Potential anti-proliferative effects of chemical constituents and hemisynthetic derivatives from Scadoxus pseudocaulus (Amarillydaceae)

Background Biological significance of Amaryllidaceae is well advocated from the literature. In Cameroon, plants from this family are routinely used for the cure of liver, cancer and cardiovascular diseases. To date, no scientific investigation corresponding to the anti-cancer activity of extracts and isolated compounds of Scadoxus pseudocaulus is available. Objective Current study is focused to elaborate the anti-proliferative effects of natural isolates (compounds 1–6, 9) and hemi-synthetic analogs (compounds 7–8) extracted from S. pseudocaulu. Methods Column chromatography of the ethyl acetate extract followed by purification of different fractions led to the isolation of seven compounds (1 – 6, 9). Esterification reaction of compound 6 was carried out using butyroyl chlorides and triethylamin to produce two derivatives (7 – 8). The cytotoxic activity was performed after staining of treated cells with florescent dye propidium iodide. Dead cells were detected using cytometer FL2 or FL3 channels/filters. Results Trans-derivative of narciclasine (a natural isolate from S. pseudocaulus), was found to be most potent among all tested compounds. Its effects were more significant on low malignant follicular lymphoma (DoHH2 cells) as compared to highly malignant (EBV infected) Burkitts lymphoma (Raji cells). Conclusion From our results, narciclasine appears to hold the potential of a lead molecule that can be used to bridge the therapeutic gaps in cancer research.


Introduction
Scadoxus pseudocaulus (I. Bjornstad and Friis), a member of the Amaryllidaceae, is used in the West Region of Cameroon, for the treatment of liver, cancer and cardiovascular diseases. Previous study demonstrated the antimicrobial, antioxidant and anti-butyrylcholinesterase activities of compounds and extracts of S. pseudocaulus. 1 Apart from these biological activities, no conclusive scientific study is reported pertaining to its anti-cancer activity. It is known African Health Sciences Vol 20 Issue 1, March, 2020 469 that alkaloids of Amaryllidaceae family (likehe non-basic hydroxylated phenanthridones) possess high cytostatic activity. [2][3][4][5][6] Some other alkaloids like galanthamine, lycorine and narciclasine can only be synthesized in plants of Amaryllidaceae family. 7 Alkaloids from this family bear a number of biological activities including anti-microbial and anti-cancer. 6 7-deoxy-trans-dihydronarciclasin or trans-dihydrolycoricidin (5) is isocarbostyrl alkaloid reported from S. pseudocaulus like narciclasine (also known as lycoricidinol) who is known for its effects on protein biosynthesis. 8 Narciclasine oil is effective in the treatment for uterine tumors. It also acts as a plant growth modulator. 9 Narciclasine's first bioactivity was observed in 1967, where it was shown to have strong mitosis blocking activity. 10 The reported biologically effects of narciclasine includes: anti-proliferative, antitumor/cytotoxic, acetylcholinesterase inhibitory, analgesic, hypotensive, antibacterial and antifungal. 11 Data from studies on HeLa cell line showed that narciclasine and other alkaloids like lycorine, hidydrolycorine, haemanthanime, pretazettine and pseudolycorine can inhibit growth and protein synthesis. 12 Not only narciclasine but also other alkaloid derivatives of Amaryllidaceae were reported to have anti-cancer effects. 13,14 However, narciclasine's group is found to be most potent and effective against cell growth and protein synthesis. 6,15,16 Narciclasine is also found active on murine (p388 lymphoma) and human cancer cell lines (e.g. A549, NS-CLC, PC3 and prostate). Narciclasine was also proposed as potential tool to cure apoptosis resistant metastasizing cancer cells. 17 A study conducted on a panel of 60 human cancer cell lines showed narciclasine's potential cytotoxic effects. 15 Another study reported anti-cancer effects of narciclasine on a variety of cancer cell lines, where fibroblasts were reported to be comparably resistant. 18 Sensitivity of cancer cells to narciclasine was also reported using HUVECs (endothelial) cells. 19 In a series of mechanistic studies, narciclasine was found to induce apoptosis driven cell death in cancer cells either mediated by the death receptors or mitochondria. 18 It was further confirmed using human promyeloic (HL-60) cells and human oral cavity squamous carcinoma (HSC-2 cells) that it can induce apoptosis even at nano-molar concentrations. 20 The above literature highlighted the significant biological effects of Amaryllidaceae family's natural isolates and hemi-synthetic derivatives and advocates further investigations to dis-cover their anti-cancer tendencies. Therefore, the current study aimed at evaluating the antiproliferative activities of isolated compounds and hemisynthetic derivatives from the whole plant of S. pseudocaulus.

