Cytotoxic Effects of Compounds Isolated from Ricinodendron heudelotii

This study was designed to explore the in vitro anticancer effects of the bioactive compounds isolated from Ricinodendron heudelotii on selected cancer cell lines. The leaves of the plant were extracted with ethanol and partitioned in sequence with petroleum ether, ethyl acetate, and n-butanol. The ethyl acetate fraction was phytochemically studied using thin layer chromatography (TLC) and column chromatography (CC). Structural elucidation of pure compounds obtained from the ethyl acetate fraction was done using mass spectra, 1H-NMR, and 13C-NMR analysis. The isolated compounds were subsequently screened using five different cancer cell lines: HL-60, SMMC-7721, A-549, MCF-7, SW-480, and normal lung epithelial cell line, BEAS-2B, to assess their cytotoxic effects. Nine compounds were isolated and structurally elucidated as gallic acid, gallic acid ethyl ester, corilagin, quercetin-3-O-rhamnoside, myricetin-3-O-rhamnoside, 1,4,6-tri-O-galloyl glucose, 3,4,6-tri-O-galloyl glucose, 1,2,6-tri-O-galloyl glucose, and 4,6-di-O-galloyl glucose. Corilagin exhibited the most cytotoxic activity with an IC50 value of 33.18 μg/mL against MCF-7 cells, which were comparable to cisplatin with an IC50 value of 27.43 µg/mL. The result suggests that corilagin isolated from R. heudelotii has the potential to be developed as an effective therapeutic agent against the growth of breast cancer cells.


Introduction
Cancer is a major cause of death across the globe, affecting all age groups [1]. The World Health Organization has predicted that cancer cases will increase from 14 million in 2012 to 22 million in the next four decades [2]. It has caused a great sense of burden both to individual lives and the society at large [3]. The absolute cure of cancer remains a big challenge despite available anticancer agents, awareness campaigns, and prevention strategies. Breast and prostate cancer have the highest incidence in women and men, respectively [4]. It is therefore pertinent to keep exploring for novel chemotherapeutic agents that can evade drug-resistant cancer cells. Natural or natural based anti-cancer drugs, such as vincristine, paclitaxel, bleomycin, and others, have been clinically used over the years [5]. Ricinodendron heudelotii (family: Euphorbiceae) plant parts have been used in traditional medicine to treat cough, stomach ailments, and pain related to child birth. Decoctions and infusions are mostly prepared from the leaves and stem bark. Traditional practitioners prescribe it for women suffering from miscarriages [6]. Phytochemical screening of the leaf extract revealed the ), 6 1,4,6-Tri-O-galloyl-β-D-glucose (6)

Bioassay
In an in vitro cytotoxicity assay, compounds 1-5 were applied to HL-60, SMMC-7721, A-549, MCF-7, and SW-480 cancer cells to assess their cytotoxic effects. Compound 3 (Corilagin) exhibited the most cytotoxic activity against MCF-7 cells with IC50 values of 33.18 and 25.81 μg/mL for HL-60 cells. The IC50 value of compound 3 in MCF-7 cells is comparable with the positive control drug (cisplatin), having an IC50 value of 27.43 (Table 1). Corilagin had 29.19, 4.91, and 49.82% inhibition for A-549, SMMC-7721, and SW-480 respectively. The inhibition rate of other compounds (gallic acid, gallic acid ethyl ester, quercetin-3-O-rhamnoside, myricetin-3-O-rhamnoside) wasless than 50% at the concentration of 40 μM ( Table 2), suggesting that they showed no significant inhibitory activity against the cancer cell lines; therefore, their IC50 values were not measured. Figures 2-4 shows the photomicrograph of the cytotoxic activities of compound 3 on breast, colon, and leukemia cancer cell lines with relatively similar activity when compared with cisplatin (control group).

Bioassay
In an in vitro cytotoxicity assay, compounds 1-5 were applied to HL-60, SMMC-7721, A-549, MCF-7, and SW-480 cancer cells to assess their cytotoxic effects. Compound 3 (Corilagin) exhibited the most cytotoxic activity against MCF-7 cells with IC 50 values of 33.18 and 25.81 µg/mL for HL-60 cells. The IC 50 value of compound 3 in MCF-7 cells is comparable with the positive control drug (cisplatin), having an IC 50 value of 27.43 (Table 1). Corilagin had 29.19, 4.91, and 49.82% inhibition for A-549, SMMC-7721, and SW-480 respectively. The inhibition rate of other compounds (gallic acid, gallic acid ethyl ester, quercetin-3-O-rhamnoside, myricetin-3-O-rhamnoside) wasless than 50% at the concentration of 40 µM ( Table 2), suggesting that they showed no significant inhibitory activity against the cancer cell lines; therefore, their IC 50 values were not measured. Figures 2-4 shows the photomicrograph of the cytotoxic activities of compound 3 on breast, colon, and leukemia cancer cell lines with relatively similar activity when compared with cisplatin (control group).

