New flavone, and cytotoxicity activity of Rauia resinosa ethanolic extract and constituents

Abstract The ethanolic extract from leaves of Rauia resinosa, Rutaceae, provided a new flavone, 5-hydroxy-5’,6,7-trimethoxy-3’,4’-methylenedioxyflavone (1), in addition to four known compounds: 3’,4’,5,5’,7-pentamethoxyflavone (2), 5,7,8-trimethoxy-3’4’-methylenedioxyflavone (3), 3’,4’,5,7,8-pentamethoxyflavone (4) and β-sitosterol (5). The structures of all compounds were established on the basis of spectroscopic methods, mainly 1D and 2D NMR, UPLC-DAD-MS and UPLC-ESI-MS/MS, involving comparison with literature data. Cytotoxicity of leaves and stems extracts, their fractions and compounds (2), (3), (4) and (5) were evaluated against T24 (bladder carcinoma), TOV-21-G (ovarian adenocarcinoma) and HepG2 (liver carcinoma) cell lines. Graphical Abstract


Introduction
Many species belonging to the Rutaceae family have been important due to their uses in traditional folk medicine for allergic diseases, treatment of ulcerations and superficial wounds, diarrhea, hemorrhages, cataracts, rheumatism and also as a stimulant, diuretic, analgesic and tonic (Rana et al. 2004;Anaya et al. 2005;Heneka et al. 2005).
There are also reports about cytotoxicity activity against cancer cell lines of this family (Pradhan et al. 2012;Velusamy et al. 2016).
According to the literature, flavones are an important source for the treatment of diseases with diverse pharmacological effects. Additionally, the screening for new flavone-based drugs with new biological properties and potential activity in vitro and in vivo continues to be an important line in the discovery of new anticancer agents (Singh et al. 2014).

Results and discussion
The phytochemical study of R. resinosa led to the isolation of a new flavone called 5hydroxy-5 0 ,6,7-trimethoxy-3 0 ,4 0 -methylenedioxyflavone compound (1) and four other known compounds ( Figure 1). Compound (1) was isolated as colorless needles, with m.p 195-197 C (CH 2 Cl 2 ). Its molecular formula C 19 H 16 O 8 was established by analysis of the UPLC-ESI-MS/MS, which exhibited a molecular ion peak [M þ H]  with carbon d C 100.8 (C-2 0 ), d H 7.08 with carbon d C 106.7 (C-6 0 ), d H 6.09 with carbon d C 102.6 (OCH 2 O). In addition, the locations of methoxyl groups were suggested by the analysis of HMBC spectrum. The methoxyl signal d H 4.00 located at C-5 0 , d H 3.97 at C-7 and d H 3.92 located at C-6 were assigned by HMBC experiments. The complete analysis of the HMBC spectrum in combination with additional 1D and 2D NMR spectral data and the total 1 H and 13 C NMR chemical shift assignments was summarized in Table S1 (Supplementary material). Thus, the new flavone was established as 5hydroxy-5 0 ,6,7-trimethoxy-3 0 ,4 0 -methylenedioxyflavone (1). The structures of the known compounds, were also determined by analysis of their spectral data (Supporting information) and comparison those reported in the literature (Machida and Osawa 1989;Arruda et al. 1993;Facundo et al. 2004;Ododo et al. 2016).
The alkaloid fraction from the ethanolic extract of Rauia resinosa leaves (RL fr Alk) showed cytotoxic activity in TOV-21-G (CC 50 ¼ 118.90 mg/mL ± 1.44) with SI ¼ 1.45 and to T24 cell line the selectivity index was below 1.0. The alkaloid fraction from the ethanolic extract of Rauia resinosa stems (RS fr Alk) showed selectivity indexes below 1.0 in T24 and HepG2 cell lines.
The dichloromethane fraction from the ethanolic extract of Rauia resinosa leaves (RLD) showed cytotoxic activity against the cell line HepG2 (CC 50 ¼ 22.92 mg/mL ± 1.27) with SI ¼ 3.04 and in the other tumoral cell lines tested the selectivity index is below 1.0. The dichloromethane fraction from the ethanolic extract of Rauia resinosa stems (RSD) exhibited cytotoxic activity for the HepG2 cell line (CC 50 ¼ 21.20 mg/mL ± 1.32) with SI ¼ 5.01, whereas in the other strains the selectivity index was also below than 1.0.
Among the substances isolated from the ethanolic extract of Rauia resinosa leaves, the flavones (2), (3) and (4) showed cytotoxic activities with a selectivity index above 1.0. Neither flavone showed cytotoxic activity against HepG2 cell line. Not enough mass of flavone (1) was obtained to perform the biological tests.

Experimental section
See supplementary material.

Conclusion
Phytochemical investigation of ethanolic extracts from Rauia resinosa led to the isolation of a new polyhydroxy flavone (5-hydroxy-5 0 ,6,7-trimethoxy-3 0 ,4 0 -methylenedioxyflavone) (1). Three other known flavones were also isolated and characterized. In vitro cytotoxicity studies employing cell cultures show that extracts from this species are potential sources of cytotoxic compounds. Additionally, it was observed that flavones present in these extracts might contribute to the cytotoxicity from the whole plant extract.