Materials and methods Plant material
Whole plant of Scadoxus pseudocaulus was collected at Dschang, Menoua Division, West Region of Cameroon, in May 2013. The plant material was identified by Mr. Victor Nana, a botanist at the National Herbarium, Yaoundé, where a voucher specimen (N° 34986/SRF/CAM) was deposited.

Extraction, isolation and hemi-synthesis of compounds
The extraction and isolation of compounds were done as previously described. 1 Briefly, S. pseudocaulus was air-dried and powdered. The powder was macerated at room temperature with MeOH to afford the MeOH extract. The CHCl3 and EtOAc fractions from the MeOH extract were collected by column chromatography. Purification of the EtOAc fraction yielded seven known compounds ( Figure 1). Esterification reaction of compound 6 was carried out using butyroyl chlorides and triethylamin to produce two derivatives. The structures of isolated compounds and derivatives were determined thrugh NMR and MS. The data thus obtained was compared with those from the literature.

Cytotoxicity assay
To assess the anti-cancer potential of natural isolates and hemi-synthetic analogs, Burkitt's and Follicular lymphoma (B lymphoma) cell lines were selected. These cell lines were a gift from Prof.

Statistical analysis Data was analyzed by one-way analysis of variance followed by Waller-Duncan Post Hoc test and Statistical
Package for Social Sciences software (SPSS, version 12.0). The results were expressed as mean ± standard deviation (SD). Differences between groups were considered significant when p < 0.05.

Cytotoxic activities
The cytotoxic activities of compounds 1-9 were evaluated against two cancer cell lines and normal non-cancer cells (Vero cells) (  (Figures 2 -3). For Raji cells, 114.47 µM concentration of compound 5 was required for the induction of death in 50% cell population, however, 134.28 µM were found sufficient for DoHH2 cells (

Discussion
The current investigation was carried out to evaluate the antiproliferative activities of isolated compounds and hemisynthetic derivatives from the whole plant of S. pseudocaulus. The findings of the present study revealed lesser sensitivity of Raji cells to compound 5 that can be due to EBV (Epstein Barr Virus) infection. This virus encodes a number of viral proteins that makes these cells resistant to apoptosis and promote growth. [29][30][31] One of these proteins is latent membrane protein (LMP2A) that is membrane bound and shares structural resemblance to B cell surface receptor protein (BCR). LMP2A is known to play role in relaying survival signal thus promoting survival of B lymphoma cells. [32][33][34] 7-deoxy-trans-dihydronarciclasin, (an enantiomer) is a well reported potent anti-neoplastic agent. 35 Trans derivative of narciclassine is found to be more active as compared to cis form. 13 7-deoxy-trans-dihydronarciclasin has significant anti-cancer effects and the detailed SAR studies elucidated that this molecule has pharmacophore moiety which induces apoptosis. 15,19,[36][37][38][39][40] The tri-hydroxyl-ated ring C of this compound is also considered to be a critical part. In fact, the substitution of tri-hydroxylated ring C with hydrophobic moieties led to decrease in anti-cancer capacity and the same was observed upon loss of three hydroxyl groups. 37,[41][42][43][44] Thus, it could be seen that 7-deoxy-trans-dihydronarciclasin has significant cytotoxic effects and can induce apoptosis in cancer cell lines making it a lead molecule in cancer research. The antiproliferative effects of sideroxylin against ovarian cancer cells are through the induction of mitochondrial dysfunction and the activation of PI3 K and MAPK signal transduction. 45 Selectivity Index (SI) of active compounds was determined in order to investigate, whether the cytotoxic activity was specific to cancer cells. The SI of the samples is defined as the ratio of cytotoxicity (IC50 values) on normal cells (Vero cells) to cancer cells: SI = IC 50 on Vero cells/IC50 on cancer cells. The Selectivity Index (SI) values of the compound 5 against the Raji and DOHH2 cells cells are 13.07 and 11.14 and could be considered as good when taking in consideration that the ratio for a good therapeutic index for a remedy or drug should be ≥10. 46 These results are consistent with the use of compound 5 for treating B lymphoma.

Conclusion
Present investigation has revealed that isolated compounds and hemisynthetic derivatives from S. pseudocaulus are active against the tested cancer cell lines and non toxic against Vero cells (non-cancer cells). The pattern of response revealed that EBV infected Burkitt lymphoma is less sensitive to 7-deoxy-trans-dihydronarciclasin as compared to Follicular lymphoma. Greater cytotoxic effect on slow growing follicular lymphoma signifies its metabolic stability which can be exploited for slow progressing malignancies. Further investigations are needed to screen them against other cancer types and human cell line of normal tissues, including bone marrow cells to justify the traditional use of S. pseudocaulus as an anticancer substance. Detailed mechanistic studies are also needed to find the mode of action of this compound on the molecular pathway(s) potentially leading to cell death.