Étude phytochimique de Vitellaria Paradoxa c. f. Gaertn (Sapotaceae), transformations chimiques et activités biologiques contre les maladies neurodégénératives et le cancer

Abstract

Our work is based on a bio guided anti-neurodegenerative and cytotoxic study of the seeds, roots and stem barks of Vitellaria paradoxa and also some chemical transformations on the active compounds in order to study their structure-activity relationship (SAR).The various parts of Vitellaria paradoxa were extracted sequentially and successively with solvents of increasing polarity (hexane, ethyl acetate, methanol and water) using a sonicator bath. These extracts were subjected to LCMS analysis and to biological screening against inflammation and cytotoxicity. The results obtained revealed that the ethyl acetate and methanol extracts were the most active and abundant in secondary metabolites. - The LCMS analysis has shown that the ethyl acetate extract is the richest in natural substances, in particular the triterpenes. - The in vivo (anti-inflammatory) activity of these extracts (ethyl acetate, methanolic and aqueous) was evaluated on acute inflammation induced by carrageenan and on chronic inflammation induced by Freund’s Complete Adjuvant (FCA) at the same dose of 150 mg/kg. On days 19-28, the maximum inflammatory percentage was between 9.60 and 8.91 for the ethyl acetate extract compared to 30.91-24.29% for the controls. All extracts significantly reduced arthritis scores but the maximum reduction was achieved with ethyl acetate extract on day 24th of experiment. - Concerning the antiproliferative activity, the crude methanol extract of stem bark is active on four cell lines NCI-H460, MCF-7, PC3 and Hela tested with GI50 values varying from 24.00 to 66.46 µg/mL. All these results prompted a systematically search for the compounds responsible for the activities observed in these extracts. The purification of these different extracts from the roots, seeds and stem barks using the usual chromatography and spectroscopic methods lead to the isolation of sixteen compounds of which ten have been fully characterized and grouped into four classes of secondary metabolites. Namely : five triterpenoids, two of which are new. It is, 1α,2β,3β,19α-tretrahydroxyurs-12-en-28-oic acid 126 (new) and 2β,3β,19α-trihydroxyurs-12-en-28-oic acid 127 (new), betulinic acid 91, 3β-acetoxy-1α,2β,19α trihydroxyurs-12-en-28-oic acid 128 and 3β-(cinnamoyloxy)-11-methoxy-urs-11,12-enoate ethyl ester 129 ; three flavonoids : epicatechin 23, catechin 22, (2R, 3S)-dihydrokaempferol 130 ; a cyclotol 131 and a fatty acid : pentadecanoic acid 132. After having characterized the isolated compounds, some triterpenes (126, 127 and 91) were evaluated for their cytotoxicity on breast cancer cell line (TNBC). The results obtained showed that betulinic acid 91 was the most active with an IC50 value of the order of 19.90 µM and 95% CI of 17.20-23.10 µM. In order to study the structure-activity relationship of these three triterpenes (91, 126 and 127) in general and betulinic acid in particular, we carried out esterification of their COOH function using diazomethane generated in situ from 1-methyl-3-nitro-1-nitrosoguanidine to obtain their corresponding methylated esters (134, 133 and 135). Esterification of the OH function of betulinic acid using valeric and heptanoic anhydrides in the presence of pyridine and DMAP afforded compounds 136 and 137, as white amorphous substances. In general, the activity of methylated esters 134 (32.90 µM), 133 (43.00 µM) and 135 (46.70 µM) against the MDA-MB-231 breast cancer cell line was more active than their initial triterpenes 91 (19.90 µM), 126 (63.50 µM) and 127 (230.00 µM) except for betulinic acid. This improvement in activity may be due to greater cell permeability of the more esters hydrophobic derivatives. The efficacy tests for betulinic acid and its methylated ester were carried out on the cancerous cells with Annexin V as reference drug. The results showed that only the cancerous cells were killed by induction of apoptosis. The present study corroborated the use of Vitellaria paradoxa in traditional medicine as an anti-inflammatory of the ethyl acetate extract and cytotoxic of isolated triterpenes. We would like to continue with an in deep to this study.