Cytotoxic potential of dihydrochalcones from Eriosema glomeratum and their semi-synthetic derivatives

Abstract In the search of cytotoxic dihydrochalcones, this investigation led to the isolation of seven compounds (1-7) from Eriosema glomeratum and the preparation of eight derivatives (8-15). The cytotoxicity of samples was evaluated against lung (A549), breast (MCF-7), and cervical (HeLa) human cancer cells. The CH2Cl2/MeOH extract of the aerial part had strong cytotoxicity against all cells [IC50 11.2 (MCF-7), 8.4 (HeLa) and 13.1 (A549) μg/mL]. A strong activity was also displayed by the n-hexane fraction on MCF-7 (IC50 11.2 μg/mL). The precursor 3 and the derivative 8 were specifically found as strong cytotoxic agents toward MCF-7 (7.6 μM) and HeLa (3.1 μM), respectively and were more effective than the positive control. Derivatives 8 (3.1 μM) and 9 (21.3 μM) against HeLa were most potent than their precursor 3 (23.7 μM). This is the first preparation of 8-14 as well as the cytotoxicity of 3, 4, 8-15, fractions, and extract. Graphical Abstract


Introduction
The genus Eriosema, a member of the Fabaceae family, contains approximately 160 species, mostly distributed in tropical regions of Africa, that are used in traditional medicine to treat various diseases (Drewes et al. 2004;Thongnest et al. 2013;Awouafack et al. 2015;2018).Flavonoids are one of the largest classes of secondary metabolites produced in different parts of the plants and those already reported from the genus Eriosema possess interesting pharmacological activities (Awouafack et al. 2015;Umereweneza et al. 2021).Among flavonoids, dihydrochalcones are very uncommon in natural resources and remain as good bioactive compounds with their derivatives (Awouafack et al. 2008;2010;Umereweneza et al. 2021).In Cameroon, the whole plant of Eriosema glomeratum is used by the Bakola Pygmy tribe to treat leprosy and venereal diseases, pulmonary troubles, and nasopharyngeal infections (Verdcourt 1970;Tabakam et al. 2021).In our ongoing search of bioactive compounds from plant species and from semi-synthesis of isolated compounds, we isolated seven compounds (1-7) from the aerial parts and roots of E. glomeratum as well as we prepared eight semi-synthetic derivatives (8-15) from erioschalcones A (3) and B (4) (Awouafack et al. 2008).Amongst these derivatives, seven (8-14) are prepared and characterized here for the first time as well as the cytotoxicity of all the derivatives (8-15) and their precursors (3 and 4), extract and fractions.

Cytotoxicity of extract, fractions, isolated compounds and semi-synthetic derivatives
Assessment of the cytotoxic effect of crude extract, fractions and isolated compounds (1-7) from E. glomeratum as well as derivatives (8-15) against a panel of three human [lung (A549), breast (MCF-7), and cervical (HeLa)] cancer cell lines were carried out and the IC 50 values obtained were classified using the cut-off point of Kuete and Efferth (2015) (Table 1).
Based on this, the CH 2 Cl 2 /MeOH extract showed strong cytotoxicity against the breast, cervical and the lung cancer cell lines with IC 50 11.2, 8.4, and 13.1 lg/mL, respectively.Strong cytotoxicity was also recorded with the n-hexane fraction toward MCF-7 (IC 50 11.2lg/mL), while moderate activity was obtained by the same fraction against the other cancer cells (IC 50 ranging from 22.7 to 41.3 lg/mL).Our results on the cytotoxicity of the CH 2 Cl 2 /MeOH extract and the n-hexane fraction are similar to those of reported CH 2 Cl 2 /MeOH extracts of E. montanum against the MCF-7 cell which has revealed the strong anticancer activity with EC 50 6.4 (stem bark) and 18 (leaves) lg/mL, respectively (Umereweneza et al. 2021).Similar results were also reported from the nhexane root extract of E. chinense against small-cell lung NCI-H187 (IC 50 12 lg/mL) and oral epidermal carcinoma KB (IC 50 9.9 lg/mL) human cell lines (Sutthivaiyakit et al. 2009).According to these authors, the presence of prenylated flavonoids and prenylated dihydrochalcones in E. chinense was responsible for the strong cytotoxicity of the n-hexane extract (Sutthivaiyakit et al. 2009).Except the moderated cytotoxicity of the EtOAc fraction (IC 50 48.0lg/mL) against MCF-7, the other fractions [i.e. the CH 2 Cl 2 , the EtOAc, and the n-BuOH fractions from the aerial part] displayed low cytotoxic activities varying from IC 50 51.5 to > 100 lg/mL against all the tested cancer cell lines.
The activity of acetylated derivatives 8 (3.1 lM) and 9 (21.3 lM) against HeLa was most potent than that of their precursor 3 (23.7 lM), as the IC 50 value of the latter is higher compare to that of its derivatives.This tends to suggest that the acetylation reaction may enhance the activity, and more specifically when a methoxy group is attached at position C-4 of the B-ring.However, the cytotoxic effect of the same derivatives (8 and 9) against MCF-7 and A549, and that of the other acetylated derivatives (10 and 11) against all the tested cell lines, has decreased compared to that of the precursors (3 and 4), respectively.From the observation above on our results, a basic model of the action of dihydrochalcones could be established.This model could then be used in the context of facilitating the development of new acetylated derivatives of the class of dihydrochalcones, which would serve as more potent cytotoxic agents against human cervical cancer.
The activity of cyclization derivatives (12 and 15) obtained from erioschalcone A (3) was higher as compared to that of 13 and 14 from erioschalcone B (4).The cyclization of the prenyl group with the 2 0 -OH of erioschalcone A (3) using DDQ occurred with a dehydrogenation of protons at a,b-position to afford the semisynthetic derivative 12 and derivative 15 without dehydrogenation of protons at a,b-position, respectively.The derivatives 12 and 15 possessed low activity on HeLa (IC 50 71.5 and 89.5 mM) and MCF-7 (IC 50 96.7 and > 100 mM), and this was less effective than that of their precursor 3 (IC 50 23.7 and 7.6 mM), respectively.The derivative 13 had low activity on MCF-7 (IC 50 > 100 mM) and this was also lower than that of its precursor 4 (IC 50 10.4 mM).All the derivatives 12-15 obtained by cyclization possessed low cytotoxicity (IC 50 ranging from 71.5 to > 100 mM) as compared to their precursors 3 (IC 50 between 7.6 and 29.0 mM) and 4 (IC 50 between 10.4 and 23.0 mM).These results allowed us to deduce that the cyclization between the prenyl at C-3 0 and the hydroxyls either at C-2 0 or at C-4 0 on the dihydrochalcone skeleton, decreases the cytotoxicity on A549, MCF-7, and HeLa cells.

