Étude des propriétés antioxydantes et anticancéreuses in vitro et in vivo des extraits bruts et fractions de Syzygium guineense (Willd.) variété macrocarpum (Myrtaceae)

Abstract

Background: One of the major concerns in the field of oncology today is the permanent research for new anticancer molecules. Indeed, the clinical application, especially the long-term application of chemotherapy, which is the most used method in the therapeutic management of cancer is hampered by the problem of chemoresistance and toxicity of the molecules used. One of the promising approaches is the use of medicinal plants that have already proven to be effective in the development of anti-cancer drugs. However, very little work has evaluated the anticancer effects of Syzygium guineense (Willd.). This work aims to study the antioxidant and anticancer properties in vitro and in vivo of extracts and fractions of the plant and to elucidate some mechanisms of action. Methods: The extracts were obtained by maceration of the leaves and barks of S. guineense harvested in the locality of Ndjie (Centre region- Cameroon) in water, ethanol and the mixture water / ethanol. The study of antioxidant properties consisted of evaluating their ability to scavenge ABTS +and DPPH• radicals on one hand and to reduce phosphomolybdate and ferric ions on the other hand. In addition, we determined the total phenolic content, as well as their identification by high performance liquid chromatography.The in vitro study consisted to evaluate the effects of the crude extracts and fractions using the sulforhodamine B method on the growth of breast cancer cells lines (MCF7 and MDA-MB231), cervical cancer (SiHa and HeLa) and leukemia cell lines (K562 and HL-60). The GI50 values (inhibitory concentrations 50), total growth inhibition (TGI), and lethal concentrations 50 (LC50) were determined. Subsequently, we investigated the mechanism of action underlying the anti-proliferative and cytotoxic effect by assessing the effects of the most active fractions on the cell cycle as well as their ability to induce apoptosis by flow cytometry. In addition, we studied the expression profile of the pro-apoptotic and anti-apoptotic proteins Bax and Bcl-2 by Western blot to determine the apoptotic pathway involved. Furthermore, we determined the phytochemical composition of the active fractions by gas chromatography coupled with mass spectrometry. The last part of the work focused on the study of anticancer effects in vivo by determining the relative tumor volume, survival percentage, and weight loss after treatment of acute myeloid leukemia xenografts obtained by transplantation of cells of KG-1 unto female NOD-SCID mice. The data were analyzed by Graphpad prism software version 5.0 and SPSS for Windows version 20.0. The results were expressed as mean ± standard deviation. The differences were considered significant at p<0.05. Results: The obtained results show that the aqueous extracts of leaves and barks had the best antioxidant power with 0.51±0.01µg/mL and 0.43±0.09µg/mL for the DPPH and ABTS+radicals respectively. In addition, the ethanolic extract of bark demonstrated the best total antioxidant capacity according to the FRAP and phosphomolydate methods with 278.88± 32.50 et 278.88 ± 5.75mEq of quercetin / g of dry matter respectively while the ethanolic extract of leaves exhibited the highest total phenolic and flavonoids contents (439.76±5.15 et 1,040±51.1 mEq of quercetin / g of dry matter. The high performance liquid chromatography analysis revealed the presence of simple phenolic acids phenolic alcohols, and flavonoids. With respect to the in vitro anticancer properties, the aqueous extract of bark and leaf ethanolic extract had the best GI50 values on all cell lines tested among all the crude extracts. Thus, for the MCF7 and MDA-MB231 lines, the values obtained were respectively 64.71 µg / mL and 46.06 µg / mL on the one hand and 184.51 and 115.26 µg / mL on the other hand. Regarding the leukemic lines, the aqueous bark extract was more active on the K562 line with a GI50 of 46.67 µg / mL against 66.34 µg / mL on the HL-60 line. Conversely, the ethanolic leaf extract demonstrated a better effect on the HL-60 line with a GI50 of 76.5 µg / mL compared to 84.5 µg / mL on the K562 line. Among the breast cancer lines, MCF7 was more sensitive to all extracts than the MDA-MB231 while SiHa was the most sensitive among cervical cancer cell lines. Similarly, K562 was more sensitive than HL-60. Fractions L4 and L5 derived from the ethanolic extract of leaves demonstrated the best anticancer effects against all the tested cell lines among the obtained fractions. In addition, the S phase arrest of cell cycle is one of the mechanisms responsible of the inhibition of the proliferation of MCF7, MDA-MB231, SiHa and HL-60 cells while the G2 / M phase arrest could explain the anti-proliferative effect demonstrated against HeLa and K562 cells. Meanwhile, the cytotoxic activity of fractions L4 and L5 exhibited against all the cell lines might be due to the induction of apoptosis. In addition to methyl disulphide hydrogen the major compound, the phytochemical composition of the fraction L4 revealed the presence of terpenes, fatty acids and sterols whereas fraction L5 is composed mainly of sulfur oxygenated and nitrogenous hydrocarbons. The study of in vivo anticancer effects showed that the maximum tolerated dose for the crude aqueous extract of barks and the L4 and L5 fractions is greater than 900 mg / kg body weight. The aqueous crude bark extract significantly inhibits tumor growth on NOD-SCID acute myeloid leukemia xenografts.Conclusion: The extracts of Syzygium guineense variety macrocarpum possess anti-oxidant and anti-cancer properties in vitro and in vivo. Nonetheless, the aqueous extract of bars and hexane/ethyl acetate fractions 60:40 and 40:60 (v: v) are good candidates for the development of new chemotherapeutic agents for acute and chronic myeloid leukemias. The mechanisms responsible of the growth inhibition of acute and chronic myeloid leukemia cell lines are the in S and G2 / M phase arrest of the cell cycle respectively and the induction of apoptosis via modulation of the expression of Bax and Bcl-2 proteins. The aqueous bark extract at a dose of 200 mg / kg body weight significantly inhibited tumor progression on xenografts of acute myeloid leukemia.