Discussion
After many years of research on cancer, cancer still remains a destructive disease responsible for over one quarter of the deaths recorded globally. It is one of the major health obstacles, representing the second largest cause of mortality [24]. This is mainly because cancer chemotherapy is often

Discussion
After many years of research on cancer, cancer still remains a destructive disease responsible for over one quarter of the deaths recorded globally. It is one of the major health obstacles, representing the second largest cause of mortality [24]. This is mainly because cancer chemotherapy is often mitigated by a high degree of toxicity to normal cells or failure of treatment due to the development of resistance to anticancer drugs. However, medicinal plants have been increasingly shown to possess anticancer properties, and this has recently become the subject of some scientific studies [25] because they are considered to be less toxic and can be effective for treatment of different diseases. To date, the therapeutic pre-clinical screening of medicinal plants against multiple cancer cells and characterization of the bioactive components in these plants are limited. Our study reports a bioactive compound with cytotoxicity effects in the leaf extracts of R. heudelotii and its potential to be developed for use for cancer treatment.
Corilagin (compound 3) was isolated and structurally identified from the ethyl acetate fraction of the ethanolic extract of R. heudelotii leaves in our study. Only compound 3 showed significant and comparable cytotoxic property relative to cisplatin. Corilagin has been previously identified in several other plants, especially those of the Euphorbiaceae family. It was firstly isolated from Caesalpinia coriaria in 1951, hence the name of the molecule [26]. It was later identified as a weak carbonic anhydrase inhibitor [27]. Studies have reported its antibacterial, hepatoprotective, anti-oxidant [28], anti-inflammatory [29], anti-hypertensive [30], and anti-tumor activities [31]. Yang et al. [32] reported that corilagin can block glioblastoma cells and stem-like cells' proliferation. In this study, corilagin was found to exhibit an inhibitory effect on HL-60, MCF-7, and SW480 cell growth by 76.23, 57.93, and 49.82%, respectively (Table 2). This could be linked to its ability to activate the Jun N-terminal kinase (JNK) signaling pathway [33]. When these pathways are turned on, it can lead to the activation of the proapoptotic β-cell lymphoma 2 (Bcl-2) protein through phosphorylation and/or upregulation of gene expression and hence mitochondrial cell death [34]. Breast cancer cells treated with corilagin showed reduced cell diameters when compared to the untreated cells, implying that the compound caused cell death of most of the cancerous cells (Plate 1). Corilagin is an ellagitannin, which have been reported to induce pro-apoptotic effects in in both MDA-MB-231 and MCF-7 breast cancer cells [35]. Kuo et al. [36] observed that ellagitannins from Terminalia arjuna bark extract induced apoptosis in A549 cells and in human breast adenocarcinoma MCF-7 cells via blockage of cell-cycle progression in the G0/G1 phase. The cytotoxic properties of tannins are characterised by the release of reactive oxygen species (ROS), which is followed by a reduction in the thioredoxin, superoxide dismutase, and in the level of redox active proteins [37]; this might explain the cytotoxic activity of compound 3. The chemopreventive activity of tannins, especially the ellagitannins and hydrolysable tannins, has been linked to activation of apoptosis [38]. According to the findings of Jia et al. [31], corilagin inhibited SKOv3ip by inducing cell cycle arrest and enhancing apoptosis in the cancer cell lines. Down regulation of cyclin B1 and phosphor-cdc 2 after corilagin treatment on cancer cells was discovered [31]. Corilagin has also been reported to cause the release of TNF-α inhibitor in inflammation [39], decrease the secretion of the transforming growth factor beta 1 (TGF-β1) in a dose dependent manner [31], and inhibit hepatitis C viral replication in vitro [40]. Corilagin enhanced apoptosis by inducing cell cycle arrest at the G2/M stage in ovarian cancer cells. Anti-cancer strategies have focused on possible ways of deactivating the G2/M checkpoint, thereby causing cancer cells to undergo mitosis, and leading to increased DNA damage and then cell death [41,42]. Ming et al. [43] also reported the inhibitory action of corilagin on hepatocellular carcinoma cells (HCC) at the G2/M phase by upregulating p53 and p21, and downregulating cyclin B1, cdc 2, and pAKT. Selectivity index (SI) signifies the differential activity of compounds; the greater the SI value, the more selective the compound [44]. The SI data, as shown in Table 3, indicate that corilagin was non-selective to HL-60, MCF-7, and SW480 cancer cell lines. This suggests its general toxicity to the cell. Adverse effects caused by treatment and increased resistance to tumor cells, leading to treatment failure, has led to a search for therapeutic alternatives. Therefore, it is important to find selective bioactive compounds that could arrest tumor cell proliferation.

Plant Sample Collection
Ricinodendron heudelotii leaves were collected within Covenant University premises Ota, Ogun, Nigeria. The plant was identified by Dr. J.O. Popoola (Botanist, Biological Science Department, Covenant University, Ota, Ogun State, Nigeria) and a voucher specimen was prepared and submitted in the Forest research Institute of Nigeria, Ibadan with voucher number, FHI 110573. Leaves were dried at room temperature (25 • C) and blended using an electric blender into a coarse powder.

Extraction and Solvent Partitioning ofthe Crude Extract
The powdered leaf samples (10 kg) were extracted using 95% ethanol by macerationat room temperature, the filtrate was further condensed under reduced temperature and pressure, and the yield of the extract obtained was 11.75%. The concentrated crude extract (1 kg) was suspended in 1 L distilled water and partitioned in sequence with petroleum ether (Pet; 8 L), ethyl acetate (EtOAc; 8 L), and n-butanol (8 L). The solvent fractions were concentrated to dryness to afford four (4) fractions as: Pet, EtOAc, n-butanol, and aqueous fractions. The EtOAc fraction was selected for subsequent isolation procedures.