General experimental procedures
MS data were measured on SHIMADZU LCMS-ITTOF spectrometer (Japan).NMR spectra were recorded on JEOL 400 and 500 MHz spectrometers (Japan), with TMS as internal reference standard.Column chromatography was performed on silica gel Merck 60 F 254 [(0.2-0.5 mm) and (0.063-0.2 mm)] 70-230 and 230-400 mesh (Darmstadt, Germany) and on Sephadex LH-20 (Sigma-Aldrich).TLC was performed on silica gel GF254 pre-coated (Merck) plates (Germany), with detection by visualization with a UV lamp at 254 and 365 nm, followed by spraying with 20% aqueous sulphuric acid and heating up to 150 C.
Semi-preparative reversed-phase HPLC was run on Agilent Technologies apparatus with UV detection at 254 nm using a Waters column C 18 -AR-II, 5 mm (250 Â 10 mm), with H 2 O (þ 0.01% TFA) (A) and MeCN (þ 0.01% TFA) (B) with the following gradient programs (flow rate 1.5 mL/min): for compounds 8 and 9 (running time: 25 min), 50% B linear to 0% A for 10 min, after 100% B isocratic over 10 min, the system was returned to its initial condition (50% A) over 3 min and equilibrated for 2 min before the next injection; and for compounds 11-13 (running time: 30 min), 50% B linear to 0% A for 10 min, after 100% B isocratic over 15 min, the system was returned to its initial condition (50% A) over 2 min and equilibrated for 3 min.

Plant materials
The roots and the aerial part of Eriosema glomeratum (Guill.& Perr.)Hook.f. (Fabaceae) (Figure S1) were collected in Bakou (GPS coordinates: Latitude 5 09 0 36 00 N, Longitude 10 11 0 14 00 E), Western Region of Cameroon, in October 2019 and 2020, respectively.This plant was authenticated by Mr. Nana, a botanist of the Cameroon National Herbarium in Yaounde where the specimens were deposited under the voucher number 2643 HNC.

Cytotoxicity assay
Cell viability in the presence or absence of our samples was determined using the standard WST-8 assay (Tominaga et al. 1999).a-Minimum essential medium with l-glutamine and phenol red (a-MEM; Wako) was used to culture the human [lung (A549), breast (MCF-7), and cervical (HeLa)] cancer cell lines.All media were supplemented with 10% fetal bovine serum (FBS; Sigma) and 1% antibiotic antimycotic solution (Sigma).For MCF-7 cells, the growth medium was supplemented with 1% of 0.1 mM non-essential amino acids (NEAA; Gibco) and 1% of 1 mM sodium pyruvate (Gibco).Each cancer cell line was seeded in 96-well plates (2 Â 10 3 cells per well) and incubated at 37 C for 24 h in the respective medium under 5% CO 2 and 95% atmospheric air.Thereafter, cells were washed with phosphate-buffered saline (PBS) and samples were added at different concentrations (1-100 lM).After incubation for 72 h, the cells were washed with PBS, and 100 lL of medium containing 10% WST-8 cell counting kit solution (Dojindo; Kumamoto, Japan) was added to each well and incubated again for 3 h.Subsequently, the absorbance of each well was measured at the wavelength of 570 nm.The viability was evaluated based on a comparison with untreated cells.The IC 50 values were calculated from a calibration curve by linear regression using Microsoft Excel.The data are mean ± SD of triplicates.The statistical analysis t-test was used to calculate the p value.
All the isolated compounds and the semi-synthetic derivatives tested had a purity range from 95 to 99% as indicated by HPLC-DAD.

Conclusion
Our results reported the cytotoxicity of extract, fractions, constituents (1-7) from the aerial part and the roots of Eriosema glomeratum as well as eight derivatives (8-15) prepared from the isolated erioschalcones A (3) and B (4).The CH 2 Cl 2 /MeOH extract had strong cytotoxicity against all the cancer cell lines whereas the n-hexane fraction inhibited significantly the growth of the MCF-7 cell.The precursor 3 and the derivative 8 were specifically found as strong cytotoxic agents toward MCF-7 and HeLa cells, respectively.The activity of acetylated derivatives 8 and 9 against HeLa was most potent than that of their precursor 3. Further investigation is needed to better understand the cytotoxicity mechanism of action of dihydrochalcones mainly the precursors erioschalcones A and B.More derivatives may also be prepared in view to discuss the cytotoxic activity relationship on erioschalcones A and B against cancer cell lines in view to have new anticancer drugs development leads.
a Isolated compounds