid chromatography, ending with flash chromatography employing silica impregnated with silver nitrate. The structures of the isolates were elucidated by NMR and mass spectrometry. 13C NMR data of the two molecules were made available for the first time in this study. BRA and ISO showed good in vitro antiHelicobacter pylori activity. Transgenic INS-Gas mice were colonized with Helicobacter felis by gavage. From 2 weeks after colonization, their drinking water was supplemented with the vehicle or HECb and given ad libitum. Equivalent uninfected groups were studied. Animals were culled 6 weeks after H. felis colonisation. Preneoplastic pathology was quantified using established histological criteria. Gastric epithelial cell turnover was quantified by immunohistochemistry for Ki67 and active-caspase 3. Cytokines levels were quantified using electrochemiluminescence assays. Vehicle treated H. felis infected mice exhibited higher gastric atrophy scores than similarly treated uninfected mice (p<0.01). The same pattern was observed following low dose HECb treated group. However, following high dose of HECb treatment, H. felis status did not significantly alter atrophy scores compared to similarly treated uninfected mice (p>0.05). Gastric epithelial apoptosis was not altered by H. felis or HECb administration. However, amongst vehicle treated mice, gastric epithelial cell proliferation was increased by 2.8 fold in the infected compared to uninfected mice (p<0.01). Administration of HECb at both the high and low doses reduced proliferation in infected mice to levels similar to the uninfected mice. Th17 polarized response to H. felis infection was observed in all infected groups. HECb at high dose attenuated IFN-γ, IL-6 and TNF production following H.felis infection (p<0.01, p<0.01 p<0.05, respectively) vs vehicle, however, HECb had no effect on IL-17 and KC-GRO levels in the gastric tissue. It may be concluded that BRA and ISO are responsible, at least in part, for the bacteriostatic anti-H. pylori activity of HECb and that the extract reduces gastric epithelial pathology following H. felis infection in INS-Gas mice. Further studies are indicated to deepen the understanding of the antipreneoplasia mechanism of action of the compounds, as well as the anti-ulcer activity of brasiliensic acid, which proved to be devoid of in vitro and in vivo toxicity.