Chemical constituents and antileukemic activity of Eugenia dysenterica

Abstract The study about Eugenia dysenterica led to the isolation of 3-acetyl-urs-12-en-28-oic (1), 3-acetyl-olean-12-en-28-oic acid (2) and isoquercetin (3) from the stem barks, and of 3-O-β-glucopyranosyl-β-sitosterol (4), methyl 3-hydroxy-4-methoxybenzoate (5), methyl 4-hydroxyphenyl propionate (6), E-methyl-4-hydroxycinnamate (7), quercetin-3-O-(6ꞌꞌ-O-galloyl)-β-d-glucopyranoside (8) and quercetin-3-O-β-d-galactopyranoside (9) from the leaves. The structures 1–9 were set through the analysis of their NMR spectroscopic data. Compounds 2, 3 and 5–8 were reported for the first time in the Eugenia genus. Compound 8 reduced cell viability and presented IC50 values 40.3 and 36.7 μM, for the CCRF-CEM and the Kasumi-1 cells, respectively.


Introduction
Eugenia dysenterica DC is a fruit species native from the Brazilian savannah (Martinotto et al. 2008). Popularly known as 'cagaita' , the tea of its leaves is commonly indicated in folk medicine to treat dysentery, diabetes and jaundice; the flowers are used to treat kidney and bladder infections, and the fruits are prescribed as laxative (Costa et al. 2000). Previous studies have described some biological activities associated to the E. dysenterica leaves such as antifungal and antidiarrhoeic attributed to the essential oil (Costa et al. 2000;Galheigo et al. 2015), and molluscicidal found in the ethanolic extract (Bezerra et al. 2000).
According the literature, several plants belonging to the Eugenia genus are rich in essential oils and many biological activities including antifungal, antileukemic, cytotoxic, antioxidant, antimicrobial and antileishmania have been related to them (Galheigo et al. 2015;Zatelli et al. 2015;Tenfen et al. 2016). Another compound generally found in this genus are flavonoids, specially polyhydroxylated flavonols, and terpenic compounds such as triterpenoids and steroids (Moresco et al. 2016). All these compounds make Eugenia a promising genus for therapeutic molecules. The aim of the present study is to report the compounds isolated from the leaves and stem barks of E. dysenterica and to investigate the biological activities promoted by their flavonoids in leukaemia cell lineages.

Biological assays
The cell viability and the cell death features were evaluated in order to set the cytotoxic potential of isoquercetin (3), quercetin-3-O-(6ꞌꞌ-O-galloyl)-β-d-glucopyranoside (8) and quercetin-3-O-β-d-galactopyranoside (9) in leukemic lineages. Only compound (8) has reduced concentration-dependent cell viability after 24 h in Kasumi-1 (acute myeloid leukemia) and CCRF-CEM (Acute T cell leukemia) cells. The CCRF-CEM lineage was more sensitive to (8) (IC 50 = 36.7 ± 1.07 μM) than the Kasumi-1 cells (IC 50 = 40.3 ± 1.15 μM) ( Figure  S13C, Supplementary Material). The cell death features were assessed through double staining using Annexin V-APC and Sytox. The Kasumi-1 cells showed the predominant label of Anx − /Sytox + and Anx + /Sytox + ( Figure S13D, Supplementary Material), and it suggested the occurrence of late apoptosis or necrosis. The CCRF-CEM cells preferentially showed the label of Anx + /Sytox − , which indicated apoptosis ( Figure S13E, Supplementary Material). The following experiments concerned the Kasumi-1 cell lineage, due to the presence of t(8;21) translocation, which is one of the most common cytogenetic alterations in human acute myeloid leukemia (Asou et al. 1991). Another feature was Ψ mit using the JC-1 dye in Kasumi-1 cells ( Figure S13F, Supplementary Material). After 24 h of treatment with (8), 49.7% of cells were reduced red fluorescence by decreased Ψ mit . A noticeable reduction in the number of cells was observed through cell death investigation, even at low concentrations. Thus, the ability of (8) to induce cell cycle arrest was investigated.
The cell cycle showed G 0 /G 1 increase and G 2 /M phases when 10.0 μM of compound (8) used in the treatment ( Figure S14A, Supplementary Material). Additionally, the Ki-67 protein, which is strictly expressed during cell proliferation (Li et al. 2015), was quantified. There was increase in the number of Ki-67 cells (cells out of cell cycle) ( Figure S14B, Supplementary Material). Thus, the myeloid differentiation of Kasumi-1 cells was treated with 10.0 μM of compound (8) a low concentration, was not able to cause cell death for three consecutive days. The differentiation status was determined through the expression of mature cell markers (Lin: CD2, CD3, CD4, CD7, CD8, CD11b, CD14, CD19, CD20, CD38) (Nogueira-Pedro et al. 2014). The expression significantly increased (1.7-fold) after the cells were treated ( Figure S15A, Supplementary Material). Furthermore, the treatment using (8) has reduced the total number of cells ( Figure S15B, Supplementary Material), which meets the cell cycle arrest. There are many quercetin glycosides that show anti-cancer activity against different cancer cells, such as, quercetin-6-C-β-d-glucopyranoside (Hamidullah et al. 2015). Our results suggest that compound (8) exerts cytotoxic effects on Kasumi-1 and on CCRF-CEM cells. In addition, the compound (8) promotes cell cycle arrest in concentrations lower than IC 50 and cell differentiation effects on Kasumi-1 cells. Therapies based on cell differentiation have shown beneficial effect on acute myeloid leukemia (Lemarie et al. 2006). This point is of great interest and indicates the potential applications in the development of new antileukemic drugs.

Conclusion
The compounds 2, 3 and 5-8 are reported for the first time in the Eugenia genus. Biological assays for the bioactive compound 8, showed cytotoxic effects against CCRF-CEM and Kasumi-1 cells lines, and antiproliferative and cell differentiation ability in Kasumi-1 cells indicated that the O-galloyl flavonoids may be potential prototype antileukemic drug.

Disclosure statement
No potential conflict of interest was reported by the authors.